Self-priming isothermal polymerization engineered in-situ copper nanoparticles generation for one-tube biomarkers...

On the one hand, for most of isothermal polymerization-based biosensing, the detection signals are uniformly originated from non-specific fluorescent staining that usually leads to high background or false positive, which limited their applications in molecular diagnostics. On the other hand, in virtue of characteristic advantages including but not limited to short preparation time (<5 min), large Stokes shift (>230 nm) and high template dependence, DNA-templated copper nanoparticles (CuNPs) enable low-cost and label-free signal transduction in low-background fluorescent sensing, which thus are ideal candidates for signal sources in promising molecular diagnostics.

Herein, we developed highly sensitive and label-free methods for one-tube biomarkers sensing based on self-priming isothermal polymerization (SPIP) engineered in-situ generation of DNA-templated CuNPs. As the core element for SPIP, a dumbbell-shaped primary DNA is rationally designed to consist of adenine/thymine (AT)-rich stem-loop structure and phosphorothioate (PS)-modified DNA hairpin. Once initiated by polymerase, enzymatic extension and self-folding alternately occurred on the primary DNA, which resulted in accumulation of numerous AT-rich templates repeatedly included in the elongated dsDNA products, and ultimately enabled rapid formation of fluorescent CuNPs. By virtue of alkaline phosphatase (ALP)-catalyzed dephosphorylation to generate 3'-OH available for polymerization, the strategy of SPIP engineered CuNPs generation was successfully adapted to sensitive ALP detection. Moreover, by utilizing apurinic/apyrimidinic endonuclease 1 (APE1)-triggered conformation transformation of probe DNA to produce primary DNA, sensitive APE1 sensing was also realized with this self-contained isothermal amplification approach.

Benefiting from the robustness and simplicity of SPIP engineered CuNPs generation, the sensing methods enabled accurate analysis of real samples including ALP assay in clinical human serum and APE1 determination in normal and cancer cells. In brief, this work provides a new vision for nano-signal amplification and a promising sensing strategy for molecular diagnostics.