Protein engineering by resurfacing is an efficient approach to provide new bio-molecular toolkits for biotechnology and bioanalytical chemistry.By resurfacing of fluorescent proteins,supercharged green fluorescent proteins(ScGFP)with high surface net charges have been developed.Owing to their intrinsic fluorescence,robust stability,abundant surface functional groups,and cell-penetrating ability,it is attractive to adopt the resurfaced GFPs as biosensors.Therefore,we developed a series of sensitive biosensors based on resurfaced GFPs:(1)Taking ScGFP as the signal reporter,a simple turn-on homogenous fluorescent method for DNA detection has been developed based on the polyionic complex of ScGFP/DNA and toehold strand displacement.(2)Combined with bisulfite conversion,the ScGFP-based assay was applied to analyze site-specific DNA methylation status of genomic DNA extracted from human colon carcinoma tissue samples with ultrahigh sensitivity.(3)A label-free fluorescent sensor was developed for Cu2+ sensing based on the unique multivalent metal ion-binding property of H39GFP and fluorescence quenching effect of Cu2+ by electron transfer.(4)Based on the unique quenching effect of graphene oxide(GO)on ScGFP fluorescence as well as the DNA-mediated ScGFP/GO interaction,a label-free fluorescent method has been established for homogenous analyzing the activity and inhibition of base excision repair enzymes.(5)A label-free and convenient strategy for the assessment of PARP-1 activity and inhibitors has been established based on fluorescence resonance energy transfer(FRET)between cationic conjugated polymer(CCP)and ScGFP.