Dissertations / Theses on the topic 'Nanozymes'

This work aimed, firstly, at the production of hydrolitic catalysts, active towards organic phosphorous diesters. This was achieved through the cooperative interaction of metal ions and organic functional groups. Secondly, gold nanoparticles coated with metal-ions-bringing thiols were realized and used for catalisys.
Realizzazione di catalizzatori idrolitici attivi nei confronti di diesteri fosforici organici mediante la cooperazione di ioni metallici e gruppi funzionali organici. In seconda istanza realizzazione di nanoparticelle d'oro ricoperte con tioli recanti complessi di ioni metallici per catalisi.

Nanomaterials offer many unique opportunities for the development of effective and rapid point-of-care (POC) devices to be exploited in many fields, including early diagnosis, health monitoring, and pollutant detection. In particular, gold nanoparticles (AuNPs) exhibit tunable catalytic and plasmonic properties, which are key enabling tools to design and develop innovative detection schemes in several sensing applications. The aim of this PhD project was the development of AuNPs-based colorimetric POCs to detect heavy metal ion contaminations and specific biomarkers in non-invasive biological fluids. First, we developed a novel strategy that exploits the combination of the plasmonic and catalytic properties of AuNPs to achieve an ultrafast (1 min) and sensitive colorimetric sensor for highly toxic methyl mercury. Taking advantage of the AuNP nanocatalyst to promote the rapid reduction of methyl mercury with nucleation on the particle surface and consequent aggregation-induced plasmonic shift, we were able to detect by naked-eye mercury contaminations as low as 20 ppb, which is relevant for food contaminations or biological fluid assessment. Moreover, an innovative and versatile platform, based on multibranched AuNPs, was developed for the detection of salivary biomarkers. Coupling etching and growing reactions in a reshaping process onto the nanostars surface, we created a customizable platform with boosted color change readout for fast detection of salivary glucose at low concentrations. The nanosensor performance was validated on samples from patients with diabetes, proving its potential as a novel non-invasive tool for frequent monitoring of glycaemia. As side project we also investigated the platinum nanoparticles enzymatic activity in a colorimetric sensor for inorganic mercury contamination monitoring in water sources.

The aim of this doctoral project has been centered on the development of innovative green synthetic methods able to govern the key physico-chemical properties of noble metal nanoparticles (NPs). To fully uncover the potential of metallic NPs, it is necessary to finely control the shape of nanomaterials while keeping the ultra-small characteristics, in order to achieve superior efficiency (per mass unit), selectivity and enhanced activity in catalytic processes. Size and shape of the nanomaterial together with the capping agents govern the surface properties and all the processes happening at the surface, such as catalysis. Different shapes offer great versatility to tune the nanocrystal (NCs) catalytic properties, which are dictated by surface facets. Green synthetic procedures have been developed to obtain pure, monodisperse, citrate-capped Pt and Pd NPs with accurate control on their size and the shape, without the use of polymers, surfactants and organic solvents. For Pd NPs, different geometrical shapes were achieved, such as icosahedrons, cubes, rods and wires while maintaining the thickness of 7 nm and length ranging from 38 to 470 nm (in the case of rods and wires) and the size (in the case of cubes and icosahedron) below 10 nm. These engineered nanomaterials exhibited good biocompatibility along with interesting enzymatic and catalytic properties, due to the absence of sticky molecules, high quality of the surface and the removal of toxic reagents. Moreover, a green synthetic procedure has been developed to obtain ultra-small Pt NCs by combining a strong and a weak reducing agent in aqueous environment in a single reaction vessel in only 10 minutes. NCs with size as low as 2.8 nm and high percentage of {111} surface domains have been achieved. These NCs have been physico-chemically and electrochemically characterized, disclosing significant perspectives for their use as innovative electrocatalysts or nanozymes in portable diagnostics.

Bio-inspired or biomimetic chemistry deals with the replication of the nature’s fundamental processes, which can help in understanding the functioning of biological systems and develop novel applications. Although a large number of researchers worked towards the replication of natural synthetic pathways through biogenetic syntheses, enzyme mimicry by the small organic molecules and inorganic complexes emerged in leaps and bounds over the years. The development of biomimetic chemistry then continued in designing the molecules that can function like enzymes. And now, with the advent of nanotechnology, nanostructured materials have been shown to exhibit enzyme-like activities (nanozymes). Interestingly, the two distinct fields, biology and materials science, have been integrated to form an entirely new area of research that has captured a great attention. Along with the pronounced application of nanomaterials as drug delivery vehicles, anticancer agents, antimicrobials, etc., research is also focused on designing nanomaterials for the biomimetic applications. The thesis consists of five chapters. The first chapter provides a general overview of the recently discovered nanozymes that mimic heme-peroxidase, oxidase, superoxide dismutase, catalase, haloperoxidase and phosphatase. This chapter also deals with the nanozymes’ application in sensing and immunoassay, and as antioxidants, neuroprotective agents. The factors affecting the nanozymes’ activity and the challenges associated with them is also covered in this chapter. Chapter 2 is divided into two parts and it deals with the biomimetic properties of graphene-based materials. In part A, the remarkable peroxynitrite (PN) reductase and isomerase activities of hemin-functionalized reduced graphene oxide (rGO) is discussed. In part B, the activity of graphene oxide (GO) as peroxide substrate for the glutathione peroxidase (GPx) enzyme is discussed. In chapter 3, the oxidant material, V2O5, is shown to exhibit significant GPx-like antioxidant activity in its nano-form. Chapter 4 deals with the oxidase-like activity of MnFe2O4 nanooctahedrons for the antibody-free detection of major oxidative stress biomarker, carbonylated proteins. In chapter 5, the phosphotriesterase mimetic role of vacancy engineered nanoceria is discussed. instead of H2O2 for glutathione peroxidase (GPx) enzyme. As partial reduction of GO was observed when treated with GPx enzyme due to the fact that large sheet-like structures cannot be accessible to the active site, we studied the reaction with some GPx mimetics (Fig. 2). Varying the concentration of cofactor glutathione (GSH) required for the reaction, GPx mimic, ditelluride, could accomplish the reduction of GO following Michaelis-Menten kinetics. As the structure of GO is elusive and under active investigation, our study highlights the presence of peroxide linkages as integral part of GO other than hydroxyl, epoxy and carboxylic groups. This study also highlights an important fact that the modification of GO by biologically relevant compounds such as redox proteins must be taken into account when using GO for biomedical applications because such modifications can alter the fundamental properties of GO. Figure 2. The GO reductase and decarboxylase activities of GPx mimetic ditelluride compound, suggesting the presence of peroxide linkages on GO. In chapter 3, we have discussed about the novel antioxidant nanozyme that combats oxidative stress. During our attempts in the investigation of antioxidant nanozymes, we surprisingly noticed that the oxidant material, V2O5, shows significant GPx-like antioxidant activity in its nano-form. The Vn readily internalize in the cells and exhibit remarkable protective effects when challenged against reactive oxygen species (ROS). Although Vn has been shown to protect cells from ROS-induced damage, cells treated with bulk V2O5 and few vanadium complexes resulted in generation of ROS and severe toxicity. Detailed investigation on the mechanism of this interesting phenomenon Chapter 4 deals with the development of novel methodology for detection of biomarkers. Inspired by the use of antibodies and enzymes for detection of a specific antigen, we have shown for the first time that the nanozymes can entirely replace antibodies and enzymes in Enzyme-linked Immunosorbent Assays (ELISA). As a specific example, we focused on the antibody-free detection of chief oxidative stress biomarker, carbonylated proteins, as our target. To achieve this, we designed MnFe2O4 nanooctahedrons that can function as oxidase enzyme and form signaling point of detection. We functionalized MnFe2O4 nanooctahedrons with hydrazide terminating groups so that carbonylated proteins can be linked to nanozymes by hydrazone linkage (Fig. 4a). Treatment of various carbonylated proteins (hemoglobin (Hb), Myoglobin (Mb), Cytochrome c (Cyt c), RNase and BSA) coated in well plate with hydrazide-terminated MnFe2O4 nanooctahedrons and then with 3,3’,5,5’-tetramethylbenzidine substrate, resulted in instantaneous detection by well plate reader (Fig. 4b). Considering the challenges and difficulties associated with the conventional methods used to detect such modified proteins, this methodology opens up a new avenue for the simple, cost-effective, instantaneous and entirely antibody-free ELISA-type detection of carbonylated proteins. Our results provide a cumulative application of nanozymes’ technology in oxidative stress associated areas and pave a new way for direct early detection of post translational modification (PTM) related diseases. Figure 4. a) Nanozyme linked to the carbonylated protein coated on a plate through hydrazone linkage. b) General bar diagram showing detection of oxidized (carbonylated) proteins by nanozymes. Synopsis Figure 5. a) A cartoon view of surface of ceria showing vacancy. b) Zoomed portion of high resolution transmission electron microscopic image showing few vacancies on the surface of nanoceria. c) Catalytic mechanism of detoxification of paraoxon at the defect site. In the final chapter, chapter 5, we have discussed about the nanomaterial that can function as phosphotriesterase enzyme. Phosphotriesterase enzyme is a bacterial enzyme that is involved in the rapid hydrolysis of sarin gas-related deadly nerve agents such as paraoxon, parathion and malathion. When encountered with these orgnaophospatetriesters, living beings tend to undergo nerve shock to cause paralysis by inhibiting an extremely important enzyme called acetylcholine esterase. They are also known to cause severe oxidative stress problems and are associated with neurodegenerative disorders. Therefore, curbing the toxic effects and detoxification of these nerve agents is a world-wide concern and many research teams have focused their attention to address this important problem. Working on the development of nanozymes for important problems, we found that nanoceria, especially the vacancy engineered one (Fig. 5a,b), can serve as active mimic of phosphotriesterase enzyme in the presence of N-methylmorpholine (acting as a distal base histidine). Vacancy engineered nanoceria has been shown to catalyze the hydrolysis of high amounts of paraoxon quiet efficiently and within few minutes with very low activation energy and high kcat. Detailed mechanistic investigation revealed that the presence of both Ce(III) and Ce(IV) is very essential for detoxification activity (Fig. 5b). The vacancies on the surface of nanoceria, were the buried Ce(III) ions are directly exposed to the reaction environment, behave as hotspots or enzyme active sites for detoxification reaction (Fig. 5b).

Bio-inspired or biomimetic chemistry deals with the replication of the nature’s fundamental processes, which can help in understanding the functioning of biological systems and develop novel applications. Although a large number of researchers worked towards the replication of natural synthetic pathways through biogenetic syntheses, enzyme mimicry by the small organic molecules and inorganic complexes emerged in leaps and bounds over the years. The development of biomimetic chemistry then continued in designing the molecules that can function like enzymes. And now, with the advent of nanotechnology, nanostructured materials have been shown to exhibit enzyme-like activities (nanozymes). Interestingly, the two distinct fields, biology and materials science, have been integrated to form an entirely new area of research that has captured a great attention. Along with the pronounced application of nanomaterials as drug delivery vehicles, anticancer agents, antimicrobials, etc., research is also focused on designing nanomaterials for the biomimetic applications. The thesis consists of five chapters. The first chapter provides a general overview of the recently discovered nanozymes that mimic heme-peroxidase, oxidase, superoxide dismutase, catalase, haloperoxidase and phosphatase. This chapter also deals with the nanozymes’ application in sensing and immunoassay, and as antioxidants, neuroprotective agents. The factors affecting the nanozymes’ activity and the challenges associated with them is also covered in this chapter. Chapter 2 is divided into two parts and it deals with the biomimetic properties of graphene-based materials. In part A, the remarkable peroxynitrite (PN) reductase and isomerase activities of hemin-functionalized reduced graphene oxide (rGO) is discussed. In part B, the activity of graphene oxide (GO) as peroxide substrate for the glutathione peroxidase (GPx) enzyme is discussed. In chapter 3, the oxidant material, V2O5, is shown to exhibit significant GPx-like antioxidant activity in its nano-form. Chapter 4 deals with the oxidase-like activity of MnFe2O4 nanooctahedrons for the antibody-free detection of major oxidative stress biomarker, carbonylated proteins. In chapter 5, the phosphotriesterase mimetic role of vacancy engineered nanoceria is discussed. instead of H2O2 for glutathione peroxidase (GPx) enzyme. As partial reduction of GO was observed when treated with GPx enzyme due to the fact that large sheet-like structures cannot be accessible to the active site, we studied the reaction with some GPx mimetics (Fig. 2). Varying the concentration of cofactor glutathione (GSH) required for the reaction, GPx mimic, ditelluride, could accomplish the reduction of GO following Michaelis-Menten kinetics. As the structure of GO is elusive and under active investigation, our study highlights the presence of peroxide linkages as integral part of GO other than hydroxyl, epoxy and carboxylic groups. This study also highlights an important fact that the modification of GO by biologically relevant compounds such as redox proteins must be taken into account when using GO for biomedical applications because such modifications can alter the fundamental properties of GO. Figure 2. The GO reductase and decarboxylase activities of GPx mimetic ditelluride compound, suggesting the presence of peroxide linkages on GO. In chapter 3, we have discussed about the novel antioxidant nanozyme that combats oxidative stress. During our attempts in the investigation of antioxidant nanozymes, we surprisingly noticed that the oxidant material, V2O5, shows significant GPx-like antioxidant activity in its nano-form. The Vn readily internalize in the cells and exhibit remarkable protective effects when challenged against reactive oxygen species (ROS). Although Vn has been shown to protect cells from ROS-induced damage, cells treated with bulk V2O5 and few vanadium complexes resulted in generation of ROS and severe toxicity. Detailed investigation on the mechanism of this interesting phenomenon Chapter 4 deals with the development of novel methodology for detection of biomarkers. Inspired by the use of antibodies and enzymes for detection of a specific antigen, we have shown for the first time that the nanozymes can entirely replace antibodies and enzymes in Enzyme-linked Immunosorbent Assays (ELISA). As a specific example, we focused on the antibody-free detection of chief oxidative stress biomarker, carbonylated proteins, as our target. To achieve this, we designed MnFe2O4 nanooctahedrons that can function as oxidase enzyme and form signaling point of detection. We functionalized MnFe2O4 nanooctahedrons with hydrazide terminating groups so that carbonylated proteins can be linked to nanozymes by hydrazone linkage (Fig. 4a). Treatment of various carbonylated proteins (hemoglobin (Hb), Myoglobin (Mb), Cytochrome c (Cyt c), RNase and BSA) coated in well plate with hydrazide-terminated MnFe2O4 nanooctahedrons and then with 3,3’,5,5’-tetramethylbenzidine substrate, resulted in instantaneous detection by well plate reader (Fig. 4b). Considering the challenges and difficulties associated with the conventional methods used to detect such modified proteins, this methodology opens up a new avenue for the simple, cost-effective, instantaneous and entirely antibody-free ELISA-type detection of carbonylated proteins. Our results provide a cumulative application of nanozymes’ technology in oxidative stress associated areas and pave a new way for direct early detection of post translational modification (PTM) related diseases. Figure 4. a) Nanozyme linked to the carbonylated protein coated on a plate through hydrazone linkage. b) General bar diagram showing detection of oxidized (carbonylated) proteins by nanozymes. Synopsis Figure 5. a) A cartoon view of surface of ceria showing vacancy. b) Zoomed portion of high resolution transmission electron microscopic image showing few vacancies on the surface of nanoceria. c) Catalytic mechanism of detoxification of paraoxon at the defect site. In the final chapter, chapter 5, we have discussed about the nanomaterial that can function as phosphotriesterase enzyme. Phosphotriesterase enzyme is a bacterial enzyme that is involved in the rapid hydrolysis of sarin gas-related deadly nerve agents such as paraoxon, parathion and malathion. When encountered with these orgnaophospatetriesters, living beings tend to undergo nerve shock to cause paralysis by inhibiting an extremely important enzyme called acetylcholine esterase. They are also known to cause severe oxidative stress problems and are associated with neurodegenerative disorders. Therefore, curbing the toxic effects and detoxification of these nerve agents is a world-wide concern and many research teams have focused their attention to address this important problem. Working on the development of nanozymes for important problems, we found that nanoceria, especially the vacancy engineered one (Fig. 5a,b), can serve as active mimic of phosphotriesterase enzyme in the presence of N-methylmorpholine (acting as a distal base histidine). Vacancy engineered nanoceria has been shown to catalyze the hydrolysis of high amounts of paraoxon quiet efficiently and within few minutes with very low activation energy and high kcat. Detailed mechanistic investigation revealed that the presence of both Ce(III) and Ce(IV) is very essential for detoxification activity (Fig. 5b). The vacancies on the surface of nanoceria, were the buried Ce(III) ions are directly exposed to the reaction environment, behave as hotspots or enzyme active sites for detoxification reaction (Fig. 5b).

博士
國立臺灣大學
化學研究所
105
Nanozymes can catalyze specific reactions. In addition, nanozymes possess unique features such as large surface area for further conjugation of multiple molecules for biorecognition. In this study, we synthesized nanozymes and applied them in the detection of biomolecules and heavy metal ions. This dissertation is structured in four chapters. In Chapter 1, the background of nanozymes including their classification, developments, and applications is reviewed. In Chapter 2, Bismuth–gold nanoparticles (Bi–Au NPs) were prepared through self-deposition of bismuth ions (Bi3+) on Au NPs as a result of their aurophilic interactions. Bi–Au NPs possess intrinsic peroxidase-like activity, which in the prescence of hydrogen peroxide (H2O2) could catalytically oxidize Amplex Red (AR) to produce highly fluorescent resorufin, applicable for the detection of H2O2. Then, the as-prepared Bi–Au NPs were further modified with fibrinogen (Fib) to form fibrinogen-adsorbed Bi–Au NPs (Fib-Bi–Au NPs). In the presence of thrombin, soluble fibrinogen converts into insoluble strands of fibrin on Bi–Au NPs'' surfaces, causing a decrease in peroxidase-like activity of Bi–Au NPs, which can be employed in the detection of thrombin and further screening of anticoagulant drugs for thrombin. In Chapter 3, we have successfully demonstrated that Au NPs can be programmed to regulate their peroxidase (POX)-, oxidase (OX)- and catalase (CAT)-like activities through deposition of various metal ions (Ag+, Bi3+, Pb2+, Pt4+, Hg2+). Furthermore, we used metal ions (i.e., Hg2+/Bi3+, Pt4+/Hg2+, Pb2+/Hg2+, and Ag+/Bi3+, respectively) as inputs and the enzyme-like activity of the Au NPs as the output for the construction of OR, AND, INHIBIT, and XOR logic gates. In Chapter 4, we used as constructed “Pt4+/Pb2+(AND)Au NPPOX” and “Bi3+/Hg2+(INHIBIT)Au NPPOX” logic gates for the selective detection of Pb2+ and Hg2+. When Pt4+ and Pb2+ co-exist, strong metallophilic interactions (between Pt and Pb atoms/ions) and aurophilic interactions (between Au and Pb/Pt atoms/ions) result in significant enhanced peroxidase-like activity (AND logic gate) which were then employed in the detection of Pb2+. High peroxidase-like activity of Au NPs in the presence of Bi3+ is a result of the various valence (oxidation) states of Bi3+ and Au (Au+/Au0) atoms on the nanoparticle’s surface. When Bi3+ and Hg2+ co-exist, strong Hg–Au amalgamation results in a large decrease in the peroxidase-like activity (INHIBIT logic gate) of the Au NPs which were then employed in the detection of Hg2+. In addition, an integrated logic circuit based on the color change (formation of reddish resorufin product) and generation of O2 bubbles from these two probes has been constructed, allowing visual detection of Pb2+ and Hg2+ in aqueous solution. In Chapter 5, we used a simple one-step synthesis of well-dispersed amorphous cobalt hydroxide/oxide-modified graphene oxide (CoOxH-GO) possessing peroxidase-like catalytic activity. Interestingly, cyanide ions (CN–) significantly inhibited the catalytic activity of CoOxH-GO nanocomposite, which allows for the construction of a probe for the detection of CN– in water samples and laboratory wastes. We fabricated a paper-based CoOxH-GO probe for the visual detection of CN– by preparing a thin film of CoOxH-GO on a positively charged and porous nylon membrane (N+M), which operates on the principle that CN– inhibited CoOxH-GO catalyzed H2O2 mediated oxidation of AR to reddish resorufin on membrane. The intensity of the red color of membrane decreases with the increase in CN– concentration, which can be easily observed with naked eye in nanomolar concentrations.

Nanomaterials possess a myriad of potential in various biomedical fields such as cancer diagnostics, biosensing, imaging, immunoassay, drug delivery and therapeutics etc. In particular, the ability of the nanozymes to modulate the catalytic activities and biological functions of natural enzymes attracts burgeoning interest for various biomimetic applications. There is a significant relevance of nanozymes which can regulate the reactive oxygen species (ROS) levels in cells, mimicking the cellular antioxidant enzymes due to their possible therapeutic potential in various oxidative stress related disorders. However, there are certain drawbacks associated with their selectivity, limited surface area due to functionalization and biocompatibility. Further, the mechanistic insights revealing the enzymatic role of nanozymes in a cellular environment are not well explored. Our study introduces novel enzyme-mimetics as artificial antioxidants for further development as a potential therapy against oxidative stress-mediated pathological conditions.

碩士
國立臺灣海洋大學
生命科學暨生物科技學系
103
The first part, We synthesized DNA-capped copper nanoparticles (Cu NPs) using simple ascorbate-mediated reduction of Cu(II) ions in TBA29-Tn templates comprising of a 29-mer thrombin-binding aptamer (TBA29) with a poly(dT) (Tn; n = 6−30) motif in both 3′ and 5′ termini. High affinity between thymine and copper resulted in greater growth of Cu NPs along the poly(dT) scaffold. In this method, the DNA acts as the template for synthesis as well as functional group on the Cu NPs. Freshly prepared TBA29-T30-template copper nanoparticles (TBA29-T30Cu NPs) exhibited much stronger fluorescence at 650 nm than that of TBA29-T6Cu NPs and TBA29-T15Cu NPs. However, the fluorescence stability of TBA29-Tn−Cu NPs was very poor due to rapid oxidation of nanoparticles to TBA29-Tn−CuO/Cu2O NPs by molecular oxygen (O2) in the aqueous solution. TBA29-T6CuO/Cu2O NPs and TBA29-T15CuO/Cu2O NPs are spherical particles of about 24 nm, whereas TBA29-T30CuO/Cu2O NPs have short rod-like shapes with a diameter of about 4.2 nm and a length of about 9.2 nm. TBA29-T30−CuO/Cu2O NPs possess stronger peroxidase-like catalytic activity for the H2O2-mediated oxidation of Amplex Red (AR) to fluorescent resorufin than TBA29-T15−CuO/Cu2O NPs and TBA29-T6−CuO/Cu2O NPs. The catalytic activity of TBA29-T30CuO/Cu2O NPs was significantly suppressed in the presence of thrombin, reflecting perturbation of interfacial electron-transfer between substrates and NPs after the specific binding of thrombin with the TBA29 units on the particles’ surfaces. The H2O2/AR–TBA29-T30CuO/Cu2O NPs probe provided a limit of detection (signal–noise ratio = 3) of thrombin in serum samples of 0.5 nM. Hence, this simple, rapid, and economical sensing system shows great potential for analyses of thrombin generation in blood. The present sensing platform was further extended to detect tumor cells using Mucin1 aptamer–templated CuO/Cu2O NPs. The second part, we demonstrate a very simple method to disperse graphene in water and in situ low-temperature synthesis of MnO2 on graphene sheets’ surface using carbon dots (C-dots) through a simple chemical approach. C-dots synthesized by pyrolysis of ammonium citrate had excellent solubility in water due to its surface functional groups and dispersed graphene in water through π–π interaction and formed a stable graphene-C-dot solution (Gr/C-dot). Graphene is highly hydrophobic and cannot disperse in water which restricts its use in many synthesis reactions and making of composites for various applications. However, our C-dots can form stable dispersion of graphene in water which is a promising material for a wide variety of application in graphene based composite synthesis and catalytic experiments. The Gr/C-dot reduced MnO4− to MnOx in aqueous solution at 75 °C, depositing the MnOx on graphene’s surface to form Gr/C-dot/MnOx composite. Since the reaction was conducted at 75 °C, the C-dots were oxidized without affecting the graphene’s graphitic carbons unlike in other hydrothermal or high temperature syntheses. In order to demonstrate our approach can be applied to the modification of other carbon materials without affecting the conductivity of the electrodes, we used dipped-coating method to synthesize C-dots/MnOx onto the graphene surface of the multilayer carbon nanotube paper (multi-walled carbon nanotubes paper; MWCNTs paper). We then applied the four-point probe measurement to measure the electrical resistance and discovered that the conductivity of the MWCNTs paper was not affected after synthesis process. The as formed amorphous MnOx on GR/C-dot exhibited good capacitance properties. Gr/C-dot/MnOx composite exhibited a specific capacitance of ~480 F g–1 at a constant charge or discharge of 0.2 A g–1. Further, the composite showed excellent stability; it retained a 95% capacitance after 5000 charge-discharge cycles. Thus, our one-pot synthesis method is green, cost effective and rapid, and has great potential in synthesis of graphene based composites in aqueous medium.

Natural enzymes are highly efficient macromolecular biocatalysts that can selectively catalyze biological reactions with high activity and substrate specificity under optimum conditions. However, natural enzymes suffer from several inherent drawbacks, such as susceptibility to denaturation, laborious preparation, difficulties in recycling, and high cost, significantly constraining their practical applications. Among the natural enzymes, laccases are an important class of oxidative enzymes belonging to the family of multicopper oxidases, which couple the monoelectronic oxidation of its substrates with the reduction of dioxygen into water. This enzyme exhibits great potential in several applications, including dye bleaching, anticancer treatment, wastewater treatment, soil bioremediation, and biocatalysts for organic synthesis. Nevertheless, the poor stability under harsh environmental conditions, high cost, and non-recyclability of the native laccase enzyme seriously restrict its practical applications. In this thesis work, we have explored the laccase like activity of Cu2O nanosphere, fabricated using one pot polyol-based microwave-assisted method. The as-synthesized Cu2O nanosphere exhibited outstanding laccase-like activity with a Michaelis−Menten rate constant (Km) value of 0.2 mM for 2,4-dichlorophenol as a substrate, which is noticeably smaller than previously reported nanozymes as well as natural laccase. The laccase-like oxidase property of the nanozyme was exploited in the effective and sensitive detection of biorelevant catechol-bearing molecules such as epinephrine and dopamine. Furthermore, a platform has been developed for the sensitive detection of Acetylcholinesterase using the Cu2O nanozyme as a probe. In general, this robust and recyclable laccase mimetic nanozyme holds great potential for biosensing, sustainable environmental protection, and biotechnology applications.

碩士
國立中山大學
醫學科技研究所
107
We have developed self-linkable magnetic graphene oxide-bPEI-prussian blue (MPP) as a peroxidase mimicking nanozyme with high oxidizability to 3,3’,5,5’-tetramethylbenzidine (TMB), which generates significant absorption intensity for the colorimetric immunosensing of apolipoprotein A1 (ApoA1) in early bladder cancer (BC) diagnosis and prognosis monitoring. The ultrasensitive immunosensor was constructed using an ApoA1 antibody (ApoA1Ab)-functionalized chip (biochipApoA1) and self-linkable magnetic graphene oxide-bPEI-prussian blue (MPP). After incubating the sample and capturing ApoA1 proteins captured on the biochipApoA1, the MPP functionalized with ApoA1Ab and mouse IgG (MPP-1), rabbit anti-mouse IgG antibody (MPP-2), and goat anti-rabbit IgG antibody (MPP-3) were added together. We envisioned that each captured ApoA1 protein would allow the retention of a large amount of MPP through a self-linking process to amplify the colorimetric signal of TMB in the presence of H2O2. The linear detection range could be obviously widened in the presence of self-linkable MPP—from 0.05 ng/mL to 100 ng/mL—compared with the group without signal amplification (from 1 ng/mL to 100 ng/mL). Our immunosensor analysis of ApoA1 in the urine of BC patients and healthy individuals was highly correlated with enzyme-linked immunosorbent assay measurements; moreover, the ApoA1 concentrations of patients with high-grade BC were significantly higher than those of patients with low-grade BC. After standard clinical treatment, a significant drop of ApoA1 concentration occurred in urine that was lower than the cut-off concentration, suggesting potential clinical applications of the new self-linkable MPP-generating colorimetric immunosensor in early BC diagnosis and prognosis monitoring.

In recent years carbon-based nanomaterials are growing rapidly in the field of science and technology, due to the tunable optical and physical properties such as electronic arrangement, photostability, flexibility and excellent biocompatibility. Considering, the emerging materials of carbon family such as graphene, graphene oxide (GO) and it derivative, carbon nanotube, fullerene, covalent organic framework (COF) carbon nitride (g-C3N4) and carbon dots (C-Dots) has been highlighted. Based on their structure and morphology, the carbon-based materials have received immense interest in the fields of catalysis, electronic, photonic devices, sensors, molecular recognition and biomedical applications. At the same time, it is also reported that the tunable size and shape of the materials (extended -conjugation, state of oxidation etc.) has shown significant attention in antibacterial activity, relative molar extinction coefficient and cellular internalization, molecular recognition etc. Hence this chapter has shortly provided the literature background of preparation and applications of carbon-based materials. Further information regarding synthesis of different type of carbon and sulfur-based luminescent materials and their functionalization are reported in the literature. Finally, a stack of literature reports towards the materials and biological applications such as biomolecular recognition, cellular imaging, sensing and stimuli responsive systems.

Infectious diseases caused by pathogenic bacteria are creating a global health problem. In the recent report of World Health Organization (WHO), it has been mentioned that around 7 lacks people are dying each year worldwide due to drug resistant microbials. After discovery of the lifesaving “wonder drug” molecule penicillin, it was extensively used for the treatment of bacterial infection diseases. However, the excessive use of antibiotics leads to the development of antimicrobial resistance in the pathogenic bacterial strains to overcome the bactericidal effect of antibiotics. The drug-resistance bacteria follow multiple pathways to show resistance towards the existing antimicrobial agents and eventually make them abortive. The prevalence of these drug resistant bacterial strains poses a serious threat to the present medical system. Therefore, there is an urgency to develop advanced antimicrobial agents which can restrict the spread of pathogenic bacteria to eradicate infectious diseases. In this context, the current advancement in the field of nanotechnology would help us to develop nanomaterial-based antimicrobial agents which could be one of the possible alternatives of conventionally used antibiotics. There are numerous reports, which established that nanomaterials such as graphene oxide, carbon nanotube, noble metal nanoparticles, metal oxides like ZnO2, MnO2 etc. have possessed antibacterial activity. In particular, the use of nanosized molybdenum disulfide (MoS2), a transition metal dichalcogenide showed a great potential to utilize for the development of potent antibacterial agents owing to its unique chemical and photophysical properties. Two-dimensional MoS2 nanosheets provide a large surface to volume ratio for the effective interaction with the bacterial cell membrane. For better biological interactions of MoS2 nanomaterials, its surface modification can be easily achieved through functionalization using thiol ligand molecule. Functionalization also enhances its aqueous dispersibility in manyfold. In this thesis work, I have utilized MoS2 nanomaterials and their nanocomposites to develop nanomaterial-based effective antimicrobial agents for the pathogenic bacterial strains using multiple strategies. To extend my work towards the development of nanomaterial-based antibacterial agents, I have explored antibacterial activity of the supramolecularly self-assembled nanosized cage molecule to eradicate drug-resistant bacteria. Apart from antibacterial activity, I have also expanded the scope of applicability of our newly developed nanomaterials in the direction of bio-analyte detection and environmental remediation such as degradation of organic pollutant and detoxification of the chemical warfare agent.

The role of mitochondria is multidimensional and ranges in vast areas, including apoptosis, cellular response towards stress, metabolism, which is regulated by a plethora of proteins, acting together to maintain cellular and organellar homeostasis. In spite of the presence of mitochondrial DNA, most of the mitochondrial proteins are nuclear encoded and translocated inside the organelle through dedicated translocases present on outer and inner membrane of mitochondria. To fulfil the cellular energy demand, mitochondria efficiently generate ATP by oxidative phosphorylation, and thus are considered as "power house of cell." There occurs a transfer of electrons from various oxidizable substrates to oxygen, which is achieved by a series of redox reactions with generation of water as a byproduct. This process is coupled with ATP synthesis, involves five protein-complexes present in the inner mitochondrial membrane. During this process, it generates extremely reactive intermediate species of oxygen as a byproduct collectively referred as Reactive Oxygen Species (ROS) through partial reduction of oxygen. These intermediate metabolites of oxygen include superoxide anion (O2-º), H2O2 and highly reactive hydroxyl radicals (OHº). Although ROS are produced by different cellular sources, such as widely expressed and evolutionary conserved NADPH Oxidases, xanthine oxidase, cyclooxygenases, lipoxygenases and cytochrome P450 enzymes but mitochondria are one of the major contributors of cellular ROS. Earlier, reactive oxygen species were considered as harmful but for past few decades, the role ROS has been appreciated as signalling molecules. Because of their high reactivity, these species can cause redox mediated modifications to cellular components and thus have an ability to participate in signalling process. The regulation of signalling pathway by ROS is governed by either alterations in cellular redox conditions or by oxidative modifications of certain residues in proteins, which are involved in signalling cascades. Reactive Oxygen Species can modify amino acid residues, interact with Fe-S clusters or other metal complexes and induce dimerization of proteins to alter protein structure and function. ROS causes modifications to critical amino acids, mainly by oxidation of cysteine residues, where oxidation of sulfhydryl group (-SH) of a single cysteine residue leads to formation of sulfenic (-SOH), sulfinic (-SO2H), sulfonic (-SO3H), or S-glutathionylated (-SSG) derivatives. Thus, by incorporating these modifications, ROS affects the function of proteins, thereby modulating the cellular signalling process. On the other hand, the accumulation of higher level of reactive oxygen species may damage cellular components causing oxidative stress. Therefore, it is necessary to maintain the ROS levels and regulation of intracellular redox homeostasis depends upon a complex network of antioxidant molecules. These antioxidants range from low molecular weight glutathione to large proteins like glutathione peroxidases. Cell has an array of antioxidants with different subcellular locations. Superoxide Dismutase which catalyzes dismutation of superoxides and converts them to H2O2, localizes in cytosol, mitochondrial intermembrane space and extracellular matrix. Different isoforms of Glutatione Peroxidases (GPx) and Peroxiredoxins (Prx) are located in cytosol as well as in mitochondria and scavenge H2O2 by using glutathione (GSH) and thioredoxin (Trx) respectively, as co-factors. During this peroxidase activity of GPx and Prx, GSH and Trx get oxidized and recycled back to the reduced form by Glutathione Reductase (GR) and Thioredoxin Reductase (TR) correspondingly, with the help of NADPH. Thus, GPx system (GPx, GR, GSH and NADPH) and Prx system (Prx, Trx, TR and NADPH) helps in maintenance of redox balance by scavenging H2O2. Catalase is present in peroxisomes for the catalytic degradation of H2O2. Along with Thioredoxin, glutaredoxin (Grx) also reduces protein disulphides and maintains the redox homeostasis. Although, reactive oxygen species are important for normal physiological process, oxidative stress caused by imbalanced ROS levels is thought to be involved in progression of many disorders. However, in most of the diseases, the role of ROS is not yet clear. Elevated oxidative stress is observed with insulin resistance and progression of type II diabetes mellitus, and the resultant high glucose levels alter mitochondrial physiology, leading to the fragmentation of organelle. However, on contrary it has also been observed that ROS improves insulin sensitivity. ROS is directly involved in progression of neurodegenerative disorders, which are characterized by oxidative stress mediated neuronal loss. Interestingly, in case of cancer ROS plays a differential role. At moderately higher levels, ROS helps cancer cells to detach from the matrix and thus assist in metastasis but the higher accumulation of ROS leads to oxidative stress mediated cell death. Thus, cancer cells have an enhanced expression level of antioxidants to maintain the optimum ROS concentration for their survival and proliferation. The role of ROS in cellular signalling and progression of diseases highlights the importance of redox regulation. Mitochondria being the major source of ROS, harbours various redox regulators such as a mitochondrial permeability transition pore (mPTP), inner membrane anion channel (IMAC), Ca++ ions, etc. In addition, certain proteins like Hsp31/DJ1 class also translocate into the organelle in a stress dependent manner to maintain redox homeostasis. These proteins are encoded by the nuclear genome and translocated in the organelle, suggesting the importance of mitochondrial import machinery in regulation of redox balance. Another such example is MIA pathway of protein import, where MIA40 regulates ROS indirectly by catalyzing folding of disulfide containing proteins such as SOD-1 in a redox coupled process. However, under most cases, the physiological disorders lead to uncontrolled production of reactive oxygen species, thereby overloading the cellular antioxidant defence machinery. The failure of the antioxidant machinery leads to enhanced disease progression. Under such disease conditions where the upheaval of redox homeostasis leads to the accumulation of ROS, artificial antioxidants can be used to protect cells against oxidative damage. Artificial systems such as Cyclodextrins, metal complexes, porphyrins, polymers, supramolecules and biomolecules such as nucleic acids, catalytic antibodies and proteins, have been created to mimic the structures and functions of natural enzymes through various approaches. In the present thesis, we have elucidated the role of two mitochondrial proteins, which are part of mitochondrial import motor, as redox regulators and the effect of artificial antioxidants in maintenance of redox homeostasis under stress. A detailed description on importance of ROS in cellular signalling and disease progression has been included in Chapter I, which gives a preface for the work mentioned in this thesis. Chapter II to chapter V elucidates the main objectives of the present thesis, which are: 1. Identification of novel human mitochondrial regulators of redox homeostasis • Role of NEF in redox sensing (Chapter II) • Evolved function of J-like protein in ROS regulation (Chapter III) 2. Characterization of potential artificial antioxidants as redox therapeutics • Organo-selenium compounds as potential artificial antioxidants (Chapter IV) • Use of nanoparticles as a natural antioxidant mimics (Chapter V) Chapter II: Mitochondrial Hsp70 (mtHsp70) plays a critical role for the import of the precursor proteins. The import activity of mtHsp70 is attributed by cyclic binding and release of precursor proteins which in turn is regulated by co-chaperones J-proteins and nucleotide exchange factor (NEF). The affinity for substrate is governed by the binding of ADP or ATP at the N-terminal nucleotide binding pocket of mtHsp70. The affinity for substrate is higher in ADP bound state as compared to ATP bound state. mtHsp70 by its ATPase activity hydrolyze ATP (low-affinity state) to ADP (high-affinity state), which is replaced back to ATP by NEF thus maintaining the mtHsp70 cycle for protein import. In the present study, we have biochemically and functionally characterized GrpEL1 and GrpEL2 as a nucleotide exchange factor for mtHsp70. We observed that like their yeast ortholog Mge1, both the mammalian NEFs interacts with mtHsp70 and exchange ADP from ATP to maintain the cycle of mtHsp70. Interestingly, we observed that both the NEFs are part of human mitochondrial import motor and are recruited at the import motor as hetero-subcomplex. The formation of GrpEL1-EL2 hetero-subcomplex is important to maintain the stability of both the NEFs. In this study, we have elucidated that the interplay between the two NEFs governs organellar response towards oxidative stress. Chapter III: Redox imbalance generates multiple cellular damages leading to oxidative stress mediated pathological conditions such as neurodegenerative diseases, diabetes, ageing and cancer progression. Therefore, maintenance of ROS homeostasis is most important, that involves well-defined antioxidant machinery. In the present chapter, we have identified for first time a component of mammalian protein translocation machinery, Magmas, to perform a critical ROS regulatory function. Magmas overexpression has been reported in highly metabolically active tissues, cancer cells and tissues of developmental origin that are prone to oxidative damage. We found that Magmas regulates cellular ROS levels by controlling its production as well as scavenging. Magmas promotes cellular tolerance towards oxidative stress by enhancing antioxidant enzyme activity, thus preventing induction of apoptosis and damage to cellular components. Magmas enhances the activity of ETC-complexes, causing reduced ROS production. Our results suggest that J-like domain of Magmas is essential for maintenance of redox balance. The function of Magmas as an ROS sensor was found to be independent of its role in protein import, underlying its dual role in human mitochondria. The unique ROS modulatory role of Magmas is highlighted by its ability to increase cellular tolerance to oxidative stress even in yeast model organism. The cyto-protective capability of Magmas against oxidative damage makes it an important candidate for future investigation in therapeutics of oxidative stress related diseases. Chapter IV: The dysregulation of antioxidant machinery in oxidative stress mediated disorders lead to accumulation of excess ROS, highlighting the importance of artificial antioxidants. For the therapeutics of oxidative stress related disorders, artificial antioxidants have been used as combination redox therapy. In order to realize potent biocompatible antioxidants with minimum toxicity, we have utilized two approaches – synthesis of organic compounds and nanoparticle based enzyme mimetics. We have synthesized novel isoselenazoles with high glutathione peroxidase (GPx) and peroxiredoxin (Prx) activities, which provide remarkable cytoprotection to human cells, mainly by exhibiting antioxidant activities in the presence of cellular thiols. The cytotoxicity of the isoselenazoles is found to be significantly lower than that of ebselen, which is being widely clinically evaluated by several research groups for the treatment of reperfusion injuries and stroke, hearing loss, and bipolar disorder. The compounds reported in this study has the potential to be used as therapeutic agents for disorders mediated by reactive oxygen species.. Chapter V: Nanomaterials with enzyme-like properties have attracted significant interest, although limited information is available on their biological activities in cells. Here, we show that V2O5 nanowires (Vn) functionally mimic the antioxidant enzyme, glutathione peroxidase by using cellular glutathione as a co-factor. Although a bulk V2O5 is known to be toxic to the cells, the property is altered when converted into a nanomaterial form. The Vn nanozymes readily internalize into mammalian cells of multiple origins (kidney, neuronal, prostate, cervical) and exhibit robust enzyme-like activity by scavenging the reactive oxygen species, when challenged against intrinsic and extrinsic oxidative stress. The Vn nanozymes fully restore the redox balance without perturbing the cellular antioxidant defense, thus providing an important cytoprotection for biomolecules against harmful oxidative damage. Based on our findings, we envision that biocompatible Vn nanowires can provide future therapeutic potential to prevent ageing, cardiac disorders and several neurological conditions, including Parkinson’s and Alzheimer’s disease.

The role of mitochondria is multidimensional and ranges in vast areas, including apoptosis, cellular response towards stress, metabolism, which is regulated by a plethora of proteins, acting together to maintain cellular and organellar homeostasis. In spite of the presence of mitochondrial DNA, most of the mitochondrial proteins are nuclear encoded and translocated inside the organelle through dedicated translocases present on outer and inner membrane of mitochondria. To fulfil the cellular energy demand, mitochondria efficiently generate ATP by oxidative phosphorylation, and thus are considered as "power house of cell." There occurs a transfer of electrons from various oxidizable substrates to oxygen, which is achieved by a series of redox reactions with generation of water as a byproduct. This process is coupled with ATP synthesis, involves five protein-complexes present in the inner mitochondrial membrane. During this process, it generates extremely reactive intermediate species of oxygen as a byproduct collectively referred as Reactive Oxygen Species (ROS) through partial reduction of oxygen. These intermediate metabolites of oxygen include superoxide anion (O2-º), H2O2 and highly reactive hydroxyl radicals (OHº). Although ROS are produced by different cellular sources, such as widely expressed and evolutionary conserved NADPH Oxidases, xanthine oxidase, cyclooxygenases, lipoxygenases and cytochrome P450 enzymes but mitochondria are one of the major contributors of cellular ROS. Earlier, reactive oxygen species were considered as harmful but for past few decades, the role ROS has been appreciated as signalling molecules. Because of their high reactivity, these species can cause redox mediated modifications to cellular components and thus have an ability to participate in signalling process. The regulation of signalling pathway by ROS is governed by either alterations in cellular redox conditions or by oxidative modifications of certain residues in proteins, which are involved in signalling cascades. Reactive Oxygen Species can modify amino acid residues, interact with Fe-S clusters or other metal complexes and induce dimerization of proteins to alter protein structure and function. ROS causes modifications to critical amino acids, mainly by oxidation of cysteine residues, where oxidation of sulfhydryl group (-SH) of a single cysteine residue leads to formation of sulfenic (-SOH), sulfinic (-SO2H), sulfonic (-SO3H), or S-glutathionylated (-SSG) derivatives. Thus, by incorporating these modifications, ROS affects the function of proteins, thereby modulating the cellular signalling process. On the other hand, the accumulation of higher level of reactive oxygen species may damage cellular components causing oxidative stress. Therefore, it is necessary to maintain the ROS levels and regulation of intracellular redox homeostasis depends upon a complex network of antioxidant molecules. These antioxidants range from low molecular weight glutathione to large proteins like glutathione peroxidases. Cell has an array of antioxidants with different subcellular locations. Superoxide Dismutase which catalyzes dismutation of superoxides and converts them to H2O2, localizes in cytosol, mitochondrial intermembrane space and extracellular matrix. Different isoforms of Glutatione Peroxidases (GPx) and Peroxiredoxins (Prx) are located in cytosol as well as in mitochondria and scavenge H2O2 by using glutathione (GSH) and thioredoxin (Trx) respectively, as co-factors. During this peroxidase activity of GPx and Prx, GSH and Trx get oxidized and recycled back to the reduced form by Glutathione Reductase (GR) and Thioredoxin Reductase (TR) correspondingly, with the help of NADPH. Thus, GPx system (GPx, GR, GSH and NADPH) and Prx system (Prx, Trx, TR and NADPH) helps in maintenance of redox balance by scavenging H2O2. Catalase is present in peroxisomes for the catalytic degradation of H2O2. Along with Thioredoxin, glutaredoxin (Grx) also reduces protein disulphides and maintains the redox homeostasis. Although, reactive oxygen species are important for normal physiological process, oxidative stress caused by imbalanced ROS levels is thought to be involved in progression of many disorders. However, in most of the diseases, the role of ROS is not yet clear. Elevated oxidative stress is observed with insulin resistance and progression of type II diabetes mellitus, and the resultant high glucose levels alter mitochondrial physiology, leading to the fragmentation of organelle. However, on contrary it has also been observed that ROS improves insulin sensitivity. ROS is directly involved in progression of neurodegenerative disorders, which are characterized by oxidative stress mediated neuronal loss. Interestingly, in case of cancer ROS plays a differential role. At moderately higher levels, ROS helps cancer cells to detach from the matrix and thus assist in metastasis but the higher accumulation of ROS leads to oxidative stress mediated cell death. Thus, cancer cells have an enhanced expression level of antioxidants to maintain the optimum ROS concentration for their survival and proliferation. The role of ROS in cellular signalling and progression of diseases highlights the importance of redox regulation. Mitochondria being the major source of ROS, harbours various redox regulators such as a mitochondrial permeability transition pore (mPTP), inner membrane anion channel (IMAC), Ca++ ions, etc. In addition, certain proteins like Hsp31/DJ1 class also translocate into the organelle in a stress dependent manner to maintain redox homeostasis. These proteins are encoded by the nuclear genome and translocated in the organelle, suggesting the importance of mitochondrial import machinery in regulation of redox balance. Another such example is MIA pathway of protein import, where MIA40 regulates ROS indirectly by catalyzing folding of disulfide containing proteins such as SOD-1 in a redox coupled process. However, under most cases, the physiological disorders lead to uncontrolled production of reactive oxygen species, thereby overloading the cellular antioxidant defence machinery. The failure of the antioxidant machinery leads to enhanced disease progression. Under such disease conditions where the upheaval of redox homeostasis leads to the accumulation of ROS, artificial antioxidants can be used to protect cells against oxidative damage. Artificial systems such as Cyclodextrins, metal complexes, porphyrins, polymers, supramolecules and biomolecules such as nucleic acids, catalytic antibodies and proteins, have been created to mimic the structures and functions of natural enzymes through various approaches. In the present thesis, we have elucidated the role of two mitochondrial proteins, which are part of mitochondrial import motor, as redox regulators and the effect of artificial antioxidants in maintenance of redox homeostasis under stress. A detailed description on importance of ROS in cellular signalling and disease progression has been included in Chapter I, which gives a preface for the work mentioned in this thesis. Chapter II to chapter V elucidates the main objectives of the present thesis, which are: 1. Identification of novel human mitochondrial regulators of redox homeostasis • Role of NEF in redox sensing (Chapter II) • Evolved function of J-like protein in ROS regulation (Chapter III) 2. Characterization of potential artificial antioxidants as redox therapeutics • Organo-selenium compounds as potential artificial antioxidants (Chapter IV) • Use of nanoparticles as a natural antioxidant mimics (Chapter V) Chapter II: Mitochondrial Hsp70 (mtHsp70) plays a critical role for the import of the precursor proteins. The import activity of mtHsp70 is attributed by cyclic binding and release of precursor proteins which in turn is regulated by co-chaperones J-proteins and nucleotide exchange factor (NEF). The affinity for substrate is governed by the binding of ADP or ATP at the N-terminal nucleotide binding pocket of mtHsp70. The affinity for substrate is higher in ADP bound state as compared to ATP bound state. mtHsp70 by its ATPase activity hydrolyze ATP (low-affinity state) to ADP (high-affinity state), which is replaced back to ATP by NEF thus maintaining the mtHsp70 cycle for protein import. In the present study, we have biochemically and functionally characterized GrpEL1 and GrpEL2 as a nucleotide exchange factor for mtHsp70. We observed that like their yeast ortholog Mge1, both the mammalian NEFs interacts with mtHsp70 and exchange ADP from ATP to maintain the cycle of mtHsp70. Interestingly, we observed that both the NEFs are part of human mitochondrial import motor and are recruited at the import motor as hetero-subcomplex. The formation of GrpEL1-EL2 hetero-subcomplex is important to maintain the stability of both the NEFs. In this study, we have elucidated that the interplay between the two NEFs governs organellar response towards oxidative stress. Chapter III: Redox imbalance generates multiple cellular damages leading to oxidative stress mediated pathological conditions such as neurodegenerative diseases, diabetes, ageing and cancer progression. Therefore, maintenance of ROS homeostasis is most important, that involves well-defined antioxidant machinery. In the present chapter, we have identified for first time a component of mammalian protein translocation machinery, Magmas, to perform a critical ROS regulatory function. Magmas overexpression has been reported in highly metabolically active tissues, cancer cells and tissues of developmental origin that are prone to oxidative damage. We found that Magmas regulates cellular ROS levels by controlling its production as well as scavenging. Magmas promotes cellular tolerance towards oxidative stress by enhancing antioxidant enzyme activity, thus preventing induction of apoptosis and damage to cellular components. Magmas enhances the activity of ETC-complexes, causing reduced ROS production. Our results suggest that J-like domain of Magmas is essential for maintenance of redox balance. The function of Magmas as an ROS sensor was found to be independent of its role in protein import, underlying its dual role in human mitochondria. The unique ROS modulatory role of Magmas is highlighted by its ability to increase cellular tolerance to oxidative stress even in yeast model organism. The cyto-protective capability of Magmas against oxidative damage makes it an important candidate for future investigation in therapeutics of oxidative stress related diseases. Chapter IV: The dysregulation of antioxidant machinery in oxidative stress mediated disorders lead to accumulation of excess ROS, highlighting the importance of artificial antioxidants. For the therapeutics of oxidative stress related disorders, artificial antioxidants have been used as combination redox therapy. In order to realize potent biocompatible antioxidants with minimum toxicity, we have utilized two approaches – synthesis of organic compounds and nanoparticle based enzyme mimetics. We have synthesized novel isoselenazoles with high glutathione peroxidase (GPx) and peroxiredoxin (Prx) activities, which provide remarkable cytoprotection to human cells, mainly by exhibiting antioxidant activities in the presence of cellular thiols. The cytotoxicity of the isoselenazoles is found to be significantly lower than that of ebselen, which is being widely clinically evaluated by several research groups for the treatment of reperfusion injuries and stroke, hearing loss, and bipolar disorder. The compounds reported in this study has the potential to be used as therapeutic agents for disorders mediated by reactive oxygen species.. Chapter V: Nanomaterials with enzyme-like properties have attracted significant interest, although limited information is available on their biological activities in cells. Here, we show that V2O5 nanowires (Vn) functionally mimic the antioxidant enzyme, glutathione peroxidase by using cellular glutathione as a co-factor. Although a bulk V2O5 is known to be toxic to the cells, the property is altered when converted into a nanomaterial form. The Vn nanozymes readily internalize into mammalian cells of multiple origins (kidney, neuronal, prostate, cervical) and exhibit robust enzyme-like activity by scavenging the reactive oxygen species, when challenged against intrinsic and extrinsic oxidative stress. The Vn nanozymes fully restore the redox balance without perturbing the cellular antioxidant defense, thus providing an important cytoprotection for biomolecules against harmful oxidative damage. Based on our findings, we envision that biocompatible Vn nanowires can provide future therapeutic potential to prevent ageing, cardiac disorders and several neurological conditions, including Parkinson’s and Alzheimer’s disease.