Multifunctional Se-Cu bimetallic nanoparticles from marine Bacillus licheniformis: targeting oxidative stress,...

Selenium-copper bimetallic nanoparticles (Se-Cu BMNPs) were synthesized using metabolic extracts from the marine bacterium Bacillus licheniformis LHG166 isolated from the Red Sea. UV-Vis spectroscopy showed maximum absorption at 208 nm. FT-IR analysis revealed bacterial proteins and polysaccharides from the bacterial extract as reducing and capping agents, showing an Amide I shift to 1646.28 cm^-1 and new Cu-O/Se-O stretching at 470.34 cm^-1. XRD patterns confirmed the presence of both orthorhombic and cubic phases of CuSe, with an average crystallite size of 27.2 nm. TEM showed spherical morphologies of 20-120 nm diameter. EDX confirmed Cu : Se atomic ratio near 1 : 1 (7.1 at% Cu, 7.5 at% Se). DLS measured the hydrodynamic diameter of 84 nm (PDI 0.26) with a zeta potential of -24.11 mV. Antioxidant testing showed DPPH scavenging up to 96.1% at maximum concentration with an IC₅₀ of 4.1 µg mL^-1 vs. ascorbic acid's 3.1 µg mL^-1, and ABTS scavenging reached 94.6% with an IC₅₀ of 10.73 µg mL^-1 compared to 2.55 µg mL^-1 for ascorbic acid. Anti-inflammatory assessment demonstrated COX-1 inhibition up to 97.3% (IC₅₀ = 7.05 µg mL^-1) and COX-2 inhibition reaching 95.3% (IC₅₀ = 12.11 µg mL^-1) vs. celecoxib's IC₅₀ values of 5.93 and 4.51 µg mL^-1, respectively. Antimicrobial screening via agar well diffusion showed inhibition zones of 28 mm for B. subtilis, 24 mm for E. faecalis, and 27 mm for C. albicans. Broth microdilution revealed MIC values ranging from 15.62 µg mL^-1 (B. subtilis, C. albicans, C. tropicalis) to 125 µg mL^-1 (S. aureus), with MBC/MFC values between 15.62-250 µg mL^-1, yielding ratios of 1.0-4.0, indicating bactericidal activity. Gram-negative bacteria required 31.25-62.5 µg mL^-1 for inhibition and 62.5-125 µg mL^-1 for complete killing, while A. niger showed complete resistance. Biofilm inhibition through microtitre plate assays demonstrated concentration-dependent effects, with 75% MBC achieving over 90% inhibition for most organisms (C. albicans 96.09%, B. subtilis 93.76%, E. coli 91.59%), though S. aureus required higher concentrations (84.33% at 75% MBC). These results demonstrated that marine bacterial metabolites produce biocompatible Se-Cu BMNPs with potent antioxidant, anti-inflammatory, antimicrobial, and antibiofilm properties suitable for biomedical applications.