Construction Of Aflatoxin B₁ Smart Hydrogel Detection Sensor And Screening Of Its Precursor Aptamer

Aflatoxin B₁（AFB₁）,the most toxic mycotoxins,is widely distributed in all aspects of food production,storage,transportation and processing,and its pollution has become an urgent problem to be solved worldwide.Therefore,the field detection,prevention and control of AFB₁is particularly important.AFB₁has low molecular weight and no immunogenicity,and the preparation of antibodies for on-site immunodetection is complicated and the variation between batches is large.Aptamers,as new recognition elements,are similar to but superior to antibodies in function,and have the characteristics of convenient preparation,good stability,and no batch difference.The aptame-based DNA smart hydrogel detection sensor not only has the characteristics of loose and porous hydrogels,but also has the characteristics of specific recognition,high-precision assembly,easy degradation,cost-effectiveness,portability and stability.However,sterigmatocystin（ST）,the precursor of AFB₁,is an indispensable part of the prevention and control of AFB₁,and its harm cannot be ignored,but its aptamer is rarely reported.Therefore,in this study,a target-responsive DNA smart hydrogel detection sensor was constructed based on the existing AFB₁aptamer.Using ST as the target,an aptamer H Seq02 with high affinity and specificity was screened by improved Capture-SELEX technology,and the binding mechanism between H Seq02 and ST was studied by circular dichroism and molecular simulation docking technology,in order to provide a reference for the field detection,prevention and control of aflatoxin B₁.The details are as follows:1.AFB₁responsive DNA smart hydrogel detection sensor:The sensor was constructed using a DNA smart hydrogel as the substrate,an AFB₁aptamer as the crosslinker and recognition element,and a dual signal amplification strategy.When AFB₁is present,it competitively binds to the aptamer,resulting in hydrogel cleavage and horseradish peroxidase（HRP）release.The addition of Exo I realized the recycling of AFB₁,promoted the more thorough cleavage of the hydrogel,and released more HRP.HRP catalyzed the color development of TMB,and finally realized the visual and quantitative detection of AFB₁by the changes in color and absorbance value.The results showed that the absorbance of the solution had a good linear relationship with the concentration of AFB₁in the range of 0-500n M,and the detection limit was 4.93 n M（S/N=3）.The sensor has been successfully applied to the detection of AFB1 in peanut oil samples,and the detection results are not significantly different from those of UPLC-HRMS.2.ST aptamer Capture-SELEX screening:The primers were fixed to the streptavidin magnetic beads by combining streptavidin with biotin-primer.The library was then indirectly immobilized to streptavidin magnetic beads by complementary base pairing.Referring to the preparation method of ST hapten,ST molecules were activated to increase its water solubility to prepare a high-concentration target solution,and the library fixed magnetic beads were put into it for incubation,so that the ss DNA with affinity for ST was dissociated from the surface of magnetic beads into the solution.With the help of magnetic separation,the PCR template was obtained for amplification,and the long and short strand method was used to prepare ss DNA before entering the next cycle.In the process of cyclic screening,the pressure of screening was gradually increased by reducing the amount of library input,reducing the target concentration,reducing the positive screening time,adding negative screening and reverse screening,and the screening process was monitored by QPCR fluorescence amplification curve.After 16 rounds of screening,the enriched library of ST was obtained.3.Sequencing and analysis:Homology and similarity analysis were performed on the sequencing results,and eight aptamer candidate sequences were preliminarily selected.The Aptamer affinity column（AAC）was constructed to directly investigate the affinity and specificity of the aptamer.The results showed that the affinity of aptamer H Seq02 was the best,and the recovery rate of 20 ng ST was 99.2%.The specificity of 17 mycotoxins for AAC based on H Seq02 and commercial Immunoaffinity column（IAC）was investigated.The results showed that the recovery rates of AAC for 16 kinds of toxins except ST were less than 5%,while the recovery rates of IAC for AFB₁,AFB₂,AFG₁,AFG₂,OTA and ST were higher than 90%,indicating that AAC was more specific than IAC.The capacity of AAC constructed by 2.3 nmol H Seq02 was up to 82.4 ng.Molecular docking results showed that H Seq02 and ST mainly bound by hydrogen bonds,and the binding sites were DA-29,DT-30,DT3-40 bases on H Seq02 and carbonyl,hydroxyl,and methoxy groups on ST molecules.The difference in molecular structure between ST and AFT mainly lies in carbonyl and hydroxyl groups,which also confirms the reason why H Seq02 has better specificity for ST recognition.