Study On Nucleic Acid Biosensing Strategies Based On Mnazyme And DNA Nanotechnology

The detection of disease associated nucleic acids is of great significance for the diagnosis,treatment,and guidance of clinical medication.In recent years,various target molecule recognition and signal amplification strategies have been extensively studied,and a variety of biosensor analysis methods have been developed for nucleic acid detection.The application of various biological enzymes,metal nanoparticles,and inorganic nanomaterials greatly improves the sensitivity of detection.However,these methods usually require rigorous reaction conditions or complicated operation steps,and the repeatability are poor.Simple,rapid and highly sensitive methods for the detection of disease associated nucleic acids are of great significance.In this study,MNAzyme and DNA nanotechnology were used to analyze and detect disease related nucleic acids using electrochemical and surface plasmon resonance?SPR?biosensors as detection platforms,providing a new path for clinical diagnosis.The research content is divided into the following two parts:1.Electrochemical DNA biosensor based on MNAzyme-mediated signal amplificationWe describe an electrochemical sensing strategy for highly sensitive and specific detection of target?analyte?DNA based on an amplification scheme mediated by a multicomponent nucleic acid enzyme?MNAzyme?.MNAzymes were formed by multicomponent complexes which produce amplified output signals in response to specific input signal.In the presence of target nucleic acid,multiple partial enzymes?partzymes?oligonucleotides are assembled to form active MNAzymes.These can cleave H₀ substrate into two pieces,thereby releasing the activated MNAzyme to undergo an additional cycle of amplification.Here,the two pieces contain a biotin-tagged sequence and a byproduct.The biotin-tagged sequences are specifically captured by the detection probes immobilized on the gold electrode.By employing streptavidinylated alkaline phosphatase as an enzyme label,an electrochemical signal is obtained.The electrode,if operated at a working potential of 0.25 V?vs.Ag/AgCl?in solution of p H 7.5,covers the 100 pM to 0.25?M DNA concentration range,with a 79 pM detection limit.This strategy is expected to be applied to the detection of nucleic acids in complex matrices.2.Highly sensitive surface plasmon resonance biosensor for the detection of HIV-related DNA based on dynamic and structural DNA nanodevicesEarly detection,diagnosis and treatment of human immune deficiency virus?HIV?infection is the key to reduce acquired immunodeficiency syndrome?AIDS?mortality.In our research,an innovative surface plasmon resonance?SPR?biosensing strategy has been developed for highly sensitive detection of HIV-related DNA based on entropy-driven strand displacement reactions?ESDRs?and double-layer DNA tetrahedrons?DDTs?.ESDRs as enzyme-free and label-free signal amplification circuit can be specifically triggered by target DNA,leading to the cyclic utilization of target DNA and the formation of plentiful double-stranded DNA?ds DNA?products.Subsequently,the dsDNA products bind to the immobilized hairpin capture probes and further combine with DDTs nanostructures.Due to the high efficiency of ESDRs and large molecular weight of DDTs,the SPR response signal was enhanced dramatically.The proposed SPR biosensor could detect target DNA sensitively and specifically in a linear range from 1 pM to 150nM with a detection limit of 48 fM.In addition,the whole detecting process can be accomplished in 60 min with high accuracy and duplicability.In particular,the developed SPR biosensor was successfully used to analyze target DNA in complex biological sample,indicating that the developed strategy is promising for rapid and early clinical diagnosis of HIV infection.In summary,this study provides new technical support for the detection of clinical disease related nucleic acids and provides new ideas for biomedical research and molecular diagnostics.