Ultrasensitive detection of NDM-1 resistant bacteria based on signal amplification with sandwich-type LNA electrochemical biochips

New Delhi metallo-beta-lactamase-1 (NDM-1) is an identified metallo-beta-lactamase that confers resistance to carbapenems and all other beta-lactam antibiotics, with the exception of aztreonam. Herein, a novel electrochemical biochip was constructed based on a zinc oxide (ZnO) and reduced graphene oxide (rGO) complex (ZnO@rGO) supported by Au nanocages (Au NCs) for signal amplification for ultrasensitive detection of NDM-1 in early clinical diagnosis. A Au NC and a sulfhydryl (SH) modified locked nucleic acid-1 (Au NC@LNA-1) probe was synthesized with stable Au-S bonds to on the biochip surface. In hybridization solution, Au NCs and LNA-2 probes modified with SH groups formed stable Au NC@LNA-2 complexes. Au NC@LNA-1 and Au NC@LNA-2 were linked with the NDM-1 to form a Au NC@LNA-1/NDM-1/Au NC@LNA-2 "sandwich-like" complex through complementary base pairing, and this complex can bind additional copies of the target sequence to further amplify it's the detection signal. Under optimized conditions, the system showed a linear range from 1 pg L-1 to 100 mu g L-1, and the detection limit was 0.042 pg L-1. The time of biochip probe-modification was 45 min, incubation was 30 min, and electrochemical scanning detection was 1 min. In addition, the results indicate that the specific LNA probe identified single base mismatches and exhibited affinity for the target DNA molecule, resulting in the direct detection of the NDM-1 in complex clinical bacteria samples without PCR amplification. In conclusion, this novel electrochemical biochip method allows for the rapid, specific, and sensitive detection of the NDM-1, indicating its potential application in disease diagnostics.