Development Of Electrochemiluminescence Aptasensor Based On CdSe/CdS Quantum Dots And Study On The Detection Of Mycotoxins In Rhubarb

Objective Mycotoxins are toxic secondary metabolites produced by fungi.Among them,aflatoxins(AFB1,AFB2,AFG1,AFG2)and ochratoxin A(OTA)are the most common and most poisonous.Chinese medicine may be contaminated by a variety of toxic fungi during the process of growth,harvest,processing,transportation and storage,which poses a great threat to the quality and safety of Chinese medicines.Establishing a rapid detection method for mycotoxins in Chinese medicines and studying the effects of mycotoxins on the quality of Chinese medicines are of great significance for controlling the quality of Chinese medicines and ensuring the safety of medicines.This subject established a simple and low-cost electrochemiluminescence aptasensor for the highly sensitive detection of OTA and investigated the influence of accumulation of AFs on the content of active ingredients in rhubarb.Methods To construct an electrochemiluminescence aptasensor based on quantum dots,the properties of CdSe/CdS quantum dots were investigated,the electrochemiluminescence mechanism of CdSe/CdS quantum dots was explored,and the scanning rate,p H value,the concentration of co-reaction reagent(K2S2O8)were optimized.Finally,the excellent and stable electrochemiluminescence signals of CdSe/CdS quantum dots were obtained.Based on CdSe/CdS quantum dots and aptamer,an electrochemiluminescence aptasensor was constructed,and the assembly process of the sensor was characterized by electrochemical methods.A series of conditions were optimized,including the concentration of chitosan,the reaction time of 2% glutaraldehyde and chitosan,the concentration of aptamer and the reaction time of aptamer and OTA.Finally,the constructed electrochemiluminescence aptasensor was used to detect OTA in rhubarb samples.An ultra-high performance liquid chromatography-photodiode array detection(UPLC-PDA)method and a high-performance liquid chromatographyfluorescence detection(HPLC-FLD)method were established to detect the contents of five active ingredients(aloe-emodin,emodin methyl ether,rhein,emodin and chrysophanol)and AFs(AFB1,AFB2,AFG1,AFG2).The "reverse culture" technology was used to infect rhubarb with Aspergillus flavus strains,and the infected and uncontaminated rhubarb samples were placed into a constant temperature incubator(temperature: 28°C;humidity: 90%).The culture period was 20 days.A part of rhubarb was taken out every 4 days to detect the content of active ingredients and AFs at the same time to explore the influence of the accumulation of AFs on the quality of rhubarb.Results The CdSe/CdS quantum dots were spherical,with relatively uniform particle size,and had good optical properties.When the scan rate was 100 m V/s,the p H was 7.4,and the K2S2O8 concentration was 0.1 mol/L,quantum dots had excellent and stable electrochemiluminescence signals.The optimal experimental conditions for the electrochemiluminescence aptasensor were determined: chitosan concentration was 1.0 mg/m L,the reaction time of 2% glutaraldehyde and chitosan was 2 h,the concentration of the aptamer was 2 μM,and the reaction time between the aptamer and OTA was 2 h.Under the best experimental conditions,the constructed electrochemiluminescence aptasensor had excellent detection performance for OTA,with a linear range of 1-100 ng/m L and a detection limit(LOD)of 0.89 ng/m L.For rhubarb samples,the recovery rate of OTA was 97.3-101.5%,RSD≤4.2%.Through continuous monitoring of the content of five active ingredients and AFs in rhubarb,the results showed that the content of aloe-emodin,rhein,emodin,chrysophanol and emodin methyl ether decreased from 1.58,3.93,1.34,7.91,1.25 mg/g to 0.27,1.41,0.35,1.35 and 0.24 mg/g as the culture time extended,while the content of AFB1 and AFB2 rose to 278.75 μg/kg and 37.87 μg/kg,respectively.There is a negative correlation between the content of AFs and the active ingredients.Conclusions With good selectivity,stability and reproducibility,the electrochemiluminescence aptasensor based on quantum dots and aptamer could realize highly sensitive detection of OTA in rhubarb,which provides a new idea for the detection of mycotoxins in Chinese medicine.With the extension of the culture time,the accumulation of AFs will lead to a decrease in the content of the main active ingredients in rhubarb.The accumulation of mycotoxins will seriously affect the quality of traditional Chinese medicine.