| Gastrodia elata Bl.is a traditional and precious Chinese herbal medicine with the effects of calming liver yang,calming wind and relieving spasm,and it has a wide range of use and a great amount of use in China.In order to achieve the purpose of anticorrosion and improve the appearance quality,sulfur fumigation is usually carried out during the drying process,but sulfur fumigation will lead to the reduction of effective components in G.elata,and the sulfur dioxide remaining in G.elata will seriously threaten the health of consumers.Our previous research results showed that p-hydroxybenzyl hydrogen sulfite(p-HS)would be produced after G.elata was fumigated with sulfur,which could be used as a landmark product to judge whether G.elata was fumigated with sulfur.However,the detection of p-HS is mainly based on instrumental method at present,which has some problems such as long detection period,high detection cost and difficulty in popularization.Therefore,it is of great significance to develop a simple,efficient and accurate method to detect p-HS for the identification of sulfur fumigation G.elata.This study uses the p-HS marker of Gastrodia elata treated with sulfur fumigation as the target,and employs the systematic evolution of ligands by exponential enrichment to screen for nucleic acid aptamers that recognize p-HS.Based on this aptamer,a visual biosensor for detecting p-HS is established,providing a new method for the identification of sulfur-fumigated Gastrodia elata.(1)Screening of p-HS Nucleic Acid Aptamers Based on Capture-SELEXUsing Capture-SELEX technology,high-affinity nucleic acid aptamers for p-HS were screened.First,an initial library was constructed with a total sequence length of76 nt,including 36 random bases in the middle.After 8 rounds of cycling selection,the enrichment of aptamer sequences that could bind to p-HS reached saturation.The final product obtained after the last round of screening was subjected to high-throughput sequencing,generating 198,170 unique sequences.Based on the enrichment of sequences,the top 20 most enriched sequences were selected for further study.Homology analysis of the obtained aptamer sequences was performed using DNAMAN software,according to the analysis results,the sequences were divided into three families.Two aptamers were selected from each family as candidate sequences.(2)Identification of the affinity of p-HS nucleic acid aptamers.Isothermal titration calorimetry was used to characterize the affinity of candidate aptamers.The results showed that only aptamers seq4 and seq6 had obvious thermal changes when they reacted with p-HS,which indicated that these two aptamers had affinity for p-HS.The dissociation constants(Kd)of these two aptamers binding to p-HS were determined by fluorescence assay,and were 32.12±2.91 nmol/L and 26.75±6.48 nmol/L,respectively.Both of them,the lower Kd value of seq6 indicates that it has higher affinity for p-HS,so it is further analyzed as the optimal aptamer..Molecular docking was used to explore the interaction mechanism between the optimal aptamer and the target molecule p-HS.The results showed that seq6 and p-HS spontaneously bind together through hydrophobic,van der Waals,and hydrogen bonding interactions.The main binding sites between p-HS and the aptamer were located at positions 24-27and 56-58 of the nucleotide sequence.(3)Establishment of a visual detection method for p-HS based on colloidal gold.A simple and efficient biosensor was developed using colloidal gold as a color indicator and the selected aptamer seq6 as the recognition element for the visual detection of p-HS,with good specificity for p-HS.To improve the sensitivity of the sensor for p-HS detection,the experimental conditions of the sensor were optimized.The optimal working concentration of Na Cl was found to be 58μmol/L,the optimal aptamer concentration was 115 nmol/L,the optimal reaction temperature was 65℃,and the optimal reaction time was 2.5 h.The standard sample of p-HS was detected using this method,and it was found that the ratio of Abs620/Abs520 had a good linear relationship with p-HS concentration in the range of 1-4μg/m L,with a linear equation of y=0.1173 x+0.5520 and a correlation coefficient R~2=0.9942,with a detection limit of 1μg/m L.Finally,the market samples of G.elata were tested,and it was found that the recoveries of p-HS were between 88.5%and 105%,indicating that this method could be used for p-HS detection in G.elata samples.To sum up,in this study,the aptamer with affinity for p-HS,a sulfur fumigation marker of G.elata,was successfully screened by SELEX technology,and a visual method for detecting p-HS was developed with the screened aptamer as recognition element.The detection method has the advantages of high specificity,simple operation and low cost,and can be directly applied to the detection of p-HS in G.elata,providing a new method for the identification of sulfur-smoked G.elata. |
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