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Study On Novel Surface Plasmon Resonance Biosensor And Its Application In Immunoassay

Posted on:2019-10-12Degree:DoctorType:Dissertation
Country:ChinaCandidate:Q WuFull Text:PDF
GTID:1368330548461989Subject:Analytical Chemistry
Abstract/Summary:PDF Full Text Request
Surface plasmon resonance?SPR?biosensors represent advanced andmature optical biosensing technology.SPR biosensors have become powerful tools for studying the kinetic properties of biomolecular interactions due to the ability of real-time measurements of biomolecule interactions without labeling.A large amount of scientific researches have focused on improving the sensitivity and selectivity of SPR biosensors to detect low-molecular weight target molecules with low concentration in complex biological matrices.In this thesis,highly sensitive and selective SPR biosensors have been constructed on the basis of nanomaterials and successfully applied in immunoassays.Because of its good biocompatibility,large specific surface area,high capacity of loading biomolecule and excellent electrical properties,graphene oxide?GO?has become an ideal material for fabricating novel biosensors.Since GO-based sensing platform shows low sensitivity,GO is often used in conjunction with metal nanoparticles to improve the performance of biosensors.A SPR sensing platform based on GO and GBPs was designed and applied to detect rabbit IgG.Firstly,GO was assembled on amine-modified gold film by electrostatic interaction.Next,SPA-modified GBPs were prepared by attaching staphylococcal protein A?SPA?to the surface of GBPs,and then the SPA decorated GBPs were covalently immobilized on the GO sheet to form GBPs-SPA-GO modified sensing membrane.The SPA on the sensing membrane surface can immobilize antibody in an orderly manner without affecting the subsequent binding of antigen.The GBPs with anisotropic structure are used to amplify the response signals and improve the detection sensitivity.The minimum detectable concentration for rabbit IgG by the fabricated biosensor is 0.15?g mL-1,16-fold lower than that of conventional MPA-based SPR biosensor.The advantages of GO-SPA-GBPs-based sensing platform in immobilizing antibodies and enhancing response signals have effectively improved the sensitivity of the assay.Silver nanocube?SNC?with eight sharp apexes shows a significant electromagnetic field enhancement effect and is very sensitive to global and local dielectric changes.Therefore,SPR biosensors based on carboxyl-functionalized GO?cGO?and SNC were designed.Carboxyls are functionalized on the surface of GO to form cGO by using 4-aminobenzoic acid?PABA?.The obtained cGO were assembled on the amine-modified gold chip through electrostatic interaction for immobolizing antibodies.SNC-antigen conjugates were prepared by attaching antigen molecules on the surface of 3-mercaptopropionic acid?MPA?modified SNCs.The introduced SNCs not only amplify the response signal generated from antigen-antibody binding by electromagnetical coupling with the gold film but also serve as labels to increase the mass reaching on the sensing film.The constructed SPR biosensor responds well to human IgG in the concentration range of 0.075-40?g m L-1 and the lowest concentration that can be detected is 32-fold lower than that of traditional MPA-based SPR biosensor.The multiple sharp branches of GNS accumulate high-intensity electromagnetic field and GNS is reported to be one of the most sensitive nanostructures currently available.Monodisperse GNSs were prepared using a surfactant-free method and then combined with target molecular pig IgG to form GNSs-pig IgG conjugates for amplifying the response signals.The performance of the developed SPR biosensors based on cGO as sensing substrate and AuNSs-antigen or GNPs-antigen conjugate as signal enhancer were investigated.The result shows that GNSs with protuberant sharps exhibit stronger amplification of response signals originating from the binding of antigen and antibody than GNPs.The SPR biosensor based on cGO and GNS responds well to pig IgG in the concentration range of 0.0375-40?g mL-1 and the lowest concentration that can be detected is 32-fold lower than that of GO-based SPR biosensor.Magnetic nanomaterials Fe3O4 NPs possess good biocompatibility,strong superparamagnetism,low toxicity,high loading capacity,simple preparation process and low cost.Gold nanostructures have played an important role in the field of SPR biosensing because of their excellent optical properties,good stability and easy modification.Combining Fe3O4 NPs with gold nanostructures to form Fe3O4-gold nanostructures hybrids will reserve properties of these two nanomareials and relieve aggregation of Fe3O4 NPs.Fe3O4-hollow gold nanoparticles?HGNPs?hybrids?FH?were prepared and the detection antibody?dAb?were biofunctionalized on the surface of FH to form magnetic immune probe?FH-dAb?.With the help of FH-dAb,the target analyte human IgG in the sample solution were selectively captured,separated and enriched.This process can significantly enhance the sensitivity of detection and effectively reduce the matrix effect.The magnetic immunoprobe serves as“vehicle”for rapidly delivering target molecules to the surface of sensor chip for specifically binding to the capture antibody?cAb?immobilized on the cGO-based sensing platform with the help of an external magnetic field.Magnetic field-driven mass transfer overcomes the disadvantage of slow diffusion limited mass transfer and matrix interference effect in regular patterns,which has been proven to be fast and efficient.In addition,the considerable increase in the total mass of FH on the sensing film and the electronic coupling interaction between the localized surface plasmon of HGNPs and the surface plasmon wave associated with the SPR gold films significantly enhance the sensitivity.A novel magnetic field-assisted SPR biosensor for the detection of human IgG was constructed on the basis of magnetic immunoprobe,which combines SPR sensing technology and immunomagnetic separation technology.The obtained detection limit is 1.88 ng mL-1,about 260-fold lower than that obtained by conventional sandwich methods.In order to improve the stability of magnetic nanoparticles,Fe3O4 NPs were dispersed on the surface of multi-walled carbon nanotubes?MWCNTs?.The obtained MWCNTs-Fe3O4 composites?MMWCNTs?were further coated by polydopamine?PDA?film.The dAb can be directly immobilized on the surface of MMWCNTs-PDA nanocomposite to form MMWCNTs-PDA-dAb immune probe by Schiff base reaction between the amine groups of biomolecules and the quinone groups of PDA.HGNPs-PDA modified gold chip provides a mild and efficient sensing platform for immobilizing cAb without needing any activation process.The interaction between localized surface plasmon resonances of HGNPs and the propagating plasmon on the surface of gold film leads to the amplification of SPR response signal.The introduction of MMWCNTs-PDA-dAb immune probe offers additional sensitivity enhancement through efficient enrichment of analytes,fast mass transfer and significant amplification of SPR response signal as labels.As a result,the detection limit of cardiac troponin molecule?cTnI?obtained by this method was 1.25ng mL-1,which was 1000-fold lower than that of PDA-based sensors.The method of separating and concentrating analytes from a sample solution using MMWCNTs-PDA-dAb immune probe realizes high sensitivity and good accuracy for detection of cTn I in human serum.Based on the above experiments,AuNPs-PDA-cAb,Fe3O4@PDA-dAb,and MWCNTs-PDA-AgNPs/secondary antibodies?MWCNTs-PDA-AgNPs/Ab2?were prepared and served as sensing platforms,immune probe,and response signals enhancer,respectively.A novel sensing mechanism combining SPR biosensor with immunomagnetic separation technology was developed to detect cTn I.The PDA-modified gold film was obtained by self-polymerization of dopamine?DA?in an alkaline environment,and then HAuCl4 was injected into the reaction cell.PDA as a reducing agent can reduce Au3+into gold nanoparticles and deposit on the surface of the PDA film to form AuNPs-PDA modified gold film.The obtained sensing platform was used for direct immobilization of capture antibody?cAb?and SPR sensing.Magnetic immune probe was prepared by attaching detection antibody?dAb?on the surface of Fe3O4 NPs coated by PDA for the precise capture,magnetic separation and enrichment of the target analyte?cTnI?from samples.After cTnI labeled by Fe3O4@PDA-dAb immune probe was captured by cAb immobilized on the sensing platform,the MWCNTs-PDA-AgNPs/Ab2 nanoconjugates were introduced into the immune system for further binding with the unreacted cTnI exposed on the immunoprobe,which realizes the secondary amplification of the response signal.The detection limit obtained by this method is 3.75 ng mL-1,320-fold lower than that obtained by PDA-modified sensing platform.This method also shows good selectivity and accuracy in the analysis of complex serum samples,indicating its potential for clinical diagnosis.
Keywords/Search Tags:Surface plasmon resonance, Graphene oxide, Metallic nanoparticles, Polydopamine, Magnetic immune probe, Signal amplification technology
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