A universal strategy based on a multifunctional DNAzyme biomineralized nanodevice for imaging low-abundance proteins

Abnormal protein expression is closely linked to the onset and progression of various diseases, making real-time in vivo monitoring of proteins highly significant for disease diagnosis. Herein, a biodegradable and cofactor self-sufficient multifunctional DNAzyme bioinspired mineralization nanodevice has been developed for catalytic amplified imaging of low-abundance protein in vivo. The nanodevice was fabricated by in situ encapsulation of two antibody-labeled nucleic acid strands containing the target protein epitope sequence and partial split DNAzyme sequences (P1 and P2), together with a molecular beacon (H1) labeled with a fluorophore and quencher, into pH-responsive manganese-doped calcium carbonate nanoparticles. After accumulating at tumor sites and being internalized by cells, the nanodevice disintegrates in the weakly acidic intracellular environment. The target protein specifically recognizes the antibody-conjugated P1 and P2, thereby generating a complete DNAzyme sequence. Moreover, Mn²⁺ ions released from the nanodevice activate the DNAzyme, which cleaves molecular beacon to restore fluorescence. The cleaved molecular beacon products can rehybridize with P1 and P2 to trigger iterative reactions, thereby converting protein amplification analysis into nucleic acid amplification analysis and enabling sensitive in vivo imaging of low-abundance proteins. Using vascular endothelial growth factor (VEGF) as a model, the detection limit of the nanodevice for VEGF reached 24.7 fM. Furthermore, in vivo fluorescence imaging of VEGF enabled clear discrimination between tumor and normal tissues. By simply replacing the corresponding aptamer sequences and antibody molecules, the developed nanodevice can be readily extended to detect other biomolecules, providing a promising tool for highly sensitive in vivo imaging of low-abundance biomolecules.