Development Of Novel Biosensing Based On Nanogold Enhancement Effect And Its Application On Detecting Food-borne Pathogenic Bacteria Virulence Gene And MiRNAs

Guaranteeing food safety and human health are two hot topics.Among them,foodborne pathogens cause food safety incidents frequently,which seriously affects the normal economic development and public health in China.Therefore,establishing highly sensitive detection methods is an urgent need to reduce the risk of foodborne pathogens and prevent infection.Virulence genes of foodborne pathogens provide pathogenic bacteria toxicity and specific genetic information.Sensing virulence genes is an important part of identifying foodborne pathogens in samples.In this study,virulence gene of foodborne pathogens and miRNAs were regarded as the targets.Based on gold nanoparticles,gold nanomaterials with different morphologies and properties and their assemblies were prepared,and they showed good biocompatibility through surface modification.With their special optical properties and good electrical conductivity,a series of optical or electrical signal enhanced nano-nucleic acid biosensors with simple,safe and visual characteristics have been developed,and they are used for rapid and sensitive detection of virulence genes of foodborne pathogens.Together with development of high sensitivity detection and visualization of intracellular toxic biomarker miRNAs,these methods lay the foundation for rapid detection of pathogenic bacterias and intracellular miRNAs.Using miRNAs to evaluate the toxicity of exogenous substances provides new ideas and theoretical basis for early screening pathogenic bacterias in the food based on the sensitive cell models with the relationship between the expression levels of miRNAs in host cells and the contamination dose of foodborne pathogens.1.Controllable synthesis of gold-silver core-shell nanorods?Au@Ag NRs?with different aspect ratios.Au@Ag NRs with different aspect ratios were synthesized by seed growth method,and the optimal amount of silver nitrate,ascorbic acid and gold seeds were determined.The growth mechanism of Au@Ag NRs was investigated by adjusting the pH value with the help of UV-vis spectrometer,transmission electron microscope and X-ray diffractometer.Our results showed that the pH value affected the position and rate of surfactant CTAB adsorbing on the surface of gold nanorods?AuNRs?,which regulated the position and rate of the Ag⁰adsorption on the surface of the AuNRs.Au@Ag NRs of different sizes and shapes were prepared,which laid the foundation for the next stage to develop biosensors using the physical and chemical properties of Au@Ag NRs.2.Development of a surface enhanced fluorescence?SEF?sensor based on gold-silver core-shell nanorods?Au@Ag NRs?.The Au@Ag NRs were prepared by using AuNRs with different aspect ratios as cores.By functionalizing the stem-loop probe and fluorophore Cy3 on the surface of Au@Ag NRs,a surface enhanced fluorescence“ON-OFF”nucleic acid sensor with Au@Ag NRs was developed to detect E.coli O157:H7 virulence gene eae A with fluorescence signal.The factors affecting the fluorescence intensity of the fluorophore molecule Cy3 in the sensor were investigated.When the distance between Cy3 molecule and Au@Ag NRs surface was 10 nm,and Au@Ag NRs with long aspect ratio was chosen,the surface enhanced fluorescence of Au@Ag NRs was the strongest for fluorophore molecule Cy3,which was beneficial to improve the detection sensitivity.When the concentration of the eae A gene was in the range of 1×10^-17 to 1×10^-11 mol/L,the fluorescence intensity of the Cy3 fluorophore molecule was linear with the logarithm of the E.coli O157:H7 eae A gene.The linear regression equation was y=95.97 x+1992?R²=0.9947?and the limit of detection was 3.33×10^-18 mol/L.The developed surface enhanced fluorescent nucle acid sensor showed good specificity and repeatability,and could differentiate mismatched sequences,the recovery of the sensor was98.36%-101.67%.3.Development of a portable sensor with electrochemical signals enhancement based on heterodimers gold nanocrosses@molybdenum disulfide?AuNCs@MoS2?.To further achieve rapid,sensitive,on-site detection of foodborne pathogenic bacteria,an electrochemical sensor using screen printed electrode?SPE?based on heterodimer AuNCs@MoS₂ was proposed to enhance the electrical signal.This was used to realize portable biosensors for in-situ sensing E.coli O157:H7 virulence gene eae A.A toehold-mediated DNA strand displacement reaction triggered by the target eae A gene was used to obtain double stranded DNA H₁/H₂,which bound to the cDNA on AuNCs@MoS₂/SPE.In the presence of[Ru?NH₃?₆]³⁺molecules,an electrochemical signal was generated to achieve quantitative detection of the target.The sensitivity of detection and electrochemical signal was greatly improved by the toehold-mediated DNA strand displacement reaction and modification of AuNCs@MoS₂ on the electrode surface.The concentration of eae A with 1-10⁵ amol/L was linear with current differential,and the linear equation was?I??A?=1.61 log[C_{eae A}?amol/L?]+2.02?R²=0.9974?with detection limit of0.096 amol/L.The sensor showed good specificity and could distinguish single base mismatched sequences.4.Development of“FRET-SEF”fluorescence signal enhancement for sensing miRNAs and intracellular imaging based on gold nanorod-gold nanocross?AuNR-AuNC?dimers.In order to achieve the goal of evaluating intracellular toxicity,the novel one-to-one assembled AuNR-AuNC dimers“FRET-SEF”probe with“OFF-enhanced ON”fluorescent-enhanced switch was further developed to detect extracellular and intracellular biomarker miRNAs.The distance between AuNC and Cy5 fluorophore molecules was 10 nm,which was beneficial to enhance the fluorescence intensity of Cy5 by AuNC.The side assembly of AuNC and AuNR was beneficial to the fluorescence resonance energy transfer of AuNR to Cy5 molecule.Through the combination of AuNR and AuNC to develop the dimers and make full use of the fluorescence enhancement effect produced by the dimers,the detection sensitivity was greatly improved.When the concentration of miRNA-21 was in the range of 0.0006-0.0016 fmol/L and 0.1-100fmol/L,the linear equations were F=1830.32 logC+6349.27?R²=0.9901?and F=244.41logC+1916.10?R²=0.9984?,respectively,and the detection limit could reach 0.5 amol/L and0.03 fmol/L,respectively.This proved that the newly AuNR-AuNC dimers probe could accurately determine the biomarker miRNA.In addition,the dimers presented good stability and low toxicity and could be used for intracellular detection of biomarker miRNA-21 in Hep G2cells.The AuNR-AuNC dimers probe was also preliminarily used for evaluating toxic levels of exogenous substances such as foodborne pathogenic toxic factors with sensitive cell model.5.Development of comet-like heterodimer sensitizing probe for sensing miRNA-34a and imaging in vivo based on gold nanoflower@graphene quantum dots?AuNF@GQDs?and FDTD Solutions.In order to achieve the goal of evaluating toxicity in vivo,a low-toxic comet-like heterodimer AuNF@GQDs probe based on toehold-mediated by target miRNA from fluorescence signal FRET“off”to DNA curcit“on”DNA strand displacement sensitizing reaction was developed to identify miRNA-34a in vitro and in vivo.The expression levels of miRNA-34a were used to assess the degree of aging cells.According to FDTD Solutions simulation,AuNF had a strong FRET effect on GQDs when the distance between AuNF and GQDs was 4 nm,which could shorten the response time and enhance the extinction efficiency during the detection process,and was beneficial to improve the detecting sensitivity.When the concentration of miRNA-34a was 0.4-4 fmol/L,the linear equation was Y=1963 x+213,R²=0.9921,and the detection limit of miRNA-34a was 0.1 fmol/L.This probe could distinguish one base mismatched sequence.This low toxic probe was also applied for in situ imaging and detection of miRNA-34a expression in rat cardiomyocytes cells?H9C2?induced by growth factor TGF-?1.When TGF-?1 concentration was 5-500 ng/mL,the intracellular fluorescence intensity was linearly correlated with the logarithm concentration of TGF-?1,and the linear equation was y=1.37 x+1.04 with R²=0.9330.In addition,the AuNF@GQDs dimers probe was injected into the left ventricle of 12-month old C57BL/6J mice to measure the expression levels of miRNA-34a,and the degree of aging in the mouse heart was evaluated successfully with no negative toxic effect of the probe on the mice.In summary,the anisotropic gold-silver core-shell nanorods and nano dimers nucleic acid probes with enhancement were developed in this thesis.The fluorescence and electrochemical enhancement mechanisms were investigated by designing the morphology and spatial distribution of the probes and examining highly sensitive detection of virulence genes in foodborne pathogens.This work would provide a basis of monitoring effective in vivo biomarkers for foodborne pathogenic bacteria infections and changes in expression levels,and would help to assess the toxicity of foodborne pathogens and the extent of host infection.