A label-free colorimetric biosensor for sensitive detection of vascular endothelial growth factor-165

Sensitive detection of a low abundant protein is essential for biomedical research and clinical diagnostics. Herein, we develop a label-free colorimetric biosensor for the sensitive detection of recombinant human vascular endothelial growth factor-165 (VEGF₁₆₅). This biosensor consists of an aptamer-based hairpin probe, an assistant DNA-trigger duplex and a linear template. In the presence of VEGF₁₆₅, the specific binding of VEGF₁₆₅ with the aptamer-based hairpin probe results in the opening of a hairpin probe and the opened hairpin probe subsequently hybridizes with the single-stranded region of the assistant DNA-trigger duplex to initiate the strand displacement amplification (SDA) to yield abundant triggers. The released triggers can further function as the primers to anneal with the hairpin probe and lead to the opening of the hairpin structure, which subsequently hybridizes with the assistant DNA-trigger duplex to initiate the next round of SDA reaction and generates more triggers. Large amounts of triggers could be generated by the synergistic operation of dual SDA reaction, and the obtained triggers can initiate a new round of SDA reaction to yield numerous G-quadruplex DNAzymes, which subsequently catalyze the conversion of ABTS^2- to ABTS˙^- by H₂O₂ to yield a color change with the assistance of a cofactor hemin. In contrast, in the absence of target VEGF₁₆₅, the hairpin probe, the assistant DNA-trigger duplex and the linear template can stably coexist in solution, and thus no color change is observed because no trigger can initiate SDA to generate the G-quadruplex DNAzyme. This biosensor has a low detection limit of 1.70 pM and a dynamic range over 3 orders of magnitude from 24.00 pM to 11.25 nM. Moreover, the biosensor shows excellent specificity toward the target VEGF₁₆₅ and the entire reaction can be carried out in an isothermal manner without the involvement of a high precision thermal cycler, making the current assay extremely cost effective.