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«Kinetic Investigations of Thiolate Protected Gold Nanoparticles: Protein Interactions, Electron Transfer, and Precursor Formation By Brian N. Turner ...»

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In conclusion, a number number of studies with diverse applications were conducted which all utilized similar nanoparticle synthesis, functionalization, and characterization techniques. The studies underscore the versatility and potential applicability of these novel nanomaterials in a variety of areas, from materials to life sciences. Additionally, new knowledge was gained on fundamental aspects of nanoparticle synthesis. The knowledge should prove useful in future attempts to exploit the underlying chemistry of nanoparticle synthesis in order to create more well-defined or completely novel nanomaterials.

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Biomimetic Nanoparticles Many aspects of the biomimetic HRSV nanoparticle project elicit further investigation or methodological improvement. These aspects include peptide design, epitope mapping strategies, nanostructural considerations, mimic detection techniques, and in vivo evaluation for antigenicity. While some of these facets are currently under investigation for different systems, the HRSV biomimetic project is not currently active even though many avenues of research remain unexplored.

The rate limiting step to designing a useful peptide for conjugation to a nanoparticle had always been the lack of any crystal structure data for either the F protein, the antibody, any complex related to the two. Now that this data is available88-90, 125, and given the results obtained in Chapter II, much more informed decisions about peptide design in the context of nanoparticle biomimetics should be forthcoming. Knowing the residues critical to Palivizumab-epitope binding and their spatial orientation should allow one to only select the most important parts of the primary sequence, or potentially add in discontinuous residues with an inert linker. As was indicated previously, the desired orientations and lengths of the two alpha helical sections known to be important to the binding interaction should be evaluated in the ELISA assay. The specific effects of adding, removing, or replacing single amino acids would allow for the selection of the smallest, most efficient peptides to be used in nanoparticle studies as supposed to the 24 amino acid sequence (not including any added linker residues) found most successful in the current study. This might also add additional insight into the binding of the native proteins, further corroborating the previously mentioned crystal structure data.

While the current research discussed the importance of peptide structural modifications beyond primary sequence variation such N and C terminal modification and linker chemical structure and length with regards to optimizing peptide efficacy in biomimetic design, the data collected was not systematic. This was largely due to the desire to obtain a working mimic as a starting point. Having found this starting point, a systematic variation of peptide structure is now appropriate. The following systematic variations are prescribed: effect of C-terminal amination and N-terminal acetylation individually or in tandem versus unmodified termini for like primary sequences, directly comparing PEG spacers to inert amino acid spacers, and comparing chemically similar spaces of different lengths.

Once ideal peptides have been designed, a number of structural changes can be investigated with regards to the final nanomaterial. Nanoparticle core size, especially in the case of dual cysteine functionalized peptides, should have a significant effect. In the case of a single cysteine containing peptide, the core size could affect the secondary structure of the peptide by modifying the interactions between neighboring ligands. In the case of a dual cysteine containing peptide, the core size is expected to have an effect on how far apart the cysteine residues space themselves (due to spacings between active sites and surface curvature versus the strain imparted to the peptide upon anchoring the cysteine thiol groups on the gold core), and, therefore, on the conformation of the formed peptide loop.110 Options for controlling the nanoparticle size are via synthetic conditions (for instance, ligand to metal ratio)63 or by post-synthetic size separation.55, 183 Another exciting possibility, especially for this specific case, is the concept of functionalizing a nanoparticle with two (or more) peptides to represent a discontinuous epitope, suggested by Rutledge and coworkers.92 In the Palivizumab epitope, one should recall that the interacting residues are located on two alpha helices separated by an amorphous loop region, and a further residue on an entirely different region of the peptide. If both helices are separately synthesized, and potentially a third peptide is synthesized with the other interacting residue, and they are subsequently place exchanged onto an appropriate sized nanoparticle, then the interacting residues could be placed at the appropriate spatial coordinates without the need for non-interacting amino acids.

Finally, it would be prudent to evaluate this system with different methodologies. In the context of the QCM, different antibody immobilization strategies should be evaluated, as spacing of antibodies in an immunoassay is critical in optimizing sensitivity.128 Also, QCM with dissipation measurement should provide additional information as to whether the binding is rigid, as would be expected for an antigen-antibody interaction.184 An instrument suited to this task is available from Q-Sense which also is equipped with an autosampler. As manual QCM experiments are time intensive (typically 2 hours) and subject to errors during sample change which lead to useless data sets, such as the introduction of air bubbles or pressure changes across the crystal face, a higher data yield might be attained with such an instrument. Another consideration should be the validation of the QCM results with an additional analytical method such as ELISA or SPR.





Wired Nanoparticles Currently, Dave Crisostomo is continuing with the research in wired nanoparticles. As with the biomimetic nanoparticle project, a great deal of systematic changes should lead to important insights on structure-function relationship. Currently, the research is focused on observing whether or not electron transfer kinetics of nanoparticles protected by alkanethiolate ligands of different lengths (for instance, hexanethiol versus octanethiol) are similarly affected by the introduction of a molecular wire ligand.

Another interesting question is how the nanoparticle core size might affect electron transfer or the ability of the molecular wire ligands to modulate the electron transfer. It is possible that a change in surface curvature could have a significant effect on the ability of the core itself to come in close proximity of an electrode surface. It might also effect the chemical environment of the molecular wire ligand itself.

Upon successful completion of these fundamental studies, integration of the “wired” nanomaterials into functional nanomolecular electronic devices is of great interest. An idea proposed by David Cliffel is to integrate wired particles with Photosystem I (PSI).

In this context, the nanoparticles could store charge from photocurrent generated by covalently attached PSI which could later be discharged to a quantum dot to produce light. The whole device is comparable to a nanoscale, battery-powered flashlight with wired nanoparticle capacitors.

Nanoparticle Precursor Formation While the determined rate law for gold(III) chloride reduction by tiopronin in methanol accurately fit the data over a sufficient concentration range (much greater than typical synthetic conditions)122, collection of more data points to verify the saturation kinetics data is crucial. Simply put, more data points are needed to prove the non-linear relationship at higher tiopronin concentrations. Verification of this rate law is crucial to suggesting a mechanism for tiopronin gold nanoparticle precursor formation. In order to measure the initial pre-reduction ligand exchange process, a spectrophotometer with a stop-flow system is required. Such an experimental set-up would also allow us to interrogate the initial rates of reduction in the bromide system.

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THERMAL GRAVIMETRY-MASS SPECTROMETRY AND ELEMENTAL

ANALYSIS OF TIOPRONIN PROTECTED GOLD NANOPARTICLES

This research was conducted at the Center for Nanophase Materials Sciences, which is sponsored at Oak Ridge National Laboratory by the Division of Scientific User Facilities, U.S. Department of Energy. This research was authorized upon successful submission and review of a rapid access proposal, ID CNMS2007-R19. The experiments were held at the Macromolecular Complex Systems Group under the supervision of Dr.

Joseph Pickel.

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Thermal gravimetric analysis is the easiest route to determining the percent organic composition of gold nanoparticles. There has been debate about the identity of the burnoff material, and often times the average approximate molecular formulas determined for certain preparations are not reasonable with the current picture of how a nanoparticle should look. Therefore, it was endeavored to characterize the thermal gravimetric plot with mass spectrometry. The technique is occasionally referred to as temperature programmed desorption mass spectrometry (TPD-MS).

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Synthesis of Au216Tiopronin129 Tiopronin MPCs were synthesized as previously described. 122 5.25 g (15.4 mmol) HAuCl4  3H2O was dissolved in 500 mL 6:1 methanol:acetic acid and chilled in an ice bath for 30 minutes, giving a yellow solution. 7.56 g (46.2 mmol) tiopronin was then added to the solution, forming a ruby red intermediate. Upon dissolution of the tiopronin,

5.84 g (154 mmol) sodium borohydride in approximately 10 mL water was immediately and rapidly added to the mixture. A violent, exothermic reaction results, leaving a black solution. The solution is stirred overnight, yielding polydisperse nanometer sized clusters. The resulting solution is rotary evaporated in vacuo to remove the methanol.

The resulting precipitate/viscous acetic acid solution is redissolved in water, pH adjusted to 1 with concentrated hydrochloric acid and placed in dialysis tubing (cellulose ester, MWCO = 10 kDa). The dialysis proceeds for 2 weeks changing the water at least twice daily until the water does visually turn a color. 1H NMR: 4.00 ppm broad singlet (tiopronin methylene on cluster), 3.76 ppm broad singlet (tiopronin CH on cluster), 1.66 ppm very broad singlet (tiopronin methyl on cluster). TEM: average particle diameter of

2.2 nm  0.6 nm. UV/Visible spectroscopy: no surface plasmon resonance band

observable. TGA: 33.134 weight percent organic (tiopronin). Average formula:

Au2167Tiopronin1294.

Synthesis of Hexanethiol Monolayer Protected Clusters

1.5 g of tetraoctylammonium bromide (a phase transfer agent) was dissolved in 80 mL of toluene. 0.31 g of HAuCl4 was dissolved in 25 mL of water. To the vigorously stirring tetraoctylammonium bromide solution, the gold solution was added slowly. The resulting orange gold-containing organic phase was isolated and chilled in an ice bath for 30 minutes and stirred vigorously. 0.38 g of NaBH4 was dissolved in 25 mL of water.

To the stirring organic solution, 260 L of 1-hexanethiol was added. Immediately, the NaBH4 solution was added over a period of 10 seconds. The resulting black solution was stirred for 30 minutes on ice, and then additionally overnight at room temperature. The organic phase was isolated and rotary evaporated to yield a black solid. The solid was dissolved in a minimum of toluene and precipitated in 400 mL of ethanol at -20 C overnight. The precipitate was collected on a fine glass frit and washed with ethanol and acetone. Additional purification was accomplished by sonicating in acetonitrile for 15 minutes, and again collecting on a glass frit and washing with ethanol and acetone. 1H NMR: 2.17 ppm, 1.45 ppm, 0.98 ppm broad peaks. TEM: 2.8 nm  0.9 nm diameter.

UV/Vis: slight surface plasmon band observed at 520 nm. TGA: 20.367 weight percent organic. Average molecular formula: Au448Hexanethiol192.

Thermal Gravimetric-Mass Spectrometric Analysis TGA with mass spectrometric analysis of the evolved gases was accomplished using a TA Instruments 2950TGA equipped with Pfeiffer Thermostar Mass spectrometer. Both ion selective trend scans and scanning bar graph scans were completed. The purge gas was argon, and the temperature was ramped from room temperature up to 1000 °C.

Elemental Analysis Samples were sent to Columbia Analytical Services for analysis of carbon, hydrogen, nitrogen, sulfur, oxygen, gold, and sodium. Carbon, hydrogen, and nitrogen were analyzed by combustion with a WO3 catalyst, and detection by TC and IR. Sulfur was analyzed by combustion with IR detection. Oxygen was analyzed by pyrolysis with IR detection. Sodium and gold were measured either by ICP-OES, ICP-MS, or AAS.

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Thermogravimetric Analysis Thermogravimetric analysis for AuTiopronin and AuHexanethiol were compared qualitatively, as in Figure 53.

Figure 53: Thermogravimetric profiles of gold hexanethiol cluster (red) and gold tiopronin cluster (black). Differences in the total magnitude of percent weight loss may be due to size dependent properties, but the step-wise nature of the tiopronin cluster weight loss is characteristic of polar MPCs Whereas alkanethiol nanoparticles have a single mass loss step when heated under nitrogen, tiopronin nanoparticles exhibit two. The shape of the curve is identical for thermal analysis of tiopronin ligand, although the temperature of each step is shifted slightly to lower temperature. The assumption had always been that any thermal loss should be attributed strictly to loss of organic material, which made theoretical sense with what was understood about thiolate gold nanoparticles if composition calculations used burn off data up to 550 °C. Once spectrum were run up to 800 °C, however, composition calculations yielded thiols in excess of hypothesized surface gold atoms, which was inconsistent with expected compositions.

Elemental Analysis The results of elemental analysis in terms of calculated molecular composition compared to results obtained with a combination of TGA and TEM by two different calculations are compared in Table 13.



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