A colorimetric and fluorometric dual-mode biosensor for point-of-care testing of β-lactoglobulin in milk based on DNAzyme and carbon dots

β-Lactoglobulin (BLG) is a major allergen in milk and has proven to be a valuable biomarker for the assessment of milk quality. Herein, a colorimetric and fluorometric dual-mode biosensor was first constructed for detecting BLG based on the G-quadruplex/hemin DNAzyme and orange-emitting carbon dots (O-CDs). The BLG aptamer was employed to generate G-quadruplex/hemin DNAzyme, which exhibits peroxidase-like (POD-like) activity and serves as a specific recognition probe. The DNAzyme efficiently catalyzes the conversion of 3,3′,5,5′-Tetramethylbenzidine (TMB) into its blue oxidation product (oxTMB) and simultaneously quenches the fluorescence of O-CDs through the internal filter effect (IFE). Upon the presence of BLG, the aptamer specifically binds to BLG, thereby destroying the structure of the DNAzyme. Consequently, the POD-like activity of the DNAzyme is decreased, leading to an inhibition of oxTMB production and a restoration of the fluorescence of O-CDs. The BLG can be accurately measured by observing the changes in the colorimetric signal of oxTMB at 370 nm and the fluorescence signal of O-CDs at 580 nm. The developed biosensor demonstrates excellent linearity over a concentration range spanning from 1 μg·mL⁻¹ to 500 μg·mL⁻¹. Integrated with the smartphone platform, it could realize convenient quantitative analysis without instruments and meet the requirements of point-of-care testing (POCT). This study holds significant potential for ensuring food security.