Electrokinetically Synchronized Polymerase Chain Reaction Microchip Fabricated in Polycarbonate

Abstract

This paper presents a novel method for DNA thermal amplification using the polymerase chain reaction (PCR) in an electrokinetically driven synchronized continuous flow PCR (EDS-CF-PCR) configuration carried out in a microfabricated polycarbonate (PC) chip. The synchronized format allowed patterning a shorter length microchannel for the PCR compared to nonsynchronized continuous flow formats, permitting the use of smaller applied voltages when the flow is driven electrically and also allowed flexibility in selecting the cycle number without having to change the microchip architecture. A home-built temperature control system was developed to precisely configure three isothermal zones on the chip for denaturing (95 degrees C), annealing (55 degrees C), and extension (72 degrees C) within a single-loop channel. DNA templates were introduced into the PCR reactor, which was filled with the PCR cocktail, by electrokinetic injection. The PCR cocktail consisted of low salt concentrations (KCl) to reduce the current in the EDS-CF-PCR device during cycling. To control the EOF in the PC microchannel to minimize dilution effects as the DNA "plug" was shuttled through the temperature zones, Polybrene was used as a dynamic coating, which resulted in reversal of the EOF. The products generated from 15, 27, 35, and 40 EDS-CF-PCR amplification cycles were collected and analyzed using microchip electrophoresis with LIF detection for fragment sizing. The results showed that the EDS-CF-PCR format produced results similar to that of a conventional block thermal cycler with leveling effects observed for amplicon generation after approximately 25 cycles. To the best of our knowledge, this is the first report of electrokinetically driven synchronized PCR performed on chip.

Stop flow lithography in perfluoropolyether (PFPE) microfluidic channels

“…4c and f). The fluorescent 30 signal of particles synthesized in PDMS devices was two times lower than the signal of particles synthesized in PFPE devices, validating that the former particles were thinner than the latter. Also, the particles synthesized in the PDMS device emitted concave fluorescent signals, indicative of deformed particles.…”

“…The composite layer was further cured at 80 ˚C for 45 min. Lastly, the A-rich SIFEL channel was combined 30 with the B-rich SIFEL coated glass at 120 ˚C for 45 min. PDMS channels are generated by pouring PDMS over an SU-8 master, and then curing 2 hrs at 60 ˚C in an oven.…”

“…Also, distinct chemical regions in particles are defined by flow patterns that can be precisely adjusted via microfluidic modulation. The 30 combination of photolithography and microfluidic techniques allows independent control over particle size, shape, and even chemical patchiness. Using this in situ fabrication method, diverse multifunctional particles have been prepared for new applications in the fields of diagnostics [7][8][9][10][11] , assembly [12][13][14] , 35 MEMS 15 , photonics 16 , and tissue engineering 17 .…”

“…Also, we used PFPE-based SFL to synthesize multifunctional particles, showing that the technique can be compatible with the particle synthesis process on the structured microflows. Of particular interest, we demonstrate that PFPE-based SFL allows for the 30 synthesis of anisotropic particles from precursors dissolved in organic solvents, a technique not possible with PDMS-based SFL. As one demonstrative application, we synthesize anisotropic particles from nonpolar organic precursors with stable dispersion of upconverting nanocrystals (UCNs).…”

Microfabricated valveless devices for thermal bioreactions based on diffusion-limited evaporation

“…There are two major types of miniaturized bioreaction systems: batch-based systems where the stationary reaction solution is heated or thermocycled inside a reaction chamber by either external heaters [3][4][5][6][7][8][9][10][11][12][13][14][15][16] or integrated on-chip heaters, [17][18][19][20][21][22][23][24] and continuous flow-based systems where the sample flows through certain temperature zones with well-defined flow rates. [25][26][27][28][29][30] Other novel approaches, such as on-chip rotary reaction, 31 noncontact infrared-mediated reaction, [32][33][34] electrokinetically synchronized reaction, 35 electrowetting-based reaction 36 and Rayleigh-Bénard convection-based reaction 37,38 have also been reported. Recent trends in miniaturized bioreaction systems are to integrate bioreactions with sample preparation, fluidic handling, and product detection to produce systems that can rapidly, conveniently, and economically extract information from raw biological samples with greatly reduced cost.…”