Application Of Aptamer Affinity Column In The Detection Of Lactoferrin And Aminoglycoside Antibiotics In Milk Samples

ObjectiveSample pretreatment is an important step in the process of analysis and detection,especially when using instrumental analysis method,too much matrix impurities will not only interfere with the detection of the substance to be measured,but also lead to column pollution and shorten the life of the column.The purpose of sample pre-treatment is to extract the target substance from the sample,at the same time,remove impurities as much as possible,reduce the matrix interference,thus reducing the detection limit and improving the detection sensitivity and accuracy.In this study,aptamer affinity columns for lactoferrin(Lf)and Aminoglycosides(AGs)were prepared by using aptamer as molecular recognition elements,which can be used for the specific recognition and enrichment of Lf and AGs in milk.MethodsLf-AAC was prepared by covalently covalent conjugation of an amino-modified aptamer with NHS-activated Sepharose.The washing buffer type and volume and the sodium chloride concentration in the elution buffer were optimized for the AAC method.The performance of the AAC was then evaluated in terms of the column capacity,stability,and reusability.The optimized Lf-AAC was used for the enrichment and purification of Lf in milk,and the quantitative analysis was carried out by HPLC.The affinity of kanamycin aptamer with AGs was verified by gold nanometer colorimetry.AGs-AAC was prepared by covalently binding NHS-activated agarose with amino modified aptamer as solid phase carrier.The working conditions of AGs-AAC,such as p H of loading solution and eluent composition,were explored,and the column capacity,specificity and reusaility of AGs-AAC were investigated.The optimized AGs-AAC was used in the pre-treatment process for the determination of four AGs residues in milk,including kanamycin A,kanamycin B,tobramycin and amikacin,and the quantitative determination of AGs-AAC was carried out by UPLC-MS/MS.ResultsLf-AAC can specifically recognize lactoferrin.The maximum bearing capacity of Lf-AAC can be up to 500±13.7 μg.The Lf-AAC can be repeated use for 10 times.After purified and enriched by Lf-AAC,the milk samples were determined by HPLC and quantified by external standard method.The recovery rate was 85.45%～115.54%.Kanamycin aptamer has good affinity with four AGs,including kanamycin A,kanamycin B,tobramycin and amikacin.AGs-AAC can be used as a class-specific aptamer affinity column.AGs-AAC has a maximum target load of8 μg and can be reused at least 20 times.UPLC-MS/MS method was established for the determination of four AGs in milk samples.The average recoveries of the four target compounds were 85.86%～96.69%.ConclusionsIn view of the wide range of aptamer recognition targets,Lf-AAC for a single target and AGs-AAC that can recognize multiple targets were prepared respectively in this study,which were used in the pre-treatment process of detection of various targets in milk samples.The pretreatment method using AAC has the advantages of simple operation,good purification effect,high specificity,high repeatability and low cost.It has a good application prospect.