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RME2013 1 21-23 January 2013, the Netherlands

FINAL PROGRAMME & ABSTRACTS OF LECTURES AND POSTERS

THE RME CONFERENCE SERIES

8TH CONFERENCE

RME2013

Food Feed Water Analysis innovations and breakthroughs!

21-23 January 2013 Noordwijkerhout, the Netherlands

2 RME2013 21-23 January 2013, the Netherlands

THE RME CONFERENCE SERIES – 8TH CONFERENCE

SILVER SPONSOR

EXHIBITORS AB SCIEX BioDot Bruker Nederland Campro Scientific / Fluid Management Systems Holomic LLC MP Biomedicals QIAGEN Scienion AG Shimadzu Europa GmbH Thermo Scientific SECRETARIAT Bastiaanse Communication P.O. Box 179 NL-3720 AD Bilthoven the Netherlands T +31 30 2294247 F +31 30 2252910 [email protected] www.bastiaanse-communication.com


CONTENTS Conference history 4 Welcome 5 Programme 6-17 Programme at a glance 6 Conference programme 7-15 Workshop programme 16-17 Mobile LC-MS/MS lab 18 Abstracts of lectures 19-97 Plenary meeting : From science to application 19-24 Parallel session 1 : Sampling and sample preparation for microbial analysis 25-32 Parallel session 2 : Sampling and sample preparation for contaminant analysis 33-39 Plenary Keynote Lectures 40-41 Parallel session 3 : Advances in microbial analysis 42-48 Parallel session 4 : Advances in contaminant analysis 49-57 Parallel session 5 : Simple, cost-effective and reliable technologies for quality and safety 58-63 Parallel session 6 : Advances in contaminant analysis 64-71 Plenary meeting : The laboratory practice – technical presentations 72-76 Plenary Keynote Lectures 77-79 Parallel session 7 : Advances in multi-analyte detection of microorganisms and contaminants 80-86 Parallel session 8 : Alternative biosensor recognition elements for microorganisms and contaminants 87-91 Plenary meeting : Coming near – testing with a smartphone 92-97 Abstracts of posters 98-134 Index 98-101 Abstracts 102-134 Key to the abstracts of lectures and posters:

abstracts of lectures and posters are grouped separately;

lectures are grouped according to the daily programme;

posters are grouped in an alphabetical order according to the corresponding author. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of RME2013. No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein.


THE RME CONFERENCE SERIES CONFERENCE HISTORY In 2004, the RME conference series started as ‘Rapid Methods Europe’, providing a reference source for anyone interested in the rapid detection of biological and chemical contaminants in food, feed and the environment. During the years it has strengthened its position as an important meeting point for academia and industry. The term ‘rapid’ is used variously in discussions of rapid methodology and instrumentation. It should be noted that ‘rapid’ is not a goal in itself. In addition to increased speed, rapid methods must also take account of other criteria such as sampling and sample preparation, multitarget screening, lower detection limits, accuracy and sensitivity, data analysis, total costs proportionate to the benefits, etc., eventually leading to methods best suited for use. The RME conference series focuses on the developments in the shift from slower, traditional measurements to more rapid methods for microbial and chemical analysis of food, feed and water analysis ensuring safety and quality. ADVISORY COMMITTEE Dr. Aart van Amerongen Food & Biobased Research, Wageningen UR, the Netherlands Dr. Daniel Barug Ranks Meel, the Netherlands Helena B. Bastiaanse Bastiaanse Communication, the Netherlands Prof.dr. Sarah De Saeger Department of Bio-analysis, Ghent University, Belgium Bram van der Gaag KWR Watercycle Research Institute, the Netherlands Prof.dr. Dick Heederik Institute for Risk Assessment Sciences, Utrecht University,

the Netherlands Dr. Lourens Heres VION Food Group, the Netherlands Dr. Gerrit Keizer Prionics Lelystad, the Netherlands Dr. Kitty Maassen National Institute for Public Health and the Environment,

the Netherlands Dr. Bert Popping Eurofins Scientific Group, UK Dr. Michele Suman Barilla, Italy


THE RME CONFERENCE SERIES – 8TH CONFERENCE WELCOME Continuing the successful series of Rapid Methods Europe conferences, RME2013 takes place in Noorderwijkerhout, the Netherlands, 21-23 January 2013. RME2013 is the 8th in a series of conferences dedicated to innovations and breakthroughs in microbiological and chemical analysis of food, feed and water. This series aims to further strengthen the academia-industry relations and to further disseminate advanced research towards practical applications in food, feed and water analysis. RME2013 presents new and cutting-edge technologies focusing on multitarget screening, lower detection limits, wider range of matrices and shorter time, with an eye toward looking for unknown compounds and moving tests out of the laboratory. As a comprehensive overview, RME2013 offers an excellent way to network and to share ideas, providing a reference source for anyone wishing to gain insight into the latest developments in microbial and chemical analysis of food, feed and water ensuring safety and quality. RME2013 features:

Plenary lectures & parallel sessions

Reviews and case studies

Poster sessions

Interactive workshops & demonstrations

Instrument & manufacturers exhibition

Mobile LC-MS/MS lab

B2B meetings As a comprehensive overview RME2013 offers an excellent way to network and to share ideas, providing a reference source for anyone wishing to gain insight into the latest developments in microbial and chemical analysis for food, feed and water ensuring safety and quality. You are cordially invited to take part in the discussions with participants from different disciplines and to meet business relations in your area. We wish you an active and fruitful meeting! On behalf of the Advisory Committee,

Dr. Daniel Barug


PROGRAMME AT A GLANCE Monday 21 January 2013

12:45 – 14:45 Plenary meeting From science to application

Instrument & manufacturers exhibition Mobile LC-MS/MS lab

15:15 – 17:30 Parallel session 1 Sampling and sample preparation for microbial analysis

Parallel session 2 Sampling and sample preparation for contaminant analysis

17:30 – 18:30 Workshops & demonstrations Poster viewing

18:30 – 19:30 RMEs’ Lounge Party

20:00 – 22:00 Excursion to Space Expo

Tuesday 22 January 2013

08:30 – 09:45 Plenary Keynote Lectures

Instrument & manufacturers exhibition Mobile LC-MS/MS lab

09:45 – 12:30 Parallel session 3 Advances in microbial analysis

Parallel session 4 Advances in contaminant analysis

12:30 – 13:30 Workshops & demonstrations Poster viewing

13:30 – 15:15 Parallel session 5 Simple, cost-effective and reliable technologies for quality and safety

Parallel session 6 Advances in contaminant analysis

15:45 – 17:15 Plenary meeting The laboratory practice

17:30 – 18:30 Poster viewing & drinks Question Hour with Prof.dr. Daniel Fung B2B meetings

20:00 Conference dinner (reservations only)

Wednesday 23 January 2013

08:45 – 09:40 Plenary Keynote Lectures

Instrument & manufacturers exhibition

09:40 – 11:00 Parallel session 7 Advances in multi-analyte detection of microogranisms and contaminants

Parallel session 8 Alternative biosensor recognition elements for microorganisms and contaminants

11:30 – 12:30 Plenary meeting Coming near: testing with a smartphone


CONFERENCE PROGRAMME

THE RME CONFERENCE SERIES 8TH CONFERENCE

RME2013

Food Feed Water Analysis innovations and breakthroughs!

MONDAY 21 JANUARY 2013 12:45 Opening of RME2013 Plenary meeting: From science to application Start-up companies that are lead by scientists are sporadically successful. Which bridges have to be built and crossed to walk the road of success? Chair: Dr. Gerrit Keizer, Prionics Lelystad, the Netherlands 13:00 Session outline by Dr. Gerrit Keizer, Prionics Lelystad, the Netherlands 13:05 Valorisation of diagnostic innovations

Prof.dr. Benedikt Sas, Chief Business Officer, Corporate & Innovation Management, Food2Know, Ghent University, Belgium

13:35 Case study: Concept to manufacture – a case study in taking innovative lateral flow

devices to market Chris Danks, Forsite Diagnostics, UK

13:55 Case study: A high tech company is like any other company: it's all about .....

(launching) clients! Jos-Willem Verhoef, Optiqua Technologies, Singapore

14:15 Keynote Presentation: Horizon 2020, the EU Framework Programme for Research

and Innovation. What's in it for me? Dieter Brigitta, DG Research & Innovation, European Commission, Belgium

14:45 Networking break & exhibition


MONDAY 21 JANUARY 2013 Parallel session 1: Sampling and sample preparation for microbial analysis While the importance of sampling and sample preparation in developing the total analytical system for microorganisms is acknowledged, it is not often understand just how challenging this issue really is. Chair: Dr. Lourens Heres, VION Food Group, the Netherlands 15:15 Implications of bacterial clustering for sampling plans

Dr. Ursula Gonzales-Barron, Biosystems Engineering, University College Dublin, Ireland

15:40 Pre-analytical sample preparation: past disappointments, present knowledge and

future hopes Prof.dr. Jeffrey Hoorfar, Division of Food Microbiology, National Food Institute, DTU Food, Denmark

16:05 Sample preparation-free detection of microorganisms in water: optical trapping and

Raman spectroscopy Dr. Jaap Caro, Department of Imaging Science and Technology, Delft University of Technology, the Netherlands

16:30 Challenges in microbial sampling in the indoor environment: fast or slow?

Prof.dr. Dick Heederik, Institute for Risk Assessment Sciences, Utrecht University, the Netherlands

16:55 Remote control strategy for combined microbiological auto-sampling and on-line

chemophysical monitoring at drinking water resources: event and time series analysis Dr. Hermann Stadler, Joanneum Research, Department for Water Resources Management, Austria

17:15 Sample preparation strategies for rapid diagnostic methods

Dr. Ronald van Doorn, Innosieve Diagnostics BV, the Netherlands 17:30 – 18:30

Workshops & demonstrations (see pp. 16-17) Poster viewing

18:30 – 19:30

RME’s Lounge Party 20:00 – 22:00

Excursion to Space Expo, Europe's first permanent space exhibition. See what it was like for the first men on the moon. Experience the simulated launch of an Ariane rocket 'live'. Discover the world of satellites with the test models of European space missions. Find out all about astronauts and see how they live and work in space.


MONDAY 21 JANUARY 2013 Parallel session 2: Sampling and sample preparation for contaminant analysis Sample preparation can be the bottleneck in analytical methods. The range of potential chemical contaminants is diverse and samples cover a wide range of physical matrix types. What are the challenges encountered? Chair: Dr. Franz Ulberth, Institute for Reference Materials and Measurements, Joint

Research Centre, Belgium 15:15 Legislative requirements for sampling

Dr. Franz Ulberth, Institute for Reference Materials and Measurements, Joint Research Centre, Belgium

15:40 Improved certainty from sampling uncertainty

Prof.dr. Michael H. Ramsey, School of Life Sciences, University of Sussex, UK 16:05 Sample preparation strategies and techniques for food contaminant analysis

Dr. Kathy Ridgway, Investigative Analysis Department, Reading Scientific Services Limited, UK

16:30 Matrix effects: the foe of fast LC-MS/MS based multi-analyte methods

Dr. Michael Sulyok, BOKU Vienna, Department IFA-Tulln, Austria 16:55 Mycotoxin analysis in grain dusts – a promising field of application for rapid test

systems Mareike Reichel, Eurofins WEJ Contaminants, Germany

17:15 On-line SPE-TOX monitoring: the end of lab toxicity testing

Dr. Varvara Kokkali, Vitens Water Technology, the Netherlands 17:30 – 18:30

Workshops & demonstrations (see pp. 16-17) Poster viewing

18:30 – 19:30

RME’s Lounge Party 20:00 – 22:00

Excursion to Space Expo, Europe's first permanent space exhibition. See what it was like for the first men on the moon. Experience the simulated launch of an Ariane rocket 'live'. Discover the world of satellites with the test models of European space missions. Find out all about astronauts and see how they live and work in space.


TUESDAY 22 JANUARY 2013

Plenary Keynote Lectures 08:30 Rapid analysis in forensic science: mayhem or magic? Prof.dr. Niamh Nic Daeid, Centre for Forensic Science, University of Strathclyde, UK 09:00 Rapid methods in microbiology: are we still getting simpler, faster, better? Prof.dr. Daniel Y.C. Fung, Department of Animal Sciences and Industry, Kansas State University, USA

Parallel session 3: Advances in microbial analysis The future of rapid methods in microbiology is very bright with many new developments and applications. This session provides an insight into selected areas of microbial analysis where there have been recent advances or where the pathogen itself has some topicality. Chair: Dr. Aart van Amerongen, Food & Biobased Research, Wageningen UR, the

Netherlands 09:45 Detection of pathogenic Escherichia coli in food: the experience of the European

Reference Laboratory for VTEC in methods' development Dr. Rosangela Tozzoli, Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare, Istituto Superiore di Sanità, Italy

10:10 Rapid detection of foodborne MRSA and ESBLs

Prof.dr. Jeffrey Hoorfar, Division of Food Microbiology, National Food Institute, DTU Food, Denmark

10:35 Abandoning the surrogates for potential cyanobacterial hazards: a plea for cyanotoxin

analysis Dr. Miquel Lürling, Aquatic Ecology and Water Quality Management Group, Wageningen UR, the Netherlands

11:00 Networking break & exhibition 11:30 Application of laser induced breakdown spectroscopy and neural network: rapid

identification and discrimination of bacterial strains Sadia Manzoor, Department of Analytical Chemistry, University of Madrid, Spain

11:55 A cylinder as a platform of multi-analyte diagnostics

Mag. Christoph Reschreiter, Anagnostics Bioanalysis GmbH, Austria 12:10 GENSPEED – a new tool for rapid and multiplexed microbial analysis

Dr. Max Sonnleitner, Lambda, Austria 12:30 – 13:30

Networking break & exhibition Workshop & demonstrations (see pp. 16-17) Poster viewing


TUESDAY 22 JANUARY 2013

Plenary Keynote Lectures 08:30 Rapid analysis in forensic science: mayhem or magic? Prof.dr. Niamh Nic Daeid, Centre for Forensic Science, University of Strathclyde, UK 09:00 Rapid methods in microbiology: are we still getting simpler, faster, better? Prof.dr. Daniel Y.C. Fung, Department of Animal Sciences and Industry, Kansas State University, USA

Parallel session 4: Advances in contaminant analysis This session provides an insight into selected areas of contaminant analysis where there have been recent advances or where the contaminant itself has some topicality. Information on analytical approaches is provided, but the main emphasis is on applications. Chair: Prof.dr. Sarah De Saeger, Department of Bio-analysis, Ghent University, Belgium 09:45 Finding the needle in the haystack: non-targeted analysis for identifying chemicals in

food and drink Dr. Adrian J. Charlton, Department for Environment, Food & Rural Affairs, The Food and Environment Research Agency, UK

10:10 Rapid methods for mycotoxins screening in the food industry – an overview of current

solutions and future perspectives Dr. Michele Suman, Food Research Labs, Barilla, Italy

10:35 Are all allergens detection methods created equal? New findings on methods

development and comparison, allergen trigger levels and processed products Dr. Bert Popping, Eurofins Scientific Group, UK

11:00 Networking break & exhibition 11:30 Single copy GMO detection using bioluminescent real time reporter (BART) of loop

mediated isothermal amplification (LAMP) Patrick Hardinge, School of Biosciences, Cardiff University, UK

11:50 Development of rapid extraction methods and hyphenated techniques for

determination of mineral oil hydrocarbons: the focus on MOSH and MOAH in foods Dr. Laura Barp, Department of Food Science, University of Udine, Italy

12:10 On-line quality control of drinking water: fact or fiction?

Dr. Bendert de Graaf, Vitens, the Netherlands 12:30 – 13:30

Networking break & exhibition Workshop & demonstrations (see pp. 16-17) Poster viewing


TUESDAY 22 JANUARY 2013 Parallel session 5: Simple, cost-effective and reliable technologies for quality and safety Food, feed and water analysis for quality and safety: the progress made in terms of costs, time and simplicity. Chair: Dr. Michele Suman, Barilla, Food Research Labs, Italy 13:30 Printed functional solutions for diagnostic platforms

Dr. Leena Hakalahti, VTT Technical Research Centre of Finland, Finland 13:50 Nitrocellulose membranes as a template for novel immunoassays: variations in

colours and shapes Dr. Denise van Rossum, Sartorius Stedim Biotech GmbH, Germany

14:10 Diagnostic applications in roll-to-roll hot-embossed microfluidics

Christina Liedert, M.Sc., VTT Technical Research Centre of Finland, Finland 14:30 Rapid microarray diagnostic platforms using carbon nanoparticles

Dr. Aart van Amerongen, Food & Biobased Research, Wageningen UR, the Netherlands

14:50 Fixing surface properties, a key process for new biobased innovations

Dr. Luc Scheres, Surfix, the Netherlands 15:15 Networking break & exhibition Plenary meeting: The laboratory practice – technical presentations Recent developments in food, feed and water analysis: sponsor & exhibitor presentations. Chair: Bram van der Gaag, KWR Watercycle Research Institute, the Netherlands 15:45 Planar protein arrays in microtiter plates – development of a new format towards

accurate, automation-friendly and affordable (A3) diagnostics Hans Dijk, Scienion AG, Germany

16:05 Determination of pesticides and organic contaminants in aqueous samples using

automated SPE Wim Traag, RIKILT-Institute of Food Safety, the Netherlands

16:25 Innovative online sample preparation techniques in food contaminant analysis

Dr. Laszlo Hollosi, Thermo Scientific, Germany 16:45 PesticideScreener: a fast and comprehensive solution for multi-target screening of

pesticides using accurate-mass UHR-QTOF mass spectrometry and an information-rich (700 compound) database Rob van der Heijden, Bruker Nederland, the Netherlands

17:05 Shimadzu total solution for quality defects in food, feed and water

Johan Scholtens, Shimadzu Benelux, the Netherlands 17:30 – 18:30

Poster viewing & drinks Question Hour with Prof.dr. Daniel Fung B2B meetings


TUESDAY 22 JANUARY 2013 Parallel session 6: Advances in contaminant analysis The story continues. Chair: Dr. Bert Popping, Eurofins Scientific Group, UK 13:30 On-line fingerprinting of fluids for quality control using coaxial stub resonator

technology Natalia Hoog-Antonyuk, M.Sc., Wetsus, the Netherlands

13:50 Characterisation of the performance of anti-gliadin antibodies

Sonia J. Miguel, DNA and Protein Department, Reading Scientific Services Ltd., UK 14:10 Challenges in POP analyses – 14 years after the Belgian dioxin chicken gate

Dr. Jean-François Focant, Department of Chemistry, University of Liège, Belgium 14:30 Developments in analytical methods for detection of biogenic amines in food

Prof.dr. Attila Kiss, Egerfood Knowledge Centre, Eszterházy Károly University, Hungary

14:50 U-HPLC-Orbitrap-MS for quantification of known and identification of unknown boar

taint contributing compounds Prof.dr. Lynn Vanhaecke, Department of Veterinary Public Health and Food Safety, Ghent University, Belgium

15:15 Networking break & exhibition Plenary meeting: The laboratory practice – technical presentations Recent developments in food, feed and water analysis: sponsor & exhibitor presentations. Chair: Bram van der Gaag, KWR Watercycle Research Institute, the Netherlands 15:45 Planar protein arrays in microtiter plates – development of a new format towards

accurate, automation-friendly and affordable (A3) diagnostics Hans Dijk, Scienion AG, Germany

16:05 Determination of organic contaminants in aqueous samples using automated SPE

Wim Traag, RIKILT-Institute of Food Safety, the Netherlands 16:25 Innovative online sample preparation techniques in food contaminant analysis

Dr. Laszlo Hollosi, Thermo Scientific, Germany 16:45 PesticideScreener: a fast and comprehensive solution for multi-target screening of

pesticides using accurate-mass UHR-QTOF mass spectrometry and an information-rich (700 compound) database Rob van der Heijden, Bruker Nederland, the Netherlands

17:05 Shimadzu total solution for quality defects in food, feed and water Johan Scholtens, Shimadzu Benelux, the Netherlands 17:30 – 18:30

Poster viewing & drinks Question Hour with Prof.dr. Daniel Fung B2B meetings


WEDNESDAY 23 JANUARY 2013

Plenary Keynote Lectures 08:30 Biosensors for food and the environment: is it all in the DNA? Dr. Valerio Beni, Department of Physics, Chemistry and Biology, Linköping University, Sweden 09:00 Does synthetic biology offer a new approach for biosensor design? Prof.dr. Paul Freemont, co-director of the EPSRC National Centre for Synthetic Biology and Innovation, Imperial College London, UK

Parallel session 7: Advances in multi-analyte detection of microorganisms and contaminants Multi-analyte testing enables the simultaneous detection of a broad range of target components. Several platforms will be presented and the pros and cons will be discussed. Chair: Dr. Kitty Maassen, National Institute for Public Health and the Environment, the

Netherlands 09:40 Recent developments in rapid multiplexed bioanalytical methods for foodborne

pathogenic bacteria detection Barbara Roda, Department of Chemistry 'G. Ciamician', University of Bologna, Italy

10:00 Sophisticated application of standard ELISAs for multi-analyte detection

Dr. Alex Räber, Research & Development Department, Prionics, Switzerland 10:20 FTIR spectroscopy: source tracking and monitoring of microbial contamination in food

industry Volha Shapaval, Nofima AS and Department of Mathematical Sciences and Technology, Norwegian University of Life Sciences, Norway

10:40 Natural receptor-based binding assays as screening tool for toxicity prediction

Bram van der Gaag, KWR Watercycle Research Institute, the Netherlands 11:00 Networking break & exhibition Plenary meeting: Coming near – testing with a smartphone Testing with a smartphone is nearer than you can imagine! Chair: Dr. Bert Popping, Eurofins Scientific Group, UK 11:30 Computational technologies toward telediagnosis and telemicroscopy

Dr. Neven Karlovac, Holomic LLC, USA 12:00 A lab-on-a-chip integrated into a smart system: a strategy to fulfil real diagnostic

needs Dr. Jesús M. Ruano-Lopez, IKERLAN, Spain

12:30 Closing of RME2013


WEDNESDAY 23 JANUARY 2013

Plenary Keynote Lectures 08:30 Biosensors for food and the environment: is it all in the DNA? Dr. Valerio Beni, Department of Physics, Chemistry and Biology, Linköping University, Sweden 09:00 Does synthetic biology offer a new approach for biosensor design? Prof.dr. Paul Freemont, co-director of the EPSRC National Centre for Synthetic Biology and Innovation, Imperial College London, UK

Parallel session 8: Alternative biosensor recognition elements for microorganisms and contaminants A lot of research effort has been dedicated to the development of novel biosensor recognition elements with improved characteristics. Learn about the latest developments. Chair: Dr. Gerrit Keizer, Prionics Lelystad, the Netherlands 09:40 Alternative biorecognition elements for food and environmental biosensors

Dr. Johan Robbens, Animal Sciences Unit, Institute for Agricultural and Fisheries Research, Belgium

10:00 Advantages of llama single-domain antibody fragments in diagnostic applications of

infectious diseases, with a focus on Campylobacter jejuni Dr. Michiel Harmsen, Central Veterinary Institute, Wageningen UR, the Netherlands

10:20 Detection of heavy metals and other contaminants with bioluminescent

microorganisms Prof.dr. Gérald Thouand, University Institute of Technology of La Roche-sur-Yon, University of Nantes, France

10:40 Integration of live cells with the organic electrochemical transistor for in vitro detection

of pathogens and toxins Dr. Róisin Owens, Department of Bioelectronics, École Nationale Supérieure des Mines de Saint-Étienne, France

11:00 Networking break & exhibition Plenary meeting: Coming near – testing with a smartphone Testing with a smartphone is nearer than you can imagine! Chair: Dr. Bert Popping, Eurofins Scientific Group, UK 11:30 Computational technologies toward telediagnosis and telemicroscopy

Dr. Neven Karlovac, Holomic LLC, USA 12:00 A lab-on-a-chip integrated into a smart system: a strategy to fulfil real diagnostic

needs Dr. Jesús M. Ruano-Lopez, IKERLAN, Spain

12:30 Closing of RME2013


WORKSHOP PROGRAMME

AB SCIEX Monday 21 January 2013 17:30 – 18:30 Workshop ‘New tools for food and environmental analysis’ This workshop will present new instrument and software which has been used to speed up and reduce analyses and increase confidence in your results. Two main topics will be covered. Part I The application of Micro LC to food analysis. This part of the workshop will cover the

combination of Micro LC with an LC-MS/MS method developed on an AB SCIEX QTRAP® 4500 system, which was applied to a screen of over 100 pesticides in QuEChERS food extracts of a chili powder, a matrix notorious for producing dirty extracts. It will discuss the technology and the merits of using MicroLC which can offer huge cost reductions for routine analysis.

Part II The second section of the workshop will highlight the use of the AB SCIEX

TripleTOF 4600 and 5600+ systems for accurate mass screening for contaminants in food and environmental samples. The workflows, system attributes and unique software that enable fast and accurate screening for contaminants in a variety of matrices will be highlighted.

AB SCIEX Tuesday 22 January 2013 12:30 – 13:30 Workshop ‘New applications in food analysis’ This workshop will present new advancements in application development from AB SCIEX. Two main topics will be covered. Part I Detection of allergens in food by LC/MSMS. The prevalence of food allergies is

estimated at around 6% for children (in the USA) and reports suggest that the number of allergies is rising. Screening for allergens is traditionally performed using enzyme-linked immunosorbent assays (ELISAs), but ELISAs can generate variable results and false-positives. This workshop will give an overview of the use of LC-MS/MS to detect allergens such as gluten, egg, milk, mustard and peanut and will discuss the feasibility of such an approach.

Part II The second section of the workshop will look at new areas of food ingredient

analysis and will give insight into new approaches to the analysis of fat and water soluble vitamins, sweeteners, amino acids and other additives or ingredients. It will be shown how recent advances in LC-MS/MS technology and instrumentation are enabling scientists to push the limits in mass spectrometry for the analysis of additives and ingredients in complex food and feed matrices.


BIODOT Monday 21 January 2013 17:30 – 18:30 Tuesday 22 January 2013 12:30 – 13:30 Workshop ‘Advances in manufacturing of lateral flow tests’ Part I BioDot will have the batch set of equipment for the production of diagnostic tests.

The basic steps will be presented of dispensing antigens/antibodies, conjugates, buffers, etc., followed by lamination and cutting. There will also be a USB microscope mounted on a platform to examine the effects of the different types dispenser, non-contact and contact, and the effects of different materials. This will be presented on a large screen TV for examination of the group.

Part II In-line processing for high throughput and improved reproducibility. A questions and

answers section, and the ability to have one-to-one hands-on the equipment. Part III Symbolics – advances in the quantification of lateral flow tests.

QIAGEN Monday 21 January 2013 17:30 – 18:30 Tuesday 22 January 2013 12:30 – 13:30 Workshop ‘PCR-based methods for successful food safety testing’ In a globalized food market with increasing demand for food research and monitoring, there is a need for streamlined testing solutions that are sensitive, accurate, and easy to use with a variety of starting materials. New technologies that deliver results significantly faster are increasingly being utilized in the food testing field. One such technology, refined in recent years to make it suitable for use in food safety testing, is polymerase chain reaction (PCR). PCR is a method that can detect pathogens in food samples by amplifying specific fragments of DNA. These fragments are either detected at the end of the amplification process (end-point PCR) or during the amplification (real-time PCR). Real-time PCR is a more widely accepted method and is more commonly used in routine food safety testing applications. Detection using PCR is much faster than traditional bacterial culture methods. Real-time PCR methods have evolved over the past 10 years from niche applications, reserved for use by experts, to modern, well-proven workflows that can be used in food testing facilities worldwide to ensure high quality food production. This workshop provides an overview of QIAGEN´s mericon product line, a complete system for sample preparation and assays that meets the demands listed above. Complementing PCR-based detection assays, QIAGEN offers the innovative QIAsymphony RGQ system, an automated workflow solution, providing standardized pathogen analyses for a broad variety of food samples. The QIAsymphony RGQ system in combination with the mericon Salmonella spp. kit has received validation from the AOAC Research Institute, an independent third party organization that evaluates analytical methods for use in food safety and other testing. PCR-based technologies will continue to evolve to provide improved means of guaranteeing safe, high-quality food. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN Kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.


THE COMPLETE MOBILE LC-MS/MS LAB MONDAY 21 JANUARY & TUESDAY 22 JANUARY 2013 Free to attend during the conference: a unique mobile LC-MS/MS laboratory with live demonstrations! Experts in LC-MS/MS solutions are on board the mobile laboratory, available to talk to the participants of RME2013 about a broad array of topics. See advertisement next page.


LECTURES VALORIZATION OF DIAGNOSTIC INNOVATIONS Benedikt Sas Chief Business Officer, Corporate & Innovation Management, Food2Know, Ghent University, Belgium [email protected] Doing research is fun and often results are published in peer reviewed papers which hopefully are read by members of the scientific community or industry. From an economical and cash flow perspective, however, it is better that research results in innovations that can be commercialized with a good return. This is called valorization. This presentation gives a short overview of the ‘from bench to market’ pathway, with a focus on the different aspects of valorization such as intellectual property (IP), market size and royalty rates.


CONCEPT TO MANUFACTURE: A CASE STUDY IN TAKING INNOVATIVE LATERAL FLOW DEVICES TO MARKET Chris Danks Forsite Diagnostics, UK [email protected] Forsite Diagnostics was founded in 2007 as a spin out from a UK government research agency. The foundation for this bold move was solely on the basis of a novel range of products known as Pocket Diagnostics. These are a range of lateral flow devices for plant pathogen disease in the field. The initial customers being government inspectors but following the success of their regulated use, the decision to make them available worldwide to the plant health industry was made and a company born. From innovative product beginnings Forsite Diagnostics has flourished to become a leading manufacturer of lateral flow devices, with exceptional facilities located on the Applied Innovation Campus in York, UK. Following two rounds of external investment and changes in ownership alongside significant changes to its business model and strategic plans, Forsite has now actively diversified from plant heath diagnostics into veterinary, food safety and most recently human healthcare diagnostic markets, through its service R&D and contract manufacturing capabilities. The worldwide financial climate has been challenging for everyone over the last decade, and no easy time for new biotechnology companies. However Forsite Diagnostics has survived, through diversification and product realisation, and this presentation hopes to share some thoughts, examples and lessons learnt on how this has been achieved, and how we aim to continue to grow through continuing innovation.


A HIGH TECH COMPANY IS LIKE ANY OTHER COMPANY: IT’S ALL ABOUT ….. (LAUNCHING) CLIENTS! Jos-Willem Verhoef Optiqua Technologies, Singapore [email protected] Optiqua Technologies provides the water industry with innovative tools for both online and sample based water quality monitoring. All Optiqua products leverage our award winning and patented Optiqua lab-on-chip technology. Based in Singapore and the Netherlands, we serve international drinking water companies to safeguard the distribution of safe drinking water. Cities invest heavily to ensure that their drinking water is safe, and while most treatment facilities keep close watch on their supply, water becomes harder to monitor once it goes into their water distribution networks. Optiqua Technologies has developed an early warning system that utilities can use to detect real time changes in the water quality throughout their networks. The system is called EventLab. The development of the EventLab application has now reached the point where the sensor technology has been successfully validated in demonstration projects with Vitens in the Netherlands and the national drinking water agency PUB in Singapore. The product is commercially available and being marketed to the water industry in collaboration with international partners. Launching partners Vitens and PUB are deploying the EventLab sensors in ever expanding sensor networks. The origin of the Optiqua technology goes back to 1993, when a scientific research programme was initiated by the Integrated Optics group at the University of Twente, the Netherlands. Between 1993 and 2001 the research team, led by Prof. Paul Lambeck, achieved spectacular results by combining multiple technologies into one integrated sensor concept. To bring this technology to a fully commercial application a separate commercial entity was set up in Enschede, the Netherlands. Where most start-up companies are led by scientists, the entrepreneurs behind Optiqua have a background in finance and marketing. The first years of operation were focused on bringing the technology from its scientific concept status into a functional prototype. The company was extended with highly skilled staff from the University of Twente. In close co-operation with the university and with access to the sophisticated cleanroom facilities of the MESA+ institute, Optiqua was able to transfer the scientific proof of principle into a robust and patented optical sensor chip. This sensor chip could be translated into multiple product concepts. We then made a strategic choice to create maximum value to all our stakeholders, not by licensing a technology to industrial partners, but to develop a commercial application ourselves. We also knew, as a high tech SME, that focus is essential. Optiqua decided to focus first on one high growth market: the global water market. Online water quality monitoring plays a pivotal role in the supply of safe drinking water and smart water management. There is a strong and growing demand for new sensor technologies that make it possible to monitor water composition in real-time and identify the location of any contamination events. This not only increases water safety control, but also feeds back important information to the water supply and distribution process and helps to minimize recovery costs. Internationally we see a growing development where water utilities have an interest and invest in intelligent water distribution networks or ‘smart grids’. We realized that we could only be successful in this market with the full commitment of a launching partner. Therefore, Optiqua engaged in a co-development and launching customer relationship with Vitens, market leader in the Dutch drinking water market. This innovative drinking water company considers real-time monitoring as a key strategic objective for the coming years. Optiqua’s sensor technology was selected as core to this strategic roadmap. In 2007 Optiqua founded a subsidiary in Singapore. Strong government support for the


development of innovative technologies, in combination with the strategic decision to develop Singapore into an international water hub, made Singapore a preferred location. In Singapore we formed a dedicated product development team with expertise in electrical and mechanical engineering, data analysis, system assembly and product calibration. Since then, Optiqua in the Netherlands focuses on development of its core sensor technologies, while in Singapore we develop these technologies into commercially viable products. In 2008, PUB joined Vitens as a launching customer. With PUB as a reference customer, Singapore acts as a gateway to the fast growing Middle Eastern and Asian regions. Our launching customers Vitens and PUB are key in the successful roll out of our products for the following reasons. Firstly, they helped us to define our business proposition (EventLab). They challenged us to broaden our development activities from our sensor technology to all the aspects of an integrated early warning system. This helped us to expand our skill base and to invest other capabilities like data analysis, algorithm development, data communication, and sensor placement selection. Secondly, in bringing science to application many hurdles have been overcome. In joint demonstration projects with Vitens and PUB, key technological challenges were overcome and test product improvements in real life situations were made. Thirdly, successful demonstration projects at Vitens and PUB helped Optiqua to gain credibility with other innovative drinking water companies with a vision for smart water grids. And finally, but certainly not least important, is the relation between launching customers and the funding of a high tech company. The road from science to application can be long, risky and often unpredictable. A successful company must, of course, secure sufficient funding and without the backing of a launching customer the potential of a high tech company is difficult to judge for an equity investor. Therefore Optiqua’s relationship with Vitens and PUB has been key in attracting long term financing that enables us to be successful in the international water industry Which brings us to the conclusion: Optiqua is like any other high tech company: it’s all about…..(launching) clients!


HORIZON 2020, THE EU FRAMEWORK PROGRAMME FOR RESEARCH AND INNOVATION. WHAT’S IN FOR ME? Keynote Presentation Dieter Brigitta DG Research & Innovation, European Commission, Belgium [email protected] Horizon 2020 is a proposal by the European Commission for an 80 billion euro research and innovation funding programme for the period 2014-2020. As a core part of the Europe 2020 strategy, Horizon 2020 aims to respond to the economic crisis by investing in future jobs and growth; to address people’s concerns about their livelihoods, safety and environment; and to strengthen the EU’s global position in research, innovation and technology. Novelty. Horizon 2020 is a single programme bringing together three existing, separate initiatives: the 7th Research Framework Programme (FP7), innovation aspects of the Competitiveness and Innovation Framework Programme (CIP), and the EU contribution to the European Institute of Innovation and Technology (EIT). By coupling research to innovation, Horizon 2020 will be able to support projects ranging from research to retail, and thus include all forms of innovation. Furthermore, Horizon 2020 will focus on societal challenges facing the EU society (e.g. health, clean energy and transport), and provide simplified access for all companies, universities, institutes in all EU countries and beyond. A clear emphasis on the participation of SMEs is also present, e.g. via a new SME instrument where participation of 1 single applicant will be possible. 3 priorities. Horizon 2020 focuses on 3 priorities:

1. Excellent science. World class science is the foundation of tomorrow’s technologies, jobs and wellbeing. Therefore, Europe needs to develop, attract and retain research talent, and give researchers access to the best infrastructures.

2. Industrial leadership. Strategic investments in key technologies (e.g. advanced manufacturing, micro-electronics) underpin innovation across existing and emerging sectors. Therefore, Europe needs to attract more private investment in research and innovation, and needs more innovative SMEs to create growth and jobs.

3. Societal challenges. The concerns of citizens and society/EU policy objectives (climate, environment, energy, transport etc.) cannot be achieved without innovation. Breakthrough solutions will come from multi-disciplinary collaborations, including social sciences & humanities. Therefore, promising solutions need to be tested, demonstrated and scaled up.

Each priority is subdivided in different themes, each with its own funding proposed by the EC. Implementation. Horizon 2020 will be implemented using different techniques and project types, ranging from Collaborative Projects, Coordination & Support Actions (as currently used in the 7th Research Framework Programme), projects carried out by the European Institute of Innovation & Technology (such as the planned 2014 theme: Food4future, sustainable food supply chain, from farm to fork), European Technology Platforms (e.g. Food4Life), European Innovation Partnerships, and public-private and public-public partnerships. Three planned public-public partnerships in the form of Joint Programming Initiatives (JPIs) require further attention, since


they are relevant for the RME2013 participants: FACCE-JPI (Agriculture, Food Security and Climate Change), the 'A Healthy Diet for A Healthy Life' JPI, and the 'Water challenges for a changing world' JPI. The objective of these JPIs is to increase the value of relevant national and EU R&D funding by concerted and joint planning, implementation and evaluation of national research programmes. In Joint Programming, Member States are expected to coordinate national research activities, group resources, benefit from complementarities and develop common research agendas, in order to face grand societal challenges. Joint Programming intends to tackle the challenges that cannot be solved solely on the national level and allows Member States to participate in those joint initiatives where it seems useful for them. Next steps? Currently, there are many on-going initiatives: the final calls under the 7th Framework Programme for research (to bridge the gap towards Horizon 2020), the European Parliament and Council negotiations on the basis of the Horizon 2020 proposals of the European Commission, but also the Parliament and Council negotiations on the overall EU budget for 2014-2020. It needs to be noted that the Horizon 2020 budget is only part of these Multi-annual Financial Framework (MFF) negotiations. The adoption of Horizon 2020 legislative acts by the European Parliament and Council are expected by mid-2013, whereas the official start of Horizon 2020 –including the launch of the first calls - is planned for January 1st, 2014.


IMPLICATIONS OF BACTERIAL CLUSTERING FOR SAMPLING PLANS Ursula Gonzales-Barron Biosystems Engineering, University College Dublin, Ireland [email protected] Sampling and testing for microorganisms in foods is a risk management strategy used to evaluate whether a food safety system achieves the appropriate level of control. Whether sampling plans to test microorganisms in foods are derived to meet the consumer’s or producer’s quality requirements, the statistical methods proposed for their design have been entirely based on quality control statistics; reason as to why two simplifying assumptions have been traditionally adopted: (i) that the log microbial concentrations among food units produced in a batch can be represented by a normal distribution, and (ii) that its measure of spread (i.e., standard deviation) is constant among batches of production. Furthermore, from these assumptions, it is implicitly understood that the normal distribution is adequate for any contamination level (i.e., low counts or high counts), and that the measure of spread is independent of the contamination level. Nevertheless, recent work has demonstrated that the normality assumption only holds for high microbial concentrations. Heterogeneous Poisson and zero-modified distributions have been proven to be more appropriate to characterise bacterial clustering, low microbial concentrations and microbial data consisting of many zero counts. On the other hand, the within-batch measure of spread is in many cases dependent on the contamination level and associated with the within-batch mean concentration. This phenomenon is very likely to be an effect of the physical agglomeration of bacterial cells. The importance of the choice of the most appropriate distribution to represent bacterial clustering resides on its critical effect on the acceptance probability for the design of sampling plans. Furthermore, the derivation of Operating Characteristic (OC) curves should ideally take into account the shifts in the within-batch measure of spread for the acceptance probability calculation as we move along the x-axis values of within-batch mean concentration. It has been shown that the Poisson-gamma assumption allowing for clustering produces stricter sampling plans than both the lognormal and the Poisson distribution for the same level of safety. Unlike the manufacturing process of non-food items that, when under control, has been proven to operate with only chance (or non-assignable) causes of variation that are an intrinsic part of the process, and therefore leading to a stable variance, in the production of food items, the batch-to-batch variability in the microbiological quality of the food product is expected to be more unstable due to phenomena inherent to the biological systems. To account for the effect of the batch-to-batch variability, a novel approach to jointly model the within-batch and between-batch variability in microbial counts based on a random-effects Poisson-gamma model has been proposed to characterise low and high microbial counts from many batches of production. Thus, in the derivation of an OC curve, the uncertainty around the within-batch spread measure for a given within-batch mean can be propagated by simulation to the probability of batch acceptance. In this way, OC curves are obtained with confidence intervals representing the uncertainty about the spread measure arising from the between-batch variability. The OC curves obtained as such lead to more conservative sampling plans than using classical statistics based on the normal distribution. Thus, new modelling trends based on more realistic assumptions are examined that consider the clustering of bacteria, the intrinsic variability among food batches and the use of past monitoring microbial data.


PRE-ANALYTICAL SAMPLE PREPARATION: PAST DISAPPOINTMENTS, PRESENT KNOWLEDGE AND FUTURE HOPES Jeffrey Hoorfar Division of Food Microbiology, National Food institute, Technical University of Denmark (DTU), Denmark [email protected] Sample preparation is a difficult and filthy job, which is why most scientists prefer to work on the detection step developing fancy analytical methods: nano-sensors and probe techniques are certainly more impressive and more prestigious to work with. But what is the use of high-tech device, when the murky diagnostic sample ‘kills’ the signal. The technology ‘cemetery’ is full of large and micro-devices that have not survived the real-life testing. This dilemma has caused many disappointments in the past. That is why an increasing number of laboratories are turning their focus on the sample preparation step in order to understand the impact of various ingredients and chemicals on subsequent molecular reactions. This is currently creating much generic knowledge with substantial scientific interest. Besides food testing, research grants to projects on bio-terror, metagenomic-based geo-microbiology, genomic anthropology, molecular evolution, etc. are helping to develop better (and hopefully simpler) sample preparation methods that can resist the tough conditions of routine testing. Here, of course the decisive element is the cost: while human cancer diagnostics can pay a rather large sum for a test, food producers are restricted by the relatively low food price. Hence, the hope is to come up with a miniaturized device that can accommodate both the sample preparation and the subsequent detection steps in a single device, indeed in a nano-volume chamber that obviates the cultivation completely. A truly viable alternative to the microbial enrichment step is the long needed revolution we all are waiting for. And there are some indications that this future is not that far away.


SAMPLE PREPARATION-FREE DETECTION OF MICROORGANISMS IN WATER: OPTICAL TRAPPING AND RAMAN SPECTROSCOPY Jacob Caro and T. van Leest Department of Imaging Science and Technology and Kavli Institute of Nanoscience, Delft University of Technology, the Netherlands [email protected] Worldwide research laboratories show many activities in developing new sensing approaches for environment, health and medicine, and drinking water technology. According to the second Strategic Research Agenda by Photonics21 [1], photonic sensors and devices should play a crucial role in this. The Agenda explicitly mentions lab-on-a-chip techniques and Raman spectroscopy in relation to medical diagnostics and biological analysis. In these fields, there is an increasing need of rapid, non-destructive and low-cost methods to identify, label-free analyze and study living human cells, bacteria and viruses, follow the phases of the cycle of single cells and provide detailed information about their metabolism, e.g. in response to drugs, contaminants and toxic molecules in the medium these bio-objects live in. In this contribution, I will present an approach to miniaturization of a laser tweezers Raman spectrometer we are exploring in Delft. As pioneered by Ashkin [2], with conventional laser tweezers a single biological cell can be trapped in the tight focus of a laser beam. In a laser tweezers Raman spectrometer the laser beam used for trapping is also used to generate the Raman spectrum of the immobilized cell, which is a fingerprint of the molecular composition of the cell and thus offers the possibility to indentify the cell. Important advantages of this Raman spectroscopy are that it (i) is non-invasive, (ii) is label-free, and (iii) has a very high discriminating power. Obstacles for widespread and easy application of laser tweezers Raman spectroscopy are the considerable size (≥big microscope) and high costs of the instrument. A further disadvantage is that the laboratory instruments used so far require operation by an expert. The only way to solve this problem is the miniaturization of the tweezers, i.e. its fabrication on a chip. Mass production of the chip enabled by integrated photonics and microfluidics will lead to a completely different cost perspective. Realization of these devices as a lab-on-a-chip advantageously connects Raman spectroscopy to well-established lab-on-a-chip techniques, such as addition of nutrients/drugs and handling of multiple fluidic channels, and does not require labelling or other preparation of the cells to be Raman-fingerprinted.

Figure 1. Artist impression of a photonic crystal with a cavity for optical trapping. The red wavelike structure depicts the strongly enhanced evanescent field of the cavity mode that bulges out of the slab, exerting optical force on a bacterium. In the figure, the cavity cannot be seen, since it is hidden under the trapped bacterium.

One approach we work on is based on a photonic crystal with a cavity, fabricated on a photonic lab-on-a-chip across which water or another medium with the bio-objects to be


analyzed can be flown [3,4]. In crystallographic terms, a cavity is a lattice defect in an otherwise highly regular periodic structure. A cavity works as a resonator for light, similar to a laser. When light is coupled into a cavity, this leads to a strongly enhanced and highly localized electromagnetic field at the cavity, which takes the role of the focus of the conventional laser tweezers. As a result, particles can be trapped on-chip by the optical forces of the cavity. Once immobilized, the local field may also be used to generate the Raman spectrum of the trapped bio-particle. Apart from these functionalities, the chip may also be used as a kind of optical conveyer belt for the propulsion and sorting of cells. An impression of a device is given in Figure 1, where a bacterium trapped at a cavity is sketched. So far, we have studied three types of cavities for trapping [3,4]. They are defined in a 220 nm thick silicon photonic crystal with a regular pattern of nanometer-scale holes. In the trapping experiments we use two types of bacteria, viz. Bacillus subtilis and Escherichia coli, well-known organisms in microbiology. B. subtilis is used in the form of both vegetative cell and spore. The spores, because of their highly defined size, shape and distribution of the refractive index, are ideal biological model particles for quantitative studies, both for experiments and simulations. We clearly observe optical trapping of single bacteria by each of the three cavities [5], when they are pumped at the resonance wavelength. In a sequence for a typical trapping event, the bacterium is supplied to the cavity by the water flow. When coming in reach of the evanescent field of the cavity mode, it becomes trapped at the cavity. The optical force is that strong that it can overcome the drag force of the flowing water. After this demonstration of the principle of optical trapping of bacteria with a photonic crystal, we are shifting our attention to the aspect of on-chip generation of the Raman spectrum. In the presentation laser tweezers Raman spectroscopy will be introduced and illustrated on the basis of our study of two types of yeast cells. Further, trapping studies with photonic crystal cavities will be illustrated in the form of movies of trapping events for bacteria. The magnitude of the optical forces derived from the analysis of the movies will be supported by simulation results for the optical properties of the cavities and the resulting optical forces acting on the bacteria. References 1. Photonics21 is the European Technology Platform representing the photonics community in

Europe. http://www.photonics21.org/download/Photonics21StrategicResearchAgenda.pdf 2. Askin, A., 1970. Acceleration and trapping of particles by radiation pressure. Physical Review

Letters 24: 156-159. 3. Van Leest, T., Heldens, J., Van der Gaag, B. and Caro, J., 2012. Photonic crystal cavities for

resonant evanescent field trapping of single bacteria. Proceedings of SPIE Vol. 8427: 84270T. 4. Caro, J., Heldens, J.T. and Van Leest, M.M., 2012. Optimization of three cavities in a silicon

photonic crystal slab for trapping of bacteria in water. Proceeding of the Symposium IEEE Photonics Society Benelux Chapter, pp. 263-266.

5. Van Leest, T. and Caro, J., unpublished data.


REMOTE CONTROL STRATEGY FOR COMBINED MICROBIOLOGICAL AUTO-SAMPLING AND ON-LINE CHEMOPHYSICAL MONITORING AT DRINKING WATER RESOURCES: EVENT AND TIME SERIES ANALYSIS Hermann Stadler1 and A. Farnleitner2

1Joanneum Research, Institute for Water, Energy and Sustainability, Department for Water Resources Management, Austria and 2Vienna University of Technology, Institute of Chemical Engineering, Department of Applied Biochemistry and Gene Technology, Austria [email protected] Water resources from alpine and other mountainous karst aquifers play an important role for water supply in many countries. World-wide approximately 25% of the population are fed by drinking water from karst aquifers. These karst regions cover 12.5% of the land surface [1]. In Austria, 25% of the people are supplied with karst-water, including Vienna [2]. As regulated in the WFD (Water Framework Directive), karstic catchments require a sustainable protection. The increasing impact to such regions and the different utilization in the watersheds of karst springs are important reasons to establish early warning systems and quality assurance networks in water supplies. These systems rely heavily on in-situ measurements and online and near real-time availability of the data. With a satellite based networking of measuring and sampling stations it was possible to carry out precipitation triggered event monitoring campaigns at different karst springs [3] combining on-line measurements of hydrological parameters with field-laboratory based analyses of microbial faecal indicators [4]. The targets in the study were (i) to investigate the dynamic of microbial faecal pollution indicators, chemical parameters and environmental isotopes at a high resolution time scale during hydrological events, and (ii) to evaluate the in previous investigations established parameter SAC254 (Spectral Absorption Coefficient at 254 nm) as an appropriate real-time pollution proxy for optimised spring water abstraction management within an early warning. System description Based on extensive technical and cost comparisons and on validation measurements [5], the ORBCOMM Low Earth Orbiting (LEO) Satellite system was chosen. ORBCOMM is a ‘little-LEO’ system, with 30 servicing satellites in 6 orbit planes of 800 km altitude. It provides bi-directional ‘short message’ data-transfer at 2.4/4.8 kbps, with data blocks preferably less than some 100 bytes. ORBCOMM operates at frequencies about 140 MHz, providing large satellite footprints, and requires only low-cost/low-power equipment. The ORBCOMM modem transmits its data to the satellite, from where down-link transmission is performed either directly to one of the Gateway Earth Stations (GES). The GES emails the data to the receiver via internet or re-transmits it to any ‘nomadic’ ORBCOMM modem again via satellite. The precipitation station (PS) is located in the catchment area of the spring, where the event sampling will be carried out. It is equipped with a tipping bucket, a data logger and a LEO-satellite modem. It can be supplemented with additional meteorological sensors and sampling devices. The monitoring and sampling site at the spring (spring sampling station, SSS) is equipped with an additional data logger, a pressure probe to register the changing of discharge, two automatic sampling units (one for the reference sample and one for the periodic samples) and a LEO-Satellite modem for real-time control and data transmission. It can be supplemented with additional hydrological or meteorological sensors. Study area and methods The investigations were carried out in different karst catchment areas of the Northern Calcareous Alps. The event at the karst spring shown in Figure 1 was caused by an aestival


thunderstorm with 40.2 mm precipitation measured in the catchment area at 1520 masl. The samples at the spring (n=157) were taken with automatic sample devices within 10 days in August 2009. They were stored at ambient spring water conditions and treated for the different analyses not later than 24 hours after sampling. Escherichia coli was analyzed by the Colilert system (IDEXX) directly at a field laboratory as previously described [4]. Hydrological in situ measured on-line parameters were collected with an increment of 15 min. To study microbial faecal pollution E. coli was chosen as indicator organism. In contrast to other standard faecal indicators, detailed previous investigations highlighted its excellent applicability as a general faecal pollution indicator in alpine karstic environment (i.e. high prevalence and abundance in human, live stock and wildlife excreta, low or non prevalence in alpine soils, half life time of E. coli in spring water in the range of the average event period length [6]). Results The integration of on-line measured data, laboratory and field laboratory analyses, all of them recovered with high time resolution, allows a deep insight to these sensitive aquatic systems. Especially the combination with environmental isotopes generates new knowledge of the dynamics, mass transport and substance transfer being of fundamental importance for the sensible use of early warning systems. As an example the correlation between SAC254 and E. coli during the course of the event is shown in Figure 1. Very important for the use of SAC254 as an early warning proxy is the lead time of SAC254 to E. coli, which enables reactions times for water abstraction management. Figure 1. Correlation of SAC and E. coli during the observed event.

Acknowledgements The investigations were financed and supported by the Vienna Waterworks and by the Austrian Federal Ministry for Transport, Innovation and Technology. References 1. Martin, J.B. and White, W.B. (eds.), 2008. Frontiers of karst research – Special Publication 13.

Karst Waters Institute, Leesburg, VA, USA. 2. Zwahlen, F., (ed.), 2003. Vulnerability and risk mapping for the protection of carbonate (karst)

aquifers. Scope – goals – results. COST Action 620, European Commission, DG Science, Research and Development, Luxemburg, 39 pp.

3. Stadler, H., Klock, E., Skritek, P., Mach, R.L., Zerobin, W. and Farnleitner, A.H., 2010. The spectral absorption coefficient at 254 nm as a near real-time early warning proxy for detecting faecal pollution events at alpine karst water resources. Water Science and Technology 62: 1898-1906.

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4. Stadler, H., Skritek, P., Sommer, R., Mach, R., Zerobin, W. and Farnleitner, A.H., 2008. Microbiological monitoring and automated event sampling at karst springs using LEO-satellites. Water Science and Technology 58: 899-909.

5. Stadler, H. and Skritek, P., 2004. Networking of automated event-sampling hydro-meteorological measuring sites using LEO-satellite communication. Proceedings of the 18th International Conference EnviroInfo Vol.2, pp. 331-334.

6. Farnleitner, A.H., Ryzinska-Paier, G., Reischer, G.H., Burtscher, M.M., Knetsch, S., Kirschner, A.K.T., Dirnböck, T., Kuschnig, G., Mach, R.L. and Sommer, R., 2010. Escherichia coli and enterococci are sensitive and reliable indicators for human, livestock and wildlife faecal pollution in alpine mountainous water resources. Journal of Applied Microbiology 109: 1599-1608.


SAMPLE PREPARATION STRATEGIES FOR RAPID DIAGNOSTIC METHODS Ronald van Doorn Innosieve Diagnostics, the Netherlands [email protected] There is an ever-increasing demand for rapid methods for the detection of (harmful) micro-organisms in different disciplines, ranging from clinical diagnostics, food safety, and environmental pest management to the evaluation of treatment efficacy. Such methods ideally provide conclusive results within one working day while maintaining an exclusively high assay specificity and sensitivity. Additionally, rapid methods should be easy to use and allow automated and/or on-site microbial detection. Because in rapid methods time consuming microbial enrichments prior to detection are greatly reduced or even eliminated, low levels of micro-organisms should be detectable in relatively large sample volumes. This combined with the fact that rapid methods are often prone to assay inhibition/disturbance by matrix related compounds, a purified and high quality input sample is required, making the analyses of practical samples challenging. Solutions to overcome these hurdles include removal of matrix related compounds and the concentration or purification of the microbial content and/or analytes (e.g. nucleic acids) from the practical samples. These issues including promising solutions will be discussed in view of rapid methods for acceptance for on-site or point of care diagnostics with equal to better performance compared to reference methods.


LEGISLATIVE REQUIREMENTS FOR SAMPLING Franz Ulberth Institute for Reference Materials and Measurements, Joint Research Centre, European Commission, Belgium [email protected] Determining whether a food in international trade complies with certain specifications, e.g. legal or contractual requirements, requires three steps: (i) sampling, (ii) testing, and (iii) conformance assessment. The first step in this sequence, i.e. selecting an appropriate set of samples to represent the whole lot, is of crucial importance. To ensure that the estimated value obtained from the laboratory sample is a good representation of the population it is necessary to develop and apply a suitable sampling plan. However, it has to be noted that no sampling plan can ensure that every item in a lot conforms to specifications. Nevertheless, sampling plans are useful for guaranteeing an acceptable quality level for stakeholders involved in trading activities (vendors, buyers, consumers). Sampling and testing in international food trade are often used for the purpose of risk management related to food safety. Sampling, testing and conformance assessment actions should be properly documented to avoid disputes and, in case of disputes, to expedite their resolution. To that end standardisation organisations such ISO, Codex Alimentarius as well as individual jurisdictions, among them the European Union, standardised and codified a set of rules related to certain commodities and analytes. For example, Commission Regulation (EC) No 401/2006 lays down the methods of sampling and analysis for the official control of the levels of mycotoxin foodstuffs; equivalent provisions are found in Codex Alimentarius Standard 193-1995 (Codex General Standard for Contaminants and Toxins in Food and Feed). The presentation will present the basic legal requirements for sampling and testing for a range of priority contaminants in foodstuffs and discuss current developments of standards related to sampling of food in international trade.


IMPROVED CERTAINTY FROM SAMPLING UNCERTAINTY Michael H. Ramsey School of Life Sciences, University of Sussex, UK [email protected] Most measurements on food, feed or water are used to make decisions, such ‘as is this material safe for human or animal consumption?’ We would like to be certain that such decisions are reliable, but this might appear to require perfectly accurate measurements of contaminant concentration. However, it is now well recognized that all measurements are wrong to some extent, in that they all have uncertainty. Furthermore, this measurement uncertainty does not arise solely from the analytical process, but also from the processes of primary sampling and physical sample preparation. This presentation gives an overview of how the total uncertainty of measurements can be estimated, using some of the methods described in a Eurachem Guide [1] applied to case studies. These methods include the ‘duplicate method’, in which as small proportion of samples (~10%) are taken in duplicate by a re-application of the same sampling protocol. Using an unbalanced experimental design has recently been shown to accurately quantify both the analytical and sampling components of the uncertainty, whilst reducing the cost of implementing this method by 33% [2]. A more advanced method uses data from sampling proficiency testing to also include the uncertainty contribution from the bias between different samplers [3]. These methods can also be designed to separately quantify the contribution to uncertainty from the physical preparation of the sample [1, p. 97]. There is increasing evidence that the processes of sampling and physical sample preparation are often the dominant sources of measurement uncertainty. Knowing this dominant source is important when deciding which part of the measurement system to improve in order to reduce uncertainty overall. It may often be much more cost effective to improve the quality of primary sampling rather than that of chemical analysis. Once a realistic value for the measurement uncertainty has been estimated, it is possible to propagate this value through calculations on whether contaminant concentration values exceed threshold safety values. This enables management decisions to be made with increased, and specified, levels of certainty [4]. References 1. Ramsey M.H. and Ellison S.L.R. (eds.), 2007. Eurachem/EUROLAB/ CITAC/ Nordtest/ AMC

Guide: Measurement uncertainty arising from sampling: a guide to methods and approaches Eurachem ISBN 978 0 948926 26 6. (http://www.eurachem.org/index.php/publications/guides/musamp)

2. Rostron, P. and Ramsey, M.H., 2012. Cost effective, robust estimation of measurement uncertainty from sampling using unbalanced ANOVA. Accreditation and quality assurance. Journal for Quality, Comparability and Reliability in Chemical Measurement 17: 7-14. (http://link.springer.com/article/10.1007%2Fs00769-011-0846-2)

3. Ramsey, M.H., Geelhoed, B., Damant, A.P. and Wood, R., 2011. Improved evaluation of measurement uncertainty from sampling by inclusion of between-sampler bias using sampling proficiency testing. Analyst 136: 1313-1321. (http://pubs.rsc.org/en/content/articlepdf/2011/AN/C0AN00705F)

4. Ramsey, M.H., 2009. Uncertainty in the assessment of hazard, exposure and risk. Environmental Geochemistry and Health 31: 205-217. (http://link.springer.com/article/10.1007%2Fs10653-008-9211-8)

http://www.eurachem.org/index.php/publications/guides/musamp

http://link.springer.com/article/10.1007%2Fs00769-011-0846-2

http://pubs.rsc.org/en/content/articlepdf/2011/AN/C0AN00705F

http://link.springer.com/article/10.1007%2Fs10653-008-9211-8


SAMPLE PREPARATION STRATEGIES AND TECHNIQUES FOR FOOD CONTAMINANT ANALYSIS Kathy Ridgway Reading Scientific Services Limited (RSSL), UK [email protected] Chemical contamination of food can originate from a variety of sources throughout the food supply chain. The term food contaminant covers a wide range of compounds and their presence may be of concern due to implications for food safety, regulatory compliance, or quality of products and consumer perception. The levels of contaminants observed can vary greatly and analytical methodology must be fit for purpose to enable accurate detection down to required levels, or be sufficient to demonstrate absence as low as reasonably practical for some banned substances. The range of compounds and complexity of food as a matrix presents a challenge to the analyst in determination of chemical contaminants in food as no one approach will be suitable for all potential contaminants [1]. Strategies need to be developed to enable an intelligent choice of technique to be employed for both known and unknown contaminant identification and quantification. Consideration should be given to the instrumentation available and the level of accuracy and sensitivity required. Sometimes this will include an initial rapid screening approach followed by more accurate fully quantitative analysis if required. There is also a requirement for development of more rapid and environmentally friendly approaches that have reduced environmental impact by using less solvent and smaller sample sizes. However, in taking smaller sample sizes it must be ensured that the sample is representative of the bulk matrix and also the potential implications on precision of the method need to be understood. To obtain exhaustive extraction, large volumes of solvent or repeat extractions may be required and invariably some clean-up or concentration will be needed for trace residues or contaminants in foods. Equilibrium based techniques such as sorptive extraction and headspace analysis can require more optimisation and careful control and the use of internal standards for such techniques is recommended. Generic extraction techniques can be useful in applications where there are a variety of matrixes to analyse and these are being increasingly used for multi-analyte approaches or determination of unknown contaminants. Techniques used for sample preparation are changing. Miniaturisation and speed are the main drivers but caution is needed, particularly for real applications in food contaminant analysis. The use of more powerful instrumentation enables selectivity at the detection stage and with software packages available, some applications require little or even no sample preparation. This presentation will cover the main techniques available and how to select the correct approach for specific applications covering both known and unknown food contaminants. References 1. Ridgway, K., Lalljie, S.P.D. and Smith, R.M., 2007. Journal of Chromatography A 1153: 36-53.


MATRIX EFFECTS: THE FOE OF FAST LC-MS/MS BASED MULTI-ANALYTE METHODS Michael Sulyok BOKU Vienna, Department IFA-Tulln, Austria [email protected] Due to the increased sensitivity and robustness of the latest generations of mass spectrometers the multi-analyte approach has become more and more popular. A large number of related methods covering hundreds of pesticides, veterinary drugs or biotoxins have been successfully developed. Since the set of target substances is often quite diverse, only an unspecific clean-up (such as QuEChERS) or even analysis of crude extracts is included in most of these methods. The selectivity of MS/MS may mislead to the assumption that all effects deriving from the matrix are effectively eliminated and that only little or even no sample clean-up is needed, thus enabling high sample throughput. In reality, co-eluting matrix components influence the ionization efficiency of the analyte, either through competition for the electrical charges in the MS source or through affecting the evaporation of the ESI-droplets and the analyte transfer to the gas phase [1]. Therefore, these so-called matrix effects decrease or (more rarely) increase the analytical signal, which negatively affects the method accuracy and precision. Unfortunately, these effects are of unpredictable nature, as their extent depends on the type of matrix and of the analyte and frequently varies between different batches of a given matrix [2]. The aim of this presentation is to discuss the common approaches for counteracting matrix effects in multi-analyte methods. These include dilution of the extract, stable isotope dilution assays, unspecific clean up of the sample. The performance these approaches is compared based on a selected example of a spiked extract of green pepper. References 1. Antignac, J.P., De Wasch, K., Monteau, F., De Brabander, H., Andre, F. and Le Bizec, B., 2005.

Analytica Chimica Acta 529: 129-136. 2. Matuszewski, B.K., Constanzer, M.L. and Chavez-Eng, C.M., 2003. Analytical Chemistry 75: 3019-

3030.


MYCOTOXIN ANALYSIS IN GRAIN DUSTS – A PROMISING FIELD OF APPLICATION FOR RAPID TEST SYSTEMS Mareike Reichel, S. Staiger and S. Biselli Eurofins WEJ Contaminants, Germany [email protected] Mycotoxins are secondary metabolites produced by fungi. Due to their acute and chronicle toxicity, they are unwanted contaminants and constitute a risk to food safety. Grains are often naturally infected with mycotoxin-producing moulds, either pre-harvest in the field or post-harvest during storage. As a staple food, grain and grain products are consumed in high amounts, so that contaminated raw materials would have a strong impact on consumers’ health. Therefore, regulation (EC) No 1881/2006 sets maximum levels for several mycotoxins in grain and grain products. Contamination with mycotoxins cannot be completely avoided. Hence, permanent industrial self-control remains the only possibility to protect consumer’s health and avoid cost-intensive rejections. The best point to control mycotoxin contamination is at the raw-material reception or at the loading point. To suit the workflow and requirements on-site, tests for mycotoxins must be rapid, cheap, easy to handle, representative for whole lots and specific enough to detect at low legal limits. In practice, hardly any test fulfils all these requirements. Hence, the major complains from grain traders and mills are that representative sampling is too complex, sample preparation is too laborious, trained staff is needed, tests are too expensive and take too much time from sampling until the final analytical result. To cope with these problems, an innovative sampling regimen was developed and tested: sampling of dusts. Such small particles from the surface of grains are abraded during threshing, transport or handling procedures. Dusts are ubiquitous and homogenously distributed in unprocessed grain lots. Using a dust-sampling equipment, samples with specific particle sizes were taken during the unloading of trucks. In this way, non-destructive samples that are representative for whole grain lots could easily be collected. As no grinding or homogenization step was needed, sample preparation could be reduced to one short extraction step. Mycotoxins strongly accumulate on small particles in food bulk. Based on the fact that the contamination of the overall sample and its dust particles is correlated, the contamination in grains was recalculated from concentrations determined in the respective dust particles. Furthermore, dust particles only contain minor levels of starch, proteins, and fats. Hence, the natural enhancement of the mycotoxin contamination in the dust and its limited matrix effects facilitated rapid analyses of mycotoxins even at low legal limits. Dust samples were analysed without prior clean-up or concentration steps by both commercial immunochemical rapid tests and HPLC-MS/MS multi toxin methods. Even if some main problems such as easy and representative sampling, fast sample preparation, and detection of mycotoxin contamination at low legal limits could be solved, dust sampling could offer further room for improvement. In the future, more rapid and cost-efficient tests might be customized to dust samples and due to the small amounts of dust samples needed for analyses, fully automated miniaturised test instruments are entirely conceivable.


ON-LINE SPE-TOX MONITORING: THE END OF LAB TOXICITY TESTING Varvara Kokkali1, B. Bajema1, W. van Delft 1and J. Appels 2 1Vitens Water Technology, the Netherlands and 2microLAN, the Netherlands [email protected] Over the last decades, the quality of surface water in the Netherlands has been improved resulting in no measurable effects on bioassays. This situation has lead to the integration of a pre-treatment step using solid phase extraction (SPE) for concentrating samples prior to toxicity assessment [1]. The National Institute for Public Health and the Environment (RIVM, Bilthoven, the Netherlands) has reported a concentration method using two synthetic resins XAD-4 and XAD-8 combined for extracting the organic micro pollutants from water samples [2]. This study aims to develop a concentration method using SPE prior to toxicity assessment with TOXcontrol (microLAN, the Netherlands) in the field, of the same sensitivity as in the laboratory where advanced equipment is applied. Initially, the most efficient combination of SPE material and pH was determined using online Symbiosis™ (Spark, The Netherlands) SPE system tandem to Q-TOF liquid chromatography high resolution-mass spectrometry analysis (LC-HR-MS) (Agilent, the Netherlands). Thirteen SPE materials and five different pH values were applied. Drinking water samples of 10 ml were spiked with 257 polar compounds including pharmaceuticals, pesticides and industrial compounds. This study resulted in Resin-SH at pH 5 and Oasis® HLB at pH 7, which were the most applicable combinations for recovering 91% of the tested compounds above 25% for both SPE materials after LC-HR-MS analysis positive mode [3]. Then, the same method was scaled up for concentrating higher volume samples spiked with the same compounds using the TOXcontrol SPE unit. TOXcontrol is an online toxicity biomonitor using luminescent bacteria (Vibrio fischeri) for monitoring toxicity. Oasis® HLB material was selected and columns were packed manually. Furthermore, an SPE patented material provided by Hocer (France) was also tested. Drinking water samples of 1 l were concentrated to 1 ml. An extra elution step of 1 ml followed in order to determine the most efficient quantity of eluting solvent for retrieving the maximum recoveries of the tested compounds. The extracts were analysed using Q-TOF LC-HR-MS for both positive and negative modes. The results demonstrated that only the second elution step gave extra 6.5 and 12% average recovery for the tested compounds when either Oasis® HLB or Hocer material were applied, respectively. Nevertheless, the comparison of the two materials Oasis® HLB and Hocer for SPE unit of TOXcontrol resulted in almost the same average recovery, 68 and 66% respectively, at the end of the two elution steps measured with LC-HR-MS analysis positive mode. In conclusion, the SPE unit of TOXcontrol with either Oasis® HLB or Hocer material demonstrated high efficiency for concentrating high sample volumes and close comparable results to the online advanced system in the laboratory given the different concentrating volumes and the cost. Acknowledgements This research was part of the 3iTOX project. 3iTOX project is part of the InnoWATOR project that is subsidized by Agentschap NL, Ministry of Economic Affairs, the Netherlands. The authors would like to thank Andre Berg and Ronny Bosch (Vitens) and Jaap van den Dries (microLAN) for their assistance in the conduction of the project and also the partners of 3iTOX project: KWR, Capilix, Waternet and Water Laboratorium Noord (WLN).


References 1. Penders, E.J.M., 2011. Development of aquatic biomonitoring models for surface waters used for

drinking water supply. Ph.D. Thesis, Wageningen UR, Wageningen, the Netherlands. 2. Durand, A.M., Rotteveel, S., Collombon, M.T., Van der Grinten, E., Maas, J.L. and Verweij, W.,

2009. Toxicity measurements in concentrated water samples. Evaluation and validation. RIVM Report. RIVM, Bilthoven, the Netherlands.

3. Kokkali, V., Bajema, B., Bosch, R., Berg, A. and Van Delft, W., 2013. Development of a solid phase extraction technique (SPE) for 257 compounds suitable for both online SPE tandem Q-TOF analysis and also for advanced toxicity bio-monitoring. In: Book of abstracts of RME2013 – 8

th

conference on innovations and breakthroughs in food, feed & water analysis, 21-23 January 2013, Noordwijkerhout, the Netherlands, p. 116.


RAPID ANALYSIS IN FORENSIC SCIENCE: MAYHEM OR MAGIC Plenary Keynote Lecture Niamh Nic Daeid Centre for Forensic Science, University of Strathclyde, UK [email protected] Peoples external perception of forensic science is very much influenced by television and crime novels. In reality forensic science as a science is conservative in nature but increasingly moves are being made to integrate technological developments into the industry. This causes stresses and potentially unforeseen consequences within and between the multifaceted communities serving the criminal justice system. This presentation will highlight some of the risks and benefits of the introduction of rapid methods for analysis into the forensic science process and discuss the potential consequences of such developments.


RAPID METHODS IN MICROBIOLOGY: ARE WE STILL GETTING SIMPLER, FASTER, BETTER? Plenary Keynote Lecture Daniel Y.C. Fung Department of Animal Sciences and Industry, Kansas State University, USA [email protected] Rapid methods and automation in microbiology is a relatively new field, but it has a lot of important implications to applied microbiology. This presentation will provide a short historical review of the field and then move into some simple but powerful tools in applied microbiology such as:

Advances in simple and effective total viable cell count methodologies.

Thin agar layer method for recovery of injured cells in foods and environment.

Ingenious ways to use a new enzyme – oxyrase – for stimulating the growth of all types of microorganisms faster so that we can detect them much more effectively;

Instantaneous results in applied microbiology; catalase and enzymes test, etc. for food residues.

The new Fung Double Tube (FDT) system which can detect and enumerate Clostridium perfringens in only four to five hours from the time of sample application to the time of seeing the black colonies without using any equipment or expensive materials. A truly simple, effective, and powerful method to detect and enumerate C. perfringens from all kinds of food and environments.

The talk will end with Fung's ten predictions of development of rapid method made in 1995 and see how the predictions held up in 2013!


DETECTION OF PATHOGENIC ESCHERICHIA COLI IN FOOD: THE EXPERIENCE OF THE EUROPEAN REFERENCE LABORATORY FOR VTEC IN METHODS' DEVELOPMENT Rosangela Tozzoli EU Reference Laboratory for E. coli including VTEC, Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare, Istituto Superiore di Sanità, Italy [email protected]

Verocytotoxin-producing Escherichia coli (VTEC) are a very heterogeneous group of pathogenic E. coli characterized by a complex array of virulence genes and an evolving epidemiologic paradigm. They can cause a wide range of diseases with symptoms varying from non-complicated diarrhoea to the life-threatening haemolytic uremic syndrome (HUS). Many E. coli serotypes have been demonstrated to produce verocytotoxins (VTs) or possessing the vtx genes, but the majority of them are rarely or never isolated from human cases of disease. Epidemiology of human infections indicates that VTEC causing more than 90% of HUS worldwide belong to a dozen of serogroups. In the EU, this observation led to the definition of pathogenic VTEC as VT-producing E. coli strains possessing the intimin-coding eae gene and belonging to the O157, O26, O111, O103 and O145 serogroups [1]. Detection of pathogenic VTEC cannot benefit from metabolic peculiarities or growth characteristics allowing the use of biochemical tests or selective culture media for differential identification. The EU RL VTEC has chaired an ad hoc group of international experts with the aim of developing a method for the detection of VTEC in food upon mandate from the European Committee for Normalization (CEN). The group defined a step-wise molecular procedure and produced an international standard, the ISO Technical Specification 13136, with the title ‘Microbiology of food and animal feed – Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens – Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups’. The methodology includes the molecular screening of enrichment cultures for the presence of genes encoding VTs (vtx). The samples positive at the screening for vtx are subjected to isolation. Enrichment cultures are also screened for the presence of the eae gene, as well as of genes associated to the main VTEC serogroups causing severe human disease. The identification of the serogroups-associated genes, despite not crucial for issuing the test report, may facilitate the isolation step by driving the selection of serogroup-specific strategies such as the immuno-magnetic enrichment. Moreover this information may represent an added value for the risk managers. The USDA has also adopted a similar approach for the control of meat products. Based on the epidemiology of the VTEC infections in the US, the spectrum of serogroups has been extended to O45 and O121. Although human infections are mainly acquired through food vehicles, environmental sources are becoming more important. The environment can be contaminated by pathogenic E. coli strains from human and animal sources and setting the possibility that new pathotypes may emerge and contaminate crops used for human consumption. It can be hypothesized that such an environmental reservoir might have favoured the emergence of the VTEC O104:H4 strain that caused the huge outbreak of HUS in Germany in 2011. The outbreak strain is an enteroaggregative E. coli (EAEC) that developed the capability to produce VT through the acquisition of a VT-converting bacteriophage. EAEC circulate predominantly in the human host and, generally, do not come into contact with the VT-phages, which are conversely common in the ruminants’ gastrointestinal tract and in the environment contaminated by ruminants’ manure. However under circumstances where wastewater treatment is not


adequate, which are common in developing countries, human EAEC are massively released in surface waters. Such an environment is shared with animals including ruminants attending the water to drink or swim. This promiscuity would allow an E. coli strain of animal origin as VTEC to encounter a human-borne pathogen such as EAEC, making it possible the exchange of genetic material, including the VT-phages. Whatever the way it originated and spread, the EAEC/VTEC O104:H4 outbreak strain challenged the definition of pathogenic VTEC. As a matter of fact, it does not possess the eae gene and does not belong to any of the serogroups included in the ISO TS 13136, setting the need for the development of an ad hoc assay. The modular nature of the international standard for VTEC, however, was flexible enough to promptly adapt it to the analytical needs. As a matter of fact, the ISO TS 13136 has been adapted to the detection of the new VTEC type by adding two additional modules for the amplification of the genes specifying the O104 serogroup and the H4 antigen. The flexibility of the ISO TS 13136 was demonstrated to be a valuable tool for facing the emergency, while the development of the new tools by the EU RL VTEC and their diffusion through the network of the European National Reference Laboratories resulted in a prompt response to the crisis. References 1. European Food Safety Authority (EFSA), 2007. Scientific Opinion of the Panel on Biological

Hazards on a request from EFSA on monitoring of verotoxigenic Escherichia coli (VTEC) and identification of human pathogenic VTEC types. The EFSA Journal 579: 1-61.


RAPID DETECTION OF FOODBORNE MRSA AND ESBLs Jeffrey Hoorfar Division of Food Microbiology, National Food institute, Technical University of Denmark (DTU), Denmark [email protected] Methicillin-resistant Staphylococcus aureus (MRSA) in livestock have been in focus due to the possibilities of zoonotic spread of livestock associated lineages. However, the methods for sampling and detection are so far not considered optimal in terms of sensitivity and practical application. MRSA was isolated in the Danish pig production for the first time in 2006, and MRSA CC398 was retrospectively found in a Danish patient in 2003. The prevalence of MRSA in Danish pig herds in 2008 was considered low compared to some other European countries. In recent years, 16% of the pig herds were found infected based on one pool of nasal swabs and 44% of the pigs at slaughter. Recent Danish studies show that MRSA detection by air sampling is easy to perform and reduces the costs and analytical time compared to existing methods. This could be suitable for initial screening of herds. Escherichia coli is the most common cause of urinary tract infection. It has been proposed that E. coli causing urinary tract infections could have an animal origin. E. coli isolates from broiler chicken meat, broiler chickens, pork, and pigs share phylogroups and antimicrobial resistance with community-dwelling humans and patients with urinary tract infection. Extended spectrum beta-lactamase (SBL)-producing organisms have recently emerged as nosocomial pathogens. ESBLs are defined as transferrable enzymes able to hydrolyze third and fourth-generation cephalosporins. Unlike MRSA, the resistance mechanisms of ESBLs are not limited to one or even two species but rather a whole family of organism, the Enterobacteriaceae. Thus, the rapid detection of ESBLs is a challenge for most microbiology laboratories because inaccurate identification of ESBL producers has important clinical implications for both antibiotic treatment and infection control.


ABANDONING THE SURROGATES FOR POTENTIAL CYANOBACTERIAL HAZARDS: A PLEA FOR CYANOTOXIN ANALYSIS Miquel Lürling1,2 and E. Faassen1

1Aquatic Ecology and Water Quality Management Group, Wageningen UR, the Netherlands and 2Department Aquatic Ecology, NIOO-KNAW, the Netherlands [email protected] The intensity and frequency of water blooms of cyanobacteria is rising in many freshwater and coastal systems world wide. These blooms present a serious threat to the environment and the health of wildlife, cattle, pets and humans because of the ability of cyanobacteria to produce potent toxins. This is also the reason why cyanobacteria are addressed explicitly in the EU Bathing Water Directive (Directive 2006/7/EC). However, implementation of this Directive in the Netherlands led to a protocol that uses indirect proxies for potential risks. We will show that there are major drawbacks in the current approach and in the proposed ‘rapid’ techniques to identify cyanobacterial hazards. Hereto, we will present some case studies and experiments that will exemplify research should be directed towards cyanotoxins analysis.


APPLICATION OF LASER INDUCED BREAKDOWN SPECTROSCOPY AND NEURAL NETWORK: RAPID IDENTIFICATION AND DISCRIMINATION OF BACTERIAL STRAINS Jorge O. Cáceres and Sadia Manzoor Department of Analytical Chemistry, University of Madrid, Spain [email protected] A method based on the combination of laser induced breakdown spectroscopy (LIBS) and neural network (NN) algorithm has been applied to strain identification of more relevant bacteria causing hospital acquired infections (HAI), i.e. Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium and Staphylococcus aureus. The aim of this study was to evaluate the capacity of LIBS/NN methodology to detect changes in the atomic composition of different strains samples which occur as a result of their genetic variations. This is of utmost importance because solely identification of bacterial specie is not sufficient to diagnose a disease. The proposed method was able to discriminate strains of the same bacterial species. The accuracy in the identification of different strains of the tested bacteria was higher than 90%. The culture medium showed no significant effect on the discrimination capacity of the method. The optimized NN models provided reliable bacterial strain identification for all samples analyzed, showing the effectiveness of the method to address the safety and social-costs of HAI-related issues.


A CYLINDER AS A PLATFORM OF MULTI-ANALYTE DIAGNOSTICS B. Ronacher, S. Eisenberger, B. Nussbaumer, and Christoph Reschreiter Anagnostics Bioanalysis GmbH, Austria [email protected] Sepsis is a systemic response to infections of the bloodstream that causes organ failure and death in severe cases. Early pathogen identification in conjunction with the determination of the inflammation status may reduce the high mortality rates. As there is a wide range of sepsis-causing pathogens, a multiplex diagnostics technology is favourable. Anagnostics hybcell is the world’s first cylindrical microarray intended for the use in (clinical) routine laboratories and therefore offering complete automation. The objectives are to evaluate novel multiplex methods in order to quickly identify pathogens as well as inflammation markers in septic patients and presentation of results for clinicians in a comprehensive format. Based on multiplex hybcell microarray technology an OnSpot Primer-Extension assay (compact sequencing) has already demonstrated to detect mutations in the KRAS, BRAF and EGFR genes. Now it is applied for the detection of bacteria and fungi DNA profile. Multiplex protein analysis of inflammation markers (compact profiling) quickly determines the inflammation status. The robust and simple approach shows the identification of a range of pathogens within less than five hours, starting from whole blood collection. Critical inflammation markers are quickly determined within 20 minutes, starting from collected plasma. In conclusion, compact sequencing and compact profiling are quick, cost efficient and accurate methods to detect and identify pathogens and determine the inflammation status from human samples in the routine diagnostic laboratory. So an integrated approach in sepsis diagnostics – from identifying the cause to monitor therapy – is feasible and will truly establish personalized diagnostics.


GENSPEED – A NEW TOOL FOR RAPID AND MULTIPLEXED MICROBIAL ANALYSIS Max Sonnleitner Lambda, Austria [email protected] Resistant bacteria are a dramatically growing public health problem with rapidly increasing prevalence of livestock associated resistances. Thus, rapid testing of humans but also animals, food and water for resistant bacteria is one of the most urgent needs in the near future. In this context, two major problems have to be solved: (i) culture-based tests are slow and have to be carried out in dedicated labs and (ii) PCR tests, which are much faster, still are quite expensive and in case of methicillin-resistant Staphylococcus aureus (MRSA) the commercially available PCR tests lack the new mecC variant that is strongly connected to livestock-associated MRSA. With the GENSPEED Testsystem and the first associated Testkit – the GENSPEED MRSA – we present a solution to both problems. The GENSPEED Testsystem provides multiplexing capabilities to rapidly test a panel of up to 8 analytes simultaneously without the need for an expert operator. The platform combines a disposable microfluidic test cartridge with a compact device for chemiluminescence (CL) based optical detection. While the use of capillary-flow based microfluidics avoids the need for complex micro-pumping schemes, CL enables sensitive and robust detection in a very compact optical architecture. In combination with multiplex PCR the system can be applied as fast and highly sensitive detection platform for multiplex-PCR-products. The GENSPEED MRSA test is the first in the product line that uses this combination for differential diagnostics or microbial analysis. It is the worldwide first rapid test for MRSA to include the mecA LGA251 (mecC) and thus allows complete and rapid MRSA testing for human diagnostics but also for microbial analysis of food or water. A second Testkit covering 6 different extended spectrum beta lactamases (ESBLs) will be launched in Q3/2013 to complete the product portfolio for rapid screening of sample-material for a variety of resistances using the GENSPEED platform.


FINDING THE NEEDLE IN THE HAYSTACK: NON-TARGETED ANALYSIS FOR IDENTIFYING CHEMICALS IN FOOD AND DRINK Adrian J. Charlton1, J. Donarski1, M. Dickinson1, F. Schoutsen2 and M. Godula3

1Food and Environment Research Agency, UK, 2Thermo Fisher Scientific, the Netherlands and 3Thermo Fisher Scientific, Czech Republic [email protected] Recent high publicity issues of economic fraud have resulted in severe food safety issues. The deliberate addition of melamine to infant formula resulted in fatalit ies. Other adulteration issues such as the presence of potentially carcinogenic dyes (e.g. Sudan I) in spices have placed a significant financial burden on the food industry, whilst clearly also presenting a threat to public health. Advances in analytical detection capability and associated informatics tools have enabled a step change in the way that emerging contamination events are detected. Traditionally, analytical chemistry is based on the so-called ‘target list’ approach. Targeted analysis involves the development of specific methodologies that allow the measurement of a particular compound or compound group. This approach is appropriate for routine monitoring purposes often achieving the lowest limits of detection for the compounds that are targeted. It is routinely used to monitor for the presence of known food contaminants such as licensed pesticides. This presentation will discuss alternative approaches to targeted analysis for the detection of toxins in food, focusing on recent developments in the fields of nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HR-MS). The complementarity of NMR spectroscopy and HR-MS will be demonstrated using a number of case studies. The main benefits of using this combined approach are the relatively simple sample preparation strategy and short analysis times. Very high mass accuracy from HR-MS compliments the highly specific data generated by NMR spectroscopy. Analysis of a variety of contaminants in relatively dirty extracts at very low concentrations can be achieved. The combined structure elucidation power of two analytical approaches is then used to categorically determine the nature of an unexpected contamination incident. Using this approach the detection of small quantities of unknown compounds in complex mixtures has been demonstrated without a priori knowledge of potential contaminants. This presentation will also discuss non-targeted analysis for the detection of toxins in food and feed. Examples will be taken from a range of studies. Other uses of non-targeted methodologies within a metabolomics context and for identifying high value compounds will briefly be discussed.


RAPID METHODS FOR MYCOTOXINS SCREENING IN THE FOOD INDUSTRY – AN OVERVIEW OF CURRENT SOLUTIONS AND FUTURE PERSPECTIVES Michele Suman

Food Research Labs, Barilla SpA, Italy [email protected] Mould infection remains nowadays a challenge worldwide problem with more than 20% of the crops affected by mycotoxins and with economic losses estimated at billions of dollars (FAO data). Poor harvesting practices, improper drying, handling, packaging, storage, and transport conditions contribute to fungal growth and increase the risk of mycotoxins production. Fungal toxins of concern are generally produced by species within the genera Fusarium, Aspergillus and Penicillium, which frequently occur in major agricultural crops, including cereals, groundnuts and various fruits; most research has focused on aflatoxins, fumonisins, trichothecenes, zearalenone, ochratoxin A and patulin. Analytical methods for the determination of mycotoxins in unprocessed raw materials and also in finished products are highly needed in order to properly assess both the relevant exposure and the toxicological risk for humans and animals (acute or chronic diseases including carcinogenesis, immune suppression and growth retardation), as well as to ensure that regulatory levels are respected. There is an increasing interest for rapid screening tests for mycotoxins to support the confirmatory/official methods (actually mainly based on HPLC or LC-MS), permitting to increase the speed at which the analysis is performed, the simplicity of the sample preparation and the low cost per analysis. There are a range of commercially available rapid test kits, which vary in sensitivity and ease of use and generate qualitative or quantitative outputs. However, there are no designated protocols or internationally harmonized rules for the assessment of the ability of a kit to generate results comparable to a reference method in a repeatable and reproducible manner. The best known rapid screening solutions for mycotoxin detection are antibody-based methods. The most common of these is the microtiter plate enzyme-linked immunoabsorbent assay (ELISA), commercially available for many years and extensively used especially for raw materials. Rapid and user-friendly lateral flow devices (LFD) are becoming progressively competitive with the common ELISA methods: sensitivity and selectivity are provided by specific antibodies, generally they do not require expensive instrumentation and additional chemicals or handling steps, providing in few minutes a semi-quantitative or quantitative result for a target mycotoxin. LFD. In the range of immunochemical strategies there is also a growing interest towards homogeneous approaches like the fluorescence polarization immunoassay (FPIA) and optical-based detection provided by surface plasmon resonance (SPR). FPIA measures interactions between a fluorescently labelled antigen and a specific antibody in solution and in the last years it has been for example optimized for the detection of deoxynivalenol (DON) in wheat food chain. In a parallel way several papers have been published on the determination of DON and T-2\HT-2 toxins in cereals by SPR. Fourier transform near-infrared (FT-NIR) spectroscopy and principal component analysis (PCA) have been recently used for the determination of DON content in durum and common wheat. Furthermore, electronic noses (e-nose) based on metal oxide sensors are emerging as potential suitable direction for the rapid, non-invasive screening of large numbers of cereal samples for mycotoxins contamination. Then, the new generation of biosensors is notable. These sensors potentially permit to achieve multiplex testing and use antibodies attached to the surface of the sensor. The antibodies interact with the mycotoxin, producing measurable

mailto:[email protected]


changes in surface properties by fluorescence, evanescent and planar wave guide (PWG) technologies, optical fibres. Another approach, with future industrial application perspectives for the determination of trichothecenes, is the one based on electrochemical detection, using screen-printed electrodes (ScPEs). The main areas for improvement in rapid methods are in increased throughput and sensitivity, reduction of matrix effect and cross reactivity, conducing to a limited number of false-positive results. Besides, considering the highly competitive environment, the continuously increasing legal contest and the cost-efficient needs, it is clear why rapid methods represent today strategic tools for mycotoxin issues management. A final ‘provocative’ question could be related to liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique that is spreading rapidly as successful solution for simultaneous screening, identification and accurate quantitative determination of a large number of mycotoxins. Should we have to consider this strategy in some way as ‘a sort of rapid method’?! Figure 1. Rapid methods for mycotoxins screening: ‘mosaic image’.

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Chemistry30: 192-203. 3. Liu, J., Zanardi, S., Powers, S. and Suman, M., 2012. Food Control 26: 88-91. 4. Lippolis, V., Pascale, M. and Visconti, A., 2006. Journal of Food Protection 69: 2712-2719. 5. Meneely, J.P., Sulyok, M., Baumgartner, S., Krska, R. and Elliott, C.T., 2010. Talanta 81: 630-636. 6. De Girolamo, A., Lippolis, V., Nordkvist, E. and Visconti, A., 2009. Food Additives and

Contaminants Part A 26: 907-917. 7. Campagnoli, A., Cheli, F., Polidori, C., Zaninelli, M., Zecca, O., Savoini, G., Pinotti, L. and

Dell’Orto, V., 2011. Sensors 11: 4899-4916. 8. Mullett, W., Lai, E.P.C. and Yeung, J.M., 1998. Analytical Biochemistry 258: 161-167. 9. Ricci, F., Pino, F., Abagnale, M., Messia, C., Marconi, E., Volpe, G., Moscone, D. and Palleschi,

G., 2009. World Mycotoxin Journal 2: 239-245. 10. Sulyok, M., Krska, R. and Schuhmacher, R., 2010. Food Chemistry 119: 408-416.

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http://www.ncbi.nlm.nih.gov/pubmed?term=Visconti%20A%5BAuthor%5D&cauthor=true&cauthor_uid=19680966


ARE ALL ALLERGENS DETECTION METHODS CREATED EQUAL? NEW FINDINGS ON METHODS DEVELOPMENT AND COMPARISON, ALLERGEN TRIGGER LEVELS AND PROCESSED PRODUCTS Bert Popping1, C. Diaz-Amigo2 and J. Heick3

1Eurofins Scientific Group, UK, 2Eurofins Scientific Group, Germany and 3Eurofins Analytik, Germany [email protected] Allergens require labelling in many jurisdictions around the world. According to Codex Alimentarius (STAN 1-1985), 7 major food allergens should be labelled in all countries to protect affected consumers:

cereals containing gluten;

crustacea;

eggs;

fish;

peanuts, soybeans;

milk (lactose included); and

tree nuts and nut products. The most common methods to detect allergens are PCR and ELISA. However, all of these methods have major drawbacks. PCR can only give an indication of the possible presence of allergens since only the (non-allergenic) DNA is detected. And at least two of the major allergens, namely egg and milk, contain little or no DNA. ELISA detects proteins, a major advantage over PCR since this is the same compound class (i.e. proteins). However, publications by Eric Garber, Stephen Taylor, Carmen Diaz and Julia Heick clearly demonstrated that substantial quantities of allergens go entirely undetected by ELSIA in processed products. This poses an immediate risk to food industry since the labelling may be incorrect – and therefore also can adversely affect allergic consumers. Allergens where ELISA has shown to fail in processed products are egg, milk and soy. A newly developed detection method, the allergen LC-MS/MS detection could demonstrate that it was able to detect even those allergens in processed products that ELISA and – due to the lack of DNA – PCR was unable to detect. This presentation discusses new developments, recent findings and impact for food industry, regulators and consumer.


SINGLE COPY GMO DETECTION USING A BIOLUMINESCENT REAL TIME REPORTER (BART) OF LOOP MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) Patrick Hardinge School of Biosciences, Cardiff University, UK [email protected] To sustain an expanding population, many crops have been genetically modified with foreign DNA to express novel traits such as increased yield and herbicide tolerance. Public resistance to these genetically modified (GM) plants has led to mandatory labelling of foodstuffs above defined thresholds of GM to non-GM. Within the European Union the accepted level of adventitious GM is currently defined as 0.9% GM in a non-GM product. In California at the end of last year, Proposition 37 (legislation on genetically engineered food labelling) was narrowly rejected, which would have resulted in zero tolerance to the presence of GM in 2019 after an introductory period of 0.5% from 2014. As a consequence of mandatory GM labelling, there is a requirement for quantitative technologies suitable for molecular detection in a variety of settings for monitoring genetically modified crops and their products through the food processing chain. A zero tolerance GM labelling policy would further require very sensitive testing procedures. Molecular diagnostic techniques for the detection of GM contamination are numerous, but the most widely used is the polymerase chain reaction (PCR). PCR requires rapid thermo-cycling to denature and amplify the double stranded DNA target which therefore requires highly specialised equipment. The discovery of DNA polymerases with strand displacement activity has resulted in the development of a number of nucleic acid amplification technologies which operate under isothermal conditions. Loop-mediated isothermal amplification (LAMP) is one of these methods. LAMP is characterised by the use of four different primers; two of which promote the formation of hairpin loops and the other two displace the newly synthesised loop structures (REF). The method is rapid and can be accelerated by the addition of loop or stem primers. It is sensitive and highly specific due to the number of primers required. The LAMP amplification produces a large quantity of inorganic pyrophosphate which can be detected by turbidity and fluorescent techniques. A bioluminescent detection method of pyrophosphate detection (bioluminescent assay in real time: BART) based on firefly luminescence allows quantitative analysis of isothermal amplifications in real time. BART monitors dynamic changes in pyrophosphate levels at a set temperature over the course of the assay [1]. During the BART assay the amplified target produces pyrophosphate which is simultaneously converted into light by a thermostable luciferase. The light output for a positive sample will increase to a peak which then rapidly declines as the luciferase is inhibited by pyrophosphate. The assay time to peak is proportional to the initial concentration of the template DNA. An isothermal amplification coupled BART assay therefore only requires a simple light detection and integration device which can be used in the field. Primers designed for the cauliflower mosaic virus 35S promoter (CaMV 35Sp) and Agrobacterium tumefaciens nopaline synthetase gene terminator (NOS-t) were used to target the GM component of certain maize events [2]. Primers were also designed to target the maize reference gene Zea mays alcohol dehydrogenase (ADH1) as a transgene quantitation standard. These GM-LAMP-BART assays have successfully determined genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material. A GM-LAMP-BART assay using the CaMV 35Sp primers and linearised plasmid template can provide single copy detection, and recent work on improvements to low copy detection will be presented.


References 1. Gandelman, O.A., Church, V.L., Moore, C.A., Kiddle, G., Carne, C.A., Parmar, S., Jalal, H., Tisi,

L.C. and Murray, J.A.H., 2010. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time. PLoS ONE 5: e14155.

2. Kiddle, G. Hardinge, P., Buttigieg, N., Gandelman, O., Pereira, C., McElgunn, C.J., Rizzoli, M., Jackson, R., Appleton, N., Moore, C., Tisi, L.C. and Murray, J.A.H., 2012. GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use. BMC Biotechnology 12: 15.


DEVELOPMENT OF RAPID EXTRACTION METHODS AND HYPHENATED TECHNIQUES FOR DETERMINATION OF MINERAL OIL HYDROCARBONS: THE FOCUS ON MOSH AND MOAH IN FOODS Laura Barp, G. Purcaro, L.S. Conte and S. Moret Department of Food Science, University of Udine, Italy [email protected] The term ‘mineral oil’ is used to indicate a wide range of products, primarily manufactured from crude petroleum through distillation processes and various refining steps. Mineral oils are mainly composed of MOSH (mineral oil saturated hydrocarbons), including n-alkanes, isoalkanes and cycloalkanes, and MOAH (mineral oil aromatic hydrocarbons), mainly consisting of alkylated PAHs. Food can get highly contaminated with mineral oil coming from packaging, especially when recycled fibres or mineral oil based printing inks are used. Depending on the complexity of the matrix to analyze, the extraction step can be more or less demanding. Traditional sample preparation methods (classical solvent extraction, saponification and acid hydrolysis) are usually applied. Rapid extraction methods, such as microwave assisted saponification (MAS) or extraction (MAE) and pressurized liquid extraction (PLE), have been recently developed. Combining elevated pressure and temperature, these methods allow for a time savings over conventional extraction methods. Some data concerning extraction methods comparison are reported. After the extraction, a purification/separation step is necessary to isolate the fractions of interest from the bulk of the matrix (mainly triglycerides). For this purpose, both off-line and on-line methods based on the ability of silica gel or silver silica gel to retain fat, are available. Off-line purification/separation can be performed using solid phase extraction (SPE) cartridges packed with silver-silica gel followed by a large volume injection and gas chromatography-flame ionization detection (GC-FID). However, the most applied method is the on-line coupling of high performance liquid chromatography to GC (LC-GC) described by Biedermann et al. [1]. Starting from this method, a fast LC-GC method that allows increasing the sample throughput and reducing solvent consumption for mineral oil determination, was recently proposed. A total time and solvent reduction of 34 and 23 %, respectively, was obtained by speeding up the gas chromatographic run and reducing the reconditioning liquid chromatographic step. The possibility to use the multi-transfer mode to further reduce the solvent consumption was also explored. A series of real samples (paperboards and pasta) were analyzed obtaining comparable results using both the reference and the proposed method. References 1. Biedermann, M., Fiselier, K. and Grob, K., 2009. Journal of Agricultural and Food Chemistry 57:

8711-8721.


ON-LINE QUALITY CONTROL OF DRINKING WATER: FACT OR FICTION? Bendert R. de Graaf Vitens, the Netherlands [email protected] For safe supply of drinking water, water quality needs to be monitored online in real time. The consequence of inadequate monitoring can result in substantial health risks, and economic and reputational damages. Therefore, Vitens N.V., the largest drinking water company of the Netherlands, set a goal to explore and invest in the development of intelligent water supply. In order to do this Vitens N.V. has set up a demonstration network for online water quality monitoring, the Vitens Innovation Playground (V.I.P.). The V.I.P. is a defined area and comprises a distribution network of 2,270km. During 2012, several (sensor) demonstration projects were performed in the V.I.P. Problem definition Water quality typically is well monitored in treatment plants and pumping stations and is mainly for process control. The parameters monitored are those that help optimize the treatment process. However, once water leaves the plant and enters the distribution network, water quality monitoring and analyses are normally conducted using grab samples and are limited to ad hoc analyses that are available. Given the spatial extent of water distribution networks this means that most of the time water quality can only be monitored intermittently. Current statistics show that 30-60% of water quality incidents were related to events in the water distribution networks [1-3], and the incidents range from water discoloration to backflow of untreated stagnant water. Additionally, water officials and professional agree that distribution systems are vulnerable to intended contaminations [4].Usually, incidents are reported by consumers when they turn on their taps, i.e. at the time when they are about to and/or after they have already consumed the water. Incident analysis shows that average response times can be a few days before action is taken by the water provider [5]. When an incident is detected, the source (position) and spread of the contamination may not be readily identified or isolated, making adequate action difficult and unnecessarily expensive. Consequences of inadequate monitoring can be translated into substantial public health risks, economic damages (recovery costs) and reputational damages and liabilities (in one case the cost to community was estimated as being $155 million [6]). There are various types of online real-time water monitoring systems on the market. Only a few of them, however, come with event detection or early warning mechanism and their cost are typically too high to be deployed in the network covering the whole water distribution grid. To solve the problem water utilities require an economically viable Early Warning System that scrutinizes their distribution network online, allows rapid response (real-time), locates the source (position) and spread of the contamination, and requires no sophisticated skills to operate (integration with existing SCADA and control systems) [7]. Current solutions/approaches Currently available solutions and water quality monitoring systems are not designed to serve as a cost effective early warning system:

Multi parameter sensor systems: typically a low density approach where multiple sensors each cover part of the contamination spectrum [8]. Such systems are too expensive to be deployed in a sensor network, but only at some strategic locations.

Use of simple online sensors: pH, conductivity and other surrogate parameters. They are relatively cheap but only cover part of contamination spectrum and could miss out important incidents.


Biosensors: there include fish, daphnia, mussels, algae, bacteria, etc. They could be highly sensitive to contaminations (and health implications) but are too expensive (in terms of consumables and labour) to be operated in a water distribution network. Typically they are deployed only at strategic locations.

Network approach Vitens has decided to set up a demonstration network for online water quality monitoring, the V.I.P. In the V.I.P. and Vitens laboratory we have tested and evaluated several new innovative sensors, i.e. the Eventlab sensor of Optiqua, the Aquascope sensor of Aquaexplorer, the Coliguard of MbOnline, the spectrolyzer and I::Scan of S::can, but also performed the integration of the Eventlab sensor data in a management software tool named IMQS. This presentation will focus on the results of these demonstration pilots and added value of online continuous network monitoring for drinking water companies. References 1. National Research Council, 2005. Public water supply distribution systems: assessing and

reducing risks – first report. The National Academies Press, Washington, DC, USA. 2. De kwaliteit van het drinkwater in Nederland in 2008 (The quality of drinking water in The

Netherlands in 2008), 2009. VROM-inspectie, The Hague, The Netherlands. 3. Significant drinking water quality events, 2009. In: Annex 3 of Drinking Water 2009, Annual Report.

Drinking Water Inspectorate, London, United Kingdom. 4. Allmann, T.P. and Carlson, K.H., 2005. Modeling intentional distribution system contamination and

detection. Journal of the American Water Works Association 97: 58-71. 5. Coomera Cross-Connection Incident, 2010. In: Health Stream, 57, Water Quality Research

Australia, pp. 1–4. 6. The Honourable Dennis R. O'Connor, 2002. Part One. Report of the Walkerton Inquiry. The events

of May 2000 and related issues. ISBN: 0-7794-2559-6. 7. Technologies and techniques for early warning systems to monitor and evaluate drinking water

quality: a state-of-the-art review, 2005. U.S. Environmental Protection Agency – Office of Water – Office of Science and Technology – Health and Ecological Criteria Division, Washington DC, USA.

8. WaterSentinel System Architecture, 2005. U.S. Environmental Protection Agency – Water Security Division, EPA-817-D-05-003, Washington, DC, USA.


PRINTED FUNCTIONAL SOLUTIONS FOR DIAGNOSTIC PLATFORMS Leena Hakalahti VTT Technical Research Centre of Finland, Finland [email protected] Low cost, miniaturised and mass-manufactured point-of-care solutions are of great interest for diagnostic research and industry. Microfluidic biosensors enable rapid assay performance in many application fields and thus offer advantages over many traditional methods. In microfluidic system the sample volume and the test performance time can be reduced significantly. Nowadays there is a great demand for transferring the traditional laboratory based methods to miniaturised lab-on-a-chip platforms which can be used in the field. Medical and environmental diagnostics are potential areas of use for low-cost disposable polymeric chips. VTT is rising to this challenge by means of roll-to-roll printing technology. Our core area of interest includes research and development of functional fluidic structures on foil and sheet materials from prototyping to mass manufacturing. One key asset of the VTT development chain is the microfluidic prototyping capability: the same foil materials and embossing tools can be used both for prototyping of a device concept and for mass manufacturing of the desired end product. Fully integrated chips can be produced with various types of functions. These include fluidic structures for liquid handling, biomolecules for analyte capture on sensor surfaces, electronics and optics for detection. With VTT’s strong knowledge of antibody discovery and engineering as well as assay development, we are providing solutions for novel diagnostic platforms. In addition of hot-embossing we use nanoimprint UV-moulding which has also shown particular potential to become a potential method for making large-scale, low-cost, high-throughput, and simple patterning of micro- and nano-structures for use in diagnostic platforms.


NITROCELLULOSE MEMBRANES AS A TEMPLATE FOR NOVEL IMMUNOASSAYS: VARIATIONS IN COLOUR AND SHAPE Denise van Rossum Sartorius-Stedim-Biotech GmbH, Germany [email protected] Point of care tests are an important part of the in vitro diagnostics market. The lateral flow immunoassay (LFIA) in particular is showing an impressive growth due to the simplicity of use and the wide spectrum of applications available at the doctor’s office as well as home. LFIA was developed over 30 years ago but since the first patents, the platform per se has not evolved much. Indeed, it is still composed of the same basic components, namely a sample pad, a conjugated pad, a nitrocellulose membrane segment and an absorbent pad. The visual inspection or analytical quantification of the signal intensity at the test and control lines give the final outcome for a specified analyte in a given sample. Various diagnostic membranes with different physical and chemical properties are available for the specific needs of a given LFIA. Nitrocellulose membranes are most widely used thus far. The dynamic of the liquid flow into the membrane is controlled by several factors including, membrane porosity, structure and surface tension. The reaction time at the test zone is regulated by the liquid flow. The combination of these factors influence the signal intensity observed the most. The compact design, convenient storage, simplicity of use and fast access to results are the main advantages of this system configuration. However, the sensitivity, the reproducibility and the semi-quantitative nature of the readouts for LFIA are more and more often not fulfilling the quest for more precise data. Different approaches are here presented to improve the sensitivity and/or reproducibility of LFIA. In a first attempt, various coloured supports were used to laminate the nitrocellulose membrane in order to increase the signal contrast. Fluorescent signals with either Cy2-, Cy3- or Cy5-labelled immunoglobulins (IgGs) were measured on nitrocellulose membranes laminated with transparent, black or white support. The white support had the biggest influence, with up to 5 and 3 fold higher fluorescent signal intensities measured for Cy5- and Cy3-IgG, respectively (Figure 1). The black support had a light quenching effect and signals observed were up to 20% lower compared to controls. The clear support had little net effect. To further pursue the positive influence of the white backing on the signal intensity, a white colour was added directly into the membrane formulation. Interestingly, the addition of the white colour did not influence significantly (up to 10%) the signal intensities generated with the Cy3- and Cy5-labelled IgGs. However, an up to 2-fold increased signal intensity was observed with Cy2-IgG. Figure 1.


In an alternative approach to improve the sensitivity of LFIA and to decrease the need in sample and reagent amounts, geometric forms and/or channel structures were inserted in the nitrocellulose segment. Multiple paths can be designed via channel structuring (Figure 2). Diverse geometric forms can modulate the transport kinetics and consequently, a longer or shorter residence time at the respective test areas which in turn can modulate the signal intensity. Figure 2.

Ultimately, the optimized flow dynamics can lead to increased signal intensity, broader linear range for quantitative readouts as well as higher consistency in the test performance.


DIAGNOSTIC APPLICATIONS IN ROLL-TO-ROLL HOT-EMBOSSED MICROFLUIDICS Christina Liedert VTT Technical Research Centre of Finland, Finland [email protected] There is a growing need for point-of-care (POC) diagnostic tests which are affordable, easy to use and rapid allowing professionals and everyman to evaluate medical or environmental condition quicker thus enabling faster decision making and timely countermeasures. Shifting the central laboratory tests into miniaturized lab-on-a-chip solutions requires suitable mass manufacturing technologies. Roll-to-roll (R2R) hot embossing of polymer microfluidic chips allows mass production of low-cost, disposable chips with high optical quality. The performance of R2R fabricated chips in diagnostic applications are shown with examples. We present a capillary electrophoresis chip for the detection of antibiotic resistance gene mecA of Staphylococcus epidermidis [1]. To perform an immunoassay on a µfluidic chip, antibodies or other biomolecules may be deposited in the µfluidic channel prior to sealing to fabricate all-printed, all-included diagnostic tests. Such an immunoassay platform was developed for the detection of C-reactive protein (CRP) as a model analyte to study elevated risk of cardiac failure, and secondly for the detection of cyanobacterial toxins. References 1. Liedert, R., Amundsen, L.K., Hokkanen, A., Mäki, M., Aittakorpi, A., Pakanen, M., Scherer, J.R.,

Mathies, R.A., Uusitalo, S., Hakalahti, L., Nevanen, T.K.Siitari, H. and Söderlund, 2012. Disposable roll-to-roll hot embossed electrophoresis chip for detection of antibiotic resistance gene mecA in bacteria. Lab on a Chip 12: 333-339.


RAPID MICROARRAY DIAGNOSTIC PLATFORMS USING CARBON NANOPARTICLES Aart van Amerongen

Food & Biobased Research, Wageningen UR, the Netherlands [email protected] Detection of pathogenic bacteria is key to the prevention and identification of problems related to health and safety. Traditional and standard bacterial detection methods are often expensive and too slow to be of any use. For this reason, over the recent years, a lot of effort has gone into the development of rapid biosensors of diverse nature. However, their performance is variable and still needs improvement. Most of the reported methods for the detection of pathogenic bacteria are applied to Escherichia coli. There are many E. coli strains of which quite a number are part of the normal microbiota in the gut. Although most of them are harmless, some of them can cause severe illnesses; one of those is the Shiga toxin-producing E. coli (STEC, also called verotoxigenic E. coli (VTEC) or enterohemorrhagic E. coli (EHEC)). STEC has emerged as a food poisoning pathogen that can cause severe and potentially fatal illness like diarrhoea, hemorrhagic colitis or haemolytic uraemic syndrome. Alternative to a serological (antibody) approach, pathogens can be detected by specifically amplified genetic material. If tagged primers are applied in PCR, the amplicons can be bound by anti-tag antibodies (PCR-Immunoassay). Preceding studies using a lateral flow format with carbon nanoparticles have shown that there is a limitation in the number of double-labelled amplicons (i.e. genes) that can be simultaneously detected (maximally five test lines) [1]. To overcome this limitation, a similar approach was done in an antibody microarray format on slides [2]. Although the number of targets that can be detected is substantially larger than in the lateral flow format, the total assay time is much longer. Therefore, the aim of this study was the development of an assay format in which the positive points of the lateral flow and the microarray formats would be combined. This lateral flow microarray immunoassay (LMIA) is a rapid and multi-analyte format with low amounts of specific antibodies per spot, a crucial factor in the total costs of an immunoassay. As a model several genes encoding virulence factors of E. coli O157 were used as diagnostic targets. Even though many STEC serotypes have been associated with human illness, the vast majority of reported outbreaks and sporadic cases of STEC infection in humans have been associated with serotype O157:H7. Target genes in this study were those encoding the cytotoxins (verotoxins; vt1 and vt2), the outer membrane protein intimin (eae) and the enterohaemolysin (ehxA). Results of the nucleic acid LMIA (NALMIA) will be shown in comparison to the conventional lateral flow and microarray formats. Acknowledgements The financial support of the Wageningen UR Kennisbasis programme KB-VII 'Technology development' is greatly appreciated. The contributions of Fimme-Jan van der Wal and Albert de Boer from Wageningen UR Central Veterinary Institute to the preceding lateral flow and microarray assays and the delivery of E. coli samples are highly valued.

References 1. Noguera, P., Posthuma-Trumpie, G.A., Van Tuil, M., Van der Wal, F.J., De Boer, A., Moers,

A.P.H.A. and Van Amerongen, A., 2011. Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli. Analytical and Bioanalytical Chemistry 399: 831-838.

2. Noguera, P.S., Posthuma-Trumpie, G.A., Van Tuil, M., Van der Wal, F.J., De Boer, A., Moers, A.P.H.A. and Van Amerongen, A., 2011. Carbon nanoparticles as detection labels in antibody microarrays. Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli. Analytical Chemistry 83: 8531-8536.


FIXING SURFACE PROPERTIES, A KEY PROCESS FOR NEW BIOBASED INNOVATIONS Luc Scheres Surfix, the Netherlands [email protected] The emerging fields of micro- and nanotechnology offer new, unique, and exciting opportunities for microbiological and chemical analysis of food, feed and water. Eventually this will lead to earlier and easier detection with higher sensitivity, improved reproducibility and control, and eventually comprehensive prevention of disease, illness or pollution. Due to on-going research and further miniaturization new microbiological and chemical sensing principles become available and device structures become smaller and smaller every day, as a result the relative surface area exposed increases rapidly and surface properties start to seriously affect the performance, quality and lifetime of a bio(chemical) device. Thus, in order to develop new reliable and accurate micro- and nanotechnology based devices control over the surface properties is essential. Surfix introduces a new enabling surface modification technology, based on innovative molecular coatings, for the micro- and nanotechnology market. Our nanocoatings, with a thickness of 1-20 nm, possess high chemical and thermal stability and allow tuning and control over the surface properties in a versatile and reproducible manner. Tailor-made surfaces for (bio)chemical applications, with for instance biocompatible, bioselective, biofouling or catalytic properties, can be prepared with relative ease, even inside complex micro-structured, microfluidic, or lab-on-a-chip devices. Figure 1. Selectively modified photonic Figure 2. Local surface modification inside biosensing device with oriented antibody a lab-on-a-chip device. attachment.


ON-LINE FINGERPRINTING OF FLUIDS FOR QUALITY CONTROL USING STUB RESONATOR TECHNOLOGY Natalia A. Hoog-Antonyuk1,3, M.J.J. Mayer2, H. Miedema3, W. Olthuis1 and A. van den Berg1

1BIOS - the Lab-on-a-Chip group, MESA+ Institute of Nanotechnology, University of Twente, the Netherlands, 2EasyMeasure B.V., the Netherlands and 3Wetsus, the Netherlands [email protected] The feasibility of an on-line flow-through physical detection system to measure the composition and other quality parameters of fluids will be discussed. The system is based on coaxial stub resonator technology and allows detailed analysis of the dielectric properties of fluids and liquid-solid interfaces in a broad frequency range. Changes in the complex dielectric permittivity of, for example, drinking water can be related to changes in the concentration and nature of both organic and inorganic components that are present in the water under investigation. Additionally, changes in dielectric properties of the liquid-solid interface of the flow-through stub resonator, or its column package, can be used as an early warning signal for corrosion or biofilm formation. The presentation will address the basic principle of the new sensor system and the results of an experimental and theoretical validation of the system. Finally, an outlook will be given on how the developed technology will be applied for multi-analyte detection. Basic principle of the sensor concept Figure 1 shows one of the many possible designs of a flow-through coaxial stub resonator to assess the dielectric properties of a liquid. Figure 2 gives a schematic overview of all the components of the sensor involved. The inner conductor inside the liquid-filled hollow tube (=outer conductor) is coupled to a coaxial transmission line that connects the function generator (FG) with the spectrum analyzer (SA). Changes in the complex dielectric permittivity of the liquid sample and / or in geometry of the resonator result in changes in the amplitude-frequency response e.g., its resonant frequency. This means that an amplitude vs. frequency plot of the resonator provides very specific information on both the dielectric (the liquid) in the sensor and any changes of the contact interface between dielectric and sensor body. A proof of principle of this concept has been demonstrated already in [1-4]. Figure 1. The experimental coaxial Figure 2. Schematic outline of the coaxial stub resonator stub resonator sensor system sensing system consisting of a function generator (FG), connected to the spectrum analyzer a spectrum analyzer (SA) and the coaxial stub resonator

(RE). The dotted Inlet and Oulet indicate that the batch resonator can be optionally used as flow-through resonator.


Acknowledgements This work was performed in the TTIW-cooperation framework of Wetsus, Centre of Excellence for Sustainable Water Technology (www.wetsus.nl). Wetsus is funded by the Dutch Ministry of Economic Affairs, the European Union Regional Development Fund, the Province of Fryslân, the City of Leeuwarden and the EZ/Kompas programme of ‘Samenwerkingsverband Noord-Nederland’. The authors thank the participants of the research theme ‘Sensoring’ for the fruitful discussions and their financial support. References 1. Hoog-Antonyuk, N.A., Olthuis, W., Mayer, M.J.J., Miedema, H., Leferink, F.B.J. and Van den Berg,

A., 2012. Extensive modeling of a coaxial stub resonator for online fingerprinting of fluids. Procedia Engineering 47: 310-313.

2. Mayer, M.J.J. and Hoog, N.A., 2011. RF antenna filter as a sensor for measuring a fluid, WO Patent 005084, 13 January 2011.

3. Hoog-Antonyuk, N.A., Olthuis, W., Mayer, M.J.J., Yntema, D, Miedema, H. and Van den Berg, A., 2012. On-line fingerprinting of fluids using coaxial stub resonator technology. Sensors and Actuators B 163: 90-96.

4. Mayer, M.J.J. and Hoog, N.A., 2012. Werkwijze en inrichting voor fingerprinting of het behandelen van een dielectricum in het algemeen en van water in het bijzonder, NL1038869, 16 August 2012.

http://www.wetsus.nl/


CHARACTERISATION OF THE PERFORMANCE OF ANTI-GLIADIN ANTIBODIES Sonia J. Miguel Reading Scientific Services Limited (RSSL), UK [email protected] From the 1st of January 2012, new European legislation will come into force that will apply to pre-packed and food sold loose labelled as gluten free or very low gluten. The levels that will apply will be 20 mg/kg or less for gluten free and 100 mg/kg or less for very low gluten which contain cereal ingredients that have been specially processed to reduce the level of gluten for very low gluten. The legislation will not specify the frequency of testing required, but good practice and due diligence will be expected. While enzyme-linked immunosorbent assay (ELISA) is the most common routine method for the analysis of food in the laboratories, the use of rapid lateral flow devices (RLFDs) as a qualitative method for rapid allergen analysis for verification of the cleaning regimes is increasingly being used in manufactories. There are currently several ELISA kits and RLFDs on the market which use different antibodies (R5, Skerrit & Hill, Anti-gliadin polyclonal and G12) to detect the levels of gliadin (total gluten concentration is derived by multiplying gliadin concentration by 2) (gliadin x2) in food. Each antibody has been raised against specific proteins (α, β and or ω-gliadins) or toxic peptides, which potentially can create a disparity between the results when a food product is analysed. Codex Alimentarius recommends the use of ELISA based on the R5 Mendez method for the analysis of gluten from wheat, rye and barley in food samples. The AOAC official method is based on the Skerrit & Hill antibody (specific to ω-gliadins) for the analysis of gluten. The G12 antibody is the latest on the market, developed after recent studies in coeliac patients showing reactivity to toxic peptides from α-2 gliadin. The aim of our study was to analyse a rice flour based cookie spiked at 1, 5, 10, 20 and 100 mg/kg total protein with wheat, rye, barley and oats using four different antibodies (R5, Skerrit & Hill, Anti-gliadin polyclonal and G12) by ELISA and RLFDs (R5, Skerrit & Hill and Anti-gliadin polyclonal) and to compare their performance.


CHALLENGES IN POP ANALYSES: WHERE ARE WE 14 YEARS AFTER THE BELGIAN DIOXIN CHICKEN GATE? Jean-François Focant CART, Organic and Biological Analytical Chemistry, Department of Chemistry, University of Liège, Belgium [email protected] When more than a million of broiler chickens suddenly and unexpectedly died in the eastern and Midwestern parts of the United States in late 1957, the first dioxin crisis record was set [1]. The 1999 Belgian dioxin chicken gate affair ultimately demonstrated the economic damage such a contamination episode could yield to and pushed the European Union (EU) to set an efficient and pro-active monitoring program to ensure proper quality of the European food and feed web and try to maintain most of the population below tolerable weekly intake [2,3]. The current European Commission (EC) strategy relies on the implementation of maximum (and action) residue levels (MRLs) for selected polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) (both families actually called ‘dioxins’), and dioxin-like polychlorinated biphenyls (DL-PCBs). To ensure the adequate production of comprehensive and reliable data on the presence of PCDD/Fs dioxins and DL-PCBs in food and feed, a screening-confirmatory approach was thus early adopted for the official control of the PCDD/F dioxins and DL-PCBs [4,5]. In practice, screening is most of the time performed using chemical activated luciferase gene expression (CALUX) bioassays, response-binding assays (RBAs) based on the aryl hydrocarbon receptor (AhR) [6], although the sole confirmatory method is gas chromatography coupled to 13C-labeled isotope dilution sector high resolution mass spectrometry (GC-IDHRMS) [7]. More than 10 years after the implementation of the screening-confirmatory approach, the analytical situation has drastically evolved because of advances in automation and hyphenation of parallel sample preparation techniques that allowed both cost and result delivery time to be significantly reduced (down to 350 EUR and 24 h, respectively) for the confirmatory GC-IDHRMS method [8]. Nevertheless, RBAs could and should now be used with non-restrictive sample preparation techniques that would allow most toxicants present in the sample to interact with the AhR to give a general persistent organic pollutant (POP) toxicity information rather than an estimation limited to dioxin regulation compliance. This would be much more biologically relevant and would allow enlarging current food safety practices to other known and unknown POPs. Sample showing high response for the biological screening should be further analyzed in the hope of identifying new compounds. The extension of the list of target compounds to more ‘exotic’ (un)suspected persistent molecules present in our food requires both chromatographic resolution and instrumental limits of detection (iLODs) to be improved. Using comprehensive two-dimensional GC (GCxGC) [9,10] or cryogenic zone compression (CZC) [11] GC coupled to HR time-of-flight MS (HRTOFMS) operating in full scan (FS) mode [12] could nicely complement the classical GC-sector IDHRMS performing in selected ion monitoring (SIM) used for target analyses. This would open the possibility to screen for other compounds (organochlorine pesticides, halogenated flame retardants, GC-amenable perfluorinated compounds, …) than the one under current regulation without compromising sensitivity. Recent advances in coupling between GCxGC and HRTOFMS nicely put this approach one step further as it allows producing elemental composition data for unknown compounds present in complex mixtures. Furthermore, when considering halogenated compounds, such a system can be granted by a tremendous improvement in sensitivity (back to low fg iLODs) by favouring resonance electron capture (REC) when operating in


negative chemical ionization (NCI) rather than electron impact (EI). Major recent analytical advances in the GC-MS of emerging GC-amenable POPs in the context of prioritization of new targets for food control will be highlighted. References 1. Firestone, D., 1973. Environmental Health Perspectives 5: 59-66. 2. Bernard, A., Hermans, C., Broeckaert, F., De Poorter, G., De Cock, A. and Houins, G., 1999.

Nature 401: 231-232. 3. Scientific Committee on Food, 2001. Report CS/CNTM/DIOXIN/20 Final.

(http://ec.europa.eu/food/fs/sc/scf/out90_en.pdf) 4. European Commission (EC), 2006. Commission Regulation (EC) No 1883/2006. Official Journal of

the European Union L364: 32-43. 5. European Commission (EC), 2009. Commission Regulation (EC) No 152/2009 Official Journal of

the European Union L54: 1-130. 6. Windal, I., Van Wouwe, N., Eppe, G., Xhrouet, C., Debacker, V., Baeyens, W., De Pauw, E. and

Goeyens, L., 2005. Environmental Science and Technology 39: 7357-7364. 7. Eppe, G., Focant, J.-F. and De Pauw, E., 2005. In: Gross, M.L., Caprioli, R.M. and Niessen,

W.M.A. (eds.) Encyclopedia of mass spectrometry, Vol. 8: Hyphenated methods, Elsevier, Amsterdam, the Netherlands, pp. 531-541.

8. Focant, J.-F., Pirard, C. and De Pauw, E., 2004. Talanta 63: 1101-1113 9. Focant, J.-F., Pirard, C., Eppe, G. and De Pauw, E., 2005. Journal of Chromatography A 1067:

265-275. 10. Focant, J.-F., Sjödin, A. and Patterson Jr., D.G., 2005. In: Gross, M.L., Caprioli, R.M. and Niessen,

W.M.A. (eds.) Encyclopedia of mass spectrometry, Vol. 8: Hyphenated methods, Elsevier, Amsterdam, the Netherlands, pp. 553-564.

11. Patterson Jr., D.G., Welch, S.M., Turner, W.E., Sjödin, A. and Focant, J.-F., 2011. Journal of Chromatography A 1218: 3274-3281.

12. Shunji, H., Yoshikatsu, T., Akihiro, F., Hiroyasu, I., Kiyoshi, T., Yasuyuki, S., Masa-aki, U., Akihiko, K., Kazuo, T., Hideyuki, O. and Katsunori, A., 2008. Journal of Chromatography A 1178: 187-198.


DEVELOPMENTS IN ANALYTICAL METHODS FOR DETECTION OF BIOGENIC AMINES IN FOOD Attila Kiss and B. Bóka Egerfood Knowledge Centre, Eszterházy Károly University, Hungary [email protected] Biogenic amines are low-molecular-weight, nitrogen-containing compounds of biological importance. Although they are essential to living organisms, consumption of food containing high amounts of them may have toxicological effects. The so-called histamine poisoning can occur after consumption of scombroid fish not properly handled, or fermented foods, such as cheese and wine. The adverse effect of tyramine, known as cheese reaction, can be observed in case of cheese products with high tyramine content. In some fermented foods, like cheese and wine higher amount of biogenic amines can be expected, but in non-fermented food like meat products they can be present as a consequence of microbial contamination and inappropriate conditions during processing and storage. Therefore, the content of biogenic amines, especially putrescine, cadaverine, histamine and tyramine can be considered as freshness markers and could be used as an indicator of microbial spoilage. The analytical methods used for the determination of biogenic amines are mainly based on chromatographic methods: thin layer chromatography (TLC), gas chromatography (GC) and high-performance liquid chromatography (HPLC). Among them, HPLC is most often used for food analysis. The large majority of assays employ fluorimetric or spectroscopic detection with pre- or post-column derivatisation techniques. These methods are time-consuming, require expensive instrumentation and chemicals, and well-trained experts. Contrary to the above mentioned methods, biosensors offer a simple, rapid and cost-effective solution. The aim of our work was to develop enzyme based amperometric biosensors for the determination of biogenic amines in food samples. Diamine oxidase (EC 1.4.3.6) from Pisum sativum, human recombinant monoamine oxidase A (EC 1.4.3.4) and putrescine oxidase (EC 1.4.3.10) from Kocurea rosea were selected. Diamine oxidase (DAO, EC 1.4.3.6) from Pisum sativum reacts with the most important biogenic amines that can be found in foods, so the total biogenic amine content could be determined with the DAO biosensor. Monoamine oxidase A (MAO A) was used for detection of tyramine and tryptamine content, while putrescine oxidase (PUO, EC 1.4.3.10) isolated from Kocurea rosea was used for the selective measurement of putrescine. The enzymes were separately immobilized on the surface of a graphite electrode in redox hydrogel with horseradish peroxidase, Os mediator and poly(ethylene glycol) (400) diglycidyl ether (PEGDGE) as crosslinker. Three-electrode configuration was employed in the wall-jet type amperometric cell, consisting of one of the bienzymatic measuring electrode, whereas Ag/AgCl electrode and platinum electrode served as reference and counter electrodes, respectively. The sensors worked in flow injection analysis system (FIA) using a potentiostat (QuadStat 164, eDAQ, USA). The linear measuring range was 0.01-0.5 mM for the PUO sensor (phosphate buffer 66 mM, pH 8.0), for MAO A electrode in tyramine equivalent (phosphate buffer 100 mM, pH 8.0) and for DAO electrode in histamine equivalent (phosphate buffer 100 mM, pH 7.0), respectively. Biosensors were investigated for the analysis of different beer, wine and cheese samples. Beer and wine samples could be measured directly after dilution. The amine content of cheese samples was extracted with 100 mM phosphate buffer, pH 7.0 for biosensor analysis. This extract could be measured after centrifugation and dilution. The developed biosensors were also tested for freshness monitoring of fish and pork meat samples stored at different


conditions. Spun drips from meat were collected from time to time with cooled centrifuge using a special plastic separator, and measured by biosensor methods after dilution. The biogenic amine content was also measured by HPLC method for validation. The biogenic amine content was also measured by HPLC method for validation. References 1. Bóka, B., Adányi, N., Virág, D., Sebela, M. and Kiss, A., 2012. Spoilage detection with biogenic

amine biosensors, comparison of different enzyme electrodes. Electroanalysis 24: 181-186.


U-HPLC-ORBITRAP-MS FOR QUANTIFICATION OF KNOWN AND IDENTIFICATION OF UNKNOWN BOAR TAINT CONTRIBUTING COMPOUNDS Lynn Vanhaecke, J. Vanden Bussche and K. Bekaert Laboratory of Chemical Analysis, Department of Veterinary Public Health and Food Safety, Ghent University, Belgium [email protected] Boar taint is an off-odour and off-flavour that can occur when meat or fat from entire male pigs is heated. Most of the analytical methods available so far were not capable of detecting the three known boar taint compounds (indole, skatole and androstenone) simultaneously, which rendered their analysis labour intensive and time-consuming as separate analyses were required. Moreover, the concentration levels of these known boar taint contributing compounds do not completely account for the occurrence of boar taint as determined by sensory analyses. A plausible explanation for this would be the contribution of other ‘unknown’ compounds to boar taint. Therefore in this research, a new U-HPLC-HR-Orbitrap-MS analysis method was developed for the quantitative determination of the three known and possibly unknown boar taint compounds in fat. The sample pretreatment involved a melting step followed by extraction with methanol and clean-up consisting of freezing the extract and solid phase extraction (HLB cartridges). The analytes were then chromatographically separated and detected with an ExactiveTM high-resolution mass spectrometer. Due to the absence of guidelines for the analysis of boar taint, Commission Decision 2002/657/EC [1] and ISO 17025 [2] were used as reference for the validation of the developed detection method. This resulted in limits of detection and limits of quantification between 2.5 and 7 μg/kg and between 5 and 10 μg/kg for the three compounds, respectively, which is far below the threshold values set at 100 μg/l for indole, 200 μg/l for skatole and 1000 μg/l for androstenone in pig fat samples. The method demonstrated for all three compounds, a repeatability (RSD) below 12.7% and a within-laboratory reproducibility (RSD) below 16.9%. The recovery of the compounds ranged between 99 and 112% and an excellent linearity (R2 ≥ 0.99) was observed. In addition, 92 boar adipose samples collected during a previous study were measured within an inter-laboratory comparison. Although these samples were stored for over 6 months and endured several freeze-thaw cycles prior to analysis, the correlations between the different analytical results for indole, skatole and androstenone amounted respectively 0.48, 0.75 and 0.92. Next, a post-acquisition re-interrogation of this dataset of boar taint positive and negative samples was performed. A targeted screening of the compounds identified during previous research (GC-Sniff analysis) and literature findings using ToxID software identified two additional compounds that were correlated (R2 resp. 0.105 and 0.075) to boar taint, namely 4,16-androstadie-3-one and isovaleric acid. Within this dataset 67% of the sensory score could be explained by skatole and androstenone. This R2 did however not increase when these new compounds were included. Therefore, an untargeted post-acquisition screening of the same dataset, using differential and OPLS-DA analyses enabled the elucidation of eight new boar taint contributing compounds, including the three established boar taint compounds androstenone, skatole and indole. In total six new compounds were identified as correlated to boar taint, with R2 ranging form 0.142 to 0.424. Skatole, 6-methoxyindole and a newly unidentified ion were able to explain 58% of the sensory score of this database, while skatole and androstenone only explained for 53% of the score. References 1. European Commission (EC), 2002. Commission Decision 2002/657/EC. Official Journal of the

European Communities L221: 8-36. 2. ISO, 2005. General requirements for the competence of testing and calibration laboratories.

ISO/IEC 17025:2005.


PLANAR PROTEIN ARRAYS IN MICROTITER PLATES – DEVELOPMENT OF A NEW FORMAT TOWARDS ACCURATE AUTOMATION-FRIENDLY AND AFFORDABLE (A3) DIAGNOSTICS Hans Dijk Scienion AG, Germany [email protected] A technology for protein microarrays in 96-well microplates has been developed as a widely adoptable platform for multiplexed protein based diagnostics. Procedures for protein microarray manufacturing, immobilization, assay optimization and optical detection methods were developed. Using a clever combination of state-of-the-art technologies, a versatile platform that works with peptides, proteins and antibodies as capture agents in a scalable format for planar arrays is described. Protein microarrays have become an important high throughput tool to investigate protein-protein interactions, because they provide a parallel, fast and straightforward analysis of as many proteins as can potentially be immobilized on a given surface. For diagnostic applications typically a chosen subset of capture agents is deposited on planar substrates, to allow the parallel analysis of two or more interactions in a single reaction. Planar protein arrays have historically been developed using inexpensive membrane formats (lateral flow-based rapid test diagnostics). A significant number of publications have described the use of glass slides for capture probe immobilization. While membrane based diagnostic tests can easily be produced and interpreted, there are some disadvantages regarding the use of slides. These disadvantages (relatively high price and difficult handling) have driven many researchers and developers of diagnostic tests to look for alternatives to glass slides. The so-called biosensor also is a planar substrate. Although biosensors can be mass produced, still their size is something that is typically unknown in diagnostic laboratories and often the interface to the real laboratory environment needs more and better ergonomic design. Another planar microarray format is the multi-well or microtiter plate (‘ELISA-plate’). These plates are well known in the diagnostics industry and cover a broad spectrum of biological, chemical and pharmacological laboratory uses. For high-throughput screening as well as diagnostic methods such plates are mostly produced in one piece by injection moulding. Especially as a result of high throughput applications auxiliary automatic and robotic instrumentation have been developed and the dimensions of these plates were standardized. Direct micro-spotting into microplates is a potentially straightforward and inexpensive way to support a diagnostic platform. As an example different antibodies directed against various antibiotics can be spotted into the wells of a microplate. After incubation with a sample (for instance milk) and a secondary antibody with a detector molecule, each individual well can be scanned. The scanned pattern of spots can be interpreted via dedicated software and a result can be obtained. This multiplex approach seems to have a promising future as an alternative to single result ELISA’s and lateral flow-based test-strips. Please visit the Scienion stand in the exhibition area. We will be pleased to welcome you and provide you full information on our products and applications.


DETERMINATION OF ORGANIC CONTAMINANTS IN WATER SAMPLES USING AN AUTOMATED SPE SYSTEM Wim Traag, A. Goossens, S. Munniks, G. van der Weg and R. Peters RIKILT-Institute of Food Safety, Wageningen UR, the Netherlands [email protected] The main mission of RIKILT is to contribute to the safety of the Dutch food supply. In addition to that, RIKILT has developed into a crisis organization not only for food and feed but also for environmental accidents, for instance in case of a fire. In such emergency situations there is a demand for a 24/7 analyses capability for samples that should be analysed for a number of hazardous compounds with a very short response time. In order to fulfil this task automation of analysis methods is a prerequisite. The analysis of polychlorinated dibenzo-p-dioxin (PCDD/Fs) and dioxin-like polychlorinated biphenyls (PCBs) has often been a subject of incidents with food, feed or fires in the past two decades. By far reaching automation, sample capacity for the determination of dioxins and PCBs has been increased resulting in shorter turnaround time of 2 to 4 days and a higher sample capacity. This automation includes extraction, clean-up and unattended extract concentration, followed by state of the art gas or liquid chromatography coupled to mass spectrometric techniques. For aqueous samples solid phase extraction (SPE) is a commonly used technique for concentration and separation of compounds of interest from complex matrices. Solid phase extraction columns are available with a variety of stationary phases, each of which can separate analytes according to their different chemical properties. However, manual application of SPE is not very practical when working with samples for which large sample volumes (>100 ml) are needed in order to meet the required limits of quantification. Therefore an automated SPE-system (PowerPrep™ SPE; Fluid Management Systems, Waltham, USA) has been evaluated. This system consists of one module allowing five samples to be processed. Since six modules can be controlled by one system, a maximum of 30 samples can be processed automatically. The system can process one sample at a time and, when dealing with large volume samples, it can take half an hour per sample to process. Since the process is fully automated, the samples can be processed overnight. In this presentation the first results will be presented using aqueous samples from a massive fire at a chemicals packaging plant in Moerdijk, south of Rotterdam. During this fire enormous clouds of smoke were emitted leading officials to declare a phase four alarm. RIKLT was requested to analyse a few hundred samples including grass, filters, soil, water and vegetables. Analysis of fire fighting water showed high amounts of organic compounds including phenoxy herbicides that were stored at the chemical packing plant. Please visit the Campro Scientific / Fluid Management Systems stand in the exhibition area. We will be pleased to welcome you and provide you full information on our products and applications.



INNOVATIVE ONLINE SAMPLE PREPARATION TECHNIQUES IN FOOD CONTAMINANT ANALYSIS Laszlo Hollosi1, K. Bousova1, E. Ates1 and M. Godula2

1Thermo Fisher Scientific, Germany and 2Thermo Fisher Scientific, Czech Republic [email protected] Liquid phase online sample preparation techniques possess lot of benefits over offline sample preparation techniques. Through online coupling, advantages of solid phase extraction (sample clean-up and pre-concentration) and direct injection (sample analysis) techniques can be achieved. Due to elimination of the most significant error source (human factor) from the sample manipulation procedure, online sample preparation techniques can significantly reduce sample preparation time, operational costs and simultaneously increase both method repeatability and reproducibility values. Online coupling of turbulent flow (TurboFlow) chromatography and conventional HPLC or (U)HPLC techniques is one possible option to perform effective online sample preparation method, in which the TurboFlow column acts as a clean-up and trap column. TurboFlow chromatography applies high flow rates (1.5-5.0 ml/min) through relatively large particle columns (50-60 μm) resulting in a turbulent flow profile. Effective separation of matrix and typically smaller target compounds can be achieved primarily based on their different diffusion coefficients which results in less matrix load on the chromatographic column and less matrix effects in the detector. In some cases, TurboFlow columns can also act in a size exclusion mode restricting further penetration of matrix compounds into the pores embedded with active reverse or normal phase extraction sites, and reducing further co-extraction of matrix components. TurboFlow chromatography is increasingly being used in clinical chemistry but only a very limited number of food contaminant applications have been published up to now. This presentation aims to fill a gap and, after a brief introduction to the technique, to report on food safety relevant multi-residue applications applying online coupled Turboflow-(U)HPLC methods. Developed methods for four high relevant food contamination classes, i.e. pesticides, veterinary drugs, mycotoxins and selected food contact materials, in different liquid or solid matrices will be discussed and established method performance criteria will be presented. Special focus will be put on method performance parameters like matrix effects, sensitivity, repeatability values and accordance with legislation requirements. Please visit the Thermo Scientific stand in the exhibition area. We will be pleased to welcome you and provide you full information on our products and applications.


PESTICIDESCREENERTM: A COMPREHENSIVE SOLUTION FOR MULTI-TARGET SCREENING OF PESTICIDES IN FOOD USING ACCURATE-MASS HIGH RESOLUTION MASS SPECTROMETRY AND AN INFORMATION-RICH DATABASE Rob van der Heijden Bruker Nederland BV, the Netherlands [email protected] Comprehensive full scan accurate mass screening becomes a valid alternative in food control, in particular if hundreds of pesticides have to be proved in a reasonable time frame. Additionally to the high number of targets the technique takes advantage of the evaluation of unknowns and retrospective analysis. The recently introduced PesticideScreener offers a complete high performance, accurate mass-based solution for multi-residue analysis as a turnkey approach to accurate and verifiable analysis of pesticides in food and feed. Enabling fast and reliable identification of selected compounds at ppb levels in complex sample matrices, with very low false positive rates, the PesticideScreener identifies compounds based on combined scoring of mass accuracy, isotopic patterns, retention times and, optionally, MS/MS qualifier ion fragment data. At the heart of the PesticideScreener is a high-quality database of over 700 target pesticides. This database is not merely a ‘list of compounds’, but includes retention times for the matched UHPLC method, adduct information, isomer information, fragment ion information, isotopic confirmation, qualifier ions for ‘one-shot’-confirmation in broad-band MS/MS mode. Processing is completely automated with our well established and comprehensive software TargetAnalysis, which screens data and delivers simple scoring against database information for at-a-glance confidence. This enables fast and reliable identification of selected compounds at ppb levels with very low false positive rates even in complex matrices. The engine driving PesticideScreener is an ultra-high resolution (UHR) bench-top QTOF mass spectrometer, the maXis Impact offering high resolution and high speed simultaneously for coupling to UHPLC, unique full sensitivity resolution (FSR) of greater than 40,000, one-shot method providing screening and confirmation in the same run, true isotope pattern (TIP) for ultimate confidence in molecular formula determination and high-resolution extracted ion chromatograms (hrXIC) at +/- 1 mDa. Excellent sensitivity, high dynamic range and mass accuracy meet the performance requirements, established in EU protocol SANCO/ 12495/2011. PesticideScreener has been developed with the sole purpose of providing food safety laboratories with a confident and comprehensive multi-target analysis solution. In this presentation the work flow of the PesticideScreener is discussed and is illustrated with numerous experimental (validation) data from a series of food matrices. Please visit the Bruker Nederland stand in the exhibition area. We will be pleased to welcome you and provide you full information on our products and applications.


SHIMADZU TOTAL SOLUTION FOR QUALITY DEFECTS IN FOOD, FEED AND WATER Johan Scholtens Shimadzu Benelux, the Netherlands [email protected] The number of food, feed and water related complaints to manufacturers and public agencies regarding contamination with foreign substances and poisoning symptoms continue to increase year after year. The types of contamination with foreign compounds vary widely and it is difficult to specify the route of contamination. Various other complaints such as a strange smell, strange taste, a different texture, etc. are also commonly received. It is very important for companies receiving reports of such complaints to investigate and ascertain the cause and to take prompt and appropriate measures in order to prevent any reoccurrences. To address the many types of problems Shimadzu offers an array of analytical instrument for detecting and analyzing quality defects. X-ray transmission instrument is useful for detection of high density contaminants. FTIR excels in analysis of contaminants that consist of organic substances, allowing qualitative analysis based on infrared spectra. TOC (total organic carbon) analyzer is ideal for monitoring drinking water and waste water, and for assisting in the management of water in manufacturing processes. HPLC and LC-MS are typically used for separation, qualitative and quantitative analysis of odour causing hydrophylic compounds in aqueous solution. Here, we have introduced techniques for evaluating characteristic flavours due to acidity, sweetness, bitterness and astringency. GC-MS can be used for the separation, qualitative analysis and quantitation of odour-causing compounds having different volatility characteristics. Furthermore, using mass spectrometry as detection system quantitation with high sensitivity has been made possible. The Texture Analyzer EZ-S is an instrument that allows evaluation of such food ‘texture’ components as crunchiness, firmness and palatability using numerical measurement data. Shimadzu Corporation, a diversified analytical and measuring instrument manufacturer, provides a comprehensive array of instruments: spectrometers, chromatography systems, X-ray and physical testing instruments.

Please visit the Shimadzu stand in the exhibition area. We will be pleased to welcome you and provide you full information on our products and applications.


BIOSENSORS FOR FOOD AND THE ENVIRONMENT: IS IT ALL IN THE DNA? Plenary Keynote Lecture Valerio Beni Biosensors & Bioelectronics Centre, Department of Physics, Chemistry and Biology (IFM), Linköping University, Sweden [email protected] Biosensor technology not only holds great promise for the healthcare market but it is also expected to have significant impacts in other areas such as environmental and food analysis. Between the different biosensor formats nucleic acid-based biosensors are gaining increasingly importance allowing the specific identification of DNA/RNA fragments and, more recently, the detection of biological or chemical species. Direct identification of DNA/RNA fragments, via hybridisation assays (genosensors), has been shown to be a very powerful tool for the detection of pathogens in environment and food, in food contamination analysis and food allergens monitoring. Recently the discovery of aptamers, synthetic receptors based on short functional DNA/RNA chains, has opened new opportunities for oligonucleotide-based biosensors and more specifically in the detection of biological and/or chemical species, such as proteins and pollutants. In this paper an overview of the most recent advances in the development and applications of nucleic acid-based biosensors for environmental and food safety/quality application will be presented with special emphasis on electrochemical and optical hybridisation genosensors and aptasensors.


DOES SYNTHETIC BIOLOGY OFFER A NEW APPROACH FOR BIOSENSOR DESIGN? Plenary Keynote Lecture Paul Freemont Centre for Synthetic Biology and Innovation, Imperial College London, UK [email protected] Synthetic biology is an application driven field attempting to apply a rational engineering approach to the re-design of existing or new biological systems. Notable progress has been made in the development of platform technologies like large-scale DNA synthesis and assembly as well as the implementation of synthetic genetic circuits in living-systems that mimic functions such as switches, oscillators, timers, pulse generators, band-pass filters and logic gates. These genetic circuits are anticipated to enable the engineering of biological systems to a complexity previously unattainable [1]. Over the past decade, synthetic biology has aided the development of novel biological systems for a range of applications from bacteria that seek and destroy herbicides to the biosynthesis of valuable drugs [2,3]. The potential economic promise of synthetic biology is such that numerous countries are developing strategies for establishing synthetic biology in both academia and industry [4]. Synthetic biology can be thought of as a natural evolution of biotechnology, as opposed to being a separate field although it does offer a novel and exciting approach. In the last 40 years biotechnology has undoubtedly produced many valuable biological products, yet current approaches tend to be firstly lengthy due to the ad hoc nature of the approach and secondly focused on the tweaking of one or a few genes [5]. In addition, there is little lateral transfer of knowledge between projects meaning knowledge gained in research and development in one area may not assist the post hoc development of other projects in other unrelated areas. Taken together, this has made the engineering of biological systems time-consuming and costly. One application area that has received significant attention from synthetic biologists is biosensors. Whilst this application area is already very mature with many devices and/or kits already exist on the market or are fully utilised in analytical laboratories worldwide, some new approaches from synthetic biology are emerging. These primarily include the application of systematic design to DNA-encoded biosensors that can operate within whole cells, as well as in vitro ‘cell-free’ systems or artificial ‘cell-like’ systems. This enables researchers to design specific but reusable genetic circuits/modules that satisfy a 3-step design strategy namely Input –sensing/amplification – Output (Figure).

In this talk I will present a ‘proof of concept’ exemplar for the detection of pathogenic bacterial biofilms to illustrate the synthetic biology approach [6]. References 1. Kitney, R.I. and Freemont, P.S., 2012. Synthetic Biology – the state of play. FEBS Letters 586:

2029-2036.


2. Sinha, J., Reyes, S.J. and Gallivan, J.P., 2010. Reprogramming bacteria to seek and destroy an herbicide. Nature Chemical Biology 6: 464-470.

3. Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J.D. and Keasling, J.D., 2003. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotechnology 21:796-802.

4. http://www.innovateuk.org/content/news/synthetic-biology-roadmap-.ashx 5. Purnick, P.E. and Weiss, R., 2009. The second wave of synthetic biology: from modules to

systems. Nature Reviews Molecular Cell Biology 10:410-422. 6. Chappell, J. and Freemont, P.S., 2011. Synthetic biology – a new generation of biofilm biosensors.

In: The Science and applications of synthetic and systems biology, Institute of Medicine of the National Academies USA, Forum on Microbial Threats Report.


RECENT DEVELOPMENTS IN RAPID MULTIPLEXED BIOANALYTICAL METHODS FOR FOODBORNE PATHOGENIC BACTERIA DETECTION Barbara Roda, C. Colliva, M. Mirasoli, M. Di Fusco, P. Reschiglian and A. Roda Department of Chemistry 'G. Ciamician', University of Bologna, Italy [email protected] Foodborne illnesses caused by pathogenic bacteria represent a widespread and growing problem to public health, and there is an obvious need for rapid detection of food pathogens. Traditional culture-based techniques require tedious sample workup and are time-consuming. It is expected that new and more rapid methods can replace current techniques. To enable large scale screening procedures, new multiplex analytical formats are being developed, and these allow the detection and/or identification of more than one pathogen in a single analytical run, thus cutting assay times and costs. A variety of strategies, such as multiplex polymerase chain reaction assays, microarray- or multichannel-based immunoassays, biosensors, and fingerprint-based approaches (such as mass spectrometry, electronic nose, or vibrational spectroscopic analysis of whole bacterial cells), have been explored [1-3]. In addition, various technological solutions have been adopted to improve detectability and to eliminate interferences, although in most cases a brief pre-enrichment step is still required. None of the proposed methods, however, is able to recognize a target bacterial species when present in bacterial mixtures, nor to distinguish between viable and non-viable cells. Here, we focus on a new integrated sensing system, in which an electronic nose is on-line coupled with a field-flow fractionation (FFF) device. FFF techniques are separation techniques suitable for the non-invasive fractionation of cells based on their morphological features [4]. In FFF the separation, which is performed within an empty channel, occurs due to the combination of two orthogonal forces: a laminar flow of mobile phase along the channel and a perpendicular field. Based on their size, shape, density and surface properties, analytes are positioned at different heights within the channel, where they encounter a fluid layer travelling at a given velocity. Analytes travelling towards the center of the channel are thus eluted faster than those travelling close to the channel walls. FFF employing earth gravity field as applied field has been widely applied for the separation of bacteria and we have previously demonstrated the possibility to on-line couple FFF with orthogonal detection techniques, with significant improvements of analytical performance. The electronic nose used in this work is equipped with six metal oxide semiconductor sensors (MOS) producing a distinct response signature for each vapour regardless of its complexity, resulting in a ‘smell fingerprint’ that can be used for sample identification after multivariable chemometric data analysis [5]. Escherichia coli O157:H7 and Yersinia enterocolitica cells were used as model samples. The combination of FFF-EOS allowed to detect selectively a given bacteria in presence of other bacteria and to distinguish live and dead cells after a simple and fast sample fractionation, thus increasing multiplexing capabilities and simplifying sample preparation procedures. The direct application to real matrices such as contaminated milk samples was also demonstrated. References 1. Magliulo, M., Simoni, P., Guardigli, M., Michelini, E., Luciani, M., Lelli, R. and Roda, A., 2007.

Journal of Agricultural and Food Chemistry 55: 4933-4939.


2. Donhauser, S.C., Niessner, R. and Seidel, M., 2011. Analytical Chemistry 83:3153-3160. 3. Parisi, D., Magliulo, M., Nanni, P., Casale, M., Forina, M. and Roda, A., 2008. Analytical and

Bioanalytical Chemistry 391:2127-2134. 4. Roda, B., Zattoni, A., Reschiglian, P., Moon, M.H., Mirasoli, M., Michelini, E. and Roda, A., 2009.

Analytica Chimica Acta 635:132-143. 5. Dutta, R., Hines, E.L., Gardner, J.W. and Boilot, P., 2002. BioMedical Engineering OnLine 1: 4.


SOPHISTICATED APPLICATION OF STANDARD ELISAs FOR MULTI-ANALYTE DETECTION Alex J. Räber1, X. Yang1, D. Zwald1, B. Kramer2, W. Schielen3 and G.D. Keizer4

1Prionics AG, Switzerland, 2Micronic Holding B.V., the Netherlands, 3European Livestock Diagnostic Center B.V., the Netherlands and 1Prionics Lelystad B.V., the Netherlands [email protected]

Despite precautions taken, the food industry struggles with high levels of bacterial and viral food contamination caused by pathogens. These pathogens form a significant risk for the quality and safety of the food supply chain, causing associated foodborne illness and economical losses. As a result, there is increasing public and regulatory pressure on the agro-food industry to reduce the numbers of pathogens in the entire food chain. Unfortunately, none of the available tests allow the detection of multiple pathogens in single assays, where animals and animal products need to be tested for several different types of pathogens. Detection of these multiple pathogens requires parallel tests for each different type of pathogen and is therefore time consuming and expensive. Hence, many animal stocks are not tested on multiple pathogens and as a result infectious-causing pathogens can spread among animals and pass into the food chain. We have developed a novel diagnostic platform, the MultiFlex technology, which is essentially based on ELISA technology but allows multiple tests to be performed simultaneously on one sample. Standard sized microtiter plates were designed in which each assay unit consists of a group of four clustered wells. The interconnection between the four wells is lowered to a height that allows individual coating of each well to the appropriate testing volume. The applied sample spills over the lowered wall and presents the sample to all four wells at the same time. Addition of sample and conjugate and subsequent washing and detection is analogous to the current ELISA based 96-well processing of assays for detection of pathogens. As a prototype assay on the MultiFlex platform, a porcine food safety test panel was developed and evaluated. The test panel consists of four pathogens in swine which present a major threat to the human food chain: Salmonella, Trichinella, Toxoplasma and Mycobacterium avium. All tests were developed as individual indirect ELISAs and were converted to the MultiFlex format by harmonising the buffers and incubation steps. Results on the performance and outlook on the evaluation of the four individual tests on the MultiFlex platform will be presented and discussed.


FTIR SPECTROSCOPY: SOURCE TRACKING AND MONITORING OF MICROBIAL CONTAMINATION IN FOOD INDUSTRY Volha Shapaval1,2, T. Møretrø1, J. Schmitt3, H.-P. Suso4, R. Talintyre5, D. Zeze6, A. Kohler2,1

1Nofima AS, Norway, 2Department of Mathematical Sciences and Technology, Norwegian University of Life Sciences, Norway, 3Synthon GmbH, Germany, 4Elopak, Norway, 5Labman Automation Ltd., UK and 6School of Engineering & Computing Sciences, Durham University, UK [email protected] The interest in FTIR spectroscopy for bacteria, yeast and moulds identification is growing; food industry and the medical sector are already making extensive use of this technique. Over the last ten years, application of infrared spectroscopy as a modality to characterize microorganisms has undergone a remarkable evolution. Many papers have been published dealing with the application of vibrational spectroscopy in microbiology, giving technical details and describing different applications [1-3]. FTIR spectroscopy is a biophysical technique providing a precise fingerprint of the overall biochemical composition of microbial cells (Figure 1). FTIR spectra of microorganisms contain absorption bands of many different chemical functional groups referring to a large number of bio-molecules. The spectra can be roughly divided into specific regions containing signatures of fatty acids, proteins, and polysaccharides, respectively. An FTIR spectrum is therefore not restricted to one specific cell characteristic like other methods used for microbial identification; it is based on the sampling of many different spectral characteristics. Another important asset of the technique is that it can be used to solve different kinds of identification problems with one and the same technique. The technique is applicable to all microorganisms that can be grown in culture. Figure 1. Typical IR spectra obtained from Aspergillus fumigatus.

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It has been recently shown that FTIR spectroscopy, when combined with high throughput liquid microcultivation, allows analyzing a high number of microorganisms and identify samples at species, sometimes even at strain level [4,5]. In addition, it allows easily building big libraries/databases, where spectra for all reference and non-reference microorganisms can be stored along with the information, obtained by other analytical techniques. In combination with large reference databases, spectra of microbial cells can be analyzed for example for storage material production, spore formation or identification at the species level and in most cases at the subspecies level. Spectra of new strains are compared to spectra in


the database for identification. When not exactly the same strain is in the database, but strains belonging to the same species, the chance for correct identification is still very high. This is due to the fact that spectra being phylogenetically similar have similar spectral characteristics. The technique can for example be used for identification of mould strains. In a recent study, 59 food-spoilage strains were analysed and identified by high-throughput FTIR spectroscopy [5]. On genus level, 94% of the strains were correctly identified, while on species level the percentage of correct identification was 93.8%. In an up-coming EU project, the combination of high-throughput FTIR spectroscopy and high-throughput cultivation will be combined in a fully automated system that can be used for microbial source tracking along production chains. Acknowledgements This work is funded by the European Commission FP7 grant contract 315271 ‘Source tracking and monitoring of mould contamination in food production’ (FUST). References 1. Mansour, F.S., Nagy, K.S., Taqi, A.A. and Askar, K.A., 2011. African Journal of Biotechnology 10:

12957-12962. 2. Naumann, D., Helm, D., Labischinski, H., Giesbrecht P., 1991. In: Nelson, W.H. (ed.) Modern

techniques for rapid microbiological analysis. VCH Publishers, New York, NY, USA, pp. 43-96. 3. Naumann, D., 2001 In: Gremlich, H.U. and Yam, B. (eds.) Infrared and Raman spectroscopy.

Marcel Dekker, New York, NY, USA, pp.323-377. 4. Shapaval, V., Møretrø, T., Suso, H.-P., Wold Åsli, A., Schmitt, J., Lillehaug, D., Martens, H.,

Boecker, U. and Kohler, A., 2010. Journal of Biophotonics 3: 512-521. 5. Shapaval, V., Schmitt, J., Møretrø, T., Skaar, I. Suso, H.-P., Wold Åsli, A., Lillehaug, D. and

Kohler, A., 2012. Journal of Applied Microbiology, accepted.


NATURAL RECEPTOR-BASED BINDING ASSAYS AS SCREENING TOOL FOR TOXICITY PREDICTION Bram van der Gaag KWR Watercycle Research Institute, the Netherlands [email protected] At present, cell/bacteria based in vitro assays (Ames II, Comet assay, CALUX bioassays) are used to determine potential toxic effects in water samples of individual chemicals and mixtures. Total analysis time is in general 3-5 days and the assays are laborious to perform. Protein binding assays however can be performed within 15 min and in series overnight. Complementary on-line MS/MS analysis can be performed for the identification of the compound causing the toxicological effect [1]. The ‘functional’ aspect of the binding assays is defined by the binding of an activated receptor molecule (by its ligand) to a signal reporter molecule of the transcriptional chain. An example of such an interaction is described by Wärnmark et al. [2]. Their article describes the monitoring of the binding of an activated oestrogen receptor (by 17ß-estradiol) to factor 2 nuclear receptor box peptides [2]. Figure 1 is a schematically representation of this assay. Figure1. Functional binding assay mechanism between oestrogen receptor α to nuclear receptor co-regulator.

At KWR we started to develop Biacore 3000 based functional toxicity binding assays in addition to cell based toxicity assays. During this presentation results will be presented for estrogenic and adrenergic toxicity assays. For the oestrogenic assay a nuclear estrogen receptor and for the adrenergic assay a G-Protein coupled receptor was used. Although several groups published Biacore based functional binding assays using activated receptors and their binding partner it is not clear yet if these assays can be performed routinely [2-4]. A possible major bottleneck is the availability or production of the binding partners. Some of the receptor molecules are over-expressed in cell lines but soluble receptors are hard to collect in a stable way. Successes and setbacks will be reported as well as the potential benefits for these types of assays. References 1. Marchesini, G.R., Buijs, J., Haasnoot, W., Hooijerink, D., Jansson, O. and Nielen, M.W.F., 2008.

Nanoscale affinity chip interface for coupling inhibition SPR immunosensor screening with nano-LC TOF MS. Analytical Chemistry 80: 1159-168.

17ß-estradiol

Monomer ERa

NR co-regulator


2. Wärnmark, A.E, Gustafsson, J.A., Hubbard, R.E., Brzozowski, A.M. and Pike, A.C., 2002. Interaction of transcriptional intermediary factor 2 nuclear receptor box peptides with the coactivaor binding site of estrogen receptor α. The Journal of Biological Chemistry 277: 21862-21868.

3. Yu, C., Chen, L., Luo, H., Chen, J., Cheng, F., Gui, C., Zhang, R., Shen, J., Chen, K, Jiang, H. and Shen, X., 2004. Binding analyses between human PPARgamma–LBD and ligands. European Journal of Biochemistry 271:386-397.

4. Yue, L., Ye, F., Gui, C., Luo, H., Cai, J., Shen, J., Chen, K., Shen, X. and Jiang, H., 2005. Ligand-binding regulation of LXR/RXR and LXR/PPAR heterodimerizations: SPR technology-based kinetic analysis correlated with molecular dynamics simulation. Protein Science 14: 812-822.


ALTERNATIVE BIORECOGNITION ELEMENTS FOR FOOD AND ENVIRONMENTAL BIOSENSORS Johan Robbens, J. Mehta, G. Vandermeersch, E.-R. Martin, K. Bekaert, B. Van Dorst and R. Blust Animal Sciences Unit, Institute for Agricultural and Fisheries Research, Belgium [email protected] A sensitive monitoring of contaminants in food, feed and environment is essential to assess and avoid risks for human health and environmental. There is a high need for sensitive, robust and cost-effective biosensors that make real time and in situ monitoring possible. Due to their high sensitivity, selectivity and versatility, affinity-based biosensors are interesting for monitoring contaminants in food and environment. Recently a lot of research effort has been dedicated to the development of novel recognition elements – as alternative to antibodies –with improved characteristics, like specificity, stability and cost-efficiency. Newly developed methods for better selection of biorecognition elements and characterization of the binding have highly impacted the field. Different innovative affinity-based recognition elements will be discussed that are of special interest for food, feed and environmental sensors.


ADVANTAGES OF LLAMA SINGLE-DOMAIN ANTIBODY FRAGMENTS IN DIAGNOSTIC APPLICATIONS OF INFECTIOUS DISEASES, WITH A FOCUS ON CAMPYLOBACTER JEJUNI Michiel M. Harmsen and F.-J. van der Wal Central Veterinary Institute, Wageningen UR, the Netherlands [email protected] Camelids and sharks produce single-domain antibodies that are naturally devoid of an antibody light chain. The single N-terminal antigen binding fragments of these antibodies, especially those derived of camelids (VHHs, nanobodies), show great promise for biotechnological applications due to their high suitability for phage display selection, high apparent physicochemical stability and amenability for production of multivalent and bispecific formats by molecular engineering. Furthermore, as with conventional recombinant antibodies, their specificity and affinity can later be improved by molecular evolution methods. We intend to use these nanobodies for immunoaffinity solid phase extraction of livestock pathogens from samples intended for diagnostic applications. In such applications the nanobodies are covalently bound to a solid support, resulting in an immunosorbent. Complex samples such as faeces, blood or meat containing the pathogens of interest can then be passed over these immunosorbents, washed, and eluted. This concentrated and cleaned sample can then be used in another assay for specific pathogen detection. Many pathogens show high antigenic variability and as a result most monoclonal antibodies will not recognize all variants of such a pathogen. However, for use in sample precleaning an immunosorbent is required that recognizes all pathogen variants. We intend to produce such immunosorbents by producing multivalent formats of VHHs by genetic fusion of several different VHH domains that recognize different pathogen variants. The use of such small recombinant antibody fragments, which is 20% of the size of an intact antibody, also offers the advantage that a higher density of functional binding sites can be present on the immunosorbent, which is important for enrichment of low amounts of pathogen from large sample volumes, i.e. for assay sensitivity. We have started such a project for detection of Campylobacter jejuni by qRT-PCR using immunosorbents containing anti-flagella nanobodies.


DETECTION OF HEAVY METALS AND OTHER CONTAMINANTS WITH BIOLUMINESCENT MICROORGANISMS Gérald Thouand University Institute of Technology of La Roche-sur-Yon, Technical University of Nantes, France [email protected] The pollution of the environment includes the biological pollution (micro-organisms, species accidentally introduced into a biotope), the chemical pollution and the physical pollution (e.g. radiation). Chemical pollutions include inorganic contaminants, primarily metals, metalloids and organic micropollutants. The former are naturally present in the earth's crust, they are limited to the elements of the periodic table, and thus the resulting pollution is due to an enrichment that will have more or less a toxic impact to organisms. Organic pollutants originate from human activities are unlimited. For example, the European Union published a list of 33 priority substances to be detected in the context of water policy in 2001 [1]. Among these substances, organotins, metals, polycyclic aromatic hydrocarbons (PAHs) and long chain alkanes found in different environments such as polluted marine waters are classified as ‘priority hazardous substances’. Other organic micropollutants not yet regulated at present are drugs (human and veterinary). They are often not degraded by the sewage treatments system and may eventually be found in the resources. A special issue in 2011 on the use of microorganisms for analysis provides a comprehensive overview of the current knowledge on biosensor microorganisms [2]. Microbial biosensors are advantageously applied in environment because they are able to account for the toxicity of an effluent or a chemical molecule (non-specific biosensor) or act as a specific biosensor for the measurement of a particular pollutant (metals, organotins, pesticides, ...). In all cases the organisms are sensitive to the bioavailable part of the pollutant, that is to say, the pollutant concentration which acts on a specific mechanism of microorganisms (respiration, DNA synthesis, ...). The orientation of our research is to develop biosensors and bioassays using bioluminescent bacteria for all the main priority substances listed in European Country legislation. So far, work has been done on organotin compounds and for heavy metals and pesticide. The group is now exploring developments of biosensor chips and microarrays for the detection of an overall toxicity or specific targets. For the detection of heavy metals, a lack of specificity of the bioreporter triggered specific research leading to the association of 5 bioluminescent strains, specific software and a new concept of biosensor using long term preservation of the bacteria and avoiding any encapsulation. It allowed to specifically quantifying a mixture of four heavy metals in environmental samples during a long time prefiguring robust and easy to use systems for on line monitoring in water [3,4]. Sea water contamination remains often an ecosystem that is seldom taken into account. We focused on the detection of the prohibited organotin compounds (tributyl- and dibutyltin) known to be highly toxic. Organotin compounds especially tributyltin (TBT) have been widely used as biocide to prevent biofouling on ship hull. Even if the major antifouling paint companies have decided to comply with this regulation there is no simple way to control the application of this regulation. We have developed a bioassay based on the use of a recombinant bioluminescent bacterium to detect TBT and dibutyltin (DBT), one of its major degradation products. The genetic construction and the localization of the lux genes in the chromosome of this bacteria have been studied [5-7]. The bioassay was tested on different commercial paint containing or not TBT, chemical analysis was done in parallel. The results show that the bacterium could detect the presence of TBT and DBT in the paint without any extraction procedure in one hour and also in waste


water from shipyards. Due to its simplicity this bioassay could be used to control the application of the legislation on antifouling paint. These two examples will be used to illustrate the approach, the limits as well as the future of the detection with microbial biosensors. References 1. European Commission (EC), 2001.Decision No 2455/2001/EC of the European Parliament and of

the Council. Official Journal of the European Communities L331: 1-5. 2. Thouand, G., 2011, Microorganisms for analysis. Analytical and Bioanalytical Chemistry 400: 893-

894. 3. Jouanneau,

S., Durand,

M.J., Courcoux, P., Blusseau, T. and Thouand, G., 2011. Improvement of

the identification of four heavy metals in environmental samples by using predictive decision tree models coupled with a set of five bioluminescent bacteria. Environmental Science and Technology 45: 2925-2931.

4. Jouanneau, S., Durand, M.J. and Thouand, G., 2012. Online detection of metals in environmental samples: comparing two concepts of bioluminescent bacterial biosensors. Environmental Science and Technology 46:11979-11987.

5. Durand, M.J., Thouand, G., Ivanova, T., Vachon, P. and DuBow, M.S., 2003. Specific detection of organotin compounds with a recombinant luminescent bacteria. Chemosphere 52: 103-111.

6. Gueuné, H., Durand, M.J., Thouand, G. and DuBow, M.S., 2008. The ygaVP genes of Escherichia coli form a tributyltin-inducible operon. Applied and Environmental Microbiology 74: 1954-1958.

7. Gueuné, H., Thouand, G. and Durand, M.J., 2009. A new bioassay for the inspection and identification of TBT-containing antifouling paint. Marine Pollution Bulletin 58: 1734-1738.


INTEGRATION OF LIVE CELLS WITH THE ORGANIC ELECTROCHEMICAL TRANSISTOR FOR IN VITRO DETECTION OF PATHOGENS AND TOXINS L.H. Jimison, S.A.Tria, M. Ramuz, A. Hama, M. Bongo, G.G. Malliaras and Rósin M. Owens Department of Bioelectronics, École Nationale Supérieure des Mines, France [email protected] The ability to measure paracellular transport provides a wealth of information about barrier tissue function. Measurement of the integrity of the apical junctional complex in epithelia and endothelia is key, not only for understanding the function of these structures but is additionally an indicator of toxicity or disease, as disruption or malfunction of these structures often arises due to the effects of pathogens and toxins. We aim to generate a novel method for measuring barrier tissue integrity as a tool for fundamental research on tight junction function, but also as means for carrying out in vitro toxicology. The organic electrochemical transistor (OECT) provides a very sensitive way to detect minute ionic currents in an electrolyte, due to inherent amplification of signal provided by the transistor. The OECT is a conducting polymer device fabricated of organic electronic materials which combines key advantages of low cost and easy fabrication which may be designed to suit the application in hand. We demonstrate the ability to measure dynamic, minute variations in barrier tissue integrity induced by toxic compounds in real time, with unprecedented temporal resolution and extreme sensitivity. Detection of several toxic species including peroxide, ethanol and the pathogenic bacterium Salmonella typhimurium was successfully demonstrated, with detection sensitivity or equal to existing methods. Our results were validated using other techniques for assessing barrier tissue integrity such as immunofluorescence staining of tight junction proteins, a permeability assay using a fluorescent tracer (Lucifer Yellow) and the Cellzscope TER measurement system. Ongoing work includes the incorporation of our device set up into an in situ measurement system with integrated electronics to allow long term measurements under conditions compatible with cell culture.


COMPUTATIONAL TECHNOLOGIES TOWARD TELEDIAGNOSIS AND TELEMICROSCOPY Neven Karlovac and O. Mudanyali Holomic LLC, USA [email protected] Our mission at Holomic LLC – in collaboration with UCLA's research laboratories of Prof. Aydogan Ozcan – is to develop low cost, portable and field-deployable instruments, enabling rapid and accurate screening of potentially hazardous biomarkers and contaminants (e.g. peanut) as well as pathogens (e.g. Escherichia coli) in water and food samples. To that end, we employ a range of technologies based on inexpensive and widely available CMOS cameras in smartphones or as standalone devices. The common theme of our work is to use back-end computational and hardware capabilities to compensate for the limitations of low cost sensors and sample preparation methods, and to use ubiquitous mobile networks for telemetry as well as centralized data collection and analysis. In this paper, we describe some of these platforms including a personalized cell-phone based food allergen testing tool, handheld readers for lateral flow immunoassays (LFIs) of food pathogens and toxins, cell-phone based fluorescent imagers for the detection of E. coli, and lens-free holographic microscopy for the detection and enumeration of biological contaminants and pathogens in water. LFI strips are most suitable for the rapid field screening of water and food samples due to their low cost and relatively simple sample preparation steps. Strips are commercially available for mycotoxins, allergens, gluten, GMO, enterotoxins, Salmonella, Giardia lamblia, and others. The Holomic rapid diagnostic reader (HRDR) utilizes a hardware attachment to the smartphone to digitally sense and evaluate the control and test lines of LFIs and provide fast, accurate and objective analysis of test results. The reader can operate as a standalone instrument and also wirelessly integrated into a global server to store and organize the digital diagnostic results/reports with other related data (e.g. RDT images, demographic data, test lot ID, etc.). Enabling real-time spatio-temporal mapping for the prevalence of various waterborne and foodborne diseases that is accessible through a web interface, this platform can be a valuable tool for monitoring the integrity of the supply chain and for tracking disease outbreaks. Smartphones are attractive as an instrument platform because of their imaging, computational, and communication capabilities, and low cost. In addition to the rapid diagnostic reader attachment for lateral flow devices, we also present two other platforms running on cell phones. One attachment performs calorimetric analysis of ‘iTubes’ that contain food samples for allergen analysis. Using a commercially available peanut-specific kit (Veratox Neogen 8430), this cell phone-based allergen monitoring tool achieves 1 ppm sensitivity for the peanut contamination in food samples (e.g. cookie). Another attachment converts the smartphone into a fluorescent microscope; using anti-E. coli O157:H7 antibody functionalized glass capillaries as solid substrates we report results for a quantum dot based sandwich assay with a detection limit of 5-10 cfu/ml. For detection of waterborne parasites in the field, we are also developing field-portable alternatives to conventional optical microscopes using lens-free holographic microscopy invented by Prof. Ozcan and his team at UCLA. These compact in-line digital holographic microscopes weighing less than 50 g do not require any mechanical alignments and the use of bulky/costly optical components, and yet achieve numerical aperture of ~0.1-0.2 over an imaging field of view of ~ 24 mm2 that is more than an order of magnitude larger than a typical 10X microscope objective. Using these lens-free microscopes, we were able to


image and automatically count Giardia lamblia and Cryptosporidium parvum at low concentrations <400/ml without use of any pre-concentration steps (e.g. membrane filtration). Imaging of E. coli is possible using additional technologies of pixel super-resolution and wetting films to improve resolution to less than 1μm. Providing compact, light-weight and low cost toolset for the identification and analysis of potentially hazardous contaminants and pathogens in water and food samples, our stand-alone and cell-phone based technologies can enable high-throughput and highly-sensitive diagnostics that can especially be useful to combat against waterborne and foodborne diseases and outbreaks.


A LAB-ON-A-CHIP INTEGRATED INTO A SMART SYSTEM: A STRATEGY TO FULFIL REAL DIAGNOSTICS Jesus M. Ruano-López IK4-Ikerlan, Spain [email protected] This work presents a lab-on-a-chip where DNA is injected, concentrated and washed in the first chamber and then eluted and transported to the second one for qPCR, using gelified reagents stored inside (Figure 1). A self-contained unit controls the lab-on-a-chip (Figures 2-4). Unlike other studies mentioned below, this lab-on-a-chip: (i) contains mastermix with fluorophores stored by gelification, allowing long-term storage and simplification of the lab-on-a-chip; (ii) has integrated valves; (iii) uses magnetic beads making the assay independent from sample volume and nature; (iv) has no clogging problems; (v) performs qPCR in a clean solution; (vi) is fabricated by three layer stack of COC polymer; and (v) is controlled by a tablet PC. . The biological method is explained in Figure1. The disposable lab-on-a-chip comprises a Luer syringe connector for sample loading, five microvalves, and two chambers. It is made of a COC substrate sealed by a polypropylene film coated with a pressure sensitive adhesive. COP discs work as valves (Figures 5 and 6). The control unit consists of an external fluorescence detector, two micropumps, two heaters and temperature sensors, two magnets, three bottles, a battery, 5 pin actuated micromotors, and an automatic lab-on-a-chip insertion mechanism. This platform is connected to a tablet PC by USB executing the automatic steps. None of the reader´s components need to be replaced/washed from assay to assay. Sample preparation and amplification have two research trends: (i) all steps in a one chamber chip, and (ii) lab-on-a-chip with several chambers. From the first trend, Ruano-López [1] fabricated a PMMA chip where reagents were stored under a paraffin layer in the same chamber where the amplification took place. Previously, DNA sample preparation and PCR on a SU8-one-chamber chip to detect Salmonella spp. in human faeces using magnetic beads was presented [2]. This chip was further used for Campylobacter spp. in chicken faeces [3] and for influenza viruses in nasopharyngeal human sample by RT qPCR [4]. Lee et al. developed a similar lab-on-a-chip for gram negative bacteria [5]. From the second trend, Gulliksen et al. [6] presented a lab-on-a-chip where RNA purification and NASBA were carried out by injecting a crude Escherichia coli cultured lysate. Landers group reported for the first time a fully integrated device capable of integrating solid-phase extraction (SPE) with PCR and electrophoresis [7]. The difference from the first trend is that our work allows DNA to be automatically concentrated and transferred into a clean second chamber. The difference from the second trend resides basically in the fact that SPE sample preparation part is more prone to clogging. Furthermore, Easley´s work did not integrate quantification. In Gulliksen’s work, the chamber was open to buffers and mixing depended on syringe pumps instead of microvalves. Our method is compatible with any lysate of raw material and magnetic beads. After the prevalidation experiments shown here (one shown in Figures 7 and 8), this instrument is being technically validated in a clinical, environmental and a food application under the EU project LABONFOIL (www.labonfoil.eu).


Figure 1. Schematic representation of the protocol: first 5 µl of DNA, 10 µl of magnetic beads, 50 µl of isopropyl alcohol are added to 995 µl of nuclease-free water; 1) DNA concentration is done by magnetic separation using two magnets placed on both sides of the concentration chamber; 2) as sample containing the magnetic beads and DNA passes through the chamber, beads are retained in the magnetic field generated by the two magnets. The impurities leave the lab-on-a-chip and only the beads and bound DNA remain inside the chamber; 3) DNA is eluted using elution buffer injected and carefully transferred to the PCR chamber; 4) DNA and PCR reagents are mixed rehydrating the gelified reagents; 5) once the amplification chamber is filled with eluted DNA, valves are closed and thermocycling begins with the fluorescence detector activated.

Figure 2. Picture of the lab-on-a-chip. Figure 3. Picture of the unit.

Figure 4. Unit schematic representation.

Figure 5. Fabrication flow process. Figure 6. Representation of the valve function; left,

diagram of the lab-on-a-chip; right, zoom and valve

in a open and close mode.

Figure 7. Graph showing the final result once the nucleic acid concentration, amplification and detection took place (fluorescence vs. cycle). The smaller graph shows the Bioanalyser result showing an amplification peak of 92 bp.

Figure 8. Left, picture of the chamber with the gelified reagents; right, pictures of the capsule covering the amplification and concentration chambers of the lab-on-a-chip; top, stored reagents with fluorophores and concentrated beads with bound DNA amplicons; bottom, mixing upon contact with eluted DNA.

Gelified Reagents


References 1. Ruano-López, J.M., 2009. Lab on a Chip 9: 1495-1499. 2. Agirregabiria, M., Verdoy, D., Olabarria, G., Aldamiz-Echevarría, P. and Ruano-López, J.M., 2007.

MicroTAS 2007, 7-11 October 2007, Paris, France. 3. Bang, D., Agirregabiria, M., Walczak, R., Dziuban, J.A., Wolff, A. and Ruano-Lopez, J.M., 2009.

MicroTAS 2009, 1-5 November 2009, Jeju, Korea. 4. Verdoy, D., Barrenetxea, Z., Fernández, L., Agirregabiria, M., Berganzo, J., Ruano-López, J.M.,

Marimón, J.M., M. Montes, M., Hammoumi, S., Albina, E. and Olabarria, G., 2009. MicroTAS 2009, 1-5 November 2009, Jeju, Korea.

5. Lee, J.-G., Cheong, K.H., Huh, N.M., Kim, S., Choi, J.W. and Ko, C., 2006. Lab on a Chip 6: 886-895.

6. Gulliksen, A., Karlsen, F., Rogne, H., Hovig, E. and Sirevåg, R., 2005. Lab on a Chip 5: 416-420. 7. Easley, C.J., Karlinsey, J.M., Bienvenue, J.M., Legendre, L.A., Roper, M.G., Feldman, S.H.,

Hughes, M.A., Hewlett, E.L., Merkel, T.J., Ferrance, J.P. and Landers, J.P., 2006. Proceedings of the National Academy of Sciences of the USA.103: 19272-19277.


LOOKING FORWARD TO

SEEING YOU AGAIN NEXT YEAR!

RME2014

SPRING 2014

THE NETHERLANDS


POSTERS P1 Bead-based suspension array for the simultanious detection of antibodies agains the

Rift Valley fever virus nucleocapsid and Gn glycoprotein René P. Achterberg, F.J. van der Wal, S.M. de Boer, H. Boshra, A. Brun, C.B.M. Maassen and J. Kortekaas Central Veterinary Institute, Wageningen UR, the Netherlands

P2 Influence of pluronic F127 on the distribution of protein molecules and resulting spot

morphology of microarrays in a porous nitrocellulose substrate L. Hamid Mujawar1,2, Aart van Amerongen1 and W. Norde1

1Laboratory of Physical Chemistry and Colloid Science, Wageningen UR, the Netherlands and 2Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen UR, the Netherlands

P3 Lanthanide chelates as a very sensitive detection principle based on time-resolved

fluorescence Aart van Amerongen, B. Beelen, T. Posthuma and J. Wichers Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen UR, the Netherlands

P4 Multiresidue mycotoxin analysis of conventional and organic agriculture commodities

by UPLC-MS/MS Martina Bolechová1,2, P. Kosubová1, J. Čáslavský2 and M. Pospíchalová1 1Central Institute for Supervising and Testing in Agriculture, Czech Republic and 2Faculty of Chemistry, Brno University of Technology, Czech Republic

P5 Determination of pyrrolizidine alkaloids in feed by UPLC-MS/MS

Martina Bolechová1,2, P. Kosubová1 and M. Mrkvicová1 1Central Institute for Supervising and Testing in Agriculture, Czech Republic and 2Faculty of Chemistry, Brno University of Technology, Czech Republic

P6 Determination of 4-methylimidazole in balsamic vinegars by GC-MS

C. Cunha, L. Senra, Sara C. Cunha and J.O. Fernandes REQUIMTE, Laboratory of Bromatology and Hydrology, Faculty of Pharmacy, University of Porto, Portugal

P7 Bisphenol A and bisphenol B in canned vegetables and fruits using QuEChERS and

dispersive liquid-liquid microextraction and analysis by gas chromatography-mass spectrometry Sara C. Cunha and J.O. Fernandes REQUIMTE, Laboratory of Bromatology and Hydrology, Faculty of Pharmacy, University of Porto, Portugal

P8 Mycotoxins poly-contamination in maize: fast and sensitive ELISA test kits for a multi-

analyte screening Francesco Gon, G. Rosar, E. Paoluzzi and F. Diana Tecna s.r.l., Italy

P9 A new fast ELISA test kit for the quantitative detection of T2 and HT2 toxins:

from the R&D development to the inter-laboratory trial G. Rosar1, Francesco Gon1, B. Puppini1, R. Baudino2, N. Rizzi3, G. Tosi4 and F. Diana1


1Tecna s.r.l., Italy, 2Associazione Provinciale Allevatori, Italy, 3A.R.A.L. Associazione Regionale Allevatori della Lombardia, Italy and 4Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Italy

P10 Evaluation of rapid detection of Alicyclobacillus acidoterrestris in energy drink by

fluorescence using EZ-Fluo Rapid detection system Marisa Hohnadel Merck Millipore, France

P11 Routine high resolution, accurate mass TOF screening for pesticide residue analysis

J. Burgess1, M. Mc Cullagh1, A. Gledhill2, Jean-Marc Joumier3, D. Philip1 and S. Stead2 1Waters, USA, 2Waters, UK and 3Waters, France

P12 Determination of increased catalase content in milk samples by means of a novel,

catalase-based amperometric biosensor P. Futo1, N. Adanyi2 and Attila Kiss1 1EGERFOOD Regional Knowledge Centre, Eszterhazy Karoly University, Hungary and 2Central Environmental and Food Science Research Institute, Hungary

P13 Novel acetylcholinesterase-based amperometric biosensor for the rapid analysis of

different carbamate and organophosphate pesticides G. Csiffáry1, Attila Kiss1 and N. Adányi2

1Egerfood Regional Knowledge Centre, Eszterházy Károly University, Hungary and 2Central Food Research Institute, Hungary

P14 Development of QCM-based direct, label-free immunosensor for rapid, cost-effective

assessment of probiotic bacteria in fermented dairy products Attila Kiss1, H. Szalontai1 and N. Adanyi2 1Egerfood Regional Knowledge Centre, Eszterházy Károly University, Hungary and 2Central Food Research Institute, Hungary

P15 Development of a solid phase extraction technique (SPE) for 257 compounds suitable

for both online SPE tandem Q-TOF analysis and also for advanced toxicity bio-monitoring Varvara Kokkali, B. Bajema, R. Bosch, A. Berg and W. van Delft Vitens Water Technology, the Netherlands

P16 Was the lupin flour contaminated with soy?

Katharina Köstlbauer, H. Schwartz, E. Gonzalez, P. Ansari Eshlaghi, K. Brunner, R. Krska and S. Baumgartner BOKU, Department IFA-Tulln, Austria

P17 Low-cost, fast, miniaturised solutions for water quality assessment

Christina Liedert, L. Hakola, H. Boer, L. Hakalahti, A. Jokinen, R. Rikkola, T. Sipilä-Malm, M. Smolander, H. Virtanen, S. Känä, J. Jokela, A. Ylinen and K. Sivonen VTT Technical Research Centre of Finland, Finland

P18 Increasing selectivity in LC-MS/MS analysis using techniques such as MRM3,

SelexION™ and high resolution LC-MS/MS Stephen Lock and J. Stahl-Zeng AB SCIEX, UK

P19 Allergen screening in food by LC-MS/MS

Stephen Lock and J. Stahl-Zeng AB SCIEX, UK





P20 Fat soluble vitamin detection in food by LC-MS/MS Stephen Lock AB SCIEX, UK

P21 The use of microflow UHPLC as a way to solvent usage in pesticide screening of food

samples by LC-MS/MS Stephen Lock AB SCIEX, UK

P22 Diagnostics method for the rapid detection and identification of low level

contamination of water systems with human pathogenic bacteria Elizabeth Minogue Molecular Diagnostics Research Group, Microbiology Department, School of Natural Sciences, National University of Ireland, Ireland

P23 In vino veritas – quantifying allergens in wine

Ronald Niemeijer, M. Richter, M. Lacorn and U. Immer R-Biopharm AG, Germany

P24 RIDASCREEN®FAST Soya (R7102) sandwich ELISA to detect traces of soya in

native as well as in processed food Ronald Niemeijer, M. Richter, M. Lacorn and U. Immer R-Biopharm AG, Germany

P25 Developing a membrane-based assay for the simultaneous screening of six

mycotoxins in different feed matrices: achievements, challenges and perspectives Emmanuel Njumbe Ediage1, Y. Guo1, I. Goryacheva2 and S. De Saeger1 1Laboratory of Food Analysis, Ghent University, Belgium and 2Department of Common and Inorganic Chemistry, Chemistry Faculty, Saratov State University, Russia

P26 A lateral flow microarray immunoassay as compared to conventional lateral flow and

slide microarray assay formats Truus Posthuma, L. Berendsen, A. Moers and A. van Amerongen

Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen UR, the Netherlands

P27 CIM monoliths as a tool for simultaneous concentration and removal of enteric viruses

from water Nejc Rački1, I. Gutierrez-Aguirre1, A. Steyer2, J. Gašperšič3, B. Brajer Humar4, M. Stražar4, A. Štrancar3 and M. Ravnikar1 1National Institute of Biology, Slovenia, 2Institute of Microbiology and Immunology, Slovenia, 3BIA Separations, Slovenia and 4Central Waste Water Treatment Plant Domžale-Kamnik, Slovenia

P28 Sample concentration methods for sensitive and reliable PCR-detection as

exemplified by Mycoplasma and Legionella Alexandra Scholz1, L. Kukuk2, T. Scheper1 and K. Pflanz3 1Leibniz University Hannover, Germany, 2Heinrich Heine University Düsseldorf, Germany and 3Sartorius Stedim Biotech GmbH, Germany

P29 Chip-based microsieves for concentrating microorganisms to enable detection

Heleen de Vogel-van den Bosch1, R. van Doorn2, M. Klerks2 and A. van Amerongen1

1Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen UR, the Netherlands and 2Innosieve Diagnostics BV, the Netherlands


P30 Rapid lateral flow detection of Legionella-specific RNA and DNA amplicons Heleen de Vogel-van den Bosch1, L. Nederhoff1, A. Martin-Pagola2, D. Verdoy2, J.-O. Axe3, S. Peters3, B. Oberheitmann3, M. Klerks4, A. Moers1 and A. van Amerongen1

1Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen UR, the Netherlands, 2Fundacion GAIKER, Spain, 3Q-Bioanalytic GmbH, Germany and 4Innosieve Diagnostics BV, the Netherlands

P31 Bead-based DNA-assay for the detection of Streptococcus suis in tonsillar specimens

of pigs Fimme Jan van der Wal1, H.J. Wisselink1, J.H. Bergervoet2, M. de Weerdt2 and R.P. Achterberg1 1Central Veterinary Institute, Wageningen UR, the Netherlands and 2Plant Research International, Wageningen UR, the Netherlands

P32 Rapid lateral flow tests for Tulip virus X in plant material by detection of a viral surface

antigen or a specific RNA sequence M. Koets1, Jan Wichers1, J. van Doorn2, M. de Kock2 and A. van Amerongen1

1Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen UR, the Netherlands and 2Applied Plant Research, Wageningen UR, the Netherlands

P33 MALDI-TOF MS as confirmation tool for pathogens in drinking water

Marsha van der Wiel, P. Willemse and G. Wubbels

WLN, the Netherlands


P1 BEAD-BASED SUSPENSION ARRAY FOR THE SIMULTANIOUS DETECTION OF ANTIBODIES AGAINS THE RIFT VALLEY FEVER VIRUS NUCLEOCAPSID AND GN GLYCOPROTEIN René P. Achterberg, F.J. van der Wal, S.M. de Boer, H. Boshra, A. Brun, C.B.M. Maassen and J. Kortekaas Central Veterinary Institute, Wageningen UR, the Netherlands [email protected] Rift Valley fever virus (RVFV) is a mosquito borne virus belonging to the Phlebovirus genus of the Bunyaviridae. RVFV was first isolated in Kenya in 1930, and has since caused devastating outbreaks throughout Africa. The first commercially available ELISAs used inactivated whole virus as antigen. More recently ELISAs based on recombinant antigen have become available, but these assays only detect antibodies against the nucleocapsid protein N. Preferably, a test also detects antibodies against other immunogenic proteins of the virus, such as the structural glycoproteins Gn and Gc as used in the virus neutralization test. The N protein and the purified ectodomain of Gn were covalently linked to paramagnetic Luminex beads. The resulting multiplex bead-based suspension array can be used for the simultaneous detection of antibodies against N and Gn of RVFV in various animal species. The assay was evaluated using ring trial sera [1] (57 sheep, 13 cattle), field sera (11 ovine, 5 bovine and 16 human), and experimental sera from six lambs vaccinated with a recombinant NDV virus expressing the RVFV Gn and Gc proteins [2]. Most of the sera positive in the ring trial also scored positive in the Gn/N IgG Luminex assay. Positive sera that scored negative in the Gn/N IgG Luminex assay were found positive in the Gn/N IgM Luminex assay. The results obtained with field sera using the Gn/N IgG Luminex assay corresponded with ELISA results. Animals, vaccinated with a Gn/Gc RVFV vaccine that lacks N, over time developed an IgG response exclusively against Gn, as confirmed with the Gn/N IgG Luminex assay. The RVFV Gn/N Luminex assay can be used for the simultaneous detection of antibodies against the RVFV Gn and N proteins in a single sample. The bead-based assay is capable of detecting IgG and IgM antibodies in multiple species and its results are in agreement with those of an ELISA ring trial. These results demonstrate that the Luminex assay may be used as DIVA test (differentiate infected from vaccinated animals) to accompany glycoprotein based RVFV vaccines. References 1. Kortekaas, J., Kant, J., Vloet, R., Cêtre-Sossah, C., Marianneua, P., Lacote, S., Banyard, A.C.,

Jeffries, C., Eiden, M., Groschup, M., Jäcket, S., Hevia, E. and Brun, A., 2013. European ring trial to evaluate ELISAs for the diagnosis of infection with Rift Valley fever virus. Journal of Virological Methods 187: 177-181.

2. Kortekaas, J., De Boer, S.M., Kant, J., Vloet, R.P., Antonis, A.F. and Moormann, R.J., 2010. Rift Valley fever virus immunity provided by a paramyxovirus vaccine vector. Vaccine 28: 4394-4401.


P2 INFLUENCE OF PLURONIC F127 ON THE DISTRIBUTION OF PROTEIN MOLECULES AND RESULTING SPOT MORPHOLOGY OF MICROARRAYS IN A POROUS NITROCELLULOSE SUBSTRATE L. Hamid Mujawar1,2, Aart van Amerongen1 and W. Norde1

1Laboratory of Physical Chemistry and Colloid Science, Wageningen UR, the Netherlands and 2Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen UR, the Netherlands [email protected] The protein microarray is an emerging application format in diagnostics. High quality microarrays are produced on various substrates. However, the distribution of printed biomolecules on these substrates is often non-homogeneous, commonly known as the “doughnut or coffee-stain effect”. We studied the influence of pluronic F127 in the printing buffer on spot morphology in porous nitrocellulose membranes (11 µm thick); a high signal-to-noise substrate. Antibodies specific for discriminating tags were printed on nitrocellulose membrane pads on slides using phosphate buffered saline (pH 7.4) and various concentrations of pluronic F127 as a printing buffer. Double-tagged amplicons were sandwiched in a one-step incubation between the printed antibodies and streptavidin conjugated flurophore. Results were analyzed by the 'Z-stack' method of the confocal laser scanning microscope. The intensity distribution in each stack was assessed. Printing antibodies with increasing concentrations of pluronic F127 up to 0.2% (v/v) showed an increasing uniformity of spot morphology. Acknowledgements The authors acknowledge the financial support of STW project 10095 ‘Biomolecule substrate topography of inkjet printed structures’.


P3 LANTHANIDE CHELATES AS A VERY SENSITIVE DETECTION PRINCIPLE BASED ON TIME-RESOLVED FLUORESCENCE Aart van Amerongen, B. Beelen, T. Posthuma and J. Wichers Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen UR, the Netherlands [email protected] Lanthanides, rare earth elements, can be used as a part of a signal principle in diagnostics. When coupled to ligands and by using chromophore-chelate complexes these metals provide strong luminescent signals that can be detected in a time-resolved fluorescence (TRF) mode. The principle takes advantage of the long fluorescence lifetime of the lanthanides and can detect less than one attomole of europium in a multiwell plate sample. Furthermore, this high sensitivity is coupled to a wide dynamic range of measurement. Labelling procedures to couple lanthanides to ligand molecules are quite easy. The labelled compounds have a high specific activity and a good stability with a minimal influence on biological activity. The technology is also very flexible. It suits both 96-well and 384-well plate formats and can be applied in coated plate and in filter assays. The fluorescence lifetime of the specific signal is several orders of magnitude longer than the non-specific background. This enables the label to be measured at a time when the background (e.g. due to auto-fluorescence of a sample) has already decayed. The large Stokes’ shift, i.e. the difference between excitation and emission wavelengths, and the narrow emission peak contribute to an excellent signal-to-noise ratio. Based on the dissociation-enhancement principle the sensitivity is increased as well: the lanthanide chelate is dissociated into a new highly fluorescent chelate consisting of a protective micelle. Suitable lanthanide metals for use as chelate labels are europium, samarium, terbium, and dysprosium. The europium ion, Eu3+, is most often used. Samarium, Sm3+, has been introduced as a second choice, and together with europium dual labelling is possible. Triple and quadruple labelling can be done with Terbium, Tb3+, and/or dysprosium, Dy3+. The lanthanide chelates do not suffer from self-quenching. Therefore, they can be packed inside a nanoparticle without degrading the fluorescence properties of the probe. Typically, these particulate labels are latex or silica-based. Furthermore, the shell protects the lanthanide chelates inside the particle from interferences, such as water molecules or metal ions. Ligand molecules can be physically or covalently attached to the particles, which enable the connection between a high amount of particle-incorporated fluorescence and down to a single binding event between a ligand and its antigen. We are exploring the potential of this signal principle by coupling functionalised europium to ligand molecules. In addition, particulate labels, having the lanthanide-chelate complexes incorporated, are being purchased or developed in our laboratory and are used to couple specific antibodies to set up several immunoassays. Initial results will be shown. Acknowledgements The authors acknowledge the financial contributions from the programmes Nano4Vitality (N4V), NanoNextNL (Project 05A.01 Microbial Food Quality and Safety) and RAAK-PRO NAT-TEST (Alternative test battery for safety and effect of natural compounds). Initial contacts with Prof.dr. Elisabeth Bouwman, Department. of Inorganic Chemistry, Leiden Institute of Chemistry, Leiden University, the Netherlands, on the synthesis of lanthanide chelate-silane derivatives is highly appreciated.

References 1. Handl, H.L. and Gillies, R.J., 2005. Lanthanide-based luminescent assays for ligand-receptor

interaction. Life Sciences 77: 361-371. 2. Kokko, T., 2009. Lanthanide chelates as donors in fluorescence resonance energy transfer:

exciting prospects for bioaffinity assay detection. PhD thesis. Painosalama Oy, Turku, Finland.


P4 MULTIRESIDUE MYCOTOXIN ANALYSIS OF CONVENTIONAL AND ORGANIC AGRICULTURE COMMODITIES BY UPLC-MS/MS Martina Bolechová1,2, P. Kosubová1, J. Čáslavský2 and M. Pospíchalová1

1Central Institute for Supervising and Testing in Agriculture, Czech Republic and 2Faculty of Chemistry, Brno University of Technology, Czech Republic [email protected] In 2011, the organic farming, which is continuously growing, reached almost 12% of the farmland acreage in the Czech Republic. The crops of great importance are cereals followed by fodder crops. The organic wine market is increasing and it is becoming popular as well. Organic products have been grown in agreement with principles of organic farming that typically excludes the use of artificial chemicals such as fertilizers, fungicides, herbicides and other types of pesticides. The study deals with the analysis of mycotoxins in organic and conventional farm products such as raw materials for feed production and grapes. The multiresidue mycotoxin method for feed analysis by ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) which has been recently developed represents useful tool for both feed and fruit analysis with certain modifications during the sample preparation. The method for determination of 17 mycotoxins (deoxynivalenol, nivalenol, HT-2 toxin, T-2 toxin, ochratoxin A, zearalenone, aflatoxins, fumonisins, beauvericin and enniatins) based on the unbuffered QuEChERS method has been validated. The study presents results of mycotoxin screening performed on 24 grape samples of conventional and organic origin produced in 2011 and 2012, and 25 raw feed material samples of conventional and organic origin produced in 2012. Acknowledgements This work was performed with support from the Central Institute for Supervising and Testing in Agriculture, MŠMT ČR, grant No. FCH-S-12-4.


P5 DETERMINATION OF PYRROLIZIDINE ALKALOIDS IN FEED BY UPLC-MS/MS Martina Bolechová1,2, P. Kosubová1 and M. Mrkvicová1

1Central Institute for Supervising and Testing in Agriculture, Czech Republic and 2Faculty of Chemistry, Brno University of Technology, Czech Republic [email protected] Alkaloids are widely distributed plant metabolites, which are grouped by typical structural characteristics into large families such as pyrrolizidine alkaloids (PAs), which include more than 350 individual heterocyclic compounds. Intoxications caused by PAs are described by hepatotoxicity with tendency to cause hemorrhagic necrosis and veno-occlusion in liver, pneumotoxicity, carcinogenity, mutagenity and genotoxicity. Thus there is a need for methods that allow determination of these dangerous plant toxins. In this study fast, reliable and sensitive approach is proposed allowing simultaneous screening, identification, and quantification of PAs in feed samples. PAs including monocrotaline, senkirkine, senecionine, seneciphylline and retrorsine were determined by ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Sample preparation is based on modified QuEChERS approach. Mean recovery, precision, matrix effects and limits of quantification (LOQ) were assessed for four matrices within the method validation. The presented method was tested on 41 various feed samples of Czech production, where presence of pyrrolizidine alkaloids was expected. The most abundant PA was seneciphylline, while monocrotaline, retrorsine, senecionine and senkirkine were found in lower quantities. Feed samples with high content of chlorophyll contained the highest levels of PAs, in general. Acknowledgements This work was performed with support from the Central Institute for Supervising and Testing in Agriculture, MŠMT ČR, grant No. FCH-S-12-4.


P6 DETERMINATION OF 4-METHYLIMIDAZOLE IN BALSAMIC VINEGARS BY GC-MS C. Cunha, L. Senra, Sara C. Cunha and J.O. Fernandes REQUIMTE, Laboratory of Bromatology and Hydrology, Faculty of Pharmacy, University of Porto, Portugal [email protected] Caramel colours are complex mixtures of compounds produced by carefully controlled heat treatment of carbohydrates (sugars). They are used in a variety of foods and beverages, e.g., carbonated beverages, dark beers, baked products, soups, soy sauce and dry mixes, as a colouring and flavouring agents. The caramel colours are divided into four classes, according to the reactants used in their manufacture: class I, plain caramel (E 150a); class II, caustic sulphite caramel (E 150b); class III, ammonia caramel (E 150c); and class IV, sulphite ammonia caramel (E 150d). In caramels type III and IV small quantities of substituted imidazoles of health concern can be presented, e.g. 4-methylimidazole (4-MeI). The presence of 4-MeI in food is undesirable because it has been shown to be a potent convulsing agent that elicits neurological signs in mice, chicks, rabbits and cattle. The aim of this work was to screen the presence of 4-MeI in balsamic vinegars commercialized in Portugal using a previously validated gas chromatography-mass spectrometry (GC-MS) method [1]. The sample preparation entails the following steps: (i) extraction with an ion-pair (di-2-ethylhexylphosphoric acid); (ii) back-extraction with 0.1 M HCl; (iii) derivatization with isobutulychloroformate; and (iv) extraction of derivatives with isooctanol. GC-MS analysis was performed in SIM mode using for quantification 2-ethylimidazole as internal standard. 4-MeI was found in all the 20 vinegar samples studied at levels that ranged from 0.630 to 4.150 mg/l. Acknowledgments This research was supported by a grant from the FCT project ‘PTDC/AGR-ALI/101583/2008’ and COMPETE FSE/FEDER/OE. S.C.C. is grateful to ‘POPH-QREN-Tipologia 4.2, Fundo Social Europeu e Fundo Nacional MCTES’. References 1. Cunha, S.C., Barrado, A.I., Faria, M.A. and Fernandes, J.O., 2011. Assessment of 4-(5-)

methylimidazole in soft drinks and dark beer. Journal of Food Composition and Analysis 24: 609-614.


P7 BISPHENOL A AND BISPHENOL B IN CANNED VEGETABLES AND FRUITS USING QUECHERS AND DISPERSIVE LIQUID-LIQUID MICROEXTRACTION AND ANALYSIS BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY Sara C. Cunha and J.O. Fernandes REQUIMTE, Laboratory of Bromatology and Hydrology, Faculty of Pharmacy, University of Porto, Portugal [email protected] Canned foods offer an easy to use and high quality food to the consumers. However, the direct contact between food and packaging materials provides the conditions for migration of undesirable compounds. Among the toxics that could leach from the canned containers into food and beverages, bisphenol A (2,2-bis(4-hydroxyphenyl)propane; BPA), a key component of epoxy resins widely used as inner coatings of metallic food and drink cans, has recently received considerable attention from both scientific community and regulatory authorities due its recognized endocrine disruptor properties. In this work a dispersive liquid-liquid micro-extraction (DLLME) combined with acetonitrile based extraction was proposed for simultaneous extraction, concentration and derivatization of bisphenol A (BPA) and bisphenol B (BPB) in canned vegetables and fruits prior to gas chromatography- mass spectrometry (GC-MS) analysis. The DLLME procedure involved the use of tetrachloro-ethylene as extractive solvent while the own acetonitrile extract obtained from QuEChERS was used as dispersive solvent, and anhydride acetic as derivatizing reagent. Besides the great enrichment factor provided, the final DLLME extractive step allowed the simultaneous acetylation of the compounds required for their gas chromatographic analysis. The optimized method showed to be accurate (> 69% recovery), reproducible (<20% relative standard deviation) and sensitive for the target analytes (method detection limits of 0.3 and 0.6 µg/kg for BPA and BPB, respectively). The screening of several canned vegetables and fruits samples commercialized in Portugal revealed the presence of BPA in more than 87% of the samples with levels ranging from 3.7 to 265.6 µg/kg, while BPB was detected in only two samples one canned fruit and one canned vegetable with levels of 3.4 and 3.0 µg/kg, respectively. Acknowledgments This research was supported by a grant from the FCT project ‘PTDC/AGR-ALI/101583/2008’ and COMPETE FSE/FEDER/OE. S.C.C. is grateful to ‘POPH-QREN-Tipologia 4.2, Fundo Social Europeu e Fundo Nacional MCTES’.


P8 MYCOTOXINS POLY-CONTAMINATION IN MAIZE: FAST AND SENSITIVE ELISA TEST KITS FOR A MULTI-ANALYTE SCREENING Francesco Gon, G. Rosar, E. Paoluzzi and F. Diana Tecna s.r.l., Italy [email protected] Mycotoxins are toxic secondary metabolites produced by several species of fungi. These compounds have been reported to be etiologic agents in several human and animal diseases. The complex ecology of fungal growth and mycotoxin production can lead to a mixed contamination of mycotoxins in food and feed. The co-occurrence of mycotoxins can affect the level of mycotoxin production itself: the presence of the aflatoxins in stored grains, for instance, may be enhanced by the co-occurrence of trichothecenes. Naturally occurring combinations of different mycotoxins could affect also the toxicity of the incurred grains. For example the affect of deoxynivalenol on weight gain in swine was shown to be synergized by T-2 toxin. To keep the risk for both humans and animals as low as possible, food producers and feed industries have to control carefully raw materials. The demand of fast, sensitive, accurate and easy-to-use analytical methods for mycotoxins has been booming and high throughput screening and rapid methods are needed. The aim of the present work is to introduce Tecna Celer line of fast ELISA test kit for the quantitative detection of aflatoxins, deoxynivalenol, fumonisins, zearalenone and T-2/HT-2 toxins in cereals, particularly in maize. The Celer test kits are characterized by two main aspects. One is the quickness of the analysis: both Celer Afla and Celer Afla B1, for the detection of total aflatoxins and aflatoxins B1 respectively, are 15 minutes long tests, while Celer DON v2, Celer ZON v2, Celer T2 and Celer FUMO, for the detection of deoxynivalenol, zearalenone, T-2/HT-2 toxins and fumonisins, have an assay time of 20 min. The second main aspect is the common sample preparation for maize. Maize samples have to be mixed, ground, extracted 1:5 in a methanol:water mixture in presence of 20% NaCl, shaked for 3 min and filtered. A further dilution could be necessary. This means that it is sufficient to run one single extraction per sample and implement the same extract in the whole ELISA panel. The Celer test kits were in-house validated in terms of specificity, sensitivity and trueness by implementing certified materials or control samples left over the materials of different proficiency tests. The limit of quantification (LOQ) of Celer AFLA was set at 2 ppb of total aflatoxins; the mean recovery of three different reference maize samples was 100+12% (n=12). For Celer Afla B1, the LOQ was set at 1 ppb of aflatoxins B1; the trueness was studied on four materials and the mean recovery was 124+18% (n=36). The LOQ for Celer DON v2 was set at 125 ppb of deoxynivalenol in maize, while the trueness was set by analysing three control samples and the mean recovery was 96+11% (n=54). Celer ZON v2 LOQ was set at 20 ppb of zearalenone; the accuracy for three different incurred reference maize materials turned to be 90+13% (n=21). The sensitivity of Celer FUMO was 1000 ppb of fumonisins; the accuracy for two samples left over the materials of FAPAS proficiency tests turned to be 113+10% (n=18). For the latest released Celer T2, the LOQ was set at 25 ppb of T-2 toxin, while the truness was established by analysing two T-2/HT-2 incurred reference materials and the mean recovery turned to be 85+5% (n=10). As a conclusion, Celer is a suitable panel of test kits for accurate and sensitive multi-analyte parallel screening of mycotoxins in maize. The common and easy sample preparation allows end-user to perform one single extraction per sample and then run the extract on all the test kits in parallel, to obtain the whole contamination profile in less than half an hour.



P9 A NEW FAST ELISA TEST KIT FOR THE QUANTITATIVE DETECTION OF T2 AND HT2 TOXINS: FROM THE R&D DEVELOPMENT TO THE INTER-LABORATORY TRIAL G. Rosar1, Francesco Gon1, B. Puppini1, R. Baudino2, N. Rizzi3, G. Tosi4 and F. Diana1

1Tecna s.r.l., Italy, 2Associazione Provinciale Allevatori, Italy, 3A.R.A.L. Associazione Regionale Allevatori della Lombardia, Italy and 4Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Italy [email protected] T-2 toxin and HT-2 toxin are trichothecenes type A mycotoxins produced by fungi of the

Fusarium genus, which are commonly found in various cereal crops and processed grains.

These toxins have been shown to cause a variety of toxic effects in both animals and

humans, including protein synthesis inhibition, immunity system alterations and

haematopoiesis inhibition. The EU recommended limits for the sum of toxins are under

discussion after EFSA (European Food Safety Authority) recent evaluations. To monitor the

contamination levels of these toxins in cereals and feed, it is mandatory to have a rapid,

easy-to-use, robust and reliable screening tool with sufficient cross-reactivity for the HT-2

metabolite. The present work shows the performances of Celer T2, a new fast ELISA test kit

for the quantitative determination of T-2 and HT-2 toxins in cereals and feed. Thanks to a

proper immunogen design, a polyclonal antibody with 100% cross-reactivity for T-2 toxin and

72% for HT-2 was obtained. The measuring range of the assay is 0.025 - 1.00 ppm and can

be extended up to 5 ppm by dilution of sample extracts. The assay was in-house validated

for whole wheat, wheat flour, oats, barley, maize and poultry feed. The limit of quantification

(LOQ) was set at 0.025 ppm for wheat and maize and 0.050 ppm for oats, barley and feed.

The mean recovery for 0.1-1 ppm spiked samples was 125±18% for whole wheat, 116±14%

for wheat flour, 114±7% for oats, 100±9% for hulled oats, 117±12% for barley, 109±13% for

maize, 113±20 % for poultry feed. The trueness was investigated on reference samples left

over the materials of different proficiency schemes and turned always to be satisfactory. The

test kit was submitted to an inter-laboratory trial. In order to verify the variability of the assay

itself, the sample preparation was omitted. Four different laboratories performed an analytical

session where three curves were run and the concentrations of two different T-2 solutions

(0.04 and 0.20 ppm) were determined. The B/Bo (%) of the calibrations curves and the

results of the solutions were analysed by Kruskal-Wallis ANOVA test: the populations of data

never turned to be different one to each other. The inter-laboratory CV of the results was 17

and 10%, respectively, for 0.04 and 0.20 ppm solutions. As a conclusion, Celer T2 showed

good performances during the whole in-house validation, both on spiked and incurred

samples. The robustness of the reagents was confirmed during the inter-laboratory trial, thus

demonstrating its suitability to a routine implementation.


P10 EVALUATION OF RAPID DETECTION OF ALICYCLOBACILLUS ACIDOTERRESTRIS IN ENERGY DRINK BY FLUORESCENCE USING EZ-FLUO RAPID DETECTION SYSTEM Marisa Hohnadel Merck Millipore, France [email protected] Alicyclobacilli have been isolated and implicated in spoilage of many beverages and are a major concern for food and beverage industry. Rapid microbiological method will allow detecting potential contaminants at early stages and avoiding financial losses. Rapid detection of Alicyclobacillus has been assessed with the EZ-Fluo Rapid detection system combining an easy to use platform with a simple microbiological test for microbial contamination in filterable samples. Based on fluorescent detection after membrane filtration and growth on traditional culture media, the system offers an early detection and enumeration of microorganisms allowing a time to result divided by three compared to the reference method. After a short and validated incubation time, the membrane is transferred and stained with the EZ-Fluo reagent kit. The reagent contains a non fluorescent marker cleaved by viable micro-organisms. The cleaved marker is accumulated inside the cells and emits fluorescence when it is excited by the EZ-Fluo reader. Enumeration is performed through the window of the EZ-Fluo reader or using the optional camera and EZ-Fluo spot counter software. After staining; the membrane can be re-incubated onto medium to continue growth allowing species identification. The EZ-Fluo Rapid detection system was challenged to detect A. acidoterrestris contamination in energy drink on potato dextrose agar pH 5.6 at 46°C. Following this protocol, A. acidoterrestris was detected by fluorescence and enumerated after 20 h. Equivalence study between reference method and alternative method was performed on the sample inoculated with 3 levels of A. acidoterrestris. The accuracy and linearity were demonstrated for fluorescence and viability by each method on the same sample. The fluorescence and the viability with EZ-Fluo method are equivalent to the reference method. The alternative method gives results which are proportional to those of the reference method. Results proved that the EZ-Fluo method reduces significantly time to result by detecting accurately contaminants after only 20 h instead of 3 days with the traditional method.


P11 ROUTINE HIGH RESOLUTION, ACCURATE MASS TOF SCREENING FOR PESTICIDE RESIDUE ANALYSIS

J. Burgess1, M. Mc Cullagh1, A. Gledhill2, Jean-Marc Joumier3, D. Philip1 and S. Stead2 1Waters, USA, 2Waters, UK and 3Waters, France [email protected] As global food trade continues to expand, so too does the challenge of meeting the requirements of an ever-changing food safety regulatory environment. In recent years, the use of accurate mass instrumentation has been at the forefront of food and environmental screening discussions. This has been reflected in the evolving Codex Alimentarius guidelines that acknowledge the complexity of screening and promote the use of accurate mass technology to address the challenges faced by industry. Integral to any chemical analysis experiment are: sampling, extraction, compound separation, data acquisition and reporting. It is the entire process of analysis that is recognised, and must be met by the Codex regulations. In this poster, the first commercially available pesticide applications solution will be presented. This applications solution encompasses from sample preparation to reporting, and includes an application-specific installation specification to ensure that the entire system solution meets the performance requirements for food and environmental laboratories prior to use. The poster will discuss the new sample preparation and standards mix (that can be used as part of a system suitability /instrument performance protocol); the superior separation that is achieved with the ACQUITY UPLC I-Class and the sensitivity performance for food environmental analysis of the Xevo G2-S Qtof MS in order to satisfy regulatory demands, and UNIFI – the software platform of the future. UNIFI delivers a complete scientific information system solution for data acquisition, data processing and data management. UNIFI also provides access to an extensive scientific library and combines the essential software elements necessary to provide an effective, comprehensive screening solution.


P12 DETERMINATION OF INCREASED CATALASE CONTENT IN MILK SAMPLES BY MEANS OF A NOVEL, CATALASE-BASED AMPEROMETRIC BIOSENSOR P. Futo1, N. Adanyi2 and Attila Kiss1 1EGERFOOD Regional Knowledge Centre, Eszterhazy Karoly University, Hungary and 2Central Environmental and Food Science Research Institute, Hungary [email protected] The inflammation of the udder of dairy cattle is a common disease, caused by mechanical, chemical effects and by the infection of different bacteria strains (Staphylococcus aureus, Pseudomonas mastitis). It causes changes in the consistence of the milk, lower lactation and decrease of quality. During inflammation, the natural level of catalase enzyme in the milk can be higher. The aim of our work was to develop a selective amperometric biosensor for the detection of higher catalase content in infected milk samples. The catalase enzyme (EC 1.11.1.6) was immobilized onto the surface of a thin-layer enzyme cell. The measurements were carried out in stopped-flow injection analysis system (0.8 ml/min flow rate, +590 mV polarization potential) in organic phase (6% sodium acetate buffer 200 mM pH 6.0, ferrocene conductive salt (7.5 mg/l)) dissolved in acetonitrile. Known amounts of hydrogen peroxide were added to the milk samples, and after a short incubation time, the samples were injected onto the enzyme cell. The changes of the concentration of hydrogen peroxide were measured. A certain amount of hydrogen peroxide was degraded by the catalase content of the milk. The degradation of the hydrogen peroxide is proportional to the infection of the milk samples. The developed system can be utilized for the fast and cost-effective pre-selection of raw milk samples.


P13 NOVEL ACETYLCHOLINESTERASE-BASED AMPEROMETRIC BIOSENSOR FOR THE RAPID ANALYSIS OF DIFFERENT CARBAMATE AND ORGANOPHOSPHATE PESTICIDES G. Csiffáry1, Attila Kiss1 and N. Adányi2

1Egerfood Regional Knowledge Centre, Eszterházy Károly University, Hungary and 2Central Food Research Institute, Hungary [email protected] Pesticides utilized in agriculture, and their residues pose an enormous risk to public health. For this reason, the determination of these substances is of vital importance. However, the classical analytical methods tend to be time consuming and costly. The use of biosensors offers an alternative approach when considering the food safety and environmental aspects of agriculture. Our goal was the elaboration of a novel amperometric biosensor for the detection of various pesticides (carbamates and organophosphates) based on the inhibition of acetylcholinesterase enzyme (AChE; EC 3.1.1.7). The enzyme was immobilized on a natural protein membrane in a thin-layer enzyme cell [1]. The electrochemically active acetylthiocholin (AcTCh) was used as substrate for the detection. The biosensor worked in flow injection analysis (FIA) system. A stopped-flow measuring setup was installed and the measuring parameters for AcTCh were optimized. The proper polarization potential was at 550 mV. We used 100 mM (pH 6.5) phosphate buffer. The optimal measuring temperature was 37-38 ºC, the e flow rate 0.66 ml/min. The residence time for the inhibition reaction was determined at 60 s in the stopped flow mode. The linear measuring range was obtained in the range of 0.1-100 μmol/l AcTCh. The inhibitory effect of dichlorvos (DDVP), carbofuran, methomyl and pirimicarb pesticides was studied on the immobilized enzyme. The linear measuring range of the analytes was 1-100, 1-100, 200-1000 and 100-10,000 nM, respectively. The detection limits were 1, 1, 200 and 100 nM for dichlorvos, carbofuran, methomyl and pirimicarb, respectively. The enzyme cell proved to be very stable and robust. It could be stored at 4°C for one month. When used, stable signals were obtained even after hundreds of injections. Food and environmental samples, i.e. apple juice and soil extract spiked with different amount of methomyl, were also studied. The recovery of the spiked methomyl was acceptable in both samples. Based on our results, it can be stated that the developed biosensor is capable of pesticide detection even in food and environmental samples. It can possibly be used as a screening device, discriminating between contaminated and non-contaminated samples. This offers a chance for the pre-selection of samples to be forwarded to analytical examination. References 1. Adányi, N., Tóth-Markus, M., Szabó, E.E., Váradi, M., Sammartino, M.P., Tomassetti, M. and

Campanella, L., 2004. Analytica Chimica Acta 501: 219-225.


P14 DEVELOPMENT OF QCM-BASED DIRECT, LABEL-FREE IMMUNOSENSOR FOR RAPID, COST-EFFECTIVE ASSESSMENT OF PROBIOTIC BACTERIA IN FERMENTED DAIRY PRODUCTS Attila Kiss1, H. Szalontai1 and N. Adanyi2 1Egerfood Regional Knowledge Centre, Eszterházy Károly University, Hungary and 2Central Food Research Institute, Hungary [email protected] Quartz crystal microbalance (QCM), optical waveguide lightmode spectroscopy (OWLS) and surface plasmon resonance (SPR) have been known as label-free biosensoric techniques suitable for the direct antibody-based detection of target molecules. The development of simple immunosensoric methods for rapid quantification of microbial cells is still a significant challenge in food analysis. QCM is efficiently applicable for immuno-recognition and provide sensitive mass detection and high sensitivity. QCM-based direct immunoassay method was developed for the real-time determination and quantification of Lactobacillus acidophilus O1132 (L. acidophilus) and Bifidobacterium bifidum O1356 (B. bifidum) in fermented milk products. Two different flow injection analysis (FIA) methods were compared. Flow-through and stopped-flow methods were used to determine the applicability of these measuring methods. To optimize the immunosensor system, model measurements were carried out with bovine serum albumin (BSA) and anti-BSA antibody. Self-assembled monolayers were created with MHDA (16-mercapto-hexadecanoic acid) and with sulfo-LC-SPDP (sulfosuccinimidyl 6-(3’-(pyridyldithio) propionamido) hexanoate) cross-linking agents [1,2] for immobilizing anti-BSA antibody onto the gold surface of the quartz wafer. After the model experiments, B. bifidum and L. acidophilus were detected by immobilizing polyclonal anti-B. bifidum IgG and anti-L. acidophilus IgG onto the sensor surface by sulfo-LC-SPDP. The probiotics were measured from buffer solution and from real samples (spiked milk samples, acidophilus and bifidus milk samples). Using the novel immunosensor the target bacteria could be detected in the range of 1.0E+3 – 1.0E+7 cfu/ml within 30 min. The cell numbers of fermented samples determined by QCM and plate counting showed a good correlation. The selectivity of a-B. bifidum and a-L. acidophilus antibody-coated sensors was also examined. References 1. Su, X.L. and Li, Y., 2004. Biosensors and Bioelectronics 19: 563-574. 2. Adányi, N., Váradi, M., Kim, N. and Szendrő, I., 2005. Current Applied Physics 6: 279-286.


P15 DEVELOPMENT OF A SOLID PHASE EXTRACTION TECHNIQUE (SPE) FOR 257 COMPOUNDS SUITABLE FOR BOTH ONLINE SPE TANDEM Q-TOF ANALYSIS AND ALSO FOR ADVANCED TOXICITY BIOMONITORING Varvara Kokkali, B. Bajema, R. Bosch, A. Berg and W. van Delft Vitens Water Technology, the Netherlands [email protected] A concentration technique has been proved necessary for lowering the detection limits of advanced toxicity bio-monitors [1]. Further analysis of toxic samples with high resolution mass spectrometer is used when an alarm is triggered. This study aimed to the development of a fully automated method based on on-line solid phase extraction (SPE) tandem Q-TOF liquid chromatography- high resolution- mass spectrometry analysis (LC-HR-MS) for the concentration and determination of 257 polar compounds, including pharmaceuticals, pesticides and industrial compounds, spiked in drinking water samples. As a result, the developed extraction method should be applicable in concentrating aqueous environmental samples as a pre-treatment step prior to toxicity monitoring and if necessary followed by LC-HR-MS screening. Subsequently, the identification of the most efficient SPE material for achieving the highest possible recoveries for the maximum of the compounds and the affect of pH on the efficiency of the SPE materials were studied during this research. Initially, 13 SPE materials were tested including Oasis® HLB, HySphere Resin-SH, HySphere Resin-GP, HySphere C18-HD, HySphere C8-EC-SE, HySphere MM Anion, HySphere MM Cation, PLRP-s, XB 55, XB 96, HySphere C2-SE, HySphere CN-SE and Isolute® NH2, using Symbiosis™ online SPE system (Spark, the Netherlands) and then the recoveries were determined using Q-TOF LC-HR-MS analysis (Agilent, the Netherlands). 10ml samples spiked with pesticides (1 μg/l), pharmaceuticals and industrial compounds (ranged between 0.04 -0.8 μg/l) were concentrated at neutral pH. The results showed that Resin-SH, Resin-GP, Oasis® HLB, MM-Anion, PLRP-s and C18-HD were the most efficient SPE materials with 89-95% of the tested compounds being recovered with percentages higher than 25%. The effect of pH of the samples on the SPE efficiency of these six materials was determined for the same 257 compounds. The pH of the drinking water samples was adjusted to 1, 3, 5, 7 and 9 and then the samples were spiked with the 257 compounds. Then, 10ml samples were analysed with Symbiosis™ online SPE system followed by Q-TOF LC-HR-MS analysis. The samples were analysed in triplicates. The results showed that Resin-SH at pH 5 and Oasis® HLB at pH 7 were the most efficient combinations for retrieving 91% of the tested compounds above 25% in both cases. Resin-SH, Resin-GP and Oasis® HLB proved the most efficient materials with 245, 242 and 239 compounds out of 257 being recovered respectively above 25% with Q-TOF LC-HR-MS analysis. The study of the pH effect showed that Resin-SH at pH 5, Oasis® HLB at pH 7 and Oasis® HLB at pH 5 had the maximum compounds being recovered (>25%) with 236 compounds for the first 2 materials and 233 compounds for the last one. In conclusion, a generic concentration technique for polar compounds would be effective when using either Resin-SH or Oasis® HLB at neutral pH. The development of this method with a broad applicability to such a great range of compounds improves the potential of lowering the detection limits of biomonitors and receiving early warning alarms. References 1. Penders, E.J.M., 2011. Development of aquatic biomonitoring models for surface waters used for

drinking water supply. PhD thesis, Wageningen UR, Wageningen, the Netherlands.


P16 WAS THE LUPIN FLOUR CONTAMINATED WITH SOY? Katharina Köstlbauer, H. Schwartz, E. Gonzalez, P. Ansari Eshlaghi, K. Brunner, R. Krska and S. Baumgartner BOKU, Department IFA-Tulln, Austria [email protected] Currently, lupin is increasingly applied in the European food industry. Lupin is rich in valuable proteins, promises health benefits and is claimed to be genetically unmodified in contrast to its close relative soy. But individuals, who are allergic to other legumes, can be pre-sensitized to lupin allergens due to cross reactivity and thus can suffer from allergic reactions against lupin. Therefore, food products containing lupin – even in trace amounts – must be compulsory declared on food labels in the European Union. The objective of this study was to find lupin specific peptide marker that can be used for the identification and quantification of lupin proteins in food products via HPLC-MS/MS. To establish such a method, we purchased white lupin seeds and lupin flour. As we screened for potential marker peptides, the two samples showed different peptide patterns. They shared some peptides but some peptides were unique. Thus we hypothesised that either the protein pattern varied between the different lupin species or the lupin flour was contaminated, probably with lupin’s closest relative with similar properties, soy. The outcome would not make any difference for persons who suffer from soy allergy, because they would risk an allergic reaction by consuming lupin as well. But we became curious about the specificity of different rapid methods for the trace analysis of allergens in food. Furthermore, the implementation of soy to our HPLC-MS/MS method for the detection of lupin allergens was the first step towards a future multi-allergen method. To ensure whether there was soy in the lupin flour, we used different techniques: immuno-analytical tests using anti-soy-, respectively anti-lupin-antibodies, a real-time PCR method for the detection of soy specific marker DNA, an HPLC-MS/MS approach to test for isoflavones, which can be found in soy beans but not in lupin seed and a second HPLC-MS/MS method supported by a Mascot search to identify lupin- and soy-specific marker peptides. Some of these techniques were actually able to detect soy in the lupin flour others were not that sensitive. Nevertheless, this study showed the impact of reference methods for the development of new techniques for the detection and quantification of allergens in food.





P17 LOW-COST, FAST, MINIATURISED SOLUTIONS FOR WATER QUALITY ASSESSMENT Christina Liedert, L. Hakola, H. Boer, L. Hakalahti, A. Jokinen, R. Rikkola, T. Sipilä-Malm, M. Smolander, H. Virtanen, S. Känä, J. Jokela, A. Ylinen and K. Sivonen VTT Technical Research Centre of Finland, Finland [email protected] The poster summarises the results of the Waterchip project which is funded by the Finnish Funding Agency for Technology and Innovation and industrial partners (MK fluidics, Orion Diagnostica, FIWA). The aim is to explore the potential of mass-manufacturing technologies in environmental applications, and to develop novel, low-cost, miniaturised and fast test concepts for water quality assessment by combining printing, hot embossing and injection moulding as manufacturing methods with biological and chemical detection of certain impurities and pollutants from water. The focus areas are:

polymer based microfluidic chip for immunochemical detection of cyanobacterial toxins (proof-of-concept demonstrator);

injection moulded prototype for chemical detection of phenolic compounds based on visual detection;

chemical and molecular test for odorous compounds of cyanobacteria; and

enzymatic test for phenolic compounds based on visual and spectrophotometric detection.


P18 INCREASING SELECTIVITY IN LC-MS/MS ANALYSIS USING TECHNIQUES SUCH AS MRM3, SELEXION™ AND HIGH RESOLUTION LC-MS/MS Stephen Lock and J. Stahl-Zeng AB SCIEX, UK

[email protected] The demand for speed and cost reduction in food analysis has meant that sample preparation is often simplified. Techniques such as liquid /liquid extractions or QuEChERS are commonly employed in food testing, but the resulting extracts are more complex leading to matrix interferences, which can in turn lead to increased number of false positives in food testing and issues with quantitation. There is therefore a need to increase the selectivity of detection in LC-MS/MS to get around the issues of matrix interferences but still maintain speed of analysis and the sensitivity needed to reach the regulatory limits. This poster will discuss several different ways to overcome these issues using various types of mass spectrometry techniques. Techniques including MRM3 and ion mobility separation using the new SelexION™ interface will be discussed and examples where these techniques have benefits will be shown. In addition the principles of high resolution LC-MS/MS will be described with examples of where this has been applied.


P19 ALLERGEN SCREENING IN FOOD BY LC-MS/MS Stephen Lock and J. Stahl-Zeng AB SCIEX, UK

[email protected] The prevalence of food allergies in the USA is estimated at around 6% for children and reports suggest that the number of allergies is rising. Screening for allergens is traditionally performed using enzyme-linked immunosorbent assays (ELISAs). ELISAs can generate variable results, and false-positive as well as false-negatives. Additionally each allergen requires a separate kit so a method that could unambiguously confirm the identification of individual allergens in a multiple screen would be invaluable. Here we present the first ever allergen method incorporating diverse allergens such as mustard, tree nuts, milk and gluten, which represent over 12 separate species from different allergenic groups. The LC-MS/MS method detects individual allergic proteins which have been reduced, alkylated and digested using trypsin. The peptides from the digested proteins are purified and these extracts analysed by LC-MS/MS and reverse phase chromatography using positive mode electrospray ionisation. The mass spectrometry methods utilises Scheduled MRM™ and information dependant acquisition so that not only are multiple peptides detected for each allergen but full scan product ion data can be collected at the same time for each peptide so that presence of allergen can unambiguously be confirmed. The method developed has been shown to be appropriate for the simultaneous quantification of numerous allergens using the same extraction conditions. The results show that a combined screening method for several allergens in a single run is now possible by LC-MS/MS. Future work is planned to expand this approach to other allergens and to test this method on a wide range of processed food.


P20 FAT SOLUBLE VITAMIN DETECTION IN FOOD BY LC-MS/MS Stephen Lock AB SCIEX, UK

[email protected] Vitamins are needed for healthy growth and fat soluble vitamin deficiency can lead to conditions such as rickets (lack of vitamin D). Traditionally individual methods have been used to screen for each vitamin so one method that is capable to screen for all fat soluble vitamins would be beneficial. Here we present some new data acquired by LC-MS/MS with a screening method which contains Vitamin K1, K2, D2, D3, A and E. Detection of these vitamins has been shown to be at the low ppb levels by LC-MS/MS and reverse phase chromatography using positive mode atmospheric pressure chemical ionisation. The mass spectrometry methods utilises Scheduled MRM™ and a small particle size HPLC column. Reference material and food samples were then extracted and analysed by LC-MS/MS to show the applicability of this method to routine sample analysis.


P21 THE USE OF MICROFLOW UHPLC AS A WAY TO SOLVENT USAGE IN PESTICIDE SCREENING OF FOOD SAMPLES BY LC-MS/MS Stephen Lock AB SCIEX, UK

[email protected] Traditionally in pesticide screening of food samples, sample are prepared using generic extraction procedures, like QuEChERS (quick, easy, cheap, effective, rugged, and safe) and then analyzed by LC-MS/MS or GC-MS/MS. Usually when analyzing samples, LC flow rates are in excess of 500 µl/min and are used in combination with high pressures with smaller particle size HPLC columns to maintain sharp peaks and fast chromatography. These flow rates produce fast speeds and excellent peak shapes and results, but have a draw back in that they require higher volumes of organic solvent. The consumption of HPLC organic solvents, such as acetonitrile and methanol, is a growing cost of analysis and its disposal has an environmental impact. Therefore, ways to reduce solvent consumption in pesticide residue testing will be beneficial to the environment and reduce running costs of a testing laboratory. Here we present new data using microflow LC (using an ExpressHT™-Ultra running at 10 µl/min) in combination with a LC-MS/MS method developed on a AB SCIEX QTRAP® 4500 system utilizing the Scheduled MRM™ algorithm in combination with fast polarity switching and acquisition of MS/MS spectra for compound identification. Initially, this approach has been applied to a screen of over 100 pesticides in QuEChERS food extracts and we will present robustness and sensitivity data on several matrices to show its applicability in food analysis.


P22 DIAGNOSTICS METHOD FOR THE RAPID DETECTION AND IDENTIFICATION OF LOW LEVEL CONTAMINATION OF WATER SYSTEMS WITH HUMAN PATHOGENIC BACTERIA Elizabeth Minogue Molecular Diagnostics Research Group, Microbiology Department, School of Natural Sciences, National University of Ireland, Ireland [email protected] Microbial analysis of contamination of pharmaceutical grade water, pharmaceutical raw materials, medical devices and patient-care equipment is routinely performed by culture incubation. Biochemical tests are subsequently performed to identify the contaminating microorganism. This analysis can take 5-7 days to achieve an end result. The aim of this study was to design and develop a rapid bacterial diagnostics method, combining culture incubation with multiplex real-time PCR, to detect and identify low level contamination of water with human pathogenic bacteria. Initially, a novel internally controlled multiplex real-time PCR diagnostics assay was designed and optimised to specifically detect and identify Pseudomonas aeruginosa and the Burkholderia genus. The multiplex real-time PCR diagnostics assay developed was determined to be 100% specific for the microorganisms tested, while an analytical sensitivity of less than 10 genome equivalents was achieved for both assays. Subsequently, sterile pharmaceutical grade water, spiked with a bacterial load ranging from 10-1000 cfu per 100 ml, was filtered and analysed for bacterial contamination using a combined bacterial culture and multiplex real-time PCR approach. This analysis demonstrated that a 4 h culture incubation was sufficient for the reliable detection and identification of low level contamination of water with these bacteria (10 cfu per 100 ml) using this diagnostics method. The diagnostics method described here is highly sensitive and specific and has a significantly shorter time to result compared to conventional culture based methodologies. Low level contamination of water with P. aeruginosa and the Burkholderia genus can be reliably detected and identified in less than 8 h. P. aeruginosa and members of the Burkholderia genus are among the most common microorganisms associated with contamination of pharmaceutical grade water, pharmaceutical raw materials, medical devices and patient-care equipment. However, a number of other microorganisms have also been reported to pose a problem in this area. As such, work has begun on developing a series of multiplex real-time PCR diagnostics assays for the detection and identification of a number of other common bacterial contaminants. Once these multiplex real-time PCR diagnostics assays have been designed, developed and optimised we intend to incorporate these assays with the methodology presented here. We propose that this rapid diagnostics method could be applied to the pharmaceutical and clinical sectors to assure the safety and quality of pharmaceutical grade water, medical devices and patient-care equipment.


P23 IN VINO VERITAS – QUANTIFYING ALLERGENS IN WINE Ronald Niemeijer, M. Richter, M. Lacorn and U. Immer R-Biopharm AG, Germany [email protected] Different egg and milk products are added to wines as clarification agents, for fine tuning of wine flavour or for conservation. According to the EU Directive 2007/68/EC these allergenic substances must be declared on the label of wine bottles from 1 July 2012 onwards. The OIV-COMEX 502-2012 resolution recommends the ELISA technology with a detection limit of 0.25 mg/l allergen to quantify allergens in wine. R-Biopharm offers different allergen ELSA test kits which allow the determination of casein, egg white proteins and egg lysozyme in a high sensitive manner. These assays can be used for determination of allergens in red and white wine after the OIV resolution. Different red and white wines from the market have been analyzed for egg and casein. During this examination only one Italian red wine contained a very low amount of egg protein. A field study with fined red wine showed that after filtering and bottling no egg protein remains in measurable amounts (below 0.5 mg/kg whole egg powder). Spike experiments with egg, casein or lysozyme in wine showed that the RIDASCREEN® ELISAs for casein, egg and lysozyme are robust and reliable methods to detect residual concentrations of fining and clarification agents in wine. A collaborative study for casein and egg was done successfully. The results will be published soon.


P24 RIDASCREEN®FAST SOYA (R7102) SANDWICH ELISA TO DETECT TRACES OF SOYA IN NATIVE AS WELL AS IN PROCESSED FOOD Ronald Niemeijer, M. Richter, M. Lacorn and U. Immer R-Biopharm AG, Germany [email protected] Soya belongs to the so called ‘big 8’ allergens and following the food labelling directive soya has to be labelled as ingredient if used in food. Parallel to this worldwide more and more soya allergies appear. This fact and the laws make a measurement of soya traces in food essential. Soya contaminations have to be prevented during food production and food samples must be checked regularly. The new sandwich ELISA RIDASCREEN®FAST Soya (R7102) is made to detect the typical allergenic soya proteins like ß-conglycinin (Gly m5), Glycinin (Gly m6) not only in untreated food but also in heated samples (e.g. sausages, bakery goods, soups, sauces, margarine, ice cream, beverages). The ELISA antibody is specific for denaturized soya proteins from raw soybean, flour, protein concentrates and other soya products. The testing of soya protein isolates and hydrolyzates is recommended with restriction. The detection limit (LOD) of this fast assay is 0.31 mg/kg (ppm) soya protein and the limit of quantification (LOQ) is 2.5 mg/kg (ppm) soya protein. The ELISA shows low cross reactivity to beans (0.0017%) and common tare (0.0003%) but not to peanut, lentil, pea and lupine.


P25 DEVELOPING A MEMBRANE-BASED ASSAY FOR THE SIMULTANEOUS SCREENING OF SIX MYCOTOXINS IN DIFFERENT FEED MATRICES: ACHIEVEMENTS, CHALLENGES AND PERSPECTIVES Emmanuel Njumbe Ediage1, Y. Guo1, I. Goryacheva2 and S. De Saeger1

1Laboratory of Food Analysis, Ghent University, Belgium and 2Department of Common and Inorganic Chemistry, Chemistry Faculty, Saratov State University, Russia [email protected] In farm animals, mycotoxins can cause among others, decreased performance, feed refusal, poor feed conversion, diminished body weight gain, immunosuppression, reproductive disorders and residues in animal food products. Many mycotoxigenic fungi can grow and produce their toxic metabolites under similar conditions. Therefore in animal feed, mycotoxins rarely occur as single contaminants. Apart from that, blends of various raw materials in compound feed can increase the risk of feed contamination with several mycotoxins. Several combinations of mycotoxins may lead to additive or synergistic toxic effects. Although the prevention of mycotoxin contamination in the field is the main goal of agricultural and food industries, under certain environmental conditions, the contamination of various commodities with mycotoxins is unavoidable. By analogy with chromatographic methods, the new trend is to develop fast screening multi-mycotoxin methods. This work is focused on the development and validation of a multiplex flow-through membrane-based assay (MBA) for the simultaneous detection of six mycotoxins in a wide variety of food and feed ingredients. Recently, the Laboratory of Food Analysis reported the successful development of a qualitative MBA for the simultaneous screening of four mycotoxins in maize, peanut cake and cassava. The MBA can be used to simultaneously screen for zearalenone, aflatoxin B1, deoxynivalenol and ochratoxin A at 175, 5, 700 and 3 µg/kg as cut-off levels, respectively. The MBA was validated within a laboratory setting and also under tropical conditions in West Africa. The false negative rate following both validation studies was <5, hence fulfilling the strict regulatory guideline as specified in Commission Decision 2002/657. Further challenging objectives will be (i) to broaden the scope of the four analyte MBA to a six analyte (zearalenone, aflatoxin B1, deoxynivalenol and ochratoxin, fumonisin B1 and T2-toxin) MBA without compromising the assay sensitivity and (ii) to achieve very low detection limits for all the target analytes which are comparable with levels achieved with conventional chromatographic methods. Furthermore, the possibility of utilizing such a tool (MBA) to screen dirty matrices such as feed ingredients and/or feed blends has not yet been exploited.



P26 A LATERAL FLOW MICROARRAY IMMUNOASSAY AS COMPARED TO CONVENTIONAL LATERAL FLOW AND SLIDE MICROARRAY ASSAY FORMATS Truus Posthuma, L. Berendsen, A. Moers and A. van Amerongen

Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen UR, the Netherlands [email protected] In food, feed and environmental analysis there is an urgent need in analysis formats that can be performed on-site. Especially in resource-limited areas and without extensive user instructions this can be added to faster screening in environmental-, food-, feed- and health-related areas. Previously, we have developed two rapid methods and used the detection of amplicons derived from genes of four virulence factors of Shiga toxin-producing Escherichia coli as a model [1, 2]. In all cases carbon nanoparticles with adsorbed neutravidin were used. In the present study we have developed this model further by combining the singleplex PCRs into a multiplex variant with a total amplification time of 30 min. Using labelled primers the amplicons derived from the virulence factors can be discriminated; the specific primer sets contain a forward primer with a unique tag and a reverse primer with biotin, both coupled to their 5’-ends. In addition, we have developed an assay format that is a combination of the (nucleic acid) lateral flow immunoassays (NALFIA; assay time 15 min) and the (nucleic acid) microarray immunoassay on slides (NAMIA; assay time ± 60 min). For NALFIA there is a limitation in the number of analytes that can be detected on one strip (max. four analytes and a control). For NAMIA the assay can be used for >50 analytes, however, the assay time is somewhat too long for a rapid assay and the assay requires more advanced laboratory facilities. In this study the sensitivity of the NAMIA was increased by using a carbon nanoparticles conjugate with a fusion protein of neutravidin and alkaline phosphatase (AP). A 30 min incubation was followed by a 10 min incubation with an AP substrate solution resulting in signal enhancement by precipitating dye. The new method, the (nucleic acid) lateral flow microarray immunoassay ((NA)LMIA) combines the better characteristics of both former assays; an assay time of 15 min and a number of 25 different antibody spots. The sensitivity of the NALMIA appeared to be similar or better as compared to the NALFIA and NAMIA assay formats. Acknowledgements The financial support of the Wageningen UR Kennisbasis programme KB-VII 'Technology development' is greatly appreciated. The contributions of Fimme-Jan van der Wal and Albert de Boer from Wageningen UR Central Veterinary Institute to the preceding lateral flow and microarray assays and the supply of E. coli samples are highly valued. References 1. Noguera, P., Posthuma-Trumpie, G., Van Tuil, M., Van der Wal, F., De Boer, A., Moers, A. and

Van Amerongen, A., 2011. Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli. Analytical and Bioanalytical Chemistry 399: 831-838.

2. Noguera, P.S., Posthuma-Trumpie, G.A., Van Tuil, M., Van der Wal, F.J., De Boer, A., Moers, A.P.H.A. and Van Amerongen, A., 2011. Carbon nanoparticles as detection labels in antibody microarrays. Detection of genes encoding virulence factors in Shiga toxin-producing Escherichia coli. Analytical Chemistry 83: 8531-8536.


P27 CIM MONOLITHS AS A TOOL FOR SIMULTANEOUS CONCENTRATION AND REMOVAL OF ENTERIC VIRUSES FROM WATER Nejc Rački1, I. Gutierrez-Aguirre1, A. Steyer2, J. Gašperšič3, B. Brajer Humar4, M. Stražar4, A. Štrancar3 and M. Ravnikar1

1National Institute of Biology, Slovenia, 2Institute of Microbiology and Immunology, Slovenia, 3BIA Separations, Slovenia and 4Central Waste Water Treatment Plant Domžale-Kamnik, Slovenia [email protected] Traditional waste water treatment usually does not remove or inactivate all of the potentially pathogen microorganisms. This is especially true for enteric viruses that are introduced into the environment through the discharge of effluent from waste water treatment plants (WWTP) [1]. Although discharged concentrations of viruses are low they can still lead to infection. For some enteric viruses ingestion of only 10-100 virus particles is enough to initiate the disease, what calls for very sensitive detection methods. It has been previously shown that CIM-quaternary amine (QA) monolithic supports are a good tool for concentration of viruses in water [2]. Here we go one step further and evaluate CIM monoliths not just for concentration of enteric viruses but also for their removal from wastewater effluent. It was chosen as a specially challenging sample to process because of its chemical and biological diversity. It contains a lot of molecules that compete with viruses for binding on a CIM QA column. Monthly monitoring of wastewater effluent was done for the presence of rotaviruses (RoV), noroviruses (NoV), astroviruses (AsV), sapoviruses (SaV) and hepatitis A virus (HAV). We showed that CIM QA based concentration step developed previously for the rotavirus can be effectively used to concentrate all the above mentioned viruses simultaneously. Moreover virus removal capabilities, of CIM QA monoliths for enteric viruses by analyzing the wastewater fraction after interacting with the monolith, were demonstrated as well. The described concentration/removal method is being developed in parallel to another physical technique such as pulsed electric field, to implement a novel water disinfection methodology (project ARRS L2-4314). References 1. Simmons, F.J. and Xagoraraki, I., 2011. Water Research 45: 3590-3598. 2. Gutiérrez-Aguirre, I., Steyer, A., Banjac, M., Kramberger, P., Poljšak-Prijatelj, M. and Ravnikar, M.,

2001. Journal of Chromatography A 1218: 2368-2373.


P28 SAMPLE CONCENTRATION METHODS FOR SENSITIVE AND RELIABLE PCR-DETECTION AS EXEMPLIFIED BY MYCOPLASMA AND LEGIONELLA Alexandra Scholz1, L. Kukuk2, T. Scheper1 and K. Pflanz3 1Leibniz University Hannover, Germany, 2Heinrich Heine University Düsseldorf, Germany and 3Sartorius Stedim Biotech GmbH, Germany [email protected] Mycoplasmas are fastidious, slow growing, small bacteria with a detection time of at least 14 days for the traditional cultivation based method. And even after 14 days of incubation Mycoplasma colonies are hardly visible to the eye. Legionella is another representative of the more slowly growing bacterial genera. The classical membrane filtration method requires an incubation time of at least five days. In both cases, cultivation time is the bottleneck using classical cultivation as detection method. Nucleic acid amplification techniques (NAT) are cultivation independent and therefore there is no need for long incubation times and cumbersome colony counting. So why not reduce the time waiting for results to mere hours instead of days or weeks? Hitherto the lack in sensitivity and the small insertable sample volume in NAT test systems have limited its use in the detection of microorganisms. Our studies present options to overcome these limitations and show two easy ways for sample concentration, sample preparation and Real-time PCR detection for sensitive and reliable results.


P29 CHIP-BASED MICROSIEVES FOR CONCENTRATING MICROORGANISMS TO ENABLE DETECTION Heleen de Vogel-van den Bosch1, R. van Doorn2, M. Klerks2 and A. van Amerongen1

1Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen UR, the Netherlands and 2Innosieve Diagnostics BV, the Netherlands [email protected] For safety and quality reasons it is important to be able to detect microorganisms. Conventional culture-based techniques are often laborious and may take several days to sometimes more than a week. In addition, often not enough cells are available for detection and the cells could be spread inhomogeneously in the matrix. Furthermore, speed could be very important. So, for the detection of pathogenic bacteria or food spoiling organisms fast tests and tests detecting low numbers of microorganisms are very desirable. Wageningen UR, Food and Biobased Research, develops several pre-treatment methods, among others using the microsieve technology. Microsieves are lithography-chip-based, inert, chemically-stable filters with homogeneous pores. Microorganisms can be trapped (concentrated) on these filters making detection possible. These trapped microorganisms can be detected using a specific fluorescent labelled antibody followed by scanning the surface of the microsieve. Hereto, an innovative LED-based optical device (MuScan) is used that allows quantification, live/dead discrimination and target specific detection of microorganisms. Furthermore, these concentrated microorganisms could be detected using (nucleic acid) lateral flow immunoassays ((NA)LFIA) with binding molecules on the lateral flow membranes and in the nanoparticle labels. Some examples using this microsieve technology will be given such as the detection of Salmonella in food products and Legionella in drinking water. Furthermore, a new concept will be showed: the microsieve-lateral flow immunoassay. This new innovative diagnostic concept aims to combine the microsieve technology with the lateral glow immunoassay detection principle and allows for the on-site detection of (pathogenic) bacteria in a simple and fast way. Acknowledgements The authors from Food & Biobased Research, Wageningen UR, acknowledge the financial contributions from the programmes Nano4Vitality (N4V) and NanoNextNL (Project 05A.01 Microbial Food Quality and Safety).


P30 RAPID LATERAL FLOW DETECTION OF LEGIONELLA-SPECIFIC RNA AND DNA AMPLICONS Heleen de Vogel-van den Bosch1, L. Nederhoff1, A. Martin-Pagola2, D. Verdoy2, J.-O. Axe3, S. Peters3, B. Oberheitmann3, M. Klerks4, A. Moers1 and A. van Amerongen1

1Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen UR, the Netherlands, 2Fundacion GAIKER, Spain, 3Q-Bioanalytic GmbH, Germany and 4Innosieve Diagnostics BV, the Netherlands [email protected] Legionella outbreaks have great sanitary, economic and social implications. The currently used diagnostic reference method to detect Legionella spp. is based on bacterial culturing, which could take more than a week to obtain conclusive results. The European seventh framework programme project ‘PINVIALEG’ addressed the detection of Legionella pneumophila type 1 within a couple of hours. NASBA and PCR amplicons were detected by a lateral flow readout platform (nucleic acid lateral flow immunoassay; NALFIA). NASBA was chosen to be able to identify viable Legionella only. However, the RNA template and the NASBA amplicon are sensitive to degradation and, therefore, difficult to work with. Hence, in this project a test based on PCR (with DNA templates and amplicons) was developed as well. In the PCR-NALFIA a primer set specific for Legionella spp. was taken as a control for comparison with conventional methods. Furthermore, these spp. primers can detect disease causing Legionella species other than L. pneumophila. The combination of molecular methods with lateral flow results in a rapid, on-site test for the detection of Legionella spp. and L. pneumophila type 1 a NASBA specific for L. pneumophila type 1 was developed yielding single strand anti-sense RNA amplicons. To detect such amplicons in a NALFIA two oligonucleotide probes were designed that hybridise specifically with these amplicons. The probes were labelled with biotin and digoxigenin, respectively. Consequently, the final product could be sandwiched between an anti-digoxigenin antibody immobilised on the nitrocellulose membrane and neutravidin that had been bound onto the surface of carbon nanoparticles (NASBA-NALFIA). Furthermore, a multiplex PCR for Legionella spp. and L. pneumophila was developed. Both tests (NASBA-NALFIA and PCR-NALFIA) have been evaluated successfully within the PINVIALEG consortium and show good specificity, inclusivity and sensitivity. Possible applications are analysis of cooling tower waters and tap waters from a variety of sources (e.g. hotels, swimming pools). Acknowledgements The authors acknowledge the financial contribution from the EU project ‘Portable microfluidic-based device for in situ detection of viable Legionella’ (PINVIALEG), Grant agreement no. 262561.


P31 BEAD-BASED DNA-ASSAY FOR THE DETECTION OF STREPTOCOCCUS SUIS IN TONSILLAR SPECIMENS OF PIGS Fimme Jan van der Wal1, H.J. Wisselink1, J.H. Bergervoet2, M. de Weerdt2 and R.P. Achterberg1 1Central Veterinary Institute, Wageningen UR, the Netherlands and 2Plant Research International, Wageningen UR, the Netherlands [email protected] Streptococcus suis is an important swine pathogen in nearly all countries with an extensive pig industry. It is associated with meningitis, arthritis, endocarditis, septicemia, pneumonia and sudden death. S. suis can be detected on the palatine tonsils of both healthy and diseased pigs. Pigs can carry multiple S. suis serotypes [1], of which presently 35 have been described. Worldwide serotype 2 strains are most prevalent, whereas serotype 1, 7 and 9 strains are also frequently isolated from diseased pigs. PCRs have been described for detection of specific S. suis serotypes and virulence-associated phenotypes [1-3], but to provide a solid basis for fast and adequate control measures a platform suitable for extensive multiplexing is desirable. Therefore, we developed a suspension array for six genes, which can serve as a scaffold for future expansion with more targets. A sixplex PCR and a subsequent target specific primer extension (TSPE) assay were designed using AlleleID and PrimerPlex 2 (PREMIER Biosoft) on four serotype specific capsular polysaccharide (cps) genes (cps1I, cps2J, cps7H, cps9H) [2,3], the extracellular virulence factor EF (epf) [4] and a general S. suis marker, i.e. glutamate dehydrogenase (gdh) [5]. DNA from reference strains with serotypes 1, 2, 7 and 9 was amplified (Qiagen Multiplex PCR Plus Kit); the amplicons were used as template for TSPE with biotinylated dCTP. The biotinylated TSPE products were hybridized to MagPlex TAG beads (Luminex), labelled with streptavidin-phycoerythrin, and subsequently analysed on a Luminex 200. Each individual PCR was capable of producing the desired amplicon. When performed as multiplex (sixplex) PCR on chromosomal DNA of reference strains, the anticipated amplicons were generated. After hybridization of the TSPE products, all multiplex PCR products produced signals on the appropriate beads. A sixplex PCR and subsequent TSPE was designed for S. suis. The proof-of-principle of the assay was demonstrated with chromosomal DNA of reference strains. Next, the assay will be evaluated with field samples, using DNA isolated from tonsillar specimens of pigs. Ultimately, this type of assay may be applicable for studying epidemiology and transmission, and could contribute to efforts aimed at control and eradication of S. suis on pig farms. References 1. Wisselink, H.J., Joosten, J.J. and Smith, H.E., 2002 Journal of Clinical Microbiology 40: 2922-

2929. 2. Smith, H.E., Van Bruijnsvoort, L., Buijs, H., Wisselink, H.J. and Smits, M.A., 1999. FEMS

Microbiology Letters 178: 265-270. 3. Smith, H.E., Veenbergen, V., Van der Velde, J., Damman, M., Wisselink, H.J. and Smits, M.A.,

1999. Journal of Clinical Microbiology 37: 3146-3152. 4. Smith H.E., Reek, F.H. Vecht, U., Gielkens, H.L. and Smits, M.A., 1993. Infection and Immunity

61: 3318-3326. 5. Okwumabua, O., O’Connor, M. and Shull, E., 2003 FEMS Microbiology Letters 218: 79-84.


P32 RAPID LATERAL FLOW TESTS FOR TULIP VIRUS X IN PLANT MATERIAL BY DETECTION OF A VIRAL SURFACE ANTIGEN OR A SPECIFIC RNA SEQUENCE M. Koets1, Jan Wichers1, J. van Doorn2, M. de Kock2 and A. van Amerongen1

1Biomolecular Sensing & Diagnostics, Food & Biobased Research, Wageningen UR, the Netherlands and 2Applied Plant Research, Wageningen UR, the Netherlands [email protected] Tulip virus X (TVX) is a pathogen for tulip. It is a filamentous, positive-stranded RNA virus, belonging to the family of Flexiviridae and the genus of potex viruses. Its natural hosts are Tulipa species. Symptoms of infection comprise chlorotic or necrotic grey–brown streaking of leaves and streaks of intensified pigment (or of necrosis) in petals. Several mechanical transmission pathways for TVX have been identified during bulb production. During bulb storage, the dry bulb mite (Aceria tulipae) is the main vector for TVX. Preventive actions focused on early recognition and removal of virus diseased plant are the best remedies to combat this virus. To support farmers and inspection services in early recognition of virus diseased plants, we developed two methods that can be applied in or near the field, both based on the well-known lateral flow immunoassay principle. In the first serological test a specific polyclonal antiserum directed against a virus coat protein was used both as capture ligand on the nitrocellulose membrane and as detection ligand immunobilised on the surface of carbon nanoparticles. In the second molecular test a double-labelled amplicon was sandwiched between an anti-digoxigenin antibody on the membrane and neutravidin on carbon nanoparticles. The double-labelled amplicon was obtained in a process in which viral RNA was converted into complementary DNA that was used as a template in a subsequent PCR procedure with a forward primer labelled with a digoxigenin and a reverse primer with a biotin molecule, both at the 5'-end. Prototypes of the serological and molecular tests were developed and optimised for performance with extracts of tulip leave material. Both tests showed good results in initial experiments. Further activities will concentrate on the validation of the tests and on the comparison of the performance characteristics with standard PCR- and ELISA diagnostics. The availability of rapid tests for TVX may initiate the development of rapid tests for other viruses in flower bulbs.


P33 MALDI-TOF MS AS CONFIRMATION TOOL FOR PATHOGENS IN DRINKING WATER Marsha van der Wiel, P. Willemse and G. Wubbels

WLN, the Netherlands [email protected] WLN is the center for water quality and water technology. WLN takes care for clean, healthy and tasty drinking water. WLN is progressive in the use of new and faster techniques. One of the newest techniques is the use of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) biotyper. With this new technique WLN can identify indicators and pathogens such as Escherichia coli, Enterococcus and Legionella within a few minutes directly from their selective media. MALDI-TOF MS is based on the chemotaxonomy of microorganisms. A single colony of a target organism is put directly on a 96 target plate. After deposition the spots were overlaid with 1 µl matrix solution (2.5 mg α-cyano-4-hydroxycinnamic solved in 50% acetonitrile, 2.5% trifluoro acetic acid, 47.5% ultra-pure water). The matrix opens the cell wall. A laser irradiates the matrix sample, to divide it in little portions of and peptides. The matrix evaporates and positive charged peptides become free. In the strong electric field the positive charged peptides are lined up. So these peptides have the same starting point, before they accelerate in the flight tube to get to their specific time-of-flight corresponding with their specific mass. Spectra are generate with the MALDI-TOF MS biotyper from Brϋker Daltonik GmbH and compared with approximately 4000 spectra in the Brϋker Daltonik GmbH database. In a log score 1 to 3 the MALDI-TOF biotyper defined the similarity of the known and unknown spectra. WLN has tested 67 reference strains and 316 samples, grown on their specific and general media. These microorganisms were confirmed with classic techniques and the MALDI-TOF MS biotyper. In Table 1, the conformity between NEN- and MALDI-TOF MS confirmation can be seen. The validation is performed according NEN-EN-ISO 16140. Legionella is divided in two stages, the genes Legionella scores 100% similarity. Legionella species have a lower score because 4 Legionella species are not present in the Bruker database. Certain peaks of the micro-organism ‘fingerprint’ are conserved, the so-called abundant proteins. These proteins are always there and make it possible to characterize microorganisms. Some micro-organisms are not detected (Legionella species) by the Bruker database, because Bruker did not introduce these microorganisms. Therefore the Dutch water laboratories will generate a collective ‘water database’. Table 1. Overview of validation results WLN, classic confirmation against MALDI-TOF MS.

Confirmation Relative accuracy Relative specificity Relative sensitivity

Escherichia coli 100% 100% 100%

Coliforms 100% 100% 100%

Enterococci 99% 100% 98%

Legionella sp. 93% 95% 92%

Legionella 100% 100% 100%

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