An electrostatic force-independent dephosphorylation-driven chemiluminescent biosensor for sensitive and rapid detection of poly (ADP-ribose) polymerase-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 centrifuga-tion 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 poly-merization 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 x 10- 7 U/mu L, 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 diag-nosis and biomedical research.