chips for isoelectric focusing and zone electrophoresis in the free-flow mode

... talk given at MSB 2003, New Orleans.

Transcript

1. chips for isoelectric focusing and
   zone electrophoresis in the free-
   flow mode
   Yi Xu, Chao-Xuan Zhang,
   Andreas Manz
   Imperial College, Dept. Chemistry,
   London UK
   

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2. What should I show you?
   Electrophoresis scaling laws !
   

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3. separation efficiency
   • Number of theoretical plates is proportional
   to voltage drop
   U
   N 
   

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4. heating problem
   •Power generated per unit length should be a
   constant
   const
   L
   I
   U
   
   
   

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5. separation efficiency
   •Number of theoretical plates is proportional
   to length / diameter of capillary
   d
   L
   N 
   

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6. separation time
   •Analysis time is proportional to length *
   diameter of capillary
   d
   L
   t 
   
   

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7. 10 fold miniaturization
   100 x faster separation
   1000 x smaller volume
   10 x lower reagent consumption
   identical quality of separation
   

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8. 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.
   electrophoresis
   by Franz von Heeren
   

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9. electrophoresis
   

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10. Agilent 2100 Bioanalyzer
    electrophoresis
    

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11. FFE
    principle
    

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12. FFE
    problem
    

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13. FFE
    problem
    el. current much larger
    heat dissipation worse
    

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14. FFE
    chip history 1994
    by Dan Raymond
    

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15. FFE
    chip history
    by Dan Raymond
    

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16. FFE
    chip history
    by Dan Raymond
    

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17. FFE
    chip history
    by Dan Raymond
    

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18. latest design
    + -
    

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20. latest design
    • volume 240 nL plus micro wells
    • 36 x 20um inlet channels
    • 72 x 20um outlet channels
    • each side 108 x 4um channels
    • separation bed 12.2 x 4.1 mm
    – 15,552 posts
    – 30 x 30 um
    

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24. diffusion…
    

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25. electrophoresis
    happens…
    

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26. very fast
    electrophoresis
    

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27. problem with posts
    

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28. 111 ms
    41 ms
    53 ms
    64 ms
    76 ms
    88 ms
    99 ms
    integrating over rectangular area
    

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29. influence of
    electric field
    separation of 2 amino acids
    

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30. 0
    200
    400
    600
    800
    0 100 200 300 400 500
    electric field [V/cm]
    migration distance [um]
    migration distance = f (E)
    

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31. isoelectric focusing (IEF)
    • establish pH gradient
    • establish electric field
    • charge of protein depends on pH
    • mobility is nil at isoelectric point
    • focusing
    

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32. IEF proof of principle
    12 mm
    0 mm
    4 mm
    4 mm = 500 ms
    angiotensin I, 1.75 kV, 10 uL/min
    

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33. IEF – two peptides
    

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34. IEF - IGF-1
    sample concentration
    10-7 M at inlet
    >10-5 M here
    

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35. IEF – IGF-1
    • sample introduced at 4.1 mm width
    • distance post-post on structure 40 um
    • apparent streamline 10 um
    • preconcentration factor is around
    100x to 400x
    

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36. IEF chip specific problem
    • side channels contribute >80% of
    voltage drop
    • part of the pH gradient will
    therefore be lost
    • buffer reservoirs have to be at
    extreme pH
    • some proteins will not stay in the
    separation area
    

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37. 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.
    comparison
    FFE
    CE
    

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38. conclusions
    • IEF is possible and very fast in
    small FFE system
    • preconcentration of over 100x is
    possible
    • problems with pH gradient and side
    channels
    • fraction collection has to be done
    • detector has to be attached
    

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39. Acknowledgment
    EU funding
    BBSRC clean room
    

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