DNA Amplification and Hybridization Assays in Integrated Plastic Monolithic Devices

Abstract

PCR amplification, DNA hybridization, and a hybridization wash have been integrated in a disposable monolithic DNA device, containing all of the necessary fluidic channels and reservoirs. These integrated devices were fabricated in polycarbonate plastic material by CO2 laser machining and were assembled using a combination of thermal bonding and adhesive tape bonding. Pluronics polymer phase change valves were implemented in the devices to fulfill the valving requirements. Pluronics polymer material is PCR compatible, and 30% Pluronics polymer valves provide enough holding pressure to ensure a successful PCR amplification. By reducing the temperature locally, to approximately 5 degrees C, Pluronics valves were liquefied and easily opened. A hybridization channel was made functional by oligonucleotide deposition, using Motorola proprietary surface attachment chemistry. Reagent transport on the device was provided by syringe pumps, which were docked onto the device. Peltier thermal electrical devices powered the heating and cooling functionality of the device. Asymmetrical PCR amplification and subsequent hybridization detection of both Escherichia coli K-12 MG1655 and Enterococcus faecalis DNAE genes have been successfully demonstrated in these disposable monolithic devices.

Self-Actuated, Thermo-Responsive Hydrogel Valves for Lab on a Chip

“…The latter types of valves operate with specialized fluids that change their viscosity in response to external stimuli such as temperatureresponsive pluronic solution [17] and a ferrofluid that responds to magnetic-field variations [18].…”

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

“…A miniaturized bioreaction system presents several advantages over the bench-top equivalent: reduced reagent, labor and equipment costs, decreased reaction time, reduced risk of contamination, and simplified sample handling. 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. 4,7,11,12,[17][18][19][20]23,39 One technical challenge in miniaturizing bioreaction systems is preventing or reducing evaporative loss during thermocycling. Although mineral oils, [4][5][6][7][8][9][10][11][12][13][14][15][16]10,26 adhesive tapes 19,22 and silicone rubber gaskets 21 have all been used in miniaturized bioreaction devices, most integrated systems reported so far have used microvalves to prevent evaporative loss.…”

“…Although mineral oils, [4][5][6][7][8][9][10][11][12][13][14][15][16]10,26 adhesive tapes 19,22 and silicone rubber gaskets 21 have all been used in miniaturized bioreaction devices, most integrated systems reported so far have used microvalves to prevent evaporative loss. These microvalves seal the reaction chamber using pneumatically or mechanically actuated diaphragms, [7][8][9]11,15,[17][18][40][41][42][43] thermally actuated phase-change pistons, 12,19,39,44 or polyacrylamide gels. 19 Because all microvalves need to provide some kind of physical confinement and most require some kind of actuations to operate, they often add complexity to the microfabrication and fluidic operations.…”

Electrochemical and Laser Deposition of Silver for Use in Metal-Enhanced Fluorescence

“…25 It is anticipated that many of these approaches will find uses in medical diagnostics and lab-on-achip-type applications. [26][27][28][29] In our recent studies of metal-enhanced fluorescence, [10][11][12] we deposited silver onto glass surfaces by the chemical reduction of silver nitrate, as was described by Ni and Cotton. 30 However, it seemed biologically valuable to devise methods for localized silver deposition without harsh reagents.…”