Enzyme-Linked Aptamer Kits for Rapid, Visual, and Sensitive Determination of Lactoferrin in Dairy Products

Lactoferrin (Lf), as a popular nutritional fortification in dairy products, has the ability regulate the body's immune system and function as a broad-spectrum antibacterial, which is of great significance to the growth and development of infants and children. Herein, an indirect competitive enzyme-linked aptamer assay (ELAA) kit was established for rapid, sensitive, and visual determination of Lf in dairy products. In the construction, the Lf aptamer was conjugated with horseradish peroxidase (HRP) as the recognition probe and aptamer complementary strand (cDNA) were anchored onto the microplate as the capture probe. The recognition probes were first mixed with a sample solution and specifically bound with the contained Lf, then added into the microplate in which the free recognition probes in the mixture were captured by the capture probe. After washing, the remaining complex of cDNA/Aptamer/HRP in the microplate was conducted with a chromogenic reaction through HRP, efficiently catalyzing the substrate 3, 3', 5, 5'-tetramethylbenzidine (TMB), therefore the color shade would directly reflect Lf concentration. Under the optimization conditions, a good linear relationship (R², 0.9901) was obtained in the wide range of 25-500 nM with the detection limit of 14.01 nM and a good specificity, as well as the reliable recoveries. Furthermore, the ELAA kits achieved the Lf determination with an accuracy of 79.71~116.99% in eleven samples, which consisted of three kinds of dairy products: including goat milk powder, cow milk powder, and nutrition drop. Moreover, the results were also validated by the high-performance capillary electrophoresis (HPCE) method. The ELAA kit provides a simple and convenient determination for Lf in dairy products, and it is highly expected to be commercialized.