| With the widespread use of antibiotics,antibiotic residues are increasingly becoming a serious problem in the field of food safety.Therefore,immediate and effective monitoring of antibiotics in foods can contribute to ensure food safety and human health.However,traditional antibiotic detection techniques have limitations in practical applications.Therefore,it is an urgent need to develop novel,rapid,sensitive and efficient analytical detection techniques.Optical biosensors have the advantages owing to their simplicity,rapidity and economy.In this study,we explored a novel assay based on polystyrene microspheres as optical signal probes and subsequently modified antibodies or complete antigen on their surfaces.After antibody-antigen recognition,the number of polystyrene microspheres could be changed after immunomagnetic separation.The concentrations of antibiotics were positively correlated with the change in the number of polystyrene microspheres.Based on the analysis of UV characteristic absorption curves of polystyrene microspheres,the chloramphenicol content in samples can be calculated by measuring the change of absorbance values at characteristic wavelengths to construct the polystyrene microsphere-mediated optical immunosensor(PMOI).PMOI was then successfully applied to determine chloramphenicol residues in milk.To achieve ultra-sensitive detection in milk,a biotin-streptavidin signal amplification system was combined with PMOI.In addition,different sizes of polystyrene microspheres were used as multivariate signal probes for simultaneous detection of multiple antibiotic residues.Combined with the partial least squares regression(PLSR)multivariate correction modeling method,the ultraviolet-visible spectra of polystyrene microspheres encoded with different particle sizes(i.e.,different signal probes)were completely separated and accurately identified.The PMOI-PLSR was used to achieve separation and accurate identification of the UV-visible spectra of polystyrene microspheres with different particle sizes(i.e.,different signal probes)and to quantify three polystyrene microspheres simultaneously.The PMOI was combined with PLSR model to analyze the optical spectrum for the simultaneous detection of three antibiotic residues(chloramphenicol,kanamycin,and neomycin)in milk.The details were as follow:1 Construction of PMOI for the detection of chloramphenicol in milkFirstly,the feasibility of the polystyrene microspheres as the optical signal was verified by measuring the UV-visible spectra of polystyrene microspheres.The linear relationship between the concentration of polystyrene microsphere probes and the absorbance at the maximum absorption wavelength were explored.Thereafter,magnetic nanoparticles coupled with chloramphenicol antibodies were used as immunomagnetic separation carriers.Meanwhile,polystyrene microspheres modified with chloramphenicol complete antigen were used as stable and efficient signal probes.In the presence of chloramphenicol,the signal of unbound polystyrene microsphere in the supernatant was calculated by measuring the absorbance after magnetic separation based on the reaction of chloramphenicol-polystyrene microsphere competitive binding to immunomagnetic separation carriers after magnetic separation.The quantitative detection of chloramphenicol was achieved by measuring the absorbance of the supernatant after magnetic separation to simulate the concentration of unbound polystyrene microspheres.The results showed that the linear range of the PMOI was 0.2 ng/m L-50 ng/m L,with the detection limit of 126pg/m L and detection time of 50 min,providing the advantages of high stability,simple operation,rapid detection and low cost with good market acceptance.2 Chloramphenicol detection by PMOI based on biotin-streptavidin signal amplification systemTo improve the sensitivity of PMOI,PMOI based on biotin-streptavidin signal amplification system was developed for the trace detection of chloramphenicol in milk.Chloramphenicol complete antigens were immobilized on the surface of magnetic nanoparticles and used as magnetic-separation carriers to specifically recognize chloramphenicol,while streptavidin-modified polystyrene microspheres were used as signal probes.In the presence of chloramphenicol,the number of antibodies to chloramphenicol bound on the magnetic particles was changed along with the competitive immunoreactions,thereby leading to the change of polystyrene microsphere probes bounded.Chloramphenicol quantification is achieved by measuring the absorbance of the signal probe in the supernatant.The PMOI based on biotin-streptavidin signal amplification system for the detection of chloramphenicol in the linear range of 10 pg/m L-5 ng/m L with a detection limit as low as 4.2 pg/m L,which is 30 times more sensitive compared with PMOI,providing a new idea for establishing a trace antibiotics detection method in foods.3 Simultaneous detection of three antibiotics in milk by PMOI-PLSRTo achieve the simultaneous detection of multiple antibiotic residues in milk,PLSR algorithm to resolve the ultraviolet-visible spectra under the conditions of multiple polystyrene microsphere probes in this chapter,which can overcome the difficulty of highly overlapping spectra of multi-grain size polystyrene microspheres.After orthogonal experimental design,the spectral information of polystyrene microspheres with different particle sizes and concentrations were obtained as the training group,and the PLSR mathematical model was established after spectral smoothing preprocessing.The prediction groups were prepared by mixing random concentration ratios.And the spectral data of polystyrene microspheres from the prediction group were substituted into the PLSR model for prediction to verify the accuracy of the established model.There were six parameters used to evaluate the analytical performance of the model,exhibiting good analytical performance.And the accuracy of the simultaneous prediction of the three polystyrene microspheres fluctuated in the range of 90%-110%.Three polystyrene microspheres with different particle sizes were selected as signal probes for the detection of chloramphenicol,kanamycin,and neomycin,respectively.Since the spectra of the polystyrene signal probes were slightly altered after coupling complete antigens,the coupled signal probes were remodeled for PLSR and analyzed for performance with excellent analytical performance.And the ultraviolet-visible spectra of the three signal probes were successfully resolved.Combining the PMOI method and the spectral signal processing function of the PLSR algorithm,the developed PMOI-PLSR can realize the simultaneous detection of three antibiotics(including chloramphenicol,kanamycin and neomycin),which greatly improves the detection efficiency.The detection limits were 0.03 ng/m L,0.07 ng/m L and 0.4 ng/m L for the simultaneous detection of chloramphenicol,kanamycin and neomycin,respectively,with a detection time of 1 h.This assay provides a good platform for the simultaneous and efficient detection of multiple antibiotic residues in the food field. |
PDF Full Text Request