Fast and automated DNA assays on a compact disc (CD)-based microfluidic platform.

摘要

Nucleic acid-based molecular diagnostics offers enormous potential for the rapid and accurate diagnosis of infectious diseases. However, most of the existing commercial tests are time-consuming and technically complicated, and are thus incompatible with the need for rapid identification of infectious agents. We have successfully developed a CD-based microfluidic platform for fast and automated DNA array hybridization and a low cost, disposable plastic microfluidic platform for polymerase chain reaction (PCR). These platforms have proved to be a promising approach to meet the requirements in terms of detection speed and operational convenience in diagnosis of infectious diseases.; In the CD-based microfluidic platform for DNA hybridization, convection is introduced to the system to enhance mass transport so as to accelerate the hybridization rate since DNA hybridization is a diffusion limited reaction. Centrifugal force is utilized for sample propulsion and surface force is used for liquid gating. Standard microscope glass slides are used as the substrates for capture probes owing to their compatibility with commercially available instrumentation (e.g. laser scanners) for detection. Microfabricated polydimethylsiloxane (PDMS) structures are used to accomplish the fluidic functions required by the protocols for DNA hybridization. The assembly of the PDMS structure and the glass slide forms a flow-through hybridization unit that can be accommodated onto the CD platform for reagent manipulation. The above scheme has been validated with oligonucleotides as the targets using commercially available enzyme-labeled fluorescence (ELF 97) for detection of the hybridization events, and tested with amplicons of genomic staphylococcus DNA labeled with Cy dye. In both experiments, significantly higher fluorescence intensities were observed in the flow-through hybridization unit compared to the passive assays. The CD fluidic scheme was also adapted to the immobilization of thiolated oligonucleotides on gold surfaces and up to a 2.5 fold increase was observed for the rate of adsorption compared to passive immobilization.; In order to reduce the reaction time for DNA amplification, a miniaturized fluidic platform was developed for rapid polymerase chain reaction (PCR). Commercially available, adhesive-coated aluminum foils and polypropylene films were laminated to structured polycarbonate films forming micro reactors in a card format. Ice valves were employed to seal the reaction chambers during thermal cycling and a Peltier-based thermal cycler was configured for rapid thermal cycling and ice valve actuation. Numerical modeling was conducted to optimize the design of the PCR reactor and explore the thermal gradient in the reaction chamber in the direction of sample depth. The PCR reactor was experimentally characterized by using thin foil thermocouples and validated by a successful amplification of 10 genome copies of E. coli ATCC 35401 tuf gene in 27 minutes. In the future, we will integrate sample preparation, PCR amplification and DNA detection into a single, centrifugal microfluidic disc that is practically affordable for molecular diagnostics.