Red fluorescence-emitting copper nanoparticles-assisted label-free bioprobe coupled with CRISPR/Cas12a for sensitive detection of T4 polynucleotide kinase

With CRISPR/Cas12a and poly T-templated copper nanoparticles (CuNPs), we built an efficient and label-free fluorescent biosensor for determinating T4 polynucleotide kinase (T4 PNK). Single-stranded DNA (ssDNA) of poly T containing 40 bases (T40) was employed as the template for the synthesis of CuNPs. As a result of the Cas12a/crRNA combining with dsDNA, the Cas12a was activated and capable of cleaving T40 and prevented the synthesis of CuNPs. However, the target T4 PNK could phosphorylate the 5 & PRIME;-OH termini of dsDNA, then dsDNA was further degraded by & lambda; exo, so the Cas12a could not be activated. Therefore, the CuNPs were formed, achieving the detection of T4 PNK in an isothermal and label-free way. As expected, the sensing platform with a low limit of detection of 0.089 U mL-1 was proved to be effective for screening and evaluating the inhibitors of T4 PNK. It is very economic to use T40 as the substrate of Cas12a instead of ssDNA probes labeled with fluorophores and quenchers. And the CuNPs can be synthesized in only five minutes. Considering the flexibility of CRISPR/Cas12a, this universal signal output strategy offers great potential to broaden its application scope for other targets assay, such as DNA, proteins, and small molecules.

Automated summary

We developed an efficient and label-free fluorescent biosensor for detecting T4 polynucleotide kinase (T4 PNK) using CRISPR/Cas12a and poly T-templated copper nanoparticles (CuNPs). The biosensor utilized single-stranded DNA (ssDNA) of poly T as a template for CuNPs synthesis. By combining Cas12a/crRNA with dsDNA, Cas12a was activated and prevented the synthesis of CuNPs. However, T4 PNK phosphorylated the 5' termini of dsDNA, degrading it and preventing the activation of Cas12a. This allowed the formation of CuNPs for the detection of T4 PNK in an isothermal and label-free manner. The sensing platform showed a low limit of detection and potential for broader applications.