Biological Synthesis of Metal / Metal Oxide Nanoparticles and Their Applications in Food Safety

Abstract

Infringement of bacterial contamination leads to devastating infectious diseases worldwide and can affect human health in two possible ways. Firstly, due to the lack of quality control in food processing sectors, food is contaminated by bacterial pathogens. Secondly, inadequate access to safe and portable clean water along with poor hygiene and sanitation facilities can lead to contamination with pathogenic bacteria—both of the cases resulting in numerous foodborne disease outbreaks. Food industries is one of the potential candidate concerned with the outbreaks of the infectious diseases as it has the susceptibilities for the pathogen contamination. The current practices for controlling the outspread of microbial infections include careful monitoring of the contamination pathway of different pathogenic bacteria by various food safety measures, such as the development of a biosensing platform for pathogen detection and application of antimicrobial agents. Conventional strategies for pathogen biosensing have several drawbacks, like being less sensitive for real sample analysis, time-consuming and laborious. Similarly, several inorganic and organic materials have been implemented as antibacterial agents, which possess some shortcomings due to the high toxicity and cost-effectiveness. The present study focused on the biological synthesis of metal and metal oxide nanoparticles using plant sources as reducing agents and finding their suitable applications in two different aspects of food safety measures, (1) antimicrobial agent, and (2) biosensing of foodborne pathogens. Two different plant extracts were utilized for the synthesis of metal and metal oxide nanoparticles- Prunus nepalensis [fruit extract for gold nanoparticles (Au NPs)] and Bauhinia purpurea [leaf extract for magnesium oxide (MgO) nanoflakes]. Then the detailed characterizations of the biogenic Au NPs and MgO nanoflakes were performed. The average size of the synthesized Au NPs and MgO nanoflakes was found to be 6 ± 1 nm and 11 nm (width), respectively, with a crystalline structure. Electron microscopy was used to investigate the morphology of the biogenic nanoparticles. Additionally, the presence of antioxidants, phenolics, and flavonoids in B. purpurea leaf extract and P. nepalensis fruit extract was studied by using different assays, which suggested the efficacy of leaf and fruit extracts as a potential reducing agent for nanoparticle synthesis. Antimicrobial activity of positively charged (+2.70 mV) MgO nanoflakes was investigated against Staphylococcus aureus, a gram-positive bacteria known to cause various infections in humans. Results suggested the high efficacy of MgO nanoflakes as a potential antibacterial agent against S. aureus at a meagre dose size of 250 μg/mL. Possible mode of action was investigated through surface morphology analysis of bacterial cells by field emission scanning electron microscopy (FESEM), and intracellular reactive oxygen species (ROS) generation by fluorescence microscopy. Another main aim of the study was to utilize biologically synthesized nanoparticles in biosensing applications for the detection of foodborne pathogens. Characterization of the peroxidase mimicking activity of the negatively charged (-20.6 mV) biogenic Au NPs in x comparison with Horseradish peroxidase (HRP, a natural enzyme), was performed. Biogenic Au NPs showed 9.64 times higher maximum reaction velocity following Michaelis–Menten kinetics at 6% H2O2 with a higher affinity towards its substrate, 3,3′,5,5 -Tetramethylbenzidine (TMB) than that of HRP. The reaction velocity was also dependent on the environmental pH, temperature, substrate, and H2O2 concentration. The Michaelis–Menten constant (Km) for biogenic Au NPs and HRP was found to be 6.9×10-2 mM and 7.9×10-2 mM, respectively, at 6% H2O2. Based on this phenomenon, two different biosensing assays were developed for the detection of Mycobacterium bovis [through indirect enzyme linked immunosorbent assay (iELISA)] and Listeria monocytogenes [through recombinase polymerase amplification (RPA)-based paper disc biosensing assay]. As a proof-of-concept for suitable biosensing applications with biogenic Au NPs, an immunoassay was developed to detect M. bovis cells using biogenic Au@1F11conjugates where Au@1F11acted as peroxidase mimic and signalling tag. It was found that the biogenic Au@1F11conjugates were capable of sensitive detection of M. bovis within a range up to 105 CFU/mL with high specificity. Then RPA based-paper disc biosensing assay was performed for the detection of L. monocytogenes where Listeriolysin O (LLO), a secreted protein encoded by hlyA gene was targeted for the detection assay. Oligonucleotide conjugated biogenic Au NPs (Au@ LM122-DP-Amino) showed intrinsic peroxidase-like activity, which acted as a detection probe for the biosensing assay. The developed RPA-paper disc biosensing assay system for L. monocytogenes detection demonstrated high sensitivity for the target pathogen. It was able to detect as low as 2 CFU/mL, or 10 fg of the genomic DNA/ 50 μL reaction. Peroxidase mimicking Au NPs played a significant role in amplifying the detection signal, which could be visualized by naked eye and could be utilized in further developments of point-of-care (POC) detection assays for various analytes, such as pathogen, disease biomarkers, and toxins for further applications.