Highly Sensitive Electrochemical Biosensors Based On Biological Amplification Technology And Functional Nanomaterial

Nucleic acids and proteins are most important biological macromolecules.Nucleic acids can transfer genetic information and protein covers all life activities.The interaction between nucleic acid and protein is helpful to research of life process so as to grasp life activity and information transfer rule and implementation of the regulation and control.MicroRNAs（miRNAs）are endogenous non-coding single-stranded RNAs and participate cell proliferation,differentiation,apoptosis,ontogenesis and virus infection process,and so on.DNA is an important genetic material.It determines the heredity and variation of biology.Deleting,replacing or inserting bases in DNA sequence will lead to the gene mutation and illness.Thrombin is a kind of formation of serine protein hydrolase by thrombin precursor,which has an important role in promoting blood clotting and regulation of blood coagulation.Therefore,detection of nucleic acid and protein has important practical significance for human disease prevention,early diagnosis,treatment and prognostic judgment.Consequently,there is a pressing need to develop high sensitivity,low detection limit,and good stability techniques to detecte miRNAs,DNA and thrombin.Currently,the detection methods of the detection of miRNAs included colorimetric method,fluorescence,chemiluminescence,surface enhanced Raman scattering,electrochemical analysis methods etc.The electrochemical analysis methods were more popular duo to the advantages of simple operation,low cost,good selectivity,good stability and high sensitivity etc.Nanomaterials have some advantages of excellent mechanical properties,high reactivity,good conductivity and high specific surface area,and more and more get the favour of the researchers in the application of biosensor.So,herein,we prepared several functional nanomaterials as electrodes material to increase large specific surface area of the electrode for fixing biomolecules so as to improve the sensitivity of the sensor.Simultaneously,we combined biologicai amplification technology application to build a variety of electrochemical biosensors,and implement high sensitivity and excellent selectivity for miRNA,DNA and thrombin detection.This article main research contents are as follows:1.We developed a sandwich-type biosensor based on magnesium oxide nanoflower and graphene oxide-gold nanoparticles hybrids coupling with enzyme signalamplification to detect microRNA.With the excellent featurea of large specific surface area of MgO nanoflower,good conductivity of AuNPs and sandwich structure,multiple signal amplification for electrochemical biosensor has been achieved.The new method showed linear relationship ranging from 1.0×10-10 to 1.0×10-16 M with a detection limit of 0.05 fM（S/N=3）.2.We developed an ultrasensitive electrochemical biosensor based on tungsten oxide-graphene composites coupling with catalyzed hairpin assembly target recycling and enzyme signal amplification for detection of miRNA.WO₃-Gr is prepared by a simple hydrothermal method and act as a sensing platform,two hairpin structure DNA fragments of H1 and H2 are introduced,and H2 can hybridize with H1 to occur catalyzed hairpin assembly target recycling in the presence of target mi RNA.Signal indicators streptavidin-conjugated alkaline phosphatase（SA-ALP）are immobilized and catalyze ascorbic acid to induce the electrochemical-chemical-chemical redox cycling to produce a strongly electrochemical response for miRNA detection in solution of ferrocene methanol and tris（2-carboxyethyl）phosphine.The method showed a detection limit of0.05 fM（S/N=3）.3.We developed a biosensor based on hollow molybdenum disulfide microcubes/AuNPs coupling with duplex-specific nuclease and enzyme signal amplification for detection of miRNA-21.Hollow molybdenum disulfide microcubes have a large specific surface area and good biological compatibility,So,increasing of the amount of DNA probe which fix on the surface of the electrode and greatly improving the current response of the sensor.Duplex-specific nuclease（DSN）will not damage to RNA of DNA/RNA hybrid duplexes,and miRNAs are released and recycled in sample solution after cleaving DNA.This experiment used materials and multiple enzymatic biocatalysis to obtain multiple signal amplification and significantly increase the sensitivity of miRNA detection.The detection limit is 0.086 fM（S/N=3）.The new method can be used for analysis of practical samples.5.We developed an ultrasensitive sandwich-type electrochemical biosensor for DNA detection based on MoSe2/SiO₂ sphere and GO–AuNPs hybrids coupling with HCR and multi-signal amplification.In our work,MoSe2/SiO₂ sphere act as a sensing platform and GO–AuNPs hybrids as carrier triggering HCR.Two biotin-hairpin probe can form many biotin polymer chains on the electrode surface.Finally,the signal was amplified byAvidin-HRP catalyze substrates.Results indicated that the sensor showed high sensitivity and good selectivity,and the linear relationship ranging from 0.1 fM to 100 pM with a detection limit of 0.068 fM（S/N=3）.The new method can be used for analysis of human blood samples.6.We developed a ultrasensitive sandwich-type electrochemical aptasensor platform based on N-GO and MoS₂/SiO₂ hybrids coupling with HCR and signal amplification for thrombin detection.The experiment with N-GO and MoS₂/SiO₂ has a larger specific surface area,which can supply more binding sites for biomolecules.Two biotin-hairpin probe can form many biotin polymer chains on the electrode surface.Finally,the signal was amplified by Avidin-HRP catalyze substrates.Results show that the electrochemical sensing platform has good selectivity,wide linear range（1.0×10-10¹.0×10-16）and low detection limit（0.027 fM）.