Immobilization-free, split-mode cathodic photoelectrochemical strategy combined with cascaded amplification for versatile biosensing.

We propose herein an immobilization-free, split-mode cathodic photoelectrochemical (PEC) strategy coupled with a cascaded amplification for versatile biosensing. Taking DNA and microRNA (miRNA) as the model targets, the hybridization between the targets and the hairpin probe triggers the digestion of the probe DNA by T7 exonuclease (T7 Exo), thus to generate G-quadruplex (G4) forming sequences, and then the released targets (DNA or miRNA) initiate the subsequent cycling processes and generate a large amount of G4 forming sequences. Subsequently, the formed G4 sequences associate with hemin to form the G4/hemin DNAzyme, which catalytically produces 1,4-bezoquinone (BQ) for conjugating onto the surface of the chitosan (CS) deposited BiOI/ITO photocathode via the quinone-chitosan conjugation chemistry (QCCC). Under photo excitation, the covalently attached quinones can act as electron acceptors of bismuth oxyiodine (BiOI), promoting the photocurrent generation and thus allowing the elegant and "signal-on" mode for probing targets of interest. Highly sensitive and selective PEC bioassays are readily realized, with the detection limits down to 2.2 fM (for DNA) and 0.2 fM (for miRNA). Since no labeling and no electrode modification processes are needed, this split-mode PEC biosensing strategy is amenable to convenient, time/labor saving, and high-throughput detections. More significantly, it provides a novel concept to design immobilization-free and label-free cathodic PEC biosensing systems, and showcases promise in general and versatile bioanalysis research.