University of Pardubice

Faculty of Chemical Technology

Department of Analytical Chemistry

DEVELOPMENT AND CHARACTERIZATION OF MAGNETIC IMMUNOSORBENTS

FOR ANALYSIS OF CLINICALLY RELEVANT BIOMARKERS IN MICROFLUIDIC SYSTEMS

Ph.D. Thesis (Annotation)

Author: Mgr. Zuzana Svobodova

Supervisor: doc. RNDr. Zuzana Bilkova, Ph.D.

2014

University of Pardubice

Faculty of Chemical Technology

Department of Biological and Biochemical Sciences

Development and characterization of magnetic immunosorbents

for analysis of clinically relevant biomarkers in microfluidic systems

Ph.D. Thesis

2014

Author: Mgr. Zuzana Svobodová

Supervisor: doc. RNDr. Zuzana Bílková, Ph.D.

This Ph.D. thesis was drawn up within the doctoral study at Department of Biological and Biochemical

Sciences, Faculty of Chemical Technology, University of Pardubice in years 2007 – 2014.

Candidate Mgr. Zuzana Svobodová

Program of study: Analytical chemistry P1419

Field of study: 1403V001

Supervisor: doc. RNDr. Zuzana Bílková, Ph.D.

Department of Biological and Biochemical Sciences

Faculty of Chemical Technology, University of Pardubice

Reviewers: Ing. František Foret, CSc.

Institute of Analytical Chemistry

Academy of Sciences of the Czech Republic in Brno

Prof. Mgr. Marek Šebela, Ph.D.

Department of Biochemistry

Faculty of Science, Palacky University in Olomouc

Defence of the Ph.D. thesis takes place on ............... 2014 at ................ in meeting place of Department

of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice before

commission for doctoral thesis defences of Analytical chemistry.

The Ph.D. thesis is available at University Library, Studentska 519, 532 10 Pardubice.

CONTENT:

1 Abbreviations ................................................................................................................................ - 4 -

2 Summary ....................................................................................................................................... - 5 -

3 Introduction................................................................................................................................... - 6 -

3.1 Magnetic microparticles and microfluidics ................................................................................... - 6 -

3.2 Alzheimer´s disease biomarkers ................................................................................................... - 7 -

3.3 Adenocarcinoma and circulating tumor cells ............................................................................... - 8 -

4 Aimes and objectives .................................................................................................................. - 10 -

5 Experimental: Results and discussion ........................................................................................ - 11 -

5.1 Magnetic immunosorbent for on-chip immunoprecipitation of amyloid β peptides ................. - 11 -

Paper I .......................................................................................................................................... - 11 -

Paper II ......................................................................................................................................... - 13 -

Paper III ........................................................................................................................................ - 14 -

5.2 Magnetic immunosorbent for on-chip immunocapture of circulating tumor cells .................... - 16 -

Paper IV ........................................................................................................................................ - 16 -

Paper V ......................................................................................................................................... - 18 -

Paper VI ........................................................................................................................................ - 19 -

6 Conclusion ................................................................................................................................... - 21 -

7 References ................................................................................................................................... - 22 -

8 List of original papers .................................................................................................................. - 24 -

9 List of conferences ...................................................................................................................... - 25 -

10 List of oral presentations ............................................................................................................ - 27 -

- 4 -

1 ABBREVIATIONS

AA Amino acid

Ab Antibody

AD Alzheimer´s disease

Ag Antigen

Aβ Amyloid β

BCA Bicinchoninic acid

BSA Bovine serum albumin

CE Capillary electrophoresis

CSF Cerebrospinal fluid

CTC Circulating tumor cell

DNA Deoxyribonucleic acid

EC European Commission

ELISA Enzyme-linked immunosorbent assay

EOF Electroosmotic flow

EpCAM Epithelial cell adhesion molecule

HSA Human serum albumin

IgG Immunoglobulin G

LOC Lab-on-chip

mAb Monoclonal antibody

MALDI-TOF Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

MCE Microchip capillary electrophoresis

MIS Magnetic immunosorbent

PAGE Polyacrylamide gel electrophoresis

PDMA-AGE Poly(dimethylacrylamide-co-allyl glycidyl ether)

PDMS Poly(dimethyl siloxane)

PEG Poly(ethylene glycol)

PGMA Poly(glycidyl methacrylate)

SDS Sodium dodecyl sulfate

SEM Scanning electron microscope

TFA Trifluoroacetic acid

μIP Microimmunoprecipitation

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2 SUMMARY

This work is intended to development of magnetic immunosorbents to be used inside a microfluidic device

for immunocapture and analysis of specific biomarkers of Alzheimer´s disease, such as 3 kDa amyloid β

peptides, and/or circulating tumor cells occurring in peripheral blood of patient´s with various

adenocarcinomas. For isolation of two different biomarkers, magnetic microparticles coated with specific

antibody against each biomarker were applied. Subsequently, such magnetic immunosorbents (MISs) were

inserted into microfluidic device designed according to requirements of each biomarker. The advantages

offered by newly developing field, microfluidics, and magnetic affinity separations were employed. During

the specific MIS development, commercial as well as newly developed polymer microparticles with

modified surface properties were utilized. The MISs were also developed according to their behavior in the

microfluidic systems. The Ph.D. thesis is composed of the “introduction” part where current state

of mentioned problematics is described and “present investigation” part where achieved results can

be found in form of 6 research articles collection with introductory summary chapters, so called “thesis

by publication”.

KEY WORDS:

Immunoaffinity separation, immunosorbent, magnetic particle, microfluidics, antibody, ligand

immobilization, dot-blot, Alzheimer’s disease, amyloid β, adenocarcinoma, circulating tumor cells

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3 INTRODUCTION

Clinical laboratory tests of various body fluids and tissues nowdays represent up to 70% of information

necessary to make the final diagnostic decision. Actually, more than 60% of performed biochemical

and/or hematological tests represent immunochemical methods. Moreover, the number of laboratory

tests per patient as well as portfolio of assays ceaselessly grows. Therefore with the aim to reduce

the costs, reagent/sample consumption and time-to-result, moreover to achieve efficient alternative

to conventional ELISA-like tests and/or to expensive automated “pipetting robots” combination

of heterogeneous bead-based immunoassay with microfluidics should be considered.

3.1 Magnetic microparticles and microfluidics

Microfluidics allow immense possibilities of microfluidic device design, number of components

incorporated, channel geometry, measurement parallelization and/or automation and analyte detection.

Thence, it enables to perform the biomarker(s) analysis from the very first steps as sample pretreatment

to the separation and final detection. Using magnetic beads in such systems might bring certain

advantages such as (i) the enrichment of target molecules from complex samples, (ii) the isolation of target

cells [1], viral particles and bacteria from a large population thus downstream processing,

and (iii) it is much easier to handle (detect, trap and transport) microparticles than single molecules

in microfluidic device [2-6]. It means that magnetic particles can act also as a label to be detected

by magnetic effect-based biosensors [7]. Also biofunctionalization of magnetic particles off-chip is much

more practical and reproducible than to modify the walls of a microchannel [8] which requires additional

micro-fabrication processing steps usually suffering from poor reproducibility and reliability [9].

Additionally, the beads could be replaced after each run eliminating the need to regenerate the solid

support which makes the device reusable and thus cheaper [10, 11]. The combination of microfluidic chips

and beads may provide namely huge analytical surface compare to flat supports (e.g. microfluidic

channels) and high surface-to-volume ratio. This enables reactions in smaller volumes which lead

to a smaller diffusion distance, shorter analysis time and thus time-to-result [8]. This also means reduction

of hazardous waste generation and minimization of potential negative environmental impacts.

The magnetic particles form a colloidal solution of solid elements in a liquid phase. They have most

frequently the spherical monodisperse shape and are made magnetisable or superparamagnetic, meaning

they are only magnetic in the presence of magnetic field. Their magnetic susceptibility has small positive

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values (0–0.01). Due to this property, they can be easily resuspended when the magnetic field is removed

[12]. Polymeric magnetic particles are nowadays widely used. They are composites of an inorganic

(magnetic) material and organic (polymer) material. The surface properties such as hydrophilicity,

functional groups, porosity and biocompatibility should be considered according to ligand being

immobilized as well as to their behavior in aqueous solutions in microfluidic channels (e.g. aggregation,

non-specific adsorption, magnetization etc.). Therefore surface coating of magnetic particles is key to their

successful implementation to microfluidic device.

Another task in magnetic immunosorbent development is appropriate antibody selection

and its attachment to functional groups of magnetic particles. Non-covalent or covalent type of binding

is possible, yet each technique in regard to orientation of ligand offers site-specific or random

immobilization. Non-covalent bindings are fairly simple to conduct, involving incubation

of the microspheres with the purified biomolecule. They typically require little optimization and reagents

may be developed relatively quickly. However, the storage and also reaction stability is much shorter

compare to covalent techniques. Those can provide needed stability when developing a commercial

reagent and/or in case of multiplexed assays, where analyte-specific bead populations are mixed. In this

work covalent site-directed immobilization was preferred because the multiple-site attachment

and random orientation was avoided and full steric availability for specific antigen achieved. This enabled

more efficient consequent immunoprecipitation and maximized bioaffinity capacity. Moreover, leakage

of ligand from the beads was minimized [13].

3.2 Alzheimer´s disease biomarkers

Alzheimer's disease (AD) is the most frequent cause of age-related dementia characterized

by a progressive fatal neurodegenerative disorder accompanied with deteriorations in cognition

and memory, a progressive impairment in the ability to carry out activities of daily living, and a number

of neuropsychiatric and behavioral symptoms [14]. The disease begins about two decades before

the symptoms or neuron death causing typical neuronal changes detectable by brain imaging techniques

onset.

Among the brain changes believed to contribute to the development of AD are (i) the accumulation

of the peptide Aβ outside neurons in the brain called Aβ plaques. This insoluble, fibrillar Aβ aggregates

consist of a deposit of this 40–42/43 amino acid peptides derived through the γ-secretase cleavage

of the amyloid precursor protein (APP) [15, 16]. Mainly soluble Aβ oligomers, metastable intermediates

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in amyloid fibril formation, are now widely recognized as key pathogenic structures in AD [17]. They inhibit

synaptic function, leading to early memory deficits and synaptic degeneration, and they trigger

the downstream neuronal signaling responsible for phospho-tau Alzheimer's pathology [18]. Such process

cause another change in brain tissue, (ii) the accumulation of an abnormal form of the protein tau inside

neurons called neurofibrillary tangles, twisted filaments of the hyperphosphorylated cytoskeletal tau

protein [16], which blocks the transport of nutrients and other essential molecules in the neuron and are

also believed to contribute to cell death. The brains of people with advanced AD show dramatic shrinkage

from cell loss and widespread debris from dead and dying neurons [19].

Currently, clinically significant biomarkers, showing the level of Aβ accumulation in the brain available

for early AD diagnosis, are believed Aβ peptides with 1-42 amino acids (AA) length [20, 21], total tau

and hyperphosphorylated tau protein in CSF [22-26].

Since number of AD patients is still growing, the situation has serious socio-economic consequences, many

research groups are trying to developed reliable tool for early diagnosis in preclinical stages of AD

and treatment which would aid to retard or stop the disease’s progression therefore prolonged

the asymptomatic period of patient and helps the affected families handling the care [25, 27].

3.3 Adenocarcinoma and circulating tumor cells

According to the World Health Organization cancer is one of the leading causes of death worldwide. 90%

of cancer-related deaths are due to metastases, primary tumors are responsible for the rest [28]. During

the process of metastasis, cancer cells detach from the solid primary tumor, enter the blood stream

and travel to different tissues of the body [29]. These cells escaped from the primary cancer and transiently

circulating in blood are known as “circulating tumor cells” [30]. The process of their dissemination

and implantation at distant sites is a complex and multi-step procedure, which is influenced by host

and tumor molecular characteristics [28].

CTCs are very rare, their representation in the blood in numbers as low as one CTC per 106-107 leukocytes

(˂0.004% of all mononucleated cells in the blood), which makes their capture and detection very

challenging [31]. Clinically relevant concentrations of CTCs in patients can range approximately from 1 CTC

per 10 ml to several hundreds of CTCs per ml. CTCs identification might be very difficult namely

because of its surface expression heterogeneity.

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Growing evidence suggests that CTCs isolation from peripheral blood may allow (i) reliable early detection

and molecular characterization of cancer at diagnosis (metastatic relapse or progression), may also provide

(ii) a minimally invasive method to guide and monitor the results of cancer therapy (e.g. efficiency

of chemotherapy after surgical removal of primary tumor) and (iii) help with identification of therapeutic

targets, moreover understanding of resistance mechanisms and/or metastatic development [31-33].

In recent years, various types of epithelial tumors (80% of all cancers), such as breast [34-38], lung [39-41],

prostate [42, 43], gastric [44, 45], colon [44, 46] and bladder cancer [47-49] were investigated for their

correlation with disease severity. It seems that the enumeration of intact CTCs and/or tumor cell fragments

(circulating tumor DNA, ctDNA) [50] can be also used to accurately predict the survival of patients

with cancer because the number correlates negatively with progression of free survival and overall survival

[32]. The techniques for CTC isolation, enumeration and characterization are being developed worldwide.

At present, only CellSearchTM system has FDA approval for CTC counting in blood of patients with brest

(since 2004), colon (since 2007) and/or prostate cancer (since 2008); in lung cancer it is still under

the research [32, 37]. This system stands as a golden standard for other methods and technologies in rare

cell detection but still analysis of CTCs is not part of the routine tumor staging in clinical practice.

Therefore, the new innovative approaches urgently have to be evaluated for reproducibility, sensitivity

and specificity in order to become applicable to clinical practice [33].

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4 AIMES AND OBJECTIVES

The goal of this work was to developed magnetic immunosorbents (MISs), that consist namely from

magnetic microspheres coated with target-specific mAbs, able to efficiently capture and analyze various

biomarkers of serious diseases, such as Alzheimer’s disease (Amyloid β) or adenocarcinomas (circulating

tumor cells), in microfluidic devices (see Fig. 1). Therefore the main objective was to thoroughly describe

process of MISs preparation and development for microfluidic purposes. The process began with mAb

source searching, magnetic particles provider selection including testing of various procedures of IgG

molecules immobilization onto microparticles and evaluation of the prepared MIS for immunocapture

efficiency. Moreover, physical aspects as aggregation and non-specific adsorption of magnetic beads

before and after Ab attachment were studied outside or inside the microfluidic devices. Subsequently,

various surface modifications (e.g. pegylation or albumin coating) were utilized if necessary. Finally,

behavior of developed MISs and their immunocapture efficiency were observed inside the microfluidic

systems designed either for cell or protein biomarker detection.

Fig. 1. Scheme of the main aims and scopes.

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5 EXPERIMENTAL: RESULTS AND DISCUSSION

5.1 Magnetic immunosorbent for on-chip immunoprecipitation of amyloid β peptides

The research described in three following papers was dedicated to development of microfluidic device able

to capture and analyze Aβ peptides in CSF of AD patients. The microfluidic devices used in this work were

designed and developed at Institute Curie, one of the European Commission (EC) project´s partner:

At the beginning the research was funded under the NeuroTAS project N° 037953 (2007-2010)

and now the research continues under the project Nadine (2010-2015). Our contribution to this project

was mainly to develop MIS able to quantitatively capture Aβ peptides (1-37, 1-38, 1-39, 1-40 and 1-42),

to perform its evaluation and implementation inside the microfluidic system. Such device could enable

Aβ peptides CSF profiling for early diagnostics of patients suspected of AD.

Paper I

B. Jankovicova, Z. Zverinova, R. Mohamadi, M. Slovakova, L. Hernychova, W. Faigle, J.-L. Viovy, Z. Bilkova

Microfluidic system with integrated magnetic carrier for immunoprecipitation of Aβ peptides

This article describes the first steps in preparation and evaluation of MIS able to capture Aβ peptides from

sample. The rabbit polyclonal IgG (Abcam, Cambridge, UK), directed against the N-terminus (1-14 AA) of Aβ

peptide, were immobilized on 1 µm in size superparamagnetic microparticles SiMAG-Hydrazide (Chemicell,

Berlin, D) through their carbohydrate chains and formation of stable hydrazone linkages. The

immunospecificity of immobilized antibodies was tested by immunoprecipitation of synthetic Aβ peptides

1-38, 1-40 and 1-42 (Apronex, Czech Republic) either in phosphate buffer or in complex biological materials

such as serum and/or CSF in batch-wise arrangement using magnetic separator under optimized

conditions. The elution fractions containing the isolated Aβ isoforms were analyzed by Tris-Tricine-SDS-

PAGE (polyacrylamide gel electrophoresis)-urea and/or MALDI-TOF-MS (matrix-assisted laser

desorption/ionization time-of-flight mass spectrometry) as confirmation techniques. The results from Tris-

Tricine-SDS-PAGE-urea confirmed that prepared immunosorbent was able to isolate Aβ peptides (1-38, 1-

40, 1-42 AA) from spiked buffer even from complex biological materials as serum or CSF (Fig. 2). Slightly

preferential capture of Aβ 1-42 was observed over other peptides (Aβ 1-38, 1-40) in CSF and/or serum.

Since batch-wise arrangement was successful the immunosorbent was subsequently tested in microfluidic

device (Fig. 3). Simple straight channel PDMS device with one inlet and outlet and two magnets arranged in

20° angel towards the channel trapping magnetic particles into a plug was applied. The ability of MIS to

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3000 3320 3640 3960 4280 4600

Mass (m /z)

694.1

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity

4700 Reflector Spec #1 MC[BP = 867.0, 11775]

4130.5

693

4151.5

947

4016.5

308

4328.5

615

4215.5

181

4513.6

465

4072.4

006

4111.5

601

3771.4

922

3981.5

574

3067.2

766

3841.6

016

3124.3

052

3012.1

689

3040.0

842

3690.3

296

3213.0

984

3938.4

470

3657.3

015

4259.5

439

3178.1

714

3527.2

764

3254.3

091

3338.1

570

4322.5

171

4402.6

699

4446.4

360

AAββ 11--4400

AAββ 11--4422

AAββ 11--3388

499.0 2399.4 4299.8 6200.2 8100.6 10001.0

Mass (m /z)

1.4E+4

0

10

20

30

40

50

60

70

80

90

100

% Inte

nsity

Voyager Spec #1[BP = 4127.4, 14075]

4127.76

4143.05

618.37

554.042068.44

578.59

596.332132.22 3812.64

794.173998.722212.68

987.52

AAββ 11--3388

I

AAββ 11--4400

AAββ 11--4422

II

isolate Aβ peptides in microfluidic arrangement on magnetic microcolumn was proved in elution fractions

that were analyzed by MALDI-TOF-MS. All spiked isoforms of Aβ were detected, see Fig. 4. Such promising

first results of anti-Aβ MIS in microfluidic device encouraged us to continue in experiments that should

confirmed our findings and also to perform more experiments in microfluidic device and also to improve

the MIS integration and work in the same range of Aβ concentrations as occurs in biological samples.

Fig. 2. Tris-Tricine-SDS-PAGE-urea analysis: Aβ peptides 1-38, 1-40, 1-42 (Apronex, Czech Republic) immunoprecipitated in batch-wise arrangement: I-from buffer, II-from serum, III-from CSF; positions: MM-Mw marker (1.4 – 26.6 kDa), OS-original sample, BF-binding fraction, W1-1

st and W2-2

nd washing fraction, E1-1

st, E2-2

nd, E3-3

rd, E4-4

th and E5-5

th elution fraction; elution

with 0.05% TFA; gel-16.5% T, 3% C, 5M urea in focusing gel, 8M urea in separation gel; silver staining.

Fig. 3. Simple straight channel PDMS device with one inlet/outlet and two magnets arranged in 20° angel towards the channel trapping magnetic particles into a plug.

Fig. 4. MALDI-TOF-MS spectra of elution fractions obtained by immunoprecipitation (IP) using SiMAG-Hydrazide immunosorbent integrated in PDMS microchip: I-IP of Aβ peptide 1-38 (Apronex, Czech Republic), elution with 0.05% TFA; II-IP of Aβ peptide mixture (1-38, 1-40, 1-42) (Apronex, Czech Republic), elution with 0.16% NH4OH.

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Paper II

Z. Svobodova, M. R. Mohamadi, B. Jankovicova, H. Esselmann, R. Verpillot, M. Otto, M. Taverna, J. Wiltfang, J. Viovy, Z. Bilkova.

Development of a magnetic immunosorbent for on-chip preconcentration of amyloid beta isoforms: Representatives of Alzheimer's disease biomarkers

In this paper, development of MIS for on-chip application is thoroughly described. Magnetic beads may

have various functional groups enabling random or site-specific covalent binding of IgG. Eight different

functional groups on silica magnetic particles were compared for their immobilization efficiency. The

immobilized amount of IgG was semi-quantitatively estimated using SDS-PAGE and/or BCA (bicinchoninic

acid) test. The high binding capacity of immobilized IgG was achieved using beads with hydrazide, cyanuric

or thiol functional group. In the end, microbeads with the hydrazide site-group were estimated to be most

suitable for further experiments because they afforded a site-specific, covalent type of IgG binding

(enabling binding through Fc fragment of IgG molecule whereas Fab fragments are freely available

for antigen) in combination with high density and reasonable binding protocol considering time, chemicals

toxicity and labor. Even immunocapture efficiency of monoclonal (mAb) and polyclonal (pAb) antibodies

were studied in batch and then in microfluidic device. Both antibodies were directed against an N-terminus

of Aβ peptide which is common for all 5 isoforms (1-37, 1-38, 1-39, 1-40 and 1-42), and hence all tested

isoforms were presumed to be immunocaptured. The ability of both anti-Aβ MISs (with pAb and/or mAb)

to capture Aβ peptides from real human CSF samples of non-AD patients was proved by immunoblotting

(Fig. 5). Both anti-Aβ MIS had higher affinity for the Aβ 1-42 isoform than for the other Aβ isoforms.

However, the anti-Aβ MIS (mAb) provided a more uniform and efficient capture of all 5 Aβ isoforms from

CSF.

The on-chip immunoprecipitation is called microimmunoprecipitation (µIP, see Fig. 6). The process

of immunocapture was studied on chip and analyzed by dot blot. Using the µIP, we were able

to preconcentrate the Ab peptide from the volume 100 µl to 0.58 µl (approx. 170 fold) and moreover

to obtain peptide in high purity presented in reagent suitable for subsequent separation and/or detection

method. Thus, µIP was found as efficient method appropriate for Aβ peptides preconcentration

in micro-volumes.

Once the Aβ peptides from the samples were immunocaptured, various subsequent analytical methods

for their detection including MALDI-TOF-MS, CE, microchip electrophoresis, and immunoblotting were

employed. For Aβ elution various elution reagents were employed according to subsequent detection

method. For MALDI-TOF-MS analysis, 0.05% TFA was used, while for CE, chip electrophoresis

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and/or immunoblotting 0.16% NH4OH was suitable. For CE, moreover, 100 mM NH4HCO3 or 0.25% formic

acid was applicable.

Fig. 5. Aβ peptides separated by Aβ-SDS-PAGE/immunoblot on polyvinylidene fluoride membrane (according to Wiltfang et al. [2002]). (a) Batchwise immunoprecipitation (IP) of CSF sample on anti-Aβ MIS (pAb) and anti-Aβ MIS (mAb). CSF = CSF sample before IP; IP (pAb) = batchwise IP on anti-Aβ MIS (pAb); IP (mAb) = batchwise IP on anti-Aβ MIS (mAb). (b) μIP of CSF sample on anti-Aβ MIS (mAb). Aβ ST = Aβ standard peptides mixture corresponding to levels in CSF; μIP = micro-immunoprecipitation on anti-Aβ MIS (mAb).

Fig. 6. Scheme of experiment setup in macro- and micro-scale. (a) Prepared magnetic anti-Aβ immunosorbent is inserted in PDMS microchip using syringe pump where they are self-assembled in affinity microcolumn. (b) Sample containing Aβ peptides in mixture with other proteins is injected into the channel with microcolumn and (c) the specific immunocapturing is performed on it. After proper washing (d) the specifically captured peptides are eluted using compatible eluting reagent according to subsequent method (e).

Paper III

M. R. Mohamadi, Z. Svobodova, R. Verpillot, H. Esselmann, J. Wiltfang, M. Otto, M. Taverna, Z. Bilkova, J-L. Viovy

Microchip Electrophoresis Profiling of Abeta Peptides in Cerebrospinal Fluid of Patients with Alzheimer´s Disease

Here, a microchip gel electrophoresis (MCE) method in polydimethylsiloxane (PDMS) chip for rapid

profiling of major Aβ peptides in cerebrospinal fluid was described. The device for MCE has the channel

cross section 100 μm (width) by 50 μm (depth), and the effective separation length was 35 mm (Fig. 7).

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Since the Aβ concentration is critically low, the µIP of the sample (described in paper II) was necessary

in order to enrich the peptides before the MCE and to dispose of fluorescence dye or abundant proteins

co-migrating with the analytes.

New double coating of separation channel by poly(dimethylacrylamide-co-allyl glycidyl ether) (PDMA-AGE)

and methylcellulose−Tween-20 was used with the aim to decrease the electroosmotic flow (EOF) in MCE.

The reason for adding 0.2% methylcellulose and 0.01% Tween-20 was to minimize the remaining

fluorescent signal of fluorescently labeled peptides which adsorbed on the surface of the chip channel.

As suitable medium for separation after multiple optimizations was evaluated 0.2% methylcellulose

in 40 mM borate buffer pH= 9.5.

Fig. 7. Scheme of microchip gel electrophoresis in polydimethylsiloxane chip with double surface treatment. The first layer is non-covalent physical coating by PDMA-AGE, and the second layer is dynamic coating by adding methylcellulose and Tween-20 in the electrophoresis buffers.

Such system was validated for the separation and detection of fluorescently labeled mixture of two Aβ

peptides (1-37 and 1-42) by Fluoprobe-488 NHS ester (Interchim, Montlucon, France). Since between

concentration ratios of the peptides and ratios of both peaks heights and peaks areas (R2 were 0.99

and 0.98 respectively) was linear correlation we started with a separation of five Aβ peptides in mixture

(Aβ 1-37, Aβ 1-38, Aβ 1-39, Aβ 1-40 and Aβ 1-42). Figure 8 shows that all peptides except Aβ 1-38 from

Aβ 1-39 were resolved. Anyway, this should be sufficient to determine the relative abundance of Aβ 1-42

for diagnostic purposes.

Fig. 8. Electropherogram from the separation of five Aβ peptides (1-37 (1), 1-38 (2), 1-39 (3), 1-40 (4), 1-42 (5)). Each peptide was labeled with Fluoprobe-488 and purified by µIP. P* stands for modifications of the main peak, F and F´ indicate the peaks related to fluorescent dye.

- 16 -

Finally, CSF samples from 4 healthy donors and from 3 patients with AD were tested. The results were

more or less similar: We were able to detect at least 2 peaks related with Aβ peptides in CSF (Fig. 9B

in paper III). By adding internal standards and filtration of the fluorescence dyes, we could identify

the peaks for Aβ 1-40 and Aβ 1-42. MALDI-TOF mass analysis was applied as confirmation method.

Fig. 9. (A) Electropherogram of CSF (250 μL) labeled with Fluoprobe-488, and the Aβ peptides in the labeled CSF were isolated using magnetic beads coated with anti-Aβ in the immunocapture chip. An electropherogram of standard Aβ 1-40 labeled with Fluoprobe-488 was superimposed to identify the peak from Aβ 1-40 in CSF. A* corresponds to the peak for aggregated or modified peptides. (B) Electropherogram of Aβ peptides immunocaptured from 250 μL of CSF which were labeled after immunocapture. An electropherogram (dashed electropherogram) of a blank control showing the peaks related with the fluorescent dye was superimposed in this electropherogram. The separation window of Aβ 1-40 and Aβ 1-42 was out of the zone for strong peaks related with the fluorescent dye.

5.2 Magnetic immunosorbent for on-chip immunocapture of circulating tumor cells

The three following papers were dedicated to EC project CAMINEMS N° 228980 (2010–2012) which

we aimed to develop new tool based on microfluidics and nanotechnologies to improve cancer diagnosis,

prognosis and therapy.

Paper IV

Svobodova, Z., B. Jankovicova, D. Horak, Z. Bilkova

Dot-ELISA Affinity Test: An Easy, Low-Cost Method to Estimate Binding Activity of Monoclonal Antibodies

Method for comparison of various monoclonal antibody clones directed against the same epitope was

presented here with the aim to select suitable clone for developed MIS. Conventional dot-ELISA technique

was improved as we enriched it with a chaotropic reagent step. We named the technique Dot-ELISA

Affinity test and its principle is described in the Fig. 10. The method was tested with anti-Aβ mAb

but the final evaluation of was performed with various 8 anti-EpCAM mAb clones.

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Fig. 10. Scheme of dot-ELISA affinity test: Protein/peptide is applied onto PVDF membrane using dot blot unit. After incubation with primary antibody, the Ag-Ab complex is created. Stability of such immune complex is tested using chaotropic reagent (NH4SCN), whereby antibodies with lower affinity are washed away whereas antibodies with higher affinity resist. Such immune complexes are detected by color development using a secondary antibody with HRP (horse radish peroxidase) in the presence of hydrogen peroxide and DAB as chromogen.

Results for 8 clones producing anti-EpCAM IgG were summarized in Tab. I. Final selection of the most

suitable clone within the 8 ones was based on affinity results and the composition of the transporting

buffer since the storage additives as BSA or gelatin were undesirable for MIS preparation. Such high quality

mAb clone for cell immunocapture was subsequently used in our next work. Moreover tests with 3 clones

that have various affinities to EpCAM molecule (high, medium and low) were applied for MIS preparation.

Such MIS was utilized for EpCAM– expressing cells isolation and the obtained results confirmed correlation

with the results from dot-ELISA affinity test. Thence, this method enabled us to predict behavior of the MIS

for cell capture efficiency.

Tab. I. Evaluation of anti-EpCAM mouse monoclonal antibodies from the 8 various clones by dot-ELISA affinity test.

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Paper V

D. Horák, Z. Svobodová, J. Autebert, B. Coudert, Z. Plichta, K. Královec, Z. Bílková, J-L. Viovy

Albumin-coated monodisperse magnetic poly(glycidyl methacrylate) microspheres with immobilized antibodies: Application to the capture of epithelial cancer cells

This paper was dedicated to development of monodisperse (4 µm) macroporous cross-linked poly(glycidyl

methacrylate) (PGMA) microspheres (Fig. 11) coated with human serum albumin (HSA) to be used for MIS

preparation that were intended for immunomagnetic cell sorting in microfluidic device.

For the microsphere fabrication, multistep swelling polymerization was applied. The microsphere

characterization concerning size, magnetic properties, surface functional groups implementation or HSA

surface coating estimation was performed using various methods, such as scanning electron microscope

(SEM), infrared (IR) spectroscopy, atomic adsorption spectrometry, Superconducting Quantum

Interference Device (SQUID). Subsequently the microspheres were applied for EpCAM-expressing cells

immunocapture, after anti-EpCAM immobilization, in batch arrangement and then in microfluidic Ephesia

chip (Fig. 12.). Thus, albumin-coated monodisperse magnetic PGMA microspheres with immobilized

anti-EpCAM proved to be promising for capture of CTCs in a microfluidic device.

Fig. 11. SEM micrograph of magnetic P(GMA-MOEAA-EDMA)-NH2 microspheres with 21.3 wt % of Fe.

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Fig. 12. Ephesia chip: (Left) Scheme of inner space inside the capture chamber of the microfluidic device. The orange columns are formed by MIS in homogenous magnetic field. (Right) Structure of the microfluidic channels inside the PDMS device. Arrows marks inlet and outlet.

Paper VI

Z. Svobodova, J. Kucerova, J. Autebert, D. Horak, L. Bruckova, J‐L. Viovy and Z. Bilkova

Application of an improved magnetic immunosorbent in an Ephesia chip designed for circulating tumor cell capture

Superparamagnetic PGMA microspheres with carboxyl groups (PGMA-COOH), 4 μm in diameter, were used

in this study since they fulfill the basic requirements concerning size, material, surface groups,

and magnetic characteristics. Nevertheless, some non-specific adsorption onto microfluidic PDMS channels

and adherence to target cells was observed. Thus, surface coating using the homobifunctional form

of polyethylene glycol (PEG), hydrazide-PEG-hydrazide, was applied (Fig. 13). Subsequently, the IgG

molecules were immobilized orientedly via carbohydrate groups on PEGylated carrier with hydrazide

functional groups.

The level of non-specific adsorption of PGMA microspheres, before and after the PEG-coating

and/or the anti-EpCAM IgG immobilization, onto the cells and/or PDMS channels inside the microfluidic

device was investigated using optical microscopy. Decrease of non-specific adsorption of PEG-PGMA

microspheres vs.

Fig. 13. Scheme of PEGylation and biofunctionalization of PGMA microspheres with anti-EpCAM. EDC, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; EpCAM, epithelial cell adhesion molecule; PGMA, polyglycidyl methacrylate; Sulfo-NHS, N-hydroxysulfosuccinimide sodium salt.

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PEG-free microspheres was observed. Capture efficiency of such improved anti-EpCAM MIS was studied

initially on EpCAM-expressing cell lines with different EpCAM densities (MCF7, SKBR3, A549 and Raji; see

Fig. 14) in a batchwise arrangement (in Eppendorf tube, on rotator and observed by microscope). These

experiments were compared with batchwise immunocapture with commercial MIS and similar results were

achieved. Finally, evaluation in a microfluidic Ephesia chip was demonstrated using the MCF7 cell line,

a model system for CTC. The microfluidic chamber filled with injected magnetic bead suspension formed

the micro-columns on the magnetic pattern when magnetic field was applied. The excess of the MIS was

washed away. Subsequently, the cells were inserted and those with EpCAM on the surface were

immunocaptured when they were passing the MIS micro-columns inside the microfluidic chamber, see

Fig. 15.

Fig. 14. (A) Graph shows amounts of immunomagnetically captured EpCAM+ cells (MCF7, average of 5 measurements) using various magnetic carriers: PEG-free-PGMA microspheres (without coating), PEG-PGMA (PEG-coated microspheres), HuIgG-PEG-PGMA, and anti-EpCAM-PEG-PGMA microspheres. (B) Graph shows amounts of immunomagnetically captured cells with various EpCAM density: MCF7 (EpCAM+++), SKBR3 (EpCAM++), A549 (EpCAM+) and RAJI (EpCAM−) using PGMA-PEG-anti-EpCAM immunosorbent, average of 3 measurements.

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Fig. 15 A-E. Micrographs of the Ephesia chip’s inner space – the immunocapture zone: (A) before adding MIS, (B) MIS flows inside (10 µl/min), (C) magnetic field is turned on (30 mT), (D) microcolumns are formed and excess of MIS is washed away, small figure shows the micocolumns of anti-EpCAM-PEG-PGMA microspheres and (E) Micrograph of MCF7 cells captured in Ephesia chip using anti-EpCAM-PEG-PGMA carrier.

6 CONCLUSION

In this work MISs suitable for their application in microfluidic devices for capture or enrichment of specific

biomarkers of serious diseases, such as Amyloid β (Aβ, Alzheimer´s disease biomarkers) and/or circulating

tumor cells (CTCs, various adenocarcinomas), were developed. The process of MISs preparation

and development for microfluidic purposes was thoroughly described and their successful application

in microfluidic systems was performed.

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8 LIST OF ORIGINAL PAPERS

Svobodova, Z., Kucerova, J., Autebert, J., Horak, D., Bruckova, L., Viovy, J‐L., Bilkova, Z. Application

of an improved magnetic immunosorbent in an Ephesia chip designed for circulating tumor cell capture.

Electrophoresis. 2014, 35, 2-3, 323-329. (IF 3.21)

Kucerova, J., Svobodova, Z., Knotek, P., Palarcik, J. et al. PEGylation of magnetic poly(glycidyl methacrylate)

microparticles for microfluidic bioassays. Mater. Sci. Eng. C. 2014, 40, 308-315. (IF 2.74)

Svobodova, Z., Jankovicova, B., Horak, D., Bilkova, Z. Dot-ELISA Affinity Test: An Easy, Low-Cost Method

to Estimate Binding Activity of Monoclonal Antibodies. J. Anal. Bioanal. Tech. 2013, 4, 3, 1000168. (IF 3.89)

Horák, D., Svobodová, Z., Autebert, J., Coudert, B., Plichta, Z., Královec, K., Bílková, Z., Viovy, J.-L. Albumin-

coated monodisperse magnetic poly(glycidyl methacrylate) microspheres with immobilized antibodies:

Application to the capture of epithelial cancer cells. J. Biomed. Mater. Res. A. 2013, 101A, 1, 23–32. (IF

2.625)

Svobodova, Z., Mohamadi, M. R., Jankovicova, B., Esselmann, H., Verpillot, R., Otto, M., Taverna, M.,

Wiltfang, J., Viovy, J.-L., Bilkova, Z. Development of a magnetic immunosorbent for on-chip

preconcentration of amyloid beta isoforms: Representatives of Alzheimer's disease biomarkers.

Biomicrofluidics. 2012, 6, 2, 024126. (IF 3.38)

Mohamadi, M. R., Svobodova, Z., Verpillot, R., Esselmann, H., Wiltfang, J., Otto, M., Taverna, M., Bilkova,

Z., Viovy, J-L. Microchip Electrophoresis Profiling of Abeta Peptides in Cerebrospinal Fluid of Patients

with Alzheimer´s Disease. Anal. Chem. 2010, 82, 7611-7617. (IF 5.70)

Skultety, L., Jankovicova, B., Svobodova, Z., Mader, P., Rezacova, P., Dubrovcakova, M., Lakota, J., Bilkova,

Z. Identification of carbonic anhydrase I immunodominant epitopes recognized by specific autoantibodies

which indicate an improved prognosis in patients with malignancy after autologous stem cell

transplantation. J. Prot. Res. 2010, 9, 10, 5171-5179. (IF 5.05)

Jankovicova, B., Zverinova, Z., Mohamadi, M. R., Slovakova, M., Hernychova, L., Faigle, W., Viovy, J.-L.,

Bilkova, Z. Microfluidic system with integrated magnetic carrier for immunoprecipitation of Aβ peptides.

Proceedings: New trends in Alzheimer and Parkinson related disorders: ADPD 2009, Medimond

International Proceedings, 239-244, ISBN 978-88-7587-528-2.

Jankovičová, B., Rösnerová, Š., Slováková, M., Zvěřinová, Z., Hubálek, M., Hernychová, L., Řehulka, P.,

Viovy, J.-L., Bílková, Z. Epitope mapping of allergen ovalbumin using biofunctionalized magnetic beads

- 25 -

packed in microfluidic channels: the first step towards epitope-based vaccines. J. Chromatogr. A. 2008,

1206, 1, 64-71. (IF 4.61)

Zvěřinová, Z., Jankovičová, B., Bílková, Z. Problematika diagnostiky a metod detekce Alzheimerovy choroby.

X. Sborník: Monitorování cizorodých látek v životním prostředí 2008, Mladkov, 295-307, ISBN 978-80-7395-

078-1.

9 LIST OF CONFERENCES

Jankovicova, B., Svobodova, Z., Hromadkova, L., Kupcik, R., Ripova, D., Bilkova, Z. Benefits

of immunomagnetic separation for epitope identification in clinically important protein antigens. ISSS 2014,

20th International Symposium on Separation Sciences, August 30 – September 2, 2014, Prague, CZ. (Poster)

Svobodova, Z., Kucerova, J., Holubová, L., Horak, D., Autebert, J., Brůčková, L., Viovy, J.-L., Bilkova, Z.

Magnetic poly(glycidyl methacrylate) particles with various coatings for on-chip immunocapture of rare

cells for diagnosis of cancer relapse. NanoBioTech-Montreux 2012, The 15th Annual European Conference

on Micro and Nanoscale Technologies for Life Sciences, November 12–14, 2012, Montreux, Switzerland.

(Poster)

Svobodova, Z., Kucerova, J., Horak, D., Autebert, J., Viovy, J.-L., Bilkova, Z. Development of Magnetic

Immunosorbent for Circulating Tumour Cells Immunocapture in Microfluidic Device. Bubble Tech to Bio App

- "LAB-ON-A-CHIP", 2nd Korea - EU Workshop on Microfluidic Technology for Chemical, Biological

and Medical Applications, October 17 – 18, 2011, Saarbrucken, Germany. (Poster)

Horák, D., Svobodová, Z., Autebert, J., Bílková, Z., Viovy, J.-L. Anti-EpCAM-immobilized albumin-coated

monodisperse magnetic poly(glycidylmethacrylate) microspheres for capture of circulating tumor cells.

IPCG Polymer Colloids Conference, June 26 – July 1 2011, University of New Hampshire, Durham, USA.

(Poster)

Svobodova, Z., Horak, D., Kralovec, K., Kucerova, J., Autebert, J., Viovy, J.-L., Bilkova, Z. Circulating Tumour

Cell Capturing by Efficient Magnetic Immunosorbent with Anti-EpCAM High Affinity Antibodies.

19th Biennial Meeting of the International Society for Molecular Recognition: Affinity 2011, June 16 – 19,

2011, Tavira, Portugal. (Poster)

Jankovicova, B., Svobodova, Z., Slovakova, M., Korecka, L., Riviere, C., Klafki, H., Le Potier, I., Taverna, M.,

Bílková, Z. Immunoprecipitation of Peptide Fragments Common to All Tau Protein Isoforms Using

- 26 -

Phospho-Insensitive Magnetic Immunosorbents for Diagnosis of Alzheimer´s Disease. MSB 2011,

26th International Symposium on MicroScale Bioseparations, May 1 – 5, 2011, San Diego, CA, USA, (Poster)

Svobodova, Z., Kucerova, J., Novotna, E., Plichta, Z., Horak, D., Bilkova, Z. First steps in preparation

of magnetic immunosorbent for CTC isolation and characterisation. XXXI. Imunoanalytické dny

a X. Mezinárodní konference CECHTUMA, May 16 – 18, 2010, Mikulov, CZ. (Poster)

Svobodová, Z., Jankovičová, B., Plichta, Z., Horák, D., Bílková, Z. Improvement in Preparation of Magnetic

Immunosorbent for Amyloid beta, Alzheimer´s Disease Marker. The 25th International Symposium

on Microscale Bioseapartions MSB, March 21 – 25, 2010, Prague, CZ. ISBN 978-80-254-6631-5. (Poster)

Bilkova, Z., Svobodova, Z., Esselmann, H., Jankovicova, B., Kucerova, J. Biofunctinalized magnetic

microparticles for efficient biomarkers capturing adapted for magnetic force-based microfluidic device.

The 25th International Symposium on Microscale Bioseapartions MSB, March 21 – 25, 2010, Prague, CZ.

ISBN 978-80-254-6631-5. (Poster)

Slováková, M., Korecká, L., Jankovičová, B., Svobodová, Z., Mohamadi, M. R., Hernychová, L., Viovy, J.-L.,

Bílková, Z. Efficient immunoaffinity carriers for in-chip isolation and pre-concentartion of alzheimer disease

biomarkers. EuroNanoForum 2009, Nanotechnology for sustainable economy, European and International

Forum on Nanotechnology, June 2 – 5, 2009, Prague, CZ. (Poster)

Svobodová, Z., Mohamadi, M R., Jankovičová, B., Esselmann, H., Viovy, J-L., Bílková, Z.

On-chip immunoprecipitation of Amyloid beta peptides for early diagnosis of Alzheimer's disease.

NanoBioTech 2009, 13th Annual European Conference on Micro and Nanoscale Technologies

for the Biosciences, November 16 – 18, 2009, Montreaux, Switzerland. (Poster)

Korecká, L., Svobodová, Z., Jankovičová, B., Mohamadi, M. R., Slováková, M., Bílková, Z., Viovy, J.-L.

Lab-on-chip analytical system with integrated self-assembled magnetic nanoparticles for efficient capturing

of biomolecules or cells from the complex biological material. EuroAnalysis 2009, September 6 – 10, 2009

Innsbruck, Austria. (Poster)

Slováková, M., Jankovičová, B., Zvěřinová, Z., Le Potier, I., Taverna, M., Bílková, Z. Biologické markery

pro včasnou diagnostiku Alzheimerovy choroby. XXX. imunoanalytické dny, April 5 – 7, 2009, Mikulov, CZ.

(Poster)

- 27 -

Jankovicova, B., Zverinova, Z., Mohamadi, M. R., Slovakova, M., Charvatova, A., Faigle, W., Viovy, J.-L.,

Bilkova, Z. First steps in development of magnetically active miniaturized analytical system for diagnosis

of alzheimer disease. ADPD 2009, March 11 – 15, 2009, Prague, CZ. (Poster)

Jankovičová, B., Zvěřinová, Z., Zdražilová, P., Minc, N., Hubálek, M., Slováková, M., Hernychová, L., Viovy,

J.-L., Bílková, Z. Immunomagnetic carriers for microfluidic analytical device to capture and concentrate

of Abeta peptides or Tau proteins, the most valid biomarkers of Alzheimer disease. 7th Interantional

Conference on the Scientific and Clinical Applicatiobns of Magnetic Carriers, May 21 – 24, 2008, Vancouver,

Canada. (Poster)

Krejčová, A., Černohorský, T., Novák, J., Zvěřinová, Z., Němcová, Z., Čápová, L., Pouzar, M. o-TOF ICP MS

Analysis of Small Biological Samples. European Symposium on Atomic Spectrometry ESAS 08,

September 28 – October 1, 2008, Weimar, Germany. (Poster)

10 LIST OF ORAL PRESENTATIONS

Svobodova, Z., Kucerova, J., Bilkova, Z. Immunomagnetic separations in Ephesia chip. 42 Months STEERING

COMMITEE Meeting of Caminems project. December 12 – 13, 2012, Paris, France.

Bilkova, Z., Svobodova, Z. Magnetic beads-based microfluidics systems: The past and the future. CECE 2012,

9th International Interdisciplinary Meeting on Bioanalysis, October 31 – November 2, 2012, Brno, CZ.

Svobodova, Z., Kucerova, J., Bilkova, Z. Developing of surface coated microparticles for immunocapture

of EpCAM cells. 36 Months STEERING COMMITEE Meeting of Caminems project. July 26 – 27, 2012,

Pardubice, CZ.

Svobodova, Z., Kucerova, J., Korecka, L., Holubova, L., Bilkova, Z. Developing of an improved strategy

for newly developed magnetic micro/nanoparticles surface coating with consequent anti-EpCAM IgG

immobilization. 24 Months STEERING COMMITEE Meeting of Caminems project. July 25 – 26, 2011, Oxford,

UK.

Svobodova, Z., Kucerova, J., Korecka, L., Holubova, L., Bilkova, Z. Methods for selection of suitable

anti-EpCAM IgG for efficient immunocapture of CTC by magnetic immunosorbent. 18 Months STEERING

COMMITEE Meeting of Caminems project. January 24 – 25, 2011, Paris, France.

- 28 -

Svobodova, Z., Kucerova, J., Bilkova, Z. Slovakova, M., Korecka, L. Developing of an improved

immobilization strategy for IgG and prepare an efficient immunosorbent with site-specific or random

orientation of immobilized specific antibodies using commercial or newly developed magnetic

micro/nanoparticles. 12 Months STEERING COMMITEE Meeting of Caminems project. July 1 – 2, 2010,

Porto, Portugal.

Zverinova, Z., Mohamadi, M. R., Viovy, J.-L., Bilkova, Z. Immunosorbents for isolation of Aβ peptides.

Steering Committee meeting of NeuroTAS project. June 14 – 15, 2009, Essen, Germany.

Zvěřinová, Z., Jankovičová, B., Bílková, Z. Preparation of magnetic nanoparticles for immunoimmobilization.

Steering Committee meeting of NeuroTAS project. June 24 – 25, 2008, Paris, France.

Zvěřinová, Z., Jankovičová, B., Bílková, Z.: Isolation of Alzheimer's ß-amyloid Peptides by Column

Immunoaffinity Chromatography. YISAC 2008, 15th Young Investigators' Seminar on Analytical Chemistry.

July 2 – 5, 2008, Ljubljana, Slovenia.