Polyion Films Research Articles

Protein film voltammetry and co-factor electron transfer dynamics in spinach photosystem II core complex

Direct protein film voltammetry (PFV) was used to investigate the redox properties of the photosystem II (PSII) core complex from spinach. The complex was isolated using an improved protocol not used previously for PFV. The PSII core complex had high oxygen-evolving capacity and was incorporated into thin lipid and polyion films. Three well-defined reversible pairs of reduction and oxidation voltammetry peaks were observed at 4 °C in the dark. Results were similar in both types of films, indicating that the environment of the PSII-bound cofactors was not influenced by film type. Based on comparison with various control samples including Mn-depleted PSII, peaks were assigned to chlorophyll a (Chl a) (Em = -0.47 V, all vs. NHE, at pH 6), quinones (-0.12 V), and the manganese (Mn) cluster (Em = 0.18 V). PFV of purified iron heme protein cytochrome b-559 (Cyt b-559), a component of PSII, gave a partly reversible peak pair at 0.004 V that did not have a potential similar to any peaks observed from the intact PSII core complex. The closest peak in PSII to 0.004 V is the 0.18 V peak that was found to be associated with a two-electron process, and thus is inconsistent with iron heme protein voltammetry. The -0.47 V peak had a peak potential and peak potential-pH dependence similar to that found for purified Chl a incorporated into DMPC films. The midpoint potentials reported here may differ to various extents from previously reported redox titration data due to the influence of electrode double-layer effects. Heterogeneous electron transfer (hET) rate constants were estimated by theoretical fitting and digital simulations for the -0.47 and 0.18 V peaks. Data for the Chl a peaks were best fit to a one-electron model, while the peak assigned to the Mn cluster was best fit by a two-electron/one-proton model.

Polysaccharide films as templates in the synthesis of hematite nanostructures with special properties

Cationic chitosan can form polyionic complexes with anionic polysaccharides such as pectin, which can be cast into films in a similar manner to that of chitosan–starch blends. We report the use of such films as templates in the synthesis of hematite nanoparticles. Mechanistically, the interaction of the iron(II) centers with the carboxyl, amino and other functional groups in the film, after their adsorption into the pores of the template is the key to subsequent synthesis of nanoparticles through calcination. The spatial separation that the specificity of interaction provides is amply made use of through controlled calcination resulting in monodisperse nanoparticles of hematite nanoparticles. This paper also highlights the specific advantages such as rhombohedral shape, ∼40nm intensity average diameter, narrow size distribution and weak ferromagnetic properties of using the polyion films at pH 2 as templates over polysaccharides either alone or in combination in solution or as blends.

Thin Film Voltammetry of Wild Type and Mutant Reaction Center Proteins from Photosynthetic Bacteria

Photosynthetic reaction centers (RC) convert light into electrical potential via a series of electron transfers between protein-bound, redox-active cofactors. Direct voltammetry was used to characterize the RC protein from Rhodobacter sphaeroides and mutants with focus on the primary electron donor (P) cofactor. Cyclic voltammetry (CV) and square wave voltammetry (SWV) of lipid and polyion films of RCs revealed similar chemically irreversible processes, and starting, switching, or preconditioning potential of -0.15 V was required to observe a well-defined P/P(+) oxidation peak at ∼0.95 V versus normal hydrogen electrode. An irreversible chemical reaction following voltammetric oxidation led to peak decreases upon multiple scans. Mutant RCs with site-directed amino acid modifications in the vicinity of P displayed shifts of oxidation peak potential correlated with those reported from redox titrations. These studies illustrate the utility of thin film voltammetry in characterizing redox properties of bound cofactors in RC proteins.

Rapid LC-MS Drug Metabolite Profiling Using Microsomal Enzyme Bioreactors in a Parallel Processing Format

Silica nanoparticle bioreactors featuring thin films of enzymes and polyions were utilized in a novel high-throughput 96-well plate format for drug metabolism profiling. The utility of the approach was illustrated by investigating the metabolism of the drugs diclofenac (DCF), troglitazone (TGZ), and raloxifene, for which we observed known metabolic oxidation and bioconjugation pathways and turnover rates. A broad range of enzymes was included by utilizing human liver (HLM), rat liver (RLM) and bicistronic human-cyt P450 3A4 (bicis.-3A4) microsomes as enzyme sources. This parallel approach significantly shortens sample preparation steps compared to an earlier manual processing with nanoparticle bioreactors, allowing a range of significant enzyme reactions to be processed simultaneously. Enzyme turnover rates using the microsomal bioreactors were 2-3 fold larger compared to using conventional microsomal dispersions, most likely because of better accessibility of the enzymes. Ketoconazole (KET) and quinidine (QIN), substrates specific to cyt P450 3A enzymes, were used to demonstrate applicability to establish potentially toxic drug-drug interactions involving enzyme inhibition and acceleration.

Control of Electrochemical and Ferryloxy Formation Kinetics of Cyt P450s in Polyion Films by Heme Iron Spin State and Secondary Structure

Voltammetry of cytochrome P450 (cyt P450) enzymes in ultrathin films with polyions was related for the first time to electronic and secondary structure. Heterogeneous electron transfer (hET) rate constants for reduction of the cyt P450s depended on heme iron spin state, with low spin cyt P450cam giving a value 40-fold larger than high spin human cyt P450 1A2, with mixed spin human P450 cyt 2E1 at an intermediate value. Asymmetric reduction-oxidation peak separations with increasing scan rates were explained by simulations featuring faster oxidation than reduction. Results are consistent with a square scheme in which oxidized and reduced forms of cyt P450s each participate in rapid conformational equilibria. Rate constants for oxidation of ferric cyt P450s in films by t-butyl hydroperoxide to active ferryloxy cyt P450s from rotating disk voltammetry suggested a weaker dependence on spin state, but in the reverse order of the observed hET reduction rates. Oxidation and reduction rates of cyt P450s in the films are also likely to depend on protein secondary structure around the heme iron.

Comparison of DNA‐Reactive Metabolites from Nitrosamine and Styrene Using Voltammetric DNA/Microsomes Sensors

Voltammetric sensors made with films of polyions, double-stranded DNA and liver microsomes adsorbed layer-by-layer onto pyrolytic graphite electrodes were evaluated for reactive metabolite screening. This approach features simple, inexpensive screening without enzyme purification for applications in drug or environmental chemical development. Cytochrome P450 enzymes (CYPs) in the liver microsomes were activated by an NADPH regenerating system or by electrolysis to metabolize model carcinogenic compounds nitrosamine and styrene. Reactive metabolites formed in the films were trapped as adducts with nucleobases on DNA. The DNA damage was detected by square-wave voltammetry (SWV) using [Formula: see text] as a DNA-oxidation catalyst. These sensors showed a larger rate of increase in signal vs. reaction time for a highly toxic nitrosamine than for the moderately toxic styrene due to more rapid reactive metabolite-DNA adduct formation. Results were consistent with reported in vivo TD(50) data for the formation of liver tumors in rats. Analogous polyion/ liver microsome films prepared on 500 nm silica nanoparticles (nanoreactors) and reacted with nitrosamine or styrene, provided LC-MS or GC analyses of metabolite formation rates that correlated well with sensor response.

Biochemical applications of ultrathin films of enzymes, polyions and DNA.

This feature article summarizes recent applications of ultrathin films of enzymes and DNA assembled layer-by-layer (LbL). Using examples mainly from our own research, we focus on systems developed for biocatalysis and biosensors for toxicity screening. Enzyme-poly(L-lysine) (PLL) films, especially when stabilized by crosslinking, can be used for biocatalysis at unprecedented high temperatures or in acidic or basic solutions on electrodes or sub-micron sized beads. Such films have bright prospects for chiral synthesis and biofuel cells. Excellent bioactivity and retention of enzyme structure in these films facilitates their use in detailed kinetic studies. Biosensors and arrays employing DNA-enzyme films show great promise in predicting genotoxicity of new drug and chemical product candidates. These devices combine metabolic biocatalysis, reactive metabolite-DNA reactions, and DNA damage detection. Catalytic voltammetry or electrochemiluminescence (ECL) can be used for high throughput arrays utilizing multiple LbL "spots" of DNA, enzyme and metallopolymer. DNA-enzyme films can also be used to produce nucleobase adduct toxicity biomarkers for detection by LC-MS. These approaches provide valuable high throughput tools for drug and chemical product development and toxicity prediction.

Direct Electrochemistry of Cytochrome P450 Reductases in Surfactant and Polyion Films

AbstractNADPH‐cytochrome P450 reductase (CPR) serves as electron donor for cytochrome P450 catalyzed monooxygenase reactions utilizing flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) as electron transfer cofactors. Here, stable films of human and rabbit CPRs with didodecyldimethylammonium bromide (DDAB), dimyristoylphosphatidyl choline (DMPC), and poly(diallyldimethylammonium) (PDDA) were made on pyrolytic graphite (PG) electrodes for comparative structural and electrochemical studies. CD and UV‐VIS absorbance spectra suggested that near native CPR conformation is retained in PDDA films, and some conformational changes occur in DMPC or DDAB films. Cyclic voltammetry of these films gave quasireversible pairs of peaks at average formal potential −0.246±0.008 V vs. NHE. In human CPR‐DDAB (H‐CPR‐DDAB), a second pair of peaks at +0.317 V vs. NHE was found that depended strongly on identity of buffer and salt. Excepting H‐CPR in DDAB, films showed similar voltammetry, formal potentials, and ks values. While CPR‐PDDA films had near native CPR structures, electrochemical parameters did not differ significantly from CPR‐DMPC films. The relative independence of film voltammetry from the influence of film materials for CPRs is in contrast with heme iron proteins that, while retaining near native structures, have formal potentials that depend significantly on identity of the film material.

Supramolecular assembly of water-soluble poly(ferrocenylsilanes): multilayer structures on flat interfaces and permeability of microcapsules.

We report on the layer-by-layer (LBL) supramolecular assembly of redox responsive, organometallic polyion films on planar and curved (spherical) substrates. Organometallic poly(ferrocenylsilane) (PFS) polyanions and polycations were first used to assemble multilayers on planar quartz, silicon and quartz-crystal microbalance (QCM) electrodes. UV/Vis spectroscopy, spectroscopic ellipsometry and quartz-crystal microgravimetry showed a linear increase of UV absorbance, film thickness and frequency shift with increasing the number of deposited bilayers. Additional ellipsometric studies showed a square-root dependence of the film thickness on solution salt (NaCl) concentration. For the preparation of multilayer films on colloidal particles (manganese carbonate, MnCO), relatively high salt concentrations (0.5 M) were employed. PFS microcapsules were subsequently obtained by colloidal template removal using ethylenediaminetetraacetic acid (EDTA). Following the removal of the spherical template, hollow microcapsules were obtained, whose wall structure-permeability characteristics received particular attention. Atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM) were used to study the wall thickness, integrity and permeability of the capsules. Capsule-wall thickness obtained from AFM indicated the existence of a linear film growth regime when the number of adsorbed bilayers was larger than four. Capsules made of PFS polyanions and rhodamine-labelled PFS polycations were directly visualized by CLSM. Using tetramethylrhodamine isothiocyanate (TRITC)-labelled dextran (∼ 4 400 g mol) as probe, CLSM showed that capsules containing more than four PFS polycation-polyanion bilayers displayed good stability and integrity. These stable capsules are excellent candidates for the investigation of polyelectrolyte microcapsule permeability control triggered by redox stimuli.

Electroactive cytochrome [formula omitted] cast polyion films on graphite electrodes

Films of electrochemically active cytochrome P 450 B M 3 were constructed on graphite electrodes using alternate assembly with polyethyleneimine (PEI). The original layer-by-layer adsorption method was slightly modified here to form so-called “cast polyion” films. The cast polyion films were elaborated by immobilizing two successive layers of PEI and protein in very large excess with respect to a monolayer, without any intermediate washing step. Following the immobilization steps by SEM showed that uniform films of a few micrometers were deposited on the graphite surface. The electrochemically activity of the immobilized cytP450 was tested with regard to the reduction of oxygen and the one-electron reduction of the heme. Cyclic voltammetry indicated surface concentration of electrochemically active cytP450 around 0.6 nmol/cm 2, which corresponded to 5% of the total amount of protein that was consumed by the immobilisation process. Adapting the procedure to a graphite felt electrode with the view of scaling up porous electrodes for large scale synthesis increased the concentration to 0.9 nmol/cm 2. Cast polyion films may represent a simple technique to immobilize high amount of electrochemically active protein, keeping the advantage of the electrostatic interactions of the regular layer-by-layer method.

Simple design of cast myoglobin/polyethyleneimine modified electrodes

Negatively charged myoglobin (at pH values above its isoelectric point) was immobilized with the positively charged polyion polyethyleneimine (PEI) on pyrolytic graphite electrodes. A modified form of the common layer-by-layer technique was proposed, which consisted in forming non-ordered cast polyion films. The modified Mb electrodes obtained by both techniques were compared by cyclic voltammetry. The cast polyion technique gave less reproducible results but enable the immobilization of higher percentages of the total quantity of protein required to prepare the electrodes. The electrochemical properties of the negatively charged myoglobin were determined, and its capability to catalyze the electrochemical reduction of oxygen and the dechlorination of trichloroacetic acid was demonstrated. The mechanisms are discussed, and the modified pathway that has been proposed to take into account the influence of pH in the Mb-catalyzed reduction of oxygen was confirmed here.

Electrolyte Effects on Charge Transport Behavior of [Os(bpy)2(PVP)10Cl]Cl and [Ru(bpy)2(PVP)10Cl]Cl Redox Polymers in Ultra-Thin Films of Polyions

Metallopolymer films have important applications in electrochemical catalysis. The alternate electrostatic layer-by-layer method was used to assemble films of [Ru(bpy)2(PVP)10Cl]Cl (denoted as ClRu-PVP) and [Os(bpy)2(PVP)10Cl]Cl (ClOs-PVP) metallopolymers onto pyrolytic graphite electrodes. Film thickness estimated by quartz crystal microbalance was 6–8 nm. The effects of pH, electrolyte species and concentration on the electrochemical properties of these electroactive polymers were studied using cyclic voltammetry (CV). Behavior in various electrolytes was compared. Also the mass changes within the ultra-thin film during redox of Os2+/3+ were characterized by in situ electrochemical quartz crystal microbalance (EQCM). The results indicate rapid reversible electron transfer, and show that both ClRu-PVP and ClOs-PVP have compact surface structures while ClOs-PVP is a little denser than ClRu-PVP. Although hydrogen ions do not participate in the chemical reaction of either film, the movement of Na+ cation and water accompanies the redox process of ClOs-PVP films.

Sensors for toxicity of chemicals and oxidative stress based on electrochemical catalytic DNA oxidation

Films of DNA, enzymes, polyions, and catalytic redox polyions of nanometer thickness on electrodes can provide active elements for sensors for screening the toxicity of chemicals and their metabolites, and for oxidative stress. The unifying feature of this approach involves layer-by-layer electrostatic assembly of films designed to detect DNA damage. Films containing DNA and enzymes enable detection of structural damage to DNA as a basis for toxicity screening. These films bioactivate chemicals to their metabolites, which can then react with DNA, mimicking toxicity pathways in the human liver. Metallopolyions that catalyze DNA oxidation can be incorporated into DNA/enzyme films leading to “reagentless” sensors. These sensors are suitable for detecting relative DNA damage rates in <5 min of the enzyme reactions. Films of the osmium polymer [Os(bpy) 2(PVP) 10Cl] + [poly(vinylpyridine), PVP] can be used to monitor DNA oxidation selectively. Such films may be applicable to determination of oxidized DNA as a clinical biomarker for oxidative stress. Inclusion of the analogous ruthenium metallopolymer in the sensor provides a monitor for oxidation of other nucleobases.

Electrochemical catalysis of styrene epoxidation with films of MnO(2) nanoparticles and H(2)O(2).

Films of polyions and octahedral layered manganese oxide (OL-1) nanoparticles on carbon electrodes made by layer-by-layer alternate electrostatic adsorption were active for electrochemical catalysis of styrene epoxidation in solution in the presence of hydrogen peroxide and oxygen. The highest catalytic turnover was obtained by using applied voltage -0.6 V vs SCE, O(2), and 100 mM H(2)O(2). (18)O isotope labeling experiments suggested oxygen incorporation from three different sources: molecular oxygen, hydrogen peroxide, and/or lattice oxygen from OL-1 depending on the potential applied and the oxygen and hydrogen peroxide concentrations. Oxygen and hydrogen peroxide activate the OL-1 catalyst for the epoxidation. The pathway for styrene epoxidation in the highest yields required oxygen, hydrogen peroxide, and a reducing voltage and may involve an activated oxygen species in the OL-1 matrix.

Development of multilayer fluorescent thin film chemical sensors using electrostatic self-assembly

Electrostatic layer-by-layer (LBL) self-assembly is an attractive method for depositing films, composed of charged molecules, on a wide variety of charged substrates. This report describes progress toward development of LBL as a platform for fabrication of fluorescent sensors based on nanocomposite multilayer ultrathin films. This study had two main features: 1) assessment of methods for immobilizing indicators using LBL and 2) demonstration of the versatility of these methods for application to templates of specific interest for optical sensing. Ruthenium-based oxygen indicators were used as model fluorophores. Three techniques for building fluorescent sensing films were considered, including direct electrostatic assembly of charged fluorescent indicators, fluorophore:polyion premixing, and conjugation of indicator to polyelectrolyte. Dye-conjugated polyion films exhibit superior linearity in growth, stability, and resistance to desorption during the assembly process. Multilayer fluorescent films containing bis(2, 2'-bipyridine) 4'-methyl-4-carboxybipyridine-ruthenium-N-succinimidyl-ester bis(hexafluoro-phosphate) (Ru(bpy)2(mcbpy)) conjugated to poly(allylamine hydrochloride) (PAH) were deposited onto glass slides, fiber-optics and polymer microspheres. Additional layers of fluorescein 5(6)-isothiocyanate (FITC) conjugated to PAH were deposited on the same templates to serve as an internal reference and allow ratiometric measurements. The fluorescence properties of films on the different substrates were similar, demonstrating that the deposition process is versatile and generally portable between templates. In all cases, the films retained oxygen sensitivity and did not exhibit significant self quenching. This work provides a basis for development of sensors for numerous biomedical sensing applications, including fiber-optic probes designed for research and clinical measurements, sensing films on tissue culture substrates, and implantable micro/nanoparticle based sensors for in vitro or in vivo monitoring.

Optimization of electrochemical and peroxide-driven oxidation of styrene with ultrathin polyion films containing cytochrome P450cam and myoglobin.

The catalytic and electrochemical properties of myoglobin and cytochrome P450(cam) in films constructed with alternate polyion layers were optimized with respect to film thickness, polyion type, and pH. Electrochemical and hydrogen peroxide driven epoxidation of styrene catalyzed by the proteins was used as the test reaction. Ionic synthetic organic polymers such as poly(styrene sulfonate), as opposed to SiO(2) nanoparticles or DNA, supported the best catalytic and electrochemical performance. Charge transport involving the iron heme proteins was achieved over 40-320 nm depending on the polyion material and is likely to involve electron hopping facilitated by extensive interlayer mixing. However, very thin films (ca. 12-25 nm) gave the largest turnover rates for the catalytic epoxidation of styrene, and thicker films were subject to reactant transport limitations. Classical bell-shaped activity/pH profiles and turnover rates similar to those obtained in solution suggest that films grown layer-by-layer are applicable to turnover rate studies of enzymes for organic oxidations. Major advantages include enhanced enzyme stability and the tiny amount of protein required.

Thin film nanofabrication via layer-by-layer adsorption of tubule halloysite, spherical silica, proteins and polycations

Abstract The microcylinders of halloysite of 50 nm diameter, 500 nm length and with 20 nm diameter hollow inner core were used in the assembly by alternate adsorption with poly(ethyleneimine) (PEI), which resulted in the formation of ordered multilayers containing from 2 up to 20 layers of the tubules glued together by polycation interlayers. The tubules in each monolayer are loosely packed; they form a network in the remaining 50% empty space. The total thickness of the (halloysite/PEI) 14 film was found to be 720 nm, i.e. 54 nm for the layer pair. The tubule/sphere superlattice films were also assembled through alternation of halloysite, 45-nm diameter silica spheres and linear polycations. An assembly of alcohol dehydrogenase (ADH) and halloysite loaded with nicotinamide adenine dinucleotide in alternation with PEI was accomplished. This assembly is designed to provide a direct supply of the ADH-cofactor to the enzyme immobilized in polyion films.

Adsorption behaviors of methyl orange to alternate polyion films as studied by in-situ absorption and second harmonic generation measurements

Abstract In-situ second harmonic generation (SHG) and total reflection absorption techniques were applied to the studies of the adsorption process of methyl orange at the polyion layer formed on the surface of a glass prism. In the case of poly(diallyldimethyl ammnoium chloride) (PDDA), adsorption isotherms obtained from the both techniques were well fitted to the Langmuir model, implying the same degree of noncentrosymmetric alignment during the adsorption process. As to poly(allylamine)hydrochloride (PAH), on the other hand, the both data resulted in different adsorption profiles; the Langmuir model was used for the absorption data, while the Brunauer–Emmett–Teller (BET) model for the SHG results.

Influence of bromide on electrochemistry of photosynthetic reaction center films on gold electrodes

A strong influence of bromide ion was found on voltammetry of layered films of photosynthetic reaction center (RC) protein and polyions on gold electrodes. Similar, but not identical, cyclic voltammetry peaks were observed for polyion films on gold with and without RC when the buffer solutions contained bromide ion. CVs of RC films were quite different in the absence of bromide. These new findings suggest that previously published results were biased by significant background peaks involving bromide ion adsorption/desorption.

Electroactive Films of Alternately Layered Polycations and Iron–Sulfur Protein Putidaredoxin on Gold

Layered, electrochemically active films of bacterial iron–sulfur protein putidaredoxin (Pdx) and poly(dimethyldiallyammonium) (PDDA) polycations were constructed on gold electrodes coated with mercaptopropane sulfonate (MPS) and on quartz slides. Second-derivative UV–vis spectra suggested similar structures of Pdx in films and solutions at pH 7. Direct electrochemistry was achieved between Pdx and gold electrodes in these films, with significantly better electrochemical reversibility than in cast Nafion–lipid–Pdx films. A formal potential dispersion model gave a good fit to square wave voltammograms by regression analysis and was used to estimate an average apparent rate constant of 4.5 s−1. Reduced Pdx in the polyion films did not react with its natural redox partner cytochrome P450cam because of unfavorable thermodynamics in the film environment.