Study On Optical Sensor Based On DNA Amplification Technology And Its Application In Detecting Myocardial Infarction Disease-related Nucleic Acid Marker

Acute myocardial infarction(AMI)is developed into a serious cardiovascular disease.Long-term ischemia can lead to tissue death and even cause heart necrosis.Due to the lack of rapid and immediate diagnostic techniques,the mortality rate remains high.Therefore,accurate analysis of the biomarkers associated with AMI is of great significance for clinical treatment and diagnosis.As for microRNA(miRNA)related to AMI(miRNA-499,miRNA-133a,etc.),the main content and research focus of this paper is to build nucleic acid sensing platform that can identify and detect nucleic acid marker in the actual environment.In addition,since miRNA is easy to be degraded and has a low content in human blood,how to construct a reliable and ultrasensitive detection biosensor is also the focus of this thesis.For the purpose of improve the sensitivity and accuracy of detecting miRNA,we combined DNA amplification technology with different types of mordern spectral technologies,and applied chemiluminescence,electrochemiluminescence and SERS technology to detect miRNAs related to AMI in patients’ serum.This paper includes the following parts:1)Spherical Nucleic Acid Enzyme(SNAzyme)Promoted Chemiluminescence(CL)miRNA Imaging with a mobile phoneAs AMI now is considered to be one of the most severe factors that threats to human health and may suddenly happens at any places.Since the expensive equipments can not be supplied to most of the places in daily life,it is of great importance for developing portable and reliable methods for the instant diagnosis and treatment of AMI.To achieve this goal,in our work we develop a biosensor for miRNA imaging by using mobile phone as the CL detector.What’s more,we realize visual inspection of AMI-related biomarkers in AMI patients;serum for the first time.We first synthesize a SNAzyme by coating the gold nanoparticle core with a closely spaced layer of G4 DNAzyme,which improves the catalytic activity to more than hundred-fold by comparing with G4 DNAzyme itself.Besides,the resistant ability to nuclease degradation in blood was also improved obviously.These distinctive characteristics enable the SNAzyme-promoted biosensor with outstanding imaging performance for sensing miRNAs in blood.This target miRNA is working as a trigger for the CHA process to generate multiple sticky dsDNA linkers which can capture the SNAzymes onto the separating well.In this way,we successfully detected target miRNA,with an imaging LDC of 1 nM and a high specificity over other miRNA mimics in patients’ blood.In consideration of the unique features of SNAzyme in blood environments,our imaging strategy holds great prospect for application in the point-of-care diagnosis of AMI.2)A Target induced Amplified Loading of Spherical Nucleic Acid Enzyme(SNAzyme)as Nanocatalyst for Ultrasensitive Electrochemiluminescence Detection of miRNACurrently,varying methods have been established for the sensing of AMI-related miRNA,but many of them require complex instruments or the detection sensitivity needs to be improved.So it is still urgent to exploit simple,easy available and highly sensitive diagnostic methods.Inspired by our previous work,we constructed an ECL miRNA detection platform with SNAzyme as the nancatalyst and signal amplification was realized through hybridization chain amplification reaction.Due to its unique anti-interference ability and strong catalytic activity,SNAzyme has a good practical application in the analysis of miRNA in blood related to AMI.First,the target miRNA complement with the probe modified on the surface of electrode to open the trigger end on the hairpin ring.Then the hybridization chain amplification reaction was promoted to produce amplification products with sticky ends,which can capture SNAzyme to the electrode.In this way,we successfully detected two miRNAs associated with AMI.This method has wide linear range,high sensitivity and the equipment is simple,so this platform has a broad application prospect in the clinical diagnosis of AMI.3)Composition-controllable Au/Ag hollow SERS Nanotags Combined with Target miRNA-Catalyzed Hairpin Assembly(CHA)for Multiple Amplification sensing of miRNAIn this work,we synthesized the Au/Ag SERS probes with controllable components and combined the probes with the targeted CHA strategy to develop a sensitive SERS biosensor for detecting the miRNA(miRNA-133a)related to AMI.The ratio of gold and silver components can be controlled through a method based on galvanic replacement and the bimetallic probe displaying strong plasmon resonance effect and high stability.Then the probes can be captured by a double chain DNA linker generated in the CHA amplification to realize signal amplification and reading.As a result,miRNA-133a was detected with a linear ranging from 1 fM to 1 nM and an ideal LDC of 0.306 fM.Besides,the biosensor shows high specificity towards other miRNAs co-existed in circulating blood.Practical assay in human serum demonstrate that the sensing platform developed by us displays robust anti-interference ability as well as ideal sensitivity in blood environments,conducive to its potential clinical applications.4)Exonuclease Ⅲ-Boosted hybridization chain amplification for ultrasensitive SERS sensing of acute myocardial infarction(AMI)Related nucleic acid markerBenefiting from some currently available nucleic acid detection technologies,here we apply enzyme amplification to boost isothermal nucleic acid amplification and couple the two-step amplification platform with SERS technique to construct a signal-on nucleic acid marker sensing platform.Tanks to the ideal cleavage efficiency and strong recognition ability of Exonuclease Ⅲ(Exo Ⅲ),plenty of trigger DNAs are generated to switch on multiple cycles of cascade amplification.Probe-labeled hairpin DNAs are linked together in series,and then the structure of the product is anchored onto the gold plasmonic chip as well as enriched to achieve ultra-sensitive SERS detection.The experimental results demonstrated that our sensing platform is very sensitive to AMI related nucleic acid marker with a detectable limit(LDC)as low as 1 fM.Besises,it can well differentiate single base mismatched nucleic acids and non-complementary sequences.This sensing strategy develops a powerful signal amplification tool for nucleic acid marker detection,which could even work in blood samples.