| Lactoferrin is an immunoactive protein derived from mammals,which has the functions of improving immunity and broad-spectrum antibacterial.It plays an important role in the growth and development of infants and young children,especially in the establishment of immune barrier,so it is often used as a nutritional enhancer in dairy products.It has been found that the specific content of lactoferrin is of great significance for the realization of its nutritional fortification function.In the face of a variety of lactoferrin dairy products in the consumer market,the traditional detection methods for the detection of lactoferrin content are very inadequate,and there is still a lack of accurate,sensitive and convenient rapid detection methods.In this study,lactoferrin was taken as the target,the lactoferrin aptamer with strong specificity and high affinity was used as a biometric element,and horseradish peroxidase was used to catalyze color production to generate visual and optical signals.An enzyme-linked aptamer(ELAA)kit was constructed and applied to the rapid detection of lactoferrin in dairy products,and then the lactoferrin aptamer was used as a capture probe.The sandwich structure was constructed to screen the new sequence of lactoferrin aptamer by heterogeneous SELEX,which provided technical support for aptamer screening and application.(1)To establish an ELAA method for the detection of lactoferrin in dairy products.The complementary DNA was coated in a 96-well plate by the reaction of streptavidin and biotin as the capture substrate,and the lactoferrin aptamer and horseradish peroxidase were prepared by the reaction of streptavitin with biotin.The absorbance value of TMB at 450 nm was measured for the qualitative and quantitative detection of lactoferrin.The encapsulation,sealing,preparation of detection probe,buffer,probe binding time,chromogenic liquid volume and termination time of the capture base in the ELAA method were optimized,and an ELAA kit was designed by using this method.The results showed that: The linear detection range of lactoferrin in dairy products was 4-24 μg/m L(50-300 n M),and the detection limit of lactoferrin in five kinds of dairy products was not more than 1.67 μg/m L(20.98 n M).When lactoferrin was added at 4 μg/m L,8 μg/m L,12 μg/m L,16 μg/m L,20μg/m L and 24 μg/m L levels,the recoveries of the ELAA kit for these 5levels were 84.42%-106.47%,the relative standard deviation was less than 2%,and the accuracy was high.The relative standard deviations of intra-day test and intra-day test were all less than 3.65%,and the overall stability was strong,which could be used for the rapid detection of lactoferrin in dairy products.(2)A high performance capillary electrophoresis(HPCE)method was applied for the detection of lactoferrin in dairy products,and the results of ELAA kit were verified.After optimization,in the positive current mode,PBS-Brij35 was used as the electrophoresis buffer,the method could quantitatively detect lactoferricin dairy products with the concentration range of 10～400 μg/m L,and the detection limit was 8.23 μg/m L.The HPCE method was used to verify the detection results of the ELAA kit.The results showed that the accuracy of the ELAA kit for the detection of nine samples was 79.71%to 146.43%,and the relative standard deviation was less than 3%,which could realize the quantitative detection of lactoferrin in dairy products.(3)A heterogeneous systematic evolution of ligands by exponential enrichment(SELEX)method for aptamer screening was developed.Aptamers were prepared as trapping substrate,and the sandwich structure was formed by adding lactoferrin protein and single-stranded DNA(ss DNA)library.The sandwich structure was used to combine with SELEX technique to screen ss DNA with affinity to lactoferrin.The key conditions of PCR amplification conditions were optimized.The annealing temperature was set as62.7℃ and 30 rounds of PCR amplification were used for 7 times screening.The results showed that: The target ss DNA can be screened and amplified by this method,which provides technical support for the application of aptamers. |
