Abstract
A microfluidic chip with an integrated planar waveguide was fabricated in poly(methyl methacrylate), PMMA, using a single-step, double-sided hot-embossing approach. The waveguide was embedded in air on three sides, the solution being interrogated on the fourth. DNA probes were covalently attached to the waveguide surface by plasma activating the PMMA and the use of carbodiimide coupling chemistry. Successful hybridization events were read using evanescent excitation monitored by an imaging microscope, which offered high spatial resolution (2 microm) and a large field-of-view (20 mm diameter field-of-view), providing imaging of the entire array without scanning. The application of the microfluidic/waveguide assembly was demonstrated by detecting low abundant point mutations; insertion C mutations in BRCA1 genes associated with breast cancer were analyzed using a universal array coupled to an allele-specific ligation assay. DNA probes consisting of amine-terminated oligonucleotides were printed inside the microfluidic channel using a noncontact microspotter. Mutant and wild-type genomic DNAs of BRCA1 were PCR (polymerase chain reaction) amplified, with the amplicons subjected to ligation detection reactions (LDRs). LDR solutions were allowed to flow over the microarray positioned on the polymer waveguide with successful ligation events discerned through fluorescence signatures present at certain locations of the array. The microfluidic/waveguide assembly could detect polymorphisms present at <1% of the total DNA content.