Bimetallic nano mixtures enable rapid ultrasensitive detection of high risk HPV for early cervical cancer diagnosis

Background High-risk human papillomavirus (HPV) infection, particularly HPV16 and HPV18, is the primary etiological factor in cervical cancer development. The oncogenic potential of these viruses stems from the action of viral E6 and E7 oncoproteins, which drive malignant transformation by degrading tumor suppressor proteins p53 and pRb, disrupting cell cycle regulation, and inducing genomic instability [1, 2]. These mechanistic insights underscore the critical need for sensitive, rapid, and reliable diagnostic tools suitable for point-of-care settings to enable early detection and intervention. Methods We designed, synthesized, and evaluated bimetallic nano mixture-based lateral flow assays (LFAs) employing Au-Cu, Ag-Cu, and Ag-Au nano mixtures for detection of HPV16 L1 and HPV18 L1 capsid proteins. Nanoparticles were characterized by dynamic light scattering (DLS), zeta potential analysis, scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, UV-Vis spectroscopy, and X-ray photoelectron spectroscopy (XPS) before and after functionalization with CTAB and monoclonal antibodies. Analytical performance including linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, specificity, and stability was systematically validated. Clinical evaluation was conducted using 100 cervical samples, with performance parameters calculated against PCR as reference standard. Additional experiments were performed to assess early infection detection capability, correlation with E6/E7 oncoprotein expression, and specificity in the presence of p53 and pRb tumor suppressor proteins. Results Physicochemical characterization revealed successful antibody conjugation across all formulations, with predictable increases in hydrodynamic size (5-7 nm) and shifts toward less negative zeta potentials (-30 to -35 mV). Among monometallic systems, Au nanoparticles demonstrated the highest analytical sensitivity (LOD = 1 ng/mL), while Cu nanoparticles showed the poorest performance (LOD = 12 ng/mL). Remarkably, bimetallic nano mixtures dramatically outperformed monometallic counterparts, with Ag-Au nano mixtures achieving the lowest LOD (0.15 ng/mL), highest linearity (R² = 0.998), and most rapid response time (10 min). Time-course experiments revealed that Ag-Au LFA detected infection as early as day 3 post-infection, 4 days before detection of integrated viral DNA. Among 20 PCR-positive clinical samples, 18 (90%) showed detectable E6/E7 oncoprotein expression, with strong correlations between LFA signal intensity and both E6 (r = 0.82) and E7 (r = 0.79) levels. No cross-reactivity was observed with p53, pRb, or E6/E7 oncoproteins. Clinical evaluation demonstrated that Ag-Au formulations achieved superior clinical performance (95% sensitivity, 91-92% specificity for both HPV targets), 96% positive predictive value (PPV), and 89-90% negative predictive value (NPV). Conclusion Bimetallic nano mixture-based LFAs, particularly the Ag-Au formulation, represent a highly sensitive, specific, and rapid point-of-care diagnostic platform for HPV detection. By enabling early identification of high-risk HPV infection-the critical initiating event in cervical carcinogenesis driven by E6/E7-mediated disruption of p53 and pRb tumor suppressor pathways-this platform addresses a crucial need in cervical cancer prevention.