Surface Plasmon Resonance Immunosensor Based On Signal Amplification Of Magnetic And Noble Metal Nanomaterial

Surface plasmon resonance(SPR)has become a mature method in monitoring molecular interactions.SPR can measure small changes in the refractive index of the noble metal surface when the analytes are combined on the sensing surface.Therefore,the SPR biosensor has been widely used for label-free detection of biomolecules in real time.Compared with the enzyme-linked immunosorbent assay(ELISA),SPR biosensor has attracted a huge amount of attention due to the advantages of simple operation,rapid detection and low sample consumption.However,label-free deteciton also brings certain shortcomings.The sensitivity of SPR biosensors is not high enough to detect low concentration substances,which limits the further development of SPR technology in the field of biosensors.To improve the sensitivity of SPR sensors,two methods have been extensively reported:(1)improving the sensing surface of SPR sensor to enhance the biocompatibility and spficific surface aera of the interface;(2)introducing the nanomaterial detection probe to achieve signal amplification detection through the sandwich immunoassay.However,the effect of improving the sensing surface of SPR sensor is not significantly enough to improve sensitivity.Therefore,the introduction of nanoparticle-based signal amplification probes has become the mainstream method.In addition,the sandwich method is also commonly empolyed to enhance the sensitivity of SPR sensors.In this paper,magnetic cobalt tetroxide nanoparticles(Co3O4 NPs),porous Ag@Au(p-Ag@Au NPs)and Au@Pd core-shell nanooctahedrons(Au@Pd NPs)were synthesized successfully.The signal amplification probes based on above nanomaterials were prepared and adopted to construct signal-amplified SPR sensors to detect human immunoglobulin G(HIgG).The specific work carried out is as follows:(1)Surface plasmon resonance immunosensor for detection of HIgG based on signal amplification of Co3O4 magnetic nanoparticlesIn this work,carboxylated Co3O4 nanoparticles(Co3O4 NPs)were synthesized,and the signal amplification probes were prepared by immobilizing antibody molecules on the surface of the Co3O4 NPs.The sandwich immunoassay mode was utilized to construct an SPR immunosensor for highly sensitive detection of HIgG based on the signal amplification strategy.The synthesized Co3O4 NPs and signal amplification probes were characterized by scanning electron microscope(SEM),transmission electron microscope(TEM),X-ray diffraction(XRD),X-ray photoelectron spectroscopy(XPS)and other technologies.The results proved the successful preparation of Co3O4 NPs and signal amplification probes.SPR immunosensor was prepared by fixing staphylococcal protein A(SPA)on the surface of SPR gold chip,and then combined with the capture antibodies.Then,the HIgG antigen solution and Co3O4 NPs signal amplification probes were injected to SPR the sensor separately.The sandwich conjugate formed by two-step incubation.Finally,the real-time reaction signal was recorded by SPR instrument.The change of SPR signal showed a linear relationship with the concentration of the antigen solution.The linear range was 0.01-50 ?g/mL,and the detection limit was 2.9 ng/mL.The SPR sensor realizes highly sensitive detection of HIgG with good specificity and reproducibility,and provides a promising platform for clinical diagnosis.(2)Porous Ag@Au signal amplification for detection of HIgG based on Surface plasmon resonance immunosensorThe Ag@Au core-shell nanoparticles were synthesized using a two-step synthesis method,and then etched by H2O2 to prepare porous Ag@Au core-shell nanoparticles(p-Ag@Au NPs).The resultant p-Ag@Au NPs were empolyed to prepare a signal amplification probe to construct a signal amplification SPR sensing strategy.The p-Ag@Au NPs were chatacterized by TEM and XRD.The TMB color reaction proved that p-Ag@Au NPs possessed peroxidase activity.The sandwich immune complexes were obtained after two-step incubation on the SPR immunosensing surface.Due to the utilization of the p-Ag@Au NPs signal amplification probe,the refractive index of the sensing surface obviously increased,causing the shift of SPR angle.Subsequently,the mixed solution of aniline and H2O2 was injected the flow cell.With the help of peroxidase activity of p-Ag@Au,aniline was polymerized on the sensor surface,leading the further increase of the SPR angle.The SPR instrument was employed to record the reaction signal.Using HIgG as the target analyte,the signal of the SPR immunosensor increased linearly with the concentration of HIgG.The linear range was 0.01-1?g/mL with a detection limit of 1.4 ng/mL.Furthermore,the sensor has excellent performance such as high sensitivity,good specificity and reproducibility.The SPR sensor based on p-Ag@Au signal amplification can detect the real serum sample and has important application value.(3)Surface plasmon resonance immunosensor for detection of HIgG based on Au@Pd nanooctahedral signal amplificationAu@Pd nano-ctahedral particles(Au@Pd NPs)with core-shell structure was synthesized through one-step hydrothermal method.The antibodies could be firmly combined with the Pd shell through the amino group to form a sigal amplificaon probe.The morphology of Au@Pd NPs was characterized through SEM,TEM and high resolution transmission electron microscope(HRTEM).The peroxidase activity was also verified by the color reaction of TMB.The SPR immunosensor was obtained by sequentially modifying SPA and Rabbit anti-HIgG(Ab1)on the surface of SPR gold chip,and blocking unreacted active sites with bovine serum albumin(BSA).Then,the antigen solution and the signal amplification probes were respectively injected the sensor for the incubation,forming sandwich immune complexes.The immune complex changed the refractive index of the SPR sensor surface,and further improved SPR angle.Subsequently,Au@Pd NPs,possessing mimetic enzyme properties,catalyzed the reaction of aniline to form a polymer on the sensing surface,further increasing the quality and prompting the second increase of SPR signal.The proposed SPR sensor based on Au@Pd nanooctahedral signal amplification achieved highly sensitive detection of HIgG in the range of 0.005-1 ?g/mL with a detection limit of 0.106 ng/mL.The SPR sensor was successfully utilized in real human serum sample analysis.This strategy shows the advantage of simple operation,low sample consumption,high efficiency and sensitivity,and has great potential in clinical application.