The Application Of New Signal Amplification Strategies Based On Biological Enzymes And Nano-functional Materials In Analytical Detection

In recent years,with the steady progress of globalization economic level,people's living standard has been greatly improved.Meanwhile,"HEALTH" has become people's primary concern.Analytical chemistry is associated tightly with life science.Among the field of analytical chemistry,medical diagnostics and food safety detection occupied the prominent position as they are closely related to people's health.In order to detect the low abundant disease biomarkers and food contaminants accurately for achieving the detection requirement of recent practical application,it's necessary to develop novel analysis methods which own high sensitivity.Signal amplification technique has been developed quickly and has become one of the research focuses in the field of analytical detection.Recently,because of the fact that the enzymes have very high catalytic activity,the nanomaterials own distinct optical properties,good conductivity,high catalytical activity,unique electrochemical activity,and large surface area,strategies of signal amplification based on enzymes and nanomaterials have been widely designed and applied in analytical field.By using enzymes,which have high catalytic activity,and functionalized nanomaterials,which can improve electron transfer and load abundant signal molecules,we developed a series of new strategies of signal amplification to improve analytical sensitivity in disease diagnostics and food safety detection.They provided strong theoretical and experimental support for the development of new-type electrochemical and optical detection methods.They have high sensitivity,and could be used to analyze practical samples.They are efficient and promising analysis methods for disease diagnostics,treatment,drug efficacy monitoring,and food safety analysis.This dissertation is mainly divided into the following five parts:?1?Vascular endothelial growth factor receptor 2?VEGFR2?is a potential cell-type biomarker in clinical diagnostics.Besides,it's the target protein of many tyrosine kinase inhibitors and its expression significantly associates with clinical performance of these inhibitors.VEGFR2 detection provides an early warning for diseases and a basis for therapy and drug screening.An electrochemical biosensing platform for VEGFR2 analysis has been proposed.By using the enzyme catalysis for signal amplification,the sensitivity was improved.The constructed immunosensor can detect the total concentrations of the VEGFR2 protein in cells lysates directly and can be used to monitor the changes of VEGFR2 expression levels induced by treatments of different inhibitors.Moreover,the inhibitor-VEGFR2 interactions are illuminated through theoretical simulation.The simulation results agreed well with the experimental data,indicating the veracity of the proposed method.The electrochemical detection methodology for VEGFR2 would be promising in clinical diagnostics and drug screening.?2?As the electrochemical signal of molybdenum can be used as the output signal,considering the fact that phosphomolybdic acid?H3PMo12O40/PMo12?is a Mo-rich molecule,PMo12 was chosen as the signal molecule for signal amplification.Based on the fact that single and double strand DNA show different capacities of interaction with graphene oxide?GO?,the vacancy on the interface of GO generated by double-stranded DNA releasing might form stable complex with polyoxometalate on account of its strong chemisorption on carbon materials.By jointly taking advantages of PMo12's native electronegativity,electrochemical activity and chemisorption with GO,a novel,simple,and label-free electrochemical biosensor was constructed.Using disease-related gene sequence?mitochondrial DNA related to maternally inherited diabetes?and thrombin as models,the platform was applied to the analysis of DNA and protein.DNA assay and quantitative determination of thrombin in human blood were realized and satisfactory results were achieved.The novel PMo12-based electrochemical biosensing platform would have a promising prospect in clinical diagnostics.?3?The principle of analyte-induced aggregation of metal nanoparticles?MNs?have been widely used in colorimetric analysis.Based on this principle,a novel concept is proposed for converting liquid-phase colorimetric assay into enhanced surface-tethered electrochemical analysis.Contamination of the environment with heavy metal ions has been an important worldwide concern for decades.Mercury,which can accumulate in vital organs and tissues,such as the liver,brain,and heart muscle,is highly toxic and can produce lethal effects in living systems.In a proof-of-concept trial,thymine-functionalized silver nanpparticles?Ag NPs?were designed as the sensing units for Hg2+ determination.Through a specific T-Hg2+-T coordination,the aggregation of Ag NPs leads to an obvious colour change,so that the functionalized sensing units-based validation system can perform well in a colorimetric Hg2+ assay.It also can be developed into a more sensitive and stable electrochemical sensor.In electrochemical analysis,the aggregation of MNs on the electrode surface assembled into the Ag NPs-based network architecture.Through detecting the unique electrochemical redox signal of Ag NPs,the Hg2+-induced formation of Ag NPs-based network architecture can be used as a strategy of elctrochemical signal amplification.This strategy can significantly improve the detection sensitivity.More importantly,those numerous and diverse colorimetric assays that rely on the target-induced aggregation of MNs,could be augmented to satisfy the ambitious demands of sensitive analysis by converting them into electrochemical assays via this approach.?4?The albumin nanoparticles which load large amounts of fluorescence molecules were synthesized for fluorescent signal amplification.The fluorescence molecules-loaded albumin nanoparticles were applied into a magneto-RNA sensing system for direct and one-step detection of probe-hybridized miRNAs from cellular or serum samples.In this system,target miRNA is specifically captured and enriched through hybridizing to a probe modified on the surface of magnetic beads.This probe:miRNA duplex can be recognized by p19 protein,a viral protein that can bind to short RNA duplex.Finally,the abundance of the duplex is quantified by introducing the newly synthesized fluorescent Alb NPs that are conjugated with p19 protein and loaded with large amounts of fluorescence molecules.The conjugation of p19 protein to Alb NPs can simplify the miRNA determination assay through the combination of the capture step with the signal production step,from a multi-step process into a one-step process.Additionally,the implication of particle enrichment,magnetic separation and fluorescent enhancement makes this system sensitive enough for miRNA analysis.MiRNA-21 is a biomarker of various carcinomas,it is a potential tool for early cancer diagnostics.This method enables a linear detection range from 10 fM to 10 nM for miRNA-21,with a detection limit of 2 fM.We demonstrated the potential of this magneto-RNA sensing system by detecting miRNA-21 in breast cancer cell lysates and serum samples.The possibility of this method for direct and one-step quantification may significantly advance the use of miRNAs as biomarkers in the clinical praxis.?5?We co-immobilized the enzyme,antibody and Cu3?PO4?₂ into a novel three-in-one hybrid protein-inorganic nanoflower.As the three-in-one nanoflower is comprised of large numbers of horseradish peroxidase?HRP?,and it has distinct hydrangea-like hierarchical structure to ensure its high specific surface area,the nanoflower shows enhanced enzyme catalytical activity.TMB can be catalyzed by the three-in-one nanoflower to produce amplified optical signal response.The prepared antibody-enzyme-inorganic nanoflower was applied in ELISA to serve as a novel enzyme-labeled antibody for Escherichia coli 0157:H7?E.coli 0157:H7?determination.E.coli 0157:H7 is an important foodborne pathogen,which can cause serious infection with low infectious dose.The detection limit is 60 CFU L-1,which is far superior to commercial ELISA systems.The three-in-one nanoflowers hold great prospect in food safety analysis.