MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING Ho Chi Minh City, August, 2022 SKL 0 0 9 1 5 7 SUPERVISOR:
INTRODUCTION
Rationale
Nanotechnology plays a crucial role in various fields, including electronics, energy, medicine, cosmetics, and particularly biomedicine, due to its ability to manipulate materials at the nanometer scale, where unique properties emerge While both chemical and physical methods for synthesizing nanomaterials have been extensively explored, these methods often incur high costs and involve toxic substances that pose risks to human health and the environment Consequently, there is a pressing need to investigate and develop eco-friendly biological methods for synthesizing nanoparticles.
Recent studies in nanotechnology have primarily focused on synthesizing gold and silver nanoparticles, with limited research on copper nanoparticles (CuNPs), particularly through green synthesis methods CuNPs are gaining attention due to their low synthesis costs, ease of use, large-scale applicability, and notable antibacterial properties comparable to those of precious metals However, bare nanoparticles are thermoelectrically unstable in solutions and tend to aggregate, which diminishes their unique properties, prompting researchers to explore various coatings for nanoparticle synthesis For instance, one study successfully synthesized copper (I) oxide nanoparticles with a particle size of 222 ± 13 nm using glucose as a reducing agent and polyvinyl alcohol (PVA) for encapsulation, demonstrating significant antibacterial efficacy against E coli at a concentration of 160 ppm after one hour at pH 8.0 Additionally, recent findings highlighted the antibacterial and antifungal activities of copper oxide/carbon (CuO/C) nanocomposites against a range of pathogens, including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, Candida albicans, and Aspergillus niger.
As science and technology advance, there is a growing focus on developing optimal preservation methods for agricultural products, particularly fruits, with biopolymer films, especially edible films, gaining prominence The superior antibacterial properties of copper nanoparticles (CuNPs) are crucial in addressing the rising threat of pathogenic bacteria to human and organismal health Consequently, researching and applying CuNPs has become a significant and urgent area of study Therefore, we have chosen to investigate the "Effects of capping agent concentration and reaction time on the antimicrobial activities of copper nanoparticles (CuNPs)."
Thesis objects
This study synthesized copper(I) oxide nanoparticles through chemical reduction, utilizing copper sulfate anhydrous as a precursor, D-Glucose as a reducing agent, and starch as a capping agent We examined the factors influencing the antibacterial efficacy of copper nanoparticle solutions, including reaction time, capping agent concentration, and storage conditions The goal was to identify the optimal conditions for synthesizing nano copper with enhanced antibacterial properties.
Limits and scope of the study
CuNPs were synthesized using a chemical reduction method, employing copper sulfate anhydrous as the precursor and D-glucose as the reducing agent, with soluble starch as a capping agent We examined the effects of reaction time, encapsulation concentration, and storage conditions of the nano solution to determine the optimal synthesis formula and process for achieving the highest antibacterial activity The study focused on the bacteria Escherichia coli and the mold Colletotrichum gloeosporioides.
Research content
In this study, we conducted the following:
- Synthesis of copper(I) oxide nanofluids
- Investigate the effect of starch capping agent concentration on the antibacterial ability of copper(I) oxide nanofluids
- The effect of copper concentration on antimicrobial resistance was investigated
- Evaluation of the size, distribution, and morphology of copper nanoparticles by TEM, SEM, and Zeta potential.
The scientific and practical significance of the topic
Nanotechnology has significantly advanced various fields, particularly in biomedical, energy, environmental, information technology, and military applications Nano copper(I) oxide, known for its strong antibacterial properties, emerges as a promising material for food preservation The use of active films in preserving food products like meat, fish, and fruits extends their shelf life while reducing the growth of harmful microorganisms Comprehensive research provides valuable insights into the characteristics of copper nanoparticles (CuNPs), facilitating the application of nano coatings on fruits to enhance post-harvest preservation, ultimately benefiting the economy.
Layout of the report
The report consists of 5 chapters:
- Chapter 3: Materials and research methods
OVERVIEW
Overview of nanotechnology
The National Nanotechnology Initiative (NNI) defines nanotechnology as a field that operates at the nanoscale (1 to 100 nm), where unique phenomena enable new applications across various disciplines, including chemistry, physics, biology, medicine, engineering, and electronics This definition highlights two key aspects: the scale of nanotechnology, which involves manipulating structures at the nanometer level, and the novelty of using nanoscale materials to develop improved materials, devices, and systems with enhanced properties By modifying matter at this scale, it is possible to create materials with increased surface area, leading to higher vibration frequencies and enhanced characteristics such as stiffness, lightness, durability, and improved electrical and thermal conductivity These advancements have paved the way for the development of antibacterial nanomaterials, marking a significant revolution in their future applications.
Nanomaterials are rapidly evolving, necessitating the continuous adaptation of grouping principles Current classification methods focus on the intrinsic properties of nanomaterials and their physiological interactions within their life cycle context A proposed methodology, DF4nanoGrouping, categorizes nanoparticles into four distinct groups: (i) soluble nanoparticles; (ii) nanomaterials with high persistence; (iii) nanomaterials with negligible biological impact; and (iv) nanomaterials exhibiting specific hazardous characteristics related to their surface This approach effectively integrates both inherent and system-dependent properties, along with the toxicological effects of nanomaterials.
Nanoparticles are categorized into organic, inorganic, and carbon groups at the nanometer scale, exhibiting enhanced physical, chemical, and biological properties compared to larger nanoparticles Additionally, nanomaterials can be classified based on their overall size and shape into four categories: zero-dimensional (0D), one-dimensional (1D), two-dimensional (2D), and three-dimensional (3D).
0-D nanomaterials are three-dimensional structures that exist uniformly at the nanoscale, which restricts electron movement and enhances their dispersion and stability These unique properties make 0-D nanomaterials highly advantageous for various applications.
0-D nanomaterials, including nanoparticles and nanospheres, have garnered significant attention due to their unique structural features and properties, such as the surface effect and small size effect Their high surface-to-unit volume ratio enhances ion adsorption, making them valuable in antimicrobial applications and energy storage Additionally, their stable structure is crucial for accommodating substantial volume expansion, leading to their use in electrochemistry and microsensor research and production.
1-D nanomaterials, such as nanofibers, nanotubes, nanowires, and nanorods, are two-dimensional materials at the nanoscale that exhibit a larger surface area and unique properties, including thermal, electrical, mechanical, optical, and magnetic characteristics These features lead to shorter ion and electron transport pathways, enhancing stress tolerance Consequently, 1-D materials show great potential for exceptional performance in energy storage and biological applications.
2-D nanomaterials possess a single spatial dimension at the nanoscale, addressing the limitations of 1-D nanoparticles, which often face challenges like limited surface area and load-carrying capacity These innovative materials are engineered to overcome the shortcomings associated with their one-dimensional counterparts.
2-D nanomaterials, characterized by their sheet-like structure, encompass films, nanocoatings, and nanosheets, and are extensively studied in electronics, optical electronics, and biomedicine due to their unique physicochemical and biological properties Their large surface area enhances ion adsorption, improving capacitance, while high conductivity and adjustable interlayer distances facilitate electron transfer and ion overlap, leading to high agent loading capacity and biocompatibility, particularly in antimicrobial applications Additionally, the reflective surface area and electrochemical properties of 2-D platforms have advanced the field of nuclear medicine Consequently, 2D materials have become increasingly favored by researchers across various disciplines.
3-D nanomaterials, which include structures such as nanoballs, nanocoils, nanoclusters, nanocones, nanopillars, and nanoflowers, differ significantly from 0-D materials Additionally, 3-D-like nanoparticles can be synthesized by integrating 0-D, 1-D, and 2-D nanomaterials Notably, complex composite materials with advanced supramolecular structures, which do not fit into the categories of 0-D, 1-D, or 2-D, such as 3-D metal–organic frameworks and covalent organic frameworks, have demonstrated significant antibacterial properties.
2.1.3 Applications of nanotechnology in food
Nanomaterials, due to their distinct physical, chemical, and biological properties, are increasingly utilized to enhance the functionality of food products Their applications span various areas of agriculture, including the development of pesticides, fertilizers, vaccines, and the detection of pathogens in plants and animals, as well as targeted genetic engineering In food processing, nanotechnology aids in the encapsulation of flavors, improves food texture and quality, and introduces new gelling and viscous agents Additionally, in food packaging, nanomaterials enable the creation of pathogen sensors and packaging that is anti-toxic, UV-resistant, and waterproof Furthermore, they enhance nutritional supplements, resulting in nutraceuticals with superior stability and bioavailability compared to their original forms.
Fig.2 2 Applications of nanotechnology in all areas of food science [21]
The application of nanocomposites and nanolaminates in food nanopackaging enhances packaging properties by modifying diffusion rates, creating barriers against water vapor, oxygen, carbon dioxide, and other volatile compounds These materials foster conditions that inhibit microbial growth, with polymeric silicate nanocomposites offering improved resistance to gas and water vapor permeation and increased heat resistance Natural nanoparticles, such as those derived from potato starch and calcium carbonate, enhance the toughness and eco-friendliness of packaging materials Additionally, both organic and inorganic nanoparticles provide antimicrobial effects, ensuring food safety by limiting microbial growth and potentially self-sterilizing to reduce re-contamination risks Nanotechnology also plays a crucial role in food microbiology, facilitating the development of biosensors for detecting diseases and toxins in food and processing environments.
G-liposomal protein nanovesicles have been utilized as a biosensor capable of detecting Escherichia coli and Salmonella typhi in food products This innovative G-liposomal-based sensor highlights the potential of nano-products, including those with antimicrobial properties, recombinant DNA, and nanotubes, in healthcare and medicine These advancements contribute to the promotion of a healthy food culture and enhance the nutritional value of food.
Introduction to copper nanoparticles
Copper, represented by the symbol Cu and atomic number 29, is a ductile metal known for its excellent electrical and thermal conductivity With an atomic mass of 64 and a density of 8.94 g/cm³, copper has a melting point that highlights its unique properties in various applications.
1083 ºC and a boiling point of 2595 ºC Copper has two stable isotopes, Cu 63 and Cu 65 , accounting for 69,2% and 30,8%, respectively [25] Elemental copper exists naturally in the environment as
7 metallic copper in the non-valent state (Cu o ), ionic copper (Cu + or Cu 2+ ), or as nanoparticles (CuNPs) [26]
Copper is crucial for the metabolism of living organisms, playing a key role in various biological activities through copper-containing enzymes that assist in oxygen transport and iron balance It is found in the skin, bones, and several organs, making it vital for plant development and essential functions in the human body Copper regulates proteins, supports photosynthetic electron transport, mitochondrial respiration, and cell wall metabolism A deficiency in copper leads to curled leaves and downward-spiraling petioles, resulting in the permanent shedding of new leaves Conversely, excessive copper levels can cause toxicity, inhibit growth, disrupt photosynthesis, and induce oxidative stress.
Nanocopper and nanocopper oxide are well-known for their effective antibacterial properties against various pathogens Compared to more expensive metals like silver, copper offers a cost-effective solution for producing copper nanoparticles (CuNPs) Additionally, the ability to oxidize and create copper oxide nanoparticles allows for easy mixing with polymers or macromolecules, ensuring both chemical and physical stability.
2.3.1 Methods for synthesizing copper nanoparticles
Bottom up and top down are the two main techniques for nano synthesis today These two techniques include three different synthesis methods: chemical, physical and biological [31]
The top-down approach in nano-generation utilizes physical effects like electric pulses, laser cutting, mechanical grinding, and solvent breakdown to reduce the size of materials Key synthesis techniques include evaporation-condensation and laser ablation, which have been extensively studied by researchers This method offers the advantage of producing uniformly distributed nanoparticles without solvent contamination, unlike other techniques However, it is often costly due to the need for expensive and energy-intensive equipment.
The Bottom-up technique, which includes chemical and biological methods, contrasts with the Top-down strategy for nanoparticle synthesis The chemical method is particularly advantageous for producing copper nanoparticles due to its simplicity, efficiency, and minimal equipment needs However, the use of hazardous compounds in the synthesis process poses significant health and environmental risks The choice of reducing agent plays a crucial role in determining the size and response time of the nanoparticles, with reduction reaction times varying from minutes to over 24 hours based on specific conditions By carefully controlling parameters such as reaction duration, pH, and substance ratios, it is possible to influence the size, growth, shape, and dispersion of the nanoparticles.
The biological method utilizes plant and animal substances as reducing agents, offering a cost-effective approach to nanoparticle production This environmentally friendly technique is simpler and more sustainable than traditional methods.
This study identifies the chemical reduction method as the optimal approach for synthesizing copper nanoparticles (CuNPs), utilizing CuSO4 as the precursor and glucose as the reducing agent For enhanced sustainability and environmental considerations, starch has been selected as the capping agent.
8 friendliness We hope to synthesize copper nanoparticles with the best antibacterial activity with this method
Fig 2 3 Methods for synthesizing copper nanoparticles [31]
2.3.2 Antibacterial mechanism of copper nanoparticles
The antibacterial activity of copper nanoparticles (CuNPs) primarily arises from the generation of reactive oxygen species (ROS) The effectiveness of CuNPs is largely influenced by their size; particles ranging from 10 to 100 nm adhere to bacterial surfaces through electrostatic attraction, leading to the release of copper ions and ROS In contrast, smaller nanoparticles (less than 10 nm) or released metal ions can penetrate directly through the cell membrane Elevated levels of ROS result in various forms of cellular damage, compromising cell membrane stability and inhibiting cellular respiration, while also causing lipid peroxidation and the oxidation of essential biomolecules like proteins.
The interaction of copper nanoparticles (CuNPs) with bacterial cell membranes is hypothesized to reduce the transmembrane electrochemical potential, which in turn affects membrane integrity.
Copper nanoparticles (CuNPs) and copper ions accumulate on bacterial cell surfaces, creating holes in the membrane that lead to the leakage of cellular components and oxidative stress, ultimately resulting in cell death Additionally, CuNPs interact with the -SH (sulfhydryl) group, causing damage to bacteria and enzymes/proteins A secondary effect of reactive oxygen species (ROS) generated by CuNPs is the direct binding to DNA regions, which degrades its activity.
Fig 2 4 Schematic illustration of the antibacterial mechanism of CuNPs [39]
Capping agent
A capping agent serves as a crucial stabilizer in colloidal synthesis, effectively preventing the overgrowth and aggregation of nanoparticles Its functionality relies on the interaction between its polar head, which engages with the surrounding environment, and its non-polar tail, which binds to the ion or metal molecule of the nanocrystal.
Fig 2 5 Nanoparticles are covalently bonded to the capping agent[40]
Capping agents are categorized into three primary subgroups based on their molecular chain structure: (i) agents with an extended straight hydrocarbon chain that are directly linked to nanoparticles and closely arranged, such as oleylamine, oleic acid, and cetyltrimethylammonium bromide; (ii) unbranched polymer chains that attach to the nano surface via functional groups on the polymer chain.
A 10-dimensional network encapsulates metal nanoparticles using materials such as poly(N-vinyl-2-pyrrolidone) and polyvinyl alcohol Additionally, a branched polymer capping, including poly(amido amine) and polyethylenimine, secures the metal nanoparticles within the gaps of the polymer chain branches.
Capping agents can be categorized based on the ligand that forms the bond at the polar end These categories include -N type agents such as hexadecylamine and octadecylamine, -O type agents like oleic acid and linolenic acid, -S type agents including 1-thioglycerol and thioglycolic acid, -P type agents such as triphenylphosphine, polymeric capping agents like polyethylene glycol, polyvinyl alcohol, and polyvinyl pyrrolidine, as well as polysaccharide capping agents including cellulose and starch.
Fig 2 6 Types of capping agents based on the structure of the molecular chain [42]
Polysaccharides are compounds that form biofilms, consisting of numerous monosaccharide units linked by glycosidic bonds Natural polysaccharides like cellulose, starch, heparin, chitosan, dextran, and glucose serve as effective stabilizers for nanoparticles.
Industrial-scale copper nanosynthesis utilizing polysaccharides as capping agents offers an eco-friendly and economical solution Polysaccharides are generally more stable than proteins, as they rarely experience irreversible denaturation, making them highly valuable in materials science Furthermore, the separation and recovery of polysaccharides are typically cost-effective.
Polysaccharides exhibit variability due to factors such as molecular composition, structural arrangement, asymmetry, hydroxyl group count, and solubility The nano production process is crucial in determining their physicochemical and biological properties, making the selection of the appropriate method essential Various techniques for producing nanoparticles include adjusting response time, temperature, and the molar ratio of ions and polysaccharides Additionally, methods like microwave heating, electrosynthesis, microemulsions, and photoinduction can further enhance the properties of the resulting nanoparticles.
Starch is a highly effective and biocompatible material for producing nanoparticles, thanks to its hydroxyl groups (–OH) that interact with nanomaterial surfaces This property has facilitated the creation of various nanoparticles, including metals and metal oxides Research suggests that starch forms a significant network of hydrogen bonding, which contributes to a protective surface for nanoparticles Furthermore, using starch as a protective agent helps mitigate risks in hazardous environments.
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that can be either aerobic or facultatively anaerobic, measuring between 2.0–6.0 mm in length and 1.1–1.5 mm in width Its structure can vary from spherical cells (cocci) to elongated rods or filaments E coli is commonly found in the digestive tracts of all food-producing animals and humans The bacterium's genetic flexibility and adaptability allow it to develop a wide range of antimicrobial resistance mechanisms in response to changing environments.
Up to 90% of E coli strains inhabit the digestive systems of warm-blooded humans and animals, typically maintaining a beneficial relationship with their hosts and rarely causing disease However, E coli is a leading cause of various bacterial diseases in both humans and animals, contributing significantly to conditions such as enteritis, urinary tract infections, sepsis, postoperative peritonitis, and newborn meningitis Globally, E coli is recognized as a major human pathogen, responsible for severe infections alongside other significant bacterial foodborne agents like Salmonella spp and Campylobacter Thus, the importance of antibiotic resistance in E coli, often perceived as a benign organism, should not be overlooked.
E coli bacteria have the potential to thrive in both aerobic and anaerobic environments, with optimal growth occurring at 37°C E coli bacteria can grow at temperatures ranging from 7°C to 50°C Although the growth of E coli is best at a pH that is close to neutral, it is possible for growth to occur at a pH as low as 4.4 under other optimum circumstances and a medium that has a minimum water activity (Aw) for growth of 0,95 Because of its widespread presence in feces, ease of culture, typically non-pathogenic features, and ability to persist in water, E coli bacteria are often used as a contamination marker of feces, and the potential presence of enteric pathogens like
S typhi in the water Diarrheal strains are classified based on their virulence characteristics: enteropathogenic E coli (EPEC), enteroinvasive E coli (EIEC), enterotoxigenic E coli (ETEC) and enterohemorrhagic E.coli (EHEC) Symptoms and pathogenic mechanisms of E.coli are shown in Table 1.1
Table 2 1 The symptoms as well as the pathogenic mechanisms that are present in some E coli bacteria [48]
Species Symptom Type of diarrhea Pathogenic mechanisms
Causes frequent abdominal pain and diarrhea with discharge in infants
Cause dehydration Parasitic in the small intestine
Diarrhea with fever in all ages
With or without blood; cause dysentery
Parasitize and compete in the intestinal mucosa, causing inflammation of the large intestine
Diarrhea with discharge occurs in some less developed countries and can be transmitted by travelers
Cause dehydration Parasitic in the small intestine, the toxin produced can be stable or decomposed by heat
Hemorrhagic colitis and hemolytic uremic syndrome at any age
Produces shiga-like toxins, irritates the large intestine, causes blood clotting
The fungus Colletotrichum gloeosporioides
Colletotrichum gloeosporioides, a significant plant pathogen belonging to the Melanconiales order, is commonly found in tropical and subtropical regions worldwide Initially identified as Vermicularia gloeosporioides by Penzig, this pathogen plays a crucial role in plant disease dynamics.
The teleomorph stage of the fungus is known as Glomerella cingulata, while its asexual stage is referred to as C gloeosporioides Among the genus Colletotrichum, only the genomes of C graminicola and C higginsianum have been fully sequenced, with ongoing studies on the genome of C gloeosporioides revealing genes related to disease and host defense systems This pathogen thrives in temperatures between 25 – 28°C and pH levels of 5.8 to 6.5, entering a dormant state during dry seasons and resuming its active life cycle when environmental conditions become favorable.
13 hemibiotrophic form of infection in which both the vegetative and necrotic stages take place in sequential order [49]
Fig 2 7 Anthracnose cycle in tropical fruit trees [50]
C gloeosporioides is the pathogen that causes anthracnose, which not only infects the fruit but also other plant organs such as the leaves, flowers, twigs, and branches of the plant It is responsible for considerable harm to various crops, including cereals, coffee, legumes, and tropical and subtropical fruits like avocados, bananas, and mangoes The spores generated in these infected tissues are subsequently released and distributed on rainy days owing to splashes of water or during a period of high humidity, thereby forming the predominant inoculum for fruit diseases in the pre- harvest stage The most apparent signs of anthracnose are deep lesions that are black or dark brown and contain masses of spores on the surface of the fruit that has been affected Many small lesions can join together to form a more extensive lesion When CO2 levels are high, there is an increase in fertility, which means more spores are generated per lesion This may make the illness more severe and cause it to spread more quickly [51] Lesions that are black or dark brown on the surface of the fruit drastically lower its market value [50]
14 Fig 2 8 Anthracnose symptoms on some tropical fruit trees (A) Guava, (B) dragon fruit,
MATERIALS AND RESEARCH METHODS
Materials…
The gram-negative Escherichia coli strain ATCC 8739 and the fungus Colletotrichum gloeosporioides was provided by the Center for Biotechnology in Ho Chi Minh City, Vietnam
Copper sulfate anhydrous (CuSO 4 5H 2 O, >99%, China), D - Glucose (C 6 H 12 O 6 H 2 O, China), Soluble starch ( (C6H10O5)n, China), Sodium hydroxide (NaOH, China), Peptone (China), Citric Acid Monohydrate (C6H8O7.H2O, China), Sodium Chloride (NaCl, China), Nutrient Borth (NB), HIMEDIA, India.
Research methods
Table 3 1 Coding convention for copper nanoparticles samples in research
Reaction time (minutes) Starch concentration (%) Sample coding
In this study, copper nanoparticles (CuNPs) were synthesized using a modified chemical reduction method with CuSO4.5H2O as the precursor and D-glucose as the reducing agent Two solutions were prepared: solution A contained 1.673 g of soluble starch and 9.9 g of glucose, while solution B contained 1.673 g of soluble starch and 1.25 g of CuSO4.5H2O Both solutions were heated to 95°C for 15 minutes with continuous magnetic stirring The pH of solutions A and B was adjusted to 13-14 by adding NaOH and citric acid Solution B was then rapidly added to solution A to facilitate the reduction of Cu²⁺ to CuO, followed by an additional 30 minutes of heating at 95°C The reaction mixture was cooled rapidly in water to room temperature and stored at 7-10°C for 24 hours in preparation for subsequent steps.
The study maintained consistent parameters, including reaction temperature, glucose concentration, CuSO4 concentration, and the concentration and volume of NaOH and citric acid We explored the effects of starch capping agent concentration and reaction time on the copper nanoparticle solution.
3.2.3 Characteristic measurement methods of CuNPs
The UV–VIS spectroscopy method, conducted using a Hitachi S-4800 device in Ho Chi Minh City, is a widely utilized spectrophotometric technique for qualitatively analyzing the concentration, size, and refractive index of nanoparticles.
3.2.3.2 Energy-dispersive X-ray spectroscopy (EDS) analysis
Energy-dispersive X-ray spectroscopy (EDS) is a chemical analysis technique commonly performed with a scanning electron microscope (SEM) This method allows for qualitative, quantitative, and semi-quantitative analysis, providing insights into the distribution of elements through mapping EDS identifies the elemental composition of the examined volume at the microscopic or nanoscale by detecting X-rays emitted from the sample when exposed to an electron beam Researchers often employ EDS to analyze surface elemental components, assess their ratios under different conditions, and create comprehensive maps of the sample.
[54] In this thesis, the EDS measurement method is performed on Quantax 75 machine of Bruker (Germany) at Ho Chi Minh City University of Technology and Education
3.2.3.3 X-ray diffraction spectroscopy (XRD) analysis
This technique identifies the lattice structure of a crystal by analyzing atomic interference at the atomic level The resulting diffraction pattern is influenced by the distance between atomic planes (d) and the angle of reflection (θ), leading to the Bragg equation: \$n\lambda = 2d\sin\theta\$ (3.1).
The X-ray diffraction (XRD) technique provides an average measurement of all crystal volumes, and the observed peak broadening can be attributed to the high quality of the crystal structure Consequently, the crystal size \( D \) can be determined using the Debye-Scherrer equation.
The full width at half maximum (FWHM) of the diffraction peak, denoted as \$\beta\$, is influenced by the angle of diffraction \$\theta\$, the X-ray wavelength \$\lambda\$, and a constant \$K\$ typically set at 0.9 Additionally, the size of the crystal is represented by \$D\$.
X-ray diffraction spectroscopy was performed on a Bruker D8 Advance Eco with measuring angle (2θ) from 5 to 80, voltage 40kV, current 45mA; Anode: Cu, Cu-Kalpha radiation 1.5406 Å Collected data were analyzed using OriginPro 8.5.1
3.2.3.4 Scanning Electron Microscope (SEM) analysis
Scanning electron microscopy (SEM) is a widely used technique for analyzing the microstructure and morphology of materials It operates by scanning a sample's surface with a low-energy electron beam, leading to various interactions that result in the emission of photons and electrons Different detectors are employed based on the SEM mode to capture the signals generated by these interactions, enabling image formation SEM can function in multiple modes for material characterization, including X-ray mapping, secondary electron imaging, backscattered electron imaging, electron channels, and Auger electron microscopy.
The size distribution of copper nanoparticles was assessed by randomly selecting 100 nanoparticles from SEM images and analyzing them using ImageJ software (version 1.49u) The average size was calculated, and a histogram was created to illustrate the distribution of the nanoparticles along with their respective sizes.
3.2.3.5 Transmission electron microscopy (TEM) analysis
Transmission electron microscopy (TEM) is a powerful technique used to obtain detailed information about the shape, crystal structure, and composition of biological materials at resolutions surpassing those of scanning electron microscopy (SEM) In TEM, electrons are emitted from an electron gun within a vacuum chamber and are focused onto the specimen using electrostatic lenses The interaction between the electrons and the sample results in some electrons being scattered or absorbed, while others pass through the sample For effective imaging, the sample must be thin enough to allow a sufficient number of electrons to transmit through it.
Transmission Electron Microscopy (TEM) provides high-resolution images at the nanometer scale, with resolutions below one nanometer achievable by using short electron wavelengths The intensity of the resulting image is influenced by the sample density; denser samples allow fewer electrons to pass through, leading to darker images However, TEM has drawbacks, including potential sample damage from the electron beam, low contrast in atomic materials, and the limitation of producing only two-dimensional images.
The size distribution of copper nanoparticles is analyzed by randomly selecting 80 nanoparticles from TEM images These images are processed using ImageJ software (version 1.49U) to calculate the average size and create a distribution chart of the nanoparticles based on their sizes.
Zeta potential measurement is a key analytical technique for evaluating the surface charge of nanoparticles in suspension It involves the Stern layer, a thin liquid layer formed when a charged particle attracts and binds a layer of opposite charge As charged particles diffuse in solution, they interact with a diffuse outer layer of loosely bound ions, creating an electrical double layer The zeta potential (ZP) is determined by measuring the velocity of these charged particles in an electric field, with typical values ranging from +100 to -100 mV The stability of colloids can be predicted by ZP, where values greater than +25 mV or less than -25 mV indicate high stability Conversely, lower ZP values can lead to aggregation due to van der Waals interparticle interactions, resulting in coagulation or flocculation.
Table 3 2 Zeta potential and the stability of the colloidal [55]
Zeta potential (mV) Stable properties of colloidal
From 0 to 5 Fast coagulation or flocculation
From 30 to 40 Maintaining a steady average
Zeta potential measurements were performed on a Zetasizer Pro from Malvern
Dynamic light scattering (DLS) is a widely utilized technique in the physicochemical characterization of metal nanoparticles, as it effectively determines both the particle size and their distribution This method specifically measures the hydrodynamic size of the nanoparticles, providing crucial insights into their properties.
This technique analyzes the fluctuation in scattering light intensity over a specific time range to study 20 particles It relies on the scattering of laser light as it passes through a colloidal solution.
RESULTS AND DISCUSSION
Characterization of CuNPs
UV-vis absorption spectroscopy is a key technique for characterizing metal nanoparticles, as the spectrum shape reveals important information about their morphology Factors such as metal size, dielectric constant, and stabilizer type significantly influence the peak location and spectrum shape The morphology of metal nanoparticles is affected by the reduction potential of reactants, stabilizers, temperature, pH, and reaction time In this study, glucose was selected as the reducing agent for synthesizing copper nanoparticles, with starch serving as a capping agent to stabilize and control their morphology The characteristic absorption peak for Cu2O typically appears around 490 nm, while an absorption peak at 570 nm indicates the presence of Cu Additionally, CuO exhibits absorption peaks in the 200 – 350 nm range UV-vis spectra of copper nanocomposites at different starch concentrations show absorption peaks between 460 – 520 nm, confirming the presence of the desired product.
Fig 4 1 UV-vis spectra of CuNPs according to different starch concentrations (%w/v) at reaction times (A) t20, (B) t30
4.1.2 Energy-dispersive X-ray spectroscopy (EDS)
The EDS examination results indicate the purity level of synthesized CuNPs, with the X-ray energy scattering spectrum shown in Fig 4.2 revealing a high copper content of 62% and oxygen at 19.98% The presence of carbon in the spectrum is attributed to the soluble starch used, while hydrogen is not detected due to its low mass Additional impurities such as calcium, silicon, and magnesium are identified, likely originating from the glass substrate of the measuring device The electron beam used in the measurement interacts with the sample, causing backscattering and detection of both sample and substrate elements A related study also reports high concentrations of copper and oxygen in EDS results, despite the inclusion of biological extracts like Piper retrofractum.
Vahl), there is still element C As a result of this, the findings of the EDS demonstrate that
Cunps are synthesized in this investigation with a relatively high degree of purity and in the form of oxide or dioxide
Fig 4 2 EDS of CuNPs and composition of the elements of the CuNPs are measured from the EDS
4.1.3 X-ray diffraction of CuNPs (XRD)
XRD analysis confirmed the crystal structure and phase composition of the nanoparticles, revealing that all produced nano solution samples displayed distinct and sharp peaks at 2θ values corresponding to the crystal planes (110), (111), (200), and (311), indicative of Cu2O nanocrystal formation, as per JCPDS No 05-0667.
The XRD spectrum of copper nanoparticles, as per JCPDS No 04-0836, shows diffraction peaks at 22.4°, 31.15°, 43.31°, 50.40°, and 74.17° corresponding to the (111), (200), and (220) planes Additionally, the XRD spectrum of CuO nanoparticles, referenced in research [69], reveals peaks at 32.41°, 35.61°, 38.81°, 48.91°, 53.31°, 58.21°, and 66.31°, which are assigned to the (110), (111), (200), (-202), (020), (202), and (022) crystal planes according to JCPDS No 45-0397 Importantly, the XRD spectrum from our study (Fig 4.3) did not show any additional peaks indicative of impurities like Cu and CuO, confirming the purity of the Cu2O synthesized under the experimental conditions.
Fig 4 3 XRD plot of samples t20 S1,15; t20 S2,29; t20 S3,06; t30 S0,76; t30 S1,91 and t30
Table 4 1 The average crystal size of nanoparticles
Samples Average crystal size (nm) t20 S1,15 7,92 ± 2,81 a t20 S2,29 7,50 ± 2,43 a t20 S3,06 8,12 ± 2,12 a t30 S0,76 8,65 ± 2,44 a t30 S1,91 9,30 ± 2,71 a t30 S2,67 9,07 ± 3,08 a
Note: Different lowercase letters in a column illustrate significant difference (p