Understanding the enzyme mimicking activity of fe3o4 nanoparticles and its applications in biosensing and antibacterial activity

Abstract

Nanotechnology, manipulation of materials at nano scale, merges disciplines like biology, material science, chemistry and physics to create advanced applications in biomedicine, biosensing, computing, electronics and multiple other technologies. Among its various applications, nanoparticles mimicking natural enzymes, termed as nanozymes gained lot of interest over biological enzymes. Interest in nanozymes largely emanates from their cost effectiveness, greater range of applications, and tolerance towards harsh biological and physical environments such as wide range of pH and temperatures. Recently nanozymes of iron oxide, with artificial enzyme like activity, are being utilised for biosensing, antibacterial activity and pollutant removal applications. Iron oxide NPs (IONPs) with unique physical and chemical properties have enabled their use in several applications such as diagnostics, biosensing, imaging, hyperthermia, magnetic separation, cell proliferation and tissue repair and drug delivery. Although nanostructures of iron, cobalt, and nickel are known to exhibit superparamagnetic properties and high magnetic susceptibility, IONPs such as magnetite (Fe3O4), hematite (α- Fe2O3) and maghemite (γ - Fe2O3), are the most studied magnetic nanoparticle type, due to their low toxicity and biocompatibility. Recently, amongst many other fascinating nanoparticles, Fe3O4 NPs have created a great impact in the area of peroxidase mimetics due to their known Fenton chemistry. The intrinsic peroxidase-like activity and electro chemical sensing (non enzymatic) ability of Fe3O4 NPs have shown tremendous potential in detection of biomolecules such as glucose, H2O2, urea, creatinine, serotonin and environmental pollutants such as phenols, mercury, nitrite and antibiotics. In general the peroxidase activity of all nanozymes, based on Fenton reaction is observed at acidic pH range of 3-5. Given the fact that physiological environment is around pH 7.4, this acted as an Achilles heel in terms of designing biosensing applications of Fe3O4 NPs. This study was designed to overcome the pH lacuna of Fe3O4 NPs and extend its peroxidase mimetic activity to physiological pH. The study also included design and demonstration of its biological applications at physiological pH. In order to accomplish the goals of the study, peroxidase like activity of Fe3O4 NPs was evaluated in different buffers like sodium acetate, citrate and PBS at pH ranging from 4 to 7.4 using TMB as substrate in presence of H2O2. After understanding the intrinsic peroxidase mimetic activity of Fe3O4 NPs in various buffers and pH, it was hypothesised that the peroxidase like activity can be enhanced in presence of nucleotides and other synergistic molecules and subsequently tested. A novel omnipresent small nucleotide like ATP was evaluated to understand its effect on peroxidase like activity of Fe3O4 NPs in different buffers at acidic to physiological pH. In addition, the synergistic mechanism of Fe3O4 NPs and ATP eliciting enhanced peroxidase mimetic activity was determined at physiological pH. Kinetic parameters of reaction such as Michaelis-Menten constant and reaction velocity were calculated and compared with the natural peroxidase enzyme and other reported nanozymes. Further, the mechanism of action of ATP as synergistic agent for peroxidase activity of Fe3O4 NPs was elucidated by tracking the formation and quantification of hydroxyl radicals at near neutral pH. To demonstrate the potential of peroxidase activity at physiological pH, glucose detection was carried out based on the peroxidase principle using TMB as substrate. A single step method was successfully designed to detect different glucose concentrations at physiological pH in PBS and spiked blood serum samples. Synergistic peroxidase activity was applied further to determine antibacterial activity against gram positive (Bacillus subtilis) and gram negative (Escherichia coli) bacteria at physiological pH. As hydroxyl radicals ( .OH) are more reactive compared to H2O2 in killing bacteria via oxidative damage, the NPs synergistic peroxidase system was utilized to enhance H2O2 efficiency in killing bacteria at neutral pH. Currently peroxidase like activity of Fe3O4 NPs is applied in biosensing for biomolecule detection, waste water treatment by oxidising organic substrates, to develop novel immuno assays for detection of proteins and for antibacterial strategies. However, the major concern is their pH confined activity. Limited studies have explored the peroxidase like activity of IONPs at physiological and alkaline pH. Thus, the demonstrated nanozyme activity at physiological pH in this thesis will open novel applications of Fe3O4 NPs in designing simpler biosensors and depicting antibacterial activity towards different microorganisms, methods for waste water management and designing immunological assays. Overall, the thesis discusses the peroxidase like activity of Fe3O4 NPs at different pH and persistence of the catalytic activity at physiological pH in presence of ATP. Further, application of peroxidase activity in detection of glucose in a single step and antibacterial activity at neutral pH is elucidated in detail.