Selection,Identification And Application Of Bispecific Aptamers Against Malachite Green And Leuco-Malachite Green

Malachite green(MG)is a forbidden drug for aquatic products,its residue will endanger food safety and export trade.Leucomalachite green(LMG)is its primary metabolite.MG and LMG usually exist in aquatic products simultaneously.However,all of rapid detections now available only can detect a single target,and fail to detect MG as well as LMG simultaneously and rapidly.Therefore,it is of great significance to develop a new molecular detection probe which can recognize MG and LMG at the same time and realize the synchronous and rapid detection of MG and LMG in aquatic products.Therefore,in this study,using MG and LMG as screening targets,magnetic bead-SELEX technique was used to screen bispecific aptamers for simultaneous recognition of MG and LMG,and using them as sensor probes,a label-free AuNPs colorimetric aptamer sensor was constructed in order to realize the synchronous and rapid detection of MG and LMG.The results are as follows:(1)Screening of bispecific aptamers for MG and LMG.MG and LMG were used as positive targets,Sulfadiazine(SDZ)and Nitrofurantoin(NFT)mixed targets as counter targets,and ssDNA library immobilized magnetic beads-SELEX technique was used for screening.Using recovered and bound ssDNA as template,ssDNA secondary library was prepared by emulsion unequal length PCR technology,ssDNA recovery rate was calculated by Q-PCR technology,and ssDNA library enrichment was analyzed according to amplification curve and recovery rate.The results showed that with the increase of the number of screening rounds,the Q-PCR amplification curve moved forward,and the recovery rate of ssDNA increased gradually.In the 11th round,the recovery rate of ssDNA was the highest,which was 21.27%.After that,the ssDNA recovery rate began to decrease,so the screening was terminated at the 13th round.At the same time,R5,R11,R12 and R13 were selected as candidate aptamer libraries,and fluorescence analysis was used to evaluate the binding ability of candidate aptamer libraries with MG and LMG.The results showed that the fluorescence intensity of each candidate library changed in varying degrees after binding to MG or LMG,among which R12 had the strongest binding to LMG and R11 had the strongest binding to MG.(2)Analysis and characterization of candidate sequences.R5,R11,R12 and R13 libraries were selected for high-throughput sequencing,and the sequences were sequenced according to the enrichment multiple,reads and the lowest free energy of the sequences.finally,9 dominant candidate aptamers were determined.The specificity of 9 dominant candidate aptamers was analyzed by AuNPs colorimetry.The results showed that A1-A9 could specifically recognize MG and LMG,but had no effect on non-target substances,and the performance of A2,A3 and A5 was significantly better than that of other candidate sequences.considering the further application in the later stage and reducing the synthesis cost,9 dominant candidate sequences were truncated properly and 11 truncated sequences were obtained.The affinity of the 11 truncated sequences was analyzed by fluorescence analysis.The results showed that except for A5-b,the other candidate sequences had a certain binding to MG and LMG,and the affinity of A2-a,A3-a and A5-a was significantly better than that of other truncated sequences.The Kd values of A2-a,A3-a and A5-a for LMG were 8.435±0.845,8.192±1.214 and 13.72±1.44 nmol/L,respectively,and for MG were 3.418±0.253,2.267±0.203 and 3.019±0.165 μmol/L,respectively.(3)Construction of label-free AuNPs colorimetric aptamer sensor.Using A3 as the sensor probe,gold nanoparticles as the signal output carrier and NaCl solution as the aggregation inducer,a label-free AuNPs colorimetric aptamer sensor was constructed,which can be applied to the synchronous and rapid detection of MG and LMG in aquatic products.The performance of the sensor(NaCl concentration,aptamer concentration)was optimized,and the sensitivity and specificity of the method were analyzed under the optimal conditions.The results showed that the optimum concentration of NaCl and aptamer was 150 mmol/L and 150 nmol/L respectively.The specificity results showed that MG and LMG can be detected specifically without cross-reactivity with SDZ and NFT.The sensitivity results showed that the limit of detection for MG and LMG were 6.93nmol/L and 6.38 nmol/L respectively.Moreover,the absorbance ratio A650 nm/A520 nm showed a good linear relationship with the molar concentration of the target in the range of 0～17.5 μmol/L.The recoveries of MG and LMG were respectively 88.60%to 93.30%and 101.80%to 107.00%in repeatability test using carassius auratus tissue.The relative standard deviations were both less than 4%.These results indicated that this method is able to provide a new method for simultaneously and rapidly detect MG and LMG in aquaculture.