Chimeric ascorbic acid phosphate-releasing nucleotide probes for isothermal signal-amplified protein detection

Protein detection is essential for disease diagnosis and biomarker analysis but often relies on antibodies and sophisticated instrumentation. Here, we report a signal-amplified protein detection strategy based on chimeric ascorbic acid-2-phosphate (AAP)-releasing nucleotide probes and strand displacement amplification (SDA). Two tetraphosphate nucleotide analogs, dAP₄AA and dTP₄AA, were synthesized by covalently linking AAP to dATP and dTTP, respectively. These probes were efficiently recognized and incorporated by DNA polymerase during DNA synthesis, resulting in the release of AAP, which was subsequently hydrolyzed by shrimp alkaline phosphatase to generate ascorbic acid (AA). The produced AA was quantified through its reaction with o-phenylenediamine (OPDA), yielding a fluorescent quinoxaline derivative. By coupling aptamer-mediated target recognition, SDA, and AA-based fluorescence transduction, an antibody-free sensing platform was established for protein analysis. This approach exhibited linear fluorescence responses over the nanomolar concentration range for streptavidin (SA) and SARS-CoV-2 nucleocapsid (N) protein with detection limits of 72.4 and 346 pM, respectively. The platform displayed excellent selectivity against non-target proteins and achieved satisfactory recoveries (97.2-104.0%) in spiked serum samples. Comparison with a commercial ELISA kit showed no statistically significant difference between the measured results. This study broadens the range of functional nucleotide probes and establishes a general strategy for coupling DNA amplification with small-molecule signal generation for protein detection.