Ultrasensitive quantum dots-based DNA detection and hybridization kinetics analysis with evanescent wave biosensing platform

Ultrasensitive DNA detection was achieved using a new biosensing platform based on quantum dots (QDs) and total internal reflection fluorescence, which featured an exceptional detection limit of 3.2 amol of bound target DNA. The reusable sensor surface was produced by covalently immobilizing streptavidin onto a self-assembled alkanethiol monolayer of fiber optic probe through a heterobifunctional reagent. Streptavidin served as a versatile binding element for biotinylated single-strand DNA (ssDNA). The ssDNA-coated fiber probe was evaluated as a nucleic acid biosensor through a DNA-DNA hybridization assay for a 30-mer ssDNA, which were the segments of the uidA gene of Escherichia coli and labeled by QDs using avidin-biotin interaction. Several negative control tests revealed the absence of significant non-specific binding. It also showed that bound target DNA could easily be eluted from the sensor surface using SDS solution (pH 1.9) without any significant loss of performance after more than 30 assay cycles. A quantitative measurement of DNA binding kinetics was achieved with high accuracy, indicating an association rate of 1.38×10(6) M(-1) s(-1) and a dissociation rate of 4.67×10(-3) s(-1). The proposed biosensing platform provides a simple, cheap, fast, and robust solution for many potential applications including clinical diagnosis, pathology, and genetics.