human perception < 1 cm is small > 10 m is big < 100 ms is immediate > 1 min is slow factor 100: 100 ms to 1 ms: not impressive 17 h to 10 min: makes a difference 10 min to 6 s: very impressive
RESULTS Serial to parallel converter : 9 plugs refilled in 10 seconds, that means 1 sample plug per second 54 samples per minute 78,000 samples per day
x x x x x x x x x SYBR green x x x x x x x x x x x x x double stranded DNA SYBR green x x x x x x x x x x x x x x x x x x x x x x x x SYBR green complex [fluorescing] x x x x 1) 2) 3)
x x x x x x x x x x x x x x x x x double stranded DNA SYBR green complex [fluorescing] concentration length of plug DNA is slowing down at moving front of SYBR green SYBR green is slowing down at moving front of DNA fluorescence length of plug
conclusions • High speed separations • Good quality separations • Very good fluid control • Small volumes • Commercial products • biggest success, so far really?
Concept chemical reactions concept.ppt the chip chemical reactions mix.ppt Bioassay chemical reactions bio.ppt Electrophoretic reaction chemical reactions electrophoretic Synthesis chemical reactions synth.ppt
drug discovery • first step to find new active molecules • composed of – synthesis of new compound – isolation, characterisation – bioassay • a significant effort in pharmaceutical industry, involving new technologies
A B C A B , A , B A B A B C , A B , C A B C A B C fluorescence detection bioassay synthesis step 1 synthesis step 2 separation separation continuous flow
chemical reaction • In the most simple case, a molecule A meets a molecule B and reacts to give AB • many reactions are diffusion controlled • reaction time of hours in conventional lab • reaction time of 30 min in micro well plate
Figure 4. Separation of several amino acids using post-column derivatization for detection. D.J.Harrison, K.Fluri,N.Chiem, T.Tang,Z.Fan University of Alberta, Edmonton,Canada Transducers’95, Proc., vol.1, pp752-755 (1995)
Mixing – Diffusion times D d t 2 2 Before laminar mixing D n d t 2 2 2 After laminar mixing n = number of branches, d = tubing diameter, D= diffusion coefficient
F. G. Bessoth, A. J. de Mello and A. Manz, Anal. Commun., 1999, 36, 213-215 Chip manifold volume 600 nL Observation channel 530 nL Distributive Micromixing Device
NO2 NO2 CHO NO2 P(Ph)3 NO2 purple Br- 2-nitrobenzy ltriphenyl- phosphonium brom ide p-nitrobenza ldehyde colourless NaOMe NO2 Me OH colourless P(Ph)3 + + Wittig reaction N + O O Cl Cl Cl Cl O O Cl Cl Cl N Enamine Chloranil blue 2,3,5-trichlor-6-(2-piperidin -1-yl)-[1,4]- benzoquinone Synthesis of a substituted aminovinyl-p-quinone SYNTHESIS
N+ H H R1 R2 Cl- H H O MeOH N+ Cl- H2 O N R3 R4 R2 R1 C R1 N R2 N R4 R3 H2 O R1 N R2 N R4 R3 O R1/R2 = -CH2 (CH2 )3 CH2 - Piperidine hydrochloride + + Piperidinium cation + R3/R4 = -CH2 (CH2 )4 CH2 - Cyclohexyl isocyanide Nitrilium intermediate -Dialkylacetamide Formaldehyde N-Cyclohexyl-2-piperidin-1-yl-acetamide (1) (2) (3) (4) (5) (6) Multicomponent Chemistries: The Ugi Reaction 0oC
conclusions • Some syntheses do work! • What is the limitation? • Very fast mixing • Higher temperatures than usual • Better selectivity • Complicated device
polymerase chain reaction • = method to amplify the amount of a specific DNA sequence in a sample • each cycle doubles the amount of DNA • most commonly used procedure in biology • commercial instruments would do 20 cycles in 50 minutes
Figure 1. Cross section of micro-PCR test device. Figure 5. Gel electrophoretic photograph indicating that similar results were obtained with a 50ul microfabricated test device (mid-right three bands) as in much lager commercial instrument (mid-left two bands). The target sequence amplified was HIV.
T.M.Woudenberg, E.S.Winn-Deen, M.Albin [Applied Biosystems, Foster City, CA] High-density PCR and beyond, u-TAS 96, p 55-59 (1996) First data from polycarbonate “chip”
Advantages of Continuous Flow PCR Chip variable volumes – 1nL to1mL low carryover high speed 12 to 60s per cycles low band-broadening High speed chemical amplifier
Monitoring the growth of bacterial cultures • The sampling of a growing culture at various time intervals (by viable counts,dry weight of the biomass or optical density measurements) • Real-time monitoring • miniaturized systems • reduction of biological waste Conventional bulk growth techniques Microbiology microfluidic
Fluorescence measurements - Plots of PMT signal versus sampling time 0 1 2 3 4 5 0 10 20 30 40 50 60 70 80 90 100 sampling t (s) PMT sig. (V) 0hr 1hr 2hr a. Over the initial two hours, the data suggests that there is not a great deal of increase in the biomass. 0 1 2 3 4 5 0 10 20 30 40 50 60 70 80 90 100 sampling t (s) PMT sig. (V) 3hr 4hr b. After 3hr that there is any appreciable increase in the signal as indicated by figure b. At 4hrs, the signal has significantly increased.
State-of-the-art • inductively coupled plasma • 1 kW power consumption • gas temperature 6,000K • safety radius 1 m • very low detection limits for metals • liquid nebulizing interfaces
Scaling laws for dc glow discharge • pressure 1/d • el.current 1 • voltage 1 • # of charged particles 1/d2 • electron temperature 1 • plasma core gas temperature 1/d
conclusions • simple layout and operation • 10-50 mW power consumption • gas temperature 400K • can be touched during operation • acceptable detection limits for volatiles • problem: liquid samples plasma liq.ppt analog computing.ppt
potentiometry selectivity Ba2+ vs Mg2+ 2.10-5 Ba2+ vs Ca2+ 3.10-3 Ba2+ vs Cu2+ 3.10-5 Ba2+ vs Na+ 4.10-3 Ba2+ vs K+ 8.10-3 M.W.Laubli, W.Simon, F.Vogtle, Anal. Chem. 57, 2756 (1985) this selectivity is not enough for Ba2+ in presence of Na+
CE combined with potentiometry resolution Ba2+ vs Mg2+ n/a Ba2+ vs Ca2+ 16 Ba2+ vs Cu2+ n/a Ba2+ vs Na+ 20 Ba2+ vs K+ 36 T.Kappes, P.Schnierle, P.C.Hauser, Anal. Chim. Acta 393, 77 (1999)
injection Imaging detection Injection separation function detection SIGNAL RECORDED Shah Convolution - FT- Detection injection Delta function convolution Shah function SIGNAL RECORDED DECONVOLUTION separation Fourier transform f (frequency) Electrophoretic mobility 1/f Electrophopherogram
Table 3: S/N vs Number of Sample Plugs Number of Sample Plugs S/N a Standard deviation 1 46 2.5 2 69 5 3 102 1 (a) average of two runs for each number of sample plugs
Very simple approach to a mathematical problem: • Provide the problem as a micro channel system • Pose the question by addressing electrodes • Get the answer visualized by plasma emission MAZES
FUNDING INSTRUMENTATION SmithKline Beecham (UK) Zeneca (UK) BBSRC, UK EPSRC, UK European Commission, B Schlumberger, UK Casect, UK Agilent, D Forensic Lab, UK Asahi Kasei, Japan Lab of the Government Chemist, UK CSEM, Switzerland Amersham Pharmacia, UK Kodak, UK Glaxo Wellcome, UK Glaxo-Wellcome Heidelberg Instruments Hybaid MICROFABRICATION Alberta Microelectronics Centre, Canada Caliper Technologies, California MESA, University of Twente, The Netherlands CSEM, Switzerland !
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