Construction Of Electrochemical Biosensor Platform For Detection Sensitive Of Aflatoxin B1 And AflR Gene

With the continuous development of traditional Chinese medicine,the efficacy of traditional Chinese medicine is recognized and valued,while its safety is also widely concerned.Chinese medicinal materials are susceptible to contamination by a variety of mycotoxins in the environment of cultivation,processing,transportation and storage,thus affecting the quality of Chinese medicinal materials.Among them,aflatoxin B1 is considered to be the most toxic and carcinogenic mycotoxins.Therefore,it is urgent to develop rapid and sensitive detection methods for aflatoxin B1.At the same time,the key regulatory genes of aflatoxin B1 biosynthesis in contaminated traditional Chinese medicine were studied,which is expected to efficiently and sensitively detect the precursor material gene（aflR gene）of aflatoxin B1 synthesis before aflatoxin B1 detection,so as to achieve the role of early warning,control and reduce the threat posed by aflatoxin B1 to health of human and reduce economic losses.Electrochemical biosensors are manufacture,simple signal generation and microminiaturization,and so it has been widely used in ultrasensitive detection.In this paper,we had been synthesized a new signal marker and metal-organic framework materials,and constructed two electrochemical biosensors based on the catalytic hairpin self-assembly（CHA）and the active double-end DNAzyme assemblies were derived from target-catalyzed CHA circuit for the detection of aflatoxin B1 and the regulatory aflR gene,the specific contents are as follows:（1）An immobilization-free electrochemical aptamer sensor was developed by using the signal amplification strategy of catalytic hairpin assembly（CHA）and a new signal marker tetraferrocene,which was successfully applied to the detection of AFB1.The entire amplification process took place in homogeneous solution.The tetraferrocene was labeled at both ends of the probe H2 and contained a large number of unhybridized T bases,which causes tetraferrocene to move closer to the electrode surface and produced a highly efficient amplified signal.Under the induction of AFB1,CHA reaction generates a large number of H1-H2 double-stranded bodies and the recycling of AFB1.Under the optimal conditions,there is a good linear correlation between the peak current of the sensor and the logarithm of AFB1 concentration（0.1μM～1 pM）,and the detection limit obtained by three times signal to noise ratio is0.255 pM.In addition,the prepared sensor has good selectivity,repeatability and stability,demonstrating a sensitive and stable detection method of AFB1.（2）Fungal genomic DNA rapid extraction kit was used to extract aflR gene,and suspected mycelium was isolated from mildewed dried tangerine and inoculated into the Czapek–Dox Medium.Through the observation of colony color and morphology and microscopic examination,the Aspergillus flavus was initially screened,and then compared with standard aspergillus flavus strain 3.4408,it was confirmed as aspergillus flavus.Taking the above strain as the test strain,the regulatory aflR gene related to aflatoxin B1 production was amplified and identified by PCR,ultraviolet spectrophotometer and agarose gel electrophoresis,and sent to the sequencing company for sequencing.The sequencing results were compared with the gene bank,and the results showed that the homology could reach 99.21%.The extracted aflR genes were determined to be stored at 4℃to provide good samples for the subsequent detection of aflR gene.（3）In order to better predict whether aflatoxin contamination exists,we selected to detect aflatoxin biosynthesis-related aflR gene.DNAzyme biocatalyst and catalytic hairpin assembly（CHA）circuit was integrated to construct an electrochemical DNA sensor based on CHA-DNAzyme strategy for sensitive detection of aflR gene.In order to simplify the detection procedure,the Mg²⁺@UiO-66（Zr）-（COOH）₂MOFs was synthesized.Here,the H3 end of the probe is labeled with the redox reporter molecule tetraferrocene.The inactive DNAzyme subunits were respectively grafted into these metastable CHA hairpin reactants that were kinetically impeded without false cross-hybridizations.The aflR gene catalyzed CHA-mediated successive assembly of dumbbell-like bis-DNAzyme,which further circularly cleaved probe H3.As a large amount of tetraferrocene left the electrode surface,the electrochemical signal was significantly reduced.The results show that the sensor has a good linear range of 0.512 pM-0.2μM,the detection limit is as low as 0.9079 fM（S/N=3）,the sensor had the high specificity for mismatched DNA,and the stability for multiple detection of different time,and the proposed E-DNA sensor that the potential application in the actual extraction of gene was evaluated.