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

Owing to their high affinity and specificity for antigen target molecules, therapeutic monoclonal antibodies (mAbs) have been increasingly used for the treatment of different diseases. The sensitive and accurate detection of mAbs is crucial for the evaluation of their efficacy and safety. With a new design of a thiophosphate-modified and self-folding hairpin primer, herein, we described the establishment of an aptamer-based, highly sensitive and simple fluorescent trastuzumab mAb assay method <i>via</i> target-induced recycling and low-temperature LAMP activation of CRISPR/Cas12a signal amplifications. Target trastuzumab molecules bound with and changed 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) segments. Cas12a/crRNA subsequently associated with these PAMs to exhibit <i>trans</i>-cleavage property for cyclically cutting ssDNA reporter molecules and yielding considerably magnified fluorescence recovery for trastuzumab detection. Owing to target-recycling, LAMP and Cas12a/crRNA-integrated signal amplifications, a low picomolar detection limit (4.17 pM) for trastuzumab was achieved. This assay could also be applied to trace trastuzumab in diluted human serums. With the distinct advantages of low-temperature LAMP activation with minimal primer involvement and the integration of an amplification cascade, this sensing methodology could be employed as a robust signal enhancement methodology for detecting various molecular biomarkers for diverse biomedical and biological applications.