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chips for isoelectric focusing and zone electro...

andreas manz
February 02, 2003

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

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

andreas manz

February 02, 2003
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  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
  2. 10 fold miniaturization 100 x faster separation 1000 x smaller

    volume 10 x lower reagent consumption identical quality of separation
  3. 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
  4. 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
  5. 111 ms 41 ms 53 ms 64 ms 76 ms

    88 ms 99 ms integrating over rectangular area
  6. 0 200 400 600 800 0 100 200 300 400

    500 electric field [V/cm] migration distance [um] migration distance = f (E)
  7. isoelectric focusing (IEF) • establish pH gradient • establish electric

    field • charge of protein depends on pH • mobility is nil at isoelectric point • focusing
  8. IEF proof of principle 12 mm 0 mm 4 mm

    4 mm = 500 ms angiotensin I, 1.75 kV, 10 uL/min
  9. 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
  10. 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
  11. 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
  12. 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