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In this presentation you will go through the introduction to nanotechnology, some basicconcepts about the nanofabrication approaches and techniques, virus display fornanowire formation, TMV as template for nanowire scaffold and a little bit about theself assembly , the advantages and disadvantages of biotemplating and future aspects.

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As we all know what is nanotechnology ‘ the study of the controlling matter on theatomic and molecular scale’. Nanotechnology is very diverse ranging from the extensionsof conventional device physics to completely new approach of self assembling. On thesimilar basis we can define nano biotechnology ‘nano bio technology is the engineeringof biological scaffolds at molecular level’. Most of the processes of nanotechnology areintegrated with biology or with the use of biological materials. We will see in the furtherslides how we can use biological materials to manufacture some regular geometries andcommercial materials.

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But we need to distinguish slightly between the nanotechnology and molecularmanufacturing (mostly miss used) nanoscale technology is the use of bigmachines to make smaller products while the molecular manufacturing is ananticipated future technology based on Feynman’s vision of factories usingnanomachinaries to build complex products. It promises to bring greatimprovements in the cost and performance of manufactured goods, whilemaking possible a range of products impossible today. Every manufacturingmethod is a method for arranging atoms. Most methods arrange atoms crudely;even the finest commercial microchips are grossly irregular at the atomic scale.Many of today’s nanotechnologies face the same limit. Chemistry and biology,however, make molecules defined by particular arrangements of atoms —always the same numbers, kinds, and bonds. Chemists do this using clever tricksthat don’t scale up well to building large, complex structures. Biology, however,uses a more powerful method: cells contain molecular machines that read digitalgenetic data to guide the assembly of large molecules (proteins) that serve asparts of molecular machines

Top-down and bottom-up are two approaches for the manufacture of products. Theseterms were first applied to the field of nanotechnology by the Foresight Institute in 1989in order to distinguish between molecular manufacturing (to mass-produce largeatomically precise objects) and conventional manufacturing (which can mass-producelarge objects that are not atomically precise). Bottom-up approaches seek to havesmaller (usually molecular) components built up into more complex assemblies, whiletop-down approaches seek to create nanoscale devices by using larger, externally-controlled ones to direct their assembly. The top-down approach often uses thetraditional workshop or microfabrication methods where externally-controlled tools areused to cut, mill, and shape materials into the desired shape and order. Micropatterningtechniques, such as photolithography and inkjet printing belong to this category.Bottom-up approaches, in contrast, use the chemical properties of single molecules tocause single-molecule components to (a) self-organize or self-assemble into some usefulconformation, or (b) rely on positional assembly. These approaches utilize the conceptsof molecular self-assembly and/or molecular recognition. Such bottom-up approachesshould, broadly speaking, be able to produce devices in parallel and much cheaper thantop-down methods, but could potentially be overwhelmed as the size and complexity ofthe desired assembly increases

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This slide just compares the scale of things made by nature or made by man. Man tookmany centuries to learn the ways of nature making the nanoscale materials but stillthere are many challenges comes across. Broadly speaking we can classifynanotechnology under three headings ‘wet’, ‘dry’ and computational nanotechnology.

Wet Nanotechnology: which is the study of biological systems that exist primarily inwater environment?

Dry Nanotechnology: which derives from surface science and physical chemistry e.g.structures of carbon, silicon etc.

Computational Nanotechnology: This permits the modeling and simulation of complexnanometer scale structures.

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The most top down fabrication technique is nano lithography. In this process, requiredmaterial is protected by mask and the exposed material is etched away.e.g.Photolithography, Electron and ion based lithography and scanning probe lithography.

Bottom up approach utilizes the concept of molecular self assembly or molecularrecognition and taking the advantage of physicochemical interactions for the hierarchicalsynthesis of orders nanoscale structures.

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There are two basic ways for nanosynthesis and these are physical and chemical methods.

In chemical methods we have :

Sonochemistry

Microwave synthesis

Hydrothermal methods

Solgel methods

Wet chemical coprecipitation etc

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At about 550 million years ago, organisms began to have their simple organic moleculesin order to grow more complex organic matrices which precisely fit form to function.One of the first ‘bioinspired’ archtectural projects was the contruction of the crystalpalace (1851).In 20th century , scientist began to take a more active interest in nano-biological world. The father of this approach was R.J.P williams (oxford University), whoinstigated a study of the detailed functional use of inorganic elements in biologicalsystems.

Mann (oxford university) gave the understanding of bio mineralization in terms ofmovement and precipitation of inorganic elements within a ‘biological’ system.

The complexity of biological structures and complex systems which give rise to them arenot easily replicated that convinced scientists to directly utilize the natural occurringmaterials.

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Biotemplating is the study of biological scaffolds at the nanoscale the important example are DNA, Viruses and bacteria.

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Bio systems have inherently developed very specific molecular recognition patterns thatcan be manipulated through genetic control. It also can be used to exert molecular scalecontrol over nucleation, growth, and stabilization of inorganic materials, analogous tothe process of biomineralization. Furthermore, due to the remarkable capability ofbiological molecules to self-assemble at multiple length scales, the opportunity exists fordesigning novel nanomaterials via genetic modification and then constructinghierarchically assembled structures. The combination of biological self-assembly andbiosynthesis of nanomaterials can enable us to create entirely new conceptsapplications and devices.

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Biotemplating seeks to either replicate the morphological characteristics and thefunctionality of a biological species or use a biological structure to guide the assembly ofinorganic materials. In the first case, the biological substrate has interestingmorphological characteristics (e.g., diatoms, butterfly wing scales, viruses) and metalreplication is used to provide a more stable and more controllable synthetic substrate.The replication process typically leads to the

generation of either a negative, positive (or hollow), or exact copy of the template.Indeed, a large variety of biological species have been used as templates: bacteria,textiles/paper, hair, cells, insect wings, spider silk, wool, and wood. The majority of thebiological structures that have been used for replication show nanoporous features (e.g.,diatoms), channels (viruses), and other complex hierarchical architectures (butterflywings). The level of precision in replicating nanoscale topographies and features is themajor challenge. In the second case concerning the biologically guided assembly ofnanomaterials, a natural biological system is used to nucleate inorganic structures andpromote pattern formation. This is ubiquitously directed by

covalent/noncovalent interactions and molecular recognition processes. For suchinteractions to take place, the biological structures must present specificphysicochemical and/or morphological attributes to direct the assembly of inorganicstructures into technologically useful platforms. Such attributes can include a secludedinner channel or inner cavity that is accessible only by molecules of specific size/charge,or the presence of a unique functional group at specific locations.

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There is already quite a long list of biological materials that have been successfullyreplicated for the formation of artificial structures, and these include cotton/cloth, pinewood, human and animal hair, silk, and wool, viruses, bacteria, DNA and proteins.

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The video is taken from :www.youtube.com (with the title virus)

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Virus Display with Inorganic Materials:

(a) A combinatorialvirus library is obtained or synthesized that expresses randompeptide fusions (color shaded areas).

(a) The virus library is exposed to a substrate (typically an inorganic single crystal) andpositive binding interaction of

the peptide fusion is allowed to occur with the substrate.

(c) After washing the virus interactions with detergents to ensure specific binding to thesubstrate, the successful binding viruses are isolated via a disruption in bindingconditions, typically using a change in pH. The isolated viruses are amplified in theirbacterial host and reintroduced to a fresh substrate surface. The process between (b)and (c) is repeated several times with the isolated and amplified viruses, mimicking anevolutionary cycle.

(d) Once the (b) to (c) cycle is complete (typically 3 rounds), the DNA from the virus isisolated and sequenced to determine the identity of the peptide responsible for bindingto the substrate.

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The M13 phage pIII constructs used in the selection experiments, with the peptidedisplayed only on one end of the filamentous phage, were used in the first M13-basednanocrystal growth studies. In particular, two phage-bound peptide sequences that wereselected for ZnS, named Z8 and A7, were shown to control ZnS particle size and shape atroom temperature, under aqueous conditions.

Wild-type clones (no peptide insert) were used as a control. Transmission electronmicroscopy (TEM), high resolution TEM (HRTEM), scanning TEM (STEM) and electrondiffraction (ED) data revealed that the addition of a ZnS-specific phage clone affectedparticle size and formed discrete ZnS crystals. Crystals grown in the presence of the Z8clone were observed to be approximately 4 nm in size of the zinc blende phase. For theA7 virus, nanocrystals grown were 4 nm by 2 nm in size of the wurtzite crystal phase.

Particles grown without the ZnS-specific phage clones or with wild type clones werenon-crystalline and were much larger (100–500 nm) in size distribution.

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Schematic diagram depicting an engineered M13 virus displaying peptides to directnucleation of inorganic materials and/or further assemble viruses into complexheterofunctional arrays.

(a) M13 virus, with peptides fused to pIX shown ingreen, to pVIII shown in orange, andto pIII shown in blue.

(b) Nanoparticles represented as spheres localized on the viruses illustrate the potentialof multiple materials engineering into one viral structure, whose length and shape canbe custom-tailored depending on the genome size engineered.

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An overall advantage to this genetic programming approach to materials engineering, in

addition to materials-specific addressability, is the potential to specify viral length andgeometry. The length of a filamentous virus is related to the size of its packaged geneticinformation and the electrostatic balance between the pVIII-derived core of the virionand the single-stranded DNA.

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Additionally, viruses can be conjugated with one-dimensional nanowires/nanotubes,two dimensional nano electrodes, and microscale bulk devices. One-dimensionalmaterials, such as nanotubes or nanowires, when conjugated with the pIII end of M13viruses, may form phase separated lamellar structures that have inorganic nanotube ornanowire layers and phage building block layers.

Two-dimensional nano-thick plate shaped electrodes can be organized. Alternativecathode and anode structures might be useful for future nanosize biofuel cells. Whenthe specific binding M13 virus is combined with micro-size objects, periodic organizationof these micro-dimensional objects is also possible

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Self-assembly is a term used to describe processes in which a disordered system of pre-existing components forms an organized structure or pattern as a consequence ofspecific, local interactions among the components themselves, without externaldirection

Distinctive features: At this point, one may argue that any chemical reaction drivingatoms and molecules to assemble into larger structures, such as precipitation, could fallinto the category of SA. However, there are at least three distinctive features that makeSA a distinct concept.

Order:First, the self-assembled structure must have a higher than the isolatedcomponents, be it a shape or a particular task that the self-assembled entity mayperform. This is generally not true in chemical reactions, where an ordered state mayproceed towards a disordered state depending on thermodynamic parameters.

Interactions:The second important aspect of SA is the key role of weak interactions (e.g.Van der waals, pi-pi attractions, hydrogen bonds) with respect to more "traditional"covalent, ionic or metallic bonds. Although typically less energetic of a factor 10, theseweak interactions play an important role in materials synthesis. It can be instructive tonote how weak interactions hold a prominent place in materials, but especially inbiological systems, although they are often considered marginally with respect to"strong" (i.e. covalent, etc.) interactions. For instance, they determine the physicalproperties of liquids, the solubility of solids, the organization of molecules in biologicalmembranes.

Building blocks: The third distinctive feature of SA is that the building blocks are notonly atoms and molecules, but span a wide range of nano- and mesoscopic structures,with different chemical compositions, shapes and functionalities. These nanoscalebuilding blocks (NBBs) can in turn be synthesised through conventional chemical routesor by other SA strategies.

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Self-assembly has a fundamental advantage over mechanically directed assembly: Itrequires no machinery to move and orient components, letting random, Brownianmotion do the job instead. Selective binding between uniquely matching surfacescompensates for the randomness of the motions that bring components together.

Molecular synthesis methods and self-assembly can be used to produce atomicallyprecise nanosystems by the billions, and even by the ton, thereby establishing atechnology base with wide-ranging applications that can drive development forward.

The architecture of biomolecular fabrication is based on the use of programmablemachines to produce the complex parts necessary for self-assembly of complex systems.The same fundamental architecture can be extended to use artificial biomolecularmachines (and then non-biomolecular machines), resulting in products made of betterand more diverse engineering materials.

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The most fundamental disadvantage of pure self-assembly is that for every product, thestructure of the parts must encode the structure of the whole. This requires thatcomponents be more complex, which tends to make design and fabrication moredifficult. Another consequence is that a self-assembled product will be partitioned bycomplex internal interfaces that have no operational function. Unless they arestrengthened after assembly, these interfaces will weak. These are major constraints.

Mechanically directed assembly avoids these constraints. Because components need notencode the structure of a product, they can be simple and standardized, and they can bechosen for their functional properties with less concern for how they are put together.This will enable more straightforward design and fabrication, but one must make thenecessary machinery — and I expect that this will be accomplished by means of self-assembly.

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Linear single-stranded DNA templates have been used to direct the ordered assembly ofAu nanoparticles tagged with Complementary oligonucleotides. But it can’t be used tomake more complex structures. Hence, synthetic DNA molecules featuring branchedjunction motifs have been designed. “Sticky ends” flanking the junctions enable the self-assembly of these novel DNA sequences into 2D and 3D architectures, such as latticesand grid. DNA nanotechnology is an area of current research that uses the bottom-up,self-assembly approach for nanotechnological goals. DNA nanotechnology uses theunique molecular recognition properties of DNA and other nucleic acids to create self-assembling branched DNA complexes with useful properties.

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There are two types of self-assembly, intramolecular self-assembly and intermolecularself-assembly. Most often the term molecular self-assembly refers to intermolecularself-assembly, while the intramolecular analog is more commonly called folding. (foldingis the process by which a molecule assumes its shape or conformation. The process canalso be described as intramolecular self-assembly where the molecule is directed toform a specific shape through noncovalent interactions, such as hydrogen bonding,metal coordination, hydrophobic forces, van der Waals forces, pi-pi interactions, and/orelectrostatic effects.)

The fig at the bottom shows the micelle formation by self assembly of detergentmolecules and protein molecules. The hydrophobic tails are arranged in such a way toform a micelle with protein core.

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Notable advantages of biotemplating in nanostructure fabrication include the sheerstructural diversity of available biological species and materials, as well as thesophisticated architectures (1D, 2D, and 3D) and degree of complexity achievable.Together, these elements provide for the creation of a diverse range of novel materialswith an unprecedented repertoire of dimensions (resolution <100 nm) andmorphologies that extend beyond what is currently possible with conventionallithography/ etching techniques. The biotemplating approach is also potentially morecost- and time-effective (parallel fabrication

approach) when compared with current serial techniques (e.g., electron beamlithography, X-ray lithography) for nanostructure fabrication. In addition, the repetitivetopochemical features and variety of functional groups found in many biologicalmaterials, can be exploited for the in situ synthesis and directed self-assembly of bothorganic and inorganic nanostructures under mild conditions without the use of harshchemical treatments. And finally, biotemplates

are also highly amenable to very (spatially) precise modifications at the molecular levelthrough rational genetic engineering and/or targeted chemical modifications. Takentogether, these attributes lead to a “biomolecular tool-kit” that offers great diversity anda facile approach for the fabrication of a variety of structures and devices. The full rangeof possibilities that biological templates have to offer has only just started to beexplored. Indeed, researchers are just beginning to grasp an understanding of the effectsof nanoscale topographies on the optical, chemical, and electrical properties ofmaterials. On the basis of these initial reports, there is clearly great potential for usingbiological materials to develop entirely new types of sensing systems

that display superior selectivity and sensitivity over existing conventional designs.

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However, for biotemplating to become more established as a reliablenanofabrication approach, several limitations that currently exist will need to beovercome. Most notably, as the biotemplating technique is a relatively newapproach, it still lacks the high yield levels and precise uniformity provided byother synthetic fabrication methods. In particular, large-scale fabrication may bean issue in some cases because of a lack of sufficient quantities of purifiedbiological material, or because of a lack of long-range order in the final productdue to intrinsic lattice/morphological defects in the biotemplate itself. Moreover,because the exact mechanisms by which biological entities form defined patternsand direct the growth of crystalline materials are not yet fully understood,biotemplating studies are often conducted in a highly empirical manner. Thisoften requires a significant amount of effort to be spent in trial and errorexperiments, with results that are in some cases neither predictable nor alwaysrepeatable. Finally, there remains a great need for scientists to develop a betterunderstanding of the biological-materials interface in general. Current surfacefunctionalization methods for the creation of engineered substrates for thedeterministic, oriented attachment of biological molecules still lack the degree ofcontrol necessary to be useable on a large scale, such that high quality and highuniformity can be reproducibly achieved.

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Biotemplating also poses a number of substantial intellectual challenges. The briefsummary of these challenges is that we do not yet know how to do it, and cannot evenmimic those processes known to occur in biological systems at other than quiteelementary levels. Although there are countless examples of Biotemplating materials allaround us--from molecular crystals to mammals--the basic rules that govern theseassemblies are not understood in useful detail, and processes cannot, in general, bedesigned and carried out "to order" and to solve these issues we need amultidisciplinary approach.

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