Electrochemical Quartz Crystal Microbalance Studies on Cytochrome c/Polyelectrolyte Multilayer Assemblies on Gold Electrodes

Polyelectrolyte multilayer assemblies containing proteins are of interest for applications such as sensors, bioreactors, and bioelectronics. A multilayer electrode was built up by the layer‐by‐layer strategy consisting of alternating layers of cytochrome c and poly(aniline sulfonic acid). The electrode showed a linear increase of redox active protein with the number of deposited layers. The principle of electrode preparation was transferred from needle electrodes to planar surfaces in order to further the understanding of electron transfer through the layer assembly by means of electrochemical quartz crystal microbalance studies. The deposition process was followed on‐line by detection of the frequency shift of the crystals and was found to be rather fast (minutes). The total mass deposited was found to correlate well with the electrochemical response of the immobilized cyt.c. Furthermore, the influence of the polyelectrolyte was investigated by addition of PSS to the PASA solution. The strong interaction of the former polyelectrolyte seemed to hinder the electron transfer although a multilayer formation was proved. Dilution of the protein solution with redox inactive apo‐cyt.c led to a strong decrease of the voltammetric signal, well beyond the percentage of apo‐cyt.c inside the assembly. Thus, arguments for an electron transfer via protein–protein interaction were found.     

Insights into the formation and operation of polyaniline sulfonate/cytochrome c multilayer electrodes: contributions of polyelectrolytes' properties

The multilayer formation of two different sulfonated polyanilines with cytochrome c is presented and mechanistic aspects of the contributions of the polyelectrolytes' properties to the characteristics of the assemblies are discussed. These two modified polymers, PASA1 and PASA2 are chemically synthesized and differ in the grade of sulfonation, substitution, and the chain length of the polymer. The influence of these properties on the multilayer assembly with cytochrome c is studied in detail by Quartz Crystal Microbalance (QCM) technique and Cyclic Voltammetry (CV). It is shown that the multilayer formation is successful, however, the redox activity of polyanilines itself has to be taken into account. In the case of a strong redox activity (PASA2) voltammetric analysis allows the separation of redox processes addressed to the polyelectrolyte and cyt c. For multilayers with PASA1 as building block electroactivity can be predominantly attributed to cyt c ensuring a high amount of electroactive protein and a low probability for interfering redox reaction, making this system suitable for biosensor applications.     

Modulation of direct electron transfer of cytochrome c by use of a molecularly imprinted thin film

We describe the preparation of a molecularly imprinted polymer film (MIP) on top of a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold, where the template cytochrome c (cyt c) participates in direct electron transfer (DET) with the underlying electrode. To enable DET, a non-conductive polymer film is electrodeposited from an aqueous solution of scopoletin and cyt c on to the surface of a gold electrode previously modified with MUA. The electroactive surface concentration of cyt c was 0.5 pmol cm^?2. In the absence of the MUA layer, no cyt c DET was observed and the pseudo-peroxidatic activity of the scopoletin-entrapped protein, assessed via oxidation of Ampliflu red in the presence of hydrogen peroxide, was only 30 % of that for the MIP on MUA. This result indicates that electrostatic adsorption of cyt c by the MUA–SAM substantially increases the surface concentration of cyt c during the electrodeposition step, and is a prerequisite for the productive orientation required for DET. After template removal by treatment with sulfuric acid, rebinding of cyt c to the MUA–MIP-modified electrode occurred with an affinity constant of 100,000 mol^?1 L, a value three times higher than that determined by use of fluorescence titration for the interaction between scopoletin and cyt c in solution. The DET of cyt c in the presence of myoglobin, lysozyme, and bovine serum albumin (BSA) reveals that the MIP layer suppresses the effect of competing proteins.     

Monolayer-protected nanoparticle film assemblies as platforms for controlling interfacial and adsorption properties in protein monolayer electrochemistry

Assembled films of nonaqueous nanoparticles, known as monolayer-protected clusters (MPCs), are investigated as adsorption platforms in protein monolayer electrochemistry (PME), a strategy for studying the electron transfer (ET) of redox proteins. Modified electrodes featuring MPC films assembled with various linking methods, including both electrostatic and covalent mechanisms, are employed to immobilize cytochrome c (cyt c) for electrochemical analysis. The background signal (non-Faradaic current) of these systems is directly related to the structure and composition of the MPC films, including nanoparticle core size, protecting ligand properties, as well as the linking mechanism utilized during assembly. Dithiol-linked films of Au225(C6)75 are identified as optimal films for PME by sufficiently discriminating against detrimental background current and exhibiting interfacial properties that are readily engineered for cyt c adsorption and electroactivity (Faradaic current). Surface concentrations and denaturation rates of adsorbed cyt c are dictated by specific manipulation of the individual MPCs composing the outer layer of the film. The use of specially designed, hydrophilic MPCs as a terminal film layer results in near-ideal cyt c voltammetry, attributed to a high degree of molecular level control of the necessary interfacial interactions and flexibility needed to create a uniform and effective binding of protein across large areas of a substrate. The electrochemical properties of cyt c at MPC films, including ET rate constants that are unaffected by the large ET distance introduced by MPC assemblies, are compared to traditional strategies employing self-assembled monolayers to immobilize cyt c. The incorporation of nanoparticles as protein adsorption platforms has implications for biosensor engineering as well as fundamental biological ET studies.     

Molecular lego for the assembly of biosensing layers

We propose a procedure to assemble monolayers of redox mediator, coenzyme, enzyme and stabilizing polyelectrolyte on an electrode surface using essentially electrostatic and complexing interactions. In a first step a monolayer of redox mediator, substituted nitrofluorenones, is adsorbed. In a second step, a layer of calcium cations is immobilized at the interface. It establishes a bridge between the redox mediator and the subsequently adsorbed coenzyme NAD⁺. In the next step we use the intrinsic affinity of the NAD⁺ monolayer for dehydrogenases to build up a multilayer composed of mediator/Ca²⁺/NAD⁺/dehydrogenase. The so obtained modified electrode can be used as a biosensor. Quartz crystal microbalance measurements allowed us to better understand the different parameters responsible for the adsorption. A more detailed investigation of the system made it possible to finally stabilize the assembly sufficiently by the adsorption of a polyelectrolyte layer in order to perform rotating disk electrode measurements with the whole supramolecular architecture on the electrode surface.     

Electroactive multilayer assemblies of bilirubin oxidase and human cytochrome C mutants: insight in formation and kinetic behavior

Here, we report on cytochrome c/bilirubin oxidase multilayer electrodes with different cytochrome c (cyt c) forms including mutant forms of human cyt c, which exhibit different reaction rates with bilirubin oxidase (BOD) in solution. The multilayer formation via the layer-by-layer technique and the kinetic behavior of the mono (only cyt c) and biprotein (cyt c and BOD) multilayer systems are studied by SPR and cyclic voltammetry. For the layer construction, sulfonated polyaniline is used. The only cyt c containing multilayer electrodes show that the quantity of deposited protein and the kinetic behavior depend on the cyt c form incorporated. In the case of the biprotein multilayer with BOD, it is demonstrated that the catalytic signal chain from the electrode via cyt c to BOD and oxygen can be established with all chosen cyt c forms. However, the magnitude of the catalytic current as well as the kinetic behavior differ significantly. We conclude that the different cytochrome c forms affect three parameters, identified here, to be important for the functionality of the multilayer system: the amount of molecules per layer, which can be immobilized on the electrodes, the cyt c self-exchange rate, and the rate constant for the reaction with BOD.     

Molecular dynamics simulations of layer-by-layer assembly of polyelectrolytes at charged surfaces: effects of chain degree of polymerization and fraction of charged monomers

We performed molecular dynamics simulations of the electrostatic assembly of multilayers of flexible polyelectrolytes at a charged surface. The multilayer build-up was achieved through sequential adsorption of oppositely charged polymers in a layer-by-layer fashion from dilute polyelectrolyte solutions. The steady-state multilayer growth proceeds through a charge reversal of the adsorbed polymeric film which leads to a linear increase in the polymer surface coverage after completion of the first few deposition steps. Moreover, substantial intermixing between chains adsorbed during different deposition steps is observed. This intermixing is consistent with the observed requirement for several deposition steps to transpire for completion of a single layer. However, despite chain intermixing, there are almost perfect periodic oscillations of the density difference between monomers belonging to positively and negatively charged macromolecules in the adsorbed film. Weakly charged chains show higher polymer surface coverage than strongly charged ones.     

Layer-by-Layer Construction of Novel Biofunctional Fluorescent Microparticles for Immunoassay Applications

A novel class of biofunctional fluorescent microparticles for application in immunoassays was constructed by using the layer-by-layer self-assembly method to deposit multiple layers of fluorescently labeled polyelectrolytes onto colloidal particles, followed by deposition of a protein (immunoglobulin G, IgG) layer. Microelectrophoresis experiments revealed alternating negative and positive zeta-potentials with deposition of each successive polyelectrolyte layer, indicating that the alternate electrostatic adsorption of polyelectrolytes of opposite charge was successfully achieved. Transmission electron microscopy images showed a change of the particle surface texture after polyelectrolyte multilayer deposition. Fluorescence microscopy image (FMI) analysis provided direct measurement of the fluorescence intensity of single microparticles. The observed systematic increase of the fluorescence intensity of individual microparticles with increasing polyelectrolyte layer number from FMI analysis further demonstrated the controlled regular adsorption of polyelectrolyte layers onto the polystyrene (PS) particles. Protein immobilization onto the polyelectrolyte multilayer-coated particles was verified by the different surface properties of the microparticles with respect to surface charge under pH conditions above and below the isoelectric point of the proteins. The assembly of IgG and fluorescein isothiocyanate-labeled IgG onto polyelectrolyte multilayer-coated PS microparticles and their potential use was ultimately confirmed by a solid phase immunotest. Copyright 2001 Academic Press.     

Cytochrome c/polyelectrolyte multilayers investigated by E-QCM-D: effect of temperature on the assembly structure

Protein multilayers, consisting of cytochrome c (cyt c) and poly(aniline sulfonic acid) (PASA), are investigated by electrochemical quartz crystal microbalance with dissipation monitoring (E-QCM-D). This technique reveals that a four-bilayer assembly has rather rigid properties. A thickness of 16.3 ± 0.8 nm is calculated with the Sauerbrey equation and is found to be in good agreement with a viscoelastic model. The electroactive amount of cyt c is estimated by the deposited mass under the assumption of 50% coupled water. Temperature-induced stabilization of the multilayer assembly has been investigated in the temperature range between 30 and 45 °C. The treatment results in a loss of material and a contraction of the film. The electroactive amount of cyt c also decreases during heating and remains constant after the cooling period. The contraction of the film is accompanied by the enhanced stability of the assembly. In addition, it is found that cyt c and PASA can be assembled at higher temperatures, resulting in the formation of multilayer systems with less dissipation.     

PET/PC共混体系的酯交换反应对其高压结晶行为的影响

利用转矩流变仪、DSC、SEM及WAXD等表征手段研究了PET/PC共混体系的酯交换反应对其高压结晶行为的影响.SEM观察表明,PET和PC熔混时的酯交换反应有利于PET/PC体系在高压结晶时生成厚度较大的伸直链晶体,且可以促进其高压下酯交换反应的发生.楔形伸直链晶体和弯曲伸直链晶体的存在证明链滑移扩散和酯交换反应两种机制对体系中聚酯伸直链晶体的增厚有贡献.拟合分峰法和War-ren-Averbach傅里叶分析法的计算结果表明,随PET/PC体系熔混时酯交换反应程度的增加,高压结晶共混物的结晶度降低,PET的平均微晶尺寸增大,点阵畸变平均值则减小,而微晶尺寸分布变宽.提出了在共聚物组分都具备结晶能力时,结晶诱导化学反应和化学反应诱导结晶两种过程在一定条件下可同时发生的观点.     

胶体颗粒在聚电解质多层膜表面的可控组装

利用原子力显微镜和扫描电子显微镜研究了磺化聚苯乙烯胶体颗粒在由聚二甲基二烯丙基氯化铵和聚苯乙烯磺酸钠层状自组装而成的多层膜表面的组装.该组装受表面性质影响,通过对多层膜的最外层的组装条件或利用盐溶液对多层膜进行后处理可以控制胶体颗粒在膜表面的组装密度.     

Dual-beam polarization interferometry resolves mechanistic aspects of polyelectrolyte adsorption

The electrostatically driven binding dynamics of a polyelectrolyte multilayer (PEMU) film was investigated in real-time using dual-beam polarization interferometry (DPI) and independently supported by quartz crystal microbalance with dissipation monitoring (QCM-D) studies. Multilayer assemblies of the polyanions poly[1-[4[(3-carboxy-4-hydroxyphenylazo)benzenesulfonamido]-1,2-ethanediyl sodium salt] (PAZO) and poly(styrene sulfonate) (PSS) were respectively constructed with the polycation poly(ethylenimine) (PEI) on anionic functionalized substrates using the layer-by-layer electrostatic self-assembly method. DPI measurements indicate that polyelectrolyte adsorption occurs in three distinct stages. In the first stage, for approximately 5 s, coil-like segments of polyanion partially tether to the surface of the oppositely charged PEI. In the second stage, these coils unfurl over a period of approximately 10 s to cover the surface resulting in an increase in average density of the film. During the final adsorption step, the surface-bound polyelectrolyte diffuses into the multilayer assembly, exposing the surface to further deposition. This last step occurs over a much longer time period and results in a highly interpenetrated film containing a charge-overcompensated region at the film surface.     

实时生物分子相互作用分析技术用于链霉亲和素-生物素化抗体的层层组装研究

使用生物分子相互作用分析（Biomolecular interaction analysis,BIA)技术实时监测了在链霉素和素表面层层组装亲和素－生物素化抗体多层膜的过程，结果表明，通过链霉素和素与生物素之间的强亲和作用，能够在表面形成均一的多层膜，并用实时BIA技术求得了每层蛋白质的表面浓度，对于生物素化抗体，单层吸附表面浓度为1.32ng/mm^2；对于链霉亲和素，单层吸附表面浓度为2.93ng/mm^2。同时对蛋白质在表面的排列状态进行了探讨。     

Molecular dynamics simulations of multilayer films of polyelectrolytes and nanoparticles

We performed molecular dynamics simulations of multilayer assemblies of flexible polyelectrolytes and nanoparticles. The film was constructed by sequential adsorption of oppositely charged polymers and nanoparticles in layer-by-layer fashion from dilute solutions. We have studied multilayer films assembled from oppositely charged polyelectrolytes, oppositely charged nanoparticles, and mixed films containing both nanoparticles and polyelectrolytes. For all studied systems, the multilayer assembly proceeds through surface overcharging after completion of each deposition step. There is almost linear growth in the surface coverage and film thickness. The multilayer films assembled from nanoparticles show better layer stratification but at the same time have higher film roughness than those assembled from flexible polyelectrolytes.     

Polyelectrolyte adsorption and layer-by-layer assembly: Electrochemical control

Thin films formed via the adsorption or layer-by-layer assembly of charged polymers are important in many sensing, energy, and biomedical applications. When the underlying substrate is a (semi)conductor, the opportunity exists to influence film formation and film properties through an applied electric potential. The recent literature on electrochemical influence of polyelectrolyte-based films is reviewed, with a focus on monolayer and multilayer film assembly and disassembly. Of particular interest are monolayer films grown to a tailored thickness on the 10–100 nm scale, and polyelectrolyte multilayer films controllably disassembled, upon application of a modest electric potential. Experimental observations are discussed in terms of governing factors such as interfacial pH and ionic composition, counter-ion correlations, charge regulation, dielectric discontinuity, and short-range polymer–polymer interactions. Recent modeling efforts are also briefly addressed.     

Layer-by-layer assembly of polyelectrolyte chains and nanoparticles on nanoporous substrates: molecular dynamics simulations

We performed molecular dynamics simulations of a multilayered assembly of oppositely charged polyelectrolyte chains and nanoparticles on porous substrates with cylindrical pores. The film was constructed by the sequential adsorption of oppositely charged species in a layer-by-layer fashion from dilute solutions. The multilayer assembly proceeds through surface overcharging after the completion of each deposition step. The substrate overcharging fraction fluctuates around 0.5 for nanoparticle-polyelectrolyte systems and around 0.4 for polyelectrolyte-polyelectrolyte systems. The surface coverage increases linearly with the number of deposition steps. The rate of surface coverage increases as a function of the number of deposition step changes when the pore is blocked. The closing of the pore occurs from the pore entrance for nanoparticle-polyelectrolyte systems. In the case of polyelectrolyte-polyelectrolyte systems, the pore plug is formed inside the pore and then spreads toward the pore ends.     

Use of polyelectrolyte multilayer systems for membrane modification

Membranes with designed surface and filtration properties were prepared by the adsorption of polyelectrolyte multilayer systems on membrane surfaces using the layer-by-layer electrostatic self assembly (ESA) technique. Microfiltration membranes with a first polyelectrolyte layer grafted onto the surface showed excellent stability during filtration process. Although a twofold higher permeate flux was observed for a three-layer polyelectrolyte complex membrane compared to a just grafted one the protein retention did not change remarkably. Additionally, a reduced protein adsorption was detected for repulsive electrostatic forces between the substrate and the protein under applied conditions. Pervaporation membranes with an anionically functionalized polyamide-6 support or Nafion^®-117 support and a dense separating layer consisting of poly(acrylic acid) and poly(ethylenimine) were prepared. Those membranes were used to separate aqueous organic mixtures. Six double layers were sufficient to obtain membranes with high water permselectivity. Membranes with similar properties but a lower number of deposited layers were obtained, when the adsorption process was carried out at 80°C.     

The Electrochemical Characteristics of Multilayer Assembly of Hemoglobin and Polystyrene Sulfonate at Self-assembled Monolayer Surface

It was well known that protein interacted strongly with natural and syntheticpolyelectrolytes mainly through electrostatic forces to form a complex. These forces maylead to the formation of amorphous precipitates, protein assembly by the alternatelyelectrostatic adsorption immobilize protein can be formed a multilayer filml'2. Bypreparation of anisotropic protein layer and precise control of the distance of active layer,sequential reaction and vectorial transfer of electron and energy become f…     

Immobilization and electrochemistry of cytochrome c on amino-functionalized mesoporous silica thin films

The immobilization and electrochemistry of cytochrome c (cyt c) on amino-functionalized mesoporous silica thin films are described. The functionalized silica films with an Im3m cubic phase structure were deposited on conducting ITO substrate by co-condensation of tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) in the presence of Pluronic F127 under acidic conditions. The high specific surface area, large pore size and functional inner surface of mesoporous silica thin films result in a high cyt c loading, and the cyt c immobilization on this silicate framework is stable. After adsorption of cyt c, the ordered cubic structure of mesoporous silica and the redox activity of immobilized cyt c are retained as demonstrated by X-ray diffraction (XRD), Transmission electron microscope (TEM) and cyclic voltammetry. The redox behavior of the cyt c/silica film-modified ITO electrode is a surface-controlled quasi-reversible process for the experimental conditions used in this work and the electron transfer rate constant is calculated is 1.33 s⁻¹. The ITO electrode modified by cyt c/silica film possesses a high stability; even cyt c retains its redox activity following immobilization for several months. Furthermore, the electrocatalytic activities of the modified ITO electrode to hydrogen peroxide and ascorbic acid have been studied. Since these behaviors are quite pronounced, the modified electrode can be used for detection of hydrogen peroxide and ascorbic acid.     

Studies on the electrochemical behavior of cytochrome c and its interaction with DNA at a Co/GC ion implantation modified electrode

The electrochemical behavior of cytochrome c (cyt c) and its interaction with DNA at a Co/glassy carbon (GC) ion implantation modified electrode were studied by linear sweep and cyclic voltammetry. In 0.005 mol dm(-3) Tris-0.05 mol dm(-3) NaCl buffer solution (pH = 7.10), a sensitive reduction derivative peak of cyt c was obtained by linear sweep voltammetry. The peak potential was 0.032 V (SCE). The peak current was proportional to the concentration of cyt c. The electrode process was quasi-reversible with adsorption. The electrode reaction rate constant k and the electron transfer coefficient a of cyt c were 4.42 s(-1) and 0.47, respectively. AES and XPS experiments showed that Co was implanted into the surface of the GC electrode (GCE). The implanted Co formed Co-C, which catalyzed the reduction of cyt c. The reaction of DNA with cyt c led to an electrochemically active complex, which resulted in an increase in the reduction current of cyt c. After adding DNA into the solution containing cyt c, the electrode process was still quasi-reversible with adsorption.