A cantilever biosensor-based assay for toxin-producing cyanobacteria Microcystis aeruginosa using 16S rRNA

Monitoring of cyanotoxins in source waters is currently done through toxin-targeting assays which suffer from low sensitivity due to poor antibody avidity. We present a biosensor-based method as an alternative for detecting toxin-producing cyanobacteria M. aeruginosa via species-selective region of 16S rRNA at concentrations as low as 50 cells/mL, and over a five-log dynamic range. The cantilever biosensor was immobilized with a 27-base DNA strand that is complementary to the target variable region of 16S rRNA of M. aeruginosa. The cantilever sensor detects mass-changes through shifts in its resonant frequency. Increase in the biosensor's effective mass, caused by hybridization of target strand with the biosensor-immobilized complementary strand, showed consistent and proportional frequency shift to M. aeruginosa concentrations. The sensor hybridization response was verified in situ by two techniques: (a) presence of duplex DNA structure postdetection via fluorescence measurements, and (b) secondary hybridization of nanogold-labeled DNA strands to the captured 16S rRNA strands. The biosensor-based assay, conducted in a flow format (∼ 0.5 mL/min), is relatively short, and requires a postextraction analysis time of less than two hours. The two-step detection protocol (primary and secondary hybridization) is less prone to false negatives, and the technique as a whole can potentially provide an early warning for toxin presence in source waters.