Sk microfluidics and lab on-a-chip-ch5


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Sk microfluidics and lab on-a-chip-ch5

  1. 1. Microfluidics and Lab-on-a-Chip for biomedical applications Chapter 5 : Lab-on-a-Chip & applications. By Stanislas CNRS Université de Lyon, FRANCE Stansan International Group
  2. 2. CONTEN T 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.
  3. 3. Labs-on-a-Chip are microfluidic systems Microfluidic systems Systems, where fluids are confined in channels with dimensions of µm Background of microfluidics : • • • • 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
  4. 4. Flux électrophorétique Flux électrosmotique
  5. 5. Lab-on-a-Chip vs. Microfluidics 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.
  6. 6. HISTOR At beginning of the Y 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 pretreatment 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.
  7. 7. The Lab-on-a-Chip concept has emerged in 1990 - 1995 Integration on a small substrate of complex analytical systems
  8. 8. 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).
  9. 9. Lab-on-aChip example : Credit-cardsized 7-layer Mylar laminate. This microfluidic system from Micronics is a miniature flow cytometer that counts blood cells and measures Hb concentration. 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.
  10. 10. Lab-on-a-Chip, why ? In the domain of bio-medical research : big amount of information must be extracted and treated progress 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).
  11. 11. 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 highthroughput 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
  12. 12. 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
  13. 13. 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.
  14. 14. Approach Separation of bio-molecules (proteins and/or DNA fragments) by electrochromatography 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
  15. 15. Capillary Electrophoresis (CE) Flux électrophorétique Flux électrosmotique
  16. 16. Lab-on-a-Chip for electrophoresis
  17. 17. Schematic presentation of the « classical » control unit for Lab-on-a-Chip investigation
  18. 18. PROBLEMS Necessity of a microscope with a complex optical system Application limited to very simple Labs-ona-Chip – a microscope has only one objective At present, the control unit is just an opposite to the miniaturization
  19. 19. Serum electrophoresis alfa-1 antitrypsine albumine alfa-2 macroglobuline haptoglobine transferrine prealbumine complement gamma-globulines
  20. 20. Lab-on-a-Chip made on Si substrate take full profit from microelectronics technologies Technologies : - well controlled very precize bath production cheep
  21. 21. Lab-on-a-Chip for electrophoresis made on Si wafer and associating photodetectors (From University of Michigan)
  22. 22. About filtration   Two types of Si filters : (in fact oxidized St) Micro(nano) machined Si 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 crosssection at the top 4) Modify the contact surface of the filter in order to prevent cells/protein sample adsorption and precipitation 5) Sample dilution/pH adjusting may help too. Porous Si
  23. 23. Microporous membrane filtration of whole blood utilizing cross flow filtration
  24. 24. Cantilevers used as deflection sensors Array of eight cantilevers used as deflection sensors for several chemical 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 each polymer responds to a given analyte.
  25. 25. Notre originalité par rapport à d'autres recherches dans le domaine des Lab-on-aChip : 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
  26. 26. 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.
  27. 27. Ion exchange Technology for integrated optics in glass substrates Substrate Cleaning Masking layer etching Masking layer deposition Photoresist removal Photoresist deposition (0.5µm) Ion exchange U.V. Photolithography Masking layer withdrawal Photoresist development Buried waveguide Guide GeeO
  28. 28. 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.
  29. 29. The flow chart of the fabrication a -substrate with integrated waveguides b - Cr layer deposition Fabrication of optical waveguides in glass substrate by ion exchange technique. c - photolithography and window opening in Cr film d - microchannel etching e - microchannel etching Near field image of the output aperture of the channel waveguide. The laser beam of 632.8 nm f - mask removal, g - PDMS cover bonding
  30. 30. 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 photolithography process. 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 to maximize the etching rate.
  31. 31. Lab-on-a-Chip for electrophoresis with integrated optical detection Optical guide Separation channel Injection channel Critical optimizations Form of intersection of microfluidic channels High voltage sequence Electroosmotic flow and protein adhesion Coupling of optical guides with microfkuidic channel Protocols with real samples and hundreds of other issues…..
  32. 32. Integration of passive optical components into Lab-on-a-Chip Principal optical components that can be integrated in Lab-on-aChip microsystems: a) straight waveguide, b) curved waveguide, c) Y-junction, direct or reverse, d) Mach–Zehnder interferometer, e) directional coupler and f) X-crossing.
  33. 33. Few illustrations Light injected into an integrates guide Microfluidic channels with a network of integrated opical guides Fluorescence excited in a microfluidic channel by an integrqted optical guide Light divided into two guides thanks to an Y junction excite fluorescence in 2 areas of an microfluidic channel
  34. 34. Experimental set-up
  35. 35. Monture pour le photodétecteur ou la caméra numérique. Cette configuration est utilisée pour une collection de la fluorescence à la verticale du substrat. Experimental set-up 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.
  36. 36. Test of the sensitivty
  37. 37. Redistribution of the excitation light in the Lab-on-a-Chip The light injected by the integrated optical guides induces a very bright fluorescence signal in the microfluidic channel filled-up with tagged biomolecules. Figure below demonstrates a possibility of the redistribution of the excitation light in the Lab-on-a-Chip using simple Y junctions. This figure shows the sensibility and the linearity of the fluorescence detection in the case of a simple Rhodamine solution excited at 532 nm,
  38. 38. Réalisation et caractérisation d’une jonction Y sur 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.
  39. 39. 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.
  40. 40. Injection d’un volume défini de biomolécules dans le canal de séparation. Canal d’injection de l’échantillon et des produits nécessaires à la séparation Agrandissement de l’intersection des 2 canaux microfluidiques et déplacement de biomolécules. Réservoir source dans lequel on dépose l’échantillon biologique Canal dans lequel la séparation des bio-moécules s’effectue
  41. 41. 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
  42. 42. Séparation d’un mélange de protéines Il par CGE de séparer par CZ des protéines possédant des rapports est impossible 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.
  43. 43. Motion of DNA in a Channel
  44. 44. 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
  45. 45. Comparaison des mobilités électroosmotiques mesurées avec les données de la littérature 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
  46. 46. 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. Introduction du liquide 1ère séparation filtrage 3ère séparation 2ème séparation 4ère séparation 3ème séparation 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.
  47. 47. Example 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. colonne de séparation excitation de fluorescence collection et retour de la fluorescence par le même guide entrée/sortie optique lumière divisée pour alimenter deux colonnes
  48. 48. Comparison between electrophoresis separation in a Lab-on-a-Chip and in ahigh class standard commercial instrument. 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 βlactoglobuline A.
  49. 49. Electrophorèse couplée à une réactions immunologiques en phase liquide. 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. Electrophérogramme de l’antigène RgpB-Cy3. L’électrolyte de migration est un tampon borax de 10 mM,
  50. 50. Photoepoxies such as SU8 are new MEMS (Micro-ElectroMechanical Systems) Materials with outstanting properties : -Layer thickness : 1μm to 500μm in one single spin (depending on the viscosity of the 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
  51. 51. 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 reversedphase LC.
  52. 52. Stationary phase for LOC chromatography
  53. 53. The term « Lab-on-a-Chip » concerns devices with the presence of a microfluidic system or a mixture of these two types
  55. 55. BioChips inside a Microfluidic Channel with Integrated Waveguide
  56. 56. EXEMPLE OF FABRICATION Machine of microprinting developed in SK group : - patent - publication
  57. 57. Integrated PCR and detection microsystem
  58. 58. Integrated PCR and detection microsystem
  59. 59. Lab-on-a-Chip evolution Detection region Waste outlet Blood sample inlet DEP separation chamber PCR chamber silicon glas s Lysis region
  60. 60. DEP Results in le ts < -flo w sa m p le -> se p a ra tio n ly sis o u tle ts < -P C R w a ste -> Successful separation and electrical lysis of cells
  61. 61. DEP2: Electrical Lysis Results DEP collection of cells from diluted whole blood in hypoosmolar buffer Successful electrical lysis of collected wbc r ffe bu blood out
  62. 62. From Concept to Lab-on-a-Chip
  63. 63. Lab-on-Chip for Molecular Dia gnostics T he long-ter m V ision Microfluidics Division PCR • 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) Company Confidential
  64. 64. 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. portatif rapide capteur jetable simple d’utilisation autonome D’un prix abordable (~ 4000€) 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.
  65. 65. Companies creating Lab-on-a-Chip
  66. 66. Examples of commercial Lab-on-a-Chip
  67. 67. THANK YOU FOR YOUR ATTENTION Any question ?