Investigation On The Preparation Of Protein-Phosphate Hybrid Nanoflower And Its Sensing Properties

Organic-inorganic hybrid nanoflowers,which are flower-like microspheres,are formed by the hybridization of biologically active molecules and nanomaterials.Organic-inorganic hybrid nanoflowers possess the specificity of biologically active molecules,the acid and alkali resistance and thermal stability of nanomaterials,and they can also exhibit unique synergistic effects.Therefore,the organic-inorganic hybrid nanoflowers have huge application potential in the field of biosensing research.Accordingly,in this thesis,ten protein-phosphate hybrid nanoflowers such as hemoglobin-copper phosphate organic-inorganic hybrid nanoflowers(Hb-Cu3(PO4)2 HNFs),Hb-Mn3(PO4)2 HNFs,etc.,were prepared by the one-pot method,and the growth mechanism of organic-inorganic hybrid nanoflowers was systematically studied,especially the interaction mechanism between organic and inorganic components was clarified.A universal synthesis method of protein-phosphate hybrid nanoflower was proposed.Four kinds of biosensors based on these nanoflowers,such as fluorescence/colorimetric dual-mode biosensing,electrochemical biosensing,colorimetric immunobiosensing,and immunobiosensing test paper have been constructed.These investigations can provide a new method for the synthesis of organic-inorganic hybrid nanomaterials,enrich the research content of biosensors,and provide reference for environmental and clinical diagnosis.The thesis was divided into five chapters,the main contributions by author are as follows:(1)The Hb-Cu3(PO4)2 HNFs were synthesized by a one-pot synthetic method,its growth mechanism was explored,and the H2O2 fluorescence/colorimetric dual-mode biosensors based on this material was constructed.The structure,composition,morphology and surface electronic structure of the materials have been studied by SEM and XPS.The results showed that the synthesized Hb-Cu3(PO4)2 HNFs are hierarchical flower-like microspheres,the microspheres are highly dispersed with particle size in the range of 10?15 ?m,and Hb active centers are exposed on the surface of nanopetals.The catalytic activity test showed that the catalytic activity of Hb-Cu3(PO4)2 HNFs is 3 to 4 times higher than that of pure Hb,and the catalytic activity of H Hb-Cu3(PO4)2 HNFs retained is 89.70%after 35 days of storage at room temperature,which is 2 times higher than that of pure Hb.The developed fluorescence/colorimetric dual-mode biosensor has excellent selectivity for H2O2 detection.The linear range of colorimetric sensor is 2?10 ppb and 20?100 ppb,and the detection limit is 0.1 ppb.Fluorescence sensor is more sensitive,the linear range is 0.2?10 ppb,20?100 ppb,and the detection limit is 0.01 ppb.This biosensor was used to detect H2O2 in rainwater,tap water and wastewater,and the average recovery of the three determination results is between 88.95%and 112.36%.Compared with pure Hb,Hb-Cu3(PO4)2 HNFs exhibits significantly enhanced stability and catalytic activity.Compared with other sensing methods of H2O2,this biosensor can synchronously output fluorescence/colorimetric signals,and the dual signals are calibrated to each other to ensure the accuracy of the detection results.(2)The Hb-Mn3(PO4)2 HNFs were synthesized by using manganese ions and phosphate as inorganic raw materials,and a H2O2 electrochemical biosensor based on Hb-Mn3(PO4)2 HNFs was constructed.The conformation of Hb in this Hb-Mn3(PO4)2 HNFs was studied by using UV-vis,CD and fluorescence spectroscopy.The results showed that the conformation of Hb was unfolded,and a large number of Hb active centers were exposed on the surface of the nanoflowers.The electrochemical studies showed that this novel sensitive material has an excellent catalytic reduction effect on H2O2.The electrochemical biosensor based on the Hb-Mn3(PO4)2 HNFs has a wide linear range of 20 nm?0.36 ?M at low concentrations,and the detection limit is 7 nm,the sensitivity is 68.95 ?A·?M-1·cm-2.This biosensor was used for the rapid detection of H2O2 in rainwater and human serum samples,and the average recovery of the five determination results is between 99.8%and 105.3%.Compared with Hb-Cu3(PO4)2 HNFs,Hb-Mn3(PO4)2 HNFs are also hierarchical flower-like microspheres,but they have a larger specific surface area of 54.73 m2/g,richer Hb active centers and better electrochemical performance.Compared with electrochemical biosensors based on other nanomaterials,the detection limit of this biosensor is reduced by 1 order of magnitude,and the sensitivity is increased by 3 orders of magnitude.(3)With chicken egg white(CEW)as the organic component,the CEW-Cu3(PO4)2 HNFs were synthesized by a one-pot synthetic method.The catalytic performance and mechanism of this nanoflowers have been studied by using ESR,XPS and UV-vis.The results showed that the CEW-Cu3(PO4)2 HNFs has both mimetic peroxidase and polyphenol oxidase biocatalytic activity,and the average reaction rate constant is 11.43×10-2 min-1,which is 5 times higher than that of Cu3(PO4)2 NSs.Biotin-N-hydroxysuccinimide ester-anti-carcinoembryonic antigen secondary antibody labeled CEW-Cu3(PO4)2(CEW-Cu3(PO4)2 HNFs@Biotin-NHS-Ab2)capture probes were prepared by using the interaction between avidin in the CEW and biotin,and then a colorimetric biosensor for carcinoembryonic antigen(CEA)based on these probes was constructed.The results of experiments showed that under weak alkaline(pH=7.5)conditions,the linear range of this biosensor is 0.05?40 ng/mL,the detection limit is 3.5 pg/mL.The recovery of this biosensor for the three parallel replicatet detections of CEA in human serum is between 97.00%and 106.00%.Compared with colorimetric biosensors based on other nanomaterials,this biosensor can achieve rapid detection of CEA under conditions close to the pH value of human serum.(4)A synthetic method for Hb-M3(PO4)2·nH2O HNFs,which was suitable for using a variety of metal ions(Ca2+,Mn2+,Fe2+,Co2+,Ni2+,Cu2+? Zn2+)as inorganic raw materials,was proposed.The chemical composition of Hb-M3(PO4)2·nH2O HNFs,the conformational changes of Hb and the interaction between organic and inorganic components were analyzed by FT-IR,UV-vis,CD and XPS.The experimental results showed that the unfolding of Hb was induced by metal ions,meantime,the metal ions coordinated with exposed amide groups,carboxyl groups and other groups.Through using the hard-soft acid-base theory,the formation mechanism of Hb-M3(PO4)2·nH2O HNFs was systematically studied.With M2+located in Hb as the nucleation site,the crystal of M3(PO4)2·nH2O grew anisotropically,and finally the hierarchical flower-like microspheres were formed.The catalytic activity test results of Hb and seven kinds of Hb-M3(PO4)2·nH2O HNFs found that under the condition of system pH=7.5,Hb-Cu3(PO4)2 HNFs has the best catalytic performance,and the average reaction rate constant is 839.05×10-3 min-1,which is 14 times higher than that of pure Hb.Compared with the synthesis method proposed by Zare et al.,this method is more universal,and the catalytic performance of the synthesized material is also improved by 3 times.(5)With filter paper as the substrate material,the non-catalytically active CEW-Ca3(PO4)2 HNFs were grown in-situ on the surface of the filter paper by a one-pot method.Meanwhile,Hb-CEW-Cu3(PO4)2 HNFs with excellent catalytic activity were prepared.Biotin-NHS-Ab1 and Biotin-NHS-Ab2 were labeled on the surface of the two hybrid nanoflowers,the immuno-biosensing test paper of in situ growth of filter paper CEW-Ca3(PO4)2 HNFs@Biotin-NHS-Ab1-CEA-Hb-CEW-Cu3(PO4)2 HNFs@Biotin-NHS-Ab2 was prepared.Based on this test paper,the visual detection of CEA was realized by using the ELISA method.The experimental results showed that the detection range of the test paper is 0.5?100 ng/mL,the detection limit is 0.5 ng/mL,and the recovery of this test paper for the three parallel replicatet detections of CEA in human serum samples is between 89.50%and 108.06%.Compared with the traditional ELISA method,this test paper has the advantages of low cost,simple operation,fast detection and on-site detection.