Highly reproducible and sensitive electrochemical biosensor for Chlamydia trachomatis detection based on duplex-specific nuclease-assisted target-responsive DNA hydrogels and bovine serum albumin carrier platform

16S ribosomal-RNA (16S rRNA) is often used as an ultrasensitive marker for Chlamydia trachomatis (CT) detection because of its species specificity and high copy number in CT. Robust methods for 16S rRNA detection must be developed to realize the early diagnosis of CT infections. In this work, a highly reproducible and sensitive electrochemical biosensor based on duplex-specific nuclease (DSN)-assisted target-responsive DNA hydrogels and bovine serum albumin (BSA) carrier platform for CT detection was developed. Target rRNA can trigger the DNA hydrogel response, which causes it to be repeatedly cleaved by DSN, ultimately leading to the release of a large amount of horseradish peroxidase-labelled streptavidin (SA-HRP) embedded in the hydrogel beforehand. The released SA-HRP was stably captured by the capture probes that were orderly loaded at the gold electrode with the help of a BSA layer. Then, SA-HRP catalyzed the redox reaction of 3,3′,5,5′-tetramethylbenzidine and H2O2, producing a current signal that can be detected. The current signal was proportional to the concentration of CT 16S rRNA from 10 fM to 25 pM with a detection limit of 5.8 fM (S/N = 3). The signal conversion function of the DNA hydrogel avoids the instability of nonhomogeneous nucleic acid hybridization on the gold electrode surface, and combined with optimization by BSA for capture probe modification, this electrochemical biosensor is highly reproducible with a relative standard deviation of 4.3% for the detection of 10 samples of the same concentration. The proposed strategy provides a highly reproducible and sensitive detection method for the extensive screening of CT.