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
2. What should I show you? Electrophoresis scaling laws !
3. separation efficiency • Number of theoretical plates is proportional to voltage drop U N 
4. heating problem •Power generated per unit length should be a constant const L I U  
5. separation efficiency •Number of theoretical plates is proportional to length / diameter of capillary d L N 
6. separation time •Analysis time is proportional to length * diameter of capillary d L t  
7. 10 fold miniaturization 100 x faster separation 1000 x smaller volume 10 x lower reagent consumption identical quality of separation
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
9. electrophoresis
10. Agilent 2100 Bioanalyzer electrophoresis
11. FFE principle
12. FFE problem
13. FFE problem el. current much larger heat dissipation worse
14. FFE chip history 1994 by Dan Raymond
15. FFE chip history by Dan Raymond
16. FFE chip history by Dan Raymond
17. FFE chip history by Dan Raymond
18. latest design + -
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
24. diffusion…
25. electrophoresis happens…
26. very fast electrophoresis
27. problem with posts
28. 111 ms 41 ms 53 ms 64 ms 76 ms 88 ms 99 ms integrating over rectangular area
29. influence of electric field separation of 2 amino acids
30. 0 200 400 600 800 0 100 200 300 400 500 electric field [V/cm] migration distance [um] migration distance = f (E)
31. isoelectric focusing (IEF) • establish pH gradient • establish electric field • charge of protein depends on pH • mobility is nil at isoelectric point • focusing
32. IEF proof of principle 12 mm 0 mm 4 mm 4 mm = 500 ms angiotensin I, 1.75 kV, 10 uL/min
33. IEF – two peptides
34. IEF - IGF-1 sample concentration 10-7 M at inlet >10-5 M here
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
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
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
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
39. Acknowledgment EU funding BBSRC clean room