Preparation Of Long-Wavelength Absorption Noble Nanomaterials And Their Application In Surface-Enhanced Raman Spectroscopy Immunochromatographic Assay

Immunochromatographic assay(ICA)based on surface-enhanced Raman spectroscopy(SERS)is a powerful analytical technique,which has been widely used in pesticide detection,biological analysis,food safety analysis,medicine and other fields in recent years due to its excellent and effective on-site rapid detection of trace analytes.The sensitivity and reproducibility of SERS-ICA are highly dependent on the properties of the SERS substrate materials,which are the core of its capability for quantitative analysis.The main research content of this thesis is to prepare noble nanoparticles with high SERS signal enhancement effect,which will be used as substrate materials for immunoprobes in SERS-ICA.Since the most of the current portable Raman spectrometers use a diode laser with an emission wavelength of 785 nm as the light source.According to the theory of electromagnetic enhancement in SERS,when the frequency of the localized surface plasmon resonance(LSPR)of noble nanoparticles is close to the frequency of the incident light source,the SERS enhancement effect will be further enhanced.Therefore,in this thesis,two noble nanoparticles with strong absorption in the long-wavelength range were prepared and characterized,and they were used as substrate materials for immunoprobes.Using 5,5’-dithiobis-(2-nitrobenzoic acid)(DTNB)and 1,4-benzenedithiol(BDT)as Raman signal molecules and anti-florfenicol(FF)and anti-5-Fluorouracil(5-FU)monoclonal antibodies(Ab)as biological recognition elements,two kinds of immunoprobes were prepared.The SERS-ICAs were established for the determination of the illegal antibiotic FF in animal feed and the anticancer drug 5-FU in human blood,respectively.The details are as follows:1.Au nanocubes(Au NCs)were synthesized and characterized as the substrate materials for immunoprobes.By mixing DTNB and anti-FF monoclonal antibodies with Au NCs,DTNB and Ab were successfully immobilized on the surface of Au NCs through chemical bonds(between Au NCs and DTNB)and electrostatic adsorption(between Au NCs and Ab)to generate the immunoprobe of DTNB-Au NCs-Ab.The coated antigen and secondary antibody were fixed on the test line(T line)and the control line(C line)of the ICA strip,respectively.When the sample is added to the sample pad,the solution moves towards the adsorption pad,driving the immunoprobe to the T line,so that FF in the sample competes with the coated antigen for the limited antibody on the immunoprobe.The higher the concentration of the analyte,the lighter the color on the T line.At the same time,the signal intensity of DTNB from immunoprobe on the T line was measured by a Raman spectrometer for quantitative detection.The whole detection process can be completed within 15 minutes.Under the optimized experimental conditions,the IC50 value and detection limit of SERS-ICA for the detection of FF were 27 pg mL-1 and 0.12 pg mL-1,respectively.Then,the selectivity experiment of SERS-ICA for FF detection was carried out.There was no cross-reactivity of the SERS-ICA with chloramphenicol,ofloxacin and lomefloxacin.but the cross-reactivity of the SERS-ICA with thiamphenicol(TAP)was found to be 50.4%,which was due to the high structural similarity between TAP and FF.The standard addition experiment showed that the recoveries of FF ranged from 88.6%to 102.4%,and the relative standard deviations of the three parallel experiments ranged from 2.3%to 7.4%.2.Au/Ag nanoshuttles(Au/Ag NSs)were synthesized and characterized,which were used as substrate materials for immunoprobes.Au/Ag NSs have excellent LSPR properties,hot spots on the surface of the fusiform tip,and strong SERS enhanced effect.By successively mixing BDT and anti-5-FU monoclonal antibody with Au/Ag NSs,BDT and Ab were successfully immobilized on the surface of the Au/Ag NSs respectively through chemical bonds and electrostatic adsorption to generate the immunoprobes of BDT-Au/Ag NSs-Ab.The detection principle and process are the same as SERS-ICA for FF.Under the optimal experimental conditions,the IC50 value and detection limit of SERS-ICA for 5-FU detection were 33 pg mL-1 and 0.10 pg mL-1,respectively.In selective experiments,it was found that there was no cross-reactivity of the SERS-ICA with cytosine,uracil and thymine,which are the pyrimidines inherently existed in human blood.SERS-ICA showed 3.5%and 3.2%of cross-reactivity with carmofluorine and 5-fluor-2’-deoxyuridine,but these two compounds are the substitutes of 5-FU and will eventually decompose into 5-FU.Spiking experiments in human serum samples showed that the recovery of 5-FU was between 91.4 99.0%,and the relative standard deviation(RSD)of three parallel experiments was 3.9 8.1%.Two noble nanomaterials prepared have characteristics of simple preparation,low cost,stable performance.LSPR in longer wavelength region and large SERS enhanced effect,which made them ideal substrate materials for immunoprobes in SERS-ICA.The established SERS-ICAs for the detection of the illegal antibiotic FF in animal feed and the anticancer drug 5-FU in human blood have the advantages of high sensitivity,strong specificity,easy portability,convenient operation and low cost.It has great application prospects of the established SERS-ICAs in food detection and cancer treatment effect monitoring.