Based On Target-induced Chain Growth Signal Amplification Aptamer-sensing Method For The Electrochemical Detection Of Aflatoxin B1

Aflatoxin B1(AFB1)exists in a variety of foods.It is a strong carcinogen,teratogen,mutagen and immunosuppressant,and poses a serious threat to animal and human health.Therefore,it is of great significance to construct a reliable method for the determination of trace AFB1.At present,a variety of methods for detecting AFB1 have been developed.Among them,electrochemical biosensors have shown very superior performance due to their simple operation,rapid and sensitive,low cost,and easy miniaturization.More and more researches have found that the use of Signal amplification can further improve the sensitivity of electrochemical biosensors.Among them,hybridization chain reaction,as a new signal amplification strategy,has been widely used in DNA determination.In this paper,we use the advantages of hybrid chain reaction,combined with electroactive substances as signal transduction molecules,and amplify the chain growth signal induced by the target,and develop an electrochemical aptamer sensor for AFB1determination.The main research contents are as follows:1.The electrochemical behavior of modified aptamers for different electrodes was investigated.When the molecular recognition element is nucleic acid,glassy carbon electrode(GCE),gold electrode(GE)and conductive glass electrode(ITO)are usually selected as signal transduction elements.In order to fix the aptamer DNA chain with the sulfhydryl group on the electrode,the nano-gold was modified to the surface of the GCE electrode and the ITO electrode by the drip coating method.Firstly,the Au NPs were synthesized by reducing HAu Cl₄by Na BH₄.The particle size and UV of Au NPs were characterized.It was found that the prepared Au NPs had a smaller particle size(about 20 nm)and a narrower particle size distribution.The UV absorption peak position was 512 nm.It shows that the prepared Au NPs have small particle size and stable performance.Then,the three electrodes were characterized by cyclic voltammetry(CV),AC impedance method(EIS)and scanning electron microscopy(SEM).It was found that the Au NPs/GCE electrode had the largest current and the fastest electron transfer rate.The GE electrode itself is easy to passivate,and its characteristic peak is more complicated,which can easily interfere with the experiment.SEM showed that Au NPs/ITO electrodes had poor conductivity.Therefore,the Au NPs/GCE electrode was selected for subsequent experiments.Finally,ferrocene(Fc)labeled AFB1 aptamer probe(Fc-P)is combined with methylene blue(MB)labeled complementary DNA(MB-c DNA)to construct and verify the sensor system,record electrochemical signals by square wave voltammetry(SWV).When AFB1 is added,it produces Proportional electrochemical double signal.Because Fc-P is close to the electrode surface,the peak oxidation current of I_Fcincreases,MB-c DNA is released from the electrode surface,and the current of I_MBdecreases.The constructed electrochemical aptamer sensor has fast detection,simple operation,low cost,and only a small amount of samples are needed for detection.2.Amplify the electrochemical aptamer sensor based on the chain growth signal to detect AFB1.On the basis of the first part,it is further expanded to introduce a more stable hairpin DNA into this conformational change.First,before introducing the target AFB1,the hairpin Hs H1 was fixed to the dual-signal sensing platform constructed in the first part.When AFB1 is present,the Fc-P/MB-c DNA double helix structure is disintegrated and MB-c DNA is released.Due to the conformational change of Fc-P,the proximity of the sensing platform causes the Fc current to increase.Then,the released MB-c DNA can hybridize with the part of the hairpin Hs H1 to develop the Hs H1.The opened Hs H1can trigger a hybridization chain reaction with two precisely designed auxiliary hairpin DNAs that are stably coexisted and modified with MB signal tags.They formed long DNA nanowires through self-assembly,and then detected the electrical signal of MB increase.Through the change of the oxidation peak current of the redox tag,the quantitative analysis of AFB1 realizes fast and sensitive detection.Under the best detection conditions,the linear range is in the range of 1 pmol/L?100 nmol/L,and the detection limit(LOD)can reach 0.62 nmol/L.The sensor was applied to the detection of tea spiked samples,and the recovery rate was 82.36%-97.2%.