Enzymatic in situ generation of covalently conjugated electron acceptor of PbSe quantum dots for high throughput and versatile photoelectrochemical bioanalysis.

Most of the photoelectrochemical (PEC) bioassays need to immobilize biomolecules on electrodes, which lead to tedious modification processes, damaged biomolecules, as well as crippled sensitivity/accuracy and low throughput of the performances. To overcome these drawbacks, we now introduce an exquisitely split-mode (which separates the bioreaction (performed in microplates) from the PEC detection (conducted in PEC cell)) cathodic photoelectrochemistry for probing versatile biocatalytic events with high throughput. Specifically, the enzymatically in situ generated 1,2-bezoquinone was covalently attached onto the PbSe quantum dots (QDs) modified indium tin oxide (ITO) (ITO/PbSe) photocathode through the connector of chitosan (CS). And the attached 1,2-bezoquinone acted as an efficient electron acceptor to promote the cathodic photocurrent of the ITO/PbSe electrode, enabling us to probe quinones-generating oxidoreductase (by taking horseradish peroxidase (HRP) as a model) coupled biocatalytic cascades including the alkaline phosphatase (ALP)/HRP and the glucose oxidase (GOx)/HRP cascades. Quantitative probing for ALP activity in a wide linear range of 5.0 × 10-3 to 10 U/L with the detection limit of 1.2 × 10-3 U/L was realized. While a wide linear range of 5.0 × 10-8 to 1.0 × 10-4 moL/L with a quite low detection limit of 1.0 × 10-8 moL/L was obtained for the glucose assay. In addition, this testing protocol was also extended to an immunoassay (taking carcinoembryonic antigen (CEA) as an example) using HRP as a catalytic tracer. The developed bioassays show high sensitivity and good selectivity for CEA detection in the linear range from 0.1 pg/mL to 100 ng/mL with a detection limit of 0.02 pg/mL. Moreover, the proposed detection has distinctive merits because it not only avoids the adverse effects of the surface confined biomolecules for crippling the signal transduction, but also it has enhanced throughput.