Microfluidics and Lab-on-a-Chip for biomedical applicationsChapter 5 : Lab-on-a-Chip & applications.

By Stanislas

CNRSUniversité de Lyon, FRANCEStansan International Group

CONTENT

Chapter 1: Introduction.

Chapter 2 : Basic principles of Microfluidics.

Chapter 3 : Basis of molecular biology and analytical tools.

Chapter 4 : Micromanufacturing.

Chapter 5 : Lab-on-a-Chip & applications.

Chapter 6 : Cancer diagnostics and monitoring.

Labs-on-a-Chip are microfluidic systems

• Fluid Flow is Laminar• No Turbulent Mixing• Mixing is By Diffusion• High Electric Fields in Microchannels are

Possible• Electric field can be used to move the entire

fluid or individual molecules

Microfluidic systemsSystems, where fluids are confined in channels

with dimensions of µm

Background of microfluidics :

Flux électrophorétique Flux électrosmotique

Microfluidics is a microtechnological field dealing with the precise transport of fluids (liquids or gases) in small amounts (e.g. microliters, nanoliters or even picoliters).

A Lab-on-a-Chip (LOC) is a device that integrates one or several laboratory functions on a single chip of only millimeters to a few square centimeters in size.

LOCs deal with the handling of extremely small fluid volumes down to less than pico liters. Lab-on-a-Chip devices are a subset of MEMS devices and often indicated by "Micro Total Analysis Systems" (µTAS) as well.

However, strictly regarded "Lab-on-a-Chip" or "µTAS" indicate generally the scaling of single or multiple lab processes to perform chemical analysis.

The term "Lab-on-a-Chip" was introduced later on when it turned out that µTAS technologies were more widely applicable than only for analysis purposes.

Lab-on-a-Chip vs. Microfluidics

At beginning of the 1990’s, the LOC research started to seriously grow as a few research groups in Europe developed micropumps, flowsensors and the concepts for integrated fluid treatments for analysis systems.

These µTAS concepts demonstrated that integration of pre-treatment steps, usually done at lab-scale, could extend the simple sensor functionality towards a complete laboratory analysis, including e.g. additional cleaning and separation steps.

A big boost in research and commercial interest came in the mid 1990’s, when µTAS technologies turned out to provide interesting tooling for genomics applications, like capillary electrophoresis and DNA microarrays. A big boost in research support also came from the military, especially from DARPA (Defense Advanced Research Projects Agency), for their interest in portable bio/chemical warfare agent detection systems.

Point of care diagnostics.

HISTORY

The Lab-on-a-Chip

concept

has emerged

in 1990 - 1995

Integration on a small substrateof complex analyticalsystems

Labs-on-a-Chip can be made on :

glass

plastics

Silicon

Example of an electrophoresis system on glass(University of Louisville, KY 40292, USA)

The dimensions of micro-fluidic canals are in the range of : 50-100 µm width, 5-20 µm deep, 20-50 mm long (typically).

It includes anti-sedimentation coils, valves, mixers, a sheath flow cell alignment device, and waste storage, and is ~1 mm thick.

They are produced by a simple CO2 laser cutting system (cost ~$25K). Time from CAD file to finished devices < 4 hrs.

Lab-on-a-Chip example :

Credit-card-sized 7-layer Mylar laminate.

This microfluidic system from Micronics is a miniature flow cytometer that counts blood cells and measures Hb concentration.

Lab-on-a-Chip, why ?

In the domain of bio-medical research : big amount of information must be extracted and

treatedprogress depends on the number of analysis by

hour and on cost by analysis

small laboratory which uses Labs-on-a-Chip = = very big laboratory

In the field of medical diagnostic and follow-up : analytical systems sufficiently simples et

automatic that they can be used in physician cabinets or even at patients place

(point of care application).

Advantages of LOCs low fluid volumes consumption (less waste, lower reagents costs and less required sample volumes for diagnostics)

faster analysis and response times due to short diffusion distances, fast heating, high surface to volume ratios, small heat capacities.

better process control because of a faster response of the system (e.g. thermal control for exothermic chemical reactions)

compactness of the systems due to integration of much functionality and small volumes

massive parallelization due to compactness, which allows high-throughput analysis

lower fabrication costs, allowing cost-effective disposable chips, fabricated in mass production

safer platform for chemical, radioactive or biological studies because of integration of functionality, smaller fluid volumes and stored energies

Disadvantages of LOCs

novel technology and therefore not yet fully developed

physical and chemical effects that become more dominant on small-scale sometimes make processes in LOCs behave more complex than in conventional lab equipment (like capillary forces, surface roughness, chemical interactions of construction materials on reaction processes)

detection principles may not always scale down in a positive way

leading to low although the absolute geometric accuracies and precision in microfabrication are high, they are often rather poor in a relative way, compared to precision engineering for instance

Examples of LOC Applications Real-time PCR ;detect bacteria, viruses and cancers.

Immunoassay ; bacteria, viruses, cancers based on antigen-antibody reactions.

Dielectrophoresis : detecting cancer cells and bacteria.

Blood sample preparation ; can crack cells to extract DNA.

Cellular lab-on-a-chip for single-cell analysis.

Lab-on-a-chip technology may soon become an important part of efforts to improve global health, particularly through the development of point-of-care testing devices.

Many researchers believe that LOC technology may be the key to powerful new diagnostic instruments. The goal of these researchers is to create microfluidic chips that will allow healthcare providers to perform diagnostic tests such as immunoassays and nucleic acid assays with no laboratory support.

ApproachSeparation of bio-molecules (proteins and/or DNA fragments) by electro-chromatography carried out in multiple microfluidic channels; this separation is coupled with nucleic acid hybridization reaction (DNA) or immunological reactions (proteins) in liquid phase or using appropriate ligands bound to the separation matrix.

Integration of new nano-structured materials into the microfluidic channels for micro/nano filtering or as new type of matrix for Improved separation techniques of bio-molecules - porous Si and nano-structured polymers.

Optical integration in the Lab-on-a-Chip, which will allow a dramatic reduction of the dimension and price of the control unit.

Integrated optics in the Lab-on-a-Chip for the redistribution of the excitation light and collection of the fluorescence signal - spectacular improvement of the performances, multiple separation columns…

heterogeneous integration of various materials (Silicon, glass, polymers) combining various functions : integrated optics, integrated microelectronics, microfluidics - micro-nano components for advanced biological functions etc..

packaging issues for future bath fabrications of such devices on large heterogeneous substrates involving silicon/glass/plastic wafers.

Operation with a drop of blood

Flux électrophorétique Flux électrosmotique

Capillary Electrophoresis (CE)

Lab-on-a-Chip for electrophoresis

Schematic presentationof the « classical » control unit for Lab-on-a-Chip investigation

Necessity of a microscope with a complex optical system

Application limited to very simple Labs-on-a-Chip – a microscope has only one objective

At present, the control unit is just an opposite to the miniaturization

PROBLEMS

albumine

alfa-1 antitrypsine

alfa-2 macroglobuline haptoglobine

transferrine

complement

gamma-globulines

prealbumine

Serum electrophoresis

Lab-on-a-Chip made on Si substrate take full profit from microelectronics technologies

Technologies :

- well controlled - very precize- bath production - cheep

(From University of Michigan)

Lab-on-a-Chip for electrophoresis

made on Si wafer and associating photodetectors

Micro(nano) machined Si

Porous Si

Two types of Si filters :(in fact oxidized St)

Role : Extraction of plasma from whole blood, extraction of white blood cells & protein filtering and size exclusion electrochromatography

How to prevent clogging of Si-filters ?

1) Increase the capacity of the filter (surface increase)2) Gradient filter - with the least dense layer at the top.3) Funnel-type geometry of the filters with greater cross-section at the top 4) Modify the contact surface of the filter in order to prevent cells/protein sample adsorption and precipitation5) Sample dilution/pH adjusting may help too.

About filtration

Microporous membrane filtration of whole blood utilizing cross flow filtration

Array of eight cantilevers used as deflection sensors for severalchemical solvent vapors. The cantilevers, measuring 500x100x1 µm (length x width x thickness), are each coated with a different polymer in order to define a particular set of responses based on how eachpolymer responds to a given analyte.

Cantilevers used as deflection sensors

Notre originalité par rapport à d'autres recherches dans le domaine des Lab-on-a-Chip :

Introduction des composants optiques intégrés dans les Lab-on-a-Chip pour distribuer la lumière excitatrice vers plusieurs colonnes de séparation et pour la détection de fluorescence :

microsystèmes plus performants, comportant plusieurs colonnes de séparation ;

miniaturisation et diminution de prix du système extérieur (élimination de la microscopie confocale classiquement utilisée).

Utilisation des matériaux nano-structuré (Si-poreux) pour :

nano-filtres ;

nouveaux types de matrice dans les colonnes de séparation

La technologie des Lab-on-a-Chip avec les guides optiques intégrés, que nous avons mise au point, est la suivante :

Substrat : lames de verre de microscope (Corning).

Guides optiques obtenu par échange ionique Na/K

Nettoyage (piranha mixture – perhydrol :H2SO4 ,95%, 1:3).

Dépôt d'une mask métallique en Chrome.

Photolithographie.

Gravure humide des ouvertures dans la couche de Chrome (commercial Merck chromium etchant).

Gravure humide du verre (commercial buffered oxide etchant -BOE, mais qq. astuces y sont nécessaires).

Gravure de la couche de Chrome.

Perçage des trous dans le verre avec des forets diamantés spéciaux.

Couverture des canaux. Deux possibilité ont été explorées :

Uutilisation d'une couche de PDMS (PolyDiMethylSiloxane) -Synthétisé à partir de Sylgard 184 kit de Corning) soumise à une "corona discharge" avant d'être mise en contact avec le verre ;

Utilisation d'une autre lame de verre qui peut être liée à la première par un recuit à haute température et à haute pression. Les conditions exactes dépendent de la planéarité des lames de verre.

Fonctionnalisation de la surface à l'intérieur des canaux microfluidiques, indispensable pour rendre ces canaux hydrophiles.

Masking layerdeposition

SubstrateCleaning

Photoresist deposition(0.5µm)

U.V. Photolithography

Photoresistdevelopment

Masking layeretching

Photoresist removal

Ion exchange

Masking layerwithdrawal

Buried waveguideGuide GeeO

Ion exchange Technology for integrated optics in glass substrates

LOCs devices on glass substrates with monolithically integrated optical components

Soda lime glass substrates were used as a substrate material, in which passive integrated optical components were fabricated by ion exchange technology ;In this technology, the sodium ions from the glass substrate are exchanged, in the desired areas, defined by the photolithography, for either potassium or silver ions. The process is carried out at about 400°C, in the solution of appropriate molten salts. As a result of the local change of the chemical composition, a slight local increase of the refractive index in glass is achieved, which opens a possibility of the fabrication of optical guides and various other integrated optical components.

Near field image of the output aperture of a planar optical guide; the laser beam of 632.8 nm was used for the excitation; radial distribution of the refractive index for the TE polarization.

a -substrate with integrated waveguides

b - Cr layer deposition

c - photolithography and window opening in Cr film

d - microchannel etching

e - microchannel etching

f - mask removal,

g - PDMS cover bonding

The flow chart of the fabrication

Fabrication of optical waveguides in glass substrate by ion exchange technique.

Near field image of the output aperture of the channel waveguide. The laser beam of 632.8 nm

LOCs devices on glass substrates with monolithically integrated optical components

The second step was the fabrication of a network of microfluidic channels.These microfluidic channels were obtained by a photolithography combined with the wet etching technology in a HF:NH4F:HCl:H2O solution.

The chromium layer of 150 nm thick (deposited by magnetron sputtering), covered by positive photoresist AZ 5214 were used during the photolithographyprocess.

The windows in chromium layer were opened by Merck wet chromium etchant.

Various proportions of the components of the HF:NH4F:HCl:H2O solution and process temperatures were tested, in order to minimize the amount of insoluble precipitates, to avoid damages of the masking layers, and tomaximize the etching rate.

Optical guide

Separation channel

Injection channel

Critical optimizations

Form of intersection of microfluidic channelsHigh voltage sequenceElectroosmotic flow and protein adhesionCoupling of optical guides with microfkuidic channelProtocols with real samples and hundreds of other issues…..

Lab-on-a-Chip for electrophoresis with integrated optical detection

Integration of passive optical components into Lab-on-a-Chip

Principal optical components that can be integrated in Lab-on-a-Chip microsystems: a) straight waveguide, b) curved waveguide, c) Y-junction, direct or reverse, d) Mach–Zehnder interferometer, e) directional coupler and f) X-crossing.

Microfluidic channels with a networkof integrated opical guides

Light divided into two guides thanks to an Y junction excite fluorescence in 2 areas of an microfluidic channel

Fluorescence excited in a microfluidic channel by an integrqted optical guideLight injected into an integrates

guide

Few illustrations

Experimental set-up

Monture pour le photodétecteur ou la caméra numérique. Cette configuration est utilisée pour une collection de la fluorescence à la verticale du substrat.

Microscope à épifluorescence intégrant une lampe à vapeur de

mercure pour une excitation verticale de la fluorescence.

Laser Yag doublé de 50 mW à 532 nm. Ce laser est couplé à une fibre monomode de 3,5 µm de diamètre de cœur au travers d’un collimateur.

Tables de déplacement micrométriques pour le couplage entre les fibres d’excitation et de collection avec les guides optiques.

Experimental set-up

Test of the sensitivty

The light injected by the integrated optical guides induces a very bright fluorescence signal in the microfluidic channel filled-up with tagged bio-molecules.

Figure below demonstrates a possibility of the redistribution of the excitation light in the Lab-on-a-Chip using simple Y junctions.

Redistribution of the excitation light in the Lab-on-a-Chip

This figure shows the sensibility and the linearity of the fluorescence detection in the case of a simple Rhodamine solution excited at 532 nm,

Réalisation et caractérisation d’une jonction Ysur un Lab-on-a-Chip

Intersection entre un canal microfluidique verticale et 2 guides issus d’une jonction Y pour distribuer la lumière laser.

Détection multi-point de la streptavidine (10 µmol/L) après distribution de la lumière excitatrice par la jonction Y.

Description du comportement microfluidique à l’intersection des canaux

On définit électriquement la quantité de biomolécules à séparer et à injecter dans le canal de séparation.

•: on focalise le fluide pour injecter un volume défini.

•: on diminue les séquences électriques afin d’avoir un « plug » plus large.

•: On amorce ensuite le « plug » définit à l ’entrée du canal de séparation.

•: On effectue la séparation.

Injection d’un volume défini de biomolécules dans le canal de séparation.

Réservoir source dans lequel on dépose

l’échantillon biologique

Agrandissement de l’intersection des 2

canaux microfluidiques et déplacement de biomolécules.

Canal dans lequel la

séparation des bio-moécules

s’effectue

Canal d’injectionde l’échantillon et des produits nécessaires à

la séparation

Electrophorèse de zone

Séparation de CY3 et de la streptavidine sous un champ électrique de 310 V/cm dans un microsystème verre/PDMS. Tampon de migration : Borax 1 mM, pH=9.2

Séparation d’un mélange de protéines par CGEIl est impossible de séparer par CZ des protéines possédant des rapports

charges/tailles similaires. Par contre, il est possible de séparer des molécules uniquement en fonction de leurs tailles par CGE.

Nous avons choisi de travailler avec une matrice de haute viscosité fournit par Beckman-Coulter (ecap SDS 14-200 gel). Il s’agit d’une formulation particulière d’oxyde de polyéthylène optimisée pour séparer des protéines présentes dans la gamme 14-200 kDa.

Séparation de la β-lactoglobuline A et de l’anhydrase carbonique dans un laboratoire sur puce par électrophorèse capillaire en gel. Les protéines migrent au travers d’une matrice (ecap SDS 14-200 gel) à 300 V/cm.

Motion of DNA in a Channel

Mesures de la mobilité du flux électroosmotique

1) méthode ampérométrique

Etude ampérométrique du flux électroosmotique. La courbe rouge correspond au remplacement d’une solution concentrée présente dans le canal par une solution plus diluée. La courbe noire représente une substitution d’une solution diluée par une solution plus concentrée.

2) par fluorescence indirecte

Type de substrat Mobilité électroosmotique (µeof)(* 10-4 cm2.V-1.s-1)

PDMS/verre (Ampèrométrie LEOM)

4.73 ± 0.05

PDMS/verre (Fluorescence Indirecte LEOM)

4.70 ± 0.1

Glass/glass314 5.45

PDMS/PDMS oxydé314, 322  4.89 < µeof < 5.7

Verre/PDMS (canal en PDMS) 301 , 309, 324 

3.7 < µeof < 4.0

PDMS/PDMS natif309 3.28

Comparaison des mobilités électroosmotiques mesurées avec les

données de la littérature

1ère séparation

2ème séparation

3ème séparation

filtrage

3ère séparation

4ère séparation

Introduction du liquide

Schéma de configuration de plusieurs colonnes de séparation et filtrage arrangées en série et en parallèle. Toutes ces séparations

peuvent être contrôlées par un réseau de guides optiques. Ces guides optiques ainsi que le circuit microfluidique d’évacuation du liquide ne

sont pas représentés.

Lab-on-a-Chip multi-canaux

Photo d’un Lab-on-a-Chip possédant 2 canaux de séparation pour l’électrophorèse sur puce.Le canal de séparation le plus court fait 5 cm de long tandis que l’autre a une longueur de 7 cm.

entrée/sortie optique

lumière divisée pour alimenter deux colonnes

excitation de fluorescence

colonne de séparation

collection et retour de la fluorescence par le même guide

nano-filtres entrée du liquide biologique

A l'intérieur de la puce, les signaux sont acheminés par un réseau de guides optiques intégrés couplés entre eux par des coupleurs directionnels rendus sélectifs en longueur d'onde par des réseaux de Bragg incorporés.

Example

Séparation de la β-lactoglobuline A et de l’anhydrase carbonique II en électrophorèse capillaire de zone. La séparation est obtenue à 278 V/cm sur un capillaire de silice non traité. Les pics numérotés 1 et 2 correspondent au CY3, le pic 3 à l’anhydrase carbonique II, le pic 4 à la β-lactoglobuline A et le pic 5 à un artefact électrique. Système P/ACE 2100 de chez Beckman-Coulter.

Séparation de l’anhydrase carbonique et de la β-lactoglobuline A dans un laboratoire sur puce verre/PDMS. La séparation s’opère à 320 V/cm dans un tampon borax à 10 mM.Les pics 1 et 2 sont attribués aux formes natives et hydrolysées du Cy3. Le pic 3 correspond à l’anhydrase carbonique et le pic 4 à la β-lactoglobuline A.

Comparison between electrophoresis separation in a Lab-on-a-Chip and in ahigh class standard commercial instrument.

Electrophérogramme de l’antigène RgpB-Cy3. L’électrolyte de migration est un tampon borax de 10 mM,

Electrophérogramme de l’anticorps monoclonal dérivé au Cy3. L’électrolyte de migration est un tampon borax de 10 mM, et le champ électrique appliqué est de 250 V/cm.

Séparation d’un mélange anticorps et antigène dans un Lab-on-a-Chip. L’électrolyte de migration est un tampon borax à 10 mM. Le pic 1 correspond à l’anticorps monoclonal. Le pic 2 est identifié comme un artefact expérimental. Le pic 3 est associé à l’antigène RgpB, tandis que le pic 4 représente le complexe anticorps-antigène formé.

Electrophorèse couplée à une réactions immunologiques en phase liquide.

Photoepoxies such as SU8 are new MEMS (Micro-Electro-Mechanical Systems) Materials with outstanting properties :

-Layer thickness : 1μm to 500μm in one single spin (depending on the viscosity ofthe material).

-High aspect ratios : up to 25 lines and trenches.

-Simple processing.

-UV- Exposure (poor man’s LIGA).

- Multilayer Stuctures.

Stationary phase for LOC chromatography

(b)SEM image of individual pillars with indentations caused by the Bosch1dry etching process.

(a)SEM image of the micromachined pillar array used by Eghbali et al. for on-chip reversed-phase LC.

Stationary phase for LOC chromatography

Stationary phase for LOC chromatography

or a mixture of these two types

The term « Lab-on-a-Chip »concerns devices with the presence of a microfluidic system

EXAMPLE OF REALIZATION

BioChips inside a Microfluidic Channel with Integrated Waveguide

Machine of microprinting developed in SK group : - patent - publication

EXEMPLE OF FABRICATION

Integrated PCR

and

detection microsystem

Integrated PCR and detection microsystem

Lab-on-a-Chip evolution

DEP separation chamber

Blood sample inlet

PCR chamber

Lysis regionWaste outlet

silicon

glass

Detection region

DEP Results

sep ara tio n

lysis

inle ts< -flo w sam p le->

o utle ts< -P C R w aste ->

Successful separation and electrical lysis of cells

DEP2: Electrical Lysis Results

DEP collection of cells

from diluted whole blood

in hypoosmolar buffer

Successful electrical lysis

of collected wbc

outblood

buffer

From Concept to Lab-on-a-Chip

Company Confidential

Microfluidics Division

Lab-on-Chip for Molecular Diagnostics

The long-term Vision

• Long term: develop all the IP needed for a full sample to analysis solution (challenge is speed of adoption in a very risk averse market)

• Short term: ST can offer the only solution today integrating amplification and detection on a single chip (there is an existing market)

PCR

Notre objectif à terme: Création d'analyseurs Lab-on-a-Chip, simples et autonomes, pour le diagnostic précoce et le suivi des cancers du poumon dans le cabinet du médecin.

L'analyseur complet sera composé d'un capteur jetable au format d'une carte de crédit, comportant le Lab-on-a-Chip et sa connectique, et d'une unité de contrôle de la dimension approximative d'un livre.

portatifsimple d’utilisation

autonome

capteur jetable

rapide

D’un prix abordable (~ 4000€)

Companies creating Lab-on-a-Chip

Examples of commercial Lab-on-a-Chip

THANK YOU FOR YOUR ATTENTION                                                                                                                                     

Any question ?