Enzyme-conjugated hybridization chain reaction for magneto-controlled immunoassay of squamous cell carcinoma antigen with pH meter

摘要

A nanoparticle-based potentiometric immunoassay was designed for the sensitive detection of squamous cell carcinoma antigen(SCCA;cervical carcinoma marker) on a portable pH meter coupling enzyme-labeled hybridization chain reaction(HCR) with two alternating hairpin DNA probes for the signal amplification. Initially, a sandwich-type immunoreaction was carried out between anti-SCCA capture antibody-conjugated magnetic bead and detection antibody/initiator strand-coated gold nanoparticle(AuNP). Then, the HCR reaction was readily executed between two glucose oxidase(GOx)-labeled hairpins through the initiator strand to form numerous GOx concatamers on the AuNP via the long nicked double-helix. The concatenated GOx oxidized glucose into gluconic acid and hydrogen peroxide, thus resulting in the pH change of the detection solution on a handheld pH meter. Several labeling protocols including GOx-antibody, GOx-AuNP-antibody and GOx-HCR-AuNP-antibody were investigated for detection of target SCCA, and improved analytical features were obtained with the immune-HCR assay. Under optimum conditions, the immune-HCR assay exhibited good pH responses for the determination of SCCA at a concentration as low as 5.7 pg/mL. Additionally, the immune-HCR assay had good precision and reproducibility, high specificity, and acceptable accuracy for analyzing human serum specimens.