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

β-lactoglobulin (β-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 β-Lg detection method to protect people who are susceptible to allergies. Here, we introduce a novel and highly sensitive fluorescent aptamer biosensor for detecting β-Lg. First, a fluorescein-based dye (FAM)-labeled β-lactoglobulin aptamer (β-Lg aptamer) is adsorbed on the surface of tungsten disulfide (WS(2)) nanosheets via van der Waals forces, resulting in fluorescence quenching. When β-Lg is present, the β-Lg aptamer selectively binds to β-Lg, causing a conformational change in the β-Lg aptamer and releasing it from the surface of WS(2) 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 β-Lg. The released β-Lg then binds to another β-Lg aptamer adsorbed on WS(2), 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 β-Lg in milk samples with satisfactory results, providing new opportunities for food analysis and quality control.