Gold Nano-ELISA Fabrication for Longitudinal Salivary MMP-9 Detection

Abstract

Matrix metalloproteinase-9 (MMP-9) is a potential biomarker & therapeutic target for OSCC since it is essential for tumor invasion, metastasis, and progression. MMP-9 has been found using traditional enzyme-linked immunosorbent tests (ELISA), although these methods have a limited sensitivity. This study aims to develop a gold nano-ELISA-based detection system for MMP-9 and investigate Nigella sativa (Thymoquinone) as a natural inhibitor of MMP-9, comparing its efficacy with Diclofenac sodium, a standard anti-inflammatory drug. Salivary MMP-9 levels in OSCC patients were measured both before and after treatment using gold-based nanoparticle-enhanced ELISA (Nano-ELISA) technology. The cytotoxic & anti-inflammatory properties of Nigella sativa extracts were assessed in comparison to Diclofenac sodium using the MTT cytotoxic assay & the protein denaturation assay. In contrast to Diclofenac, the binding stability and inhibitory capability of Nigella sativa bioactive components (Thymoquinone) with MMP-9 were evaluated using molecule docking evaluation, molecular dynamics (MD) simulations, and energy decomposition analysis. Utilizing the ADME (Absorption, Distribution, Metabolism, & Excretion) analysis, the pharmacokinetics and drug-likeness of Nigella sativa components were assessed. The gold nano-ELISA method displayed great specificity and sensitivity for detecting salivary MMP-9, exhibiting a significant drop in levels post-treatment, indicating the possibility of using as a non-invasive biomarker for OSCC progression & therapeutic monitoring. Nigella sativa exhibited superior cytotoxicity compared to Diclofenac sodium, reducing OSCC cell viability more effectively. However, Diclofenac showed stronger anti-inflammatory activity in the protein denaturation assay. Molecular docking analysis revealed that Nigella sativa (Thymoquinone) had a stronger binding affinity (-8.5 kcal/mol) and a lower inhibition constant (Ki = 580 nM) than Diclofenac (-7.2 kcal/mol, Ki = 5.3 µM). MD simulations confirmed greater stability for Nigella sativa within the MMP-9 active site, with lower RMSD (1.8 Å) and higher ligand interaction retention (85%) compared to Diclofenac (68%). Energy decomposition analysis further reinforced stronger electrostatic and Van der Waals interactions for Nigella sativa, confirming its higher inhibitory potential. The ADME analysis indicated that Nigella sativa has better pharmacokinetics, with high gastrointestinal absorption and lower CYP450 inhibition, reducing potential drug interaction risks compared to Diclofenac. A promising technique for early OSCC evaluation and treatment monitoring, this study shows that gold nano-ELISA offers an efficient, non-invasive method for MMP-9 detection. The promise of Nigella sativa as a natural medicinal substitute is further supported by the fact that it has a higher binding affinity and inhibits MMP-9 more effectively than Diclofenac sodium. Nevertheless, additional in vivo confirmation and clinical research are required to prove Nigella sativa can serve as a useful pharmacological treatment for OSCC.