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

•A label-free fluorescent biosensor was built for the determination of T4 PNK.•The poly-T ssDNA was utilized as the substrate of Cas12a instead of F-Q probes.•Cas12a combined with T40 can be utilized as a universal signal output tool.•There is a high speed of signal output by using poly-T templated CuNPs.•Red-emitting CuNPs can avoid the background fluorescence in biological matrix.