Simple and efficient co-synthesis of gold nanoparticles and nanoclusters for HPV-16 detection

The detection of HPV-16 is essential for cervical cancer molecular diagnostics at the point of care and in the early stages. Current diagnostic methods, however, find it difficult to balance cost-effectiveness with good analytical performance. We have created a unique nanobiosensor that uses colorimetric and fluorometric techniques to identify the HPV-16 L1 gene sequence in order to overcome this difficulty. By using particular probe-polyadenine (A₂₀) strands, we rapidly synthesized a mixed population of anisotropic gold nanoparticles (AuNPs) and ultrasmall gold nanoclusters (AuNCs) in situ, both produced through the same DNA-templated reduction process. Gel electrophoresis, UV-Vis spectroscopy, transmission electron microscope (TEM), fluorescence spectroscopy, dynamic light scattering (DLS), and visual inspection were used to characterize the nanobiosensor. These analyses confirmed that the Poly(A)₂₀ strand facilitates the formation of anisotropic AuNPs and AuNCs, resulting in a dense layer of functionalized DNA on each nanoparticle and the formation of polyvalent (p)DNA-AuNP-AuNC, while also confirming the detection of the HPV-16 L1 gene sequence. The sensor successfully detected pUCm-T containing the HPV-16 L1 insert across a concentration range of 0.78 nM to 15.6 nM, with a limit of detection (LOD) of 3.5 nM via direct AuNP signaling and 3.11 nM through indirect peroxidase-like activity. The colorimetric assay showed clear and distinguishable color changes upon target DNA binding, highlighting the peroxidase-like activity of the AuNPs as a reliable detection signal. Meanwhile, the fluorometric assay provided a strong fluorescent signal with high contrast between test and control samples, confirming its effectiveness in differentiating positive samples from negative ones. Overall, the nanobiosensor demonstrated robust sensitivity, excellent repeatability, high specificity toward HPV-16, and a reliable operational lifespan.