Development Of Aptamer-based Lateral Flow Fluorescent Assay For Fast Screening Of Aflatoxin B₁

Various instrumental methods for AFB₁ detection require long extraction time and tedious sample preparations,making it difficult to use them for on-site and real-time determination.Although lateral flow immunoassay as a promising solution shows rapidity and simplicity,antibodies are regarded as a highly costly animal-source biorecognition element without long-term stability.Lateral flow aptasensors benefited from aptamer and lateral flow strips represent a promising tool in food safety,environmental monitoring and clinical diagnostics.However,only a few LFAs have been reported until now and a possible reason is that the affinity of most aptamer/complementary strand duplexes is stronger than the aptamer/target affinity,which is difficult to find suitable truncated complementary strands to achieve a balance between reduced affinity to aptamer and high efficiency of hybridization with aptamer.To address this problem,a novel dual-competitive LFA was first designed in this study by taking the advantages in the reaction specificity of both aptamer-target and aptamer-complementary strand.In this LFA assay,AFB₁ in the sample competes with AFB₁-hapten immobilized on the test line（T line）above the nitrocellulose（NC）membrane to bind with limited fluorescence-labeled aptamer,which serves as the first competition-inducing line where the fluorescent intensity decreases.On the control line（C line）,the aptamer complementary strand competitively binds to the aptamer in the AFB₁/aptamer complex migrating from the T line due to the stronger affinity of the DNA duplex,which serves as the second competition-inducing line where the fluorescent intensity increases.The dual-competitive LFA greatly improved the detection sensitivity.The specific works are as follows:Firstly,a Cy5 labeled aptamer based strip was designed for AFB₁ detection.A series of components including the membrane material,sample pad,absorbent pad and plastic backing pad were optimized for preparation of LFA strips.To obtain a rapid and sensitive strip for AFB₁ detection in feedstuffs,the determination conditions was performed in series.We found the following conditions to give best results:252μg/mL of AFB₁-hapten was immobilized on the test line,3μM of aptamer complementary strand was immobilized on the control line,PolyA 12 was selected as the linker for Cy5-labeling at the 5’end of the AFB₁ aptamer and the PB binding buffer contains 7.5%BSA.Finally,evaluation for the application of the LFA was successfully completed.The limit of detection（LOD）for this technique was 0.1 ng/mL of AFB₁,which could be achieved within the linear range from 0.1 ng/mL to 1000 ng/mL,and the intra-assay and inter-assay values were measureddemonstrating high reproducibility and satisfactory precision.The method displays high selectivity over other mycotoxins and targets.The calibration curves at different sample matrixes were obtained after 5-fold diluted samples were spiked at five different concentrations（0.5-5000μg/kg）and the coefficients of determination（R²）for these samples were greater than 0.95,showing good linearity in the analytical range.The LFA was compared with a commercial ELISA kit to validate its effectiveness by blindly analyzing the obtained food and feed samples（AFB₁ concentrations ranging from 0 to 554.9μg/kg）,and the two methods were highly agreed with the regression equation was:y=-1.929x+5.2667 and a large correlation coefficient（R²）being 0.9989.In this study,successfully integrated with the lateral flow test strip platform,dual competition including target-aptamer-antigen and target-aptamer-complementary strand was employed to detect AFB₁.Besides,this LFA detection principle can be further extended to other food contaminants,disease biomarkers and a variety of other biomedical applications,having a promising foreground for point-of-care tests.