Long-wavelength emissive N-doped carbon dots as a fluorescent probe for sensitive detection of pyrophosphate and cellular imaging

Nitrogen-doped carbon dots (N-CDs) exhibiting long-wavelength fluorescence were successfully synthesized via a one-step hydrothermal method using neutral red and thiosemicarbazide as precursors, specifically to address limitations in pyrophosphate (P 2 O 7 4− , PPi) detection. As an essential adenosine triphosphate (ATP) hydrolysis byproduct and disease biomarker, PPi quantification remains challenged by costly instrumentation and complex procedures in conventional methods. The synthesized N-CDs demonstrated optimal excitation/emission at 520/600 nm with a quantum yield (QY) of 4.8%, enabling rapid (1 min response time), selective PPi detection through fluorescence quenching. Quantitative analysis revealed a linear detection range of 1.38–85.60 μmol/L (R 2  = 0.9983) and low detection limit of 0.42 μmol/L. Practical validation in milk samples yielded excellent recovery rates of 95.26–105.74% with ≤1.86% relative standard deviation, confirming reliability in complex matrices. Critically, the N-CDs' deep-tissue penetration capability facilitated real-time monitoring of intracellular PPi dynamics in HeLa cells, while maintaining high biocompatibility. This work establishes multi-element doped carbon dots as both a cost-effective analytical alternative and a versatile platform for biomedical imaging applications.