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lab on a chip

3014362bc816c0e34f9bb270d226e31c?s=47 andreas manz
August 08, 2001

lab on a chip

... general lecture. Note: it was used interactively, and never shown as a whole.

3014362bc816c0e34f9bb270d226e31c?s=128

andreas manz

August 08, 2001
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  1. . …

  2. What’s up in the Manz lab Andreas Manz Imperial College,

    London UK
  3. None
  4. contents Why small? why small.ppt Chip technology microfabrication.ppt [Electrophoresis] electrophoresis.ppt

    Chemical reactions chemical reactions.ppt [PCR] pcr.ppt Bioreactor antibiotics.ppt Detection methods detection methods.ppt Analog computing analog computing.ppt
  5. None
  6. why minitiaturize volume of 1µL 1nL 1pL (1mm)3 (100µm)3 (10µm)3

    600,000,000 600,000 600 25 / cm2 2500 / cm2 250 ,000/ cm2 17 min 10s 100ms 1.5 /min / cm2 250 /s / cm2 2,500,000 /s / cm2 # molecules (1nM solution) # volumes In array diffusion time # reactions (diffusion controlled) is a cube of
  7. 10 fold miniaturization 100 x faster reactions / bioassays 100

    x faster separation 1000 x smaller volume 10 x lower reagent consumption
  8. human perception < 1 cm is small > 10 m

    is big < 100 ms is immediate > 1 min is slow factor 100: 100 ms to 1 ms: not impressive 17 h to 10 min: makes a difference 10 min to 6 s: very impressive
  9. -TAS electronics recorder pre-treatment sensor electronics recorder sampling electronics recorder

    carrier reagent mobile phases hydraulic control waste ideal sensor total analytical system -TAS
  10. None
  11. established semiconductor fabrication techniques light-source mask (from DWL) photo-resist on

    substrate developing, etching 3-dimensional structure bonding sealed microfluidic device microfabrication technique
  12. Capillary electrophoresis on chip Jed Harrison, Carlo Effenhauser, Norbert Burggraf,

    Luc Bousse
  13. blinkermuh

  14. 13-Nov-13 blinkermuh fluorescence [arb. units] time [s] 0 40 80

    120 160 1 2 3 4 5 6 cycle # 7 8 t 7 s synchr. fluorescence [arb. units] time [s] 0 40 80 120 160 1 2 3 4 5 6 cycle # 7 8 t 7 s synchr. fluorescence [arb. units] time [s] 0 40 80 120 160 1 2 3 4 5 6 cycle # 7 8 t 7 s synchr.
  15. CE on chip • Scaling laws electrophoresis scaling.ppt • [Short]

    electrophoresis video.ppt • History electrophoresis carlo.ppt • Serial to parallel electrophoresis caliper 1.ppt • Parallel separations electrophoresis caliper 2.ppt
  16. separation efficiency • Number of theoretical plates is proportional to

    voltage drop U N 
  17. heating problem •Power generated per unit length should be a

    constant const L I U  
  18. separation efficiency •Number of theoretical plates is proportional to length

    / diameter of capillary d L N 
  19. separation time •Analysis time is proportional to length * diameter

    of capillary d L t  
  20. None
  21. None
  22. None
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  24. None
  25. None
  26. None
  27. SERIAL 1 2 3 4 1 2 3 4 CONVERTER

    ? PARALEL
  28. SERIAL 2 3 4 1 1 CONVERTER PARALEL

  29. None
  30. 80 SEPARATION CHANNELS INJECTION CHANNEL

  31. None
  32. RESULTS Serial to parallel converter : 9 plugs refilled in

    10 seconds, that means 1 sample plug per second 54 samples per minute 78,000 samples per day
  33. None
  34. `

  35. None
  36. None
  37. double stranded DNA separation ele caliper 2 extended.ppt

  38. reaction with intercalating dye

  39. None
  40. x x x x x x x x x SYBR

    green x x x x x x x x x x x x x double stranded DNA SYBR green x x x x x x x x x x x x x x x x x x x x x x x x SYBR green complex [fluorescing] x x x x 1) 2) 3)
  41. x x x x x x x x x x

    x x x x x x x double stranded DNA SYBR green complex [fluorescing] concentration length of plug DNA is slowing down at moving front of SYBR green SYBR green is slowing down at moving front of DNA fluorescence length of plug
  42. blinkermuh

  43. 13-Nov-13 Agilent 2100 Bioanalyzer

  44. conclusions • High speed separations • Good quality separations •

    Very good fluid control • Small volumes • Commercial products •  biggest success, so far really?
  45. Concept chemical reactions concept.ppt the chip chemical reactions mix.ppt Bioassay

    chemical reactions bio.ppt Electrophoretic reaction chemical reactions electrophoretic Synthesis chemical reactions synth.ppt
  46. drug discovery • first step to find new active molecules

    • composed of – synthesis of new compound – isolation, characterisation – bioassay • a significant effort in pharmaceutical industry, involving new technologies
  47. COMBINATORIAL CHEMISTRY QUALITY CONTROL BIOASSAYS 100 educts A 100 educts

    B 10,000 products AB 10,000 assays 10,000 assays ??? 10,000 products AB
  48. CHEMICAL MICROPROCESSOR SYNTAS  educt A educt B is this

    a hit? yes/no specific reaction specific bioassay
  49. chemical reaction batch Time continuous flow Length

  50. A B C A B , A , B A

    B A B C , A B , C A B C A B C fluorescence detection bioassay synthesis step 1 synthesis step 2 separation separation continuous flow
  51. A A A A A A A B B B

    B B A B , A , B A B A +B A +B solvent solvent solvent solvent solvent R E A C TO R S S E P A R A T O R S S T O R A G E
  52. pressure induced flow local minimum for bandbroadening defines optimum flow

    rate How about a sequence of injected samples?
  53. electroosmotic flow minimum for bandbroadening at maximum speed

  54. pressure electroosmosis

  55. device for parallel lamination Fiona Bessoth

  56. chemical reaction • In the most simple case, a molecule

    A meets a molecule B and reacts to give AB • many reactions are diffusion controlled • reaction time of hours in conventional lab • reaction time of 30 min in micro well plate
  57. Figure 4. Separation of several amino acids using post-column derivatization

    for detection. D.J.Harrison, K.Fluri,N.Chiem, T.Tang,Z.Fan University of Alberta, Edmonton,Canada Transducers’95, Proc., vol.1, pp752-755 (1995)
  58. Y -shaped junction: 1:1 fluorescein-to-rhodamine B flowrate ratio (0.5 :

    0.5 mL/min)
  59. None
  60. Mixing – Diffusion times D d t 2 2 

    Before laminar mixing D n d t 2 2 2  After laminar mixing n = number of branches, d = tubing diameter, D= diffusion coefficient
  61. Distributive Micromixing Device: Schematic F. G. Bessoth, A. J. de

    Mello and A. Manz, Anal. Commun., 1999, 36, 213-215
  62. 16 channels 256x faster !

  63. Distributive Micromixing Chip

  64. F. G. Bessoth, A. J. de Mello and A. Manz,

    Anal. Commun., 1999, 36, 213-215 Chip manifold volume 600 nL Observation channel 530 nL Distributive Micromixing Device
  65. fused silica capillary glue glass Si glass

  66. 6 ms 14 ms 38 ms 94 ms 54 ms

    78 ms 0 ms
  67. Fluorescein and Rhodamine B; Flow rate = 50 L min-1;

    Time from point of confluence to beginning of long channel = ca. 9 ms laminar flow visualisation
  68. None
  69. fast fluorescence quenching 0 ms 6 ms

  70. Mixing * + further downstream reaction incomplete reaction incomplete reaction

    complete reaction complete reaction complete
  71. horseradish peroxidase assay Fiona Bessoth

  72. horseradish peroxidase assay 0 1 2 3 4 5 6

    7 8 0 0.02 0.04 0.06 0.08 0.1 0.12 concentration HRP [g/mL] chemiluminescence signal [V] assay time 30 minutes  400 ms “incubation time” 400 ms
  73. conclusions • interesting • very preliminary • surface is the

    problem
  74. electrophoretic mixing Luc Bousse, Andreas Manz Caliper Technologies Inc, Mountain

    View, USA
  75. synthesis of small organic molecules Michael Mitchell, Valerie Spikmans

  76. Fast Reaction

  77. NO2 NO2 CHO NO2 P(Ph)3 NO2 purple Br- 2-nitrobenzy ltriphenyl-

    phosphonium brom ide p-nitrobenza ldehyde colourless NaOMe NO2 Me OH colourless P(Ph)3 + + Wittig reaction N + O O Cl Cl Cl Cl O O Cl Cl Cl N Enamine Chloranil blue 2,3,5-trichlor-6-(2-piperidin -1-yl)-[1,4]- benzoquinone Synthesis of a substituted aminovinyl-p-quinone SYNTHESIS
  78. N+ H H R1 R2 Cl- H H O MeOH

    N+ Cl- H2 O N R3 R4 R2 R1 C R1 N R2 N R4 R3 H2 O R1 N R2 N R4 R3 O R1/R2 = -CH2 (CH2 )3 CH2 - Piperidine hydrochloride + + Piperidinium cation + R3/R4 = -CH2 (CH2 )4 CH2 - Cyclohexyl isocyanide Nitrilium intermediate -Dialkylacetamide Formaldehyde N-Cyclohexyl-2-piperidin-1-yl-acetamide (1) (2) (3) (4) (5) (6) Multicomponent Chemistries: The Ugi Reaction 0oC
  79. None
  80. Inlet capillaries Syringes Rheodyne injection valve Injection loop outlet capillary

    Micromixer chip / PTFE interface
  81. Simultaneous Observation of Reactants, Intermediates, Products and By-products 20 Lmin-1

    50 nL injection loop Room temperature
  82. conclusions • Some syntheses do work! • What is the

    limitation? • Very fast mixing • Higher temperatures than usual • Better selectivity • Complicated device
  83. Continuous-flow polymerase chain reactor Martin Kopp, Marco Luechinger

  84. polymerase chain reaction • = method to amplify the amount

    of a specific DNA sequence in a sample • each cycle doubles the amount of DNA • most commonly used procedure in biology • commercial instruments would do 20 cycles in 50 minutes
  85. Figure 1. Cross section of micro-PCR test device. Figure 5.

    Gel electrophoretic photograph indicating that similar results were obtained with a 50ul microfabricated test device (mid-right three bands) as in much lager commercial instrument (mid-left two bands). The target sequence amplified was HIV.
  86. T.M.Woudenberg, E.S.Winn-Deen, M.Albin [Applied Biosystems, Foster City, CA] High-density PCR

    and beyond, u-TAS 96, p 55-59 (1996) First data from polycarbonate “chip”
  87. None
  88. 95oC Melting 77oC Extension 60oC Annealing PCR - Continuous Flow

    Chip 20 identical cycles Time ratio of 4:4:9 (melting:annealing:extension) Theoretical amplification factor of 220
  89. PCR - Continuous Flow Chip

  90. Cold start PCR Tricine (pH 8.4) 10 mM Tween 20

    0.01% (w/v) KCl 50 mM NTP 20 M each MgCl2 1.5 mM PVP 1.4 M Primer 1 M Taq polymerase 0.25 U/L Template ca. 108 copies
  91. PCR - Continuous Flow Chip

  92. None
  93. Efficiency of amplification On the chip: Template 108 copies Product

    5.1011 copies Factor 5,000 = 1.5320 commercial thermocycler: Factor 7,030 = 1.5620
  94. None
  95. Advantages of Continuous Flow PCR Chip variable volumes – 1nL

    to1mL low carryover high speed 12 to 60s per cycles low band-broadening High speed chemical amplifier
  96. Bioreactor on a chip Paul Monaghan

  97. Bioreactor on a chip • Permits the growth of a

    bacterium on-chip • Goal: antibiotics screening • Uses PDMS device for gas permeability
  98. Monitoring the growth of bacterial cultures • The sampling of

    a growing culture at various time intervals (by viable counts,dry weight of the biomass or optical density measurements) • Real-time monitoring • miniaturized systems • reduction of biological waste Conventional bulk growth techniques Microbiology microfluidic
  99. Fused silica capillaries Graphite ferrules Temp. sensor Heating Block PDMS

    device Fluidic connections a b PDMS Device and Set-up
  100. Photomicrographs of Cell Growth on-Chip 0hr 1hr 2hr 3hr 4hr

    5hr
  101. Fluorescence measurements - Plots of PMT signal versus sampling time

    0 1 2 3 4 5 0 10 20 30 40 50 60 70 80 90 100 sampling t (s) PMT sig. (V) 0hr 1hr 2hr a. Over the initial two hours, the data suggests that there is not a great deal of increase in the biomass. 0 1 2 3 4 5 0 10 20 30 40 50 60 70 80 90 100 sampling t (s) PMT sig. (V) 3hr 4hr b. After 3hr that there is any appreciable increase in the signal as indicated by figure b. At 4hrs, the signal has significantly increased.
  102. 0.10 1.00 10.00 0.0 1.0 2.0 3.0 4.0 5.0 6.0

    Time (hr) Average PMT signal (V) 0.5mg/L 1mg/L 2mg/L Control antibiotics testing, chloramphenicol
  103. conclusions • Simple design • Gas permeability • Optical interrogation

    • A little bit faster than conventional
  104. plasma emission C:\WINDOWS\Desktop\talk 03-01\plasma.ppt electro-chemiluminescence C:\WINDOWS\Desktop\talk 03-01\ecl.ppt potentiometry Fourier transform

    methods C:\WINDOWS\Desktop\talk 03-01\scoft.ppt
  105. Plasma emission detector Jan Eijkel, Herbert Stoeri, Omar Naji, Fiona

    Bessoth, Gareth Jenkins, Darwin Reyes
  106. State-of-the-art • inductively coupled plasma • 1 kW power consumption

    • gas temperature 6,000K • safety radius 1 m • very low detection limits for metals • liquid nebulizing interfaces
  107. Scaling laws for dc glow discharge • pressure 1/d •

    el.current 1 • voltage 1 • # of charged particles 1/d2 • electron temperature 1 • plasma core gas temperature 1/d
  108. None
  109. None
  110. detector volume 50 nL

  111. Spectroscopy, carbon 300 400 500 600 700 800 -5000 0

    5000 Emission (AU) Wavelength (nm) helium helium + methanol ~ ~ ~
  112. Calibration curve for methane • Detection limit 2·10-14 g/s C

    10 100 1000 104 1 10 100 1000 emission intensity minus background / A.U. CH 4 concentration / ppm 3*Noise
  113. Calibration for hexane 10 100 1000 104 10 100 1000

    104 Plasma chip peak height (AU) FID peak height (pA)
  114. 0 500 1000 1500 2000 2500 3000 3500 4000 4500

    200 300 400 500 600 700 800 900 Wavelength (nm) Emission Intensity (AU) CH2 Cl2 CCl Cl CH He C/C2 C2 He H He Spectroscopy, Cl
  115. 0 500 1000 1500 2000 2500 3000 3500 4000 4500

    200 300 400 500 600 700 800 900 Wavelength (nm) Emission Intensity (AU) Background (He) Bromopropane 470.2 478.2 481.5 Bromopropane Br Spectroscopy, Br
  116. conclusions • simple layout and operation • 10-50 mW power

    consumption • gas temperature 400K • can be touched during operation • acceptable detection limits for volatiles • problem: liquid samples plasma liq.ppt analog computing.ppt
  117. liqid samples ? 50 nL of gas (1 atm) corresponds

    to 50 pL of liquid spraying not succesful  sputtering off sample surface
  118. Schematic of Electrolyte as Cathode Discharge (ELCAD) Optical Emission Detector

    Chip
  119. Cathode Connection Gas / sample outlet Spectrometer connection / plasma

    chamber H.V. Anode Gas inlet (Argon or Helium) Sample Inlet
  120. Experimental Setup of ELCAD Optical Emission Detector Chip

  121. Top trace : 0.1M CuSO4 in 1M HCl Bottom trace

    : 1M HCl
  122. Top trace : 0.1M CuSO4 in 1M HCl Bottom trace

    : 1M HCl Possible CuI bands at 485nm & 488nm Cu lines at 511nm, 515nm & 522nm
  123. electro-chemiluminescence Arun Arora

  124. None
  125. None
  126. None
  127. 6 x 10-12 4 x 10-12 2 x 10-12 0

    40 20 0 concentration / mol dm-3 emission intensity / a.u. figure 4
  128. Sensitivity of electrochemiluminescence detector for Ru(bpy)2 Detector cell volume 100

    nL concentration number of molecules light intensity 5.10-13 M 30,000 2.1 + 0.5 1.10-12 M 60,000 5.2 + 0.4 2.10-12 M 120,000 11.1 + 0.7 4.10-12 M 240,000 28.0 + 0.6 5.10-12 M 300,000 34.1 + 0.4
  129. floating electrodes pH changes indicate ox and red

  130. electrode electrode Pt Pt Pt ox red ox red ox

    red ox red ox red ox red red ox 1-2 V applied voltage
  131. None
  132. ECL and CE chip glass device with Pt electrode

  133. ECL and CE chip

  134. None
  135. None
  136. ECL and CE chip Ru (bpy)3 light emission increases with

    voltage
  137. ECL and CE chip direct measurement of Ru2+

  138. TBR -3 -2 -1 0 1 -6.5 -6 -5.5 -5

    -4.5 -4 -3.5 -3 Log C (M) Log Signal (arbitrary units) Calibration Curve
  139. indirect detection possible

  140. conclusions • interesting and simple • problem: electrolysis of water

    • not satisfactory detection limit
  141. potentiometric detector Ratna Tantra

  142. potentiometry

  143. potentiometry liquid chromatography S.Muller, D.Scheidegger, C.Haber, W.Simon, J. High Res.

    Chromatogr. 14, 174 (1991)
  144. potentiometry selectivity Ba2+ vs Mg2+ 2.10-5 Ba2+ vs Ca2+ 3.10-3

    Ba2+ vs Cu2+ 3.10-5 Ba2+ vs Na+ 4.10-3 Ba2+ vs K+ 8.10-3 M.W.Laubli, W.Simon, F.Vogtle, Anal. Chem. 57, 2756 (1985) this selectivity is not enough for Ba2+ in presence of Na+
  145. CE combined with potentiometry resolution Ba2+ vs Mg2+ n/a Ba2+

    vs Ca2+ 16 Ba2+ vs Cu2+ n/a Ba2+ vs Na+ 20 Ba2+ vs K+ 36 T.Kappes, P.Schnierle, P.C.Hauser, Anal. Chim. Acta 393, 77 (1999)
  146. CE combined with potentiometry FIA, potentiometry CE, potentiometry Ba2+ Ba2+

    Na2+, Ba2+ Na2+
  147. CE potentiometry chip glass microstructure with PDMS wells

  148. potentiometry chip glass microstructure with PDMS wells

  149. potentiometry chip

  150. potentiometry chip

  151. potentiometry CE chip conc [arb. units] 0 1 2 3

    4 5 6 0 10 20 30 40 50 60 time [s] EMF [V] 0 0.05 0.1 0.15 0.2 0.25 0 10 20 30 40 50 60 time [s]
  152. potentiometry CE chip main problem •membrane liquid is moved out

    when voltage is applied • reproducibility • lifetime
  153. Shah convolution, Fourier transform velocimetry John Crabtree, Toby Jeffery, Yien

    Kwok, Jan Eijkel
  154. Shah Convolution - FT- Detection Typical detections - single point

    injection detection e.g. Fluorescence Electrochemical Conductance …. separation
  155. injection Imaging detection Injection separation function detection SIGNAL RECORDED Shah

    Convolution - FT- Detection injection Delta function convolution Shah function SIGNAL RECORDED DECONVOLUTION separation Fourier transform f (frequency) Electrophoretic mobility 1/f Electrophopherogram
  156. Shah Convolution - FT- Detection µ-TAS device – ideal geometries

    - LIF 55 Slits – 700µm spacing, 300µm transparent Channel 15µm deep, 50µm wide slit array Cr layer sample carrier electrolyte waste waste injection detection area
  157. Shah Convolution - FT- Detection

  158. detection during re-mixing detector signal A (t) mobility spectrum X

  159. multiple injection detector signal B (t) mobility spectrum X scoft

    multi inj.ppt scoft particles.ppt
  160. None
  161. 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 16

    20 24 28 32 36 40 time (sec) PMT Signal (V) 1st 2nd 1st 2nd 3rd 2-plugs 3-plugs
  162. 0 150 300 450 600 750 900 0.5 1 1.5

    2 2.5 3 frequency (Hz) FT Magnitude (arb. units) 1.675 Hz fundamental 1.8 Hz fundamental 2-plugs 3-plugs -0.5 0.5 1.5 2.5 3.5 4.5 5.5 0 5 10 15 20 25 30 35 time (sec) PMT Signal (V) 2-plugs 3-plugs Fourier Transform
  163. Table 3: S/N vs Number of Sample Plugs Number of

    Sample Plugs S/N a Standard deviation 1 46 2.5 2 69 5 3 102 1 (a) average of two runs for each number of sample plugs
  164. None
  165. SR SW BW Electrophoretic Channel Cr film with micromachined slits

    40 m wide slit 30 m gap
  166. 0 0.02 0.04 0.06 0.08 0.1 50 55 60 65

    70 time (s) PMT Signal (V)
  167. 0 5 10 15 20 25 30 5 7 9

    11 13 15 17 Frequency (Hz) FT Magnitude (arb. units)
  168. 0 30 6.4 7 7.6 Frequency (Hz) FT Magnitude (arb.

    units) are these fine lines single particles?
  169. wavelet transform 7Hz area time [s] 20 0 frequency [Hz]

    single particle 10
  170. 7 Hz 14 Hz

  171. mobility spectrum X detector signal C (t) frontal analysis scoft

    frontal.ppt
  172. None
  173. 0 2 4 6 0 10 20 time (sec) PMT

    Signal (V) 50 100 150 200 0.5 1 1.5 2 frequency (Hz) FT(Magnitude) (arb. units) FT ~1 Hz fundamental (A) (B) -10 190 390 590 0 1 2 frequency (Hz) FT(Real-B)^2 (arb. units) 1 Hz fundamental (C) FT -0.004 0 0.004 0.008 0.012 0 10 20 time (sec) Differentiated PMT Signal (arb. units) (D) Differentiation 0 1 2 3 0 1 2 frequency (Hz) FT(Magnitude) (arb. units) FT (E) 1 Hz fundamental
  174. conclusions • Not clear what advantages are • Interesting •

    Hope to increase resolution [?]
  175. Analog computing Darwin Reyes

  176. 2 + 2 = ? What is the optimum linear

    velocity for minimum band broadening? What is the optimal street modification for London’s daily traffic jam?
  177. Very simple approach to a mathematical problem: • Provide the

    problem as a micro channel system • Pose the question by addressing electrodes • Get the answer visualized by plasma emission MAZES
  178. given: the most simple maze question: What is shortest connection

    between A and B? A B He in-, outlet electrodes
  179. None
  180. None
  181. Increased mathematical complexity same time to find solution

  182. None
  183. None
  184. None
  185. Victoria station Imperial College

  186. Interesting paper by Whitesides group on “maximum clique” problem solving

    by particle counting in microfluidic device PNAS 2001 …
  187. conclusion • interesting • fun ! • search the problem

    for a solution • not clear, how useful
  188. Acknowledgment Coworkers and Ph.D. students Jan Eijkel Chao-Xuan Zhang Michael

    Mitchell Fiona Bessoth Omar Naji Darwin Reyes postdocs Arun Arora Yien Kwok Gareth Jenkins Silvia Valussi Nicole Pamme Oliver Hofmann Paul Monaghan Melanie Fennah Valerie Spikmans Nils Goedeke Dimitrios Iossifidis Pierre-Alain Auroux
  189. FUNDING INSTRUMENTATION SmithKline Beecham (UK) Zeneca (UK) BBSRC, UK EPSRC,

    UK European Commission, B Schlumberger, UK Casect, UK Agilent, D Forensic Lab, UK Asahi Kasei, Japan Lab of the Government Chemist, UK CSEM, Switzerland Amersham Pharmacia, UK Kodak, UK Glaxo Wellcome, UK Glaxo-Wellcome Heidelberg Instruments Hybaid MICROFABRICATION Alberta Microelectronics Centre, Canada Caliper Technologies, California MESA, University of Twente, The Netherlands CSEM, Switzerland !