Target-induced recycling and self-folding hairpin primer-mediated LAMP activation of CRISPR/Cas12a for highly sensitive aptamer-based therapeutic antibody assay

Abstract

Owing to the high affinity and specificity for antigen target molecules, therapeutic monoclonal antibodies (mAbs) have been increasingly used for the treatment of different diseases. And, the sensitive and accurate detection of mAbs is crucial for the evaluation of their efficacy and safety. With the new design of a thiophosphate-modified and self-folding hairpin primer, we describe here the establishment of an aptamer-based, highly sensitive and simple fluorescent trastuzumab mAb assay method via target-induced recycling and low temperature LAMP activation of CRISPR/Cas12a signal amplifications. Target trastuzumab molecules bind and change the conformation of the hairpin aptamer probes to trigger Bst polymerase-mediated recycling and LAMP reactions with the assistance of hairpin primers to form long dsDNAs containing many protospacer-adjacent motif (PAM) segements. Cas12a/crRNA subsequently associate with these PAMs to exhibit trans-cleavage property to cyclically cut ssDNA reporter molecules to yield considerably magnified fluorescence recovery for trastuzumab detection. Owing to target-recycling, LAMP and Cas12a/crRNA-integrated signal amplifications, low picomolar detection limit (4.17 pM) for trastuzumab is achieved. And, such assay can be applied to trace trastuzumab assay in diluted human serums. Having the distinct advantages of low temperature LAMP with minimal primer involvement, as well as the integration of amplification cascade, such sensing methodology can be employed as robust signal enhancement methodology for detecting various molecular biomarkers for diverse biomedical and biological applications.