Screening Of Nucleic Acid Aptamers Of Borax And Its Visual Detection Method

In the process of food production,manufacturers would add the appropriate amounts of food additives to meet consumers’requirements for senses and flavors.However,some traders were accustomed to overuse food additives or use illegal additives to obtain greater profits,resulting in food safety problems which have a huge damage to society and human health.In order to standardize the rational use of food additives and eliminate the abuse of illegal additives,there is an urgent need for a strategy for direct,rapid and accurate detection of food additives in the market safety sector.Borax is considered as a highly toxic and a strong carcinogen,which due to its properties as a toughening and brightening,is illegally added to the food processing.But it can cause cumulative damage to human stomach,kidney,liver,lung and brain.The lethal dose of adult is 15 g and the infant is 2-3 g.In the national standard“Food Hygiene Law of PRC”and“Food Additives Health Management Measures”,borax is excluded from the statutory food additives.In addition,the currently available methods only determine whether borax is present by detecting the decomposition products of borax,namely boric acid or detecting boron.Therefore,it is a urgent mask of developing a method for direct detection of borax.At first,the screening study of borax-specific aptamers based on the Systematic Evolution of Ligands by Exponential Enrichment(SELEX)was carried on.In the SELEX process,borax was conjugated to the epoxy-activated sepharose 6B as an affinity column,and incubated with a random ssDNA library in vitro.The screening of aptamers was determined at the end of the tenth round by monitoring the ssDNA recovery rate for each round.Afterwards,the final round of screening products were cloned and sequenced,and then the secondary structure analysis of the obtained aptamer sequences showed that the stem-loop structure was the main recognition structure.By the circular dichroism spectroscopy,the structure of aptamers was not susceptible to salt ion solutions.The characterizations of aptamers were verified by Enzyme-Linked Oligonucleotide Assay(ELONA),indicating that aptamer Bor-A01and Bor-B02 had high specificity and affinity,with the K_D value of 22.07±2.730 nM for Bor-A01 and the K_D value of 21.64±2.732 nM for Bor-B02.Besides,the truncation experiments showed that the full-length sequence of aptamers had the highest specificity and affinity.Using the principle of gold nanoparticles(AuNPs)to accumulate in high-salt ion solution,a visual detection method based on aptamers was established to realize the rapid detection of borax in food.The feasibility of the visual detection method was verified by Nuclear Magnetic Resonance(NMR).By comparing the binding forces between the aptamers Bor-A01 and Bor-B02 with the difference particle sizes of AuNPs,the aptamer Bor-A01 was selected for subsequent optimization experiments.After optimization,the detection of borax can complete within 1 h,with a visual detection limit of 0.20μg/mL.An unspecific interaction of the non-target borax analogs(rhodamine B,methamidophos,rongalite,boric acid)was not detectable.In addition,the visual detection method has good stability.For a long-term storage,the AuNPs solution should be stored at 4°C and the aptamer Bor-A01 should be stored at-20°C.To evaluate the accuracy and practicability of the visual detection method in detecting borax.Seven kinds of food which may contain borax were purchased at random from different shops in three markets.In order to demonstrate the reliability of this assay,we also conducted the gold standard method simultaneously,ethylhexanediol-trichloromethane extraction turmeric colorimetry,and the specific process was referred to the Chinese national standard(GB/T 21918-2008).The results of the two detection methods were concordant with each other,which indicated the high accuracy of the visual detection method.In addition,the matrix effects of different samples purchased from different markes were studied.The visual detection limits of yuba,dried tofu,rice noodle,noodle,fish ball,shrimp ball and meatball were0.40,0.40,0.30,0.30,0.40,and 0.50μg/mL.Compared with the national standard method,the visual detection method can realize the direct detection of borax,and has high sensitivity,simple operation,short use time,and no need for testing instruments.In summary,nucleus aptamers of borax were successfully screened and a visual detection method based on AuNPs with the aptamer Bor-A01 as component was developed.The method has been applied to the direct detection of borax in food,which provides a novel technical strategy to ensure the safety of the food market.