A chemical toxicity sensor based on the electrochemiluminescence quantification of apurinic/apyrimidinic sites in double-stranded DNA monolayer

Abstract

a b s t r a c tA new chemical toxicity sensor was developed based on the electrochemiluminescence (ECL) quantification of apurinic/apyrimidinic sites (AP sites) in a DNA monolayer with a covalent aldehyde reactive probe (ARP). In the sensor, a uracil-containing DNA duplex was first immobilized on a gold electrode by self-assembly. The duplex was then reacted with uracil-DNA glycosylase (UDG) to convert uracils into AP sites. ARP was employed to tag the AP site with a biotin. After reacting with a ruthenium complex labeled streptavidin, ECL was measured for quantitative analysis. The DNA monolayer was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and chronocoulometry, and its density was measured to be 2.89 × 10 −12 mol/cm 2 . Characterization of the reaction product between ARP and DNA AP sites in solution by nondenaturing polyacrylamide gel electrophoresis and mass spectrometry confirmed successful biotinylation. ECL intensity of the labeled DNA monolayer on the electrode was found to correlate with the number of AP sites, and the detection limit was estimated to be about 1 lesion in 512 DNA bases, which meant that 8.5 fmol AP bases on the electrode were detected. ECL response of the DNA monolayers containing either 8-oxodGuo or methylated bases was very low, indicating that ARP-based AP sites detection method was highly selective. The sensor successfully detected the AP sites in normal DNA induced by methylmethane sulfonate, a carcinogenic chemical. The novel combination of covalent probe and ECL measurement in a sensor configuration therefore provides unique advantages in selectivity and sensitivity, and can be potentially employed in the screening of chemicals for their genetic toxicity.

Ultrasensitive recognition of AP sites in DNA at the single-cell level: one molecular rotor sequentially self-regulated to form multiple different stable conformations

“…9,10 Thus, developing active substrate molecules that can take part in the relevant chemical reactions induced by the AP site is immensely conducive for such site-specic recognition in a DNA sequence. 11,12 To date, combined with nuclear magnetic resonance (NMR) spectrometry, 13,14 mass spectrometry 15,16 and optical techniques, [17][18][19][20] some substrate molecules have been successfully implemented for quantifying AP sites in DNA. [13][14][15][16][17][18][19][20] NMR spectrometry for detecting AP sites is characterized by highly selective signals.…”

“…15,16 Still other substrate molecules with optical signals, such as UV-visible and uorescence signals, only exhibit a single optical signal change at one wavelength. [17][18][19][20] Thus, the stability of the recognition signal (especially uorescence recognition signal) for the AP sites is relatively poor in living cells. In addition, inherent interference from complex living organisms (e.g., pH, hydrophilicity and hydrophobicity) for the stability of the recognition signal becomes difficult to eliminate.…”

Ultrasensitive Apurinic/Apyrimidinic Site-Specific Ratio Fluorescent Rotor for Real-Time Highly Selective Evaluation of mtDNA Oxidative Damage in Living Cells

“…At present, AP site-specific molecules have been developed on different chemical reactions (see Figure ). ,− These molecules have been successfully implemented to quantitate AP sites before mtDNA isolation through optical signal or mass spectrometry signal. However, these derivatives emitting a mass spectrometry signal could not in situ monitor and evaluate mtDNA damage in living cells by detecting AP sites. ,,, Other derivatives possessing optical signal only exhibit the response signal at one wavelength, for example, emitting a green fluorescence for AP sites in DNA. ,,, Thus, they are less selective and sensitive for AP sites in living cells, and inherent interference becomes difficult to eliminate from complex living organisms. ,,, In addition, none of these derivatives can locate the mitochondria to specifically monitor and evaluate mtDNA damage by detection of AP sites in mtDNA.…”