Aptamer Sensing Of Aflatoxin B₁ Based On Hybridization Chain Reaction

Aflatoxin B₁（AFB₁）residues in food pose a great threat to public food safety.In view of the low allowable limit and serious toxicity of AFB₁,in order to control or solve the serious harm caused by AFB₁,it is very important to establish a practical and sensitive AFB₁ analysis method for food safety.In recent years,with the rapid development of DNA molecular technology,aptamer（Apt）sensor based on isothermal nucleic acid amplification has been widely used in AFB₁ detection.Hybridization chain reaction（HCR）is a simple and effective isothermal signal amplification technique,which can achieve efficient amplification of specific targets at constant temperature,simplify the instrument,shorten the reaction time,and better meet the needs of sensitive detection of AFB₁.Silica nanoparticles（SiO₂NPs）have stable properties and easy surface modification,so using SiO₂NPs as DNA carrier can achieve sample enrichment and greatly improve the detection efficiency of the sensor.Gold nanoparticles（AuNPs）have the advantages of low toxicity and unique optical properties,and by changing their particle size and shape,they can change the color of the solution and achieve the output of the colorimetric signals.In this study,Apt was used as the recognition element,SiO₂NPs was used as carrier for detected probe and AuNPs was used as the substrate for signal output,two kinds of fluorescence and colorimetric sensor were developed based on HCR to achieve highly sensitive detection of AFB₁.The main contents are as follows:（1）SiO₂NPs-assisted HCR cascade amplification for AFB₁ fluorescence detection.First of all,SiO₂NPs was used as the DNA carrier to achieve the first step of signal amplification.The HCR initiation chain was modified on the surface of SiO₂NPs by the carboxylic ammonia binding,and then hybridized with AFB₁ Apt to prepare the detection probe.Target AFB₁combined with the probe and freed in the microwell plate.Then the supernatant of the microplate was taken the Nb.Bbv Cl enzyme was added to release the initiation chain on the probe surface for HCR second step amplification.After the addition of SYBR Green I,the fluorescence value of the solution at 520nm was detected.The detection limit of this method for AFB₁ was as low as 6.5448×10^-17 g/mL.It has good linearity in the range of 1×10^-16-1×10^-12g/mL and has good anti-interference to other toxins,which can meet the detection needs of trace AFB₁ residues in food.（2）AuNPs-assisted HCR for AFB₁ fluorescence/colorimetric detection.First of all,in a96-well polystyrene microplate,the Apt-initiator chain（Apt-Ini）and the coated antibody jointly recognize the target AFB₁ and form a sandwich structure to initiate HCR.The fluorescence quantitative detection was achieved by the fluorescence detection of SYBR Green I at 520 nm.The detection limit was 2.471×10^-15g/ml.At the same time,after the occurrence of HCR in the microwell plate,the remaining DNA hairpins in the supernatant were taken out and added with AuNPs and salt solution.AuNPs showed different colors at different degrees of self-polymerization.The color change of AuNPs was judged by naked eye and its absorbance was detected by multimode reader.The detection limit of AFB₁ was1.333×10^-14 g/ml.This strategy can detect the target by both fluorescence and colorimetric signals at the same time,and both signals have good sensitivity.The dual signals corroborate each other and improve the accuracy and practical value of detecting AFB₁ in food.