Biogenic Synthesis of Silver Nanostructures from Propolis and Bee Pollen: Antibacterial, Antioxidant, and Biosensing Applications

Abstract

The increasing threat of antimicrobial resistance (AMR) demands innovative solutions against pathogenic bacterial infections and biofilms. Oxidative stress, linked to numerous chronic diseases, highlights the need for effective antioxidants. Additionally, early and accurate detection of hydrogen peroxide (H₂O₂) is crucial for disease diagnosis and environmental monitoring. This study aimed to develop an eco-friendly synthesis of silver nanostructures (Ag NSs) using honeybee collected Pollen (Pol-Ag NSs) and Propolis (Pro-Ag NSs), characterize their physicochemical properties, evaluate their antimicrobial and antioxidant potential, and explore the sensing potentials of palladium-decorated Ag NSs (Pd@Ag NSs) for H₂O₂ detection. Pol-Ag NSs and Pro- Ag NSs were synthesized by reducing silver nitrate (AgNO₃) with ethanol extracts of pollen and propolis, respectively. Optimization experiments determined that a 1:10 ratio of 5 mM AgNO₃ to extract for Pol-Ag NSs and 1:1 for Pro-Ag NSs, reacted at 80°C for 2 hours, yielded stable nanostructures with sharp surface plasmon resonance (SPR) peaks at 411 nm for Pol-Ag NSs and 424 nm for Pro-Ag NSs, indicating narrow size distributions. Fourier-transform infrared (FTIR) spectroscopy elucidated key functional groups involved in reduction and stabilization. Pol-Ag NSs displayed characteristic peaks at 3410 cm⁻¹ (O-H, polyphenols), 1635 cm⁻¹ (C=O, flavonoids), and 1080 cm⁻¹ (C-O, polysaccharides), while Pro-Ag NSs showed additional peaks at 1510 cm⁻¹ (aromatic C=C) and 1380 cm⁻¹ (C-N, amines), confirming their roles as reducing and capping agents. X-ray diffraction (XRD) analysis verified a face- centered cubic (FCC) crystal structure, with crystallite sizes of 12.1 nm for Pol-Ag NSs and 33.76 nm for Pro-Ag NSs. Scanning electron microscopy (SEM) revealed distinct morphologies, with Pol-Ag NSs exhibiting spherical shapes and Pro-Ag NSs displaying rod-like structures. Antibacterial assays demonstrated robust activity against selected Gram-positive (Staphylococcus aureus, Enterococcus faecalis) and Gram- negative (Escherichia coli, Pseudomonas aeruginosa) bacteria via disc diffusion test and determination of minimum inhibitory concentration (MIC), and Minimum bactericidal concentration (MBC). Pol-Ag NSs and Pro-Ag NSs exhibited significant antimicrobial activity, with inhibition zones up to 17.33 ± 1.15 mm at concentrations (50-100 μg/mL). Antioxidant capacity was assessed using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. Both nanostructures demonstrated potent antioxidant effects, with half maximal inhibitory concentrations (IC₅₀) of 48.58 ± 1.04 μg/mL (Pol-Ag NSs) and 45.54 ± 0.57 μg/mL (Pro-Ag NSs). In the context of electrochemical applications, Pd@Ag NSs were synthesized, structurally characterized by XRD and SEM, and subsequently evaluated for non-enzymatic H₂O₂ detection via cyclic voltammetry (CV), chronoamperometry (CA), and electrochemical impedance spectroscopy (EIS). Pd@Ag NSs showed excellent electro catalytic performance, with a linear H₂O₂ detection range of 5-75 mM (R² = 0.9947) and efficient charge transfer in EIS. CV revealed a reduction peak at -0.5 V (vs. Ag/AgCl), where CA reveals a limit of detection (LOD) of 1.3 μM. Honeybee-derived Ag NSs offer a sustainable, multifunctional platform for antibacterial, antioxidant, and biosensing applications. Future research should focus on optimizing biocompatibility, scalability, and practical deployment in medical and environmental technologies.