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Articles 91 - 120 of 155
Full-Text Articles in Biophysics
Dynamic Control Of Particle Separation In Deterministic Lateral Displacement Separator With Viscoelastic Fluids, Yuke Li, Hongna Zhang, Yongyao Li, Xiaobin Li, Jian Wu, Shizhi Qian, Fengchen Li
Dynamic Control Of Particle Separation In Deterministic Lateral Displacement Separator With Viscoelastic Fluids, Yuke Li, Hongna Zhang, Yongyao Li, Xiaobin Li, Jian Wu, Shizhi Qian, Fengchen Li
Mechanical & Aerospace Engineering Faculty Publications
We proposed an innovative method to achieve dynamic control of particle separation by employing viscoelastic fluids in deterministic lateral displacement (DLD) arrays. The effects of shear-thinning and elasticity of working fluids on the critical separation size in DLD arrays are investigated. It is observed that each effect can lead to the variation of the critical separation size by approximately 40%. Since the elasticity strength of the fluid is related to the shear rate, the dynamic control can for the first time be easily realized through tuning the flow rate in microchannels.
The Inverse And Direct Hofmeister Series Of Hen Egg White Lysozyme At Ph Below The Isoelectric Point (Pi) As Seen By Small Angle X-Ray Scattering(Saxs), Pawan Koirala
Open Access Theses & Dissertations
The structural changes of Hen Egg White Lysozyme (HEWL) in the intermediate stages leading up to Hofmeister anion-induced aggregation were measured using small-angle x-ray Scattering (SAXS). We used three concentrations of HEWL: 10mg/ml, 60mg/ml and 200mg/ml and two concentrations of salts: 0.1M and 1.0M, mostly in a Tris-buffer solution with a pH of 9.0. For 200mg/ml in Di-water solution, the scattering signal was best fit using an ellipsoidal form factor with equatorial radius of 28.4±0.1 Å and polar radius of 11.3±0.1 Å, and a screened Coulomb repulsive structure factor with an effective radius of 19.9±0.1 Å and protein surface charge …
Thermodynamic And Kinetic Aspects Of Hen Egg White Lysozyme Amyloid Assembly, Tatiana Miti
Thermodynamic And Kinetic Aspects Of Hen Egg White Lysozyme Amyloid Assembly, Tatiana Miti
USF Tampa Graduate Theses and Dissertations
Deposition of protein fibers with a characteristic cross-β sheet structure is the molecular marker associated with human disorders ranging from Alzheimer's disease to type II diabetes and spongiform encephalopathy. Given the large number of non-disease related proteins and peptides that have been shown to form amyloid fibrils in vitro, it has been suggested that amyloid fibril formation represents a generic protein phase transition. In the last two decades it has become clear that the same protein/peptide can assemble into distinct morphologically and structurally amyloid aggregates depending on the solution conditions. Moreover, recent studies have shown that the early stage, oligomeric …
The Behavior Response Of Antlion Larvae To Alternating Magnetic Fields, Lindsey Wagner, Caleb L. Adams
The Behavior Response Of Antlion Larvae To Alternating Magnetic Fields, Lindsey Wagner, Caleb L. Adams
Annual Symposium on Biomathematics and Ecology Education and Research
No abstract provided.
Differential Uptake Of Gold Nanoparticles By 2 Species Of Tadpole, The Wood Frog (Lithobates Sylvaticus) And The Bullfrog (Lithobates Catesbeianus), Lucas B. Thompson, Gerardo L.F. Carfagno, Kurt Andresen, Andrea J. Sitton, Taylor B. Bury, Laura L. Lee, Kevin T. Lerner, Peter P. Fong
Differential Uptake Of Gold Nanoparticles By 2 Species Of Tadpole, The Wood Frog (Lithobates Sylvaticus) And The Bullfrog (Lithobates Catesbeianus), Lucas B. Thompson, Gerardo L.F. Carfagno, Kurt Andresen, Andrea J. Sitton, Taylor B. Bury, Laura L. Lee, Kevin T. Lerner, Peter P. Fong
Biology Faculty Publications
Engineered nanoparticles are aquatic contaminants of emerging concern that exert ecotoxicological effects on a wide variety of organisms. We exposed cetyltrimethylammonium bromide–capped spherical gold nanoparticles to wood frog and bullfrog tadpoles with conspecifics and in combination with the other species continuously for 21 d, then measured uptake and localization of gold. Wood frog tadpoles alone and in combination with bullfrog tadpoles took up significantly more gold than bullfrogs. Bullfrog tadpoles in combination with wood frogs took up significantly more gold than controls. The rank order of weight-normalized gold uptake was wood frogs in combination > wood frogs alone > bullfrogs in combination …
A Numerical Study Of The Interaction Between One Dimensional Carbyne Chain And Single Stranded Dna, Zeina Salman
A Numerical Study Of The Interaction Between One Dimensional Carbyne Chain And Single Stranded Dna, Zeina Salman
Graduate Theses and Dissertations
resolution at the single nucleotide level when developing DNA sequencers. The purpose of this research was to numerically study the electrical properties associated with the interaction between 1D carbon chain, known as carbyne, and ssDNA. First, the electrical properties of the carbyne chain were calculated. Second, the electrical properties of the carbyne chain were calculated in the presence of different ssDNA bases. Analyzing the differences between the two cases led to determining the effects of these different bases on the electrical properties. The numerical simulation approach conducted in this research was based on the first-principle simulation. The first-principle simulation was …
Perspective: The Physics, Diagnostics, And Applications Of Atmospheric Pressure Low Temperature Plasma Sources Used In Plasma Medicine, M. Laroussi
Electrical & Computer Engineering Faculty Publications
Low temperature plasmas have been used in various plasma processing applications for several decades. But it is only in the last thirty years or so that sources generating such plasmas at atmospheric pressure in reliable and stable ways have become more prevalent. First, in the late 1980s, the dielectric barrier discharge was used to generate relatively large volume diffuse plasmas at atmospheric pressure. Then, in the early 2000s, plasma jets that can launch cold plasma plumes in ambient air were developed. Extensive experimental and modeling work was carried out on both methods and much of the physics governing such sources …
Physical Principles Governing Colloidal Particle Deposition At Low Reynold’S Number: Applications To Microbial Biofilms, Sophia Wiedmann
Physical Principles Governing Colloidal Particle Deposition At Low Reynold’S Number: Applications To Microbial Biofilms, Sophia Wiedmann
Macalester Journal of Physics and Astronomy
Biofilms formed from the adhesion of microbes to a surface hold great relevance to public health and wastewater management. However, the physical principles underlying the attachment stage of biofilm formation, when individual microbes first come into contact with a substrate, are not well understood. Here I report on a model of colloidal particle attachment to a surface that incorporates the effects of diffusion, advection, gravity, and the hydrodynamic lift and drag forces experienced by polystyrene beads at low Reynold’s number. The simulation predicts attachment rates of 1.04x10^(-8)m/s, 0.73x10^(-8)m/s, and 1.29x10^(-8)m/s for beads of radius 0.25 µm, 0.55 µm, and 0.90 …
Characterization And Analysis Of Real-Time Capillary Convective Pcr Toward Commercialization, Xianbo Qiu, Shiyin Zhang, Lanju Mei, Di Wu, Ke Li, Shengxiang Ge, Xiangzhong Ye, Ningshao Xia, Michael G. Mauk
Characterization And Analysis Of Real-Time Capillary Convective Pcr Toward Commercialization, Xianbo Qiu, Shiyin Zhang, Lanju Mei, Di Wu, Ke Li, Shengxiang Ge, Xiangzhong Ye, Ningshao Xia, Michael G. Mauk
Mechanical & Aerospace Engineering Faculty Publications
Almost all the reported capillary convective polymerase chain reaction (CCPCR) systems to date are still limited to research use stemming from unresolved issues related to repeatability, reliability, convenience, and sensitivity. To move CCPCR technology forward toward commercialization, a couple of critical strategies and innovations are discussed here. First, single- and dual-end heating strategies are analyzed and compared between each other. Especially, different solutions for dual-end heating are proposed and discussed, and the heat transfer and fluid flow inside the capillary tube with an optimized dual-end heating strategy are analyzed and modeled. Second, real-time CCPCR is implemented with light-emitting diode and …
Code For "Noise-Enhanced Coding In Phasic Neuron Spike Trains", Cheng Ly, Brent D. Doiron
Code For "Noise-Enhanced Coding In Phasic Neuron Spike Trains", Cheng Ly, Brent D. Doiron
Statistical Sciences and Operations Research Data
This zip file contains Matlab scripts and ode (XPP) files to calculate the statistics of the models in "Noise-Enhanced Coding in Phasic Neuron Spike Trains". This article is published in PLoS ONE.
Micro-Spectroscopy Of Bio-Assemblies At The Single Cell Level, Jeslin Kera
Micro-Spectroscopy Of Bio-Assemblies At The Single Cell Level, Jeslin Kera
Honors Undergraduate Theses
In this thesis, we investigate biological molecules on a micron scale in the ultraviolet spectral region through the non-destructive confocal absorption microscopy. The setup involves a combination of confocal microscope with a UV light excitation beam to measure the optical absorption spectra with spatial resolution of 1.4 μm in the lateral and 3.6 μm in the axial direction. Confocal absorption microscopy has the benefits of requiring no labels and only low light intensity for excitation while providing a strong signal from the contrast generated by the attenuation of propagating light due to absorption. This enables spatially resolved measurements of single …
Imaging Live Drosophila Brain With Two-Photon Fluorescence Microscopy, Syeed Ehsan Ahmed
Imaging Live Drosophila Brain With Two-Photon Fluorescence Microscopy, Syeed Ehsan Ahmed
Open Access Theses & Dissertations
Two-photon fluorescence microscopy is an imaging technique which delivers distinct benefits for in vivo cellular and molecular imaging. Cyclic adenosine monophosphate (cAMP), a second messenger molecule, is responsible for triggering many physiological changes in neural system. However, the mechanism by which this molecule regulates responses in neuron cells is not yet clearly understood. When cAMP binds to a target protein, it changes the structure of that protein. Therefore, studying this molecular structure change with fluorescence resonance energy transfer (FRET) imaging can shed light on the cAMP functioning mechanism. FRET is a non-radiative dipole-dipole coupling which is sensitive to small distance …
Investigation Of Cellular Microenvironments And Heterogeneity With Biodynamic Imaging, Daniel Alexander Merrill
Investigation Of Cellular Microenvironments And Heterogeneity With Biodynamic Imaging, Daniel Alexander Merrill
Open Access Dissertations
Imaging of biological tissue in a relevant environment is critical to accurately assessing the effectiveness of chemotherapeutic agents in combatting cancer. Though many three-dimensional (3D) culture models exist, conventional in vitro assays continue to use two-dimensional (2D) cultures because of the difficulty in imaging through deep tissue. 3D tomographic imaging techniques exist and are being used in the development of 3D efficacy assays. However, most of these assays look at therapy endpoint (dead or living cancer cell count) and do not capture the dynamics of tissue response.
Biodynamic imaging (BDI) is a 3D tomographic imaging and assay technique that uses …
Long-Range Acoustic Interactions In Insect Swarms: An Adaptive Gravity Model, Dan Gorbonos, Reuven Ianconescu, James G. Puckett, Rui Ni, Nicholas T. Ouellette, Nir S. Gov
Long-Range Acoustic Interactions In Insect Swarms: An Adaptive Gravity Model, Dan Gorbonos, Reuven Ianconescu, James G. Puckett, Rui Ni, Nicholas T. Ouellette, Nir S. Gov
Physics and Astronomy Faculty Publications
The collective motion of groups of animals emerges from the net effect of the interactions between individual members of the group. In many cases, such as birds, fish, or ungulates, these interactions are mediated by sensory stimuli that predominantly arise from nearby neighbors. But not all stimuli in animal groups are short range. Here, we consider mating swarms of midges, which are thought to interact primarily via long-range acoustic stimuli. We exploit the similarity in form between the decay of acoustic and gravitational sources to build a model for swarm behavior. By accounting for the adaptive nature of the midges' …
Using Zinc Finger Proteins As A Diagnostic Tool For The Detection Of A Cancer Biomarker, Anu Kini
Using Zinc Finger Proteins As A Diagnostic Tool For The Detection Of A Cancer Biomarker, Anu Kini
Masters Theses & Specialist Projects
RASSF1A is a tumor suppressor gene which loses its function due to methylation of CpG islands on its promoter region. Detection of methylation leads to early diagnosis of cancer.
Zinc finger proteins are capable of detecting a specific DNA sequence and Methyl binding domain can bind to the methyl group on the CpG, using this idea mCpG SEER- Lac system makes use of a split protein, β-lactamase. Lac A attached to the ZFP and Lac B attached to the MBD protein. On binding to the DNA, the Lac A and Lac B come in close proximity with each other causing …
Inquiry Of Lipid Membranes Interacting With Functional Peptides And Polyphenol Drug Molecules, Chian Sing Ho
Inquiry Of Lipid Membranes Interacting With Functional Peptides And Polyphenol Drug Molecules, Chian Sing Ho
USF Tampa Graduate Theses and Dissertations
Cellular membranes are important targets for many membrane-active peptides and drug compounds. Here we are interested in deciphering how lipid membranes are perturbed by several membrane-active molecules, including the transmembrane domain of the influenza M2 protein (M2TM), aggregates formed by a synthetic polyglutamine peptide, and three polyphenol compounds (i.e., tamoxifen, genistein, and verapamil). We employ phase-separated ternary lipid model membranes in the form of giant unilamellar vesicles (GUVs) to simulate raft-like structures that have been proposed to govern many important processes in plasma membranes (e.g., intracellular singling and trafficking). Specifically, we use fluorescent microscopy to interrogate how those membrane additives …
Analysis Of Gompertzian Growth In Aggregating Multicellular Tumor Nodules, Gwendolyn A. Deger
Analysis Of Gompertzian Growth In Aggregating Multicellular Tumor Nodules, Gwendolyn A. Deger
Graduate Masters Theses
Past studies have shown that tumor growth generally follows an exponential growth function or, with a limiting growth constraint, the sigmoid Gompertzian function, where a terminal tumor size is reached at late times. The classical Gompertzian description of tumor growth applies in the case of two-dimensional (2D) in vitro cell studies due to the effect of physical limitations on possible growth area. This project asked whether Gompertzian form applies to the in vitro growth of multifocal 3D tumor nodules, whose size is determined by aggregation events as well as cell proliferation. Previous reports have indicated that these three-dimensional (3D) spheroids …
Protein Adsorption Using A Lattice Toy Model, Ari J. Weiland
Protein Adsorption Using A Lattice Toy Model, Ari J. Weiland
Macalester Journal of Physics and Astronomy
Protein adsorption is an important subfield of Biophysics particularly relevant in medical science. Using a computational simulation with a basic but configurable two-dimensional square lattice model of approximate amino acid interactions, I investigated the entropic effects of protein adsorption on a weakly attractive surface. These simulations allow for a precise calculation of the partition functions of these complex systems, from which I can then analyze other thermodynamic properties.
A 'Tissue Model' To Study The Barrier Effects Of Living Tissues On The Reactive Species Generated By Surface Air Discharge, Tongtong He, Dingxin Liu, Han Xu, Zhichao Liu, Dehui Xu, Dong Li, Qiosong Li, Mingzhe Rong, Michael G. Kong
A 'Tissue Model' To Study The Barrier Effects Of Living Tissues On The Reactive Species Generated By Surface Air Discharge, Tongtong He, Dingxin Liu, Han Xu, Zhichao Liu, Dehui Xu, Dong Li, Qiosong Li, Mingzhe Rong, Michael G. Kong
Bioelectrics Publications
Gelatin gels are used as surrogates of human tissues to study their barrier effects on incoming reactive oxygen and nitrogen species (RONS) generated by surface air discharge. The penetration depth of nitrite into gelatin gel is measured in real time during plasma treatment, and the permeabilities of nitrite, nitrate, O3 and H2O2 through gelatin gel films are quantified by measuring their concentrations in the water underneath such films after plasma treatment. It is found that the penetration speed of nitrite increases linearly with the mass fraction of water in the gelatin gels, and the permeabilities of …
Thermal Analysis Of Borosilicate Glass For Its Biological Applications, Gregory Humble
Thermal Analysis Of Borosilicate Glass For Its Biological Applications, Gregory Humble
Symposium of Student Scholars
Borosilicate glass doped with varying wt% of cerium oxide is investigated for biological applications. Thermal studies of each formulation were conducted using an SDT Q600 differential scanning calorimeter. 30mg samples of 350 - 425 μm particle size of each type of glass were heated to 1200°C in order to obtain the glass transition, crystallization, and melting temperatures. Samples were then heated to 900°C at several heating rates, ranging from 2°C/min to 100°C/min, then compared against each other as well as against an undoped borosilicate glass.
Biophysical Investigation Of Amyloid Formation And Their Prion-Like Self-Replication, Mentor Mulaj
Biophysical Investigation Of Amyloid Formation And Their Prion-Like Self-Replication, Mentor Mulaj
USF Tampa Graduate Theses and Dissertations
Growth and deposition of amyloid fibrils, polymers of proteins with a cross beta-sheet structure, are associated with a significant number of human pathologies including Alzheimer’s disease, Parkinson’s disease, prion diseases, type II diabetes, and senile systematic or dialysis-related amyloidoses. The broader objective of my research is to identify the basic mechanisms regulating nucleation and growth of amyloid fibrils. There is increasing evidence that amyloid formation may proceed along at least two distinct assembly pathways for the formation of rigid fibrils. One pathway involves the nucleated polymerization of the characteristic rigid fibrils from partially denatured monomers and the other proceeds via …
Bayesian Signal Detection And Source Separation In Simulated Brain Computer Interface Systems, Muhammad Asim Mubeen
Bayesian Signal Detection And Source Separation In Simulated Brain Computer Interface Systems, Muhammad Asim Mubeen
Legacy Theses & Dissertations (2009 - 2024)
The problems of signal detection and source separation are important in many fields of science and engineering. In many cases, a target signal needs to be detected in real time and is contaminated by noise. Sometimes the level of noise is on the order of the signal itself. The real time detection of a target signal is of key importance in problems such as the brain computer interface systems. In brain computer interface systems, the neural activity (electric signals) of the brain is detected using sensors (electrodes) on the surface of the brain or the scalp. This signal is contaminated …
Two-Photon Excitation Based Photochemistry And Neural Imaging, Kevin Andrew Hatch
Two-Photon Excitation Based Photochemistry And Neural Imaging, Kevin Andrew Hatch
Open Access Theses & Dissertations
Two-photon microscopy is a fluorescence imaging technique which provides distinct advantages in three-dimensional cellular and molecular imaging. The benefits of this technology may extend beyond imaging capabilities through exploitation of the quantum processes responsible for fluorescent events. This study utilized a two-photon microscope to investigate a synthetic photoreactive collagen peptidomimetic, which may serve as a potential material for tissue engineering using the techniques of two-photon photolysis and two-photon polymerization. The combination of these techniques could potentially be used to produce a scaffold for the vascularization of engineered three-dimensional tissues in vitro to address the current limitations of tissue engineering. Additionally, …
Biophysical Characterization Of A De Novo Elastin, Kelly Nicole Greenland
Biophysical Characterization Of A De Novo Elastin, Kelly Nicole Greenland
Dissertations, Theses, and Capstone Projects
Natural human elastin is found in tissue such as the lungs, arteries, and skin. This protein is formed at birth with no mechanism present to repair or supplement the initial quantity formed. As a result, the functionality and durability of elastin's elasticity is critically important. To date, the mechanics of this ability to stretch and recoil is not fully understood. This study utilizes de novo protein design to create a small library of simplistic versions of elastin-like proteins, demonstrate the elastin-like proteins, maintain elastin's functionality, and inquire into its structure using solution nuclear magnetic resonance (NMR).
Elastin is formed from …
Mirror Buckling Transitions In Freestanding Graphene Membranes Induced Through Scanning Tunneling Microscopy, James Kevin Schoelz
Mirror Buckling Transitions In Freestanding Graphene Membranes Induced Through Scanning Tunneling Microscopy, James Kevin Schoelz
Graduate Theses and Dissertations
Graphene has the ability to provide for a technological revolution. First isolated and characterized in 2004, this material shows promise in the field of flexible electronics. The electronic properties of graphene can be tuned by controlling the shape of the membrane. Of particular interest in this endeavor are the thermal ripples in graphene membranes. Years of theoretical work by such luminaries as Lev Landau, Rudolf Peierls, David Mermin and Herbert Wagner have established that 2D crystals should not be thermodynamically stable. Experimental research on thin films has supported this finding. Yet graphene exists, and freestanding graphene films have been grown …
Evaluating Breast Cancer Stem Cell Response To Antiangiogenic Therapy, Connor J. Holloway
Evaluating Breast Cancer Stem Cell Response To Antiangiogenic Therapy, Connor J. Holloway
Open Access Theses
Angiogenic inhibitors function by blocking tumor cell signals used to recruit host tissue vasculature to the tumor site, thereby depriving the cancer of the nutriment needed for further expansion. The development and implementation of angiogenic inhibitors in conjunction with standard chemotherapy agents has increased progression-free survival but not overall patient survival. It is hypothesized that chronic exposure to large doses of AAT drugs worsens hypoxic conditions within the tumor mass, selectively stimulating aggressive cancer stem cell populations to grow and proliferate. ^ In this study, the expression of the CSC biomarkers ALDH1, DLL1, and EpCAM were evaluated in breast cancer …
Quantitative Mrna Detection With Advanced Nonlinear Microscopy, Jing Liu
Quantitative Mrna Detection With Advanced Nonlinear Microscopy, Jing Liu
Open Access Dissertations
Cell-specific information on quantity and localization of key mRNA transcripts in single-cell level are critical to the assessment of cancer risk, therapy efficacy, and effective prevention strategies. While current techniques are not capable to visualize single mRNA transcript beyond the diffraction limit. In this thesis, two nonlinear technologies, second harmonic super-resolution microscopy (SHaSM) and transient absorption microscopy (TAM), are developed to detect and quantify single Human edimer receptor 2 (Her2) mRNA transcripts. The SHaSM is used to detect single mRNA transcript beyond the diffraction limit, while the TAM is employed to detect mRNA without the interference of fluorescence background. The …
The Effect Of Macromolecular Crowding On The Structure Of The Protein Complex Superoxide Dismutase, Ajith Rathnaweera Rajapaksha Mudalige
The Effect Of Macromolecular Crowding On The Structure Of The Protein Complex Superoxide Dismutase, Ajith Rathnaweera Rajapaksha Mudalige
Open Access Dissertations
Biological environments contain between 7 - 40% macromolecules by volume. This reduces the available volume for macromolecules and elevates the osmotic pressure relative to pure water. Consequently, biological macromolecules in their native environments tend to adopt more compact and dehydrated conformations than those in vitro. This effect is referred to as macromolecular crowding and constitutes an important physical difference between native biological environments and the simple solutions in which biomolecules are usually studied.^ We used small angle scattering (SAS) to measure the effects of macromolecular crowding on the size of a protein complex, superoxide dismutase (SOD). Crowding was induced using …
De Novo Design And Engineering Of Functional Metal And Porphyrin-Binding Protein Domains, Bernard Howard Everson
De Novo Design And Engineering Of Functional Metal And Porphyrin-Binding Protein Domains, Bernard Howard Everson
Dissertations, Theses, and Capstone Projects
In this work, I describe an approach to the rational, iterative design and characterization of two functional cofactor-binding protein domains. First, a hybrid computational/experimental method was developed with the aim of algorithmically generating a suite of porphyrin-binding protein sequences with minimal mutual sequence information. This method was explored by generating libraries of sequences, which were then expressed and evaluated for function. One successful sequence is shown to bind a variety of porphyrin-like cofactors, and exhibits light- activated electron transfer in mixed hemin:chlorin e6 and hemin:Zn(II)-protoporphyrin IX complexes. These results imply that many sophisticated functions such as cofactor binding and electron …
Dynamics Of Biopolymers On Nanomaterials Studied By Quasielastic Neutron Scattering And Mdsimulations, Gurpreet Dhindsa
Dynamics Of Biopolymers On Nanomaterials Studied By Quasielastic Neutron Scattering And Mdsimulations, Gurpreet Dhindsa
Wayne State University Dissertations
Neutron scattering has been proved to be a powerful tool to study the dynamics of biological systems under various conditions. This thesis intends to utilize neutron scattering techniques, combining with MD simulations, to develop fundamental understanding of several biologically interesting systems. Our systems include a drug delivery system containing Nanodiamonds with nucleic acid (RNA), and two specific model proteins, β-Casein and Inorganic Pyrophosphatase (IPPase).
RNA and nanodiamond (ND) both are suitable for drug-delivery applications in nano-biotechnology. The architecturally flexible RNA with catalytic functionality forms nanocomposites that can treat life-threatening diseases. The non-toxic ND has excellent mechanical and optical properties and …