An Examination Of Synthesis On The Nanoscale Biology Essay


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The rapid expansion of the field of nanotechnology is being driven by the emergence of a wide variety of techniques that enable researchers to control the size, shape and chemical composition of structures which have at least one dimension in the 1-100 nm length scale.

In particular, metallic structures within this size range are opening up a myriad of new research possibilities ranging from electronics to medical diagnostics. This project explores various "bottom up" and possibly "top down" methods for the directed growth of gold and silver structures and subsequent modification of the surface chemical properties. Valuable experience in a number of advanced optical and physical characterization techniques was also gained.

1. Introduction

1.1 Nano-proliferation

The effective utilization of metal nanostructures and their properties is becoming more and more apparent as we acquire further knowledge for manipulating, modifying and improving synthetic techniques to produce the best shapes, sizes and substances most befitting emerging applications.

[The applications engendered by this rapidly developing field range from self-cleaning coatings that render fabrics stain-resistant, to additives that improve fuel efficiency, reducing pollution and harm to the environment; from infinitesimal computer chips and futuristic circuitry, to wonderful, noninvasive medical diagnostic and therapeutic utensils that will revolutionize the study and treatment of horribly debilitating diseases such as cancer, HIV, Alzheimer's, heart disease and many others.

For the successful advancement and implementation of such far reaching promises, three premises are revealed to be of vital importance: development of chemical synthesis and suitably sophisticated apparatus capable of surface analysis and manipulation of matter on a nanometer scale; determination of the physical and chemical changes of materials which have undergone nanoscale miniaturization; and the capitalization of these generally unusual physical and chemical properties of miniaturization in the development of superior technologies.

1.2 Optical Properties of Metallic Nanoparticles

Influence of Oxygen on the Optical Properties of Silver Nanoparticles

Silver nanoparticles in sol-gel silica films were obtained by annealing in hydrogen atmosphere and subsequently in oxygen atmosphere, Their properties were measured by UV-vis spectroscopy, transmission electron microscopy (TEM), high-resolution transmission electronic microscopy (HRTEM) and X-ray diffraction analysis. Samples prepared in a reducing atmosphere exhibited a surface plasmon resonance (SPR) located at 399 nm. Silver nanoparticles in an oxidizing atmosphere exhibited a red shift and damping of the SPR. These optical properties were explained due to the oxidation on the surface of silver nanoparticles to silver oxide yield in an oxygen atmosphere. Silver core-silver oxide shell nanostructures were observed by HRTEM. The average size of the metallic nanoparticles obtained by TEM was used for modeling the UV-vis spectra by using the Gans theory. Good fits to the spectra under an oxidizing atmosphere were obtained considering variable refractive indexes coming from the silver oxide shells surrounding to the nanoparticles. Therefore, the interaction between oxygen and the metallic surface of the nanoparticles, sensitively alters their optical properties.

1.3 Localized Surface Plasmon Resonance

1.3.1 [The remarkable property of LSPR is bestowed when the wavelength of incident light striking the surface of a nanoparticle is longer than the particle itself. The incident light induces conduction electrons of nanoparticles to oscillate at a resonance frequency characteristic of their shape, size and composition. This results in extremely intense absorption and scattering of light; so intense that single nanoparticles can be visualized by the human eye via dark-field (optical scattering) microscopy. LSPR phenomena enable noble-metal nanoparticles (such as gold and silver) to function in varied and improved dynamic labeling roles such as labels for immunoassays, biochemical sensing and surface-enhanced Raman spectroscopies. The use of nanoparticle plasmonics could have further application in the field of optical switches, waveguides, light sources, microscopes and lithographic tools.

(up to one million times greater than the fluorescence of a fluorescein molecule) giving dynamic labeling capabilities.

Most LSPR spectroscopy is performed using bulk concentrations of nanoparticles i.e. of a colloidal nature. However, recent research has revealed that individual nanoparticles could potentially serve as independent sensors.11,12,53. This would allow for heightened absolute detection limits (total number of molecules detected) and give improved signal to noise resolution for detection within e.g. cells and tissues.

The extinction and scattering spectra profiles of a nanoparticle - particularly of the λmax peak- depends on not only the outlined architectural parameters of composition, size, shape and orientation but also on the local dielectric environment of the nanoparticle. So, by close attention and guidance, the path of nanoparticle fabrication can be used to fine tune the LSPR via a variety of chemical syntheses.]6

Although this study mainly focuses on the unique localized surface plasmon resonance (LSPR) properties of silver and gold, LSPR is theoretically possible in any metal, for example aluminium; which can offer distinct advantages in refractive index sensitivity, alternative surface chemistries, as well as ultraviolet LSPR, a region where many organic molecules are active.

1.3.215[ The Plasmon-ruler (And other animals)

In a recent study investigating the relationship between nanoparticle separation and red-shift of LSPR, electron beam lithography was used to form nanodisc dimers that could be fixed at differing separation distances on fabricated nanoarrays. The results of this study allowed the derivation of an empirical "plasmon ruler" equation relating LSPR shift to separation between nanoparticles.

The LSPR frequency exhibited by coupled particles is different to that of single particles i.e. it is shifted. For example, the aggregation of gold nanoparticles in solution displays a red-shifted plasmon peak when related to their isolated nanoparticle counterparts, demonstrated by a change in colour from red to purple. Along with interparticle distance, the magnitude of LSPR shift is concurrently affected by the strength of interparticle interaction. Hence the shift can be used as a measure of particle separation. One of the plasmon ruler's prerequisites is that the spectral shift must be systematically calibrated and standardized as a function of interparticle separation, making it a less well established distance measurement technique than traditional fluorescence resonance energy transfer (FRET) methods. Advantageously though, the plasmon ruler allows for longer distance ranges and better photostability when undertaking measurement. Using electron beam lithography, metal nanoparticle structures with highly ordered morphology can be created while their separation is tightly controlled, making it an ideal tool for the study of plasmon coupling and its dependence on interparticle separation. P. K. Jain et al used lithographically fabricated Au nanodisc pairs to investigate how the plasmonic shift effect related to interparticle separations.

Figure 2. SEM image of 88 nm diameter and 25 nm thick nanodisc dimers with a gap of 12 nm used in the study of LSPR coupling and shifts as a function of interparticle separation.

The group discovered that the plasmonic shift decayed in an exponential fashion with interparticle distance when polarization took place parallel to the interparticle axis. Discrete dipole approximation simulations were used to also conclude that the trend in plasmon shift with respect to the interparticle separation becomes independent of nanodisc diameter when the shift is scaled by the single particle plasmon wavelength; and the separation gap is scaled by the nanodisc diameter. It was also discovered that the decay constant for this system is similar for varying shapes and different dielectric media; leading to the inference of universal scaling behavior of interparticle plasmon coupling distance decay.

To conduct the investigation, two-dimensional 80 μm 80 μm arrays were fabricated, featuring 88 nm diameter Au nanodiscs that were shifted progressively closer in each sample array, hopping from 212 nm to 27, 17,12, 7, and finally 2 nm. The LSPR obtained using microabsorption spectroscopy showed that under parallel polarization, the plasmon resonance was strongly red-shifted upon reduction of the interparticle gap while, conversely, there was only a faintly detectable blue-shift with decreasing gap when the direction of polarization was orthogonal to the interparticle axis. In the case of the parallel polarization, resulting in plasmonic frequency reduction i.e. a red-shift, the resonance shift is thought to result from an attractive dipole-dipole interaction. Conversely, the resulting increase in plasmon frequency (blue-shift) caused by orthogonal polarization is the product of repulsive interaction between dipoles. The larger wavelength shifts for parallel polarization are indicative of much stronger positive interparticle interactions than negative orthogonally polarized interactions.

Figure 3. Microabsorptioin spectra of Au nanodisc pairs for varying interparticle separation gap for incident light polarization direction (left) parallel and (right) perpendicular to the interparticle axis.

When the shift in plasmon extinction maximum was plotted against the interparticle separation gap for the parallel polarization results, the plot follows a near-exponential decay of length of 15.5 nm ± 3.0 nm.

Figure 4. Shift in the plasmon wavelength maximum of a pair of Au nanodiscs as a function of the interparticle separation gap. The curve is a least-squares fit to single-exponential decay, yielding a decay length l of 15.5 nm ± 3.0 nm (R2 = 0.985).

When calculating plasmon shift as a function of interparticle separation for discs of different diameters it was discovered that the magnitude of the plasmon shift for a fixed interparticle distance increased. Additionally, the coupling decay length also increases with increasing nanodisc diameter. However, when the fractional plasmon shift was plotted against the ratio of interparticle separation scaled by the nanodisc diameter, the decay trend appeared to become independent of nanoparticle size. This resulted in a plasmon coupling decay over a length approximately 0.2 times the diameter of the nanodisc.

Figure 5. Calculated fractional plasmon shift vs the ratio of interparticle gap to nanodisc diameter, showing that the scaled data points for the different disc sizes follow a common trend, with single-exponential decay with a = 0.14 ± 0.01and τ = 0.23 ± 0.03.

This universal scaling behavior allowed the plasmon ruler equation to be derived.

where is the fractional plasmon shift, s is the interparticle separation, and D is the particle diameter. This equation can be used in the estimation of interparticle separation from experimentally observed plasmon shifts, for example, distances in biological systems.]15

[1.4 Potential Applications of Gold Nanocages

1.4.1 Targeting Cancer Cells with Au Nanocages

In order to act successfully as a cancer diagnosis and treatment agent, the residence of Au nanocages within the body must be prolonged at specific sites of interest i.e. cancerous cells. Their suitability in the field of nanomedicine is attributable to their compactness and lack of bioactivity. Another of their advantages lies in their highly customizable surface chemistry: coatings such as poly(ethylene glycol) (PEG) or cancer-targeting moieties (e.g., antibodies or peptides) can be surface-layered using Au-thiolate chemistry.47

Skrabalak et al have shown that Au nanocages can be made to target the breast cancer cell line - SK-BR-3 - responsible for the overexpression of epidermal growth factor receptor 2 (EGFR2 or HER2). To specifically target this cell line, the surface of the Au nanocages was modified with anti-HER2 antibodies via a two step bioconjugation process: (i) Au nanocages were PEG-ylated by breaking the internal disulfide bond of succinimidyl propionyl poly(ethylene glycol) disulfide to form a Au-S linkage; then (ii) a PEG-antibody complex was formed through standard coupling chemistry.48

Using SEM, flow cytometry, elemental analysis and microscopy, the group determined that the antibody-modified Au nanocages attached and accumulated preferentially to the surface of SK-BR-3 cells. Conversely, it was discovered that unmodified Au nanocages exhibit no such preferential cell attachment, highlighting the specificity of such bioconjugationally amalgamated 'immuno-Au nanocages'.

1.4.2 Au Nanocages as Contrast Enhancement Agents

One of the most important factors in the ongoing battle against cancer is its initial detection and diagnosis. Evolution of conventional imaging techniques used to detect malignancy has helped reduce mortality rates in recent years; but with the integration of Au nanocages with tunable absorption/scattering dimensions, proficient and noninvasive in vivo detection could be introduced, helping to make invasive procedures such as biopsies a thing of the past.

Two of the most promising noninvasive optical imaging techniques are optical coherence tomography (OCT) and spectroscopic optical coherence tomography (SOCT) which allow the distinction between cancerous and healthy tissue to be made to the micrometer [in a way that is analogous to ultrasonic pulse-echo imaging.]10

These systems are based on a Michelson interferometer, which measures the interference signal between the backscattered light of a sample and a reference. Major image contrast arises primarily from the light scattered and absorbed by tissue, but by integrating Au nanocages, with their large absorption/scattering crosssections, the effect could be greatly enhanced.

In a recent demonstration carried out by the Skrabalak group, Au nanocages (LSPR tuned to 716 nm - a wavelength commonly used in OCT imaging) were incorporated into one half of a tissue phantom at a nanomolar concentration. [The phantom was made of gelatin embedded with TiO2 granules at a concentration of 1 mg/ml, mimicking the scattering background of average biological samples. OCT and SOCT were conducted using a 7-fs Ti:sapphire laser with a center wavelength of 825 nm and a bandwidth of 155 nm. As the TiO2 particles have negligible absorption at near-infra red wavelengths, the extinction cross-section measured from the sample without Au nanocages was assumed to be the same as the scattering cross-section of TiO2. OCT measurements indicated that Au nanocages <40 nm have a moderate scattering cross-section of ~ 8.10 x 10-16 m2 but a very large absorption cross-section of ~ 7.26 x 10-15 m2, a ratio of 8.97. Conventional imaging dyes such as Indocyanine Green (ICG) are approximately 5 orders of magnitude less absorbent, with an absorption cross-section of 2.90 x 10-20 m2 at 800 nm. These results demonstrate the potential utility of Au nanocages as OCT contrast enhancement agents. Early In vivo studies have begun.]11

1.4.3 Au Nanocages for Photothermal Therapy

As noted previously, Au nanocages exhibit large cross-sectional absorption of near infrared wavelengths. This leads to the presumption that the cages should be photothermally active, i.e. inbound photons should be converted into lattice vibrations (phonons) within the cages, which should cause a localized rise in temperature.

When targeted to a specific biological entity e.g. cancerous cells, the heat generated from absorption by the Au nanocages should be dissipated from their surface by thermal conduction into the surrounding cells, providing a therapeutic effect.

It has recently been demonstrated in vitro that with immuno-Au nanocages61 45 nm in edge length targeted breast cancer cells could be photothermally destroyed. SK-BR-3 cells were treated with immuno-Au nanocages (LSPR 810 nm) then irradiated for 5 min using an 810 nm laser at a power density of 1.5 W/cm2. These cells had been stained using calcein-AM and ethidium homodimer-1, allowing the distinction between live and dead cells to be made as the former fluoresced green and the latter fluoresced red. The results showed that the affected cells were confined to areas limited by the point of laser exposure. Conclusively, cells sans immuno-Au nanocages identically irradiated maintained viability. The power density of 1.5 W/cm2 was found to be the threshold for effective SK-BR-3 cell destruction, below which, the photothermal activity of the immuno-Au nanocages was insufficient to incur therapeutic effect. Compared with other immuno-Au nanostructures tested for cancer treatment effectiveness, the acuteness of Au nanocages becomes clear: with Au nanoshells (35 W/cm2) and Au nanorods (10 W/cm2) requiring much more power to produce therapeutic activity. It is proposed that this is most likely due to the larger absorption cross-section of Au nanocages or their greater concentration on cell surfaces.]9

1.5 Shapes and Sizes

1.6 Potential Applications of Silver Nanocubes

1.7 Surface Enhanced Raman Scattering Spectroscopy

1.7.1 Improving Upon the Raman Spectrum

When a molecule is exposed to laser light, most of the incident photons scattered will be done so by elastic Rayleigh scattering where there is no change between incident and scattered photon frequency. However, one in approximately a thousand photons is subject to inelastic scattering. This is registered as a fluctuation in vibrational energy of the molecule under investigation and gives rise to the weak Raman spectrum. If the molecule being investigated is situated near a single silver or gold nanoparticle this weak signal can be enhanced, which gives rise to the technique of Surface-enhanced Raman scattering (SERS): a vibrational spectroscopy technique which can greatly increase the Raman scattering cross-section of molecules within the proximity of a metallic nanostructure. [The magnitude of the Raman enhancement is thought to rely on three contributing factors: surface plasmon resonance (SPR); charge-transfer resonance (CTR); and molecular resonance. The metallic nanoparticle and the molecule whose Raman signal it is responsible for enhancing should be considered as one single system. The SPR is mostly a property of the metal nanoparticle; the molecular resonance is a property obviously belonging to the molecule being investigated; and the CTR states are properties shared by both molecule and metal nanoparticle. So in order to fully explain the SERS effect all three phenomena need consideration. However, at any single laser excitation frequency, quantification of the contribution from each resonance form can prove difficult. For the contribution from each resonance type to be more clearly identified, a wide variety of laser wavelengths need to be used to compare excitation, either by varying the potential applied to the system or by affecting SPR location through particle size control, or by interparticle distance. ]14

Enhancement of the weak Raman signal can be even more pronounced if the molecule under investigation is situated between two closely spaced silver or gold nanostructures, as opposed to an individual nanometallic entity. It is believed that the extremely high local electromagnetic fields associated with coherent conduction electron oscillations in the nanoparticles could enhance the weak vibrational Raman effect by a magnitude of 1014. This has been the basis for renewed investigation into the phenomenon of Surface Enhanced Raman Spectroscopy (SERS). It has been found that the potential for Raman enhancement relates strongly to the distance separating silver and gold nanoparticles. If this distance is optimized it is possible to create what are known as SERS 'hot-spots' which are capable of enhancing the Raman signal of molecules contained therein to such an extent that even an individual molecule can be detected. [Some of the more commonly used methods for producing SERS substrates involve salt induced aggregation which results in a rather uncontrollable SERS formulation; what with possible polydispersion and shape irregularity making it fairly tricky to correlate detection of scattering enhancement to any specific hot spot. An ideal system for quantitative investigation of hot spots has been implemented in a recent study by Xia et al in which individual dimers of Ag nanocubes had their hot spot isolated and probed. An individual silver nanocube dimer was functionalized with molecules of 4-methylbenzenethiol (4-MBT) creating a monolayer on the surface of the nanocubes, including the surface area in the narrow hot-spot gap between the two nanoparticles. When the dimer was plasma etched in a plasma cleaner/sterilizer (Harrick Scientific Corp., PDC-001) operated at 60 Hz and 0.2 Torr air for 2 min, the monolayer coating the dimer outwith the hot-spot was removed due to the hot-spot acting as a multilayer resist. The Ag nanocubes used had an edge length of 100 nm and had approximately 200 layers of 4-MBT residing in the hot-spot region which required a far longer plasma etching exposure to be removed compared to those coating the remaining surface area of the dimer. This demonstrates the usefulness of plasma etching for hot-spot isolation. The SERS signals obtained using nanocube dimers which exploited the hot-spot compared to single nanocubes was appreciable: the results indicated that the enhancement factor for the detection of 4-MBT by the nanocube dimer was 37 times greater than that experienced using individual nanocubes. The improved sensitivity of detection offered by exploitation of the hot-spot is shown in the spectra of figure 1.

Figure 1. The Raman Signals for 4-MBT

using a single silver nanocube (top) and

using a silver nanocube dimer (bottom).

Insets show the direction of laser polar-

ization. ]7

[Such hot-spot enhancement effects are not specific to dimers of nanocubes alone: numerous dimeric systems have been investigated as SERS substrates, including rounded nanoparticles, nanoshells, nanowires, and nanowires decorated with various shapes and sizes of nanoparticles. The cut and thrust of these studies suggest that enhancements in field strength within hot-spot regions strongly depend on the orientation of laser polarization, with the largest enhancement factors occurring when the direction of laser polarization was parallel to the hot-spot axis, i.e. across the junction between nanoparticles.]13

1.7. Non-destructive Pigment Identification in Art Conservation

In the field of art conservation, various analytical techniques such as chromatography, spectroscopy and microscopy are routinely used to identify the chemical constituents of paints and dyes for purposes of preservation and restoration. However, these techniques require relatively large sample quantities to be removed from what tends to be unique and irreplaceable works of art. By instead using surface enhanced Raman spectroscopy, artwork can be analyzed without sampling and in a non-destructive fashion. Such analysis is not limited to paintings; sketches, tapestries, pottery, sculptures and textiles have all been previously analysed.

[Recent work undertaken by Marco Leona1 and John R. Lombardi used SERS techniques to identify berberine (a yellow colored alkaloid dye) in a historical Tibetan textile. As the berberine molecule is positively charged, it was expected to show great affinity for Ag nanoparticles, thus making it particularly appropriate for SERS analysis. SERS spectra were obtained directly from minute fiber samples without prior extraction of the dye. Microscopic samples of silk thread believed to be coated in berberine were pretreated with HCl vapor in a microchamber for 15 min before being treated with 10 μl of citrate reduced Ag colloid on a microscope slide. The laser beam was focused on the Ag colloid cluster deposited on the surface of the fibers to obtain the Raman spectra which matched standards of berberine. Significantly, the identity of the berberine dye was established using only one 50th of the sample quantity required for standard HPLC analysis.

What their fiber study demonstrates is that the limit of analyte detection is hampered only by the volume and quality of the colloid used. Coupling advancements in sample handling techniques with improved Ag colloids and deposition practice will, before long, allow for even smaller samples to be used in SERS analysis.]12

Anthraquinones (reds) and flavonoids (yellows) have also been shown to be readily detected using SERS techniques. The silver nanoparticles can be deposited on the object of interest in the form of miniscule colloidal droplets that appear invisible to the naked eye; or a polymer gel can be used to extract tiny amounts of dyes present which can then be analysed by SERS.

The technique is still in its infancy in the art world and has yet to prove that it can tackle more difficult subjects such as oil paintings - where the minute quantities of organic materials are drenched in an oil binder and other substances which can interfere with the interactions between the silver nanoparticles and the organic molecules.

Post nanoscale miniaturization, there is a noticeable deviation in the functionality of materials. The most relevant example of such stark changes in property, following 'nano-turization', is exhibited by liquid colloids of metals. One of the benefits of this branch of 'bottom-up' chemistry nests in its architectural versatility, where the size, shape, surface and composition of the metal under investigation can be controlled, even tailored, to fit whatever requirement may be necessary. [Some among many of the varied and considerable repertoire of nanostructures assembled thus far include spheres, discs, rods, wires, stars, prisms, pyramids, tubes, dendrites and cubes.]1. These can all be created using various techniques of crystal seed growth with control over their rate of atomic addition in order to customize the aforementioned architectural parameters; with each parameter playing a role in the performance of any given nanomaterial.]OZIN

Silver constructs are at the leading edge of such study. [When reduced from a bulk composition, silver no longer displays its characteristic reflective luster, but exhibits specific optical properties spanning the visible and near-infrared regions of the electromagnetic spectrum; [which does not come as a complete surprise, given its long standing use in the field of photography.]]OZIN, 3

[In addition to these intriguing optical properties, silver is also the most electrically and thermally conductive of all metals, making it especially viable as an investigative platform to breach the gaps between emerging applications, such as conducting adhesives.]3

[Although this study mainly focuses on the unique localized surface plasmon resonance (LSPR) properties of silver and gold, LSPR is theoretically possible in any metal, for example aluminium; which can offer distinct advantages in refractive index sensitivity, alternative surface chemistries, as well as ultraviolet LSPR, a region where many organic molecules are active. The remarkable property of LSPR is bestowed when incident light induces the conduction electrons of the nanoparticles to oscillate at a resonance frequency characteristic of their shape, size and composition. This results in extremely intense absorption and scattering of light (up to one million times greater than the fluorescence of a fluorescein molecule) giving dynamic labeling capabilities. [This property lies at the heart of Surface Enhanced Raman spectroscopy (SERS) - a technique that amplifies the signal of inelastically scattered photons from molecules close to silver nanoparticles. The greatest Raman signal enhancement arises in localized 'hot-spots' - junctions or gaps between two or more nanoparticles in which interaction between surface plasmon oscillations occur, causing enormous electromagnetic enhancement compared with standalone nanoparticles. This phenomenon allows for the detection of single molecules.]7 [However, most common methods of Ag SERS substrate creation rely on largely uncontrolled aggregation techniques which can result in shape and size polydispersion of the particles leading to variation in SERS activity, with the random formation of 'hot-spots' accounting for much of this. The factors that control SERS profiles can be more closely investigated by controlling and reproducing assemblies of silver nanoparticles at pre-determined separation distances and orientations.]8

Most LSPR spectroscopy is performed using bulk concentrations of nanoparticles i.e. of a colloidal nature. However, recent research has revealed that individual nanoparticles could potentially serve as independent sensors.11,12,53. This would allow for heightened absolute detection limits (total number of molecules detected) and give improved signal to noise resolution for detection within e.g. cells and tissues.

The extinction and scattering spectra profiles of a nanoparticle - particularly of the λmax peak- depends on not only the outlined architectural parameters of composition, size, shape and orientation but also on the local dielectric environment of the nanoparticle. So, by close attention and guidance, the path of nanoparticle fabrication can be used to fine tune the LSPR via a variety of chemical syntheses.]6

[One such synthesis is the polyol synthesis. This is used to generate various forms of metal colloids from their salts by combination with a suitable polymeric capping agent heated in a polyol, such as ethylene glycol (EG). Of the various nanostructures, some of the greatest promise lies in silver nanocubes and their conversion to gold nanocages for use in photothermal treatment. Where silver nanostructures are concerned, AgNO3 and poly(vinyl pyrrolidone) (PVP) serve as the salt precursor and polymeric capping agent respectively. As well as acting as the solvent for the reaction, the EG also acts as the reductant for AgNO3 by undergoing oxidation in atmospheric O2 to form glycoaldehyde under heating:

As the reductant is continuously generated within the reaction vessel over time there are no problems arising from change in nanostructure growth kinetics.

Up until recently the challenges of synthesizing monodispersed nanocubes were compounded by the factor of time, with a typical polyol synthesis requiring a whole day before any significant agglomeration of silver atoms occurred. It was found that by mediating the polyol reduction of silver nitrate with a trace amount of sodium sulfide (Na2S) improved the production rate of silver nanocubes extensively i.e. within half an hour. The mechanism for such increased productivity is thought to rely on the formation of highly insoluble crystallites of Ag2S - a well known catalyst for the reduction of Ag+, as it significantly reduces the reduction potential compared to that of free Ag+. [In order to synthesize high quality monodispersed silver nanocubes, it is this reduction potential that has to be fine-tuned. This is achieved by optimizing the concentration of sulfide ions, with 28 - 30 µM presenting ideal reduction conditions. This has to be balanced with the reaction temperature in order to combat oxidative etching of newly forming silver seeds.]1 [If there is too much oxygen present during a synthesis, the oxidative etching of the small nuclei at early stages could greatly slowdown the growth of Ag nanocubes. It is also proposed that the formation of catalytic Ag2S crystallites could be disrupted, hindering the reduction of AgNO3. To combat the etching of nuclei the reaction can be carried out under an atmosphere of nitrogen or argon which allows undisturbed nucleation and formation of up to 97% single-crystal nanocubes.]2 The LSPR of these cubes - whose edge lengths can be varied from approximately 40 to 65 nm - becomes blue-shifted as the edge length decreases and a spherical shape is approached.

Nanocubes can be used as sacrificial templates for the generation of gold nanocages via galvanic replacement between Ag and HAuCl4:

The LSPR of the hollow gold cages can be fine tuned to any position in the visible and near-infrared regions simply by adding more or less aqueous HAuCl4 solution to a boiled suspension of Ag nanocubes. Depending on the intended purpose of the Au nanocages, the reaction can be stopped at any time by halting the addition of HAuCl4 to give nanocages with specific porosity matching a unique LSPR. At the conclusion of the reaction, even when the LSPR is shifted into the near-IR region, the Au cages will still contain minute traces of Ag as complete stoichiometric conversion to Au results in cage fragmentation. Au nanocages have great potential for biomedical applications such as cancer diagnosis and treatment; contrast enhancement agents and photothermal therapy.

Project Aims:

To create differently monodispersed batches of silver nanocubes and investigate how any changes in monodispersion are related to controllable experimental parameters e.g. sulfide concentration.

To convert monodispersed silver nanocube samples into their correspondingly monodispersed gold nanocage counterpart via galvanic replacement.

To compare and contrast LSPR peaks exhibited by various shapes and sizes of nanoparticle; including Ag spheres, cubes and porous Au nanocages.

To conduct preliminary Raman measurements with Ag nanocubes and Au nanocages.

Controlling the assembly of silver nanocubes through selective functionalization of their faces - Get a few references in there.

Gold nanocages: synthesis, properties, and applications - Talk more about the properties of the gold cages.

Get DPN information from Ozin.

[from the bottom up rather than the top down. Though they do not entirely disregard the latter approach e.g. nanolithography.]OZIN

Experimental Section



Ethylene glycol (J.T. Baker, cat. no. 9300) m CRITICAL Select Lot no.'s with low

Fe and Cl content; for example, Lot no.'s B25B15 with Cl o 1 p.p.m. and Fe = 0.04 p.p.m., C42B27 with Cl o 0.1 p.p.m. and Fe ¼ 0.12 o p.p.m. and

C46B29 with Cl o 1 p.p.m. and Fe ¼ 0.2 p.p.m. have all yielded high-quality

Ag nanocubes. Also note that ethylene glycol is extremely hydroscopic; we

recommend replacing the bottles approximately every month or sealing a fresh

bottle of ethylene glycol with an air-tight dispenser (e.g., VWR Labmax Bottle-

Top Dispenser) for repeated, reliable use.

AgNO3 (more than 99%; Sigma-Aldrich, cat. no. 209139)

PVP, powder, average Mr E 29,000 or 55,000 (Sigma-Aldrich, cat. no.

234257 or 856568)

Na2S.9H2O (J.T. Baker, cat. no. 3910)

Acetone (reagent grade)

Ethanol (reagent grade)

18.1 MO cm E-pure water

HAuCl4 _ 3H2O, 99.9+% (Sigma-Aldrich, cat. no. 520918) m CRITICAL Store

in a foil-wrapped desiccator to avoid light-induced decomposition

Sodium chloride (NaCl)crystal (J.T. Baker, cat. no. 3624)


Stirring hotplate with temperature controller (e.g., Corning 6795-420D

digital display hotplate, 5 in _ 7 in, 60-1,100 r.p.m., and the corresponding

Corning 6795PR temperature controller; Corning)

Crystallization dish, 100 mm _ 50 mm (e.g., Ace Glass Inc., cat. no.


Silicone fluid (e.g., Thomas Scientific, cat. no. 6428-R15)

Vial holder, custom-made (see EQUIPMENT SETUP)

Disposable 6 drams (24 ml) borosilicate vials with paper-lined plastic caps

(VWR, cat. no. 66011-143) m CRITICAL It is important to use VWR, cat. no.

66011-143 vials as switching to other vials has altered the shape of the final

product, presumably due to differences in reaction mixing and the rate of

ethylene glycol dehydration.

Four rubber O-rings, inner diameter approximately 23 mm

Teflon-coated, egg-shaped magnetic stir bars (dimensions: 5/8 in _ 1/4 in;

VWR, cat. no. 58949-010) m CRITICAL It is important to use VWR, cat. no.

58949-010 stir bars (or those with exactly the same dimensions and shape)

as other stir bars have altered the reaction, presumably due to differences in

reaction mixing.

Two micropipettes (ranges: 10-100 ml and 100-1,000 ml) with appropriate

disposable tips

50-ml, 24/40 round bottom flask with single short neck (e.g., Ace Glass Inc.,

cat. no. 4120-21)

Bushing-type adaptor, 10/30 female within 24/40 male (e.g., Ace Glass Inc.,

cat. no. 5021-09)

Septum for covering the bushing-type adaptor hole


Poly(propylene) centrifuge tubes, capacity 50 ml (e.g., CLP-PGC Scientifics,

cat. no. 2553)

Poly(propylene) micro-centrifuge tubes, capacity 1.5 ml (e.g., Fisherbrand,

cat. no. 05-406-16)

Ultrasonic cleaning bath (e.g., Branson Ultrasonic, cat. no. CPN-952-116)

Programmable syringe pump with digital display (e.g., KD Scientific

Single-Syringe Infusion Pump; KD Scientific, cat. no. KDS100 230)

Optional: heating mantle (e.g., Fisher Three-in-One Heating Mantle; Fisher,

cat. no. 12-142-3)

Disposable plastic syringe, volume 10 ml (e.g., BD Vacutainer, cat. no.

309604 EMD) with poly(vinyl chloride) (PVC) tubing (e.g., VWR brand

'Select Grade' PVC tubing, size: 1/32 in _ 3/32 in _ 1/32 in; VWR,

cat. no. 60985-501) for solution delivery and any necessary adaptors

Scanning electron microscope (SEM); for characterization

Doped-silicon wafer chips; for SEM sample preparation

Transmission electron microscope (TEM); for characterization

Carbon-coated copper TEM grids; for TEM sample preparation

UV-visible spectrometer; for recording absorbance spectra

Synthesis of Ag nanocubes

All stirring bars and glass vials used during the preparation of all nanostructures were cleaned thoroughly before use by soaking in aqua regia for 1 - 2 hours. After which, they were rinsed with distilled water which was then neutralized with sodium carbonate and washed down the sink with excess water. The stirring bars and vials were oven dried until use.

For Ag nanocubes, with constant stirring, an oil bath was heated to 150 °C using a heating mantle. Using a micro-pipette, 6 ml of ethylene glycol (EG) was transferred into each of four 24 ml glass reaction vials, also adding one of the small cleaned stirring bars to each. The vials were immersed in the oil bath with their caps loosely placed, and the EG allowed to react to form glycoaldehyde for 1 hr.

During this hour, a poly(vinylpyrrolidone) (PVP; MW ~ 55000) in EG solution of concentration 20 mg/ml was created. The 7 ml of EG added to the fresh vial preceded the addition of PVP to prevent adhesion to the glass.

Also, a 3 mM Na2S in EG solution was prepared. First, approximately 0.01 g sodium sulfide was placed into a new vial and then the necessary volume of EG required to make up a 30 mM solution was added. From this solution, 100 μl was transferred to another disposable vial, on top of which was added a further 900 μl EG to produce the final 3 mM solution. Due to evaporation and degradation of sulfide species the solutions were required to be as fresh as possible, ideally being prepared within fifteen minutes of use.

An AgNO3 in EG solution was also required. For this solution a concentration of 48 mg/ml AgNO3 was necessary, so 0.12 g AgNO3 was transferred into a fresh vial followed by 2.5 ml EG. The solution vial was wrapped in tin foil and stored in darkness until required.

Just before the EG had finished pre-heating for 1 hr, the Na2S solution was agitated using a vortex mixer. After agitation 70, 80, 90 and 100 μl of the 3 mM Na2S solution was transferred via micropipette to each of the four vials, respectively. The caps were re-placed loosely and approximately 9 min allowed to elapse. 1.5 ml of the PVP solution was then added into each vial in two 0.75 ml aliquots, followed immediately by 0.5 ml of the AgNO3 solution to each vial. The caps were again loosely re-placed on the vials, and the reaction was allowed to proceed until the media turned a dark green with a red tint visible throughout (15 - 20 min).

The reaction was quenched by placing the vials in a cold water bath at 5 - 7 °C. After cooling, the contents of each vial were transferred into their designated 50 ml centrifuge tube. Each vial was rinsed with double the reaction volume of acetone, and the washings rinsed into their respective centrifuge tube. Each tube was spun down at 2000g for 30 min. The supernatant was removed and discarded.

Approximately 2 ml of deionized water was then added to each centrifuge tube and agitated via sonication to re-disperse the products. All attainable products were transferred to 1.5 ml volume centrifuge tubes and spun down at 9000g for 10 min.

The supernatant was removed and discarded, the products re-dispersed in deionized water, and the process repeated twice more to obtain as clean a product as possible.

The triple washed nanocubes were transferred to a clean scintillation vial, diluted to 4 ml with 18.1 MΩ cm E-pure water, sealed and wrapped in tin foil. This should be sufficient to ensure the preservation of the nanocubes for approximately 2 - 3 months.

Synthesis of Ag nanocubes - Scaled-up

Using multiple 6 dram reaction vials for the synthesis of Ag nanocubes presented problems in terms of both the quantity and quality of the cubes created, mainly as a result of the innate lack of precise control over Na2S concentrations. With perhaps the main focus for Ag nanocube application lying in the ease with which their surface plasmon resonance peaks (SPR) can be tailored via galvanic replacement reaction to form gold nanocages, the utilization of a more reliable and productive synthesis was necessary. While the previous small scale reaction was carried out under an atmosphere of air and worked reasonably well, this was only due to the small surface area of reaction media exposed to the oxygen in the atmosphere for short periods, which was quite controllable. In an up scaled set-up, the oxygen-surface interface would be far greater, and so too the likelihood of chemical etching occurring to newly forming nanocube seeds.

Synthesis of Au nanocages

Prepare at least 10 ml of a 9 mM PVP solution in milipore. For each titration 5 ml is required.

Also, prepare at least 10 ml of 0.1 mM HAuCl4 in milipore. Prepare this from a 10 mM stock solution by adding 0.1 ml of the stock to a 10 ml volumetric flask and then filling to the line with milipore. Both solutions should be wrapped in tin foil.

Pipette a 5 ml aliquot of the PVP solution into a 50 ml round bottomed flask to which a clean stirring bar has also been added. Pipette 100 μl of the stored Ag nanocubes into the PVP solution; attach the bushing-type adaptor to the top of the round bottomed flask, covering the adaptor's hole loosely with a rubber septum. Heat to a mild boil for approximately 10 min.

Load the HAuCl4 into the disposable plastic syringe equipped with PVC tubing and put it into the syringe pump. Remove the rubber septum from the adaptor. With the pump add a specific volume of the HAuCl4 solution to the reaction flask at a rate of 0.75 ml of solution per minute. Stop the addition of HAuCl4 solution when the desired colour is reached, record the volume and then allow to reflux for an additional 10 min before allowing to return to room temperature.

Naming format for UV - Vis: "Ag nanocubes Xx dilution in EG - uncleaned/cleaned"

24/11/09 - First reasonably successful synthesis of Ag nanocubes achieved. Orange in colour. 100 μL Na2S added. UV - Vis of uncleaned sample taken at 3x dilution in EG. Clean sample yet to be taken.

26/11/09 - Two very different samples obtained: the first was brown with the majority of the silver becoming plated on the interior of the reaction vial. 200μL Na2S was added. The UV - Vis was conducted undiluted and was atrocious. Sample to be discarded.

The second synthesis - after an initial addition of 100 μL Na2S - appeared ever so faintly orange, even after ten minutes. So, during the reaction a further 400 μL Na2S was added in four 100 μL aliquots. This really seemed to kick start the reaction, which subsequently went to completion within a further 8 - 10 minutes, finishing up as a pale green with a red tinted meniscus. The UV - Vis of the uncleaned sample was taken at 16.67x dilution. The cleaned sample I do believe was centrifuged at the wrong r.p.m. and as a result the clean sample consists fairly strongly of just about everything, providing a less than ideal UV - Vis curve.

4/12/09 - Two out of four reaction vials went to completion: one with addition of 90 μL of Na2S, the other with 100 μL. The UV - Vis for both uncleaned samples was carried out revealing the almost exclusive creation of cubes. The UV - Vis of the cleaned samples has yet to be taken; as well as one of some remaining uncleaned 90 μL to examine how much the passage of time affects the deterioration of the sample prior to cleaning. Possibly do the same with the 100 μL sample.

7/12/09 - The creation of cubes was today successfully scaled up (more or less by a factor of ten). So, providing the cubes are of decent enough quality, we can trot along and galvanically replace them with gold cages! The reaction was carried out in a 250 ml round bottomed flask with 0.7 ml Na2S, 15 ml PVP, and 5 ml AgNO3 EG solutions being added to the initially heated 60 ml EG. Dirty sample still to have UV - Vis analysis. Clean sample tomorrow and run analysis.

10/12/09 - Just for fun, an attempted synthesis of highly branched dendrites was undertaken. Today (14/12/09) during SEM analysis it was found that the synthesis had not quite worked according to plan, with a single, dense, mountainous sheet being formed as opposed to multiple branched structures.

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