Construction And Application Of Circulating MicroRNAs Electrochemical Biosensing Technology Based On Catalytic Hairpin Assembly And 3D DNA Nanonet Structure

Background and purposeCirculating micro RNAs are classified as small non-coding RNAs secreted from living cells into the internal environment,remaining relatively stable in various bodily fluids,such as blood,urine,bile,cerebrospinal fluid,and pancreatic juice.Owing to their advantages such as tissue-specific origin,rich information content,circulating micro RNAs have been proven to serve as ideal non-invasive biomarkers for cancer and other diseases.Traditional circulating micro RNA detection methods such as PCR,require reverse transcription into c DNA,strict temperature cycling conditions,and low specificity.Therefore,it is urgent to establish a simple and efficient isothermal detection method for circulating micro RNA.While considering the low abundance of circulating micro RNAs in internal environment,circulating micro RNAs biosensor requires more efficient signal amplification strategies to achieve low Limit of detection(LOD).Compared with strategies based on complex material changes,DNA nanomaterials have significant advantages.DNA is not only a genetic information carrier,but also a natural programmable biomaterial that can be used for cascade signal amplification strategies.DNA nanomaterials have better biocompatibility under physiological environment due to their notable self-assembly ability and precise recognition capability.In this study,we proposed a DNA nanomaterial-based dual signal synergistically amplified electrochemical biosensor using cascade of catalytic hairpin assembly(CHA)and three-dimensional(3D)DNA nanonet structure for high-sensitivity and high-specificity detection of circulating micro RNA.Methods1.Design the hairpin sequences and the three ss DNA sequences of X-shaped DNA(X-DNA),use www.NUPACK.Com to analyze the structure of these sequences.2.Validate and analyze the 3D DNA nanonet structure by Polyacrylamide gel electrophoresis(PAGE)and blue dextran solution.3.Validate the feasibility of mi R-21 to initiate CHA and hybridized to 3D DNA nanonet structure in solution by PAGE.4.Construct the rapid and efficient circulating micro RNA electrochemical biosensor based on CHA and 3D DNA nanonet structure.5.Verify the feasibility of the biosensing strategy by differential pulse voltammetry(DPV),and characterize the stepwise fabrication of the biosensor by cyclic voltammetry(CV)and electrochemical impedance spectroscopy(EIS),to prove the dual signal amplification by CHA and 3D DNA nanonet structure.6.Optimize the experimental parameters of electrochemical biosensor based on CHA and 3D DNA nanonet structure:(1)Optimize the concentration of hairpin H1 and H2.(2)Optimize the reaction time and temperature of CHA.(3)Optimize the concentration and incubation time of 3D DNA nanonet structure.7.Assess the detection performance of electrochemical biosensor based on CHA and 3D DNA nanonet structure for target mi R-21:(1)Assess the linear range and LOD of electrochemical biosensors.(2)Assess specificity,stability and repeatability of electrochemical biosensors.(3)Evaluate the clinical application of the electrochemical biosensor.ResultsPAGE and blue dextran solution were used to confirm and analyze the 3D DNA nanonet structure.Subsequently,the biosensor was successfully constructed in solution and electrochemical platforms,which confirmed the feasibility of the dual signal amplification strategies.The optimal experimental parameters of the biosensor were: 2 ?M of concentration of hairpin H1 and H2,1.5 h of CHA reaction time,37 ?of CHA reaction temperature,0.5?M of concentration of 3D DNA nanonet structure,1.5 h of 3D DNA nanonet incubation time;The biosensor with a wide linear range from 10 f M to 1 n M,a low LOD of 3.6 f M.The biosensor had the ability to recognize single-base mutation and other interfering micro RNAs,good stability,repeatability,and practical application potential.ConclusionIn this study,we have proposed a DNA nanomaterial based dual-signal amplification electrochemical biosensor by integration of target-assisted CHA and 3D DNA nanonet structure.The biosensor detected circulating micro RNA effectively and sensitively,which has successfully broadened the application of DNA nanotechnology for bioassays.