Optical DNA-sensor chip for real-time detection of hybridization events

An optical sensor system based on evanescent field excitation of fluorophor e-labeled DNA-targets specifically binding to immobilized DNA probes has be en developed, thus enabling for real-time analysis of hybridization events. Oligonucleotide probes are directly immobilized on the surface of the disp osable sensor chip via biotin/neutravidin linkage and hybridize to compleme ntary Cy5-labeled target DNA in the sample; this is recorded as an increase in the fluorescence signal. Under optimized conditions the hybridization r ate was constant and directly proportional to the target concentration. Whe n an 18mer oligonucleotide was used as a probe a linear calibration curve w as obtained for a 56mer single-stranded DNA target derived from the neomyci n phosphotransferase gene, a selection marker in a variety of genetically m odified plants, with an estimated lower limit of detection of 0.21 nmol L-1 . No cross-hybridization to a 51 mer actin DNA target was observed and even a single-nucleotide mismatch led to a negligible signal. A shutter in the readout device enabled separate detection of targets hybridizing to probes immobilized at the inlet and outlet sides, respectively, of the flow channe l. This opens a route toward a real-time DNA array formal with analysis tim es as short as 1-2 min. As a realistic sample a Cy5-labeled 56 bp PCR produ ct was measured after separation of the double-stranded DNA by simple heat denaturation with a detection limit clearly lower than that of traditional gel electrophoresis.