Microfluidic hydrogel arrays for direct genotyping of clinical samples.

ABSTRACT

A microfluidic hydrogel DNA microarray is developed to overcome the limitations of conventional planar microarrays such as low sensitivity, long overnight hybridization time, lack of a melting verification of proper hybrid, and complicated sample preparation process for genotyping of clinical samples. Unlike our previous prototype hydrogel array which can analyze only single-stranded DNA (ssDNA) targets, the device is the first of its type to allow direct multiplexed single nucleotide polymorphism (SNP) detection of human clinical samples comprising double-stranded DNA (dsDNA). This advance is made possible by incorporating a streptavidin (SA) hydrogel capture/purification element in a double T-junction at the start of the linear hydrogel array structure and fabricating ten different probe DNAs-entrapped hydrogels in microfluidic channels. The purified or unpurified polymerase chain reaction (PCR) products labeled with a fluorophore and a biotin are electrophoresed through the SA hydrogel for binding and purification. After electrophoretic washing, the fluorophore-labeled DNA strand is then thermally released for hybridization capture by its complementary probe gel element. We demonstrate the precise and rapid discrimination of the genotypes of five different clinical targets by melting curve analysis based on temperature-gradient electrophoresis within 3h, which is at least 3-fold decrease in incubation time compared to conventional microarrays. In addition, a 1.7 pg (0.024 femtomoles) limit of detection for clinical samples is achieved which is ~100-fold better sensitivity than planar microarrays.