The focus of this Bioengineering Research Partnership (BRP) Organization is to bring together a multi-disciplinary team to fabricate novel micro-instrument platforms targeted for detecting mutations in genes associated with certain cancers (colon and breast). Specifically, the mutational analysis tool that we will be developing these miniaturized tools for is PCR/LDR in collaboration with Prof. Francis Barany (Cornell Medical College), which can detect point mutations at a level of 1 mutant DNA in approximately 100 normal DNAs. Our design approach will be to build sub-systems that will carry out the following functions: (1) PCR amplification of the target genes; (2) ligation detection reaction; (3) micro-capillary gel electrophoretic separation of the ligation reaction products; (4) addressable zipcode arrays for detection of ligation products; (5) synthetic preparation of ultra-bright near-IR fluorescent dyes; (6) fabrication of fluorescence readout scanners composed of simple diode lasers and avalanche photodiodes. The devices proposed in this application will be fabricated using high aspect ratio micromachining in plastics (polymethylmethacrylate, PMMA) via LIGA processing. Our expertise in LIGA and the extensive micromachining resources located on-site will allow us to make microstructures with exquisite dimensions. In addition, the choice of substrate material in which we will be fabricating our devices gives us a great deal of flexibility in both machining techniques (X-ray lithography, hot embossing, injection molding) as well as chemistries for immobilizing DNAs not obtainable using conventional glass-based substrates. For example, using PMMA as the substrate material for DNA micro-arrays, amide-type immobilization chemistries are used, which produces a bond that is very robust and stable toward most thermal and chemical denaturation steps used in many hybridization assays. Also, PMMA substrates can be used for electrophoretic analyses of DNAs without the special requirement of wall coatings, extending the operational lifetime of the micro-device. And most importantly, PMMA micro-devices can be fabricated using simple injection molding, which will allow the fabrication of devices for minimal costs at high production rates. The ability to effectively use these type of substrate materials is driven by the use of near-IR fluorescence readout, which alleviates to a great extent high background signals arising from autofluorescence of the substrate or matrix interferences and is conducive to miniaturization. Since the ability to fabricate such devices depends on a number of different expertises, we have assembled a team of researchers (Chemists, Engineers, Life Scientists) that can meet the challenges of this diverse research venture.
