Exonuclease I-Hydrolysis Assisted Electrochemical Quantitation of Surface-Immobilized DNA Hairpins and Improved HIV-1 Gene Detection

Abstract

The complete formation of stem-loop (i.e., hairpin) configuration on chip surface is of particular importance for the application of hairpin DNA (hpDNA) in building biosensors for various analytes with optimized performance. We report herein a convenient electrochemical protocol for evaluating the yield of hairpin DNA conformations upon self-assembly on electrode surface. As of the different hydrolysis capability of Exonuclease I (Exo I) toward single-stranded DNA (ssDNA) and hpDNA, we can selectively remove ssDNA from electrode but retain hpDNA strands; based on the changes in the cyclic voltammetric (CV) responses using [Ru(NH)] as redox indicators, we can then determine the fraction of hairpin configurations in mixed DNA self-assembled monolayers (SAMs). It was discovered that the molar fraction of hairpin configuration formed on the surface is considerably lower than that in the binary deposition solution (containing both ssDNA and hpDNA). The accuracy of the Exo I-assisted electrochemical quantitative protocol has been validated by standard DNA hybridization experiments; the relationship between the overall DNA packing density and the yield of hairpin configurations was also evaluated. More importantly, taking HIV-1 gene detection as a trial system, the hpDNA-based biosensor shows significantly improved detection limit and broadened response range upon the background reduction by Exo I-catalyzed hydrolysis.

Enrichment–Stowage–Cycle Strategy for Ultrasensitive Electrochemiluminescent Detection of HIV-DNA with Wide Dynamic Range

“…The method not only had a wide dynamic range spanning 6 orders of magnitude, but also had a low detection limit of 39.81 fM, which was more sensitive than other reported HIV-DNA detection methods (Table S2, SI). ,,,, The higher sensitivity and wider dynamic range was attributed the following: (1) Due to its superparamagnetism, strong magnetic response, and large specific surface area, MBs could not only increase the load of H1 molecules, but also achieve efficient enrichment and separation of HIV-DNA from complex samples; (2) Multiple QDs could be assembled on the surface of each nanosphere, and the prepared ENs could enhance the ECL signals by about 11.3-fold, effectively improving the detection sensitivity as signal labels; (3) The integration of SDA could realize cyclic amplification of the targets and further improved the ECL signals by about 3.77-fold, which was beneficial for low-abundant target detection. The same electrode was repeatedly scanned seven times during signal acquisition (Figure S6, SI).…”

“…Therefore, it is significant to develop sensitive, simple, rapid, and low-cost methods to detect HIV. Until now, many means have been proposed for HIV measurement, such as colorimetric method, electrochemistry, , fluorescence, , and surface plasmon resonance (SPR) . In addition to such methods, ECL is a better alternative analysis tool due to its high sensitivity, simple optical setup, excellent temporal and spatial controllability, and low background signal without the need of a light source.…”

An electrochemical label-free DNA impedimetric sensor with AuNP-modified glass fiber/carbonaceous electrode for the detection of HIV-1 DNA

“…Various electrochemical methods have been developed for the detection of the HIV-1 gene recently. Square wave voltammetry (Zhang et al 2010 ), differential pulse voltammetry (Li et al 2014 ), amperometry (Gao et al 2018 ), electrogenerated chemiluminescence (Poorghasem et al 2016 ) and electrochemical impedance spectroscopy (EIS) (Gong et al 2015 ) techniques were employed to design ultra-sensitive HIV-1 DNA biosensors. Most of them resulted in a limit of detection values of below pM concentrations for pM–nM detection ranges.…”

Photoresponsive Electrochemical DNA Biosensors Achieving Various Dynamic Ranges by Using Only-One Capture Probe

“…Due to their rapid response, simple fabrication, high sensitivity and selectivity, and reliability, electrochemical methods have attracted great attention for the detection of DNA sequences. − E-DNA biosensors have been employed extensively to analyze DNA in clinical diagnoses. − The dynamic range of a biosensor describes the range of target concentrations corresponding to receptor occupancies between 10% and 90% where the electrochemistry can sensitively and specifically recognize the target . E-DNA biosensors with a tunable dynamic range is critical to meet the desirable demand of DNA biomarker detection in clinical samples with different concentrations.…”