| A favored assay type in bioassay is enzymatic analysis.Enzyme-linked immunosorbent assay(ELISA)is the one kind of enzymatic analysis,as it combines the high selectivity of immunoassays with enzyme-mediated signal amplification.However,only one enzyme binds to one analyte molecule in a standard ELISA,which often leads to limitations in sensitivity.Here,nanomaterials can elegantly contribute to the improvement of this concept,as they can bind many recognition and enzyme molecules on only one nanoparticle that binds to the analyte.In this dissertation,the study focused on the organic-inorganic hybrid nanomaterials based on CaHPO4,which were applied to the analysis and detection of Escherichia coli,Salmonella and thrombin.This dissertation can be divided into five chapters:In the first chapter,nanomaterials and biosensor were introduced.Then,application of nanomaterials in the detection of pathogen and protein was discussed,and finally a brief introduction of the background and content of this dissertation was given.In the second chapter,Con A-GOx-CaHPO4(CGC)organic-inorganic hybrid nano materials was synthesized,using concanavalin A(Con A)as recognition unit,glucose oxidase(GOx)as a signal amplifying unit,dicalcium phosphate(CaHPO4)as the inorganic carrier.This facile method were demonstrated by no extreme harsh conditions,no any toxic elements involved and complicated purification processes required,only one-step mild preparing process,integrating all essential functions of biological recognition and signal amplification.For the first time,this hybrid nanomaterials were combined with a handheld pH meter and applied to a portable detection of Escherichia coli(E.coli)0157:H7.Detection limit was 10 CFU/mL.The detection range was 10-106 CFU/mL.In the third chapter,using invertase as the signal amplifying unit and take advantage of Ca2+ to promote the activity of invertase,the Con A-Invertase-CaHPO4(CIC)organic-inorganic hybrid nanomaterials was synthesized.The prepared Con A-invertase-CaHPO4 hybrid nanomaterials improved the stability of Con A and invertase and there was a 203.5%increase in activity of immobilized invertase in CIC.For the first time,this hybrid nanomaterials were combined with glucose meter to carry out a portable detetion for E.coli 0157:H7.Detection limit was 10 CFU/mL.The detection range was 10-105 CFU/mL.In the fourth chapter,using antibody(Ab)as recognition unit and horseradish peroxidase(HRP)as a signal amplifying unit,Ab-HRP-CaHPO4(AHC)organic-inorganic hybrid nanomaterials were synthesized.This hybrid nanomaterials were successfully applied to the detection of Salmonella enteritidis.The application of organic-inorganic hybrid nanomaterials based on CaHPO4 has been extended.It was possible to apply this kind of organic-inorganic hybrid nanomaterials to all the immunoassay-based detection.Taking the advantage of immunomagnetic separation and chemiluminescence method,it reduced the operation steps and the detection time,and improved the detection sensitivity.Detection limit was 1 CFU/mL.The detection range was 1-105 CFU/mL.In the fifth chapter,using the aptamer as recognition unit,the Aptamer-HRP-CaHPO4(APHC)organic-inorganic hybrid nanomaterials were synthesized.This organic-inorganic hybrid nanomaterials effectively immobilized aptamer and protein together in one step and successfully applied to the detection of thrombin,further extending application in the aptamer-based dectetion.Because the synthesis of aptamer was cheap,and it was not easy to be affected by temperature,pH and other factors,the cost of the preparation of this hybrid nanomaterials was greatly reduced,and the stability of hybrid nanomaterials was also improved.The detection limit of this method was 9 pM.The detection range was 0.01-137 nM.In summary,in this dissertation,the study focused on the organic-inorganic hybrid nanomaterials based on CaHPO4,which were successfully applied to the bioanalysis and detection of Escherichia coli,Salmonella and thrombin. |
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