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

Abstract

An optical sensor system based on evanescent field excitation of fluorophore-labeled DNA-targets specifically binding to immobilized DNA probes has been developed, thus enabling for real-time analysis of hybridization events. Oligonucleotide probes are directly immobilized on the surface of the disposable sensor chip via biotin/neutravidin linkage and hybridize to complementary Cy5-labeled target DNA in the sample; this is recorded as an increase in the fluorescence signal. Under optimized conditions the hybridization rate was constant and directly proportional to the target concentration. When an 18mer oligonucleotide was used as a probe a linear calibration curve was obtained for a 56mer single-stranded DNA target derived from the neomycin phosphotransferase gene, a selection marker in a variety of genetically modified plants, with an estimated lower limit of detection of 0.21 nmol L(-1). No cross-hybridization to a 51mer 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 channel. This opens a route toward a real-time DNA array format with analysis times as short as 1-2 min. As a realistic sample a Cy5-labeled 56 bp PCR product 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.

Electrochemical Sensors for DNA Interactions and Damage

“…Electrochemical DNA biosensors are expected to find wide application in clinical praxis, food analysis, monitoring of the environment, etc. Utilization of the results of contemporary research in the field (reviewed in this article) in connection with flowthrough [77] and microfluidic [157] systems and/or with DNA chip technology [158] in construction of inexpensive, versatile, highly sensitive and selective biosensing systems can be expected in the near future.…”

Genetic and OtherDNA‐Based Biosensor Applications

“…Selection markers of genetically modified plants were used to investigate the usefulness of a piezoelectric sensor for label-free detection, 49,50 while an optical sensor was described that monitors hybridization events with fluorescent-labeled target sequences in real time. 51 In another study, an electrochemical detector was optimized using a specific ssDNA target of transgenetic corn. 52…”

Single Molecular Detection via Micro-Scale Polymeric Opto-Electro-Mechanical Systems

“…TIRFM enables the determination of surface molecular dynamics of single molecules through the detection of a thin evanescent wave at a glass/liquid interface. TIRM has been successfully utilized in detecting real time DNA surface attachment and hybridization [57,58] and recording molecular motor activity [59].…”