Flap
endonuclease 1 (FEN1) is a structure-specific nuclease that
cleaves the 5′ single-stranded protrusion (also known as 5′
flap) during Okazaki fragment processing. It is overexpressed in various
types of human cancer cells and has been considered as an important
biomarker for cancer diagnosis. However, conventional methods for
FEN1 assay usually suffer from complicated platform and laborious
procedures with a limited sensitivity. Here, we developed a dual-signal
method for sensitive detection of FEN1 on the basis of duplex-specific
nuclease actuated cyclic enzymatic repairing-mediated signal amplification.
Once the 5′ flap of the double-flap DNA substrate was cleaved
by target FEN1, the cleaved 5′ flap initiated strand-displacement
amplification to produce plenty of G-rich DNA (G) sequences. These
G sequences that self-assembled into G-quadruplexes in the presence
of hemin revealed horseradish-peroxidase-like catalytic activities
as well as fluorescence enhancement of thioflavin T. The UV–vis
signal showed a good linear relationship with the logarithm of FEN1
activity ranging from 0.03 to 1.5 U with a detection limit of 0.01
U. The fluorescence signal correlated linearly with the logarithm
of FEN1 activity ranging from 0.001 to 1.5 U with a detection limit
of 0.75 mU. In addition, FEN1 can be visualized not only by colorimetry
but also by fluorescence (under ice–water mixture conditions).
This reliable, accurate, and convenient method would be a potential
powerful tool in point-of-care testing applications and therapeutic
response assessment.
Poly(ADP-ribose) polymerase-1 (PARP-1) is a highly conserved nuclear enzyme, which binds tightly to damaged DNA and plays a key role in DNA repair, recombination, proliferation, and genomic stability. However, due to the poor electrochemical and optical activity of PARP-1 and its product PAR, only a few studies on its activity detection method have been reported. Herein, we report a simple and sensitive colorimetric strategy to monitor PARP-1 activity based on enzyme-initiated auto-PARylation-controlled aggregation of hemin-graphene nanocomposites (H-GNs). PARP, activated by dsDNA, catalyzed its substrate nicotinamide adenine dinucleotide (NAD+) to polymerize as a poly(ADP-ribose) polymer (PAR). PAR possesses several negative charges, and its charge density is twice that of a single-stranded DNA, which greatly impacts the dispersibility of H-GNs; due to their peroxidase-like catalytic activities, H-GNs can catalyze the chromogenic reaction of TMB and H2O2. As a result, in the presence of different PARP-1 activities, the supernatant of the corresponding solution contained different amounts of dispersed H-GNs and showed different colors after the chromogenic reaction that could be discerned easily by the absorbance or the color changes of the solution. The method was simple, sensitive, and reliable. The proposed method displays a linear range from 0.05 to 1 U with a detection limit of 0.03 U. In addition, this new method has been successfully applied to detect PARP-1 activity in human serum and different cancer cells and evaluate PARP-1 inhibitors.
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