pH-Sensitive blue and red N-CDs for L-asparaginase quantification in complex biological matrices

[Display omitted] •Novel dual carbon dot-based ratiometric fluorescence assay for L-asparaginase.•Wide linear range (20–2000 U L-1) with low detection limit (6.95 U L-1).•pH-dependent opposing fluorescence responses enhance sensitivity.•Excellent selectivity and recovery (96.15–99.75%) in human serum.•Promising method for clinical and food industry L-asparaginase detection. A novel fluorometric method for the determination of L-asparaginase, an enzyme crucial in cancer therapy and food industry applications, is presented. This sensitive and selective approach utilizes L-asparagine and two pH-sensitive carbon dots (blue-N-CDs and red-N-CDs) as probes. The interaction between L-asparagine and L-asparaginase liberates ammonia, causing an increase in pH. This pH change simultaneously decreases the fluorescence of blue-N-CDs while enhancing the emission of red-N-CDs, enabling ratiometric detection of L-asparaginase. Comprehensive characterization of both carbon dots and investigation of their response mechanism towards L-asparaginase were conducted using ultraviolet–visible spectrophotometry, fluorescence spectroscopy, and transmission electron microscopy (TEM) imaging techniques. The designed approach demonstrates outstanding linearity (20 to 2000 U L-1) and a low detection limit (6.95 U L-1) for L-asparaginase quantification. Moreover, when tested to human serum samples, the detection system exhibits outstanding selectivity and high recovery rates (96.15% to 99.75%) with low standard deviation, underscoring its suitability for practical implementation in clinical diagnostics.