Highly Sensitive β-Lactoglobulin Fluorescent Aptamer Biosensors Based on Tungsten Disulfide Nanosheets and DNase I-Assisted Signal Amplification

beta-lactoglobulin (beta-Lg) is a protein found in milk that can cause severe allergic reactions, including rash, vomiting, and diarrhea. Thus, it is crucial to develop a sensitive beta-Lg detection method to protect people who are susceptible to allergies. Here, we introduce a novel and highly sensitive fluorescent aptamer biosensor for detecting beta-Lg. First, a fluorescein-based dye (FAM)-labeled beta-lactoglobulin aptamer (beta-Lg aptamer) is adsorbed on the surface of tungsten disulfide (WS2) nanosheets via van der Waals forces, resulting in fluorescence quenching. When beta-Lg is present, the beta-Lg aptamer selectively binds to beta-Lg, causing a conformational change in the beta-Lg aptamer and releasing it from the surface of WS2 nanosheets, which restores the fluorescence signal. Simultaneously, DNase I in the system cleaves the aptamer bound to the target, producing a short oligonucleotide fragment and releasing beta-Lg. The released beta-Lg then binds to another beta-Lg aptamer adsorbed on WS2, initiating the next round of cleavage, resulting in significant amplification of the fluorescence signal. This method has a linear detection range of 1-100 ng mL(-1), and the limit of detection is 0.344 ng mL(-1). Furthermore, this approach has been successfully used for detecting beta-Lg in milk samples with satisfactory results, providing new opportunities for food analysis and quality control.