Fabrication of Nanostructures with Bottom-up Approach and Their Utility in Diagnostics, Therapeutics, and Others
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Nanofabrication has been a critical area of research in the last two decades and has found wide-ranging application in improvising material properties, sensitive clinical diagnostics, and detection, improving the efficiency of electron transport processes within materials, generating high energy densities leading to pulse power, novel therapeutic mechanisms, environmental remediation and control. The continued improvements in the various fabrication technologies have led to realization of highly sensitive nanostructure-based devices. The fabrication of nanostructures is in principle carried out primarily using top-down or bottom-up approaches. This chapter summarizes the important bottom-up nanofabrication processes for realizing nanostructures and also highlights the recent research conducted in the domain of therapeutics and diagnostics.
KeywordsNanofabrication Bottom-up approach Diagnostics and therapeutics
Nanotechnology can be defined as the design, characterization, and fabrication of engineered nanostructures or nanodevices with at least one dimension less than 100 nm (Biswas et al. 2012; Abu-Salah et al. 2010; Wang et al. 2016). A reduction in the overall size of a structure to the nanometer scale results in a substantial change in its properties, e.g., chemical, physical, thermal, mechanical, which may differ entirely from their macroscale equivalents. These nanoparticles possess a high surface-to-volume ratio providing higher binding site density for the adsorption of various biomolecules (Arruebo et al. 2009). Nanoparticles conjugated with antibodies or other biological moieties (e.g., low molecular weight ligands, peptides, proteins, DNA, plasmids) provide highly specific and selective recognition characteristics. One of the distinguished features of nanoparticles is the variation of their physical or chemical properties dependent on their size and shape. For example, by varying the size of metal nanoparticles their radiation and excitation wavelength can be tuned. This unique characteristic can be attributed to an optical phenomenon known as localized surface plasmon resonance (LSPR). LSPR occurs due to the interaction of the incident light with the surface electrons present in the conduction band (Petryayeva and Krull 2011). The phenomenon is generated by entrapped light waves in the conductive metal nanoparticles. Hence, nanoparticles offer specific physical and chemical properties that enable their utilization in a variety of domains like biomedical, energy and environment, manufacturing.
In general, there are three broad classifications of nanomaterials that are, natural,incidental and engineered (Hu et al. 2010). Natural nanomaterials are formed through natural processes and are governed by natural laws. Incidental nanomaterials are the by-products of industries (e.g., coal dust, particulates). Engineered nanomaterials are complex in shape and require specific processes for their fabrication. Based on the number of dimensions of the features, these nanomaterials can be classified into four types: 0-D, 1-D, 2-D, and 3-D (Chopra et al. 2007; Ciesielski et al. 2010; Pashchanka et al. 2010; Song et al. 2010). Zero-dimensional nanostructured materials have nanoscale dimensions in all directions, e.g., nanoparticles, nanospheres, quantum dots. One-dimensional nanostructures have non-nanoscale dimensions in a single direction such as nanorods, nanotubes, nanowires, nanobelts, nanoribbons, nanostars (Kumar et al. 2017). Two-dimensional nanostructures possess two dimensions having non-nanometric size range, e.g., graphene nanosheets, nanoplates, nanobelts, nanodiscs. Three-dimensional nanostructures contain non-nanoscale features in any three dimensions, e.g., nanotetrapods, nanoflowers, nanocombs.
This chapter presents a brief review of the bottom-up fabrication techniques used for fabrication of different shaped nanostructures and nanocomposites. It also covers the recent advancements in fabrication of ZnO-based nanostructures, DNA-based nanostructures, polymer-based nanostructures, and metal-based nanostructures and their widespread applications in the field of diagnostics, therapeutics, and others.
2 Fabrication Techniques
Nanostructures, nanomaterials, and nanocomposites can be fabricated using two different techniques, top-down and bottom-up (Bellah et al. 2012). The top-down approach involves lateral patterning of bulk materials by either subtractive or additive methods to realize nano-sized structures. Several methods are used to fabricate nanostructures using the top-down approach such as photolithography, scanning lithography, laser machining, soft lithography, nanocontact printing, nanosphere lithography, colloidal lithography, scanning probe lithography, ion implantation, diffusion, deposition. (Chi 2010; Kumar et al. 2013a). Although the top-down approach has been playing a vital role in the fabrication of nanostructures, it has several limitations such as development of imperfections in processed materials, high cost (lithographic processes), requirement of high surface finished materials, longer etching times. (Mijatovic et al. 2005; Biswas et al. 2012). In the bottom-up approach, nanostructures are fabricated by building upon single atoms or molecules. In this method, controlled segregation of atoms or molecules occurs as they are assembled into desired nanostructures (2–10 nm size range). In general, there are two basic methods utilizing the bottom-up approach, i.e., gas-phase synthesis and liquid-phase formation. Some of the methods used in bottom-up approach include plasma arcing, chemical vapor deposition process, metal organic decomposition, laser pyrolysis, molecular beam epitaxy, solgel method, wet synthesis, and self-assembly processes.
2.1 Plasma Arcing
Plasma is one of the fundamental states of matter comprising of electrons and molecules in ionic states. It maintains a condition of overall neutrality, although there may be a net positive or negative charge on certain particles. Plasma arcing method requires an ionized state of gas atoms, for which high energy is necessary to peel off the electron from its valence shell to obtain a positively charged atom. An electrical arrangement consisting of an anode and cathode is developed providing sufficient amount of electric field to transform the atoms into ions. Electrodes used are usually made up of conducting materials or mixtures of conducting and non-conducting materials. Generation of contracted plasma uses inert gas as a heat source. Emission of electrons takes place from one electrode due to the presence of high potential difference causing an electrical breakdown. A sudden avalanche of electrons results in the formation of an arc in the zone between the electrodes. Positively charged ions travel at a high velocity and are driven by the applied bias voltage toward the cathode and get deposited as nanoparticles. It is ensured that the depth of deposition consists of a few layers of atoms with each particle of the order of more than 1 nm and all particles so formed are mutually separated. The average temperature of the arc in cold plasmas is generally higher than 104 K.
2.2 Chemical Vapor Deposition (CVD)
Chemical vapor deposition process is mostly used in the semiconductor industry for depositing thin films of various materials. The process involves exposure of the substrate to one or more volatile precursors. These precursors decompose the substrate and react with it to produce the desired deposit. In the process, vaporized precursors are first adsorbed onto a substrate at a high temperature, which then react with one another or decompose and produce crystals. There are three main steps involved in the process: (i) Reactants are transported onto the growth surface by a boundary layer, (ii) chemical reactions take place on the growth surface, and (iii) by-products formed by the gas-phase reaction are removed from the growth surface. Homogeneous nucleation takes place in gas phase, whereas heterogeneous nucleation takes place in the substrate.
2.3 Molecular Beam Epitaxy (MBE)
Molecular beam epitaxy is a physical evaporation process with no chemical reactions involved. The basic difference between MBE and other epitaxy systems is that the former does not involve any chemical reactions and is instead a simple physical evaporation process. This method works on the principle of vacuum evaporation where thermal molecular and atomic beams are directly impinged on a heated substrate under ultra-high vacuum conditions (Cho and Arthur 1975). The first major advantage of the MBE process is it being a comparatively low-temperature process as compared to vapor phase epitaxy. The low-temperature characteristic of this process enables it to reduce autodoping. The second advantage of MBE is that one can have precise control over the doping process. One can achieve a growth rate as low as 0.01 µm per minute up to a maximum of 0.3 µm per minute, allowing for ultra-precise control of layer growth. With the advent of VLSI technology, it is critical to reduce all dimensions to atomic levels and thus the thickness of the epitaxial layer may also reduce further in future.
2.4 Solgel Synthesis
In the solgel process, dispersed solid nanoparticles (sols with diameter of 1–100 nm) are mixed in a homogeneous liquid medium and agglomerated to form a continuous three-dimensional network (gel) with pore diameter in the sub-micrometer domain in the liquid phase (Hench and West 1990). A sol is a liquid in which solid colloidal particles are dispersed, e.g., black inkjet ink (carbon black is dispersed in water), while a gel is a wet solid-like rigid network of interconnected nanostructures in a continuous liquid phase. Generally, there are three approaches that have been employed to fabricate solgel film: (i) gelation of a solution of solid colloidal particles, (ii) hydrolysis and polycondensation of alkoxides followed by hypercritical drying of gels, and (iii) hydrolysis and polycondensation of alkoxide followed by aging and drying under ambient conditions. Several steps are involved in the process like mixing (formation of suspended colloidal solution by mixing of nanoparticles in water), casting of sol, gelation (formation of three-dimensional network), aging (for increasing the life of cast objects immersed in liquid), drying (removal of liquid from the interconnected continuous pore network), dehydration or chemical stabilization (to improve stability), and densification (heating the solgel at higher temperatures to eliminate pores and enhance the density, e.g., densification of alkoxide gels carried out at a temperature of 1000 ℃) (Hench and West 1990). The properties of solgels depend on important parameters such as pH, type of solvent, temperature, time, catalysts and agitation mechanisms.
2.5 Molecular Self-Assembly
In general, four strategies are used for chemical synthesis of nanoparticles, i.e., sequential chemical synthesis, covalent polymerization, self-organizing synthesis, and molecular self-assembly. Molecular self-assembly (MSA) process is an ensemble of the properties of each of the above methods. MSA is a process in which atoms or molecules assemble together in equilibrium conditions to form a stable and well-defined nanophase by non-covalent bonds (Whitesides et al. 1991). All natural materials (organic or inorganic) are processed through a self-assembly route; e.g., in a natural biological process, a DNA double helix is formed through self-assembly. This approach can be used as a basic structuring mechanism to fabricate complex nanostructures (Mijatovic et al. 2005). The molecular self-assembly process is highly capable of fabricating nanostructures in the range of 1–100 nm. In order to create complex nanostructures using self-assembly process, critical parameters such as, the well-defined geometry and the specific interactions between the basic units requisite significant consideration (Rothemund 2005).
2.6 DNA Nanotechnology
Deoxyribonucleic acid (DNA) nanotechnology is the method to fabricate artificial nucleic acid nanostructures which can be utilized as nanofilters, biological scaffolds, fast performing nanowire devices, etc. Owing to its excellent physical and chemical properties, DNA has become the most widely used material for construction of nanostructures. Using nucleotide sequence-directed hybridization, DNA is able to produce duplexes and other secondary structures (Feldkamp and Niemeyer 2006). This property allows DNA molecules to self-assemble and formulate nanoscale structures which can be employed in scaffolds, nanostructures, and nanodevices. DNA nanotechnology also utilizes the self-recognition properties of a DNA molecule to fabricate nanostructures in a desirable manner. A novel approach known as ‘the DNA origami method’ has been developed to fabricate two-dimensional DNA nanostructures of arbitrary shapes (Rothemund 2005).
3 Design and Synthesis of Nanostructures
3.1 ZnO-Based Nanostructures
In the recent years, various metal and metal oxide nanoparticles (MONPs) have been synthesized. Among these nanomaterials, the synthesis of metal oxides, especially zinc oxide, tin oxide, titanium dioxide nanostructures has been very prominent. The zinc oxide system in particular has shown many diverse applications owing to its relatively high and customizable band gap. Zinc oxide has been exploited for various applications like sensors (gas, bio, chemical, visible light, and ultraviolet), cosmetics, optical devices, optoelectronic devices, electrical devices, photochemical applications, solar cells, light-emitting displays, optical storages, drug delivery systems. (Gupta et al. 2013, 2014b, 2015a, b; Kumar et al. 2013b; Yao et al. 2002; Vaseem et al. 2010; Tian et al. 2003). ZnO is a semiconductor with a wide band energy gap of 3.37 eV at room temperature and a binding energy of 60 meV (Djurišić et al. 2012; Kumar et al. 2013b). The crystalline structure of ZnO is wurtzite containing hexagonal unit cells. ZnO nanostructures provide large surface area, high aspect ratio, high catalytic activity, and higher number of adsorption sites on their surfaces (Chen and Tang 2007). Also, a numerous variety of electronic and optical properties can be obtained using different ZnO nanostructures because of their rich defect chemistry (Djurišić et al. 2012).
3.2 Polymer-Based Nanostructures
Polymers are the most extensively used biomaterials in the medical field for applications in implantation, medical devices, medical coatings, tissue engineering, and prostheses, owing to their biocompatibility with human tissues and cells (Jagur-Grodzinski 2003). Polymers are generally categorized as natural or synthetic (Broz 2010). Natural polymers extracted from the Mother Nature are biodegradable and offer excellent biocompatibility. Silk, wool, proteins (Dutta et al. 2004) (e.g., collagen, gelatin), cellulose, and DNA are some examples of naturally occurring polymers. Due to their complex structures, modification of natural polymers is challenging. While synthetic polymers are fabricated using petroleum oils as the main constituent, there are mainly four types of synthetic polymers which include thermoplastics, thermosets, elastomers, and synthetic fibers (Peacock 2000). Examples of synthetic polymers include polydimethylsiloxane (PDMS), nylon, polypropylene, polyvinyl chloride, polystyrene, Teflon.
Ferroelectric polymer nanostructures have also been synthesized using flexible polyethylene terephthalate substrates (Song et al. 2015). A low-pressure reverse nanoimprint lithography technique has been developed that uses soft polycarbonate molds derived from recordable DVDs to fabricate nanostructures. These nanostructures are highly stable and exhibit switchable piezoelectric response and good crystallinity.
3.3 Metal-Based Nanostructures
Recently, many research fields have focused on the development of metallic nanostructures with complex shapes and various compositions in order to exploit their distinctive qualities (Gentile et al. 2016; Xia et al. 2009). Due to the high surface-to-volume ratios, metal-based nanostructured materials have been used in various domains such as catalysis, sensing, fuel cells, mechanical actuators, electrodes, point-of-care diagnostics, medicine. (Gentile et al. 2016; Jiang et al. 2012).
3.4 DNA-Based Nanostructures
Deoxyribonucleic acid (DNA) is a genetic molecule in which hereditary information is encoded. It has an antiparallel double-stranded helical structure which enables its use in fabrication of nanostructures and nanodevices through a self-assembly process (Seeman 2010; Sun and Kiang 2005; Yan et al. 2003). The diameter of each strand of DNA is about 2 nm, and the helical pitch is about 3.5 nm. DNA is composed of a nitrogen-containing nucleobase (adenine, cytosine, guanine, and thymine), a sugar molecule, and a phosphate group. It has several specific characteristics that make it a preferable choice for fabrication of engineered biological nanostructures. First, DNA molecules segregate by self-assembly process so that complex structures can be fabricated with a nanometer resolution (Yan et al. 2003). Second, since the genetic information is encoded by chemical coding process, the intermolecular interaction of molecules can be easily programmed (Sun and Kiang 2005). Third, although double-stranded DNA (dsDNA) is a flexible polymer, it acts as a rigid polymer below the 50 nm size (Feldkamp and Niemeyer 2006). Therefore, the nanostructures (<50 nm) made from dsDNA can be used as rigid nanomaterials. Fourth, single-stranded DNA (ssDNA) is very flexible in comparison with dsDNA. It can be twisted to about 180° and is even capable of forming tight loops in the nanometer size. By combining the properties of dsDNA and ssDNA, complex artificial DNA nanocomposites can be fabricated. The rigidity and flexibility of these tailored nanomaterials can be controlled easily. Fifth, DNA has superior physicochemical stability as compared to proteins. The nanostructures fabricated by DNA exhibit features such as robustness and can be easily processed and synthesized.
Nanostructured materials have been used in a wide range of nanotechnology fields such as nanoelectronics, optoelectronics, bioelectronics, nanochemistry, sensing, nanofluidics, point-of-care diagnostics, nanomachines, therapeutics, advanced energy storages. On account of the increasing requirements of real-time sectors such as medical and health care, the diagnostic and therapeutic applications are being developed rapidly.
4.1 Diagnostic Applications
A novel biosensor based on ZnO–Au nanocomposite has been developed for rapid and sensitive detection of microorganisms in food and water samples. Nanoporous silica film has been prepared by the traditional porogen method using poly(methylsilsesquioxane) (PMSSQ) (empirical formula: (CH3SiO1.5)n) as the matrix and poly(propylene glycol) (PPG) (empirical formula: (CH(CH3)CH2O)n and molecular weight: 20,000 g/mol) as the porogen. Propylene glycol methyl ether acetate was used as a solvent for the preparation of nanoporous silica. ZnO nanostructures were grown using wet chemical synthesis, and the ZnO–Au nanocomposite was prepared by mixing the ZnO and Au nanoparticles.
For detection of biomarkers in human blood serum, a hybrid ZnO nanorod poly(oligo(ethylene glycol) methacrylate-co-glycidyl methacrylate (POEGMA-co-GMA) polymer brush has been reported (Hu et al. 2015). The sensitivity and specificity of an antibody microarray has been improved significantly by development of polymeric nanostructures. ZnO nanorods grown on glass slide behave as the backbone substrate over which polymer brush grows. Also, ZnO nanorods amplify the fluorescence intensity facilitating the detection of biomarkers. The POEGMA-co-GMA nanobrush is utilized to retain antibodies with higher densities. The limit of detection (LOD) of biomarkers in human blood serum was reported to be as low as 100 fg mL−1.
In order to perform colorimetric detection of uric acid in human serum, graphene oxide-based gold nanoparticle-embedded networks have been utilized. A paper-based sensing platform has been developed that provides rapid results within 5 min and exhibits a high sensitivity of 4 ppm (Kumar et al. 2016).
A nonpathogenic insect, baculovirus, has been detected using a nanoelectromechanical cantilever beam (Ilic et al. 2004). Arrays of nanomechanical free standing microcantilevers, coated with polycrystalline silicon and antibodies, were used to sense the binding of varying concentrations of baculovirus. Mechanical resonance on microcantilever-based DNA detection using gold nanoparticles has been reported (Su et al. 2003). A change in the mass of the microcantilever is induced by the DNA hybridization leading to the shift in resonance frequency of the microcantilever beam. This change is further measured. The hybridization is seen to occur through the binding of the gold nanoparticles on the microcantilever surface resulting in a chemical amplification as a result of nucleation of silver. The method reports the limit of detection of target DNA at a concentration of 0.05 nM.
Clinical diagnostic devices utilizing nanostructures have shown promise owing to their increased sensitivity, speed, portability, and inexpensive nature as compared to conventional diagnostic techniques.
4.2 Therapeutic Applications
Self-assembled amphiphilic polymer nanostructures have become efficient nanocarriers for targeted delivery of anticancer therapeutics (Wiradharma et al. 2009). Polysaccharide chitosan-based polymeric nanostructures have been employed in delivery of hydrophilic and lipophilic drugs onto the eye surfaces (de la Fuente et al. 2010). Therapeutic efficiency against pancreatic tumor has been enhanced by using a bundled assembly of helical polymeric nanostructures laden with platinum drugs (Mochida et al. 2014). Due to their structural diversity, biocompatibility, and uniformity in structures, DNA-based nanostructures have been extensively used for therapeutic applications. The growth of cancer cells has been inhibited by combining AS1411 aptamers into DNA pyramids without using any transfection reagents (Charoenphol and Bermudez 2014).
Electroporation is a commonly used method for gene delivery, which requires high voltage (~100–500 k V/m) for DNA transfection. However, a major portion of the exposed cells are completely destroyed as a result of this high voltage. In order to overcome this problem, molecular delivery using shock wave-assisted methods has been widely employed (Lauer et al. 1997; Kodama et al. 2002). These shock waves can be generated using nanoenergetic materials, which can further be utilized for gene transfection purposes (Gangopadhyay et al. 2011; Patel et al. 2015).
The small size of the nanostructures provides the requisite potential in this domain, which is enablement of easy access to the internal parts of the body without affecting other body functions. Although they can be utilized for therapeutics, the materials that they are made of possess a potential danger to the human body. Hence, further research is required to critically analyze the biocompatibility and biodegradability of these materials.
4.3 Miscellaneous Applications
Apart from the mainstream applications of nanostructures, nanostructures have been largely explored for various other outlying areas, e.g., environmental protection, bio-imaging, water purification. For example, ZnO nanoparticles possess capabilities to remove organic dyes and hazardous materials from polluted water.
Studies have yielded fascinating results in these domains for nanostructures made of single as well as composite nanomaterials. Such varied applications of nanostructures have enabled the advancement of the nanotechnology sector to great extents.
In this chapter, the fabrication of nanostructures by employing bottom-up approach has been described. Some basic processes used in the bottom-up approach have been discussed in detail. The latest fabrication technologies for the fabrication of ZnO nanostructures, DNA nanostructures, polymer-based nanostructures, and metal-based nanostructures have also been discussed. It is seen that these nanostructures have wide applications as, sensors (gas, bio, chemical, visible light, and ultraviolet), cosmetics, optical devices, optoelectronics, electrical devices, photo-chemistry, solar cells and light-emitting displays separation, optical storages, and drug delivery. DNA nanostructures have been utilized in drug delivery, nanoswitches, computing, etc. Owing to characteristics like high sensitivity, inexpensive, non-hazardous, and faster response to analytes, nanostructures have been employed for sensing, detection, screening of viruses, etc. It is evident that nanotechnology is an emerging field with potential to revolutionize the therapeutics and diagnostics industry. Advancements in the area of nanostructure fabrication have allowed to achieve development of highly sensitive and specific sensors. The integration of these nanostructures for therapeutic and diagnostic purposes could facilitate the engineering of improved nanostructures specific to required biomedical applications.
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