Study On Rapid Detection Of Mycotoxins By Aptamer Strips

In recent years,frequent food safety problems have seriously endangered people's health,which has caused widespread concern.Among them,mycotoxin has a wide range of contamination,a great variety,high toxicity,and it has the characteristics of small molecular weight,stable structure,and difficult to be decomposed.The food contaminated by mycotoxins has seriously affected the health of the general public.Therefor mycotoxin has been the focus of food testing.Generally,the traditional detection methods of mycotoxin contain instrumental methods and enzyme-linked immunoassays.Among them,the methods by instruments are expensive and always require professional operations to perform on-site inspections.Enzyme-linked immunosorbent assay results are greatly affected by the external environment,the preparation cycle of the required antibody is longer,the cost is higher,and there are lot-to-lot differences,furthermore,the immunogenicity of mycotoxins requires the synthesis of artificial antigens,which also limits the development of immunoassay in the field of mycotoxin detection.Therefore,it is urgent to research and establish a rapid,sensitive,accurate,and inexpensive suitable for on-site detection method now.Aptamers,a kind of recognition molecules,is widely used in the field of detection in recent years,and have affinity and specificity comparable to antibodies,and have the advantages of wide target range,simple preparation process,and stability and easy preservation.Currently,the development of aptamers in food safety detection has attracted the attention of researchers.As an important rapid detection method,lateral flow chromatography is widely used in the detection of food hazards such as agricultural and veterinary drugs and pathogenic bacteria.As identification materials,the application of aptamers in chromatography strips can greatly increase the stability of test strips,reduce production costs,and expand the scope of application.In addition,as emerging fluorescent nanomaterials in recent years,up-conversion luminescent nanomaterials have been widely used in the development of optical sensors.The up-conversion nanomaterials have optical properties such as large Stokes shift,stable photochemical properties,low toxicity,no background interference,high signal-to-noise ratio,and no photobleaching.By further studying,as signal label probes,upconverting nanomaterials have been used in chromatography strips with aptamer recognition,in which superior performance can be obtained in terms of detection sensitivity,accuracy,and the like.Firstly,a novel zearalenone colloidal gold chromatographic strip based on aptamer recognition was constructed.The thiolated zearalenone aptamer was coupled with colloidal gold,which was used as a signal probe for the chromatographic strip;the complementary strand of recognition sequence and non-recognition sequence of the aptamer were fixed on the surface of the nitrocellulose membrane by streptavidin to form a test line and a control line.Through using the principle of competition,this method detect the target by competing for the aptamer attached to the probe between complementary strands on the detection line and the target.In the experiment,the conditions such as the salt concentration and the aptamer concentration during the preparation of the gold standard aptamer probe,the ratio of the streptavidin on the detection line to the biotinylated aptamer on the control line,and sample loading,were optimized,which improves the sensitivity and stability of the test strip.Under optimal experimental conditions,zearalenone test strips utilize the high affinity and high specificity of the aptamer.The visualization line can reach 20 ng/mL,and the linear relationship is goog in the range of 5 ng/mL to 200 ng/mL.good.The method was applied to the detection of commercial corn samples.The recovery rate was 93.4%to 114.2%,and it had good specificity.It can be used for the detection of actual samples.Secondly,we developed a novel up-converting nanomaterials chromatographic strips for ochratoxin A based on aptamer recognition.In order to further improve the detection sensitivity and signal stability of the test strips,we synthesized NaYF₄:Yb,Er up-converting nanomaterials,which could produce fluorescence signals in the visible light region excited by near-infrared light.What's more,polyacrylic acid was used as a ligand exchanger to modify the carboxyl group on the surface of the up-conversion through ligand exchange method,so that the strips have better biocompatibility.The aminated aptamer of ochratoxin A was coupled to the modified up-conversion material.And the complex was used as a probe for the chromatographic strip.With the binding ability of streptavidin,the recognition sequence and non-recognition sequence of the aptamer were immobilized on the surface of the nitrocellulose membrane respectively,which formed the detection line and the control line.Ochratoxin A competes with the complementary sequence on the detection line to bind with the aptamer on the probe.Then,the probes could produce fluorescence signal,helping to quantitatively detect ochratoxin A.In this experiment,the preparation of up-conversion materials,the surface modification methods,the pore size of the nitrocellulose membrane,and the sample volume were optimized to ensure the flowability of the signal probe on the test strip and avoid background interference.On the basis of high affinity and high specificity of aptamers,up-conversion materials are used as labels on test strips.With its unique optical properties,the sensitivity and stability of test strips were further improved.Under the optimal conditions,the detection limit of ochratoxin A was 1.86 ng/mL,and the linear response for ochratoxin A in a concentration range from 5 ng/mL to 100 ng/mL.The method was applied to the detection of commercially available beer samples with the recoveries of 95.2%¹15.6%.The method has good specificity and can be used for the detection of actual samples.