Micro-Nano Technology for Genomics and Proteomics BioMEMs - Ozkan

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Magnetic field

alternating current i

frequency f

Induced current in metal

Metal piece	solution of gold nanocrystals
a	b

FIGURE 15.4. a) General schematic for induction heating of metal parts, b) induction heating of nanoparticles attached to biomolecules in solution.

particle directly and not the biomolecule or solution, it is a way to directly control the attached biomolecule. Heat dissipation of proteins into solution is fairly rapid [41], and for limited powers control of biomolecules reversibility is possible.

Nanoparticles are ideal systems for antennas for many reasons. First, the size of nanoparticles is about the size of the biomolecules to control, proteins and DNA (nanometers). Secondly, nanoparticles are soluble due to the solution phase synthesis used to synthesize particles. As a result, the nanoparticle-biomolecule hybrid is soluble, so the protein or DNA molecule can carry its nanoparticle antenna inside of a cell, which is like solution (albeit much more crowded/higher concentration [31]). This has important ramifications for in vivo feasibility. Thirdly, the chemistry to link nanoparticles to biomolecules has been explored. A covalent link between the two is required, not simply electrostatic adsorption because the protein may become detached when nanoparticle is heated. Fortunately, there are many options for achieving this chemical link, and there is extensive previous work in which nanoparticles have been successfully attached to proteins and DNA without perturbing the function, both in vitro and in vivo. Finally, nanoparticles have been studied for their interesting size and material dependent properties [4, 7, 28, 55], and some of these properties may be exploited in developing antennas.

This technique is ideal for controlling biological function because nearly all biomolecules denature with heat, giving this the potential to be universally applicable. In addition, magnetic fields can be used in tissue. Typically tissue creates a problem for optical techniques as it blocks out wavelengths shorter than 800 nm, the majority of visible spectrum where most dyes absorb and emit. However, some have been able to circumvent this obstacle by using chromophores that that absorb and emit in the infrared (IR). Core shell nanostructures of gold shells on and silica nanoparticles are excitable in the IR due to their novel structures [30]. Also, nanocrystals composed of semiconductors with small band gaps (CdTe, GaAs) absorb and emit in the IR. These IR fluorescent nanocrystals are biocompatible and have been used successfully for imaging through tissue.

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Nanoparticles have already been incorporated into biological systems for many other purposes. Typically they are employed as sensors, where they can give an optical readout of the state of hybridization of DNA or indicate whether or not a species has bound to its receptor. Metal nanoparticles have a surface plasmon resonance that is sensitive to its immediate surroundings, so a change such an antibody binding to its target or DNA hybridizing to its complement [22, 57] can shift this resonance. In some formats, nanoparticles provide a highly sensitive means of detection, prohibiting the need for PCR, which is not an immediate readout and may introduce errors. Semiconductor nanocrystals have been used as fluorescent tags that are more robust than organic dyes [39, 51]. Other forms of using nanoparticles to control processes in biological systems have been in hyperthermia, where magneticl particles are collected in tissue and heated similarly by an alternating magnetic field to burn tumors. In addition, magnetic particles have been utilized as “magnetic tweezers” to pull on cell surfaces to determine mechanics of the membranes. In addition, magnetic particles have served as sensors of DNA hybridization [34, 49].

There are three general schemes of using RFMF heating of nanoparticles to control the activity of biological molecules (Figure 15.5). First, heat from the nanoparticles can be used to directly change the conformation of a protein, turning off activity (Figure 15.5a).

	RFMF
Protein active	Protein inactive
	RFMF
Protein assembled:	Protein disassembled
active	into subunits: inactive
	RFMF

Nucleic acid double stranded

Nucleic acid single stranded

FIGURE 15.5. Three general schemes for controlling the activity of biomolecules. a) directly controlling the conformation of a biomolecule, b) using nanoparticle heating to break up non covalent bonds between subunits of the protein, altering activity, c) using nanoparticle heating to denature nucleic acids, altering activity.

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Alternatively, it can be used to control proteins made of subunits which are held together by noncovalent bonds (Figure 15.5b). Upon heating by the RFMF, the nanoparticle breaks these noncovalent bonds, separating the subunits and turning the protein off (or on). For control of proteins, the nanoparticle must be labeled in a specific spot on the protein. Thirdly, the nanoparticles can be used to break the hydrogen bonds in nucleic acids and denature the pair (Figure 15.5c). This last example will be described in detail below.

15.2.1. Technical Approach

Nanoparticles of high crystalline quality, soluble, and well-defined sizes are required for their use as antennas. There are many routes for solution phase synthesis [10, 13, 14, 33, 45, 64], which typically yields particles that are soluble in organic solvents. However, water solubility is key for utility in biology, so methods of ligand exchange and surface functionalization have been developed [33, 45].

The linkage between nanoparticle antenna and the biomolecule to be controlled must be a covalent link. Although electrostatic adsorption has been used successfully to link proteins to inorganic nanoparticles, it may not be sufficient here as heating the particle may cause detachment from the biomolecule. In addition, precise placement of nanoparticles is desirable for control of proteins. Fortunately, multiple chemistries for attaching gold nanoparticles to biomolecules have been developed. The most commonly used chemistry is through a thiol attached to the DNA or protein which can react directly with gold to form a gold-thiol bond, which has been used successfully in many instances [22, 66]. Secondly, one can change the ligand on the surface of the nanoparticle to a moiety that can react with a group on a biomolecule to form covalent bond [29]. For example, the reaction between a primary amine with a sulpho N-hydroxysuccinimide (NHS) ester can form a peptide bond [25, 50]. Thus, the DNA can be appended with the primary amine on the 5’ or 3’ end, and the particle would have a ligand that contains the NHS ester. Alternatively, the DNA can be functionalized with a thiol group, which can react with a maleimide ligand on the nanoparticle to form a thioether bond. In general, reaction with a ligand on the surface is a method that is not specific to gold nanoparticles, and can be utilized with particles of different materials. Replacement of ligands on nanoparticle surfaces can be done by standard ligand exchange [45]. In addition, advances in DNA synthesis have facilitated these chemistries, as reactive groups can conveniently be incorporated during solid-phase synthesis. Furthermore, nanoparticles with reactive ligands are commercially available (Nanoprobes, Inc.).

Purification of the nanoparticle-bioconjugate species from unlabeled DNA/protein and free nanoparticles can be achieved by agarose gel electrophoresis. Gel electrophoresis has been established as a technique which can separate strands of DNA according to size with a resolution of a few base pairs. Consequently, it is an appropriate technique to separate DNAand protein-nanoparticle conjugates [59, 66]. In order to obtain the purified conjugates in solution, bands were cut out of the gel matrix and the DNA-gold conjugates were extracted by centrifugation of the isolated bands through spin filters. A simpler method to purify the bioconjugate from free nanoparticles is by ethanol precipitation. If the nanoparticles have suitable surface ligands, only the DNA will precipitate from solution, permitting separation. In this case the nanoparticles must be in large excess of the DNA, as unlabeled DNA will also precipitate.

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Here the system for induction heating of the particles in solution is similar to those used for industrial induction heaters, which typically consists of a generator that sends alternating currents through a coil, creating alternating magnetic fields [49, 67]. The current was applied to coils that had multiple turns (101 to 102), which were constructed of wire wrapped around a plastic cuvette/tube holder. The holder had an open structure to maximize light passage through the sample for optical experiments, and had a cross section of 1cm2 so that microfuge tubes could fit inside. An RF signal generator (Hewlett Packard 8648C) generated currents with frequencies from 30kHz to 1GHz with an output power of 1 mW, which was then amplified to result in an output power from 0.4 W–4 W. This is an estimate for the ultimate output power as losses can occur from setup architecture. In order to eliminate effects from heating of the sample by the coil, the entire coil was placed in a large water bath at room temperature (T = 22 ◦C, volume 1 L). Samples were either in PCR tubes or quartz cuvettes if spectroscopic measurements were being performed, and sample volumes were typically in the range of 180–200 µL.

15.2.2. Dehybridization of a DNA Oligonucleotide Reversibly by RFMF Heating of Nanoparticles

The first molecule to be controlled was a DNA hairpin dehybridization, to demonstrate that the nanoparticle can be heated and denature an attached biomolecule. The DNA loop hairpin (also known as a molecular beacon [58]), which is a single stranded nucleic acid that is self complementary on the ends (Figure 15.6a). The loop/hairpin is utilized because the self-complementary structure facilitates rehybridization on rapid timescales [9], so a test of reversibility is feasible. The 1.4 nm gold nanoparticle was attached to the DNA via a functional group that was appended to one of the bases in the loop region. This group was an amine, which can react with an NHS ester on the surface of the nanoparticles. Here one needs a way to monitor the hybridization state of the molecule and determined if it is in singleor double-stranded form. In order to do so, DNA hyperchromicity can be exploited, which is the property of DNA by which the optical absorption of the bases increases when going from double stranded to single stranded form [16]. This occurs at 260nm, the wavelength characteristic of base absorption. Thus, the sample is put in a coil in an optical absorption spectrometer and the optical absorption at 260 nm is monitored while turning on and off the radio-frequency magnetic field at 15 second intervals.

The resulting optical absorption values for a 0.1µM solution of the nanoparticlelabeled molecular beacon are shown in Figure 15.6b, upper curve. When the RFMF is turned on, the optical absorption increases, and when it is turned off, the optical absorption decreases to its original value. This indicates that the DNA is dehybridizing with application of the field, and rehybridizing in the absence of the field. As a result, this shows that the control of the state of hybridization of the DNA is fully reversible. Furthermore, control experiments in which the same oligo without a nanoparticle is exposed to the field show no response (Figure 15.6b, lower curve), demonstrating that neither the solution nor the DNA molecule itself is responsive to the RFMF.

15.2.3. Determination of Effective Temperature by RFMF Heating of Nanoparticles

Since the nanoparticle produces some sort of heating, it is essential to get an estimate on what temperature it creates in its local environment. It should be an appropriate temperature

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a	NH2
GTGG A
TG	AT
G	G

GC

T G

CA AA GTACT

T-A

C-G

C-G

C-G

G-C

C-G

G-C

b					Au
	0.26	on	off
	0.25
(O.D.)	0.24
	0.23
260	0.22
A	0.21
	0.20
	0.19
	0	1	2	3	4

time (min)

FIGURE 15.6. Using RFMF heating of nanoparticles to control denaturation of a molecular beacon. a) schematic of the molecular beacon and its attachment point for the nanoparticle (amine), b) optical absorption of nanoparticlemolecular beacon in the RFMF (upper curve) and only the molecular beacon in the RFMF (lower curve).

for denaturation of DNA or proteins. Based on the above results, the nanocrystals evidently create a higher temperature which may be localized to the DNA oligo. In order to determine this effective temperature increase, samples that were exposed to the alternating magnetic field were compared to those exposed to fixed global temperatures. In this case, a two-phase system was utilized in order to avoid temperature dependent effects on optical properties (Figure 15.7a). Nanocrystals were linked to a 12mer of DNA which had the 5’ end functionalized with a fluorophore, which served as a means to count the oligo. It was hybridized to a solid support. This was achieved by using a complement that had a biotin on one end, which could be captured onto streptavidin coated agarose bead. Because the beads were large (diameter 100µm), they settled to the bottom of the tube, comprising the solid phase. The supernatant above it acted as the solution phase. If the oligo was denatured by either heat or the RFMF, it could diffuse into the supernatant, where it could be removed from the solid phase and quantified by fluorescence spectroscopy.

One sample of the two phase system was prepared and aliquoted into separate tubes. Each tube was exposed to a fixed global temperature using a 1L water bath. One tube

CONTROL OF BIOMOLECULAR ACTIVITY BY NANOPARTICLE ANTENNAS

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	FAM	Au		FAM
			Au
	Au	B	B	Au
		SA
		B	B
	FAM			FAM
		agarose bead

intensity (counts/s)

2.0x106

	70°C
	50°C
1.5x106	RF
	30°C
	22°C
1.0x106
5.0x105

480 500 520 540 560 580 600

wavelength (nm)

integrated intensity

8x107

7x107

6x107

5x107

4x107

3x107

20 30 40 50 60 70 80 temperature (°C)

FIGURE 15.7. Determining the effective temperature of RFMF heating of a nanoparticle. a) schematic of the two phase system utilized. The species to be denatured is the DNA 12mer with an nanoparticle on the 3’ end and a fluorophore (FAM) on the 5’ end. b) fluorescence spectra of the supernatant c) integrated areas of the

fluorescence spectra of the thermally heated samples. The RFMF peak (dotted line) extrapolates to a temperature of35◦ C.

was placed in the coil with the RFMF at room temperature. Following heat or RFMF, the samples were spun down and the supernatant was removed. Each supernatant was then measured by fluorescence spectroscopy, which yielded a relative concentration of DNA present in the sample. Figure 15.7b shows the spectra from the supernatant of each aliquot. The fluorophore, FAM, has an emission maximum at 515nm. For samples exposed to higher temperatures, the fluorescence intensity increased as more DNA was denatured. The sample that was exposed to the RFMF (thick black line) had a fluorescence intensity between that of the 30◦C and 50◦C samples. In order to quantify the effective temperature of the RFMF sample, the fluorescence intensity peaks were integrated and plotted as a function of incubation temperature (dots), shown in Figure 15.7c. The curve is fit to a sigmoidal (line) characteristic of DNA melting curves [17]. The intensity for the RFMF sample was extrapolated (dotted line) to a temperature of 35◦C, or +13◦C above ambient temperature. This is the effective temperature increase the DNA oligo experiences from the RFMF. It is important to note that this temperature jump is sufficient to partially denature many proteins in biology. However, using this technique to dehybridize longer strands of DNA which have elevated melting temperatures may be difficult using the parameters utilized in the experiments described here. One way to achieve denaturation of longer oligos would be to increase the delivered power to the coil, which would increase the heating of the nanoparticle. Also, using multiple nanocrystals on the DNA can increase heating and permit control of longer oligos. However, functionalization becomes difficult as the only available points in the middle of the chain are off the bases themselves and nanoparticles attached to these sites may interfere with formation of the hybrid pair.

15.2.4. Selective Dehybridization of DNA Oligos by RFMF Heating of Nanoparticles

This technique will be useful only if can be used selectively, i.e., control the molecule with the nanoparticle antenna and while not affecting surrounding molecules that have no

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a

KIMBERLY HAMAD-SCHIFFERLI

FAM		TMR	Au	FAM
	Au	B	B	TMR
		SA
		B	B
FAM				FAM

acrylic bead

b

intensity (counts/s)

2.0x105					RFMF		6						70 °C
							2.5x10
1.5x105							2.0x106
							1.5x106
1.0x105
							1.0x106
5.0x104							5.0x105
0.0							0.0
480	500	520	540	560	580	600		480	500	520	540	560	580	600
	wavelength (nm)								wavelength (nm)

FIGURE 15.8. Determining selective denaturation of DNA by RFMF heating of nanoparticles. a) schematic of the two phase experiment utilized. The two species to be denatured is a nanoparticle labeled 12mer (identical to that in Figure 15.7) and another labeled with tetramethylrhodamine on the 3’ end. b) Fluorescence spectra (points) and integrated areas (dotted lines) of the sample thermally heated to 70◦ C (right) and the one heated by the RFMF (left).

nanoparticles attached. In order to do so, we used a similar twophase system described above, but used a mixture of oligos with and without nanoparticles attached (Figure 15.8a). These oligos are identical in sequence to the one with the nanoparticles (identical 12mer, same melting temperature) but instead of a nanoparticle on the 3’ end, there is another dye that is spectroscopically distinct from FAM. The oligo to be denatured was mixed with another oligo that was identical in length and sequence and melting temperature but had a fluorophore instead of a nanoparticle attached to the 3’ end. The ratio of oligos with nanoparticles to those without was roughly 1:1. The fluorophore for this second oligo was tetramethylrhodamine (TMR), which is spectroscopically distinct from FAM (λabsorption = 555 nm, λemission = 563 nm) and thus can be distinguished in fluorescence spectroscopy (Figure 15.8). One sample was immersed in a 70◦C water bath, and the other was exposed to the RFMF at room temperature. Fluorescence spectra are shown in Figure 15.8b where the ratio of the fluorescence intensity at 515nm and 563nm are compared. The globally heated sample had a higher ratio of TMR: FAM, while the sample that was exposed to the RFMF had a lower ratio. Integration of the peaks to quantify the ratios showed that the sample exposed to the global heating has approximately 50:50 gold vs. TMR labeled oligos, while

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the RFMF sample has a ratio of 80:20. This indicates that denaturation of the gold labeled oligo was enhanced in the RFMF. However, its denaturation is not completely exclusive. One possible explanation is that is that streptavidin on the bead is tetrameric, and thus has the capacity to bind four biotins and thus four complementary oligos. Consequently, each streptavidin on average would possess a mixture of gold-labeled and TMR-labeled oligos. As a result, the TMR-labeled oligos were estimated to be on the order of 10nm or less away from a gold-labeled oligo. At these distances it is expected that through-solution heating will occur, resulting in non-specific denaturation.

The experiment shows that selectivity is limited by the degree of heat localization around the nanoparticle. This presents a challenge for implementation in cells which have extremely crowded environment, with protein concentrations on the order of 300mg/mL. As a result, the space between proteins is on the order of nanometers [37], with not many solvent layers between them. Therefore, heat localization will have to be on order of nanometers for it not to affect other proteins in solution. The study of heat localization around nanoparticles in solution is currently a work in progress. Classical heat transfer equations indicate that for a point source embedded in a medium, heat by conduction is fairly localized [8], but these descriptions are based on continuum approaches, and may not necessarily apply to nanoscale systems. Experimentally it has been determined that heat conductivity coefficients for solutions of metal nanoparticles are much higher than bulk systems, and cannot simply be described in terms of volume fractions. [15, 18, 35] As a result, this problem is currently of interest to the heat transfer field as these solutions of nanoparticles could be used as heat management fluids that remove heat from electronic devices.

CONCLUSIONS AND FUTURE WORK

Control of biomolecular activity by heating of nanoparticles by RFMF has been shown to be reversible and selective for the simple case of DNA denaturation. Nanoparticle antennas are promising as a tool for studying and utilizing biological systems as they can be universally applied to biological systems and have foreseeable compatibility in vivo. Future work includes control of proteins that perform complex processes. This will be a challenge as specific placement of the nanoparticle on the protein is required. This is easily achievable when labeling DNA since end functionalization or incorporation of unique bases is possible during solid phase synthesis for short oligos. However, proteins are much more complex than DNA, and knowledge of the three-dimensional structure is crucial for application of this technique. For proper control, the nanoparticle must be close enough to the active site so that heat from it changes its structure, but on the other hand it must not perturb the protein’s function when no field is applied. Furthermore, control of the chemistry so that the nanoparticle is at one amino acid site and not at all of them is essential. Other issues like protein denaturation and sticking to the surface of the nanoparticle must also be addressed, most likely through manipulation of the nanoparticle surface chemistry. Clearly, this technique is much more powerful if applicable in cells or organisms. While this has not yet been tested, introduction of nanoparticles into cells and organisms has been successful in various instances. Nanoparticles have been ingested by cells, injected into tissues, and have had no effect on the functioning of the organism. Consequently, this is a promising new tool that has potential to control a broad range of biological functions in

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relevant biological environments. It could have important ramifications for diagnostic tools and pharmaceutical applications, as it may enable reversible and specific control of disease related species remotely. It represents but one step towards fully utilizing the richness and complexity of the machines of Nature.Hope

REFERENCES

[1]P. Abrahams, A.G.W. Leslie, R. Lutter, and J.E. Walker. Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria, Nature, 370:621–628, 1994.

[2]L. Addadi and S. Weiner. Interactions between acidic proteins and crystals: Stereochemical requirements in biomineralization. Proceedings of the National Academy of Sciences, Vol. 82, pp. 4110–4114, 1985.

[3]L.M. Adleman. Molecular Computation of Solutions to Combinatorial Problems. Science, 266:1021–10–24, 1994.

[4]A.P. Alivisatos. Semiconductor clusters, nanocrystals, and quantum dots. Science, 271, 933–937, 1996.

[5]H. Asanuma, X. Liang, T. Yoshida, A. Yamazawa, and M. Komiyama. Photocontrol of triple-helix formation by using azobenzene-bearing oligo(thymidine), Angewandte Chemie-International Edition In English, 39:1316–1318, 2000.

[6]H. Asanuma, T. Yoshida, T. Ito, and M. Komiyama. Photo-responsive oligonucleotides carrying azobenzene at the 2’-position of uridine. Tetrahed. Lett., 40:7995–7998, 1999.

[7]M.G. Bawendi, M.L. Steigerwald, and L.E. Brus. The quantum mechanics of larger semiconductor clusters (Quantum Dots). Ann. Rev. Phys. Chem., 41:477–496.

[8]A. Bejan. Heat Transfer, New York, John Wiley & Sons, Inc., 1993.

[9]G. Bonnet, O. Krichevsky, and A. Libchaber. Kinetics of conformational fluctuations in DNA hairpin-loops.

Proceedings of the National Academy of Sciences, Vol. 95, pp. 8602–8606, 1998.

[10]L.O. Brown and J.E. Hutchison. Controlled growth of gold nanoparticles during ligand exchange. JACS, 121:882–883, 1999.

[11]S. Brown. Protein-mediated particle assembly. Nano Lett., 1:391–394, 2001.

[12]S. Brown, M. Sarikaya, and E. Johnson. A Genetic Analysis of Crystal Growth. J. Mol. Biol., 299:725–735, 2000.

[13]M. Brust, J. Fink, D. Bethell, D.J. Schiffrin, and C. Kiely. Synthesis and reactions of functionalized gold nanoparticles. J. Chem. Soc., Chem. Commun., 16:1655, 1995.

[14]M. Brust, M. Walker, D. Bethell, D.J. Schiffrin, and R. Whyman. Synthesis of thiol-derivatized gold nanoparticles in a two-phase liquid-liquid system. J. Chem. Soc., Chem. Commun., 1994:801, 1994.

[15]D.G. Cahill, W.K. Ford, K.E. Goodson, G.D. Mahan, A. Majumdar, H.J. Maris, R. Merlin, and S.R. Philpot. Nanoscale thermal transport. J. Appl. Phys., 93:793–818, 2003.

[16]C.R. Cantor and P. Schimmel. Biophysical Chemistry Part I: The conformation of biological macromolecules. San Francisco, W.H. Freeman, 1980a.

[17]C.R. Cantor and P. Schimmel. Biophysical Chemistry Part II: Techniques for the study of biologial structure and function. San Francisco, W. H. Freeman, 1980b.

[18]G. Chen. Particularities of heat conduction in nanostructures. J. Nanopart. Res., 2:199–204, 2000.

[19]J. Chen and N.C. Seeman. The synthesis from DNA of a molecule with the connectivity of a cube. Nature, 350:631–633, 1991.

[20]J.H. Collier and P.B. Messersmith. Phospholipid strategies in biomineralization and biomaterials research. Ann. Rev. Mat. Res., 31:237–263, 2001.

[21]T. Douglas and M. Young. Host–guest encapsulation of materials by assembled virus protein cages. Nature, 393:152–155, 1998.

[22]R. Elghanian, J.J. Storhoff, R.C. Mucic, R.L. Letsinger, and C.A. Mirkin. Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles. Science, 277:1078– 1081, 1997.

[23]M.B. Elowitz and S. Leibler. A synthetic oscillatory network of transcriptional regulators. Nature, 403:335– 338, 2000.

CONTROL OF BIOMOLECULAR ACTIVITY BY NANOPARTICLE ANTENNAS

475

[24]T.S. Gardner, C.R. Cantor, and J.J. Collins. Construction of a genetic toggle switch in Escherichia coli. Nature, 403:339–342, 2000.

[25]J.F. Hainfield and R.D. Powell. New frontiers in gold labeling. J. Histochem. Cytochem., 48:471–480, 2000.

[26]K. Hamad-Schifferli. DNA Hybridization: Electronic control of. In J.A. Schwarz, C. Contescu, and K. Putyera (eds.), Encyclopedia of Nanoscience and Nanotechnology. New York, Marcel Dekker, 2003.

[27]K. Hamad-Schifferli, J.J. Schwartz, A. Santos, S. Zhang, and J.M. Jacobson. Remote electronic control of DNA hybridization through inductive heating of an attached metal nanocrystal. Nature, 415:152–155, 2002.

[28]J.R. Heath and J.J. Shiang. Covalency in semiconductor quantum dots. Chem. Soc. Rev., 27:65–71, 1998.

[29]G.T. Hermanson. Bioconjugate Techniques, San Diego, Academic Press, 1996.

[30]L.R. Hirsch, R.J. Stafford, J.A. Bankson, S.R. Sershen, B. Rivera, R.E. Price, J.D. Hazle, N.J. Halas, and J.L. West. Nanoshell-Mediated Near-Infrared Thermal Therapy of Tumors Under Magnetic Resonance Guidance.

Proceedings of the National Academy of Sciences. Vol. 100, pp. 13549–13554, 2003.

[31]T. Hugel, N.B. Holland, A. Cattani, L. Moroder, M. Seitz, and H.E. Gaub. Single-molecule optomechanical cycle. Science, 296:1103–1106, 2002.

[32]D.A. James, D.C. Burns, and G.A. Woolley. Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues. Protein Eng., 14:983–991, 2001.

[33]N.R. Jana and X. Peng. Single-phase and gram-scale routes toward nearly monodisperse Au and other noble metal nanocrystals. JACS, 125:14280–14281, 2003.

[34]L. Josephson, J.M. Perez, and R. Weissleder. Magnetic nanosensors for the detection of oligonucleotide sequences. Angewandte Chemie-International Edition In English, 40:3204–3206, 2001.

[35]P. Keblinski, S.R. Phillpot, S.U.S. Choi, and J.A. Eastman. Mechanisms of heat flow in suspensions of nano-sized particles (nanofluids). Internat. J. Heat Trans., 45:855–863, 2002.

[36]J.R. Kumita, O.S. Smart, and G.A. Woolley. Photo-Control of Helix Content in a Short Peptide. Proceedings of the National Academy of Sciences, 97:3803–3808, 2000.

[37]H. Kuthan. Self-organisation and orderly processes by individual protein complexes in the bacterial cell.

Prog. Biophys. Mol. Biol., 75:1–17, 2001.

[38]T.H. LaBean, H. Yan, J. Kopatsch, F. Liu, E. Winfree, J.H. Reif, and N.C. Seeman. Construction, analysis, ligation, and self-assembly of DNA triple crossover complexes. JACS, 122:1848–1860, 2000.

[39]D.T. Larson, W.R. Zipfel, R.M. Williams, S.W. Clark, M.P. Bruchez, F.W. Wise, and W.W. Webb. Watersoluble quantum dots for multiphoton fluorescence imaging in vivo. Science, 300:1434–1436, 2003.

[40]S.-W. Lee, C. Mao, C.E. Flynn, and A.M. Belcher. Ordering of quantum dots using genetically engineered viruses. Science, 296:892–895, 2002.

[41]T. Lian, B. Locke, Y. Kholodenko, and R.M. Hochstrasser. Energy flow solute to solvent probed by femtosecond IR spectroscopy: malachite green and heme protein solutions. JPC, 98:11648–11656, 1994.

[42]D. Liu, J. Karanicolas, C. Yu, Z. Zhang, and G.A. Woolley. Site-specific incorporation of photoisomerizable azobenzene groups into Ribonuclease S. Bioorg. Medi. Chem. Lett., 7:2677–2680, 1997.

[43]C.J. Loweth, W.B. Caldwell, X. Peng, A.P. Alivisatos, and P.G. Schultz. DNA-Based assembly of gold nanocrystals. Angewandte Chemie-International Edition In English, 38:1808–1812, 1999.

[44]C. Mao, D.J. Solis, B.D. Reiss, S.T. Kottmann, R.Y. Sweeney, A. Hayhurst, G. Georgiou, B. Iverson, and A.M. Belcher. Virus-based toolkit for the directed synthesis of magnetic and semiconducting nanowires. Science, 303:213–217, 2004.

[45]C.B. Murray, D.J. Norris, and M.G. Bawendi. Synthesis and characterization of nearly monodisperse CdE (E = S, Se, Te) semiconductor nanocrystallites. JACS, 115:8706–8715, 1993.

[46]C.M. Niemeyer. Progress in “engineering up” nanotechnology devices utilizing DNA as a construction material. Appl. Phys. A., 68:119–124, 1999.

[47]C.M. Niemeyer. Nanoparticles, proteins, and nucleic acids: biotechnology meets materials science. Angewandte Chemie-International Edition In English, 40:4128–4158, 2001.

[48]M. Orfeuil. Electric Process Heating: Technologies/ Equipment/ Applications. Columbus, Ohio, Battelle Press, 1987.

[49]J.M. Perez, L. Josephson, T. O’Loughlin, D. Hogemann,¨ and R. Weissleder. Magnetic relaxation switches capable of sensing molecular interactions. Nat. Biotechnol., 20:816–820, 2002.

[50]J.E. Reardon and P.A. Frey. Synthesis of undecagold cluster molecules as biochemical labeling reagents. 1. Monoacyl and mono[N -succinimidooxy)succinyl] undecagold clusters. Biochem., 23:3849–3856, 1984.