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[Display omitted] • A new chemiluminescent biosensor is constructed for poly (ADP-ribose) polymerase-1 (PARP-1) assay. • This biosensor is characterized by electrostatic force independence and rapidity. • This biosensor can screen PARP-1 inhibitors and quantify PARP-1 at single-cell level. • This biosensor can rapidly detect PARP-1 in human breast tissues. Poly (ADP-ribose) polymerase-1 (PARP-1) is a multi-domain nuclear enzyme that mediates gene transcription and DNA repair. Accurate detection of PARP-1 is essential to clinical diagnostic. However, conventional PARP-1 assays rely on electrostatic force with the involvement of lengthy and complex construction processes and false positive. Herein, we construct an ultrasensitive chemiluminescent biosensor for rapid detection of PARP-1 in human breast tissues by integrating PARP-1-directed hyperbranched poly(ADP-ribose) (PAR) formation with alkaline phosphatase (ALP)-3-(2′-spiroadamantyl)-4-methoxy-4- (3′'-phosphoryloxyphenyl)-1,2-dioxetane (AMPDD)-mediated chemiluminescence system. Upon activation by dsDNA, PARP-1 cleaves nicotinamide adenine dinucleotide (NAD+), initiating the repeated polymerization of biotinylated ADP-ribose to form the dsDNA- PAR-biotin complex. The subsequent assembly of dsDNA-PAR-biotin complexes onto AuNPs through Au-S covalent bond results in the construction of the AuNPs-dsDNA-biotin nanostructure. AuNPs-dsDNA-biotin nanostructure subsequently captures SA-ALP to form the AuNPs-dsDNA-ALP nanostructure. After centrifugation and separation, the AuNPs-dsDNA-ALP nanostructure initiates the dephosphorylation of AMPPD to produce a high chemiluminescent signal. In this assay, only PARP-1 activated by dsDNA can cleave NAD+, efficiently eliminating background signal. The introduction of frozen method greatly shortens the assay time. Benefiting from high precision of the PARP-1-catalyzed NAD+ cleavage, high efficiency of biotinylated ADP-ribose polymerization reaction, and high signal-to-noise ratio of ALP-AMPPD chemiluminescence system, this biosensor can rapidly and sensitively detect PARP-1 with a detection limit of 2.94 × 10−7 U/μL, accurately screen PARP-1 inhibitors, and quantify PARP-1 at single-cell level. Most importantly, it can differentiate PARP-1 expression between breast cancer patient tissues and healthy person tissues, with promising applications in clinical diagnosis and biomedical research. [ABSTRACT FROM AUTHOR]

Telomerase is an important reverse transcriptase that is in charge of maintaining telomeres length, and the dysregulation of telomerase activity is closely associated with cancers. Accurate measurement of telomerase activity is critical to clinical diagnosis and therapeutics. Herein, by taking advantage of in vivo repair mechanisms and single-molecule detection, we demonstrate the enzymatic DNA repair cascade-driven fluorophore encoding for sensitively sensing telomerase activity in cancer cells. The presence of telomerase enables repetitive addition of (TTAGGG) n to the 3′-OH end of telomerase substrate (TS) primer to generate long telomeric products. Telomeric product then hybridizes with the apurinic (AP) probe to induce the cleavage of AP site in dsDNA by APE1, yielding a ssDNA with a biotin and an OH group at 5′ and 3′ ends and simultaneously releasing the telomeric product which in turn hybridizes with new AP probes to yield numerous ssDNAs. By adding streptavidin-coated MBs, the ssDNAs are captured and subsequently functioned as primers to initiate template-free TdT-catalyzed random incorporation of dATPs and Cy5-dATPs into 3′-OH ends, producing long poly-A chains with multiple Cy5 molecules encoding and finally forming Cy5-ssDNA-MB complexes. After magnetic separation, Cy5-ssDNA-MBs are digested by Exo I, releasing large numbers of Cy5 molecules which can be measured by single-molecule detection. This strategy exhibits excellent specificity and high sensitivity with a limit of detection (LOD) of 1 cancer cell. Moreover, it can be employed to screen inhibitors and discriminate different cancer cell lines from normal cell lines, holding enormous potential in clinical diagnosis and therapeutics. • We demonstrate the enzymatic DNA repair cascade-driven fluorophore encoding for sensing telomerase activity. • This method is based on in vivo repair mechanism and single-molecule detection. • This method exhibits good specificity. • This method exhibits high sensitivity with a limit of detection of 1 cancer cell. • This method can be employed to screen inhibitors and discriminate cancer cell lines from normal cell lines. [ABSTRACT FROM AUTHOR]