| Immunoassay refers to the qualitative and quantitative analysis of chemical substances（such as hormones,drugs,and specific proteins）using highly specific binding between antibodies and antigens or haptens.Its excellent accuracy and operability provide reliable data support for in-depth research within the fields of disease diagnosis,food safety,and environmental protection.Surface-enhanced Raman scattering（SERS）has attracted extensive attention in physics,chemistry,biology,and other fields because of its high sensitivity,good selectivity,and rich molecular structure information.Electrochemical analysis methods,on the other hand,have the advantages of low detection limit,low price,easy miniaturization,and automatic analysis and detection.Therefore,using SERS and electrochemistry as reliable readout methods for immunoassay of proteins and small molecule substances overcomes the shortcomings of the existing readout methods,such as low sensitivity,poor resolution,and difficulty in multi-channel detection.In this thesis,a class of gold and silver array substrates and microelectrode arrays with two-dimensional ordered cavity structures are constructed,combined with a hybridization chain reaction technique and immune recognition to form a "super sandwich" immune complex.Various immunosensors are constructed for the SERS and electrochemical detection of actual samples.The main research contents and results are as follows:（1）Au/Ag bimetallic nanoparticles as SERS immunoprobes were prepared by a simple method.During the preparation process,the excess reducing agent in the reaction system was fully utilized,and through the fine control of the reaction conditions,silver-rich Au/Ag nanoparticles（BNPs）with a clean surface were obtained.A systematic study of the formation mechanism of BNPs proved that the construction process of this kind of nanoparticles was the interaction between the initial GR reaction,the co-reduction of Ag+with citrate,and the interaction between alloying and dealloying.At the same time,due to the enrichment of Ag in the nanostructure,these BNPs had a considerable SERS activity,and the enhancement factor can reach 2.3×106.Using the BNPs as nanoprobes,antibodies and probe molecules were introduced into the complex immune system.Through combined with gold staining,an Au/protein immune complex/Au/Ag island membrane with "super sandwich" structure was then constructed to complete a SERS-based immunoassay.Rabbit IgG can be detected in a wide concentration range,and the detection limit is as low as 10 pg·L-1.（2）A series of SERS substrates with good enhancement effect and high reproducibility were constructed.Polystyrene（PS）microspheres of different diameters（500,600,700,800,1000 nm and 1.2,1.5,2 μm）were arranged into two-dimensional close-packed templates through self-assembly at the gas-liquid interface.Metal（gold,silver,and gold-silver bimetallic）cavity arrays were prepared by electrodeposition to fill the interstices between PS spheres.The cavity’s morphology（aperture and depth）can be controlled by changing the amount of electricity in the electrodeposition process.The correlations among substrate morphology,localized surface plasmon resonance（LSPR）,and SERS enhancement were investigated,and the theoretical explanation was given.（3）On the surface of the highly ordered,LSPR adjustable golden cavity array（GCA）,a competitive SERS-based immunosensor for the determination of salbutamol（SAL）and brobuterol（BRO）was constructed.The negatively charged phosphate backbone of the double-stranded long-chain HCR concatemers could be used to adsorb the positively charged labeled molecules（Nile blue（NB）and 3,3’,5,5’-tetramethylbenzidine（TMB））by electrostatic interaction.This strategy dramatically increases the loading of the labeled molecule and plays the role of signal amplification.Through combining immune recognition reaction and silver staining,the SERS intensity of the characteristic peaks of labeled molecules(NB:600 cm-1,TMB:955 cm-1)is inversely proportional to the analyte concentration,which can be used for both SAL and BRO qualitative and quantitative analysis.The detection limits of the method for SAL and BRO were 1.0 pg·mL-1 and 2.0 pg·mL-1,respectively.When the target analytes were added to pork,liver,and human urine samples,the recovery rates of SAL and BRO were 96.7-105.3%,and the relative standard deviation（RSD）was 4.9-8.7%.（4）Based on the previous work,we prepared a gold/silver bimetallic cavity array（BMCA）and developed a competitive immunoassay that can achieve SERS and electrochemical double readout for simultaneous detection of β-adrenergic agonists.By changing the molar ratio of gold to silver in the precursor,the morphology and composition of this BMCA can be easily modulated.When the ratio of gold to silver is 6:4,the prepared BMCA displays regular morphology,excellent SERS enhancement,and signal reproducibility.Three different labeling molecules with clearly distinguishable electrochemical and Raman characteristic peaks are carefully selected for simultaneous qualitative and quantitative detection of salbutamol（SAL）,ractopamine（RAC）,and phenylethanolamine A（PA）.The intensity of the characteristic Raman peak and the peak current of the square wave pulse are inversely proportional to the analyte concentration.In a wide concentration range from 1 pg·mL-1 to 200 ng·mL-1,the detection limits of this immunosensor for SAL,RAC,and PA are 0.8,0.4,and 1.3 pg·mL-1,respectively.Finally,the method is suitable for analyzing real samples,the recovery rate is 95.0%-108.5%,and the RSD is 6.9-10.7%.（5）Using the close-packed two-dimensional silica spherical cavity as a template,gold nanoparticles were electrodeposited on the bottom of the spherical cavity.A type of SiO2 cavity isolated gold nanoelectrode array（GMA）with adjustable center distance was prepared.The array electrode exhibits remarkable microelectrode characteristics,including high signal-to-noise ratio,high current density,high signal resolution,etc.,thereby transmitting enhanced electrochemical signals.Subsequently,FC was used as a label molecule to provide endogenous electrochemical signals,and the recognition antibody（anti CEA,Ab2）loaded on AuNPs provides biological detection ability.Fc-AuNP-anti CEA probe loaded a large number of Fc molecules through sulfhydryl self-assembly to achieve the purpose of signal amplification,so as to improve the sensitivity of the immunosensor,and the detection limit is 0.01 ng·mL-1.The sensor also showed good recovery in the test of actual serum samples. |
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