A self-calibrated tri-emission fluorescent probe based on carbon dots assembly for in vitro pepsin detection

A novel self-calibrating triple-emission fluorescent probe (CDs/BSA-SQ-Cy7) was engineered through the strategic integration of blue-emitting carbon dots (CDs) with covalently assembled near-infrared fluorescent dyes (BSA-SQ and BSA-SQ-Cy7) for accurate in vitro pepsin detection. To validate the enhanced performance of the tri-emissive design, a dual-emission control probe (CDs/BSA-SQ) was developed. BSA-SQ-Cy7 undergoes hydrolysis upon interaction with pepsin, resulting in concentration-dependent quenching of the red fluorescence, where the blue CDs serve as a reference emission during the process. The CDs/BSA-SQ-Cy7 probe demonstrated a robust linear correlation with pepsin concentrations ranging from 0.018 to 30 μg/mL (determined by the lowest detection limit). The detection limits were found to be 0.182 μg/mL (I₄₉₅/I₆₅₉), 0.033 μg/mL (I₄₉₅/I₈₀₈), and 0.018 μg/mL ((I₄₉₅-I₈₀₈)/I₆₅₉), respectively. Additionally, the probe displayed excellent stability and reproducibility over a 72-h period, and its three-channel emission signal ratios can be utilized for self-calibration, thereby improving assay accuracy. This self-calibrated tri-emission probe is distinguished by its stability, reproducibility and high selectivity, offering an effective method for the accurate in vitro detection of pepsin.