In vitro selection and optimization of high-affinity aptamer for milk allergen α-lactalbumin and its application in dual-mode detection

Abstract

Milk is one of the most common sources of protein in people’s daily lives, and it is also recognized by the World Health Organization (WHO) as one of the eight categories of food allergies to human beings. α-lactalbumin (α-La) is the main cause of milk allergy. In this study, a single-stranded DNA aptamer with high binding affinity to α-La were selected using systematic evolution of ligands by exponential enrichment (SELEX) method. Compared with the full-length sequence, the binding affinity of the truncated aptamer LA-1t for α-La was increased six times using fluorescence analysis. Circular dichroism (CD) indicated that the secondary structure of LA-1t contained a typical hairpin structure. Through the docking simulation of LA-1t and α-La, these experimental results were further explained theoretically, and the recognition mechanism was explained. Finally, the colorimetric and fluorescence signal of boron nitride quantum dots anchored to porous CeO2 nanorods (BNQDs/CeO2) were modulated by FAM-labeled LA-1t to achieve highly selective and sensitive determination of α-La. This dual-mode sensing strategy displayed sensitive recognition for α-La in a linear range of 5–4,000 ng/ml with the LOD was 3.32 ng/ml (colorimetry) and 0.71 ng/ml (fluorescence), respectively. Simultaneously, the colorimetry/fluorescence dual-mode sensing strategy was applied for detecting α-La in spiked real samples and demonstrated good stability and reliability.