| Nelumbo Semen(lotus seed)derives from the dried seed of Nelumbo nucifera Gaertn.,with the effects of nourishing the heart and soothing the nerves,invigorating the kidney and astringing essence.It is a kind of commonly-used traditional Chinese medicine(TCM)with important medicinal and edible functions.Affected by the internal factors,such as the large amount of starch and oil contained therein and external environmental conditions,such as temperature and humidity,it will be easily infected with fungi and highly toxic mycotoxins if it is not handled properly during the harvest,processing,storage and transporation processes.Of them,aflatoxin B1(AFB1)is one of the most common-found mycotoxins with strongest toxicity.Therefore,the development of sensitive,accurate and universally applicable methods to evaluate the pollution and residual level of trace AFB1 in large quantities of lotus seeds is of great significance for ensuring the quality and safety of this TCM,as well as the physical and mental health of the consumers.Chromatographic and the combined technologies are the common analysis methods for AFB1 detection.However,due to the limitations of expensive equipment,complex sample preparation and high cost,it is difficult to achieve low-cost and sensitive detection of AFB1 in a large number of samples.With the development of immunoassay technology and the introduction of nanomaterials,the immunosensors based on luminescent nanomaterials as labels with good specificity and high sensitivity has attracted more and more attention.Among these luminescent nanomaterials,quantum dots(QDs)with the advantages of wide absorption spectrum,narrow emission spectrum,high fluorescence intensity,etc.,are widely used in the construction of immunosensors.QDs-based immunoassay strip technology does not require complex sample preparation processes,large and expensive detection equipment,and professional laboratory personnel,which can achieve rapid on-site detection of mycotoxins in a large number of TCMs with low cost.In addition,QDs-based electrochemiluminescence(ECL)sensors with the advantages of simple operation,low background interference,high sensitivity,wide linear range,suitable for portable instruments can achieve low-cost and ultra-sensitive detection of trace mycotoxins in TCMs.Therefore,this study introduced high fluorescence performance of QDs fluorescent probes to develop a simple and low-cost QDs-fluorescent microsphere immunochromatographic test strip and a sensitive and accurate ECL immunosensor,respectively,for low-cost on-site and ultra-sensitive quantitative detection of AFB1 in lotus seeds.The main contents are listed as follows:1.To investigate the immunochromatographic applicability of QD-based microbeads(QBs)and ECL properties of QDs for to providing effective support for the construction of QBs lateral flow test strip and ECL immunosensors.We firstly investigate the optical properties of QBs and the best conditions for electrochemiluminescence detection of QDs.The results showed that CdSSe@ZnS QBs have a wide and continuous UV-visible absorption spectrum,and a strong fluorescence signal at 619 nm.After coupling with anti-AFB1 antibody,the optical properties of CdSSe@ZnS QBs remained unchanged with strong fluorescence signal,which can be used to construct sensitive and accurate QBs lateral flow test strip for rapid on-site detection of AFB1.In addition,through the electrochemical luminescence experiments,K2S2O8 was determined as the co-reactant of ZnCdS@ZnS QDs,and the reduction potentials of these QDs and the co-reactant were-1.17 V and-0.68 V,respectively.Based on systematic optimization of experimental parameters,it could be found that the ZnCdS@ZnS QDs could excite a strong and stable ECL signal(around 18000 au)when K2S2O8(pH 7.4,0.1 M)was selected as the co-reactant with the sweep rate of 100 mV/s and the operating start voltage of-0.7 V,which could be used to build new QDs based ECL immunosensors for ultra-sensitive detection of AFB1.In summary,CdSSe@ZnS QBs exhibited strong immunochromatographic applicability,and the core-shell type ZnCdS@ZnS QDs displayed excellent ECL properties,providing effective support for the next research.2.To establish a CdSSe@ZnS QBs-based immunochromatographic test strip for on-site rapid detection of AFB1 in large quantities of lotus seed samples.Based on lateral flow immunochromatographic technology,a sensitive and rapid CdSSe@ZnS QBs based immunochromatographic test strip was developed for on-site detection of AFB1 in lotus seeds.Carboxylated QBs were used as the fluorescent labels,and the fluorescent probe was prepared by coupling QBs with anti-AFB1 antibody through the 1-ethyl-3-(3-dimethylamino-prophl)carbodiimide hydrochloride/N-hydroxysuccinimide(EDC/NHS)activation method.Bovine serum albumin(BSA)-AFB1 and Goat anti-mouse IgG antibody were coated on the nitrocellulose(NC)membrane to prepare test(T)and control(C)lines,respectively,to establish the QBs-based immunochromatographic test strip.Under the optimal conditions,the test strip can detect AFB1 in lotus seeds within 15 min with a wide linear range of 1-19 ng/mL(2-38μg/kg)and a limit of detection(LOD)of 1 ng/mL(2μg/kg).The recovery rates from the spiked lotus seed samples with three levels(2.5,5 and 10 μg/kg)of AFB1 were 116.0%-94.0%,with the relative standard deviation(RSD)less than 8.5%.Real-world analysis for 14 batches of lotus seeds showed that AFB1 was found in 3 samples with the positive incidence of 21.4%.All the positive samples were confirmed by liquid chromatography tandam mass spectrometry method.The newly-estbalished QBs-based immunochromatographic test strip with the advantage of simple operation,low cost and high sensitivity can meet the needs of on-site rapid and quantitative detection of AFB1 in primary screening test of large quantities of lotus seeds.3.To develop a ZnCdS@ZnS QDs-based ECL immunosensor for sensitive detection of AFB1 in lotus seeds.Through introducing Nafion membrane solution as the fixing agent to assemble a large number of ZnCdS@ZnS QDs onto the surface of the Au electrode as ECL signal probes,and using the highly-specific anti-AFB1 antibody as the capture probe,we developed a ZnCdS@ZnS QDs-based label-free ECL immunosensor for sensitive detection of AFB1 in lotus seeds for the first time.Based on the generated ECL signal resulting from the redox reaction between 32O82-in the electrolyte and the QDs on the electrode surface,the decreased ECL signal intensity △IECL caused by the presence of the target AFB1 in the sample wass recorded for quantitation of AFB1.Regarding the proposed ECL immunosensor,each step for the construction was evaluated by electrochemical impedance spectroscopy(EIS)and ECL measurements.After systematic optimization of some crucial parameters,the linear regression equation of the ECL immunosensor for AFB1 was △IECL=48.817c+1750.9 with the correlation coefficient(R)of 0.9975,along with a wide linear range of 0.05-100 ng/mL and a LOD of 0.01 ng/mL.The recovery rates from the spiked lotus seed samples with three levels(5,10 and 20 ng/mL)of AFB1 were 99.2%-101%.All these evaluations have confirmed the high specificity,satisfactory stability and good repeatability of the ECL immunosensor,which can achieve ultra-sensitive detection of trace AFB1 in lotus seeds,andfurther can be popularized in many fields such as food and TCM safety evaluation,environmental monitoring.Based on the excellent optical properties of QDs,the developed CdSSe@ZnS QBs based immunochromatographic test strip and ZnCdS@ZnS QDs-based label-free ECL immunosensor can realize low-cost on-site and ultra-sensitive detection of trace mycotoxins in large quantities of complex TCM matrices,which can reduce the dependence on large instruments,providing reliable technological support for the efficient monitoring of various mycotoxins in lotus seeds and other TCMs.It is of great significance to ensure the quality and safety of TCMs regarding clinical medication and import-export trades. |
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