Electrochemical Biosensing Of MicroRNA Based On Multiple Signal Amplification Strategy

Cancer is now one of the leading causes of human death.Many cancer patients have no obvious symptoms in the early stage of the disease,and the diagnosis is already in the middle and late stages,which greatly increases the difficulty of treatment and the mortality.Early detection and treatment of cancer has been recognized as the most effective way of cancer prevention and control,which is of great significance.Microribonucleic acid(mi RNA)plays an important role in the formation of human cancer,which is secreted even in the very early state of cancer cells.Abnormal expression of mi RNA has been confirmed to be highly correlated with the development of most types of cancer,and is an important marker of early diagnosis of cancer.Electrochemical biosensor technology is a kind of technology that combines electroanalytical chemistry and biosensor technology.The detection technology has simple operation steps and efficient detection and analysis results,which has been widely concerned by researchers.However,due to the short sequence and low content of mi RNA,the electrical signals generated by electrochemical detection are weak that cannot be detected accurately.Therefore,signal amplification technology is needed to improve the detection effect of the sensor.Signal amplification technologies based on signal amplification materials and nucleic acid amplification are widely used in electrochemical detection.In order to further improve the sensor sensitivity,researchers have developed a biosensor technology that combines multiple signal amplification technologies to effectively improve the detection effect of mi RNA.Therefore,this paper combines a variety of amplification techniques to construct two electrochemical biosensor strategies with high sensitivity and specificity for mi RNA detection.The specific research contents are as follows:1.A label-free homogeneous electrochemical biosensor based on hybridization chain reaction(HCR)was designed to detect mi RNA-21.Firstly,the target chain initiates the HCR amplification on the surface of the magnetic nanoparticles to form long DNA double strands,to which large amount of the beacon molecule Ruhex is adsorbed through the electrostatic action.Then the reaction solution is removed by magnetic separation that can avoid the signal error caused by the excess Ruhex in the reaction solution.And then the separated magnetic nanoparticles are dispersed in 1x Ne Buffer,exonuclide?(Exo?)was added to cut the long DNA strands to release Ruhex,which combined with the negatively charged Indium tin oxide(ITO)electrode to generate an electrochemical detection signal to achieve the quantitative detection of mi RNA-21.2.A DNAzyme-driven 3D DNA Walker and hyperbranched HCR-DNAzyme cascading signal amplification strategy were designed,and a sensitive electrochemical biosensor for detection of mi RNA-141 was constructed to activate 3D DNA Walker while recognizing the target mi RNA.The free hanging chain of DNAzyme in walker drives the recognition-shear-release cycle to generate more intermediate output chains for the first signal amplification.The released output chain then triggers nonlinear HCR reactions on the electrode surface,forming hyperbranched nanostructures,providing more DNA folds embedded in methylene blue(MB)molecules for secondary signal amplification.,More hemin/G-quadruplex DNAzyme is generated as the HCR reaction goes on,which helps to catalyze the decomposition of hydrogen peroxide(H₂O₂),accelerates the oxidation of MB molecules,and further improves the electrochemical signal strength to achieve highly sensitive detection of the target mi RNA-141.The sensor performs well in the detection of mi RNA-141 in different cell lysates,showing great potential in biosensing and bioanalysis.