An ultrasensitive DNA biosensor based on enzyme-catalyzed deposition of cupric hexacyanoferrate nanoparticles

An ultrasensitive electrochemical biosensor is demonstrated for the detection of a synthetic single-stranded DNA (ss-DNA) with a detection limit down to femtomolar levels. The synthetic ss-DNA with sequence associated with the influenza A virus RNA was used as our model target DNA. The detection scheme was based on a sandwich structure involving a capture probe, the target DNA and a detection probe. An avidin-labeled glucose oxidase (GOx) was attached to a biotinylated detection probe via biotin–avidin interaction. Subsequently, the DNA hybridization event was transduced and amplified by an in-situ enzyme-catalyzed deposition of cupric hexacyanoferrate (CuHCF) nanoparticles in the presence of glucose, cupric ions and ferricyanide. The peak current in differential pulse voltammetry of the deposited CuHCF nanoparticles directly correlated to the concentration of the target DNA. DNA was therefore quantified in the concentration range from 1.0 fM to 10 pM with a detection limit of 1.0 fM and a relative standard derivation of ≤11% at 100 fM. Excellent mismatch discrimination was demonstrated in the proposed biosensor.