Chapter CHAPTER MICROFABRICATED DEVICES FOR NUCLEIC ACID AMPLIFICATION AND ELECTROPHORETIC SEPARATION 2.1 Introduction The interest in electrophoretic separations on microfabricated devices has grown dramatically over the past few years due to the many advantages it offers The short plugs, good dissipation of Joule heating, and high field strengths result in extremely rapid separations that consume only picoliter sample volumes 2.1.1 Theory of Electrophoretic Based Separations Electroosmotic Flow As shown in Figure 2.1, the inner walls of fused silica capillaries possess an intrinsic negative charge due to the presence of weakly acidic silanol groups (-SiOH) (pKa ~ 5.3).1 Cations in solution build up near the capillary surface to balance this charge, thus forming an electrical double layer Upon the application of an electric field across the length of the capillary, the cations in the diffuse portion of the double layer migrate towards the cathode Since these cations are hydrated, they induce a bulk flow of solution within the capillary towards the cathode The magnitude of this electroosmotic flow (EOF) is generally described by the Scholuchowski equation: µ =− εζ η Where ε and η are the dielectric constant and viscosity of the solvent, and ζ is the zeta potential The zeta potential is the potential slightly off the silica surface at the plane of 22 Chapter shear, and is a function of the deprotonation of the silanols, ion adsorption onto the surface and the ionic strength of the buffer + + + + + + + + + _ N + N _ + adsorbed compact layer layer N _ + + + _ N N _ N + + _ _ + + diffuse layer Figure 2.1 Schematic representation of the electrical double layer at the capillary inner wall Positive, negative and N signs respectively represent cations, anions, and non-charged species Electrophoretic Flow The basis for the electrophoretic process is the differential migration of sample ions relative to solvent molecules under the influence of an externally applied electric field There are two main contributors to the ion mobility, the applied electric force (Fe) and the frictional force (Ffr) that the molecules experiences as it moves through the buffer solution The applied electric force (Fe) depends on the charge q of the particular ion and electric field E: Fe = qE The frictional force (Ffr) depends on the viscosity of the buffer (η), the velocity of the ion (ν, cm/s) and the size of the molecule (radius of ion, r): Ffr= 6πηνr When the two forces are counterbalanced the ions move with a steady-state velocity: ν = qE / 6πηr 23 Chapter The mobility is then the electrophoretic velocity normalized to the electric field strength: µ = ν/E = q/6πηr 2.1.2 Electrophoretic Separations in Microfabricated Devices 2.1.2.1 General Description The design of microchips for capillary electrophoresis has undergone significant development from simple single-channel structures to increasingly complex ones In the most basic design, a microchip is formed of two intersecting channels, the injection channel and the separation channel, as shown in Figure 1.1 The primary means of material transport on chips are the electrokinetic phenomena, i.e electrophoretic and/or electroosmotic effects Buffer and sample flows within the channel manifold are precisely controlled through potentials applied to the reservoirs The intersection between the injection and separation channels has been called the injection cross since its volume defines the sample volume injected into the separation channel The integrated injectors are usually either cross-channel injectors, formed by orthogonally intersecting the separation channel with a channel connecting the sample to waste as shown in Figure 1.1, or twin-T injectors, where the two arms of the sample to waste channel are offset to form a larger injection region as shown in Figure 2.2 24 Chapter Sample reservoir Twin-T injector Buffer reservoir Buffer waste Separation Channel Sample waste Figure 2.2 Schematic layout of a microchip electrophoretic device with a twin-T injector CE chips are mainly fabricated using various glass substrates from inexpensive soda lime glass to high quality quartz Glass substrates are the most common substrates because of their good optical properties, well-understood surface characteristics, and well-developed fabrication methods adapted from the microelectronics industry Much of the technology developed for the semiconductor industry can be transferred directly to chip fabrication using insulating substrates.2 Although slight variations in glass fabrication techniques exist, the general fabrication aspects are similar (Figure 2.3) Generally a positive photoresist is spin-coated on top of the substrate, and the channel design is transferred to the substrate using a photomask Following exposure and development of the photoresist, the channels are etched into the substrate in a dilute HF/NH4F bath Because the photoresist used to mask wet etches does not adhere well to glass, a sacrificial layer that adhere well to both glass and photoresist is often used as intermediate layer (Figure 2.3) The access holes can be drilled on the etched substrate or another blank glass wafer To form the closed network of channels, the cover plate is bonded to the substrate over the etched channels Finally cylindrical reservoirs, to hold buffers and samples, are affixed onto the extremity of the channels 25 Chapter Sacrificial layer Glass substrate coated with sacrificial layer Photoresist Photoresist coated UV light photomask Photoresist exposed Photoresist and sacrificial layer removed in mask pattern Glass etched Photoresist and sacrificial layer removed Bonded with another piece of glass Figure 2.3 Schematic diagram of the photolithographic process used for making chips Although most microchip devices fabricated to date use glass, there have been several reports of devices fabricated from a variety of polymeric substrates including poly(dimethylsiloxane) (PDMS),3 poly(methyl methacrylate) (PMMA),4 acrylic,5 and polycarbonate.6 The interest in polymeric microfluidic devices stems primarily from the fact that plastic chips are less expensive to produce, and can be disposed after single use 2.1.2.2 Injection Methods Sample injection, by default, was the first functionality integrated into single substrates by fabricating intersecting channels at right angles The formed cross was intended to create a geometrically defined sample plug with a volume close to that of the 26 Chapter intersection cross Later on, various sample injection methods were developed making use of both EOF and electrophoretic effects Floating Injection For the floating injection mode, only two electrodes are used Voltage is applied between the sample reservoir and the sample waste reservoir (Figure 2.4, left) Buffer and buffer waste reservoirs are left floating Consequently, the sample solution is pumped past the injection cross to the sample waste reservoir If the injection potential is applied long enough to ensure that even the slowest moving component passes through the injection cross, the injection plug will have a composition representative of the sample to analyze To introduce the sample plug into the separation channel, the potentials are switched from the sample loading to the separation mode of operation, i.e potential is applied between buffer reservoir and buffer waste reservoir (Figure 2.4, right) However, since the potential in the separation channel is left floating during injection, the analyte is free to diffuse into the separation channel This problem is particularly important with small ions and molecules having high diffusion coefficients 27 Chapter Buffer reservoir Sample reservoir Buffer reservoir Sample Sample waste reservoir Buffer waste Sample waste Buffer waste Figure 2.4 Schematic of floating injection Pinched Injection The pinched injection is achieved by spatially confining the sample in the cross intersection before dispensing it into the separation channel.7,8 The sample flow between the sample reservoir and sample waste is electrokinetically confined by the incoming buffer streams from the buffer reservoir and buffer waste (Figure 2.5, left) The extent of sample focusing is regulated by the electric field strength in separation channel versus injection channel Once the sample flow has reached a steady state, the electric field is switched to a dispensing step serving also as the separation step (Figure 2.5, right) In this step, to prevent sample leakage into the separation channel and achieve a short axial extent sample plug, an electric field is also applied to sample buffer and sample waste to draw sample back from the intersection 28 Chapter Buffer reservoir Sample reservoir Buffer reservoir Sample Sample waste reservoir Buffer waste Sample waste Buffer waste Figure 2.5 Schematic of pinched injection When the pinched and floating injections are compared, the pinched sample loading is superior in two areas: temporal stability and plug length The pinched sample injection is independent of time, electrophoretic mobility, and electric field strength On one hand a smaller plug length leads to higher efficiency but on the other hand can be detrimental to the sensitivity as less analyte is injected Gated Injection In order to increase the amount of analyte injected into the separation channel, the gated injection was developed.9,10 The gated valve has a loading/separation mode where the sample flows from sample reservoir to the sample waste while the buffer flows from the buffer reservoir to the buffer waste to prevent sample leakage and provide continuous buffer supply into the separation (Figure 2.6, left) To make an injection, the field in the buffer reservoir is set to zero allowing a plug of sample to move into the separation channel (Figure 2.6, middle) In the subsequent separation step (Figure 2.6, right), the field is switched back to the loading/separation step The 29 Chapter buffer flow cuts the sample plug, and the injection valve returns to its original state The length of the injection plug is therefore a function of both the time of the injection and the electric field strength Sample reservoir Sample reservoir Buffer reservoir Sample waste Sample Buffer waste reservoir Float Buffer waste Sample reservoir Buffer waste Sample waste Buffer reservoir Buffer waste Figure 2.6 Schematic of gated injection The gated injection differs from the pinched injection on several counts With the gated injector, the sample migrates electrophoretically down the separation column and is cleaved by restoring the flow of buffer from the buffer reservoir The pinched sample loading pumps the sample through the injection cross, and the plug, which is injected onto the separation channel, is the sample which resides in the injection cross With the pinched sample loading, the amount loaded onto the separation column is time independent and has no electrophoretic bias The gated injector is both time dependant and electrophoretically biased, but allows for injecting more analyte and is therefore appropriate for samples of low concentrations 30 Chapter 2.1.2.3 Advantages Both electrophoretic migration of ions and electroosmotic flow velocity are linearly dependent on the axial electric field strength applied While in the case of pressuredriven flow the external force is applied across the whole cross section of the tube leading to a parabolic flow profile, in electroosmosis the external force can only be exerted to a thin sheet of fluid close to the wall, thus leading to a plug-flow profile The short plugs, good dissipation of Joule heating, and high field strengths result in extremely rapid separations that consume only picoliter sample volumes 2.1.3 Combined Microchip Based Electrophoretic Separation and Micro Polymerase Chain Reaction Micro CE dramatically increases the speed of nucleic acid separation However, prior to separation, if present in low amounts, the nucleic acid of interest should be amplified by Polymerase Chain Reaction (PCR) to obtain enough material for detection PCR has revolutionized bioscience due to its ability to exponentially and specifically amplify DNA templates from very small starting concentrations Miniaturization offers improved thermal energy transfer compared to conventional macrovolumes, resulting in a greatly increased speed of thermal cycling and reduced amount of expensive reagents used A PCR-CE combination reduces the contamination problem, decreases the risk of infection, and allows for faster execution of the analysis through reduced manual manipulations There have been several reports on integration of DNA amplification and electrophoretic separation on a single microfabricated chip These devices contain small reaction wells, which were thermocycled to generate amplicons, followed by the injection/separation/detection steps in the interconnected microchannel network 31 Chapter 2.2.3 Nucleic Acid Amplification and Separation on Microfabricated Devices The optimization of micro PCR conditions were mostly performed in Prof Lim Tit Meng’s laboratory, Department of Biological Sciences, NUS 2.2.3.1 Functional Integration of Electrophoretic and Micro PCR Devices Micro PCR chamber (10 µl volume) and micro CE were coupled through a PDMS gasket with the same thickness as that of the micro PCR chamber glass cover and with a center hole of the same diameter to that of the microchip sample reservoir (Figure 2.23) The microchip device has the sample reservoir hole pierced through for connecting with the micro PCR from below An illustration of the integrated device is shown in Figures 2.24 and 2.25 Due to the good adhesion properties of PDMS to glass surfaces, a good seal was obtained and no apparent leak was observed throughout the experiments For the operation of the integrated system, µPCR-µchip devices were pre-filled in advance with the required reagents To transfer the DNA PCR amplified product to the microchip sample reservoir, a small pressure was applied to the inlet hole of the PCR chamber Once the PCR product was in the sample reservoir, the entire µPCR-µchip device was placed in the detection system and electrophoresis was performed Figure 2.23 Photography of the integration of the 10 µL micro PCR device with microfabricated CE 50 Chapter Pipette/Stopper Pipette/Stopper PCR in Gasket CE channel Gasket Pin PCB PCB PCR-out PCR channel PCR sample PCR sample PCR-out Pin CE channel Figure 2.24 Integrated µPCR-CE: Integrated setup at the time of PCR reaction Pressure PCR sample PCR sample CE channel Gasket PCR channel PCR-out CE channel Figure 2.25 Integrated µPCR-CE: Integrated setup at the time of CE separation 2.2.3.2 Nucleic Acid Amplification and Separation The integrated device eliminates the need for sample handling after PCR reaction, which increases analysis speed and reduces the amount of sample needed The integrated devices developed can be disassembled, cleaned and reused several times Different combinations in terms of size or length of the channel can be made according 51 Chapter to the analytical needs, which altogether make this type of integration very practical and versatile The transfer of PCR product to the electrophoretic channels is easily performed by applying a slight pressure on the inlet hole of the PCR chamber This was performed by hand but could be automated as well The amplification conditions were first developed using plasmid DNA Consequently, following the developed conditions, real sample analysis of amplified genomic DNA was performed The analysis results obtained when working under integration conditions are equivalent to 12 11 350 Voltage [mV] those working in non-integration conditions (Figure 2.26 and 2.27) 10 Migration time [minutes] Figure 2.26 Electrophoretic separation of micro PCR amplified male chicken genomic DNA on microfabricated devices 52 350 12 11 368 Voltage [mV] Chapter 10 Migration time [minutes] Figure 2.27 Electrophoretic separation of micro PCR amplified female chicken genomic DNA on microfabricated devices The system and analytical conditions developed could be applied to other bird species and sample of interest as well The following figures show the results obtained for the Voltage [mV] analysis of pigeon µPCR amplified genes (Figures 2.28, 2.29) 12 11 10 8 Migration time [minutes] Figure 2.28 Electrophoretic separation of micro PCR amplified male pigeon genomic DNA 53 Voltage [mV] Chapter 80 60 40 20 Migration time [minutes] Figure 2.29 Electrophoretic separation of micro PCR amplified female pigeon genomic DNA 2.2.3 Summary Separation conditions of Z and W genes were optimized with conventional capillary electrophoresis Various coating materials were investigated; coating of the capillaries with PVP gave similar efficiencies compared to covalent coating with PA to reduce the EOF In addition, PVP is advantageous for separations on microfabricated devices since acrylamide is toxic and covalent coatings are prone to deterioration, and a dynamic coating with PVP was easy to perform on chips Various materials were tested for on-chip separation, and separation on silicon microchips gave better results than with PDMS microchips The separation conditions developed with conventional CE were transferred to microfabricated CE The separation of genes of interest was performed in less than minutes on glass chips Microfabricated CE chips were integrated with microfabricated PCR for fast amplification and separation This system is extremely versatile, could be applied to other genes or samples of interest and could 54 Chapter easily be combined with other microscale devices for the development of an integrated “lab-on-a-chip” 2.3 Experimental Section 2.3.1 Instrumentation 2.3.1.1 Conventional Capillary Electrophoresis CE was performed using a PRINCE CE instrument (PRINCE Technologies, Emmen, The Netherlands) equipped with a LIF detector system using a 488 nm argon ion laser (Omnichrome, Palo Alto, CA, USA) Separation was performed in the reverse polarity mode with the injection side at the cathode end of the capillary The instrument was controlled by a personal computer using PRINCE and DAX data acquisition softwares (PRINCE Technologies, Emmen, The Netherlands) The temperature was maintained at 25 ± 1°C Prior to each individual run, the capillary was flushed with the polymer solution Electrokinetic injection of the DNA sample or ladder were performed at 2.3 kV for 10 s, irrespective of the electric field of the separation run Capillaries (70 cm long, 50 µm ID, 365 µm OD, Polymicro Technologies Inc, Phoenix, AZ, USA) were coated with linear polyacrylamide using a modification of the procedure published by Hjerten.36 55 Chapter 2.3.1.2 Microfabricated Electrophoretic Devices Fabrication 2.3.1.2.1 Glass Microfabricated devices The separation channel was 8.55 cm in length (0.5 cm from buffer reservoir to the injection cross and cm from the injection cross to the buffer waste reservoir) The effective separation length was cm The injection channel was cm (0.5 cm from the sample reservoir to the injection cross and 0.5 cm from the sample waste reservoir to the injection cross) The separation channel width was 50 µm and the injection channel width was 50 µm All the channels were 20 µm deep The chip used had an injection cross offset of 100 µm 2.3.1.2.2 Poly(dimethylsiloxane) Electrophoretic Microfabricated Devices The silicon wafers (5” wafers with a positive relief pattern serving as molding templates) were fabricated using standard photolithographic and wet chemical etching process The silicon wafers were silanized in mM octadecyltrimethylcholorosilane (OTS) in toluene for hours to allow easy removal of the PDMS replica from the master The silicon wafers were thoroughly rinsed with toluene and ethanol after silanization The master wafer was then carefully dried with a stream of dry nitrogen and mounted onto a glass petri dish A 10: mixture of polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning, Midland, USA) and its curing agent was stirred thoroughly and then degassed The prepolymer mixture was poured over the wafer (Figure 2.30) After curing at 90 °C for two hours, the PDMS replica was carefully peeled off the silicon master wafer In order to form a closed network, a thin slab of PDMS was permanently bonded to the structure: a small amount of fresh and uncured PDMS was spread across the thin slab of PDMS evenly by simply using a ruler or pen- 56 Chapter knife to brush across the surface This was done to smoothen and remove excess PDMS from the surface Holes that served as reservoirs and provide access to the channels were punched through the blank PDMS slabs before placing on top of the PDMS slabs with channels The whole device was then placed in an oven for hours to cure at a temperature of 90 °C After curing, a tight and irreversible bond was formed between the slabs of PDMS and a closed network was formed Master generation by standard lithographic techniques Silanization Pouring PDMS sol.+ curing agent over the mast Curing PDMS in oven Releasing of channels with master’s topography Sealing the channels with a second flat PDMS sheet Figure 2.30 Process flow for fabrication of the PDMS microchips 2.3.1.2.3 Detection systems Sensitive detection schemes are essential in microfabricated devices for CE due to the extremely small size of the detection cell Laser Induced Fluorescence (LIF) is so far the most popular detection method for CE chips due to its sensitivity Separations were monitored using a laser-excited confocal fluorescence detection system as shown in Figure 2.31 A Leica episcopic fluorescence microscope (DAS Mikroskop DMLS, Leica, Heerbrugg, Switzerland) and its accessories were modified for this purpose The 57 Chapter excitation radiation source was the line of a 488 argon ion laser (Omnichrome, Palo Alto, CA, USA) It was directed perpendicular to the microchannels by a 510 nm dichroic beam splitter and focused within the detection point of the channel using a Leica PL FLUOTAR long working distance objective (magnification, 63; numerical aperture, 0.70) A barrier filter (BA 515 nm) filtered the fluorescence emission collected perpendicular to the channels through the same objective The detector, a Hamamatsu photomultiplier was assembled and loaned from CE Resources Pte Ltd (Singapore) The analogue signal was sampled at 10 Hz A computer controlled high voltage power supply capable of delivering four independent potentials was obtained from CE Resources Pte Ltd (Singapore) Electrical contact from the power supply and the electrolyte solution was performed through platinum electrodes inserted in the reservoirs Photomultiplier Convex lens High pass filter Dichroic mirror Objective lens Argon ion laser 488 nm Microchannel Figure 2.31 Schematic representation of an epi-fluorescence detection system 2.3.1.3 Micro PCR The micro PCR chip shown in Figure 2.24 was fabricated at the Institute of MicroElectronics (Singapore) using standard lithographic techniques Micro PCR chamber (10 µl volume) and micro CE were coupled through a PDMS gasket with the 58 Chapter same thickness to that of the µ-PCR chamber glass cover and with a center hole of the same diameter to that of the microchip sample reservoir The microchip device has the sample reservoir hole made through for connecting with the µPCR from below (Figures 2.24 and 2.25) Due to the good adhesion properties of the PDMS to glass surfaces, a good seal was obtained and no apparent leak was observed throughout the experiments 2.3.2 Chemicals and Materials 2.3.2.1 Conventional Capillary Electrophoresis The capillaries used for DNA separation were coated following the protocol initially described by Hjertén.36 Channels were sequentially flushed with 1.0 M NaOH for h, water for 45 min, and a solution of 0.4 v/v % aqueous solution of γmethacryloxypropyltrimethoxysilane (γ-MTMS) for h The pH of the solution was adjusted to 3.5 with acetic acid and sonicated for two minutes After reaction, the channel was rinsed with water for Linear polyacrylamide was attached to the silanized surface by flushing a solution of 0.4 w/v % aqueous solution of acrylamide with 0.1 w/v % of ammonium persulfate and 0.01 v/v % of TEMED The solution was allowed to react in the channel for h The unreacted polyacrylamide was removed by flushing the channel with water for Channels prepared in this manner were flushed dried and stored overnight before use The run buffer consisted of 89 mM Tris (Bio-Rad), 89 mM acetic acid, mM disodium EDTA (Bio-Rad) (1×TAE), 0.75 % (w/v) hydroxyethylcellulose (type “Celllosize-WP-40”; 75-125 mPa × s for a % w/w aqueous solution, 20 ºC) (HEC) (Fluka) and 0.25 µg/mL Thiazole Orange (Aldrich, Milwaukee, MI, USA), pH 8.2 59 Chapter The run buffer was filtered through a 0.45 µm filter (Millipore, Bedford, MA, USA) and sonicated before use The 25 bp ladder was purchased from Life Technologies Inc (Gaithersburg, MD, USA) It consisted of fragments ranging from 25 to 500 bp at a concentration of µg/µL and was diluted with water to a final concentration of 10 ng/µL 2.3.2.2 Microchip-based Capillary Electrophoresis Separations For microchip-based experiments the same coating protocol and separation buffer were used Polyvinylpirrolidone (PVP) was also used as dynamic coating, in this case the separation buffer consisted of 89 mM Tris (Bio-Rad), 89 mM acetic acid, mM disodium EDTA (Bio-Rad) (1×TAE), 0.5% w/v hydroxypropylmethyl cellulose (HPMC), 0.5 % PVP and 0.4 % manitol and 0.25 µg/mL Thiazole Orange (Aldrich, Milwaukee, MI, USA), pH 8.2 The run buffer was filtered through a 0.45 µm filter (Millipore, Bedford, MA, USA) and sonicated before use 2.3.2.3 Micro PCR Amplification Micro PCR experiments were performed by Prof Lim Tit Meng’s lab, Department of Biological Sciences, NUS The micro PCR condition for both male and female chicken DNA are as follows (in final concentrations): buffer: 50 mM KCl, 10 mM Tris-HCl (pH 8.8 at 25oC), and 0.1% Triton X-100, MgCl2: 3.0 mM, dNTP: 0.2 mM each primers: µM each, Taq DNA polymerase: 0.025 U/µl and DNA template: 20-30 ng/µl The µ PCR thermal cycling profile for both male and female bird genomic DNA is: initial denaturation at 94oC for 120 sec, 36 cycles of 94oC for 35sec, 48oC for 50 sec, 72oC for 50 sec and a final extension at 72oC 480 sec 60 Chapter 2.3.2.4 Integrated Micro Capillary Electrophoresis Separations and micro PCR Amplification For the operation of the integrated system, µPCR-µchip devices were pre-filled in advance with the micro PCR mix with a thin layer of oil above the PCR mix to minimize evaporation The micro CE chip was filled with the sieving buffer described above To transfer the DNA PCR amplified product to the microchip sample reservoir, a small pressure was applied to the inlet hole of the PCR chamber Once the PCR product was in the sample reservoir, the entire µPCR-µchip device was placed in the detection system and electrophoresis was performed 2.4 References Schwer, C.; Kenndler, E Anal Chem 1991, 63, 1801 Chen, Y.; Pepin, A Electrophoresis, 2001, 22, 187 Anderson, J R.; Chiu, D T.; Jackman, R J.; Cherniavskaya, O.; McDonald, J C.; Wu, H.; Whitesides, S H.; Whitesides, G M Anal Chem 2000, 72, 3158 Martynova, L.; Locascio, L E.; Gaitan, M.; Kramer, G W.; Christensen, R G.; MacCrehan, W A Anal Chem 97, 69, 4783 McCormick, R M.; Nelson, R J.; Alonso-Amigo, M G.; Benvegnu, D J.; Hooper, H H Anal Chem 97, 69, 2626 Roberts, M A.; Rossier, J S.; Bercier, P.; Girault, H Anal Chem 97, 69, 2035 Jacobson, S C.; Hergenröder, R.; Koutny, L B.; Warmack, R J.; Ramsey, J M Anal Chem 1994, 66, 1107 61 Chapter Jacobson, S C.; Hergenröder, R.; Koutny, L B.; Ramsey, J M Anal Chem 1994, 66, 1114 Jacobson, S C.; Koutny, L B.; Hergenröder, R.; Moore, A W.; Ramsey, J M.; Anal Chem 1994, 66, 3472 10 Jacobson, S C.; Hergenröder, R.; Moore, A W.; Ramsey, J M Anal Chem 1994, 66, 4127 11 Woolley, A T., Hadley, D.; Landre, P.; deMello, A J.; Mathies, R A.; Northrup, M A Anal Chem 1996, 68, 4081 12 Oda, R P.; Strausbauch, M A.; Huhmer, A F R.; Borson, N.; Jurrens, S R.; Craighead, J.; Wttstein, P J.; Eckloff, B.; Kline, B.; Landers, J P Anal Chem 1998, 70, 4361 13 Waters, L C.; Jacobson, S C.; Kroutchinina, N.; Khandurina, J.; Foote, R S.; Ramsey, J.M Anal Chem 1998, 70, 5172 14 Khandurina, J.; McKnight, T E.; Jacobson, S C.; Waters, L C.; Foote, R S.; Ramsey, J.M Anal Chem 2000, 72, 2995 15 Kopp, M U.; De Mello, A J.; Manz, A Science 1998, 280, 1046 16 Burns, M A.; Johnson, B N.; Brahmasandra, A S N.; Handique, K., Wesbter, J R.; Krishan, M.; Sammarco, T S.; Man, P S.; Jones, D.; Helsinger, D.; Mastrangelo, C H.; Burke, D T Science, 1998, 282, 484 17 Lagally, E T.; Medintz, I.; Mathies, R A Anal Chem 2001, 73, 565 18 Northrup, M A.; Benett, B.; Hadley, D.; Landre, P.; Lehew, S.; Richards, J.; Statton, P.; Anal Chem 1998, 70, 918 19 Belgrader, P.; Benett, W.; Hadley, D.; Richards, J.; Statton, P.; Mariella, R.; Milanovich, F Science, 1999, 284, 449 62 Chapter 20 Giordano, B C.; Ferrance, J.; Swedberg, S.; Hühmer, A F R.; Landers, J P Anal Biochem 2001, 291, 124 21 Nagai, H.; Murakami, Y.; Yokoyama, K.; Tamiya, E Biosens Bioelectron 2001, 16, 1015 22 Zhu, H.; Clark, S M.; Benson, S C.; Rye, H S.; Glazer, A N.; Mathies, R A Anal Chem 1994, 66, 1941 23 Lee, L G.; Chen, C H.; Chiu, L A Cytometry 1986, 7, 508 24 Rye, H S.; Quesada, M A.; Mathies, R A.; Glazer, A N Nucleic Acids Res 1990, 19, 327 25 McCord, B R.; McLure, D L.; Jung, J M J Chromatogr A 1993, 652, 75 26 Marino, M A.; Devaney, J M.; Davis, P A.; Smith, J K.; Girard, J E Anal Chem 1998, 70, 4514 27 Schwartz, H E.; Ulfeder, K J Anal Chem 1992, 64, 1737 28 Berka, J.; Pariat, Y F.; Muller, O.; Hebenbrock, K.; Heiger, D N.; Foret, F.; Karger, B L Electrophoresis 1995, 16, 377 29 Clark, C M.; Mathies, R A Anal Chem 1997, 69, 1355 30 Ogston, A G J Chem Soc., Faraday Trans 1954, 58, 54 31 Rodbard D.; Chrambach, A Proc Natl Acad Sci U.S.A 1970, 65, 970 32 Manabe, T.; Chen, N.; Terabe, S.; Yohda, M.; Endo, I Anal Chem 1994, 66, 4243 33 Grossman, P D.; Soane, D S Biopolymers 1991, 31, 1221 34 Lerman, L S.; Frisch, H L Biopolymers 1988, 21, 995 35 DeGennes, P G Scaling Concepts in Polymer Physics, Cornell University Press, Ithaca, NY, USA, 1979 36 Hjerten, S J J Chromatogr 1985, 347, 191 63 Chapter 37 Song, L.; Liu, T.; Liang, D.; Fang, D.; Chu, B Electrophoresis, 2001, 22, 3688 38 Pang, H.-M.; Pavski, V.; Yeung, E S J Biophys Methods, 1999, 41, 121 39 Duffy, D C.; McDonald, J C.; Schueller, O J A.; Whitesides, G M Anal Chem 1998, 70, 4974 40 Effenhauser, C S.; Bruin, G J M.; Paulus, A.; Ehrat, M Anal Chem 1997, 69, 3451 64 ... SiOH Si O Si O CH2 + (MeO)3 Si Si CH3 CONH2 OMe (CH2)3 O (CH2)3 CH2 O CH2 O Si O Si CONH2 CH2 OMe (CH2)3 CH3 TEMED, Persulfate CH3 CH Si SiOH O C CH2 CH3 CH2 CH CONH2 Figure 2. 14 Reaction scheme... 150 20 0 -6 -6.05 -6.1 -6.15 -6 .2 -6 .25 -6.3 Figure 2. 12 Ln(µ) as function of the number of base pairs (N) 40 25 0 Chapter u (*10E4cm2V1s-1) 1.9 1.85 1.8 1.75 1.7 1.65 1.6 1.55 1.9 2. 1 2. 3 2. 5 2. 7... throughput PCR amplification in a silicon based array .21 2. 2 Results and Discussion 2. 2.1 Method Development Based on Conventional Capillary Electrophoresis 2. 2.1.1 Optimization of Separation Condition