<h3>Excerpt</h3> <div><div> &lt;h2&gt;CHAPTER 1&lt;/h2&gt; &lt;p&gt;&lt;b&gt;BIOLOGICAL APPLICATIONS OF MULTIFUNCTIONAL MAGNETIC NANOWIRES&lt;/b&gt;&lt;/p&gt; &lt;p&gt;Edward J. Felton and Daniel H. Reich&lt;/p&gt; &lt;br&gt; &lt;p&gt;&lt;b&gt;1.1 INTRODUCTION&lt;/b&gt;&lt;/p&gt; &lt;p&gt;Nanoscale magnetic particles are playing an increasingly important role as tools in biotechnology and medicine, as well as for studying biological systems. With appropriate surface functionalization, they enable the selective application of magnetic forces to a wide range of cells, subcellular structures, and biomolecules, and have been applied to or are being developed for areas including magnetic separation, magnetic biosensing and bioassays, drug delivery and therapeutics, and probes of the mechanical and rheological properties of cells. Despite these successes, however, the structure of the magnetic particles in common use limits the range of potential applications. Most biomagnetic particles available today are spherical, with either (a) a "core-shell" structure of concentric magnetic and nonmagnetic layers or (b) magnetic nanoparticles randomly embedded in a nonmagnetic matrix. These geometries constrain the range of magnetic properties that can be engineered into these particles, as well as their chemical interactions with their surroundings, because such particles typically carry only a single surface functionality. A new and more versatile approach is to use asymmetric, multisegment magnetic nanoparticles, such as the metal nanowires shown in Figure 1.1. The multisegment architecture of these particles, along with the ability to vary the aspect ratio and juxtaposition of dissimilar segments, allows the nanowires to be given a wide range of magnetic, optical, and other physical properties. In addition, differences in the surface chemistry between segments can be exploited to selectively bind different ligands to those segments, enabling the development of magnetic nanoparticle carriers with spatially resolved biochemical functionality that can be programmed to carry out multiple tasks in an intracellular environment.&lt;/p&gt; &lt;p&gt;This chapter provides an overview of recent results of a research program, centered at Johns Hopkins University, that is aimed at development of multifunctional magnetic nanowires for biotechnology applications. Section 1.2 provides a brief introduction to the fabrication process, and this is followed in Section 1.3 by an overview of the physical properties of the nanowires that are important in a biotechnological context. Sections 1.4&#8211;1.6 describe our development of the needed "tool-kit" for biological applications: manipulation of the nanowires in suspension, chemical functionalization, and self-assembly techniques. Section 1.7 discusses prospects for magnetic biosensing using nanowires, and Sections 1.8 and 1.9 discuss the major biological applications of the nanowires explored to date: novel approaches to magnetic separations, new tools for cell positioning and patterning, and new carrier particles for drug and gene delivery.&lt;/p&gt; &lt;br&gt; &lt;p&gt;&lt;b&gt;1.2 NANOWIRE FABRICATION&lt;/b&gt;&lt;/p&gt; &lt;p&gt;Nanowires are fabricated by electrochemical deposition in nanoporous templates. Originally developed for fundamental studies of the electrical and magnetic properties of modulated nanostructures, this method offers control of both nanowire size and composition and thus allows the nanowires' magnetic and chemical properties to be tailored for specific biological applications. To make the nanowires, a copper or gold conductive film is sputtered on one side of the template to create the working electrode of a three-electrode electrodeposition cell. Metal is then deposited from solution into the template's pores to form the wires. The nanowires' diameter is determined by the template pore size and can range from 10 nm to approximately 1 m. The wires' length is controlled by monitoring the total charge transferred and is only limited by the thickness of the template. After the nanowire growth is complete, the working electrode film is etched away and the template is dissolved, releasing the nanowires into suspension.&lt;/p&gt; &lt;p&gt;Ferromagnetic nickel nanowires were commonly used in the work reported here.&lt;/p&gt; &lt;p&gt;Grown in commercially available 50 &#181;m-thick alumina templates, they have a radius of 175 &#177; 20 nm and lengths ranging from 5 to 35 &#181;m. An SEM image of 15 &#181;m-long nickel nanowires is seen in Figure 1.1b. The high pore density of the alumina templates (3 &#215; 10&lt;sup&gt;8&lt;/sup&gt; cm&lt;sup&gt;-2&lt;/sup&gt;) enables fabrication of large numbers of nanowires. In addition to single-component nanowires such as these, nanowires comprised of multiple segments can be made by changing the deposition solution during growth. This technique has been used with alumina templates to create two-segment Ni&#8211;Au nanowires. Alternatively, multisegment nnanowires of certain materials can be grown from a single solution by varying the deposition ppotential. One example is the alternating ferromagnetic and nonmagnetic layers of the Ni&#8211111;Cu nanowire shown in Figure 1.1c. Nanowires incorporating two metals can also be synthesized as alloys. In one example, this technique has been used to produce high-surface area nanoporous Au wires by selectively etching away the Ag from Au&#8211;Ag alloy nanowires, as shown in Figure 1.1d.&lt;/p&gt; &lt;br&gt; &lt;p&gt;&lt;b&gt;1.3 PHYSICAL PROPERTIES&lt;/b&gt;&lt;/p&gt; &lt;p&gt;The elongated architecture of the nanowires and the flexibility of the fabrication method permit the introduction of various magnetic and other physical properties. The magnetic properties can be tuned and controlled through the size, shape, and composition of magnetic segments within the wires. For example, due to their high magnetic shape anisotropy, single-segment magnetic nanowires form nearly single-domain states with large remanent magnetizations for a wide range of nanowire lengths. This is illustrated in Figure 1.2, which shows magnetic hysteresis curves for 175 nm-radius nickel nanowires of different lengths. The shape of the hysteresis curves is nearly independent of nanowire length, with coercive field &lt;i&gt;H&lt;sub&gt;C&lt;/sub&gt;&lt;/i&gt; ~ 250 Oe and remanent magnetization &lt;i&gt;M&lt;sub&gt;R&lt;/sub&gt;&lt;/i&gt; ~ 0.8 &lt;i&gt;M&lt;sub&gt;S&lt;/sub&gt;&lt;/i&gt;, where &lt;i&gt;M&lt;sub&gt;S&lt;/sub&gt;&lt;/i&gt; is the saturation magnetization. These large, stable, and well-aligned moments make such nanowires useful for low-field manipulations of cells and biomolecules, as discussed in Section 1.8. As seen in the inset, &lt;i&gt;M&lt;sub&gt;S&lt;/sub&gt;&lt;/i&gt; scales linearly with the wire length, and at high fields the nanowires have moment per unit length &#181;/&lt;i&gt;L&lt;/i&gt; = 3.9 215; 10&lt;sup&gt;-11&lt;/sup&gt; emu/&#181;m. For comparison, Figure 1.2 also shows the magnetic moment of commercially available 1.5 &#181;m-diameter magnetic beads. Note that while the volume of the longest nanowires shown here is only 1.5 times that of the beads, their high-field moment is 20 times that of the beads. Thus the nanowires can provide significantly larger forces per particle in magnetic separations and other high-field applications.&lt;/p&gt; &lt;p&gt;There are, of course, biomagnetic applications in which large magnetic moments in low field are not desirable. These include situations in which it is important to control interactions among particles to reduce agglomeration in suspension. The remanent magnetization of multisegment nanowires such as those shown in Figure 1.1c can be tuned by controlling the shape of the magnetic segments. If the magnetic segments within a multisegment nanowire have an aspect ratio greater than unity, shape anisotropy favors the adoption of a high-remanence state with the segments' moments parallel to the wire axis, even if they are short compared to the length of the nanowire, as shown in Figure 1.3a. In contrast, if the magnetic segments are disk-shaped (aspect ratio &lt; 1), the shape anisotropy of the individual segments favors alignment of their moments perpendicular to the nanowire axis. Dipolar interactions between the segments then favor antiparallel alignment of the moments of neighboring segments, leading to a low-moment state in zero field, as shown in Figure 1.3b.&lt;/p&gt; &lt;p&gt;In addition to defining the magnetic properties, the segment composition can be exploited for other purposes. For example, the high-surface-area nanoporous gold segments previously mentioned (Figure 1.1d) can be used for efficient chemical functionalization, or for biosensing applications. Optical properties of the nanowires can also be controlled. Differences in reflectivity in AuAg multisegment nanowires are being exploited for "nano-barcoding" of molecules and subcellular structures, and oxide segments with intrinsic fluorescence can also be introduced.&lt;/p&gt; &lt;br&gt; &lt;p&gt;&lt;b&gt;1.4 MAGNETIC MANIPULATION OF NANOWIRES&lt;/b&gt;&lt;/p&gt; &lt;p&gt;The large and tunable magnetic moments of nanowires allow precise manipulation of molecules and bound cells, with applications ranging from cell separations to two-dimensional cell positioning for diagnostics and biosensing, and to the potential creation of three-dimensional cellular constructs for tissue engineering. The approaches we have developed for these applications take advantage of nanowirenanowire interactions, as well as their interactions with lithographically patterned micromagnet arrays and external fields. To illustrate these capabilities, we first discuss manipulation of the nanowires themselves.&lt;/p&gt; &lt;p&gt;In liquid suspensions, the nanowires readily orient with their magnetic moments parallel to an applied field. Single-segment and multisegment nanowires with long magnetic segments align with the wire axis parallel to the field, and multisegment wires with disk-shaped segments align perpendicular to the field. When magnetized, the nanowires interact through dipoledipole magnetic forces. Self-assembly of the nanowires can be achieved, either in suspension or by allowing the wires to settle on flat substrates. This process can be controlled by an external field. Without an applied field, the nanowires are randomly oriented in the suspension, and they will assemble into random collections due to the anisotropy of the dipolar interaction. Application of a small field suppresses this random aggregation by prealigning the nanowires parallel to each other. The nanowires then form end-to-end chains as they settle out of solution, as shown in Figure 1.4. The addition of descending nanowires to chains settled on the substrate can yield chains that extend over hundreds of micrometers.&lt;/p&gt; &lt;p&gt;The motion of both bare nanowires and nanowires bound to cells in suspension is governed by low Reynolds number hydrodynamics, and a nanowire's velocity is given by v = F/&lt;i&gt;D&lt;/i&gt;, where F is the net force due to external fields, neighboring nanowires, and gravity, and &lt;i&gt;D&lt;/i&gt; is the appropriate viscous drag coefficient. Integrating this equation of motion allows precise prediction and modeling of the nanowires' dynamics.&lt;/p&gt; &lt;p&gt;For example, Figure 1.5 shows an analysis of a video microscopy study of nanowire chaining dynamics. For all the events shown in Figure 1.5 the wires or chains are nearly collinear. In this case, the force between two wires or chains of lengths &lt;i&gt;L&lt;/i&gt;&lt;sub&gt;1&lt;/sub&gt; and &lt;i&gt;L&lt;/i&gt;&lt;sub&gt;2&lt;/sub&gt; is&lt;/p&gt; &lt;p&gt;[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]&lt;/p&gt; &lt;p&gt;where &lt;i&gt;r&lt;/i&gt; is the end-to-end separation. The nanowires are described very accurately in this and in all subsequent modeling discussed below as extended dipoles with magnetic charges &#177; &lt;i&gt;Q&lt;sub&gt;m&lt;/sub&gt;&lt;/i&gt; = &#177;&lt;i&gt;M&lt;/i&gt;&#960;&lt;i&gt;a&lt;/i&gt;&lt;sup&gt;2&lt;/sup&gt; separated by &lt;i&gt;L&lt;/i&gt;, where &lt;i&gt;M&lt;/i&gt; is the wire's magnetization. The solid curves are fits to r(t) based on the (somewhat involved) analytic form determined from the one-dimensional equation of motion &lt;i&gt;dr/dt&lt;/i&gt; = [??]&lt;i&gt;f(r)&lt;/i&gt;, where [??] = &lt;i&gt;D&lt;/i&gt;&lt;sub&gt;1&lt;/sub&gt;&lt;i&gt;D&lt;/i&gt;&lt;sub&gt;2&lt;/sub&gt;/(&lt;i&gt;D&lt;/i&gt;&lt;sub&gt;1&lt;/sub&gt; + &lt;i&gt;D&lt;/i&gt;&lt;sub&gt;2&lt;/sub&gt;) is the reduced drag coefficient. Full details are given in Ref. 21. These results demonstrate that quantitative predictions of the nanowirenanowire interactions and dynamics can be made.&lt;/p&gt; &lt;p&gt;Another important manipulation tool involves using the strong local fields generated by micrometer-size magnetic features patterned by microlithography on substrates to capture and position nanowires and cells. This "magnetic trapping" process works because the nanowires are drawn into regions of strong local field gradients produced by the patterned micromagnets, such as those at the ends of the Ni ellipses shown in Figure 1.6. The snapshots show video frames from a trapping event, and the trace shows the distance &lt;i&gt;z(t)&lt;/i&gt; of the wire from the trap versus time. Analysis of the force produced on the wire by the micromagnets again yields a simple model that can be integrated to obtain &lt;i&gt;z(t)&lt;/i&gt; (solid curve). A SEM image of a nanowire trapped by micromagnets is presented in Figure 1.7.&lt;/p&gt; &lt;br&gt; &lt;p&gt;&lt;b&gt;1.5 CHEMICAL FUNCTIONALIZATION&lt;/b&gt;&lt;/p&gt; &lt;p&gt;The ability to chemically functionalize nanowires enhances their utility in biological applications. Selectively binding ligands to the surface of nanowires allows additional control of interactions between nanowires, between nanowires and surfaces, and between cells and nanowires, as well as control of the wires' optical characteristics.&lt;/p&gt; &lt;p&gt;We have built on prior knowledge of surface chemistry on planar metallic films to selectively functionalize both single- and multicomponent nanowires. Functionalization of nickel utilizes binding between carboxylic acids and metal oxides, in this case the native oxide layer present on the nanowires' surface, while gold functionalization makes use of the well-known selective binding of thiols to gold. It is therefore possible to attach various molecules possessing a compatible binding ligand to a particular metallic surface. This has been demonstrated with single-segment nickel nanowires that have been functionalized with hematoporphyrin IX dihydrochloride (HemIX), a fluorescent molecule with two carboxylic acid groups, as well as 11-aminoundecanoic acid and subsequently a fluorescent dye (Alexa Fluor 488 or fluorescein-5-isothiocyanate (FITC)). Single-segment gold nanowires have been functionalized with thiols including the thioacetate-terminated thiol P-SAc and 1,9-nonanedithiol with the fluorescent dye Alexa Fluor 546.&lt;/p&gt; &lt;p&gt;Multisegment nanowires are attractive because the differences in surface chemistry between different segments makes possible spatially resolved chemical functionalization with multiple molecules on the same nanowire, with different ligands directed to different segments. Our work with two-segment Ni&dash;Au nanowires serves as an example of this spatially resolved functionalization. In one scheme, after exposure to HemIX, Ni&dash;Au nanowires showed strong fluorescence from the Ni segments, and the Au segment exhibited weak fluorescence due to nonspecific HemIX adsorption. However, after simultaneous functionalization with HemIX and Au-specific nonylmercaptan, only the Ni segments showed fluorescence, indicating that the nonylmercaptan had attached to the Au segment to prevent nonspecific binding of HemIX. Bauer and co-workers also reacted Ni&dash;Au nanowires with 11-aminoundecanoic acid and nonylmercaptan, and then subsequently with Alexa Fluor 488, which binds only to the 11-aminoundecanoic acid. Selective functionalization caused only the Ni segment to fluoresce, as shown in Figures 1.8a and 1.8b. Conversely, reacting the Ni&dash;Au wires with 1,9-nonanedithiol and palmitic acid (for specific binding to Ni), and then with Alexa Fluor 546, which binds only to the 1,9-nonanedithiol, resulted in fluorescence of only the Au segment. Lastly, exposing Ni&dash;Au nanowires to both 11-aminoundecanoic acid and 1,9-nonanedithiol, and then adding the fluorescent markers Alexa Fluor 488 and 546, resulted in fluorescence of both segments.&lt;/p&gt; &lt;p&gt;Selective surface functionalization of nanowires has also been accomplished with biomolecules. In one study, single-segment nickel and gold nanowires were functionalized with palmitic acid and an ethylene glycol-terminated alkanethiol, respectively, to render the nickel hydrophobic and the gold hydrophilic. Two-segment Ni&dash;Au nanowires were exposed to both reagents. The nanowires were then exposed to Alexa Fluor 594 goat anti-mouse IgG protein, an antibody with an attached fluorescent tag. It is known that proteins are able to attach noncovalently to hydrophobic surfaces, but are prevented from such binding to hydrophilic surfaces. As seen in Figures 1.8c and 1.8d, only the nickel surfaces showed fluorescence, confirming that they had been selectively functionalized with the protein.&lt;/p&gt; &lt;p&gt;Other experiments involving functionalization with biomolecules have used two-segment NiAu nanowires as synthetic gene-delivery systems. DNA plasmids encoding fluorescent proteins were bound to the nickel segment through a carboxylic acid intermediary, while the cell-targeting protein transferrin was bound to the gold segment through a thiolate linkage. This application of biomolecule-functionalized nanowires to gene delivery is detailed in Section 1.9. </div></div><br/> <i>(Continues...)</i> <!-- Copyright Notice --> <div><blockquote><hr noshade size="1"><font size="-2">Excerpted from <b>Biomedical Applications of Nanotechnology</b> by <b>Vinod Labhasetwar</b>. Copyright © 2007 by John Wiley & Sons, Ltd. Excerpted by permission of John Wiley & Sons.<br/>All rights reserved. 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