NANOMATERIALS FOR SENSING, HEATING AND APPLICATIONS IN COMPOSITES AND EMULSION BIOFOULING

Nanoscale materials often exhibit fascinating physical, chemical, and biological properties which are considerably different from those of their macro-and/or microscale counterparts. These unique characteristics have led to considerable growth over the last decade in nanomaterial-enabled applications in areas such as biomedicine, environmental sensing and remediation, and energy production and storage. In this dissertation, techniques are presented to create new nanomaterials relevant to these areas. Gold nanostructures are an example of a nanomaterial with great interest in biomedical therapy and sensing applications due to their high chemical and physical stability, ease of synthesis and surface functionalization and their unique optical properties. High photothermal heat generation efficiency and their biocompatibility have made them an effective tool for thermal destruction of cancer cells. Magnified electromagnetic field on their surfaces allows gold nanostructures to be used as ultrasensitive nano-sensors for analyte detection using surface-enhanced Raman scattering (SERS). There remains significant interest in further improving the synthesis and design of gold nanostructures to maximize their potential applications. In biomedicine, for instance, the absorbance spectra of gold nanostructures need to be stable and tuned to the near-infrared (NIR) window (650–1350 nm) where biological tissues have minimal light absorption, while yet it is also desirable for the nanostructures to degrade to avoid accumulation in the body after treatment. Likewise in SERS applications, it is important that the nanostructures are stable and resist surface fouling, while yet analytes with low affinity for the metal surface must be enriched at the surface to overcome poor detection limits. A versatile templating strategy was developed to create gold nanoshells on different dielectric cores for photothermal heating and sensing applications. The strategy allows for tunable NIR absorbance and high degradation capabilities upon laser irradiation using soft templates. When carbon nanomaterials are used as the template, the carbon improves the affinity of different analytes to the surface of the hybrid nanostructures, yielding sensitive SERS-active materials for monitoring aqueous pollutants. In the area of environmental remediation, nanoparticles with mixed wettability properties have been recently explored as alternative oil spill dispersants to help avoid the negative effects of surfactants on marine species. Adequate dispersing qualities and low toxicity are generally considered as the criteria for a good oil dispersant. However, their impact on the oil biodegradation process is still poorly understood. The attachment of bacteria to the oil/water interface, as the first step in oil biodegradation, was investigated to study the potential inhibitory effect of two commercially available dispersants, Corexit-9500 and Tween 20, and also carboxyl-terminated carbon black nanoparticles on biodegradation process. Finally, conductive nanoparticles were used to enhance the electrical conductivity of polymer nanocomposites. While graphene, with a high aspect ratio and excellent conductivity, seems a promising filler to induce conductivity into a polymer, dispersing it uniformly within a polymer network remains a challenge due to their strong attractive forces. To assist dispersion, carbon black nanoparticles were used as a secondary filler to prevent restacking the graphene sheets within the polymer matrix. As a result, the electrical conductivity of the composite was significantly increased and sustained even at high nanoparticle loading.

dissertation, techniques are presented to create new nanomaterials relevant to these areas.
Gold nanostructures are an example of a nanomaterial with great interest in biomedical therapy and sensing applications due to their high chemical and physical stability, ease of synthesis and surface functionalization and their unique optical properties. High photothermal heat generation efficiency and their biocompatibility have made them an effective tool for thermal destruction of cancer cells. Magnified electromagnetic field on their surfaces allows gold nanostructures to be used as ultrasensitive nano-sensors for analyte detection using surface-enhanced Raman scattering (SERS). There remains significant interest in further improving the synthesis and design of gold nanostructures to maximize their potential applications. In biomedicine, for instance, the absorbance spectra of gold nanostructures need to be stable and tuned to the near-infrared (NIR) window (650-1350 nm) where biological tissues have minimal light absorption, while yet it is also desirable for the nanostructures to degrade to avoid accumulation in the body after treatment. Likewise in SERS applications, it is important that the nanostructures are stable and resist surface fouling, while yet analytes with low affinity for the metal surface must be enriched at the surface to overcome poor detection limits.
A versatile templating strategy was developed to create gold nanoshells on different dielectric cores for photothermal heating and sensing applications. The strategy allows for tunable NIR absorbance and high degradation capabilities upon laser irradiation using soft templates. When carbon nanomaterials are used as the template, the carbon improves the affinity of different analytes to the surface of the hybrid nanostructures, yielding sensitive SERS-active materials for monitoring aqueous pollutants.
In the area of environmental remediation, nanoparticles with mixed wettability properties have been recently explored as alternative oil spill dispersants to help avoid the negative effects of surfactants on marine species. Adequate dispersing qualities and low toxicity are generally considered as the criteria for a good oil dispersant. However, their impact on the oil biodegradation process is still poorly understood. The attachment of bacteria to the oil/water interface, as the first step in oil biodegradation, was investigated to study the potential inhibitory effect of two commercially available dispersants, Corexit-9500 and Tween 20, and also carboxyl-terminated carbon black nanoparticles on biodegradation process.
Finally, conductive nanoparticles were used to enhance the electrical conductivity of polymer nanocomposites. While graphene, with a high aspect ratio and excellent conductivity, seems a promising filler to induce conductivity into a polymer, dispersing it uniformly within a polymer network remains a challenge due to their strong attractive forces. To assist dispersion, carbon black nanoparticles were used as a secondary filler to prevent restacking the graphene sheets within the polymer matrix. As a result, the                  When the size of a metallic particle decreases to smaller than the wavelength of a photon, the oscillating electric field of incident light polarizes the conduction electrons over the whole volume of the nanoparticle. The displacement of electron cloud from the positively charged lattice generates a restoring Coulombic force to pull back the polarized electrons. 9 This collective oscillation of free electrons in resonance with incident light is called localized surface plasmon resonance (LSPR) 10   induced heat is generated to kill the surrounding cancer cells. 17 The incident light which excites the plasmons is absorbed by the gold nanoparticles and causes vibrations of the metal lattice that is converted to heat. The high photothermal conversion efficiency of gold nanoparticles, as well as their tunability to near-infrared (NIR) wavelengths, has made them attractive for photothermal therapy. 18 Biological tissue and blood show maximum optical transmissivity in the 650-1350 nm wavelength range (NIR window). 19 Plasmonic nanoparticles can be accumulated into tumors via the enhanced permeability and retention (EPR) effect 20 and then illuminated with NIR laser light. The heat generated from the absorption of the NIR light leads to a temperature increase above body temperature (37°C), causing irreversible photothermal destruction of tumor tissue while avoiding damage to healthy cells (see Figure 1.2). 21 Recently, gold nanoparticle-mediated photothermal therapy in combination with other therapeutic approaches such as chemotherapy, immunotherapy and gene regulation has been explored as an enhanced multimodal approach for cancer treatment. [22][23][24] Optimal size and structure of nanoparticles play critical roles in their performance. Small particles(<20nm) are rapidly cleared through the renal system while larger particles accumulate into the body after treatments since they are nonbiodegradable. 25 Gold nanoparticle shape can also be engineered to transport drugs which enable them to be used in the targeted photo-mediated drug delivery. 26 While loading drugs onto the exterior surface of particles is an easy approach through Au-thiol bonding, the drugs are susceptible to immune recognition and degradation. 27 The first use of gold nanoparticles in targeted photothermal therapy was conducted using NIR responsive gold nanoshells formed on silica templates. 28  LSPR excitation also results in an amplification of local electric field at the surface of gold nanoparticles that can enhance the inherently weak Raman signal from molecules in close proximity to the surface. 31 This phenomenon, called surfaceenhanced Raman scattering (SERS) was first observed in the spectra of pyridine on a roughened silver surface in 1974. 32 After the enhancement mechanism was discovered in 1977, the interest in SERS has grown exponentially. It is becoming one of the most popular ultrasensitive spectroscopic techniques in medicine, biology and environmental monitoring. 33 Raman scattering is an extremely weak process in which only one in every 10 8 photons scatters inelastically. 34 The intensity of the scattered radiation is

LIST OF TABLES
proportional to the square of the magnitude of the electromagnetic field of light incident on the analyte (I α E0 2 ). 35 In SERS due to the evanescent oscillation of surface plasmons, the electromagnetic field is the field amplified by the LSPR. Thus, the SERS intensity is enhanced compared to the Raman intensity of the analyte by an enhancement factor (EF) as: where, and are the magnified local fields generated by the incident light and the scattered light, respectively. 35 The maximum enhancement occurs in narrow gaps between gold nanoparticles and at sharp nanoscale tips and crevices within individual anisotropic nanoparticles due to the associated high confinement of the electrons. 7 Various anisotropic nanoparticles such as nanorods, nanocubes, nanostar, nanoplates, and nanoflowers have been synthesized with regions of high curvature to intensify local electromagnetic field, termed as "hot spots". 36 In addition to the number of hot spots per particle, the wavelength that nanoparticles are excited can affect the magnitude of enhancement. The best spectral location of the LSPR for maximum enhancement is in the close proximity of the laser excitation wavelength. 35 In this regard, gold nanoshells can be an ideal candidate as reproducible SERS substrates because of the ease of their LSPR peak adjustment to the desired wavelength. 37 Wang et al. 38 showed that individual gold nanoshells unlike gold nanospheres, are SERS active due to the interaction of inner and outer shell surfaces and can be used for sensing applications.
However, surface plasmons have evanescent wave character and the enhanced electromagnetic field penetrates just a short distance from the metal-dielectric interface (up to 5nm). 39 So the presence of probe or analyte molecules in close proximity to the metal surface is a critical factor in Raman enhancement that makes the detection of those molecules with low affinity for metal surfaces such as polycyclic aromatic hydrocarbons (PAHs) a big challenge. Some current techniques introduce various metal surface functionalization to capture such analytes for the surface of nanoparticles. 40 However, the inherent complication of this approach, as well as the possibility for the surface passivation by surface ligands, restrict the broad applicability of this method.
Combination of gold nanoparticles with different carbon nanomaterials (such as graphene, carbon nanotubes and graphene oxide) have also been investigated as an effective alternative approach in selective trapping aromatic molecules. 41 A focus of this dissertation is to develop a novel templating strategy for the synthesis of gold nanoparticles with different functionalities. Gold nanostructures are formed on different dielectric cores and optimized for photothermal heating and sensing applications. Photothermal efficiencies for hollow gold -liposome nanostructures and also the SERS detection of a variety of analytes in aqueous solutions using gold-carbon nanoparticles, are studied.

Bacterial Oil Biodegradation
Oil biodegradation is the major natural process for the treatment of oil pollution in seawater. Due to the existence of naturally occurring hydrocarbons in all marine environments, numerous microorganisms have evolved to utilize hydrocarbons as a major source of energy for growth. 42 These microorganisms are ubiquitous in nature and dominate microbial communities after an oil spill. 43 Alcanivorax spp., for example, were shown to compose 70-90% of the prokaryotic cells within 1-2 weeks of entering oil to seawater. 44 The initial step in the biodegradation process is to transform the terminal carbon into a primary alcohol through membrane-bound oxygenase 45 , so direct contact with the hydrocarbon substrates is an essential step for bacterial oil degradation.
However, the low solubility of many oils in water has made the oil bioavailability a limiting factor in bacterial oil degradation. 43 One biological strategy to enhance the bioavailability of water-insoluble hydrocarbons is emulsification of the hydrocarbon by microbially-synthesized surfactants. 46 All strains growing on oil as the sole source of carbon and energy produce a broad range of biosurfactants ranging from low molecular weight lipopeptides and glycolipids to high molecular weight compounds such as polysaccharides, lipopolysaccharides, lipoproteins. 47 However, in an oil spill where large volumes of petroleum hydrocarbon enter into an open system, bio-emulsification never reaches a high enough value to effectively emulsify oil. 42 Dispersants are routinely applied to oil-contaminated waters during a response to marine oil spills. In the Deepwater Horizon oil spill in 2010, which resulted in the release of about 5 million barrels of crude oil, nearly 1.8 million gallons of Corexit 9500 were used to emulsify oil emanating from the seafloor, as well as break up surface slicks. 48 Toxicity effects such as disruption of cell membranes, interference with cell membrane surface receptors, reactions with cellular components or irreversible blockage of enzyme active sites resulting from the uptake of dispersants by the bacteria have been reported for different dispersants. 49 However, their impact on the oil biodegradation process is still poorly understood. At first, the dispersants were believed to promote biodegradation rate based on their ability to generate micron-sized droplets and enhancing the oil-water interface area. However, many studies have shown that dispersants practically either make no difference or even inhibit biodegradation. 50,51 Efforts to develop safer and more effective dispersant are ongoing. In this regard, colloidal particles such as carbon-based nanoparticles, silica, and clay nanoparticles have recently been examined as effective alternative marine oil spill dispersants. [52][53][54] Adequate dispersing qualities and low toxicity to marine species have generally been assumed as the criteria for a good oil dispersant. While attachment is a vital step for bacteria to degrade the oil, potential inhibitory effects of a dispersant is a critical factor that should be considered when developing alternative dispersants. Therefore, a focus of this dissertation is to explore the way in which an oil-degrading bacterium interacts with an oil-water interface populated with different dispersants, by using fluorescence microscopy and cryogenic scanning electron microscopy (cryo-SEM) to image cells at the oil-water interfaces.  Various conductive fillers such as carbon blacks, graphite, carbon fibers and nanotubes, metal particles have been used to impart conduction to polymers. 59, 60 The selection of appropriate filler materials is essential to achieve the desired properties of the composites. Metal particles have high intrinsic electrical conductivity but show a low tendency to form a conductive network. Fractal structured carbon black nanoparticles at high volume loading are extensively used in rubber composites as a filler to improve the mechanical performance and to impart electrical conductivity to prevent static charge buildup. 61 Providing a low loading at percolation is a critical aspect for easier processing, lowering the final cost and also for mechanical properties, as fillers can act as nucleation sites for crack growth. 62 Fillers with high surface area and easier distribution into a non-conductive matrix provide high conductivity enhancement at lower loading levels. 63

Electrically Conductive Composites
Graphene is a one atom-thick sheet of hexagonally arrayed sp 2 -bonded carbon atoms, first fabricated in 2004. 64 Since then, graphene has attracted considerable attention as an ideal nanofiller to modify different polymers owing to its outstanding properties; their high aspect ratio and exceptional in-plane electrical conductivity has enabled them to provide conductivity to the polymer with minimum loading. If graphene sheets are modeled as ideally dispersed and randomly rotated disks of aspect ratio AR (AR = disk diameter/thickness), the percolation threshold ϕc is given by ϕc = 1.5(ϕsphere/AR). 65 Here, ϕsphere is the percolation threshold for monodispersed spheres, i.e., ϕsphere = 0.29. Considering AR value of around 10 4 for graphene, the percolation threshold ϕc can be theoretically lowered as low as 0.001; but their high surface area also promotes their agglomeration by van der Waals interactions. 66 Their uniform dispersion in a host polymer is still a challenge.
The key goal of this project is to fabricate conductive graphene-polymer composite by developing a method to enhance the dispersion of graphene sheets. A third component was used as a secondary filler which is dispersed throughout the polymer network during processing to prevent graphene sheets restacking. The result of this study should be useful in lowering the graphene loading in conductive composites and consequently lowering the final cost.

Introduction
Gold nanostructures have attracted significant interest in medicine because of their biocompatibility, localized surface plasmon resonance (LSPR), and facile conjugation to a variety of biomolecular ligands. [1][2][3][4][5] Their LSPR can be tuned to near infrared (NIR) wavelengths by engineering their morphologies. 6-8 NIR radiation can penetrate into body tissue and blood to depths of several millimeters, 9 making these particles useful for biomedical imaging and photothermal therapy. [10][11][12] Averitt et al. 13 synthesized spherical nanoparticles consisting of a gold shell and a dielectric core. Reducing the shell thickness led to increased interactions between the inner and outer surface plasmons that produced a significant red shift in the absorption spectrum. 9,14,15 Moving the SPR peak from ultraviolet-visible (UV-Vis) to NIR wavelengths by adjustment of the shell thickness was also shown by Neeves et al.. 16 By irradiating gold-silica nanoshells with a NIR laser, Hirsch et al. 17 demonstrated photothermal destruction of targeted cancer cells. 18 Although the formation of shells over hard cores leads to uniform shell thicknesses, the key disadvantages of these structures for therapy are that they are nonbiodegradable and not easily cleared through the renal system. 2 One strategy for making plasmon resonant nanostructures with good biocompatibility and degradation capabilities is to use liposomes as templates, rather than hard inorganic cores. Troutman et al. 19 developed biodegradable plasmon resonant structures that had a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposome core and a gold shell. Upon NIR irradiation, the lipid membranes were disrupted and the gold shells degraded into 5−8 nm nanoparticles. 2,19,20 Thus, hydrophilic and/or hydrophobic drugs can be encapsulated within these gold-coated liposomes, and these drugs can be released by NIR triggering. [21][22][23][24] The gold nanoparticles produced after NIR irradiation can be easily cleared through the renal system. 25 Reduction of gold ions on liposome templates is a facile one step process. To avoid salt induced aggregation of the liposomes, the gold ion concentration in the surrounding solution must be limited.
Rather than a continuous gold shell, this leads to arrays of gold nanoparticles on the liposome surface. 26 We propose that a cationic polyelectrolyte overlaid on a liposome surface can significantly enrich that surface either with anionic gold precursor ions, or with anionic gold nanoparticles that act as heterogeneous nucleation sites for further growth. The intermediate polyelectrolyte also removes direct interaction of gold nanoparticles with lipids, stabilizing the liposome structure upon absorption of the charged gold nanoparticles. [27][28][29][30] We used poly-l-lysine (PLL)-coated liposomes, referred to as layersomes, as templates to prepare gold-layersome nanoshells. The shells were synthesized using two techniques, outlined in Figure 2 was then added. The Au seeds act as heterogeneous nucleation sites, and the slow formation of metallic gold upon addition of AA to this growth solution allowed a shell to be formed on the uncoated parts of the surface. Although this two-step process has been used previously to create nanoshells on hard templates, [16][17][18]31 this is the first report of the seeded growth method being used to obtain gold nanoshells on a polyelectrolyte-  (NaOH) and ascorbic acid (AA) were purchased from Sigma-Aldrich. All materials were used as received.

Preparation of layersomes
A 10 mM solution DOPC:DOPG at a molar ratio of 1:1 was formed in chloroform.
The solvent was removed in a rotary evaporator, and a thin lipid film formed on the sides of a round-bottom flask. The dry film was hydrated with deionized water and then vigorously vortexed at 50°C, well above the gel-fluid transition temperature for either lipid. 1 ml aliquots of this suspension were extruded 11 times at room temperature in an Nano ZS was used to determine the hydrodynamic diameter of the liposomes and layersomes, and the zeta potentials of the liposomes, layersomes, and Au seeds.
Absorption spectra were collected using a Perkin-Elmer Lambda 1050 UV-Vis-NIR scanning spectrophotometer over wavelengths from 500 nm to 1200 nm. All samples were diluted four times in water for absorption measurements. Absorption from deionized water was used as the reference.

Photothermal behavior
The photothermal behavior of the nanoshell suspension was probed using an 810 nm NIR laser with a power rating of 20 W/cm 2 . The experimental setup is shown in  We note that the sizes of the layersomes shown in Figure 2.3d are not representative of the overall distribution.       molar ratio of 0.1:1, the spectrum is similar to that from a gold nanoparticle suspension.

Gold-layersome nanoshells-direct reduction
As gold deposits on the seeds, they form islands on the layersome surfaces, as shown in Figure 2.6b, and their interparticle distance is too large to produce plasmon coupling.
As the [HAuCl4]:[lipid] molar ratio increases to 1.5:1, more gold is deposited, the interparticle distance decreases, and the absorbance peak red shifts until the shell is complete (Figures 2.6c,d). Energy-dispersive X-ray spectroscopy (EDS) displays colocalization of phosphorous, coming from the liposome, and gold, confirming the presence of gold on the surface of the layersomes (Figure 2.6c). Beyond a [HAuCl4]:[lipid] molar ratio of 1.5:1, additional gold formation increases the shell thickness ( Figure 2.6e), and the absorbance spectrum blue-shifts, because of decreased coupling between surface plasmons in the inner and outer surfaces of the shell. 9,35 We followed standard procedures for sample preparation for cryo-SEM to further examine these particles. These structures, shown in Figure 2 Kabs and h were obtained first by doing a two-parameter fit for the data from the sample prepared through the seeded growth method, giving Kabs = 0.37 and h = 34 W/m 2 K. Keeping h fixed, Kabs for the direct reduction sample was determined by fitting that experimental data to Eq. (2). Using these fitted values for Kabs, 37% and 29% of the laser power is being converted to heat in the gold-layersome nanoshell suspensions prepared by the seeded growth and direct reduction methods respectively. Figure 2.8b shows greater absorption at 810 nm for the seeded growth sample, in agreement with the trend in the transient temperature. We note that about 5% of the laser power is absorbed by just the water, the vial and thermocouple. This is a small effect compared to absorption in the presence of the gold-coated layersomes. nanoparticles. This degradation was confirmed by TEM images of samples after NIR irradiation, shown in Figure 2.8e,f.

Conclusion
Two methods have been used for the synthesis of gold nanoshells on layersome surfaces. In the direct reduction approach, reducing tetrachloroauric acid with ascorbic acid leads to the covering of the layersome surface with gold nanoparticles. Absorbance spectra exhibit red shifts as the tetrachloroauric acid to lipid ratios are increased as a result of the greater proximity of gold nanoparticles formed on the surface. When gold nanoparticles were used as heterogeneous nucleation sites, the absorption spectra were broad and red-shifted at low gold growth solution concentrations as complete shells formed. At higher gold growth solution concentrations, the spectrum showed a blue shift because the increased shell thickness resulted in less coupling between plasmons at the inner and outer surfaces. NIR illumination at 810 nm for the nanostructure suspensions showed a distinct temperature rise, indicating that the gold−layersome nanoshells absorb strongly at that wavelength and dissipate that energy as heat. Repeated NIR irradiation degraded the gold coating on the surfaces of the layersomes into 5−10 nm gold nanoparticles that can be cleared through the renal system. Thus, the NIR-triggered gold−layersome nanoshell suspension has the potential to be used in photothermal therapy and photothermally targeted drug delivery as well as in imaging.

Introduction
Engineering the shape and surface topology of gold nanostructures allows tuning of their localized surface plasmon resonance (LSPR) wavelength, making them attractive for sensing, photothermal applications and imaging. [1][2][3][4] Upon excitation at appropriate wavelengths, the collective oscillation of surface plasmons can result in strong absorption of the incident energy. 5,6 If the surface topology of these particles has nanoscale features, such as sharp tips and edges, the electric field near these features, termed hot spots, is enhanced significantly due to the high confinement of electrons. 7 The amplified electric field enhances the inherently weak Raman signal from molecules in close proximity to the surface, a phenomenon called Surface Enhanced Raman Scattering (SERS). 8 As a result, it is possible to detect organic, inorganic and biological molecules at low concentrations with SERS. [9][10][11] Different size and shaped particles, such as nanorods, nanotriangles, nanocages and nanostars have been synthesized for SERS applications. [12][13][14] The maximum Raman signal enhancement can be expected when the excitation wavelength matches the peak LSPR wavelength for the particle. 15 Core-shell structures are promising as SERS substrates as their LSPR peak can be adjusted to a desired wavelength by varying the size and/or the shell thickness. [16][17][18] The development of robust SERS substrates with high enhancement factors for the detection of a wide range of molecules and ions is an active area of research, [19][20][21][22] and is the focus of this work.
The evanescent wave character of surface plasmons in metal nanoparticles implies that the local electric field decays exponentially away from the surface, with a typical decay length of approximately 5 nm. 23,24 Probe molecules must therefore be in close proximity to the metal surface to provide detectable Raman signals. Many types of molecules such as polycyclic aromatic hydrocarbons (PAHs) cannot be analyzed using conventional SERS substrates due to their poor affinity for metal surfaces. 25 To address the issue, various surface functionalization strategies have been introduced that promote capture of specific analytes on the metal surface. [26][27][28] Usually, each ligand is specific only to a small subset of analytes restricting the broad applicability of this method. 29 A combination of gold nanoparticles with carbon nanomaterials (e.g., graphene, graphene oxide and carbon nanotubes) is an alternative approach for physisorption of diverse molecules for higher SERS enhancement. [30][31][32][33] The high specific surface area of several carbon nanomaterials and their affinity for a range of molecules have made them effective adsorbents. 34 This adsorbing property of underlying carbon can be exploited for bringing molecules in close proximity to metal surfaces. 35 We describe a simple strategy to produce gold-carbon nanoparticles that are effective SERS substrates for the detection of a wide range of analytes in aqueous solution. We show the potential of these gold-carbon nanoparticles for the detection of two groups of important anthropogenic water contaminants -organic dyes and nitrate ions. The persistence of dyes in the environment has potential carcinogenic and mutagenic effects. 36

Preparation of PLL-coated CB NP suspensions
In a typical synthesis of PLL coated CB nanoparticles, 1 mL of a 0.015wt% CB suspension was added dropwise to 4cc of a 0.019wt% aqueous solution of PLL, and stirred for 30 min. The suspension was then centrifuged at 17,000g for 60 min. The supernatant was removed, and the pellet was redispersed in 5 mL of DIW using a vortex mixer. Centrifuging removed unbound PLL from the suspension. Suspensions prepared with PLL:CB weight ratios ranging from 0.5:1 to 10:1 were used to monitor the adsorption of PLL on the CB surfaces by zeta potential measurements. Two suspensions were prepared with a PLL:CB weight ratio of 5:1 (PLLCB5 NPs) for subsequent precipitation of gold. One was centrifuged to remove unbound PLL and the other was used without centrifugation. The presence of unbound PLL has a strong impact on the morphology of the gold forming on the CB particles.

Preparation of structured gold nanoparticles
Aqueous solutions of 50 mM HAuCl4 and 75 mM ascorbic acid were used for the synthesis of structured gold nanoparticles. An ascorbic acid to HAuCl4 molar ratio of 1.5:1 was used for all experiments.
Gold-coated CB nanoparticles were prepared using the steps outlined in Figure 3

Preparation of SERS-active substrates and detection experiments
Dispersions of Au-PLLCBCF, Au-PLLCB and Au-PLL NPs were pelleted by centrifugation at 3000g for 10 min and resuspended in 400 l deionized water (DIW) using vortex mixing to reduce the colloidal suspension to ~ 35% of the initial volume.

Characterization
Nanoparticles were imaged using transmission electron microscopy (JEOL JEM-2100) at an accelerating voltage of 200 kV, and by scanning electron microscopy (Zeiss Sigma VP FESEM). Particles were examined on a Rigaku Ultima IV X-Ray diffractometer. An Oxford Inca energy dispersive X-ray (EDX) system was used for composition analysis. A Malvern Zetasizer Nano ZS was used to determine the zeta potentials of the CB and PLLCB NPs. Three different samples were analyzed, and three measurements were made for each sample; the average zeta potentials and standard deviations from these measurements were reported. Absorption spectra were collected using a UV−vis−NIR scanning spectrophotometer (Jasco, Tokyo, Japan) over wavelengths from 400 to 1300 nm. Absorption from deionized water was used as the reference. Raman spectra were recorded by using a Sierra portable system (Snowy Range Instrument) that has a 785 nm laser operating at 100 mW. The operational wavenumber range is 200-2000 cm −1 with a resolution of 8 cm −1 . The spot size on the sample was approximately 30 µm in diameter. By rastering the focused laser beam over the sample, a 20 mm 2 area was interrogated without a loss in resolution. 38 Each SERS spectrum was accumulated for 10 s.     Gold tetrachloride anions were also reduced in PLL solution only (no CB; Figure   3.4b). PLL adsorbs on nucleated gold particles, modulating the shape of the growing particles, and led to the formation of flower-like nanoparticles. 42 These results are consistent with those from other groups that have studied the structure directing roles of surfactants and polymers by selective binding to crystal facets. 43,44 This morphological variation, with and without CB present, shifts the LSPR peak from 770 nm for Au-PLLCB to 1179 nm for Au-PLL NP (Figure 3.4c).
The Raman spectra of Au-PLLCB show three distinct peaks; the D-band (1324 cm −1 ), which is a disorder-activated Raman mode, and the G-band (1595 cm −1 ), which is characteristic of sp 2 -hybridized carbon atoms (Figure 3.4d). The sp 2 -hybridized carbon is important for the adsorption of aromatic molecules through - interactions. 45 The low intensity peak at 1446 cm −1 for both Au-PLLCB and Au-PLL NPs is due to bending mode of CH2 in the lysine side chain of adsorbed PLL on the surface 46

Evaluation of the SERS signals from the particles
The assignments of the Raman bands for 4-NBT, congo red, crystal violet and nitrate ion are shown in Table 1.     (Figure 3.6c). The 16-fold higher signal from Au-PLLCB is due to the higher SERS activity of Au-PLLCB compared to Au-PLL. On the other hand, the greater enhancement effect of the Au-PLLCB for CV (65 times higher than from Au-PLL) can be attributed to the combination of its higher SERS activity and higher adsorption affinity to the surface by - interactions, which concentrate more probe molecules on the surface of the nanoparticles. Polycyclic aromatic compounds with short alkyl chains can be selectively adsorbed by carbon black nanoparticles. 53 Our results suggest that - interactions are more significant than electrostatic repulsion for CV.

Detection of nitrate ions
Given the effectiveness of Au-PLLCB for detection of CR and CV, we explored its potential to detect nitrate ions in water. As shown in Figure 3.7a, a 100 M sodium nitrate a pronounced Raman band at 1048 cm -1 , coming from the N-O stretching mode of nitrate ion. Intensity of the peak at 1048 cm -1 was then used for quantitative detection of sodium nitrate (Figure 3.7b). The SERS signal intensity increases with increasing to be 15M. We note that for environmental purposes, 10 ppm or ~117M concentration has been reported to be the desired detection limit of nitrate ions. 51

Conclusion
A novel template precipitation approach has been used for the hybridization of gold with carbon nanomaterials to generate highly active SERS particles for the detection of a wide variety of analytes. The adsorbed cationic polyelectrolyte, PLL, on carbon black concentrates the AuCl -4 anions on the surface, providing an enriched interface for reducing the anions to gold nanostructures. Equally important was the ability to further tailor the surface topography of the gold nanoparticles using PLL as a shape-directing agent, producing sharp tips and edges that enhance incident electric fields. As a result, the Au-PLLCB substrate was capable of detecting of both cationic and anionic aromatic dyes due to the dual adsorbing role of the underlying carbon template via - interactions and the PLL coating via charge attraction. Furthermore, the substrate was able to provide quantitative detection of nitrate ions at and below environmentally relevant concentrations. These results suggest that Au-PLLCB hybrid particles have great potential as sensitive SERS-active materials for detection of a wide range of analytes in aqueous solutions. emulsion stabilizer, they provide important insights on bacteria adhesion to oil, a critical step in the oil degradation process following a marine spill.

Introduction
Bacteria play an essential role in the degradation of crude oil following marine spills. 1 Alcanivorax borkumensis (AB) is a marine bacterium that dominates bacterial communities around many oil spills because it uses the oil as a nutrient source, and enzymatically degrades a wide range of alkanes, including linear and branched aliphatics, and isoprenoid hydrocarbons. They comprise up to 80-90% of the oildegrading microbial community within 1-2 weeks of oil entering sea water. 2 Because of the pivotal role of AB in oil-spill remediation it serves as a model organism to understand the alkane biodegradation process. [3][4][5] Dispersants have been widely applied to oil-contaminated waters during a response to marine oil spills. These dispersants help emulsify the oil, typically into 1-100 m diameter droplets. The enhanced oil-water interface area compared to having a surface slick promotes the availability of the oil to AB, thus enhancing degradation. 6  Mixed wettability particles have also been examined as dispersants to help avoid some of the potential negative effects of surfactants on microbial species. 8,9 High energy, usually through vortexing or homogenization is typically required for these mixed wettability particles to breach oil-water interfaces. However, unlike surfactants, these particles remain at the oil-water interfaces even after extreme dilution, due to the high energy required for their desorption. [10][11][12] Particle-stabilized emulsions are therefore typically more stable than emulsions stabilized by surfactants, making particles an attractive alternative to surfactants for this application.
The presence of dispersants at oil-water interfaces can affect the bacterial degradation process. 8 Dispersants can disrupt bacterial cell membranes, interfere with cell surface receptors, react with cellular components, or block enzyme active sites. 13 Their presence at oil-water interfaces can affect bacteria attachment and thus the degradation process. 8,14,15 Some studies suggest that dispersants enhance biodegradation, 16,17 while others conclude that dispersants either make no difference or even inhibit biodegradation. [18][19][20] These conflicting results require a fuller understanding of the degradation process.
Due to the low solubility of many oils in water, and because bacteria are present in the water, bacteria must attach to the oil-water interfaces before degradation gets initiated. 5 During aerobic degradation, the terminal hydrocarbon is transformed into a primary alcohol through membrane-bound oxygenase. 3 The direct contact of AB with the oil surface can significantly increase the rate of hydrocarbon uptake into the cells, thereby accelerating degradation as well as enhancing bacteria growth. 21 Bacterial attachment to the interface precedes biofilm formation. 22 The biofilm allows bacterial cohesion and acts as a nutrient sink which is useful when the AB responds to stressors such as shear or changes in pH. 23 In this work we seek to understand the first critical step in the degradation process -the attachment of AB to oil-water interfaces. We use fluorescence microscopy and cryogenic scanning electron microscopy (cryo-SEM) to image AB cells at the oil-water interfaces and in the aqueous phase. We chose hexadecane as the oil because it is degraded by AB and has a low solubility (910 -8 M at 25ºC) in water.

Materials
A. borkumensis (ATCC ® 700651 TM ) was obtained from the American Type Culture

Sample preparation
To train the bacteria to respond specifically to hexadecane, AB was deliberately

Sample visualization
To image AB cells, 100 μL of the bacteria dispersion was diluted by a factor of 50 with ASW and passed through a 0.08 μm pore filter. The cells on the filter were fixed with 4% glutaraldehyde and then stained with a 2% osmium tetroxide solution. The specimen was dehydrated through a graded ethanol series (35,50,75,95,95, 100, 100% ethanol). After each step, the sample was washed twice with ASW. AB cells were then air dried using HMDS as a drying agent. 24,25 The dried AB cells were sputtered with gold, then imaged in a Zeiss Sigma VP field emission scanning electron microscope.  These rod-shaped bacteria are approximately 1.0−1.5 μm long and have a diameter of ∼0.5 m. 26,27 The cells in aggregates are connected by strands of exopolymeric substances (EPS), identified by blue arrows in the inset. 28,29 During alkane metabolism AB excretes anionic glucolipids that act as surfactants. 30 Thus, even though no dispersants were added, emulsions formed after following the procedure shown in Figure 4.1a. The droplet diameters range from 2 µm -100 µm.
Because these are hexadecane-trained AB cells, the bacteria attach to the hexadecane-ASW interfaces within 3 days (Figure 4.3a), as expected. 5, 31   When Corexit was present in the hexadecane and samples were prepared using the procedure outlined in Figure 4.1b, no bacteria were found to attach to the hexadecane droplets after 3 days (Figure 4.4a). In addition, most bacteria are dead by day 3. No    Following the procedure outlined in Figure 4.1c, we exposed a Tween 20-stabilized hexadecane-in-ASW emulsion to AB in ASW. shown that Tween can absorb strongly and create a stable monolayer at oil-water interfaces. 37 The monolayer can persist even under dilution, 37 and the adsorption of other surfactants to oil−water interfaces containing pre-adsorbed Tween surfactants is significantly inhibited. 38 The absence of AB cells at the hexadecane-water interfaces is suggestive of a similar inhibition mechanism, compounded by the presence of Tween as a nutrient within the ASW. 39, 40 Carbon black particles have recently been studied as potential dispersants for marine oil spills. 10, 41 We followed the procedure outlined in Figure 4.1c and incubated AB with emulsions stabilized by CB. Figure 4.8a shows that a compact layer of live cells was formed around the oil droplets in 3 days. CB particles are also seen at the interfaces.
The presence of CB does not prevent the gram-negative bacteria from attaching to the oil droplets. Cryo-SEM images, shown in Figure 4.8b, provide a more detailed picture of the bacteria and carbon black particles at the hexadecane-ASW interfaces. The interfacial layer contains bacterial cells and carbon black aggregates, as seen in the insets of Figure 4.8b. We conducted additional experiments and did an analysis to provide further insight into these experimental observations. The details are shown in the Supporting Information file. We observed that AB cells get lysed in DIW, and there is essentially no absorption of AB to the CB particles in that medium ( Figure S.1a). Therefore, we focus our discussion on the interactions of AB and CB in ASW, where we observed some attachment ( Figure S.1b). We measured the zeta potentials of CB and AB in ASW.
We then used a Derjaguin−Landau−Verwey−Overbeek (DLVO) analysis along with the Derjaguin approximation to estimate the force between an AB cell and a CB particle, idealized as spheres of diameters 1600 and 120 nm, respectively. The interaction between the AB and CB is weakly attractive through ASW, as shown in Figure S

Conclusions
We have examined the interaction of hexadecane-trained AB cells with surfactantor particle-decorated hexadecane droplets. Corexit and Tween 20 were used as chemical dispersants, while surface-modified CB was used as a particle-based "Pickering" emulsifier.
Without any added dispersant, the hexadecane droplets are covered by a large number of AB cells, forming a dense bacterial film at the oil surface. When Corexit was used as the emulsion stabilizer, no AB attachment was observed after 3 days of incubation, and most AB cells in the ASW were dead. After 7 days, some AB cells moved to the oil water interface. The presence of some live bacteria in the ASW indicates that AB cells metabolizes one of the components of Corexit. Tween 20 forms a stable and packed layer at the oil−water interface that makes it difficult for the bacteria to contact the oil. However, AB cells survive in ASW by using free Tween 20 in the aqueous phase as a nutrient. CB did not interfere with AB attachment to the oil droplets.
AB cells and CB aggregates were observed at the interfaces. A model that considers only electrostatic and van der Waals interactions between the AB cells at the surfactant or particle-covered oil water interfaces would not predict all of our observations. The attachment of these bacterial cells to hexadecane droplets is a complex interplay between physicochemical interactions and biochemical processes.
a sharp increase in electrical conductivity is known as the percolation threshold. 3,4 Filler content is a critical aspect in the production of conductive polymer composites.
Providing a low loading at percolation has a significant benefit for mechanical properties, as filler materials can act as nucleation sites for crack growth. 5 The geometric features of conductive fillers and also their distribution in a matrix have significant roles in lowering the critical limit for making conductive composites. 3,6 Graphene, due to its remarkable electrical, thermal, stiffness and strength properties, is a promising nanofiller in composite materials. 7,8 If graphene sheets are modeled as ideally dispersed and randomly rotated disks of aspect ratio AR (AR = disk diameter/thickness), the percolation threshold ϕc is given by ϕc = 1.5(ϕsphere/AR). 9 Here, ϕsphere is the percolation threshold for spheres, i.e., ϕsphere = 0.29 (ϕsphere = 0.29 is for monodispersed spheres; that number is lower if there is polydispersity, but remains of the same order of magnitude). Graphene nanosheets have large surface areas that promote agglomeration by van der Waals interactions. Their dispersion in a host polymer remains a challenge. 10,11 One way to prevent restacking of MLG is to incorporate a second filler that acts as a spacer between the sheets. 12 Using nonconducting silica nanoparticles as a second filler resulted in an enhanced dispersion of graphene sheets, leading to a remarkable increase of the electrical conductivity of the composite as the loading of the non-conducting filler was increased. 13 In this work, multilayer graphene (MLG) was used as a primary filler. MLGs are several microns in lateral dimensions and are of 8-10 carbon layers thick, providing a lateral dimension to thickness ratio of the order of 10 3 . We utilized carbon black nanoparticles (CB) as a secondary filler. CB is largely used in rubber composites as a filler not only to improve the mechanical performance but also to impart electrical conductivity to prevent static charge buildup. 14  and was detected by a thermoelectrically-cooled charge coupled device (CCD) camera.
A total of 2400 individual spectra were collected over a 30  20 m 2 area of each composite. The integration time was 2.5 s.

Result ad discussion
The electrical conductivity versus the CB content (vol %) for the composites filled composite remarkably by six orders of magnitude. Since this volume loading of CB is well below its percolation threshold, the observed conductivity enhancement reveals that the CB particles acted as spacers between MLG sheets, instead of forming their own network. The conductivity increases further as the loading of the carbon black is increased up to 12 vol%, and then starts to plateau.
The conductivity values for PS-MLG2.5-CB composites with different loading of CB are compared with previously reported data for PS composites with the same loading of MLG but using silica nanoparticles as the secondary fillers (PS-MLG2.5-silica). 13 Interestingly, at volume loadings of the secondary fillers up to 8 vol %, both conducting and non-conducting nanoparticles resulted in similar conductivity enhancement of composite. Above 8 vol% content, CB nanoparticles enhance the conductivity one order of magnitude beyond that of the silica nanoparticles at equal volume loadings.
Conducting CB connects the MLG particles. 16 However, it is not until 12% by volume loading of secondary filler that there is significant difference in the magnitude of electrical conductivity between PS-MLG2.5-CB and PS-MLG2.5-silica composites.
Beyond a loading of 12%, , excessive silica particles start to break the connectivity of the MLG network, leading to a significant drop in the electrical conductivity to approximately 10 -3 S/m. 13 In contrast, using CB, the enhancement of electrical conductivity of polystyrene is sustained to a higher value of 442 S/m. is a second order Raman mode of graphene with no disorder. 18 The dispersion of MLG in PS with different loading of CB was obtained by Raman mapping taken in an area of each sample, by focusing on the 2D-band of multilayer graphene at 2700 cm -1 .   can be calculated using the measured full width at half maximum of graphite peak using Scherrer equation. 12 Figure 5.3f shows that the MLG crystallite size decreases as CB loading increases, indicating improved dispersion of the MLG with increasing loading of CB.

Conclusion
The electrical conductivity of PS-MLG2.5-CB composites was studied for 2.5 vol% MLG and different loading of CB. Loading of CB filler below its percolation threshold significantly increases electrical conductivity of the composite by acting as spacers to separate the MLG sheets. At higher loadings of CB, establishment of additional conductive pathways by CB results in sustained and higher conductivity values compared to using non-conductive secondary filler. The enhanced dispersion of MLG sheets throughout polymer network at different loadings of CB was also confirmed by Raman spectroscopic images and wide-angle X-ray diffraction.

S1-Physicochemical (DLVO) model for the interaction of AB with CB particles
The para-aminobezoic acid (PABA) -terminated CB particles in DI-water carry a negative surface charge at neutral pH and in deionized water. However, in ASW many carboxylate groups on the surface of the CB are salted out by cations. AB are gramnegative bacteria with an outer membrane consisting of a lipid bilayer containing lipopolysaccharides. 1 The dissociation of carboxyl and phosphate groups in the peptidoglycan and lipopolysaccharides on the cell membrane surface imparts a negative charge to the surfac, 2 in deionized water (DIW). In ASW, the zeta potential is reduced.
Measured zeta potentials of AB and CB in DIW and ASW are reported in Table S1. We caution that there is some aggregation of the CB in ASW so the zeta potential numbers may not be accurate but are qualitatively correct. The Gouy-Chapman model for electrostatic interaction for two surfaces with unequal potentials, is given by: