Research On Electrochemical Detection Method For Pathogenic Microorganims Based On Nanoporus Gold

Infectious diseases are caused by pathogenic microorganisms such as fungi,bacteria,viruses,and viroids.It is a serious global problem.It not only causes casualties but also causes huge economic losses.The rapid detection and identification of pathogenic microorganisms are of great significance in the fields of medical and food safety.Electrochemical sensors have the characteristics of miniaturization,flexible construction as well as low cost,and are very attractive in many fields that require high sensitivity,simple operation,and fast response.In the past ten decades,due to the rapid development of biological science and material science,a variety of biomarkers,enzymes,antibodies,and many nanomaterials and composite materials with good electrical conductivity and/or catalytic activity have been developed.This has greatly improved the types,detection limits,and specificity of electrochemical sensors.With these advantages,electrochemical sensors have great potential in the field of pathogenic microorganism detection,and are widely regarded as a cheaper and more effective alternative method than traditional pathogenic microorganism detection methods.Nanoporous gold(NPG)has the advantages of large specific surface area,good electrical conductivity,high biocompatibility,and catalytic activity to a variety of substances.It is an ideal immobilized carrier material for constructing electrochemical sensors.In this study,pathogenic microorganisms such as Hepatitis B virus(HBV)and Listeria monocytogenes are selected as models,and suitable biorecognition elements are selected based on the different biomarkers of pathogenic microorganisms.Combined with the excellent characteristics of NPG,different types of electrochemical sensors are constructed for the selected models.The application value of NPG-based electrochemical sensors in the field of pathogenic microorganism sm detection are explored.The research content and results are listed as follows:1.Electrochemical immunosensor for HBe antigen detection based on a signal amplification strategy:the co-catalysis of horseradish peroxidase and NPGHepatitis B e antigen(HBeAg),as an important serum marker for the diagnosis of Hepatitis B virus(HBV)infected diseases,will appear in serum during the early phase of chronic hepatitis B(CHB)with high infectivity.Therefore,the reliable detection of HBeAg is helpful for the diagnosis and prediction of CHB.NPG with unique properties is selected as an ideal carrier for biorecognition elements immobilization.In addition,as a nanoporous metal material,NPG has excellent electrical conductivity and high-efficiency catalytic activity for many substrates such as catechol.Therefore,a signal amplification strategy based on the co-catalysis of horseradish peroxidase(HRP)and NPG is used to fabricate an electrochemical immunosensor for the determination of HBeAg.Based on the signal amplification strategy,the immunosensor constructed in this study showed a good linear relationship between the peak current density and the HBeAg concentration.The linear range is 1 pg mL-1～1 ng mL-1 with a high sensitivity of 23.51 μA mL ng-1 cm-2 and a low detection limit of 0.064 pg mL-1.In addition to the wide detection range,the immunosensor proposed in this study showed selectivity and strong anti-interference ability.For the detection of HBeAg in human serum,the concentrations detected by the immunosensor are in good agreement with the standard concentrations added in the human serum samples.Based on the good performance,the immunosensor could be a good choice for HBeAg detection.Meanwhile,the signal amplification strategy based on the co-catalysis of enzyme and nanomaterial maybe provides a new idea for other researchers in the construction of an immunosensor.2.Construction of liposome sensor and its application in detection of pathogenic microorganisms producing CDCs toxinCholesterol-dependent cytolysins(CDCs)are a class of pore-forming toxins,which is generally considered to be a pathogenic factor for some gram-positive pathogens.When the pathogen invades the host cell,the CDCs produced by the pathogen can bind to cholesterol and anchor on the host cell membrane,and then the CDCs monomers bind to each other,forming pores on the cell membrane surface to help the pathogen escape from the cell and spread in the host.Therefore,the detection of CDCs-producing pathogenic microorganisms can be achieved by detecting CDCs.The pore-forming ability of CDCs is used to construct liposome biosensors.The detection principle is:use the pore-forming ability of CDCs to destroy the artificial liposomes loaded on the surface of NPG,causing the release of catechol,the content inthe liposomes,and the released catechol can be catalyzed by NPG to generate an electric signal.The electric signal intensity is positively correlated with the concentration of CDCs.To explore the sensing performance of the liposome biosensor,Listeriolysin O(LLO)protein,α-hemolysin(ALN)protein,and cereolysin O(CLO)protein,which are representative of the CDCs family,were selected as the test models.Using L.monocytogenes,Staphylococcus aureus ATCC 6538,and Bacillus anthracis genomic DNA as templates,the three proteins were expressed and purified in Escherichia coli BL21(DE3)respectively.The results show that the liposome sensor can sensitively respond to the three proteins of LLO,ALN,and CLO in the CDCs protein family,with the detection ranges of 0.5～12.0 μg mL-1,0.5～8.0 μg mL-1,and 1.0～10.0 μg mL-1.At the same time,L.monocytogenes is selected as the actual sample detection model to explore the actual detection ability of the constructed liposome biosensor on the CDCs-producing protein family bacteria.The result proves that the detection result of the liposome biosensor on the number of CDCs-producing bacteria is consistent with the plate counting method,and it has good anti-interference ability against non-CDCs-producing bacteria.Based on the above excellent performance,the liposome sensor has strong application potential in the detection of CDCs-producing Gram-positive pathogenic bacteria.3.Construction of a biosensor for simultaneous detection of L.monocytogenes marker genes hly and iapListeriosis is a disease caused by the ingestion of food contaminated with L.monocytogenes,which affects immunocompromised patients,pregnant women,and newborns.After ingesting contaminated food,L.monocytogenes will cross the intestinal barrier,blood-brain barrier,and placental barrier,causing gastroenteritis,meningitis,miscarriage,pregnancy complications,etc.,which seriously endanger human health.Therefore,the reliable,sensitive,and accurate detection of L.monocytogenes in food is of great significance for the prevention of infection.The identification of L.monocytogenes by detecting a certain marker gene is a commonly used identification method in traditional detection methods,but the detection based on single marker genes is prone to false positives,and the instruments and equipment used in traditional molecular biology detection methods are relatively expensive and complicated to operate.In this study,two marker genes of hly and iap are selected as the target genes,and thiolated capture probes complementary to hly or iap are selected as the recognition element.NPG is used as the immobilized vector for the recognition element to construct the hly-iap nucleic acid biosensor.The specific detection principle is as follows:the target genes hly and iap can be combined with the capture probe fixed on the surface of the electrode by complementary base pairing.Subsequently,signal probes carrying different signal tags are combined with the corresponding target genes.The signal probe used to detect the hly gene carries the MB electroactive label,and the detection of the hly gene is realized by detecting the electrochemical signal generated by the MB;HRP combines with iap signal probe through biotin-streptavidin interaction,which can detect iap gene by detecting the electrical signal generated by HRP catalysis H2O2.The constructed hly-iap nucleic acid biosensor has a wide detection range from 1.0 × 104 to 1.0 × 108 CFU mL-1.At the same time,the hly-iap nucleic acid biosensor has a good anti-interference ability against a variety of bacteria and realizes the detection of L.monocytogenes in actual samples.In addition,the simultaneous detection of hly and iap marker genes can avoid inaccurate or false-positive results that may occur when detecting a single gene.The results obtained from the detection of the two genes can be mutually confirmed,which enhances the reliability of the detection results.These unique characteristics make the hly-iap nucleic acid biosensor have certain application potential in the detection of L.monocytogenes.4.Construction of a three-function paper sensor for detection of L.monocytogenes based on different level markersIn this study,different markers from the nucleic acid level(hly gene),the small molecule metabolites level(acetoin),and the protin level(LLO protein)are selected to construct a three-function paper sensor forL.monocytogenes detecting.NPG,which can catalyze a variety of substrates(such as NADH),was selected as the immobilization carrier.The detection principle is as follows:the thiolated capture probe(ssDNA)is synthesized according to the hly gene sequence and fixed on the surface of NPG by Au-S.The target gene hly can be matched with the capture probe by complementary base pairing.As an electroactive tag,MB can be inserted into two consecutive base pairs in the double-stranded structure,and then generate an electrochemical signal.In addition,it is reported that acetoin can be used as a biomarker for the detection of L.monocytogenes and the concentration of acetoin is positively correlated with the concentration of L.monocytogenes.For acetoin detection,AR could catalyze acetoin to 2,3-butanediol in the presence of NADH as a cofactor.Combined with the electrochemical catalysis of NADH by NPG,the quantitative detection of acetoin is achieved by measuring the consumption of NADH using the three-function paper sensor.Thus,the concentration of L.monocytogenes is calculated indirectly by the quantitative relationship between the production of acetoin and the concentration of L.monocytogenes.For the detection of LLO protein,a liposome containing catechol was selected as the recognition element,and the pore-forming ability of the LLO protein on the surface of the liposome was used to detect LLO protein.Based on the above detection principle,the feasibility of detecting hly gene and acetoin is evaluated by using the common three electrode system,and the electrochemical behavior and the ability of L.monocytogenes detection are explored.On this basis,a three-function paper sensor was constructed using a disposable screen-printed electrode with four working electrodes to further optimize the portability of the paper sensor.The results of real samples detected by the three-function paper sensor are compared with the results of qPCR,and the practical application potential of the three-function paper sensor for the detection of L.monocytogenes is further explored.The results prove that the constructed three-function paper sensor has a wide linear range from 1.0 × 1 04 to 1.0 × 109 CFU mL-1.At the same time,the three-function paper sensor has good selective detection ability for hly and acetoin,and a certain selective detection ability for LLO protein,and realizes the reliable detection of L.monocytogenes in actual samples.Compared with the standard identification methods-GB 4789.30-2016,the multi-biocatalyst platform has the following advantages:1)Although the pre-enrichment process is needed,the simple detection process of three markers at different levels replace the complex identification process(such as dynamic test,catalase test,sugar fermentation,hemolytic properties,etc.)of GB 4789.30-2016 method;2)The detection of three markers at different levels not only ensures the reliability of the L.monocytogenes identification results,but also shortens the long identification procedure time of 2～5 days for the GB 4789.30-2016 method to about 2 h.Compared with the qPCR method based on marker detection at nucleic acid level,the multi-biocatalyst platform not only exhibit a similar detection performance,but also can reduce the probability of false positive by detecting three markers at different levels.Further,the pre-enrichment process required for the multi-biocatalyst platform provides the guarantee for the elimination of false negative in L.monocytogenes identification.These unique features make the multi-biocatalyst platform a good choice for the reliable detection of L.monocytogenes.