Double-loop Hairpin Probe And Doxorubicin-loaded Gold Nanoparticles For The Electrochemical Sensing Of MicroRNA

MicroRNA(miRNA)is endogenous,about 21 nucleotides in length,noncoding RNA molecule,which involved in the regulation of gene expression in various life process.Especially in malignant tumors,the expression levels of mi RNA are closely related to tumor progression.Abundant and stable miRNAs are found in blood and other body fluids.The miRNAs expression profiles in body fluids undergo a characteristic change,as well as in cells,in tumor and other pathological conditions.These specific miRNAs expression profiles can constitute "fingerprints" of tumors and other diseases,and become a new noninvasive diagnostic marker for disease prediction,diagnosis and prognosis.Objective:In this study,using miRNA let-7d as a model analyte,a duplex specific nuclease signal amplification(DSNSA)strategy based on the double-loop hairpin probe(DHP)and doxorubicin-loaded gold nanoparticles(AuNPs@Dox)biocomposites are developed for an ultrasensitive and specific electrochemical miRNA biosensor.Methods:1.Preparation of electrochemical biosensor: an electrochemical analysis platform by combining DHP-based DSNSA strategy and doxorubicin-loaded gold nanoparticles(AuNPs@Dox)for ultrasensitive miRNA detection was constructed.Electrochemical impedance spectroscopy(EIS),cyclic voltammetry(CV),transmission electron microscopy(TEM),zeta potential,UV–vis absorption spectra,fluorescence spectrophotometry,and agarose gel electrophoresis were used to characterize each step of the sensor construction.2.Feasibility analysis of electrochemical biosensor and optimization of experimental conditions: the feasibility of biosensor,the volume ratio of AuNPs to Dox,the concentration of MCH and the hybridization time of Cp and S1,the reaction time,concentration and reaction temperature of DSN were studied.3.Performance evaluation of electrochemical biosensor: in the optimized conditions,the detection linear range,sensitivity,stability,reproducibility,selectivity and recovery tests of the biosensor were evaluated.Results:1.The realization of DHP-based DSNSA strategy was verified by agarose gel electrophoresis and fluorescence spectrophotometry.The TEM,UV–vis absorption spectra and zeta potential was adopted to confirm the successful fabrication of AuNPs@Dox biocomposites.The EIS and CV were applied to approve the success of the modification and reaction of the probe on the electrode.2.Under optimal conditions,mi RNA let-7d of different concentration were detected by square wave voltammetry(SWV).The linear correlation between the signal and logarithm of target concentration in the range from1 pM to 10 nM with R2 = 0.993.The limit of detection of 0.17 pM(S/N = 3).3.The fabricated biosensor could retain about 92.6% of its initial current response after two-week storage at 4 °C.The miRNA sensors in the same batcha were used to detect target of different concentrations(10 pM,50 pM,and 200 pM)for 3 times,and the relative standard deviation were 4.55%,4.31%,and 5.03%,respectively.The calculated recoveries of different concentrations were in the range of 92.95–109.84%.Conclusions:This multifunctional and label-free DHP presented excellent selectivity,stability and strong resistance to DSN in DSNSA system.Moreover,this biosensor combined DHP-based DSNSA strategy with the advantages of AuNPs@Dox biocomposites to realize the target-based dual signal amplification.It had improved dramatically detection sensitivity and enabled the ultrasensitive and specific miRNA detection.The novel hairpin probe and advanced nano-biocomposites used in this study would create a great potential for the nucleic acid detection and biosensor fabrication.