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microfluidics based chemical separation and reaction schemes

microfluidics based chemical separation and reaction schemes

... talk given at Xi'an. 3rd International Symposium on Instrumentation Science and Technology, ISIST, August 18-22, 2004

andreas manz

August 18, 2004
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  1. Microfluidics based chemical
    Microfluidics based chemical
    separation and reaction schemes
    p
    Andreas Manz
    Andreas Manz
    I S A S INSTITUTE FOR ANALYTICAL SCIENCES
    I S A S INSTITUTE FOR ANALYTICAL SCIENCES
    Dortmund and Berlin

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  2. l tit l h i t id tif
    analytitcal chemistry = identify
    and quantify molecules
    and quantify molecules
    •human genome 3,000,000,000 bases
    g , , ,
    •proteomics > 100,000 different proteins
    •metabolomics > 10,000,000 different
    . small molecules
    •target: single cell analysis (femtoliter)

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  3. l tit l h i t id tif
    analytitcal chemistry = identify
    and quantify molecules
    and quantify molecules
    largest impact in near future: stem cells,
    g p ,
    cryobiological librairies, tissue engineering,
    organ engineering completely new medicine
    organ engineering, completely new medicine

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  4. l tit l h i t id tif
    analytitcal chemistry = identify
    and quantify molecules
    and quantify molecules
    example: cell culture
    p
    99% of protein mass is < 20 proteins
    1% is the remaining 100 000
    1% is the remaining 100,000
     difficult

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  5. View Slide

  6. vision
    • identify and quantify all compounds in a
    mixture („...omics“)
    • as a function of time (monitoring)
    ... as a function of time (monitoring)
    • ... as a function of space (imaging)

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  7. information
    t t
    vision
    content
    „...ome“
    complex mixture
    mixture
    1 d ti l
    mixture
    time
    1 times
    1 location
    1 compound continuously
    1/s
    1/min
    1d
    2d
    3d
    space
    3d

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  8. information
    content proteomics
    glucose sensor
    most analytical
    methods
    time
    methods
    NMR tomography
    space

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  9. vision
    time
    space

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  10. View Slide

  11. View Slide

  12. View Slide

  13. View Slide

  14. How can we do it ?
    What will it cost ?
    h i d i k
    What time does it take ?

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  15. why miniaturize
    why miniaturize
    volume of 1µL 1nL 1pL
    (1mm)3 (100µm)3 (10µm)3
    is a cube of
    600,000,000 600,000 600
    # molecules
    (1nM solution)
    25 / cm2 2500 / cm2 250 ,000/ cm2
    # volumes
    In array
    17 min 10s 100ms
    diffusion time
    1.5 /min / cm2 250 /s / cm2 2,500,000 /s / cm2
    # reactions
    (diffusion controlled)
    (diffusion controlled)

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  16. How about 100 nm scale (cube)?
    •Limitation to >mM concentration
    •Limitation to molecular weight <
    100 000
    100,000
    •Time scale is fast enough at 1 um
    g
    scale

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  17. What do we have now?
    What do we have now?
    [example 1]
    [example 1]
    • Electrophoresis chips - Caliper
    Electrophoresis chips Caliper,
    Agilent, Hitachi, Shimadzu etc.
    i l d f A f
    • mainly used for DNA fragment
    sizing
    • protein separations
    bi
    • bioassays

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  18. 10 fold miniaturization
    100 x faster separation
    p
    1000 x smaller volume
    10 x lower reagent consumption

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  19. electrophoresis
    p
    c
    e [
    a
    r b
    .
    u n
    i
    t
    s ]
    1 2
    3
    4 c
    y
    c l
    e
    #
    t
    7 s
    s
    y
    n
    c
    h
    r
    .
    c
    e [
    a
    r b
    .
    u n
    i
    t
    s ]
    1 2
    3
    4 c
    y
    c l
    e
    #
    t
    7 s
    s
    y
    n
    c
    h
    r
    .
    c
    e [
    a
    r b
    .
    u n
    i
    t
    s ]
    1 2
    3
    4 c
    y
    c l
    e
    #
    t
    7 s
    s
    y
    n
    c
    h
    r
    .
    f l
    u
    o r
    e
    s
    c e
    n
    0 4
    0 8 0 1
    2
    0 1
    6 0
    5
    6
    7 8
    f l
    u
    o r
    e
    s
    c e
    n
    0 4
    0 8 0 1
    2
    0 1
    6 0
    5
    6
    7 8
    f l
    u
    o r
    e
    s
    c e
    n
    0 4
    0 8 0 1
    2
    0 1
    6 0
    5
    6
    7 8

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  20. electrophoresis
    p

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  21. nano channels & single
    molecules
    80 x 80 nm channel bulk DNA
    L.C.Campbell, M.J.Wilkinson, A.Manz, P.Camilleri, C.J.Humphreys

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  22. electrophoresis
    Agilent 2100 Bioanalyzer
    p

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  23. What do we have now?
    What do we have now?
    [example 2]
    [example 2]
    • Reactor chips - Upchurch, Ehrfeld
    etc.
    etc.
    • Mainly used for solvent
    di t i h t h
    gradients in chromatography
    • Chemical synthesis
    y
    • Bioassays

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  24. chemical reactor

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  25. F. G. Bessoth, Dissertation, Imperial College, 2000.
    F. G. Bessoth, A. J. deMello, A. Manz, Anal.
    Commun., 1999, 36, 6, 213-215.

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  26. Continuous flow method

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  27. chemical reactor
    0 ms
    6 ms

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  28. chemical reactor
    Micromixer chip /
    PTFE interface
    PTFE interface
    Inlet capillaries
    Syringes
    Injection loop
    Rheodyne injection valve
    Injection loop
    outlet capillary

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  29. H
    R1 O
    M OH Cl-
    R1
    Multicomponent Chemistries: The Ugi Reaction
    N+
    H
    H
    R
    R2
    Cl-
    H H
    MeOH
    N+
    Cl
    H2
    O
    R2
    R1/R2 = -CH2
    (CH2
    )3
    CH2
    -
    + +
    Piperidinium cation
    Formaldehyde
    0oC
    R3
    R /R CH2
    (CH2
    )3
    CH2
    Piperidine hydrochloride
    Piperidinium cation
    y
    (1)
    (2) (3)
    N
    R4
    C
    R3
    R3
    R3/R4 = -CH2
    (CH2
    )4
    CH2
    -
    R1
    N
    R4
    H2
    O
    R1
    N
    N
    R4
    O
    +
    2
    ( 2
    )4 2
    Cyclohexyl isocyanide
    (4)
    N
    R2
    R2
    Nitrilium intermediate
    -Dialkylacetamide
    N-Cyclohexyl-2-piperidin-1-yl-acetamide
    Nitrilium intermediate
    (5)
    (6)

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  30. Simultaneous Observation of Reactants, Intermediates,
    Products and By-products
    20 L i 1
    20 Lmin-1
    50 nL injection loop
    Room temperature

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  31. Ubiquitin Native/A state
    Methanol
    Methanol
    pD=2
    Native state A state

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  32. Set-up for NMR measurement
    NMR
    Detection coil
    Micromixer
    (200m i.d.)
    Syringe pumps
    y g p p
    250m i.d. 75m i.d.

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  33. Picture of detection coil
    Reservoir
    Capillary
    1cm
    Sweedler group, Univ. of Illinois

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  34. NMR set-up
    NMR
    NMR
    3m
    Capillary
    Syringe pump
    Sweedler group, Univ. of Illinois

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  35. Hi 68
    Tyr59
    A
    His68
    N N
    A
    10 L/min
    (24sec)
    N N
    A
    40 L/min
    (6sec)
    A
    (6sec)
    Sweedler
    group,
    group,
    Univ. of
    Illinois

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  36. DNA hybridisation assays
    DNA hybridisation assays
    contribution to Fluorescence
    contribution to Fluorescence
    Intercalating dye alone low
    DNA oligomers low
    Oligomer dimers medium
    dsDNA high
    dsDNA high

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  37. influence of DNA sequence on
    influence of DNA sequence on
    kinetics
    kinetics
    Order:
    t hi 1
    matching, 1
    mismatch, 2
    mismatches
    Sequence-dependent responses from two different experiments.

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  38. Quick Decision: Exploiting
    Quick Decision: Exploiting
    Photobleaching Effects
    Photobleaching Effects

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  39. microfluidic DNA assays
    1 d t d i i
    • 1 second to decision
    • no complicated surface chemistry
    • no complicated surface chemistry
    • sensitivity 100-200nM
    sensitivity 100 200nM
    • could be competing with DNA
    arrays

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  40. What would I address?
    What would I address?
    [example 1]
    [example 1]
    protein separations
    by free-flow electrophoresis
    y p
    … isoelectric focusing

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  41. free-flow electrophoresis -
    proteins
    proteins
    + -

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  42. IEF hi
    IEF chip
    • 36 x 20 um inlet channels
    • 72 x 20 um outlet channels
    h id 108 4 h l
    • each side 108 x 4 um channels
    • separation bed 12.2 x 4.1 mm
    p
    – 15,552 posts
    30 x 30 um
    – 30 x 30 um

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  43. free-flow electrophoresis
    p

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  44. very fast
    l h i
    electrophoresis

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  45. comparison
    p
    FFE
    FFE
    c
    e [
    a
    r b
    .
    u n
    i
    t
    s ]
    1 2
    3
    4 c
    y
    c l
    e
    #
    t
    7 s
    s
    y
    n
    c
    h
    r
    .
    c
    e [
    a
    r b
    .
    u n
    i
    t
    s ]
    1 2
    3
    4 c
    y
    c l
    e
    #
    t
    7 s
    s
    y
    n
    c
    h
    r
    .
    c
    e [
    a
    r b
    .
    u n
    i
    t
    s ]
    1 2
    3
    4 c
    y
    c l
    e
    #
    t
    7 s
    s
    y
    n
    c
    h
    r
    .
    f l
    u
    o r
    e
    s
    c e
    n
    0 4
    0 8 0 1
    2
    0 1
    6 0
    5
    6
    7 8
    f l
    u
    o r
    e
    s
    c e
    n
    0 4
    0 8 0 1
    2
    0 1
    6 0
    5
    6
    7 8
    f l
    u
    o r
    e
    s
    c e
    n
    0 4
    0 8 0 1
    2
    0 1
    6 0
    5
    6
    7 8
    CE

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  46. I l t i f i
    Isoelectric focusing
    • charge of protein molecule depends on pH
    g p p p
    • low pH (acidic): protein is cation
    hi h H (b i ) t i i i
    • high pH (basic): protein is anion
    • generate a pH gradient across electric field
    g p g
    – ions move until overall charge is zero
    isoelectric point is different for each protein
    – isoelectric point is different for each protein

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  47. IEF f f i i l
    IEF proof of principle
    12 mm angiotensin I, 1.75 kV, 10 uL/min
    g o e s , .75 V, 0 u /
    4 mm = 500 ms
    4 mm = 500 ms
    4 mm
    0 mm

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  48. IEF t i
    IEF - proteins
    angiotensin I, 1.75 kV, 10 uL/min
    g o e s , .75 V, 0 u /

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  49. IEF IGF 1
    IEF - IGF-1

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  50. IEF hi
    IEF chip
    • volume 240 nL plus wells
    p
    • at 10 uL/min
    1 4 d (ti t i f ti )
    – 1.4 seconds (time to information)
    • preconcentration 100 - 400x

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  51. What would I address?
    What would I address?
    [example 2]
    [example 2]
    air quality monitor
    by plasma emission spectroscopy
    y p p py
    gas chromatography

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  52. plasma emission
    p

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  53. detector
    plasma emission
    detector
    volume
    50 L
    p
    50 nL
    J.C.T.Eijkel, H.Stoeri,
    A.Manz, J. Anal. At.
    Spectrom. 15, 297-300
    p ,
    (2000)

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  54. plasma emission CH2
    Cl2
    p
    4000
    4500
    CH2
    Cl2
    CH He He H He
    3500
    4000
    Cl
    2500
    3000
    n s ity (A U )
    2000
    is s io n In te n
    CCl
    C/C2
    1000
    1500
    Em
    0
    500 C2
    0
    200 300 400 500 600 700 800 900
    Wavelength (nm)

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  55. calibration for methane
    calibration for methane
    J.C.T.Eijkel, H.Stoeri, A.Manz Anal. Chem. 71, 2600-2606 (1999)
    d i li i 2 10 14 / C
    • detection limit 2·10-14 g/s C
    4
    U.
    104
    ound / A.
    1000
    us backgro
    100
    nsity minu
    sion inten
    3*Noise
    10
    1 10 100 1000
    emiss
    CH
    4
    concentration / ppm

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  56. triethyl phosphate headspace

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  57. View Slide

  58. CONCLUSIONS
    • Reactions and separations benefit from
    h t ti l i i h
    shorter timescales, micro is enough
    • Detection schemes usually suffer, but to
    y
    different extents, nano is quite difficult
    • Integration / small volumes advantageous,
    Integration / small volumes advantageous,
    below 1 nL doesn’t matter
    • More commercial products in near future
    • More commercial products in near future,
    but micro scale

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  59. Acknowledgment
    g
    my Imperial College team
    my new ISAS team
    Joachim Franzke
    Norbert Jakubowski
    Jan Eijkel Gareth Jenkins
    Norbert Jakubowski
    Philip Day
    Yi Xu
    Chao-Xuan Zhang
    Valerie Spikmans
    Martin Heule
    Jörg Baumbach
    Volker Deckert
    g
    Michael Mitchell
    Fi B h
    Dirk Janasek
    L C b ll
    Roland Hergenröder
    N b E
    Fiona Bessoth Lucy Campbell Norbert Esser
    Kay Niemax, Prof.

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  60. Acknowledgment
    funding
    g
    Glaxo SmithKline (UK)
    Glaxo SmithKline (UK)
    AstraZeneca (UK)
    EU research grant
    Casect Ltd. (UK)
    ( )
    Swiss National Science Foundation (Switzerland)
    EPSRC grant (UK)
    Leopoldina (Germany)

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