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

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
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)
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
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
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)
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
8. information content proteomics glucose sensor most analytical methods time methods NMR tomography space
9. vision time space
14. How can we do it ? What will it cost ? h i d i k What time does it take ?
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)
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
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
18. 10 fold miniaturization 100 x faster separation p 1000 x smaller volume 10 x lower reagent consumption
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
20. electrophoresis p
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
22. electrophoresis Agilent 2100 Bioanalyzer p
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
24. chemical reactor
25. F. G. Bessoth, Dissertation, Imperial College, 2000. F. G. Bessoth, A. J. deMello, A. Manz, Anal. Commun., 1999, 36, 6, 213-215.
26. Continuous flow method
27. chemical reactor 0 ms 6 ms
28. chemical reactor Micromixer chip / PTFE interface PTFE interface Inlet capillaries Syringes Injection loop Rheodyne injection valve Injection loop outlet capillary
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)
30. Simultaneous Observation of Reactants, Intermediates, Products and By-products 20 L i 1 20 Lmin-1 50 nL injection loop Room temperature
31. Ubiquitin Native/A state Methanol Methanol pD=2 Native state A state
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.
33. Picture of detection coil Reservoir Capillary 1cm Sweedler group, Univ. of Illinois
34. NMR set-up NMR NMR 3m Capillary Syringe pump Sweedler group, Univ. of Illinois
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
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
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.
38. Quick Decision: Exploiting Quick Decision: Exploiting Photobleaching Effects Photobleaching Effects
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
40. What would I address? What would I address? [example 1] [example 1] protein separations by free-flow electrophoresis y p … isoelectric focusing
41. free-flow electrophoresis - proteins proteins + -
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
43. free-flow electrophoresis p
44. very fast l h i electrophoresis
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
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
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
48. IEF t i IEF - proteins angiotensin I, 1.75 kV, 10 uL/min g o e s , .75 V, 0 u /
49. IEF IGF 1 IEF - IGF-1
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
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
52. plasma emission p
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)
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)
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
56. triethyl phosphate headspace
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
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.
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)