Porous GNPs assisted LAMP-CRISPR/Cas12a amperometric biosensor as a potential point of care testing system for...

A simple and ultrasensitive amperometric biosensor is introduced which has the potential to be applied as a point of care test for SARS-CoV-2 monitoring. It was prepared by integrating the reverse transcription loop-mediated isothermal amplification (RT‑LAMP) and CRISPR/Cas12a nuclease activity on a modified gold screen-printed electrode (GSPE). The GSPE is modified with double-end thiolated oligonucleotide reporters conjugated to porous gold nanoparticles (PGNPs) and inserted into a homemade poly-methyl methacrylate cartridge. This biosensor was integrated with a low-cost electronic kit to make a platform with the potential to be applied as a point-of-care testing system. The PGNPs on the reporters create a dense, negatively charged barrier that repels the redox couple of [Fe(CN)₆]^3-/4- from the GSPE surface. Upon the addition of a real sample, followed by LAMP amplification and Cas12a nuclease activity on disposable GSPE, in the presence of SARS-CoV-2, the single-guide RNA binds to the target sequence and activates Cas12a. The activated Cas12a then cleaves the reporters, releasing the PGNPs. This removal of electrostatic hindrance allows the redox couple of [Fe(CN)₆]^3-/4- to approach the positively charged GSPE, enhancing the amperometric signal. This biosensor offers an outstanding detection limit of 143 zM (~ 86 copies/mL) and a linear response from 4.7 to 7062 aM for SARS-CoV-2 real samples. By using double-end thiolated reporters and porous GNPs, this novel testing system makes it possible to minimize the required sample volume and reagent costs.