Functionalized Gold Nanoparticles and Halogen Bonding Interactions Involving Fentanyl and Fentanyl Derivatives.

Fentanyl (FTN) and synthetic analogs of FTN continue to ravage populations across the globe, including in the United States where opioids are increasingly being used and abused and are causing a staggering and growing number of overdose deaths each year. This growing pandemic is worsened by the ease with which FTN can be derivatized into numerous derivatives. Understanding the chemical properties/behaviors of the FTN class of compounds is critical for developing effective chemical detection schemes using nanoparticles (NPs) to optimize important chemical interactions. Halogen bonding (XB) is an intermolecular interaction between a polarized halogen atom on a molecule and e--rich sites on another molecule, the latter of which is present at two or more sites on most fentanyl-type structures. Density functional theory (DFT) is used to identify these XB acceptor sites on different FTN derivatives. The high toxicity of these compounds necessitated a "fragmentation" strategy where smaller, non-toxic molecules resembling parts of the opioids acted as mimics of XB acceptor sites present on intact FTN and its derivatives. DFT of the fragments' interactions informed solution measurements of XB using 19F NMR titrations as well as electrochemical measurements of XB at self-assembled monolayer (SAM)-modified electrodes featuring XB donor ligands. Gold NPs, known as monolayer-protected clusters (MPCs), were also functionalized with strong XB donor ligands and assembled into films, and their interactions with FTN "fragments" were studied using voltammetry. Ultimately, spectroscopy and TEM analysis were combined to study whole-molecule FTN interactions with the functionalized MPCs in solution. The results suggested that the strongest XB interaction site on FTN, while common to most of the drug's derivatives, is not strong enough to induce NP-aggregation detection but may be better exploited in sensing schemes involving films.

Mechanistic Elucidation of Nanomaterial-Enhanced First-Generation Biosensors Using Probe Voltammetry of an Enzymatic Reaction.

The incorporation of nanomaterials (NMs) into biosensing schemes is a well-established strategy for gaining signal enhancement. With electrochemical biosensors, the enhanced performance achieved from using NMs is often attributed to the specific physical properties of the chosen nanocomponents, such as their high electronic conductivity, size-dependent functionality, and/or higher effective surface-to-volume ratios. First generation amperometric biosensing schemes, typically utilizing NMs in conjunction with immobilized enzyme and semi-permeable membranes, can possess complex sensing mechanisms that are difficult to study and challenging to understand beyond the observable signal enhancement. This study shows the use of an enzymatic reaction between xanthine (XAN) and xanthine oxidase (XOx), involving multiple electroactive species, as an electrochemical redox probe tool for ascertaining mechanistic information at and within the modified electrodes used as biosensors. Redox probing using components of this enzymatic reaction are demonstrated on two oft-employed biosensing approaches and commonly used NMs for modified electrodes: gold nanoparticle doped films and carbon nanotube interfaces. In both situations, the XAN metabolism voltammetry allows for a greater understanding of the functionality of the semipermeable membranes, the role of the NMs, and how the interplay between the two components creates signal enhancement.

Nanomaterial-Doped Xerogels for Biosensing Measurements of Xanthine in Clinical and Industrial Applications.

First-generation amperometric xanthine (XAN) biosensors, assembled via layer-by-layer methodology and featuring xerogels doped with gold nanoparticles (Au-NPs), were the focus of this study and involved both fundamental exploration of the materials as well as demonstrated usage of the biosensor in both clinical (disease diagnosis) and industrial (meat freshness) applications. Voltammetry and amperometry were used to characterize and optimize the functional layers of the biosensor design including a xerogel with and without embedded xanthine oxidase enzyme (XOx) and an outer, semi-permeable blended polyurethane (PU) layer. Specifically, the porosity/hydrophobicity of xerogels formed from silane precursors and different compositions of PU were examined for their impact on the XAN biosensing mechanism. Doping the xerogel layer with different alkanethiol protected Au-NPs was demonstrated as an effective means for enhancing biosensor performance including improved sensitivity, linear range, and response time, as well as stabilizing XAN sensitivity and discrimination against common interferent species (selectivity) over time-all attributes matching or exceeding most other reported XAN sensors. Part of the study focuses on deconvoluting the amperometric signal generated by the biosensor and determining the contribution from all of the possible electroactive species involved in natural purine metabolism (e.g., uric acid, hypoxanthine) as an important part of designing XAN sensors (schemes amenable to miniaturization, portability, or low production cost). Effective XAN sensors remain relevant as potential tools for both early diagnosis of diseases as well as for industrial food monitoring.

On-Site Detection of Neonicotinoid Pesticides Using Functionalized Gold Nanoparticles and Halogen Bonding.

Neonicotinoid (NN) pesticides have emerged globally as one of the most widely used agricultural tools for protecting crops from pest damage and boosting food production. Unfortunately, some NN compounds, such as extensively employed imidacloprid-based pesticides, have also been identified as likely endangering critical pollinating insects like honey bees. To this end, NN pesticides pose a potential threat to world food supplies. As more countries restrict or prohibit the use of NN pesticides, tools are needed to effectively and quickly identify the presence of NN compounds like imidacloprid on site (e.g., in storage areas on farms or pesticide distribution warehouses). This study represents a proof-of-concept where the colloidal properties of specifically modified gold nanoparticles (Au-NPs) able to engage in the rare intermolecular interaction of halogen bonding (XB) can result in the detection of certain NN compounds. Density functional theory and diffusion-ordered NMR spectroscopy (DOSY NMR) are used to explore the fundamental XB interactions between strong XB-donor structures and NN compounds, with the latter found to possess multiple XB-acceptor binding sites. A fundamental understanding of these XB interactions allows for the functionalization of alkanethiolate-stabilized Au-NPs, known as monolayer-protected gold clusters (MPCs), with XB-donor capability (f-MPCs). In the presence of certain NN compounds such as imidacloprid, the f-MPCs subsequently exhibit visual XB-induced aggregation that is also measured with absorption (UV-vis) spectroscopy and verified with transmission electron microscopy (TEM) imaging. The demonstrated f-MPC-aggregation detection scheme has a number of favorable attributes, including quickly reporting the presence of the NN target, requiring only micrograms of suspect material, and being highly selective for imidacloprid, the most prevalent and most important NN insecticide compound. Requiring no instrumentation, the presented methodology can be envisioned as a simple screening test in which dipping a cotton swab of an unknown powder from a surface in a f-MPC solution causes f-MPCs to aggregate and yield a preliminary indication of imidacloprid presence.

Monolayer-Protected Gold Nanoparticles Functionalized with Halogen Bonding Capability─An Avenue for Molecular Detection Schemes.

The use of functionalized nanoparticles (NPs) and their aggregation in the presence of a targeted analyte is a well-established molecular detection strategy predicated on harnessing specific molecular interactions to the NP periphery. Molecules able to specifically interact with the functionalized NPs alter the unique optical and electrochemical properties of the NPs as a function of interparticle spacing. While many intermolecular interactions have been successfully exploited in this manner in conjunction with aqueous NP systems, the use of non-aqueous NPs in the same capacity is significantly less explored. A fundamental interaction that has not been previously investigated in NP schemes is halogen bonding (XB). XB is an orthogonal, electrostatic interaction between a region of positive electrostatic potential (δ+) on a halogen atom (i.e., XB donor) and a negative (δ-) Lewis base (XB acceptor) molecule. To couple XB with NP systems, ligands featuring a molecular structure that promotes XB interactions need to be identified, optimized, and synthesized for subsequent attachment to NPs. Herein, density functional theory (DFT) and NMR techniques are used to identify a strong XB-donor moiety (-C6F4I) and a synthetic scheme for a thiolate ligand featuring that functionality is devised and executed with high purity/yield (78%). Ligand-exchange reactions allow functionalization of non-aqueous alkanethiolate-protected gold NPs or monolayer-protected clusters (MPCs) with the XB-donor ligands. Functionalized MPCs (f-MPCs), within both assembled films and in solution, are shown to engage in XB interactions with target XB-acceptor molecules. Molecular recognition events, including induced aggregation of the f-MPCs, are characterized with UV-vis spectroscopy, cyclic voltammetry, TEM imaging, and diffusion-ordered spectroscopy NMR with limits of detection of 50-100 nM for strong XB acceptors. While fundamental exploration of XB interactions is ongoing, this study represents a step toward utilizing XB within molecular detection schemes, an application with implications for supramolecular chemistry, forensic, and environmental chemical sensing.

Evaluating Halogen-Bond Strength as a Function of Molecular Structure Using Nuclear Magnetic Resonance Spectroscopy and Computational Analysis.

Halogen bonding (XB) is a highly directional, non-covalent intermolecular interaction between a molecule (XB donor) presenting a halogen with an electron-deficient region or sigma hole (σ-hole) and an electron-rich or Lewis-base molecule (XB acceptor). A systematic, experimental, and theoretical study of solution-phase XB strength as a function of the molecular structure for both XB donor and acceptor molecules is presented. The impact of specific structural features is assessed using 19F and 1H nuclear magnetic resonance (NMR) titrations to determine association constants, density functional theory calculations for interaction energies and bond lengths, as well as 19F-1H HOESY NMR measurements of intermolecular cross-relaxation between the interacting XB donor-acceptor adducts. For XB donor molecules (perfluoro-halogenated benzenes), results indicate the critical importance of iodine coupled with electron-withdrawing entities. Prominent structural components of XB acceptor molecules include a central atom working in conjunction with a Lewis-base atom to present high electron density directed at the σ-hole (e.g., tributylphosphine oxide). Additionally, larger surrounding aliphatic R groups (e.g., butyl and octyl) were found to significantly stabilize strong XB, particularly in solvents that promote the interaction. With a more thorough understanding of structure-optimized XB, one can envision harnessing XB interactions more strategically for specific design of optimal materials and chemical applications.

Sintering-Based In-Situ Synthesis and Characterization by TEM of Noble Metal Nanoparticles for Ceramic Glaze Color Control.

Gold and silver salt mixtures are incorporated in ceramic glazes for in situ development of mixtures of gold and silver nanoparticles (NPs) that subsequently allow for a wide spectrum of low metal loading color control within ceramic materials. Prior work has shown that gold NPs can be used to create vibrant, color-rich red pigments in high-temperature ceramic and glass applications, though the achievable diameter of the gold NP ultimately limits the available range of color. The current study significantly expands color control from traditional gold nanoparticle red through silver nanoparticle green via the alteration of gold-to-silver salt ratios incorporated in the glaze formulations prior to sintering. Nanoparticle-based coloring systems are tested in both oxidative and reductive firing atmospheres. While the oxidation environment is found to be prohibitive for silver NP stability, the reductive atmosphere is able to form and sustain mixtures of gold and silver NPs across a wide color spectrum. All glazes are analyzed via reflectance spectrometry for color performance and samples are characterized via TEM and EDS for composition and sizing trends. This study creates new groundwork for a color-controlled NP system based on noble metal ratio blends that are both nontoxic and achieved with radically lower metal pigment loading than traditional glazes.

Adaptable Xerogel-Layered Amperometric Biosensor Platforms on Wire Electrodes for Clinically Relevant Measurements.

Biosensing strategies that employ readily adaptable materials for different analytes, can be miniaturized into needle electrode form, and function in bodily fluids represent a significant step toward the development of clinically relevant in vitro and in vivo sensors. In this work, a general scheme for 1st generation amperometric biosensors involving layer-by-layer electrode modification with enzyme-doped xerogels, electrochemically-deposited polymer, and polyurethane semi-permeable membranes is shown to achieve these goals. With minor modifications to these materials, sensors representing potential point-of-care medical tools are demonstrated to be sensitive and selective for a number of conditions. The potential for bedside measurements or continuous monitoring of analytes may offer faster and more accurate clinical diagnoses for diseases such as diabetes (glucose), preeclampsia (uric acid), galactosemia (galactose), xanthinuria (xanthine), and sepsis (lactate). For the specific diagnostic application, the sensing schemes have been miniaturized to wire electrodes and/or demonstrated as functional in synthetic urine or blood serum. Signal enhancement through the incorporation of platinum nanoparticle film in the scheme offers additional design control within the sensing scheme. The presented sensing strategy has the potential to be applied to any disease that has a related biomolecule and corresponding oxidase enzyme and represents rare, adaptable, sensing capabilities.

Halogen Bonding Interactions for Aromatic and Nonaromatic Explosive Detection.

Improved sensing strategies are needed for facile, accurate, and rapid detection of aromatic and nonaromatic explosives. Density functional theory was used to evaluate the relative binding interaction energies between halogen-containing sensor model molecules and nitro-containing explosives. Interaction energies ranged from -18 to -14 kJ/mol and highly directional halogen bonding interactions were observed with bond distances ranging between 3.0 and 3.4 Å. In all geometry optimized structures, the sigma-hole of electropositive potential on the halogen aligned with a lone pair of electrons on the nitro-moiety of the explosive. The computational results predict that the strongest interactions will occur with iodine-based sensors as, of all the halogens studied, iodine is the largest, most polarizable halogen with the smallest electronegativity. Based on these promising proof-of-concept results, synthetically accessible sensors were designed using 1,4-dihalobenzene (X = Cl, Br, and I) with and without tetra-fluoro electron withdrawing groups attached to the benzene ring. These sensing molecules were embedded onto single walled carbon nanotubes that were mechanically abraded onto interdigitated array electrodes, and these were used to measure the responses to explosive model compounds cyclohexanone and dimethyl-dinitro-benzene in nitrogen gas. Amperometric current-time curves for selectors and control molecules, including concentration correlated signal enhancement, as well as response and recovery times, indicate selector responsiveness to these model compounds, with the largest response observed for iodo-substituted sensors.

Functionalized carbon nanotube adsorption interfaces for electron transfer studies of galactose oxidase.

Modified electrodes featuring specific adsorption platforms able to access the electrochemistry of the copper containing enzyme galactose oxidase (GaOx) were explored, including interfaces featuring nanomaterials such as nanoparticles and carbon nanotubes (CNTs). Electrodes modified with various self-assembled monolayers (SAMs) including those with attached nanoparticles or amide-coupled functionalized CNTs were examined for their ability to effectively immobilize GaOx and study the redox activity related to its copper core. While stable GaOx electrochemistry has been notoriously difficult to achieve at modified electrodes, strategically designed functionalized CNT-based interfaces, cysteamine SAM-modified electrode subsequently amide-coupled to carboxylic acid functionalized single wall CNTs, were significantly more effective with high GaOx surface adsorption along with well-defined, more reversible, stable (≥ 8 days) voltammetry and an average ET rate constant of 0.74 s-1 in spite of increased ET distance - a result attributed to effective electronic coupling at the GaOx active site. Both amperometric and fluorescence assay results suggest embedded GaOx remains active. Fundamental ET properties of GaOx may be relevant to biosensor development targeting galactosemia while the use functionalized CNT platforms for adsorption/electrochemistry of electroactive enzymes/proteins may present an approach for fundamental protein electrochemistry and their future use in both direct and indirect biosensor schemes.

First Generation Amperometric Biosensing of Galactose with Xerogel-Carbon Nanotube Layer-By-Layer Assemblies.

A first-generation amperometric galactose biosensor has been systematically developed utilizing layer-by-layer (LbL) construction of xerogels, polymers, and carbon nanotubes toward a greater fundamental understanding of sensor design with these materials and the potential development of a more efficient galactosemia diagnostic tool for clinical application. The effect of several parameters (xerogel silane precursor, buffer pH, enzyme concentration, drying time and the inclusion of a polyurethane (PU) outer layer) on galactose sensitivity were investigated with the critical nature of xerogel selection being demonstrated. Xerogels formed from silanes with medium, aliphatic side chains were shown to exhibit significant enhancements in sensitivity with the addition of PU due to decreased enzyme leaching. Semi-permeable membranes of diaminobenzene and resorcinol copolymer and Nafion were used for selective discrimination against interferent species and the accompanying loss of sensitivity with adding layers was countered using functionalized, single-walled carbon nanotubes (CNTs). Optimized sensor performance included effective galactose sensitivity (0.037 µA/mM) across a useful diagnostic concentration range (0.5 mM to 7 mM), fast response time (~30 s), and low limits of detection (~80 µM) comparable to literature reports on galactose sensors. Additional modification with anionic polymer layers and/or nanoparticles allowed for galactose detection in blood serum samples and additional selectivity effectiveness.

Structure-function relationships affecting the sensing mechanism of monolayer-protected cluster doped xerogel amperometric glucose biosensors.

A systematic study of the structure-function relationships critical to understanding the sensing mechanism of 1st generation amperometric glucose biosensors with an embedded nanoparticle (NP) network is presented. Xerogel-based films featuring embedded glucose oxidase enzyme and doped with alkanethiolate-protected gold NPs, known as monolayer protected clusters (MPCs), exhibit significantly enhanced performance compared to analogous systems without NPs including higher sensitivity, faster response time, and extended linear/dynamic ranges. The proposed mechanism involves diffusion of the glucose to glucose oxidase within the xerogel, enzymatic reaction production of H2O2 with subsequent diffusion to the embedded network of MPCs where it is oxidized, an event immediately reported via fast electron transfer (ET) through the MPC system to the working electrode. Various aspects of the film construct and strategy are systematically probed using amperometry, voltammetry, and solid-state electronic conductivity measurements, including the effects of MPC peripheral chain length, MPC functionalization via place-exchange reaction, MPC core size, and the MPC density or concentration within the xerogel composite films. The collective results of these experiments support the proposed mechanism and identify interparticle spacing and the electronic communication through the MPC network is the most significant factor in the sensing scheme with the diffusional aspects of the mechanism that may be affected by film/MPC hydrophobicity and functionality (i.e., glucose and H2O2 diffusion) shown to be less substantial contributors to the overall enhanced performance. Understanding the structure-function relationships of effective sensing schemes allows for the employment of the strategy for future biosensor design toward clinically relevant targets.

Functional layer-by-layer design of xerogel-based first-generation amperometric glucose biosensors.

Xerogel-based first-generation amperometric glucose biosensors, constructed through specific layer-by-layer assembly of films featuring glucose oxidase doped xerogel, a diffusion-limiting xerogel layer, and capped with both electropolymerized polyphenol and blended polyurethane semipermeable membranes, are presented. The specific combination of xerogels formed from specific silane precursors, including propyl-trimethoxysilane, isobutyl-trimethoxysilane, octyl-trimethoxysilane, and hydroxymethyl-triethoxysilane, exhibit impressive dynamic and linear ranges of detection (e.g., ≥24-28 mM glucose) and low response times, as well as significant discrimination against common interferent species such as acetaminophen, ascorbic acid, sodium nitrite, oxalic acid, and uric acid as determined by selectivity coefficients. Additionally, systematic electrochemical and contact angle studies of different xerogel silane precursors, varying in structure, chain length, and/or functional group, reveal that sensor performance is more dependent on the tunable porosity/permeability of the layered interfaces rather than the hydrophobic character or functional groups within the films. While the sensing performance largely exceeds that of existing electrochemical glucose sensing schemes in the literature, the presented layered approach establishes the specific functionality of each layer working in concert with each other and suggests that the strategy may be readily adaptable to other clinically relevant targets and is amenable to miniaturization for eventual in situ or in vivo sensing.

Multi-technique quantitative analysis and socioeconomic considerations of lead, cadmium, and arsenic in children's toys and toy jewelry.

A wide spectrum and large number of children's toys and toy jewelry items were purchased from both bargain and retail vendors and analyzed for arsenic, cadmium, and lead metal content using multiple analytical techniques, including flame and furnace atomic absorption spectroscopy as well as X-ray fluorescence spectroscopy. Particularly dangerous for young children, metal concentrations in toys/toy jewelry were assessed for compliance with current Consumer Safety Product Commission (CPSC) regulations (F963-11). A conservative metric involving multiple analytical techniques was used to categorize compliance: one technique confirmation of metal in excess of CPSC limits indicated a "suspect" item while confirmation on two different techniques warranted a non-compliant designation. Sample matrix-based standard addition provided additional confirmation of non-compliant and suspect products. Results suggest that origin of purchase, rather than cost, is a significant factor in the risk assessment of these materials with 57% of toys/toy jewelry items from bargain stores non-compliant or suspect compared to only 15% from retail outlets and 13% if only low cost items from the retail stores are compared. While jewelry was found to be the most problematic product (73% of non-compliant/suspect samples), lead (45%) and arsenic (76%) were the most dominant toxins found in non-compliant/suspect samples. Using the greater Richmond area as a model, the discrepancy between bargain and retail children's products, along with growing numbers of bargain stores in low-income and urban areas, exemplifies an emerging socioeconomic public health issue.

Monolayer-protected nanoparticle doped xerogels as functional components of amperometric glucose biosensors.

First-generation amperometric glucose biosensors incorporating alkanethiolate-protected gold nanoparticles, monolayer protected clusters (MPCs), within a xerogel matrix are investigated as model systems for nanomaterial-assisted electrochemical sensing strategies. The xerogel biosensors are comprised of platinum electrodes modified with composite films of (3-mercaptopropyl)trimethoxy silane xerogel embedded with glucose oxidase enzyme, doped with Au225(C6)75 MPCs, and coated with an outer polyurethane layer. Electrochemistry and scanning/transmission electron microscopy, including cross-sectional TEM, show sensor construction, humidity effects on xerogel structure, and successful incorporation of MPCs. Analytical performance of the biosensor scheme with and without MPC doping of the xerogel is determined from direct glucose injection during amperometry. MPC-doped xerogels yield significant enhancement of several sensor attributes compared to analogous films without nanoparticles: doubling of the linear range, sensitivity enhancement by an order of magnitude, and 4-fold faster response times accompany long-term stability and resistance to common interfering agents that are competitive with current glucose biosensing literature. Ligand chain length and the MPC/silane ratio studies suggest the MPC-induced enhancements are critically related to structure-function relationships, particularly those affecting interparticle electronic communication where the MPC network behaves as a three-dimensional extension of the working electrode into the xerogel film, reducing the system's dependence on diffusion and maximizing efficiency of the sensing mechanism. The integration of MPCs as a functional component of amperometric biosensor schemes has implications for future development of biosensors targeting clinically relevant species.

Nanoparticle film assemblies as platforms for electrochemical biosensing--factors affecting the amperometric signal enhancement of hydrogen peroxide.

Factors affecting the enhanced amperometric signal observed at electrodes modified with polyelectrolyte-gold nanoparticle (Au-NP) composite films, which are potential interfaces for first-generation biosensors, were systematically investigated and optimized for hydrogen peroxide (H2O2) detection. Polyelectrolyte multilayer films embedded with citrate-stabilized gold nanoparticles exhibited high sensitivity toward the oxidation of H2O2. From this Au-NP film assembly, the importance of Au-NP ligand protection, film permeability, the density of Au-NPs within the film, and electronic coupling between Au-NPs (interparticle) and between the film and the electrode (interfacial) were evaluated. Using alternative Au-NPs, including those stabilized with thiols, polymers, and bulky ligands, suggests that the amperometric enhancement of H2O2 is optimized at poly-L-lysine-linked film assemblies embedded with Au-NPs possessing small, charged, and conductive (conjugated) peripheral ligands. As a potential application of these Au-NP film assemblies, an enhanced amperometric signal for H2O2 oxidation was shown for modified "needle" electrodes. The overall aim of this research is to gain a greater understanding of designing electrochemical sensing strategies that incorporate Au-NPs and target specific analytes.

Synthesis, assembly, and characterization of monolayer protected gold nanoparticle films for protein monolayer electrochemistry.

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM). In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (¹H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.

Electrochemical analysis of azurin thermodynamic and adsorption properties at monolayer-protected cluster film assemblies - evidence for a more homogeneous adsorption interface.

Thermodynamic and adsorption properties of protein monolayer electrochemistry (PME) are examined for Pseudomonas aeruginosa azurin (AZ) immobilized at an electrode modified with a networked film of monolayer-protected clusters (MPCs) to assess if nanoparticle films of this nature offer a more homogeneous adsorption interface compared to traditional self-assembled monolayer (SAM) modified electrodes. Specifically, electrochemistry is used to assess properties of surface coverage, formal potential, peak broadening, and electron transfer (ET) kinetics as a function of film thickness. The modification of a surface with dithiol-linked films of MPCs (Au(225)C6(75)) provides a more uniform binding interface for AZ that results in voltammetry with less peak broadening (<110mV) compared to SAMs (>120-130mV). Improved homogeneity of the MPC interface for protein adsorption is confirmed by atomic force microscopy imaging that shows uniform coverage of the gold substrate topography and by electrochemical analysis of film properties during systematic desorption of AZ, which indicates a more homogeneous population of adsorbed protein at MPC films. These results suggest MPC film assemblies may be used in PME to provide greater molecular level control of the protein adsorption interface, a development with applications for strategies to study biological ET processes as well as the advancement of biosensor technologies.

Distance dependence of electron transfer kinetics for azurin protein adsorbed to monolayer protected nanoparticle film assemblies.

The distance dependence and kinetics of the heterogeneous electron transfer (ET) reaction for the redox protein azurin adsorbed to an electrode modified with a gold nanoparticle film are investigated using cyclic voltammetry. The nanoparticle films are comprised of nonaqueous nanoparticles, known as monolayer-protected clusters (MPCs), which are covalently networked with dithiol linkers. The MPC film assembly serves as an alternative adsorption platform to the traditional alkanethiolate self-assembled monolayer (SAM) modified electrodes that are commonly employed to study the ET kinetics of immobilized redox proteins, a strategy known as protein monolayer electrochemistry. Voltammetric analysis of the ET kinetics for azurin adsorbed to SAMs of increasing chain length results in quasi-reversible voltammetry with significant peak splitting. We observed rate constants (k degrees (ET)) of 12-20 s(-1) for the protein at SAMs of shorter alkanethiolates that decays exponentially (beta = 0.9/CH(2) or 0.8/A) at SAMs of longer alkanethiolates (9-11 methylene units) or an estimated distance of 1.23 nm and is representative of classical electronic tunneling behavior over increasing distance. Azurin adsorbed to the MPC film platforms of increasing thickness results in reversible voltammetry with very little voltammetric peaks splitting and nearly negligible decay of the ET rate over significant distances up to 20 nm. The apparent lack of distance dependence for heterogeneous ET reactions at MPC film assemblies is attributed to a two-step mechanism involving extremely fast electronic hopping through the MPC film architecture. These results suggest that MPC platforms may be used in protein monolayer electrochemistry to create adsorption platforms of higher architecture that can accommodate greater than monolayer protein coverage and increase the Faradaic signal, a finding with significant implications for amperometric biosensor design and development.

Polyelectrolyte-linked film assemblies of nanoparticles and nanoshells: growth, stability, and optical properties.

Multi-layer films of nanoparticles and nanoshells featuring various polymeric linkage molecules have been assembled and their optical properties characterized. The growth dynamics, including molecular weight effects, and stability of the various nanoparticle film constructions, using both single polymer as well as combinations of alternating charge polyelectrolytes as linking mechanisms, are presented. The polymeric linkers studied include poly-L-lysine, poly-L-arginine, poly(allylamine hydrochloride), and polyamidoamine dendrimers. Significantly air stable films were achieved with the use of multi-layered polymeric bridges between the nanoparticles and nanoshells. Optical sensitivity normally observed with these nanomaterials in solution was observed for their corresponding film geometries, with the nanoshell films exhibiting a markedly higher ability to report their local dielectric environment.