Constructing A Sandwich Structure Electrochemical DNA Sensor Based On RGO/AuNPs Composite Nanomaterials For Viral Nucleic Acid Detection

Viral infectious diseases represented by viral hepatitis,AIDS and COVID-19（Corona Virus Disease 2019）pose a serious threat to human health.Therefore,rapid,sensitive and accurate pathogen detection is very important for effective prevention and control of viral infectious diseases.Point-of-care testing（POCT）based on viral nucleic acid has the advantages of simple operation,short detection time and weak dependence on testing sites,playing an important role in the early diagnosis and epidemic prevention and control of viral infectious diseases.Electrochemical DNA sensors is a kind of biosensor that fixes DNA probes on substrate surface to identify complementary target sequences and converts sequence recognition hybrid signals into electrical signals through signal conversion device.In the aspect of virus nucleic acid detection,POCT technology has become the most promising technology for its high sensitivity,simple operation,low cost and easy miniaturization.Nucleic acid detection strategy based on"sandwich"structure has gradually become the mainstream detection method for biological detection and clinical diagnosis due to its high sensitivity and specificity.The detection basis of electrochemical DNA sensors is the hybridization reaction between probes and target nucleic acid sequences.The hybridization efficiency between probes and target sequences directly determines the specificity and sensitivity of electrochemical DNA sensors.In the sandwich hybridization strategy,the target sequences to be examined bridge the capture probes（CP）and signal probes（SP）in a seamless manner to form the sandwich hybrid complexes.The length of probes（CP and SP）significantly affected the hybridization efficiency.At the same time,the hybridization of two oligonucleotides（CP and SP）in successive series with longer complementary single-stranded nucleic acids provides additional stability due to base stacking effect.Optimizing the length of probes（CP and SP）to obtain the maximum base stacking effect can further improve the hybridization efficiency.Therefore,how to select and design appropriate probes for specific target sequences of different viruses to obtain the maximum hybridization efficiency and improve the sensitivity and specificity of"sandwich"structure electrochemical DNA sensors is worth further exploration.The detection performance of electrochemical DNA sensors is also closely related to the conduction interface,which has an important influence on the thermodynamics and kinetics of biomolecule assembly,binding and signal transduction.Nanostructured interfaces modified by nanomaterials have large specific surface area and excellent electrical properties,which can provide favorable dynamic hybridization conditions for nucleic acid hybridization.Therefore,they are widely used in electrochemical DNA sensors to effectively improve the performance of the sensors.Graphene and its derivatives,graphene oxide（GO）and reduced graphene oxide（rGO）,are widely used in electrochemical biosensors because of their large specific surface area,excellent electrical conductivity,strong mechanical and thermal strength,high chemical stability,and good biocompatibility.Compared with graphene and graphene oxide（GO）,rGO has-COOH,-OH and-NH₂groups on its surface,which can provide higher electrochemical activity,more reaction sites and more efficient charge transfer.rGO can be used as a modification material on sensor electrode surface to improve the detection sensitivity by accelerating electron transfer rate and increasing the immobilization of bioactive molecules.Gold nanoparticles（AuNPs）are often used to modify the sensing interface and as a substrate for bioactive molecules due to their excellent specific surface area,high electrical conductivity,good stability and easy synthesis.When AuNPs is loaded on rGO,the composite nanomaterial modified sensing interface can obtain better detection performance than the single nanomaterial modified sensing interface.In this study,we constructed a sandwich structure electrochemical DNA sensor using rGO/AuNPs composite nanomaterials to detect the Rd Rp gene,a highly conserved sequence of the COVID-19 pathogen SARS-COV-2.Firstly,the optimal combination of specific CP and SP for SARS-Co V-2 Rd Rp sequence was screened on a planner Au electrode based on software simulation and experimental verification,and the feasibility of"sandwich"hybrid combined enzyme amplification strategy（HRP-TMB）for specific detection of SARS-Co V-2 Rd Rp was preliminally verified.Furthermore,the rGO/AuNPs composite nanomaterials modified screen-printed carbon electrode（SPCE）was used to construct an electrochemical DNA sensor for detecting SARS-Co V-2 Rd Rp based on the optimal CP/SP combination obtained in Part 1（11 nt CP/14 nt SP）.Then the interface characterization,electrochemical performance test and optimization of a series of detection conditions were carried out for the SPCE/rGO/AuNPs sensors.The detection limit of the optimized sensor for SARS-COV-2 Rd Rp is as low as 7.13 f M,and it has single-base resolution for highly homologous viral RNA sequences.In addition,the sensor can specifically detect SARS-COV-2 Rd Rp in complex biological samples,so it has certain clinical application potential.