Abstract
In this work, a simple but sensitive electrochemical DNA biosensor for nucleic acid detection was developed by taking advantage of exonuclease (Exo) I-assisted cleavage for background reduction and zirconia-reduced graphene oxide-thionine (ZrO2-rGO-Thi) nanocomposite for integral DNA recognition, signal amplification, and reporting. The ZrO2-rGO nanocomposite was obtained by a one-step hydrothermal synthesis method. Then, thionine was adsorbed onto the rGO surface, via π-π stacking, as an excellent electrochemical probe. The biosensor fabrication is very simple, with probe DNA immobilization and hybridization recognition with the target nucleic acid. Then, the ZrO2-rGO-Thi nanocomposite was captured onto an electrode via the multicoordinative interaction of ZrO2 with the phosphate group on the DNA skeleton. The adsorbed abundant thionine molecules onto the ZrO2-rGO nanocomposite facilitated an amplified electrochemical response related with the target DNA. Since upon the interaction of the ZrO2-rGO-Thi nanocomposite with the probe DNA an immobilized electrode may also occur, an Exo I-assisted cleavage was combined to remove the unhybridized probe DNA for background reduction. With the current proposed strategy, the target DNA related with P53 gene could be sensitively assayed, with a wide linear detection range from 100 fM to 10 nM and an attractive low detection limit of 24 fM. Also, the developed DNA biosensor could differentiate the mismatched targets from complementary target DNA. Therefore, it offers a simple but effective biosensor fabrication strategy and is anticipated to show potential for applications in bioanalysis and medical diagnosis.
Highlights
Sensitive detection of nucleic acid analytes is always pursued by researchers to accommodate the ever-increasing demands in disease diagnosis, environmental and food monitoring, and forensic identification (Debouck and Goodfellow, 1999; Liu et al, 2008; Zhang et al, 2013; Chen et al, 2019a)
The fabrication principle of the electrochemical nucleic acid biosensor by using ZrO2-rGO-Thi nanocomposites is illustrated in Scheme 1
The zirconia-reduced graphene oxide (ZrO2rGO) nanocomposites were firstly prepared by a hydrothermal synthesis method, with the use of GO and ZrOCl2·8H2O as reactants
Summary
Sensitive detection of nucleic acid analytes is always pursued by researchers to accommodate the ever-increasing demands in disease diagnosis, environmental and food monitoring, and forensic identification (Debouck and Goodfellow, 1999; Liu et al, 2008; Zhang et al, 2013; Chen et al, 2019a). Enzyme-free nucleic acid assembly strategies such as catalytic hairpin assembly, hybridization chain reaction, and DNA-fueled target recycling are usually based on the cascade toehold-mediated strand displacement reactions for signal amplification toward target DNA recognition events (Lv et al, 2015; Ding et al, 2018; Karunanayake Mudiyanselage et al, 2018) These nucleases or enzyme-free DNA strategies could substantially improve the detection limit of the target DNA, the relatively complex or rigorous sequence design or the use of too much DNA fragments or various nucleases for signal amplification increases the assay cost and the risk for error readouts. Development of simple and effective signal amplification means for electrochemical nucleic acid detection is still in high demand
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