Assay protocol
The detection strategy of the electrochemical-based sensors is based on sandwich hybridization of capture and detector probes with target 16S rRNA. The capture probe is anchored to the gold sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). Capture and detector probes are designed to hybridize to species- and group-specific regions of the 16S rRNA molecule that are accessible for hybridization with oligonucleotide probes. Each sensor on the chip is functionalized with a specific pair of capture and detector probes. When a substrate such as 3,3′,5,5′-tetramethylbenzidine (TMB) is added to an electrode with capture target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to the HRP, producing enzymatic signal amplification of current flow in the electrode. The concentration of the target captured on the sensor surface can be quantified by the current obtained through the redox reaction between the TMB and HRP. Amperometric measurement of the catalyzed HRP reaction is obtained at a fixed potential applied by the built-in potentiostat in the GeneFluidics Lab Automation System between the working and reference electrodes of all 16 sensors. Species-specific genetic sequences can be quantified without the use of PCR on the same sensor array chip with this universal detection approach.
