se0c00264_si_001.pdf (302.5 kB)
Single-Particle Assay of Poly(ADP-ribose) Polymerase‑1 Activity with Dark-Field Optical Microscopy
journal contribution
posted on 2020-04-03, 14:18 authored by Duoduo Zhang, Kan Wang, Wei Wei, Songqin LiuPoly(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.