Green Nanobiotechnology : Synthesis and Application

It is for PG students of Biological Science, Material Science or Nanotechnology who want to learn Green Nanobiotechnology

1. Green Nanotechnology Its easier than you think Dr. Saheli Pradhan Mitra

2. Synthesis of nanoparticles using biological samples Characterization of nanoparticles (FTIR, SEM, TEM, Scanning Tunneling Microscope, Atomic Force Microscope, UV-Vis Spectrophotometer). Contents

3. What is Nanotechnology? Nanoscience is a branch of science that comprises the study of properties of matter at the nanoscale, and particularly focuses on the unique, size-dependent properties of solid-state materials Nanotechnology is the branch that comprises the synthesis, engineering, and utilization of materials whose size ranges from 1 to 100 nm, known as nanomaterials

4. The birth of nanoscience and nanotechnology concepts is usually linked to the famous lecture of Nobel laureate Richard Feynman at the 1959 meeting of the American Physical Society, ‘‘ There’s Plenty of Room at the Bottom ’’. However, the use of nanotechnology and nanomaterials goes back in history long before that.

5. History Long before the era of nanotechnology, people were unknowingly coming across various nanosized objects and using nano-level processes. In ancient Egypt, dyeing hair in black was common and was for a long time believed to be based on plant products such as henna. However, recent research on hair samples from ancient Egyptian burial sites showed that hair was dyed with paste from lime, lead oxide, and water. In this dyeing process, galenite (lead sulfide, PbS ) nanoparticles are formed. The ancient Egyptians were able to make the dyeing paste react with sulfur (part of hair keratin) and produce small PbS nanoparticles which provided even and steady dyeing. Probably the most famous example for the ancient use of nanotechnology is the Lycurgus Cup (fourth century CE). This ancient roman cup possesses unusual optical properties; it changes its color based on the location of the light source. In natural light, the cup is green, but when it is illuminated from within (with a candle), it becomes red. The recent analysis of this cup showed that it contains 50–100 nm Au and Ag nanoparticles, which are responsible for the unusual coloring of the cup through the effects of plasmon excitation of electrons .

6. Why Nanoscale? A nanometer (nm) is one thousand millionth of a meter. People are interested in the nanoscale because at this scale physical and chemical properties of materials differ significantly from those at a larger scale.

7. Nanoparticles are small particles with sizes ranging from 1 to 100 nanometers. These materials have gained importance and interest in recent years owing to their large number of applications, because the matter at this scale presents a more compact arrangement of atoms and molecules, generating phenomena and acquiring or enhancing mechanical, electrical, magnetic, optical, catalytic, and antibacterial properties that are completely different from those of their macroscopic counterparts.

8. Why nanomaterials are different? Today, due to their unique properties, nanomaterials are used in a wide range of applications, such as catalysis, water treatment, energy storage, medicine, agriculture, etc . Two main factors cause nanomaterials to behave significantly differently than the same materials at larger dimensions: surface effects and quantum effects . These factors make nanomaterials exhibit enhanced or novel mechanical, thermal, magnetic, electronic, optical, and catalytic properties. Nanomaterials have different surface effects compared to micromaterials or bulk materials, mainly due to three reasons; dispersed nanomaterials have a very large surface area and high particle number per mass unit, the fraction of atoms at the surface in nanomaterials is increased, and the atoms situated at the surface in nanomaterials have fewer direct neighbors. Because of each of these differences, the chemical and physical properties of nanomaterials change compared to their larger-dimension counterparts. Larger surface areas and larger surface-to-volume ratios generally increases the reactivity of nanomaterials due to the larger reaction surface, as well as resulting in significant effects of surface properties on their structure.

9. The dispersity of nanomaterials is a key factor for the surface effects. The strong attractive interactions between particles can result in the agglomeration and aggregation of nanomaterials , which negatively affects their surface area and their nanoscale properties. Agglomeration can be prevented by increasing the zeta potential of nanomaterials (increasing the repulsive force), optimizing the degree of hydrophilicity/hydrophobicity of the nanomaterial, or by optimizing the pH and the ionic strength of the suspension medium

10. Nanomaterials display distinct size-dependent properties in the 1–100 nm range where quantum phenomena are involved. When the material radius approaches the asymptotic exciton Bohr radius (the separation distance between the electron and hole), the influence of quantum confinement becomes apparent. In other words, by shrinking the size of the material, quantum effects become more pronounced, and nanomaterials become quantum. Those quantum structures are physical structures where all the charge carriers (electrons and holes) are confined within the physical dimensions. As a result of quantum confinement effects, for instance, some non-magnetic materials in bulk such as palladium, platinum, and gold become magnetic in the nanoscale. Quantum confinement can also result in significant changes in electron affinity or the ability to accept or donate electrical charges, which is directly reflected on the catalytic properties of the material. For example, the catalytic activity of cationic platinum clusters in N 2 O decomposition is dictated by the number of atoms in the cluster. 6–9, 11, 12, 15, and 20 atom-containing clusters are very reactive, while clusters with 10, 13, 14, and 19 atoms have low reactivity

11. Classification of Nanomaterials Zero-dimensional nanomaterials (0-D): the nanomaterials in this class have all their three dimensions in the nanoscale range. Examples are quantum dots, fullerenes. One-dimensional nanomaterials (1-D): the nanomaterials in this class have one dimension outside the nanoscale. Examples are nanotubes, nanofibers, nanorods, nanowires. Two-dimensional nanomaterials (2-D): the nanomaterials in this class have two dimensions outside the nanoscale. Examples are nanosheets, nanofilms, and nanolayers. Three-dimensional nanomaterials (3-D) or bulk nanomaterials: in this class the materials are not confined to the nanoscale in any dimension. This class contains bulk powders, dispersions of nanoparticles, arrays of nanowires and nanotubes, etc.

12. Synthesis of Nanomaterials

13. Chemical reduction: This method involves the reduction of metal ions in solution using chemical reagents such as sodium borohydride or sodium hydroxide to form nanoparticles. Coprecipitation: Synthesis involves mixing two or more solutions containing metal ions. When the solutions are mixed, metal ions precipitate out of the solution and form nanoparticles. Sol-gel: The process requires mixing a metal salt with a solvent and gelling agent. The solvent is evaporated leaving behind the gel. The gel is then heated, causing it to solidify and form nanoparticles. Microemulsion: This method needs surfactants, water-soluble compounds, and oil-soluble compounds. The mixture forms small droplets that contain the metal ions. When droplets are heated, metal ions precipitate out of the solution and form nanoparticles. Sonochemical /electrochemical synthesis: This process uses ultrasound or an electrical current to break down metal salts into nanoparticles. Solvothermal/hydrothermal synthesis: This reaction involves heating a solution of metal ions in water or an organic solvent under high pressure. High pressure and temperature cause metal ions to precipitate out of the solution and form nanoparticles.

14. Environmental Limitations in Nanoparticle Synthesis Traditional methods for synthesizing nanoparticles have several limitations. Using organic reagents can harm the environment, humans, and animals, causing illnesses, such as liver damage. In addition, wastewater generated from nanoparticle synthesis can contain harmful chemicals. The low yield is another disadvantage: Only a small percentage of the starting materials is converted into nanoparticles, generating raw material waste. The high cost of the starting materials, equipment, labor required, long-time synthesis, and the inability to control the size and shape can limit their applications.

15. Strategies to Overcome Barriers to Nanoparticle Synthesis Several strategies can be used to overcome the disadvantages of nanoparticle synthesis, such as the use of environmentally friendly solvents, reagents, and processes. Using water, ionic liquids, and supercritical fluids are examples of eco-friendly solvents or one can even perform solvent-free synthesis, eliminating the need for hazardous chemicals and reducing the environmental impact of nanoparticle synthesis. Many nanoparticle synthesis methods are not scalable, which limits their application. Therefore, it is necessary to develop cost-effective and efficient processes to obtain large quantities of nanoparticles. Multipurpose nanoparticles can be used to improve their performance in a variety of applications and fields. For example, biocompatible nanoparticles are used in biomedicine or as stable nanoparticles for long-term applications. The characterization of nanoparticles is important for understanding their size, shape, surface properties, and chemical composition. This information can be used to understand how nanoparticles interact with their environment and ensure they are safe.

16. Green Synthesis of Nanoparticles

17. This involves minimizing or, if possible, eliminating the pollution produced in the synthesis processes, avoiding the consumption and wastage of nonrenewable raw materials, using hazardous or polluting materials in product manufacturing, and reducing the synthesis time. Paul J. Anastas , considered the father of green chemistry, defined it as “ a work philosophy that involves the use of alternative tools and pathways to prevent pollution ,” referring to both the design of the synthetic strategy and the treatment of possible secondary products originating from that route. Green synthesis aims to promote innovative chemical technologies to reduce or eliminate the use and production of hazardous substances in the design, manufacture, and use of chemical products.

18. The synthesis of nano-sized materials employing “green” processes is relatively cost-effective and does not harm the surrounding environment, as non-toxic chemicals are employed throughout the entire process. Therefore, the usage of stabilizers and reducing agents that possess a biological origin, such as microbial entities, fauna and various other resources, is a sustainable way to produce nano-sized materials. Although being cost-effective and environment-friendly are key factors motivating the green synthesis of nano-sized materials, the “stability” of the produced material has attracted the attention of researchers across the globe. Although the methods involved in green synthesis are relatively diverse, the living entities that are involved in the synthesis usually simply react with different salts (metallic) and reduce them to nano-sized materials, which can be utilized for different purposes only following appropriate characterization. Both microbe- and plant-mediated approaches are employed to synthesize nano-sized materials. Microbe-mediated construction products involve their inherently sophisticated biochemical machinery, which leads to well-defined nanoparticles of different chemical compositions, shapes and sizes.

19. Scaling up may, however, sometimes prove challenging with regard to microbial preparations. This drawback can be easily overcome by using plant-based extracts, and the production rates can be amplified as a consequence. Plant extracts are more efficient than microbes with regard to the production rate. They reduce metal-ions faster than microbial entities and produce nano-sized materials, which are also very stable. Plants contain various compounds (i.e., alkaloids, flavonoids, phenol, tannin, alcohol) with the capability to reduce metallic ions to nanoparticles with decent stability. Two approaches can be used to generate nanoparticles. “Top-down” approach: In which nanoparticles are produced using physical techniques such as grinding or abrasion of a material. “Bottom-up” approach: Where nanoparticles are generated from “building blocks” of atoms or molecules, resulting in more complex assemblies. Three alternatives are identified using this approach.

20. Chemical synthesis: The method of producing molecules or particles by the reaction of substances used as raw materials. Self-assembly: A technique in which atoms or molecules self-order through physical and/or chemical interactions. Positional assembly: The atoms, molecules, and aggregates are deliberately manipulated and positioned individually. However, this method is extremely laborious and unsuitable for industrial applications. The “bottom-up” approach is preferred over the “top-down” approach because specialized equipment is not required and the time to obtain nanoparticles is shorter. Green synthesis is gaining relevance in producing nanoparticles within the “bottom-up” approach. The use of plant species, algae, or microorganisms such as bacteria or fungi is one of the most commonly used resources for this procedure. Various compounds from plants or microorganisms, including terpenes, polyphenols, alkaloids, carbohydrates, proteins, and genetic materials, play an important role in the synthesis of nanoparticles by acting together. In addition to the biological resources used to perform the synthesis, other factors influence the shape and size of nanoparticles, such as the concentration of the metal ion, pH, reaction time, and temperature.

21. In general, the phases for the green synthesis of nanoparticles include- Initial phase: Obtaining the reaction medium, which is the aqueous extract of one or several parts of the plant species or the culture media for the growth of microorganisms, in addition to the precursor salt, which is the source of metal ions. Activation phase: Chemical reduction of metal ions and generation of nucleation centers occur where nanoparticles emerge and grow.

22. Growth phase: Small adjacent nanoparticles spontaneously fuse into larger particles, forming aggregates, which are influenced by factors such as temperature, concentration, and type of compounds, pH, and reaction time. Termination phase: The final shape of the nanoparticles is determined, and the compounds that participate in the reaction help stabilize and enhance their properties.

23. Biological Resources for the Green Synthesis of Nanoparticles

24. Bacteria Nanoparticle synthesis using bacteria is performed both extracellularly and intracellularly. Intracellular: The synthesis is carried out inside the living microorganism, using its growth conditions to favor synthesis, known as “nanoparticle micro-factories.” To recover nanoparticles, bacteria must be destroyed. Extracellular: The components released by the bacteria after lysis are used. The synthesis is performed by adding a metal salt precursor to the medium in which these components are located. Extracellular synthesis has the advantage of being faster because it does not require additional steps to recover nanoparticles from microorganisms. Enzymes, such as reductases, which catalyze the reduction of metal ions into nanoparticles, participate in the synthesis. Even components of the genetic material participate in this process.

25. 2. Fungi Fungi contain active biomolecules, such as proteins or enzymes, that participate in nanoparticle synthesis, improving their yields and stability. Some fungal species can synthesize nanoparticles using extracellular amino acids. For example, glutamic and aspartic acids on the surface of yeast or the reductase enzyme in the cytosol of fungi reduce metal ions to form nanoparticles. This is facilitated by the presence of hydroxyl groups in the mycelium, which donate electrons to the metal ion and reduce it to form nanoparticles. Aliphatic and aromatic amines or some proteins act as coating agents to stabilize them.

26. 3. Algae Algae are used in nanotechnology because of their low toxicity and their ability to bioaccumulate and reduce metals. Nanoparticle synthesis can be intracellular, with the metal ion entering the alga, or extracellular, and involves compounds such as polysaccharides, proteins, and pigments that direct the reduction of metal ions and coat the newly formed nanoparticles. These particles are subsequently released from the cell in the form of colloids.

27. 4. Plant Species The use of plants in nanoparticle synthesis is one of the most widely used methods because of its environmentally friendly nature, as it avoids the use of toxic or harmful substances. It is also one of the fastest and most economical methods because it involves fewer steps. This makes it highly efficient in the nanoparticle production process compared to synthesis using microorganisms. Plants contain several compounds (terpenes, flavonoids, polyphenols, alkaloids, proteins, etc.) that reduce metal ions and stabilize the resulting nanoparticles. This type of synthesis can be performed using intracellular, extracellular, and phytochemical-mediated methods. Intracellular: The synthesis is carried out inside the plant cell, and the nanoparticles are recovered by breaking down the structure, which is very similar to the intracellular method using microorganisms. Control of the growth factors of plant species is required so that they do not interfere with synthesis.

28. Extracellular: This method is the most commonly used because of its ease and speed. The process begins by obtaining a plant extract, which is generally water-based, to which a metal salt precursor is added. Owing to the action of the different compounds present in the extract, nanoparticles are generated and stabilized in a single step. Phytochemically mediated: This is based on the extracellular method, but with the difference that isolated phytochemical compounds are used and other substances are added to stabilize the nanoparticles. There is greater control over the synthesis, but more components and steps are involved.

29. Factors Involved in the Green Synthesis of Nanoparticles Temperature: This is one of the most influential factors, as the shape (spherical, prismatic, flakes, triangular, octahedral, etc.), size, and synthesis depend on temperature. As the temperature increases, the reaction rate and the formation of nucleation centers increase, resulting in higher yields. Different temperatures promote different interactions between the reactants, giving rise to various shapes; the larger the temperature increase, the larger the size of the nanoparticles. pH: This influences the nucleation centers, generating more centers at higher pH values. Another important influence of pH is that some nanoparticles can only be synthesized in acidic or alkaline media. For example, magnetic nanoparticles are synthesized at an alkaline pH, and metal oxide nanoparticles are generally synthesized at an acidic or neutral pH. Time: This parameter plays an important role in defining the size of the nanoparticles. It has been observed that longer reaction times favor an increase in the size of the nanoparticles and higher yields, owing to the prolonged interaction time between reactants

30. Mechanisms Involved in the Green Synthesis of Nanoparticles The plant extract or organism used for the synthesis is an important factor that influences the morphology and size of nanoparticles because different concentrations of metabolites or cellular components give rise to differences in the nanoparticles.

31. Proteins and enzymes facilitate the formation of nanoparticles from metal ions. Because of their high reducing activity, proteins and enzymes can attract metal ions to specific regions of a molecule responsible for reduction, facilitating the formation of nanoparticles; however, their chelating activity is not excessive. The amino acids of a protein can greatly influence the size, morphology, and quantity of nanoparticles generated, thus playing a very important role in determining their shape and yield. Removing a proton from amino acids or other molecules results in the formation of resonant structures capable of further oxidation. This process is accompanied by the active reduction of metal ions followed by the formation of nanoparticles. Flavonoids are a large group of polyphenolic compounds that can actively chelate and reduce metal ions because they contain multiple functional groups capable of forming these structures. Structural transformations of flavonoids also generate protons that reduce metal ions to form nanoparticles; therefore, they are involved in the nucleation stage, their formation, and further aggregation. Saccharides can also play a role in nanoparticle formation. Monosaccharides, such as glucose, can act as reducing agents, as the aldehyde group of the sugar is oxidized to a carboxyl group through the addition of hydroxyl groups, which in turn leads to the reduction of metal ions and the synthesis of nanoparticles.

32. The mechanism of green synthesis of nanoparticles has been associated with the action of polyphenols, which act as ligands. Metal ions form coordination compounds, in which the fundamental structural unit is the central metal ion surrounded by coordinated groups arranged spatially at the corners of a regular tetrahedron. The aromatic hydroxyl groups in polyphenols bind to metal ions and form stable coordinated complexes. This system undergoes direct decomposition at high temperatures, releasing nanoparticles from the complex system. Flavonoids, amino acids, proteins, terpenoids, tannins, and reducing sugars have hydroxyl groups that surround the metal ions to form complexes. After this process, the hydroxyl ions are oxidized to carbonyl groups, which stabilize the nanoparticles. Synthesis is favored if the participating molecules have at least two hydroxyl groups at the ortho- and para-positions. Amino acids influence the size, morphology, and yield of nanoparticles generated, depending on the specific amino acids present in the extract and their concentration, along with the reaction conditions that give rise to nanoparticles with different shapes.

33. The 12 principles of green synthesis are fulfilled with the biological approach to produce nanoparticles.

34. In a Nutshell Nanoparticles have emerged as a versatile and promising class of materials with unique properties that can be harnessed for various applications. The use of green synthesis utilizing natural resources and biologically active compounds to produce nanoparticles is an area of continuous research to improve processes, reduce environmental damage, and meet the increasing demand for the application of these nanostructures. Utilizing biological resources, the synthesis of nanoparticles is inexpensive, faster, and considered a one-step synthesis while preserving or even improving the physical and chemical properties of the nanoparticles. With the great potential of this method and the sustainable and efficient production of nanoparticles, different sizes and shapes can be obtained, which makes it a very attractive option not only for the synthesis of nanostructures, but also for the application of this technique in the synthesis of other compounds.

35. Characterization of Nanoparticles

36. Typically, engineered materials with dimensions in the nanometer scale are intermediates between isolated small molecules and bulk materials. Nanomaterials, which are similar to biological moieties in scale, can be used as diagnostic and therapeutic nanomedicines. Compared to their bulk material counterparts, the distinct physicochemical properties of the nanomaterials, such as size, surface properties, shape, composition, molecular weight, identity, purity, stability and solubility, are critically relevant to physiological interactions. These physiological interactions may provide benefits in medical applications, including improvements in efficacy, reduction of side effects, prevention and treatment. Impact of nanomaterials on their physiological behaviors will influence the therapeutic efficacy and/or diagnostic accuracy of nanomedicines. In this context, it is important to understand how the different physicochemical characteristics of nanomaterials affect their in vivo distribution and behavior. This demands reliable and robust techniques for studying the different physicochemical characteristics of nanomaterials in general and nanomedicines in particular

37. Size of the Nanoparticles In engineered nanomaterials, size is a crucial factor that regulates the circulation and navigation of nanomaterials in the bloodstream, penetration across the physiological drug barriers, site- and cell-specific localization and even induction of cellular responses. The relationship of size and/or shape to nanoparticle toxicity or nanomedicine efficacy has to be investigated on a case-by-case basis, because of the wide differences in the behavior of different nanomaterials.

38. Surface Properties of the Nanoparticles Many characteristics of nanomaterial interfaces are functions of atomic or molecular compositions of the surfaces and the physical surface structures that respond to the interactions of the nanomaterial with surrounding species. From the aspect of nanomedicine, these characteristics are considered the elements of surface properties in the environment of biological fluid. Among the different surface properties, surface composition, surface energy, wettability, surface charge and species absorbance or adhesion are commonly considered important parameters. Surface composition is intrinsically relevant to the superficial layers but not to the bulk materials. Surface energy is relevant to the dissolution, aggregation and accumulation of nanomaterial.

39. Surface charge, with potential effect on receptor binding and physiological barrier penetration, governs the dispersion stability or aggregation of nanomaterials and is generally estimated by zeta potential. Species absorbance or adhesion potentially alters the surface of nanomaterial as well as the conformation and the activity of the attached species.

40. Shape of the Nanoparticles Efficiency of drug delivery carriers was highly influenced by controlling the shapes of the carriers, while phagocytosis of drug delivery carriers through macrophages was also dependent on carrier shape. Furthermore, flow and adhesion of drug delivery carriers throughout the circulatory system and the in vivo circulation time of the nanomedicine can be controlled by modulating the shapes of drug-loaded nanomaterials In addition to size and surface properties, the shape of nanomaterial can play an important role in drug delivery, degradation, transport, targeting and internalization.

41. The shape of nanomaterial affects cellular uptake, biocompatibility and retention in tissues and organs. Additionally, the disposition and translocation of nanomaterials in the organism may be influenced by their shape, accompanying size and state of agglomeration. One example is an in vitro study of silica NPs demonstrating shape-driven agglomeration as a potential trigger in the pulmonary pathogenesis.

42. Composition and Purity of the Nanoparticles A broad variety of nanomaterials are utilized in the production of approved or potential nanomedicines. These nanomaterials can be categorized by their structural types, such as NP and its derivatives, liposome, micelle, dendrimer/ fleximer , virosome, emulsion, quantum dot, fullerene, carbon nanotube and hydrogel, and each type may consist of polymers, metals and metal oxides, lipids, proteins, DNA or other organic compounds. Composition of a nanomaterial affects transport, delivery and biodistribution. In biomedical applications of nanomaterials, there may be a need to combine two or more types of nanomaterials to form a complex such as a chelate, a conjugant or a capsule. Consequently, chemical composition analysis of the nanomaterial complex is more complicated than that for a single entity.

43. Composition and Purity of the Nanoparticles A broad variety of nanomaterials are utilized in the production of approved or potential nanomedicines. These nanomaterials can be categorized by their structural types, such as NP and its derivatives, liposome, micelle, dendrimer/ fleximer , virosome, emulsion, quantum dot, fullerene, carbon nanotube and hydrogel, and each type may consist of polymers, metals and metal oxides, lipids, proteins, DNA or other organic compounds. Composition of a nanomaterial affects transport, delivery and biodistribution. In biomedical applications of nanomaterials, there may be a need to combine two or more types of nanomaterials to form a complex such as a chelate, a conjugant or a capsule. Consequently, chemical composition analysis of the nanomaterial complex is more complicated than that for a single entity.

44. Stability of the Nanoparticles Pharmaceutical stability refers to retaining the same properties for a period of time after the pharmaceutical is manufactured. Similar to conventional single-molecule pharmaceuticals, the stability of nanomedicines may be affected by one or more factors, such as temperature, moisture, solvents, pH, particle/molecular size, exposure to different types of ionizing and non-ionizing radiation, enzymatic degradation and even the presence of other excipients and impurities.

45. Interaction between Nanomaterials and Biological Environment When nanomaterials are introduced into biological environments or integrated in biomaterials, many undesirable effects such as aggregation, coagulation and non-specific absorption can occur. These may be due to a variety of intermolecular interactions occurring at the interfaces of nanomaterials with biomolecules and interaction-mediating fluids. While the surface properties of nanomaterials in a given medium are characterized by their physicochemical properties, including chemical composition, shape, surface geometry and crystallinity, porosity, heterogeneity and hydrolytic stability, other properties, such as surface charge, dissolution, hydration, size distribution, dispersion stability, agglomeration and aggregation of nanomaterial, are mainly governed by ionic strength, pH, temperature and the presence of biological or organic macromolecules. Thus, appropriate physicochemical characterization of nanomaterials should be profiled based on different physical states of the nanomaterials, such as solution, suspension or dry powder, as well as before and after exposure to the in vitro or in vivo test environment. Techniques for determining the shelf life of nanomaterial formulations are essential before considering the manufacture and use of nanomedicines.

46. Scanning Electron Microscopy (SEM)

47. Biological Sample Preparation for SEM

48. In contrast to optical microscopy, which uses light sources and glass lenses to illuminate specimens to produce magnified images, electron microscopy (EM) uses beams of accelerated electrons and electrostatic or electromagnetic lenses to generate images of much higher resolution, based on the much shorter wavelengths of electrons than visible light photons. Scanning Electron Microscope (SEM) is a surface imaging method in which the incident electron beam scans across the sample surface and interacts with the sample to generate signals reflecting the atomic composition and topographic detail of the specimen surface. The incident electrons cause emissions of elastic scattering of electrons, referring to backscattered electrons, inelastic scattering of electrons named low-energy secondary electrons, and characteristic X-ray light called cathodoluminescence from the atoms on the sample surface or near-surface material . Among these emissions, detection of the secondary electrons is the most common mode in SEM and can achieve resolution smaller than 1 nm. The size, size distribution and shape of nanomaterials can be directly acquired from SEM; however, the process of drying and contrasting samples may cause shrinkage of the specimen and alter the characteristics of the nanomaterials.

49. In addition, while scanned by an electron beam, many biomolecule samples that are nonconductive specimens tend to acquire charge and insufficiently deflect the electron beam, leading to imaging faults or artifacts. Coating an ultrathin layer of electrically conducting material onto the biomolecules is often required for this sample preparation procedure. Because a cryogenic freezing method is often required in EM to image surface groups attached to NPs, the size of nanomaterial cannot be investigated in physiological conditions. An exception is environmental SEM (ESEM), through which samples can be imaged in their natural state without modification or preparation. Because the sample chamber of ESEM is operated in a low-pressure gaseous environment of 10–50 Torr and high humidity, the charging artifacts can be eliminated, and coating samples with a conductive material is no longer necessary. Still, most of the EM techniques, including SEM, possess the disadvantage of a destructive sample preparation, prohibiting its analysis by other modalities. In addition, biased statistics of size-distribution of heterogeneous samples is unavoidable in SEM due to the small number of sample particles in the scanning region.

50. Principle of SEM The scanning electron Microscope uses emitted electrons. The Scanning electron microscope works on the principle of applying kinetic energy to produce signals on the interaction of the electrons. These electrons are secondary electrons, backscattered electrons, and diffracted backscattered electrons which are used to view crystallized elements and photons. Secondary and backscattered electrons are used to produce an image. The secondary electrons are emitted from the specimen play the primary role of detecting the morphology and topography of the specimen while the backscattered electrons show contrast in the composition of the elements of the specimen.

51. The source of the electrons and the electromagnetic lenses are from tungsten filament lamps that are placed at the top of the column, and it is similar to those of the transmission electron Microscope. The electrons are emitted after thermal energy is applied to the electron source and allowed to move in a fast motion to the anode, which has a positive charge. The beam of electrons activates the emission of primary scattered (Primary) electrons at high energy levels and secondary electrons at low-energy levels from the specimen surface. The beam of electrons interacts with the specimen to produce signals that give information about the surface topography and composition of the specimen. The specimen does not need special treatment for visualization under the SEM, even air-dried samples can be examined directly. However, microbial specimens need fixation, dehydration, and drying in order to maintain the structural features of the cells and to prevent collapsing of the cells when exposed to the high vacuum of the microscope. The samples are mounted and coated with thin layer of heavy metal elements to allow spatial scattering of electric charges on the surface of the specimen allowing better image production, with high clarity.

52. Scanning by this microscope is attained by tapering a beam of electrons back and forth over a thin section of the microscope. When the electrons reach the specimen, the surface releases a tiny staw of electrons known as secondary electrons which are then trapped by a special detector apparatus. When the secondary electrons reach and enter the detector, they strike a scintillator (a luminescence material that fluoresces when struck by a charged particle or high-energy photon). This emits flashes of light which get converted into an electric current by a photomultiplier, sending a signal to the cathode ray tube. This produces an image that looks like a television picture that can be viewed and photographed. The quantity of secondary electrons that enter the detector is highly defined by the nature of the specimen i.e., raised surfaces to receive high quantities of electrons, entering the detector while depressed surfaces have fewer electrons reaching the surface and hence fewer electrons enter the detector. Therefore, raised surfaces will appear brighter on the screen while depressed surfaces appear darker.

53. Transmission Electron Microscopy (TEM)

54. TEM provides direct images and chemical information of nanomaterials at a spatial resolution down to the level of atomic dimensions. In the conventional TEM mode, an incident electron beam is transmitted through a very thin foil specimen, during which the incident electrons interacting with specimen are transformed to un-scattered electrons, elastically scattered electrons or inelastically scattered electrons. The magnification of TEM is mainly determined by the ratio of the distance between objective lens and the specimen and the distance between objective lens and its image plane. The scattered or un-scattered electrons are focused by a series of electromagnetic lenses and then projected on a screen to generate an electron diffraction, amplitude-contrast image, a phase-contrast image or a shadow image of varying darkness according to the density of un-scattered electrons .

55. TEM has advantages over SEM in providing better spatial resolution and capability for additional analytical measurements. There are certain drawbacks accompanying the advantages of TEM. A significant tradeoff is that a high vacuum and thin sample section are required for electron-beam penetration in TEM measurement. Sample destruction and measurement in unnatural/non-physiological conditions are common to all EM techniques. In general, high resolution EM imaging enables examination of a minute part of the specimen over a certain period of time and results in poor statistical sampling. Interestingly, wet TEM can be used for determining the particle size, dispersion, aggregation/agglomeration and dynamic displacement of nanomaterials in an aqueous environment.

56. Principle of TEM A heated tungsten filament in the electron gun produces electrons that get focus on the specimen by the condenser lenses. Magnetic lenses are used to focus the beam of electrons of the specimen. By the assistance offered by the column tube of the condenser lens into the vacuum creating a clear image, the vacuum allows electrons to produce a clear image without collision with any air molecules which may deflect them. On reaching the specimen, the specimen scatters the electrons focusing them on the magnetic lenses forming a large clear image, and if it passes through a fluorescent screen, it forms a polychromatic image. The denser the specimen, the more the electrons are scattered forming a darker image because fewer electron reaches the screen for visualization while thinner, more transparent specimens appear brighter.

57. Preparation of Specimen for Visualization by TEM Electrons are easily absorbed and easily scattered on solid elements, showing poor visualization for thick specimens. And therefore, very thin specimens are used for accurate and clear visualization forming a clear image as well. The specimen should be about 20-100nm thin and 0.025-0.1nm diameter, as small as that of a bacterial cell. Thin specimens allow interaction with electrons in a vacuumed space, are able to maintain their innate structure. To get thin slice specimens, the specimen is first fixed on a plastic material with glutaraldehyde or osmium tetraoxide . These chemical agents stabilize the structure of the cell and maintain its originality. The addition of an organic solvent like alcohol such as ethanol will dehydrate the cell completely for embedding the specimen to the plastics. The specimen is then permeated by adding an unpolymerized liquid epoxy plastic making it hardened like a solid block. This is where thin sections are cut from using a glass knife with a piece of special equipment known as an ultramicrotome. The specimen is then stained appropriately (with the appropriate stain) for the uniform scattering of electrons.

58. The thin sections are then soaked in heavy metallic elements such as lead citrate and uranyl acetate allowing the lean and aluminum ions to bind to the cell structures. This forms an opaque layer against the electrons on the cell structures to increase contrast. The stained thin sections are then mounted on copper grids for viewing. Freeze-itching Treatment: To reduce the possible dangers of artifacts, freeze-itching is used especially for the treatment of microbial cells, unlike chemical fixation, dehydration, and embedding, where most specimens get contaminated. Microbial cell organelles undergo special treatment known as Freeze-itching whereby the specimens are prepared with liquid nitrogen and then warmed at -100°C in a vacuum chamber. The sections are then cut with a precooled knife in liquid nitrogen at -196°C. After warming up the sectioned specimen in a high vacuum for about 2 minutes, it can then be coated with platinum and carbon layer forming replicas. These are then be viewed under the TEM displaying more detailed internal structures of the cell in 3D. This step of treatment with Liquid nitrogen is known as freeze-itching.

59. Scanning Tunneling Microscopy (STM)

60. STM uses quantum tunneling current to generate electron density images for conductive or semiconductive surfaces and biomolecules attached on conductive substrates at the atomic scale. Adapting the generic principle for all SPM techniques, i.e. bringing a susceptible probe in close proximity to the surface of an object measured to monitor the reactions of the probe, the essential components of an STM include a sharp scanning tip, an xyz -piezo scanner controlling the lateral and vertical movement of the tip, a coarse control unit positioning the tip close to the sample within the tunneling range, a vibration isolation stage and feedback regulation electronics.

61. As the tip–sample separation is maintained in the range of 4–7 Å, a small voltage applied between the scanning tip and the surface causes tunneling of electrons by which variation of the responding current can be recorded while the tip moves across the sample in the x–y plane to generate a map of charge density. Alternatively, keeping the responding current unchanged by adjusting the tip height through the use of feedback electronics can generate an image of tip topography across the sample.

62. As for characterization of biomolecules using STM or EM techniques, the samples are usually embedded into a matrix to preserve their original conformations, followed by coating the samples with a thin metallic layer, such as gold, before acquiring images. It is impossible to image these biomolecules in their native conditions using conventional EM techniques that usually accompany a time-consuming sample preparation procedure. STM, on the other hand, can not only diminish the disadvantages of the EM techniques but also provide an image with atomic scale resolution by, for example, using a Pt– Ir tip with a very sharp end. Although the high spatial resolution of STM should benefit the characterization of nanoscale biomaterials such as size, shape, structure, and states of dispersion and aggregation, only few studies using gold or carbon as substrates have been reported. The practical obstacles are mainly due to requirements of the conductive surface of the sample and detection of the surface electronic structure. Unfortunately, most biomaterials are insulating, and a simple connection of the sample's surface electronic structure with its surface topography may not necessarily exist. Still, STM is a preferred tool for investigating conductive atomic structures of, for example, carbon nanotubes, fullerenes and graphene.

63. Atomic Force Microscopy (AFM)

64. Unlike STM, AFM does not require oxide-free, electrically conductive surfaces for measurement and is a SPM imaging tool consisting of a micro-machined cantilever (typically made of silicon or silicon nitride) with a sharp tip at one end to detect the deflection of the cantilever tip caused by electrostatic and van der Waals repulsion, as well as attraction between atoms at the tip and on the measured surface. The oscillating cantilever then scans over the surface of specimen to generate an image with a vertical resolution of around 0.5 nm. Like SEM and TEM techniques, AFM can be used for investigating the size, shape, structure, sorption, dispersion and aggregation of nanomaterials — the different scanning modes employed in AFM studies include noncontact mode (also called static mode), contact mode and intermittent sample contact mode (also called dynamic mode and tapping mode). In addition to probing the sizes and shapes of nanomaterials under physiological conditions, AFM is capable of characterizing dynamics between nanomaterials in biological situations, such as observing the interaction of nanomaterials with supported lipid bilayers in real time, which is not achievable with current EM techniques.

65. AFM is gaining importance due to its capability for imaging biomaterials without causing appreciable damage to many types of native surfaces. The main strength of AFM is its capability to image a variety of biomaterials at the sub-nanometer scale in aqueous fluids. However, a major drawback is that the size of the cantilever tip is generally larger than the dimensions of the nanomaterials examined, leading to unfavorable overestimation of the lateral dimensions of the samples. Unlike fluorescence techniques, AFM lacks the capability of detecting or locating specific molecules; however, this disadvantage has been eliminated by recent progress in single-molecule force spectroscopy with an AFM cantilever tip carrying a ligand, a cell adhesion molecule or chemical groups, which can probe or detect single functional molecules on cell surfaces.

66. AFM microscopes operate on the principle of surface sensing using an extremely sharp tip on a micromachined silicon probe. This tip is used to image a sample by raster scanning across the surface line by line, although the method varies dramatically between distinct operating modes. The two primary groups of operating modes are widely defined as contact mode and dynamic, or tapping, mode . The underlying principle of AFM is that this nanoscale tip is attached to a small cantilever which forms a spring. As the tip contacts the surface, the cantilever bends, and the bending is detected using a laser diode and a split photodetector. Principle of AFM

67. This bending is indicative of the tip-sample interaction force. In contact mode, the tip is pressed into the surface and an electronic feedback loop monitors the tip-sample interaction force to keep the deflection constant throughout raster scanning. Tapping mode limits the contact between the sample surface and the tip to protect both from damage. In this mode, the cantilever is caused to vibrate near its resonance frequency. The tip subsequently moves up and down in what is described as a sinusoidal motion. This motion is reduced by attractive or repulsive interactions as it comes near the sample. A feedback loop is used in a similar fashion to contact mode, except it keeps the amplitude of this tapping motion constant rather than the quasistatic deflection. By doing so, the topography of the sample is traced line by line. Principle of AFM

68. Fourier Transform Infra-red (FTIR) Spectroscopy

69. The electromagnetic spectrum consists of different regions corresponding to different energy (E), frequency (ѵ), and wavelength (λ) ranges. The unit for near-, mid-, and far-infrared, the wavenumber (cm -1 ), is derived from the inverse relationship between wavelength and frequency. FTIR spectroscopy takes advantage of how IR light changes the dipole moments in molecules that correspond to a specific vibrational energy. Vibrational energy corresponds to two variables: reduced mass (μ) and bond spring constant (k). For k constant, we can look at C-C, C=C, and C≡C showing an increase of 800 cm -1 across the series. Substituting atoms in a C-C bond with nitrogen and oxygen causes a shift of 100 cm -1 . By looking at the two series, it can be seen that bond strength alters the wavenumbers more than mass.

70. For nanomaterial applications, Fourier transform infrared (FTIR) spectroscopy is commonly employed to use the expression of characteristic spectral bands to reveal nanomaterial–biomolecule conjugation, e.g. proteins bound to NP surfaces, and to illustrate the conformational states of the bound proteins.

71. A recently developed technique called attenuated total reflection (ATR)–FTIR spectroscopy uses the property of total internal reflection in conjunction with IR spectroscopy to probe the structure of adsorbed/deposited species at a solid/air or solid/liquid interface, while avoiding the drawbacks of sample preparation complexity and spectral irreproducibility in conventional IR. In an ATR–FTIR system, the total internal reflectance, occurring within the equipped internal reflection element (IRE) crystal, which has a high refractive index at certain angles, forms evanescent waves that extend from the IRE crystal–sample interface into the sample with penetration depth of micrometers (0.5–5 μm ), and the intensity of the evanescent waves decays exponentially from the interface. ATR–FTIR can provide IR absorption spectra to investigate, for example, changes in surface properties as well as identification of chemical properties on the polymer surface when sample on the IRE–sample interface absorbs the evanescent IR waves with frequencies matching the vibrational modes of the sample. Although ATR–FTIR spectroscopy can be implemented to study the surface features of nanomaterials, it is not a very sensitive surface-analysis method at nanometer scale because the penetration depth of ATR–FTIR has the same order of magnitude as the incident IR wavelength.

72. UV-Vis Spectrophotometer

73. The UV-visible absorbance spectrum is highly dependent on nanoparticle geometry. The shapes of the two spectra are quite different despite the two types of nanoparticles having similar dimensions and being composed of the same material. UV-visible absorbance spectra of 50 nm diameter gold nanospheres (A) and 25 nm diameter, 60 nm length gold nanorods (B).

74. Using UV-Visible Spectroscopy to Determine Nanoparticle Aggregation States The UV-visible absorbance spectrum is also dependent on the aggregation state of the nanoparticles. When nanoparticles are in close proximity to each other, their plasmons couple, which affects their LSPR and thus their absorption of light. Dimerization of nanospheres causes a “red shift,” a shift to longer wavelengths, in the UV-visible absorbance spectrum as well as a slight increase in absorption at higher wavelengths. Unlike dimerization, aggregation of nanoparticles causes a decrease in the intensity of the peak absorbance without shifting the wavelength at which the peak occurs ( λmax ). UV-visible absorbance spectrum of 50 nm gold nanosphere dimers with a reference spectrum of single gold nanospheres (A) and UV-visible absorbance spectrum of 50 nm gold nanospheres exposed to various concentrations of NaCl (B).

75. Thank You