Riboflavin-Mediated Photooxidation of Gold Nanoparticles and Its Effect on the Inactivation of Bacteria.

Photodynamic inactivation (PDI) of microorganisms, based on the ability of photosensitizers to produce reactive oxygen species (ROS) under adequate irradiation, emerges as a promising technique to face the increasing bacterial resistance to conventional antimicrobials. In this work, we analyze the combined action of Riboflavin (Rf) and pectin-coated gold nanoparticles (PecAuNP) on Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) as suitable PDI strategy. We demonstrate that gold ions can be generated upon Rf-photosensitized oxidation of PecAuNP. Transient absorption spectroscopy shows that the Rf cationic radical can accept an electron from the nanoparticles to yield Au(I) ions, which in aqueous medium is disproportionate to yield Au0 and Au(III). Microbiological assays showed that the presence of PecAuNP enhanced the antibacterial activity of photoirradiated Rf toward S. aureus and P. aeruginosa, in line with the well-known antibacterial activity of gold ions. Moreover, the irradiation of Rf solutions containing about 100 µM PecAuNP enabled the solutions to be bactericidal against both bacteria.

Photodynamic inactivation induced by carboxypterin: a novel non-toxic bactericidal strategy against planktonic cells and biofilms of Staphylococcus aureus.

Microbial related contamination is of major concern and can cause substantial economic losses. Photodynamic inactivation (PDI) has emerged as a suitable approach to inhibit microorganism proliferation. In this work, PDI induced by 6-carboxypterin (Cap), a biocompatible photosensitizer (PS), was analyzed. The growth inhibition of Staphylococcus aureus exposed to artificial UV-A radiation and sunlight in the presence of Cap was investigated. After UV-A irradiation, 50 µM Cap was able to decrease by three orders (with respect to the initial value) the number of S. aureus cells in early biofilms. However, this concentration was 500 times higher than that needed for eradicating planktonic cells. Importantly, under solar exposure, 100 µM Cap was able to suppress sessile bacterial growth. Thus, this strategy is able to exert a bactericidal effect on sessile bacteria and to eradicate planktonic cells by exposing the Cap-containing sample to sunlight.

New findings for the composition and structure of Ni nanoparticles protected with organomercaptan molecules.

Here we explore the synthesis of alkanethiol-coated Ni NPs following the one-phase reaction method by Brust et al. The reduction of NiCl2 with NaBH4 in the presence of dodecanethiol (C12SH) yields a complex product that is difficult to identify as illustrated in the figure of merit. We synthesized Ni(II) dodecanethiolate (C12S) (without the addition of NaBH4) for comparison and performed an exhaustive characterization with TEM, HR-TEM, AFM, MFM, XPS, XRD, UV-vis, magnetism, and FT-IR. It is found that the organic coating is not quite a well-organized self-assembled monolayer (SAM) surrounding the Ni cluster as previously reported. XPS and XRD data show slight differences between both syntheses; however, Ni(II) thiolate appears to be more stable than reduced Ni when exposed to ambient air, indicating the propensity of metallic Ni to oxidize. It has been shown that irradiating with TEM electrons over various metal thiolates leads to nanoparticle formation. We irradiated over Ni(II) thiolate and observed no evidence of NP formation whereas irradiating a reduced Ni sample exhibited an ~3.0 nm nanoparticle diameter. Magnetism studies showed a difference between both samples, indicating ferromagnetic character for the reduced Ni sample. According to our results, the product of the synthesis is comprised of ultrasmall metallic clusters embedded in some form of Ni(II) C12S. In this work, we open a discussion of the chemical nature of the core and the shell in the synthesis of Ni NPs protected with organomercaptan molecules.

Antimicrobial-Loaded Polyacrylamide Hydrogels Supported on Titanium as Reservoir for Local Drug Delivery.

Arthroplasty is a highly successful treatment to restore the function of a joint. The contamination of the implant via bacterial adhesion is the first step toward the development of device-associated infections. The emerging concern about antimicrobial resistance resulted in a growing interest to develop alternative therapeutic strategies. Thus, the increment in the incidence of bacterial periprosthetic infections, the complexity of treating infections caused by organisms growing in biofilms, together with the rise in antibiotic resistant bacteria, expose the need to design novel surfaces that provide innovative solutions to these rising problems. The aim of this work is to develop a coating on titanium (Ti) suitable for inhibiting bacterial adhesion and proliferation, and hence, biofilm formation on the surface. We have successfully prepared polyacrylamide hydrogels containing the conventional antibiotic ampicillin (AMP), silver nanoparticles (AgNPs), and both, AMP and AgNPs. The release of the antibacterial agents from the gelled to aqueous media resulted in an excellent antibacterial action of the loaded hydrogels against sessile S. aureus. Moreover, a synergic effect was achieved with the incorporation of both AMP and AgNPs in the hydrogel, which highlights the importance of combining antimicrobial agents having different targets. The polyacrylamide hydrogel coating on the Ti surface was successfully achieved, as it was demonstrated by FTIR, contact angle, and AFM measurements. The modified Ti surfaces having the polyacrylamide hydrogel film containing AgNPs and AMP retained the highest antibacterial effect against S. aureus as it was found for the unsupported hydrogels. The modified surfaces exhibit an excellent cytocompatibility, since healthy, flattened MC3T3-E1 cells spread on the surfaces were observed. In addition, similar macrophage RAW 264.7 adhesion was found on all the surfaces, which could be related to a low macrophage activation. Our results indicate that AMP and AgNP-loaded polyacrylamide hydrogel films on Ti are a good alternative for designing efficient antibacterial surfaces having an excellent cytocompatibility without inducing an exacerbated immune response. The approach emerges as a superior alternative to the widely used direct adsorption of therapeutic agents on surfaces, since the antimicrobial-loaded hydrogel coatings open the possibility of modulating the concentration of the antimicrobial agents to enhance bacterial killing, and then, reducing the risk of infections in implantable materials.

Multifunctional Titanium Surfaces for Orthopedic Implants: Antimicrobial Activity and Enhanced Osseointegration.

The use of implants in orthopedics and dental practice is a widespread surgical procedure to treat diverse diseases. However, peri-implantitis due to infections and/or poor osseointegration can lead to metallic implant failure. The aim of this study was to develop a multifunctional coating on titanium (Ti) surfaces, to simultaneously deal with both issues, by combining antibacterial silver nanoparticles (AgNPs) and regenerative properties of lactoferrin (Lf). A simple and cost-effective methodology that allows the direct multifunctionalization of Ti surfaces was developed. The modified surfaces were characterized by atomic force microscopy (AFM), X-ray photoelectron spectroscopy, and contact angle measurements. Additionally, in vitro preosteoblast cell adhesion, cell viability, and differentiation were evaluated. The antibacterial capability of the surfaces was tested against Staphylococcus aureus as a prosthesis infection model strain. Our results showed that Lf adsorbed on both Ti surfaces and Ti surfaces with adsorbed AgNPs. Simultaneously, the presence of Lf and AgNPs notably improved preosteoblast adhesion, proliferation, and differentiation, whereas it reduced the bacterial colonization by 97.7%. Our findings indicate that this simple method may have potential applications in medical devices to both improve osseointegration and reduce bacterial infection risk, enhancing successful implantation and patients' quality of life.

Mono- and Bis-Alkylated Lumazine Sensitizers: Synthetic, Molecular Orbital Theory, Nucleophilic Index and Photochemical Studies.

Mono- and bis-decylated lumazines have been synthesized and characterized. Namely, mono-decyl chain [1-decylpteridine-2,4(1,3H)-dione] 6a and bis-decyl chain [1,3-didecylpteridine-2,4(1,3H)-dione] 7a conjugates were synthesized by nucleophilic substitution (SN 2) reactions of lumazine with 1-iododecane in N,N-dimethylformamide (DMF) solvent. Decyl chain coupling occurred at the N1 site and then the N3 site in a sequential manner, without DMF condensation. Molecular orbital (MO) calculations show a p-orbital at N1 but not N3 , which along with a nucleophilicity parameter (N) analysis predict alkylation at N1 in lumazine. Only after the alkylation at N1 in 6a, does a p-orbital on N3 emerge thereby reacting with a second equivalent of 1-iododecane to reach the dialkylated product 7a. Data from NMR (1 H, 13 C, HSQC, HMBC), HPLC, TLC, UV-vis, fluorescence and density functional theory (DFT) provide evidence for the existence of mono-decyl chain 6a and bis-decyl chain 7a. These results differ to pterin O-alkylations (kinetic control), where N-alkylation of lumazine is preferred and then to dialkylation (thermodynamic control), with an avoidance of DMF solvent condensation. These findings add to the list of alkylation strategies for increasing sensitizer lipophilicity for use in photodynamic therapy.

Immobilization of alkyl-pterin photosensitizer on silicon surfaces through in situ SN2 reaction as suitable approach for photodynamic inactivation of Staphylococcus aureus.

The tuning of surface properties through functionalization is an important field of research with a broad spectrum of applications. Self-assembled monolayers (SAMs) allow the surface tailoring through the adsorption of molecular layers having the appropriate functional group or precursor group enabling in situ chemical reactions and thus to the incorporation of new functionalities. The latter approach is particularly advantageous when the incorporation of huge groups is needed. In this study, we report the immobilization of pterin moieties on 11-bromoundecyltrichlorosilane-modified silicon substrates based on the in situ replacement of the bromine groups by pterin (Ptr), the parent derivative of pterins, by means of a nucleophilic substitution reaction. The modified surface was structurally characterized through a multi-technique approach, including high-resolution XPS analysis, contact angle measurements, and AFM. The designed synthesis method leads to the functionalization of the silicon surface with two compounds, O-undecyl-Ptr and N-undecyl-Ptr, with a higher proportion of the N-derivative (1:8 ratio). The alkyl-pterins immobilized via the proposed strategy, retain their photochemical properties, being able to inhibit Staphylococcus aureus growth under irradiation (84.3 ± 15.6 % reduction in viable cells). Our results open the possibility for the modification of several materials, such as glass and metal, through the formation of SAMs having the proper head group, thus allowing the design of photosensitive surfaces with potential microbiological self-cleaning properties.

How Functionalized Surfaces Can Inhibit Bacterial Adhesion and Viability.

Device-associated infections (DAI) remain a serious concern in modern healthcare. Bacterial attachment to a surface is the first step in biofilm formation, which is one of the main causes of DAIs. The development of materials capable of preventing or inhibiting bacterial attachment constitutes a promising approach to deal with this problem. The multifactorial nature of biofilm maturation and antibiotic resistance directs the research for multitargeted or combinatorial therapeutic approaches. One attractive strategy is the modification or the engineering of surfaces in order to provide antiadhesive and/or antimicrobial properties. Currently, several different approaches that involve physical and chemical surface modification deliver some possible alternatives to achieve this goal. The engineered surfaces can be coated with molecules capable of inhibiting the bacterial adhesion or with active agents that kill microorganisms. In addition, surfaces can also be modified in order to be stimuli-responsive, responding to a particular trigger and then delivering the consequent antimicrobial outcome. Here, we review the prevailing strategies to modify surfaces in order to create an antimicrobial surface and discuss how different surface functionalization can affect bacterial adhesion and/or viability.

Two-step biocompatible surface functionalization for two-pathway antimicrobial action against Gram-positive bacteria.

The use of indwelling devices has emerged as a frequent and often life-saving medical procedure. However, infection in prosthetic surgery is one of the most important and devastating complications. Once the biofilm has been formed, its eradication is extremely difficult, due to an increased resistance to host defense and conventional antimicrobials. Thus, the design of novel strategies for inhibiting the bacterial adhesion on implantable devices is a key point for successful surgical procedures. In this work, the development of a simple two-step protocol to prepare surfaces able to prevent the bacterial growth was successfully achieved. The surface-modification design includes a combined approach involving the multi-functionalization of Ti surfaces with silver nanoparticles (AgNPs) and/or ampicillin (AMP). The surface chemistry involved in AMP adsorption on titanium and silver surfaces was elucidated for the first time, thus establishing the basis for the further anchoring of other antibacterial compounds having similar functional groups. Our results show that the antibiotic binds to the titanium surface through covalent interactions between the COOH groups in AMP and the OH groups of the native TiO2 on the surface, although electrostatic interactions between protonated AMP and negatively charged TiO2 can also contribute to the antibiotic anchoring to the surface. The AMP immobilization on the AgNPs is carried out by thiolate-like bonds. The ß-lactam ring functionality is preserved after the adsorption process, since the Ti-AgNPs-AMP surface was able to decrease the bacterial viability in more than 80%. Moreover, the antimicrobial capacity is maintained over time due to a two-pathway antibacterial mechanism: death by contact (AMP) and death by release (AgNPs). The effect of AMP prevails on AgNPs at early stages of bacterial adhesion, while AgNPs are responsible for sustaining the relatively low but steady release of Ag(I), preserving the bacteriostatic activity of the surface over time. This effect would contribute to prevent infections due to sessile cells on indwelling devices, powering the action of the immune system and the conventional antibiotics usually dosed in implanted patients.

Resveratrol enhancement staphylococcus aureus survival under levofloxacin and photodynamic treatments.

Reactive oxygen species (ROS) are an efficient tool to eradicate micro-organisms owing to the capacity of these species to damage almost all types of biomolecules and to kill cells. The increase in mechanisms of antimicrobial resistance has led to the exploration of new strategies to eliminate micro-organisms that involve the production of ROS such as superoxide anion (O2•-) and hydrogen peroxide (H2O2). ROS are produced during several antimicrobial treatments, including antibiotic and photodynamic therapies. Among the natural antioxidants, resveratrol (RSV) is efficient at preventing damage from ROS, and every day more people incorporate it as a dietary or cosmetic supplement. However, the consequences of the administration of RSV during antimicrobial treatment are unknown. To investigate possible antagonistic or synergistic effects of RSV during antibiotic therapy (levofloxacin) or photodynamic therapy (visible radiation and methylene blue), killing of planktonic Staphylococcus aureus bacteria was evaluated in the presence of RSV. The results showed that the antimicrobial capacity of these therapies is significantly diminished when levofloxacin or methylene blue are co-administered with RSV, indicating that consumption of RSV during antimicrobial treatment must be, at least, cautioned. Moreover, considering the ROS antimicrobial activity of antibacterial agents, the topical addition of RSV may also affect the control of pathogens of the human body. The results presented in this article highlight the importance of the evaluation of possible antagonistic effects when an antimicrobial agent with ROS-mediated action is co-administrated with RSV.

Highly Stabilized Nanoparticles on Poly-l-Lysine-Coated Oxidized Metals: A Versatile Platform with Enhanced Antimicrobial Activity.

The increasing incidence of infections in implantable devices has encouraged the search for biocompatible antimicrobial surfaces. To inhibit the bacterial adhesion and proliferation on biomaterials, several surface functionalization strategies have been developed. However, most of these strategies lead to bacteriostatic effect and only few of these are able to reach the bactericidal condition. In this work, bactericidal surfaces were designed through the functionalization of titanium surfaces with poly-l-lysine (PLL) as the mediator for the incorporation of antimicrobial silver nanoparticles (AgNPs). This functionalization influences the adsorption of the particles on the substrate impeding the agglomeration observed when bare titanium surfaces are used, leading to a homogeneous distribution of AgNPs on the surfaces. The antimicrobial activity of this surface has been tested against two different strains, namely, Staphylococcus aureus and Pseudomonas aeruginosa. For both strains and different AgNPs sizes, the surface modified with PLL and AgNPs shows a much enhanced antimicrobial activity in comparison with AgNPs deposited on bare titanium. This enhanced antibacterial activity is high enough to reach bactericidal effect, a condition hard to achieve in antimicrobial surfaces. Importantly, the designed surfaces are able to decrease the bacterial viability more than 5 orders with respect to the initial bacterial inoculum. That means that a relative low load of AgNPs on the PLL-modified titanium surfaces reaches 99.999% bacterial death after 24 h. The results of the present study are important to avoid infections in indwelling materials by reinforcing the preventive antibiotic therapy usually dosed throughout the surgical procedure and during the postoperative period.

Self-sterilizing ormosils surfaces based on photo-synzthesized silver nanoparticles.

Medical device-related infections represent a major healthcare complication, resulting in potential risks for the patient. Antimicrobial materials comprise an attractive strategy against bacterial colonization and biofilm proliferation. However, in most cases these materials are only bacteriostatic or bactericidal, and consequently they must be used in combination with other antimicrobials in order to reach the eradication condition (no viable microorganisms). In this study, a straightforward and robust antibacterial coating based on Phosphotungstate Ormosil doped with core-shell (SiO2@TiO2) was developed using sol-gel process, chemical tempering, and Ag nanoparticle photoassisted synthesis (POrs-CS-Ag). The coating was characterized by X-ray Fluorescence Spectroscopy (XRF), Field Emission Scanning Electron Microscopy (FE-SEM), Atomic Force Microscopy (AFM) and X-ray Photoelectron Microscopy (XPS). The silver free coating displays low antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, in opposition to the silver loaded ones, which are able to completely eradicate these strains. Moreover, the antimicrobial activity of these substrates remains high until three reutilization cycles, which make them a promising strategy to develop self-sterilizing materials, such as POrs-CS-Ag-impregnated fabric, POrs-CS-Ag coated indwelling metals and polymers, among other materials.

Molding and replication of ceramic surfaces with nanoscale resolution.

The design of reproducible and more efficient nanofabrication routes has become a very active research field in recent years. In particular, the development of new methods for micro- and nanopatterning materials surfaces has attracted the attention of many researchers in industry and academia as a consequence of the growing relevance of patterned surfaces in many technological fields, ranging from optoelectronics to biotechnology. In this work we explore, discuss, and demonstrate the possibility of extending the well-known molding and replication strategy for patterning ceramic materials with nanoscale resolution. To achieve this goal we have combined physical deposition methods, molecule-thick anti-sticking coatings, and nanostructured substrates as master surfaces. This new perspective on an "old technology", as molding is, provides an interesting alternative for high-resolution, direct surface-relief patterning of materials that currently requires expensive and time-consuming lithographic approaches.

Functional nicotinic acetylcholine receptor reconstitution in Au(111)-supported thiolipid monolayers.

The insertion and function of the muscle-type nicotinic acetylcholine receptor (nAChR) in Au(111)-supported thiolipid self-assembled monolayers have been studied by atomic force microscopy (AFM), surface plasmon resonance (SPR), and electrochemical techniques. It was possible for the first time to resolve the supramolecular arrangement of the protein spontaneously inserted in a thiolipid monolayer in an aqueous solution. Geometric supramolecular arrays of nAChRs were observed, most commonly in a triangular form compatible with three nAChR dimers of ∼20 nm each. Addition of the full agonist carbamoylcholine activated and opened the nAChR ion channel, as revealed by the increase in capacitance relative to that of the nAChR-thiolipid system under basal conditions. Thus, the self-assembled system appears to be a viable biomimetic model to measure ionic conductance mediated by ion-gated ion channels under different experimental conditions, with potential applications in biotechnology and pharmacology.

Self-assembly of flagellin on Au(111) surfaces.

The adsorption of flagellin monomers from Pseudomonas fluorescens on Au(111) has been studied by Atomic Force Microscopy (AFM), Scanning Tunneling Microscopy (STM), X-ray Photoelectron Spectroscopy (XPS), Surface Plasmon Resonance (SPR), and electrochemical techniques. Results show that flagellin monomers spontaneously self-assemble forming a monolayer thick protein film bounded to the Au surface by the more hydrophobic subunit and exposed to the environment the hydrophilic subunit. The films are conductive and allow allocation of electrochemically active cytochrome C. The self-assembled films could be used as biological platforms to build 3D complex molecular structures on planar metal surfaces and to functionalize metal nanoparticles.

Citrate-capped silver nanoparticles showing good bactericidal effect against both planktonic and sessile bacteria and a low cytotoxicity to osteoblastic cells.

A common problem with implants is that bacteria can form biofilms on their surfaces, which can lead to infection and, eventually, to implant rejection. An interesting strategy to inhibit bacterial colonization is the immobilization of silver (Ag) species on the surface of the devices. The aim of this paper is to investigate the action of citrate-capped silver nanoparticles (AgNPs) on clinically relevant Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria in two different situations: (i) dispersed AgNPs (to assess the effect of AgNPs against planktonic bacteria) and (ii) adsorbed AgNPs on titanium (Ti) substrates, a material widely used for implants (to test their effect against sessile bacteria). In both cases, the number of surviving cells was quantified. The small amount of Ag on the surface of Ti has an antimicrobial effect similar to that of pure Ag surfaces. We have also investigated the capability of AgNPs to kill planktonic bacteria and their cytotoxic effect on UMR-106 osteoblastic cells. The minimum bactericidal concentration found for both strains is much lower than the AgNP concentration that leads to cytotoxicity to osteoblasts. Planktonic P. aeruginosa show a higher susceptibility to Ag than S. aureus, which can be caused by the different wall structures, while for sessile bacteria, similar results are obtained for both strains. This can be explained by the presence of extracellular polymeric substances in the early stages of P. aeruginosa biofilm formation. Our findings can be important to improving the performance of Ti-based implants because a good bactericidal action is obtained with very small quantities of Ag, which are not detrimental to the cells involved in the osseointegration process.

Reduction of the "burst release" of copper ions from copper-based intrauterine devices by organic inhibitors.

BACKGROUND: The copper intrauterine device is a contraceptive method that is based on the release of copper ions from a copper wire. Immediately after insertion, the dissolution of copper in the uterine fluid is markedly higher ("burst release") than that necessary for contraception action, leading to a variety of harmful effects. STUDY DESIGN: Pretreatments with organic compounds [thiourea (TU) and purine (PU), 10(-4)-10(-2) M concentration range, 1- and 3-h immersion times] were tested. The dissolution of copper with and without pretreatments in TU and PU solutions was analyzed by conventional electrochemical techniques and surface analysis. RESULTS: Pretreatments in PU solutions reduced the initial corrosion rate of copper in simulated uterine solutions, with inhibitory efficiencies that depend on the PU concentration and on the immersion time assayed. Inhibitory efficiency values higher than 98% for pretreatments with ≥10(-3) M PU were found. Conversely, after TU pretreatments, a high copper release was measured. CONCLUSIONS: It was concluded that 10(-3) M PU pretreatment is a promising strategy able to reduce the "burst release" of copper and to ensure contraceptive action.

Organization of Pseudomonas fluorescens on chemically different nano/microstructured surfaces.

This paper describes bacterial organization on nano/micropatterned surfaces with different chemical properties, which show different interactions with the biological systems (inert, biocompatible, and bactericide). These surfaces were prepared by molding techniques and exposed to Pseudomonas fluorescens (P. fluorescens) cultures. Results from atomic force microscopy and optical imaging demonstrate that the structure of P. fluorescens aggregates is strongly dependent on the surface topography while there is no clear linking with the physical-chemical surface properties (charge and contact angle) of the substrate immersed in abiotic culture media. We observe that regardless of the material when the surface pattern matches the bacterial size, bacterial assemblages involved in surface colonization are disorganized. The fact there is not a relationship between surface chemistry and bacterial organization can be explained by the coverage of the surfaces by adsorbed organic species coming from the culture medium. Viability assays indicate that copper behaves as a toxic substrate despite the presence of adsorbed molecules. The combination of surface traps and biocidal activity could act synergistically as a suitable strategy to limit bacterial spreading on implant materials.

Submicron trenches reduce the Pseudomonas fluorescens colonization rate on solid surfaces.

Bacterial adhesion and spreading on biomaterials are considered key features of pathogenicity. Roughness and topography of the substrate have been reported to affect bacterial adhesion, but little is known about their effect on spreading. Submicron row and channel tuning with bacterial diameter (S2) were designed to test bacterial motility on these surfaces. Random nanometer-sized structures (S1) were used as controls. Optical microscopy and AFM were employed to detect biological and surface pattern details in the micro- and nanoscale, respectively. Results showed that motility strategies (flagella orientation, elongation, aggregation in rafts, formation of network structures, and development of a bacterial frontier) were affected by the presence of submicropatterns. Importantly, the rate of bacterial spreading on S2 was significantly reduced and influenced by the orientation of the submicropatterns. Consequently, submicroengineered substrates could be employed as a tool to downgrade bacterial colonization. Such patterns could impact on the design of proper engineered structures to control biofilm spreading on solid surfaces.

Electrocatalytic and magnetic properties of ultrathin nanostructured iron-melanin films on Au(111).

We have prepared ultrathin, nanostructured melanin films on Au(111) by means of electrochemical self-assembly. These films were characterized by using Auger electron spectroscopy, X-ray absorption near-edge structure spectroscopy, scanning tunneling microscopy, magnetic force microscopy, and electrochemical techniques. Two types of nanostructures are present in the film: melanin nanoparticles and Fe(3)O(4) nanoparticles. The melanin nanoparticles contain Fe bonded to oxygen-containing phenolic groups in an octahedral configuration similar to that found in Fe(2)O(3). The inorganic-organic composite exhibits magnetic properties and catalyzes the electroreduction of hydrogen peroxide in alkaline and neutral electrolyte solutions. The electrocatalytic activity depends on the Fe-bound melanin and appears to be similar to that found for Fe-porphyrins.