Single-Particle Assay of Poly(ADP-ribose) Polymerase-1 Activity with Dark-Field Optical Microscopy

Poly(ADP-ribose) polymerase-1 (PARP-1), over expression in vast majority of cancer cells, is a potential biomarker for clinical diagnosis. However, very limited detection methods have been developed so far, especially for in situ intracellular imaging. Here, we developed a spectral-resolved single-particle detection method for detection of PARP-1 in vitro and in situ intracellular imaging with dark-field microscopy (DFM). A gold nanoparticle (50 nm) modified with active DNA duplex (Au₅₀-dsDNA) was used as a scattering probe. Under the function of active dsDNA, PARP-1 catalyzed to synthesize the hyperbranched poly (ADP-ribose) polymer (PAR) by using nicotinamideadenine dinucleotide as substrates, forming Au₅₀-dsDNA@PAR. Then, negatively charged PAR adsorbed positively charged AuNPs (8 nm) to form Au₅₀-dsDNA@PAR@Au₈. As a result, a notable red shift occurred in localized surface plasmon resonance scattering spectra of Au₅₀, accompanying with obvious color change. Thus, PARP-1 has been detected with a linear range from 0.2 to 10 mU based on the scattering spectra change. The detection limit was 2 orders of magnitude lower than previously reported methods. Probes showed distinct different colors in cancer cells and normal cells, realizing in situ imaging of intracellular PARP-1 at a single-particle level. Compared with previously reported fluorescence imaging methods, the proposed strategy avoided sophisticated label procedures, which has great potential to be used for clinical diagnosis and PARP-1 inhibitor research.