| Point-of-care testing(POCT)technology is of great significance for rapid on-site testing in the fields of disease diagnosis,environmental monitoring and food safety testing.The lateral flow immunochromatography assay(LFIA)using gold nanoparticles(GNPs)as a marker has many advantages such as simplicity,speed,low cost,stability and reliability,and it has become one of the most widely used POCT technologies.However,gold immunochromatographic assay(GICA)mainly relies on visual colorimetric detection,of which the detection sensitivity is generally lower than other immunological methods based on fluorescent labeling,radioactive labeling,and enzyme-labeled colorimetric,and it can not realize quantitative detection of analyte.On the basis of retaining the convenient and quick characteristics of lateral immunochromatography,research on new LFIA nanomarker detection methods has become a new trend to improve the sensitivity of LFIA detection methods.The gold nanocage is a cubic hollow structure with adjustable surface plasmon optical properties.By adjusting its wall thickness and edge length,it can efficiently absorb light in the range of 400-1200 nm.Based on the excellent photothermal conversion efficiency and good modifiability of the gold nanocage,it is expected to research and develop a new method of lateral immunochromatography based on the gold nanocage label.In this paper,based on the excellent photothermal effect of gold nanopcages,a new method of gold nanocage calorimetric lateral flow immunoassay(CLFA)was studied.Two immunochromatographic rapid detection methods of double-antibody sandwich method and indirect competition method were established.It is used to detect proteins and small molecule antigens,and research the CLFA photothermographic theorem processing algorithm.The main contents and results are as follows:(1)Gold nanocages with uniform particle size,strong absorption at 808 nm,and good photothermal properties were prepared;the study used thiol-polyethylene glycol-succinimide ester(HS-PEG-NHS)as a coupling agent to obtain stable labeled immunogold nanocage GNCs-Ab1.By optimizing the calorimetric detection conditions,the photothermal properties of GNCs were tested on aqueous solutions and chromatography test strips respectively,and compared with the photothermal properties of spherical gold nanoparticles,it was confirmed that the calorimetric analysis tof gold nanocages has advantages in the high sensitivity detection.Combining GNCs calorimetric analysis with immunochromatography,a new method for detecting AFP antigen based on double-antibody sandwich immunochromatography was established.The comparison test results with the commercial kit of AFP colloidal gold immunochromatography showed that the gold nanocage CLFA calorimetric method improved the detection sensitivity of AFP by about 5 times.In the detection experiment of AFP in clinical serum samples,the gold nanocage CLFA method obtained similar detection results to the clinical commercial enzyme-linked immunonsorbent assay(ELISA).The relative error of the detection values of the two methods was in the range of-3.0% to 7.9%,the technical feasibility and reliability of the new gold nanocage CLFA calorimetric method were further verified,and the fast and highly sensitive quantitative detection of AFP was achieved.(2)Using the high sensitivity of GNCs calorimetric detection,the research of immunocompetitive chromatography detection technology for small molecule antigens was developed to study the content of zearalenone(ZEN)in feed by GNCs CLFA.Benefiting from the dual advantages of better precision and higher sensitivity of the GNCs calorimetric method,the signal discrimination of CLFA is better than the traditional colloidal gold VLFA method in both low and high concentration ranges,and the detectable concentration of the method is improved.The range is from 20 to 200 ng/m L of GNPs VLFA to 5 to 500 ng/m L.In further study GNCs CLFA method was be used to detect the content of methamphetamine(MET)in saliva and fentanyl(FEN)in hair.Compared with the GNPs VLFA method,the detection limit of the GNCs CLFA method is reduced by about 2-3 times.Using quantum dot fluorescence immunoassay as a control method,the actual measurement results of hair samples of suspected drug users show that the GNCs CFLA method is basically consistent with the quantum dot fluorescence detection results,reaching the same level.The highly sensitive detection technology based on GNCs CLFA calorimetric method is not only applicable to the detection of large molecule antigens by the double-antibody sandwich method,but also applicable to the detection of small molecule antigens by the indirect competition method.(3)A new region growing algorithm combined with fast peak detection(RGFPD)for segmenting CLFA images is proposed.The implementation of RGFPD mainly includes two steps.In the first step,peak detection is used to obtain the information of the signal area,which provides seed points and growth conditions for regional growth,and can effectively distinguish interfering substances and signals.The second step uses the seed points and growth conditions are used to grow the area to obtain a binary image,and then the signal area is extracted from the binary image,and finally quantitative analysis is performed.Compared with the advantages and disadvantages of fuzzy c-mean(FCM)and cellular neural network(CNN),the results show that the new method is faster and more practical,and it solves the problems such as blur signal boundary and uneven laser spot in thermal image of the calorimetric lateral flow immunoassay(CLFA)method better.So it is of practical significance for reducing human error,improving the accuracy and reproducibility of CLFA quantitative results,and realizing on-site real-time detection. |
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