Pyramidal, rodlike, spherical gold nanostructures for direct electron transfer of copper, zinc-superoxide dismutase: application to superoxide anion biosensors

It is the first time that direct electron transfer of copper, zinc-superoxide dismutase (Cu, Zn-SOD) is realized at nanospherical, nanorodlike, and nanopyramidal gold nanostructures, without any mediators or promoters. Thermodynamic and kinetic parameters of the electron transfer vary with the morphology of the electrodeposited gold nanostructures, suggesting the morphology-dependent electrochemistry of SOD. Experimental results reveal that SOD is strongly confined onto the nanostructured gold surfaces and processes its inherent enzymatic activity after being adsorbed on all three kinds of gold nanostructures, which also enable the direct electron transfer of SOD itself. A combination of the facilitated direct electron transfer and the bifunctional enzymatic catalytic activities of the SOD substantially offers a dual electrochemical approach to determination of O2(*-), in which O2(*-) could be detected both anodically and cathodically. In both the oxidation and reduction regions, the present O2(*-) biosensors display excellent analytical performance, such as wide linear range, low detection limit, quick response time, and good stability and reproducibility, while not being limited by interferences, for instance, uric acid, ascorbic acid, and hydrogen peroxide.     

Gold sputtered electrode surfaces enhance direct electron transfer reactions of human cytochrome P450s

We immobilized human cytochrome P450 (CYP), a membrane-bound enzyme, onto both smooth and nanostructured surfaces of gold electrodes via a naphthalene thiolate monolayer film. Rapid electron transfer of CYP with an electrode as a redox partner took place when the enzyme was immobilized onto an electrode surface with nanostructures. This structure was easily prepared by conventional sputtering techniques. A well-defined pair of peaks was observed at ? 0.175 V (vs. SHE) with the largest heterogeneous electron transfer rate constant of 340 s^? 1 for human CYP. The positive redox potential shift of 45 mV upon drug (testosterone) binding was clearly detected, which corresponded to a change in the spin states of heme iron in CYP. The present study showed that gold sputtered surfaces are very useful for direct electron transfer reactions of human CYP isoforms.     

Lightweight ITO Electrodes Decorated with Gold Nanostructures for Electrochemical Applications

Gold nanostructures were fabricated on a transparent indium tin oxide (ITO) coated PET substrate by an electrodeposition technique from a potassium gold (III) chloride solution for two different types of applications. It was found that the optical transparency of lightweight ITO electrodes could be maintained by depositing isolated gold nanostructures while opening up the use of these electrodes for inner sphere electron reactions, such as hydroquinone oxidation, which are not possible at ITO electrodes. For practical applications the adhesion of gold to the ITO electrode was improved by modifying the ITO surface with 3‐mercaptopropyl‐trimethoxysilane (MPS). Compared to Au/ITO, the Au/MPS/ITO electrode showed vastly improved electrochemical activity toward various electron transfer reactions when subjected to mechanical stress. The biosensing properties of the Au/MPS/ITO electrode was also investigated by studying the detection of immobilized DNA on the Au/MPS/ITO electrode via electrochemical impedance spectroscopy (EIS).     

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.     

甲烷氧化菌素功能化金纳米层层自组装修饰电极上过氧化氢的催化还原

借助巯基试剂,在纳米金颗粒表面修饰生物活性物质Mb,制备保持有Mb生物活性的功能化金纳米巯基乙胺-Au NPs-Mb.采用UV-Vis、FTIR光谱和投射电镜表征其结构,该纳米颗粒分布均匀且粒径均一,并显著改善了金纳米颗粒团聚现象.以Mb功能化金纳米为基元,采用单层自组装及层层自组装方式将其修饰到裸金电极表面.各Mb或Mb-Cu电极的电化学测试并未借助电子传递媒介.配位Cu~(2+)后,修饰有Mb的单层及层层自组装修饰的催化还原能力均显著提升.其中Cu~(2+)配位的{巯基乙胺-Au NPs-Mb}3/Au修饰电极作为一种新型H2O2生物传感器,响应时间大约为2 s,米氏常数KappM为0.787 mmol/L,表现出了较强的还原H2O2的催化活性,且稳定性较好.     

基于纳米金自组装膜固定辣根过氧化物酶的生物传感研究

将辣根过氧化物酶(HRP)通过纳米技术和自组装技术固定于电极表面,制得了酶修饰电极.纳米金与HRP形成了静电复合物并高效地保持了HRP的生物活性,以对苯二酚作为电子媒介体,差示脉冲伏安法(DPV)研究生物酶电极测定H2O2的线性范围为5.0×10-6～1.0×10-3 mol/L,检测限为2.5×10-6 mol/L,线性方程为△I=0.34765+4.05553CH2O2(mM).酶电极的表观米氏常数(K(app))为0.0675 mmol/L.实验同时证明该生物酶电极具有良好的稳定性和使用寿命.     

Ionic liquid modified GC electrode for the direct electrochemistry of chloroperoxidase

Chloropcroxidase （CPO） was immobilized by konjac glucomannan （KGM） on the 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIM][BF4]/Nafion modified glassy carbon eloctrode. The electrochemical behaviors of the immobilized CPO were investigated by cyclic voltammetry. The results showed that CPO was successfully immobilized on the GCE and underwent fast direct electron transfer reactions with the formal potential at -0.3 V vs. SCE. The modified electrode showed a good catalytic activity for elcctrocatalytical reduction of O2 and H2O2.     

石墨烯-聚多巴胺纳米复合材料制备过氧化氢生物传感器

通过合成具有仿生功能的石墨烯-聚多巴胺纳米材料,将其与辣根过氧化酶组装到电极表面,以对苯二酚为电子媒介体制备H2O2传感器.此修饰电极对H2O2具有良好的电催化活性,检测的线性范围为5.0×10-7～3.3×10-4 mol/L;线性回归方程为Y=29.69x+ 0.04577,相关系数为R=0.9995;检出限为3.7×10-7 mol/L(S/N=3).     

金电极表面聚赖氨酸固定微过氧化物酶-11的电化学研究

通过聚赖氨酸修饰将微过氧化物酶-11(MP-11)固定在金电极表面,制备成MP-11修饰电极.修饰在电极表面上的MP-11的血红素活性中心与电极之间可进行直接的电子传递反应,其氧化还原式电位为-0.39V.该修饰电极对氧的还原具有电催化活性.当MP-11与咪唑发生轴向配位反应时,其氧化还原式电位发生负移,此时对氧的还原不再具有电催化活性.     

Electrocatalysis of oxygen at hemoglobin-Au colloid-1,4-benzenedimethanethiol modified electrode

A novel renewable O2 sensor based on the direct electron transfer of hemoglobin (Hb) is proposed. Hb was immobilized on a gold nanoparticles (GNP) associated with a 1,4-benzenedimethanethiol (BDT) monolayer which were modified the electrode. The direct electrochemistry of Hb was investigated by electrochemical methods and cyclic voltammetric showing a pair of redox peaks of Hb. The high efficiency of the Hb/GNP/BDT modified gold electrode towards the catalytic electro-reduction of oxygen has been observed and the potential application of Hb/GNP/BDT modified gold electrode as biosensors to monitor O2 is proposed. The electrocatalytic response showed a linear dependence on the O2 concentration ranging from 2.0 to 40.0 micromol/L.     

Direct electrochemistry of cytochrome c on nanotextured diamond surface

Nanotextured diamond surfaces with geometrical properties close to protein dimensions were used for the realization of direct electron transfer of cytochrome c (cyt c) without any covalent bonding. The peroxidase activity of native and denatured cyt c was also investigated. Cyclic voltammograms of native cyt c show quasi-reversible electron transfer reactions, while no heme redox activity is detected for denatured cyt c. Unfolding (denaturation) of cyt c can be achieved in the presence of hydrogen peroxide. Partially or fully denatured cyt c showed higher peroxidase activity than native cyt c. This is because denatured cyt c loses its tertiary structure and hydrogen peroxide is easier to access the heme redox center. The apparent Michaelis–Menten constant K_m for native and denatured cyt c has been determined to be 0.23 mM and 0.08 mM.     

A Simple Fabrication Method for Three‐Dimensional Gold Nanoparticle Electrodes and Their Application to the Study of the Direct Electrochemistry of Cytochrome c

A simple method for constructing gold nanoparticle‐modified electrodes with three‐dimensional nanostructures is demonstrated. The electrodes were prepared by casting citrate‐reduced AuNPs onto polycrystalline gold electrodes. The resultant electrodes had a large surface area‐to‐volume ratio, adequate for high protein loading and conferring high stability. The gold nanoparticle electrodes were covered with a self‐assembled monolayer of 11‐mercaptoundecanoic acid for electrostatic immobilization of cytochrome c (cyt c). At the electrode, direct, reversible electron transfer from cyt c was observed with remarkable stability. Moreover, an extremely high surface coverage of electrochemically active cyt c, 167 fully packed monolayers, was obtained through use of the electrode.     

Using scanning electrochemical microscopy (SECM) to measure the electron-transfer kinetics of cytochrome c immobilized on a COOH-terminated alkanethiol monolayer on a gold electrode

Cytochrome c was electrostatically immobilized onto a COOH-terminated alkanethiol self-assembled monolayer (SAM) on a gold electrode at ionic strengths of less than 40 mM. Scanning electrochemical microscopy (SECM) was used to simultaneously measure the electron transfer (ET) kinetics of the bimolecular ET between a solution-based redox mediator and the immobilized protein and the tunneling ET between the protein and the underlying gold electrode. Approach curves were recorded with ferrocyanide as a mediator at different coverages of cytochrome c and at different substrate potentials, allowing the measurement of k(BI) = 2 x 10(8) mol(-1) cm3 s(-1) for the bimolecular ET and k degrees = 15 s(-1) for the tunneling ET. The kinetics of ET was also found to depend on the immobilization conditions of cytochrome c: covalent attachment gave slightly slower tunneling ET values, and a mixed CH3/COOH-terminated ML gave faster tunneling ET rates. This is consistent with previous studies and is believed to be related to the degree of mobility of cyt c in its binding configuration and its orientation with respect to the underlying electrode surface.     

Characterization of nanoporous gold electrodes for bioelectrochemical applications

The high surface areas of nanostructured electrodes can provide for significantly enhanced surface loadings of electroactive materials. The fabrication and characterization of nanoporous gold (np-Au) substrates as electrodes for bioelectrochemical applications is described. Robust np-Au electrodes were prepared by sputtering a gold-silver alloy onto a glass support and subsequent dealloying of the silver component. Alloy layers were prepared with either a uniform or nonuniform distribution of silver and, post dealloying, showed clear differences in morphology on characterization with scanning electron microscopy. Redox reactions under kinetic control, in particular measurement of the charge required to strip a gold oxide layer, provided the most accurate measurements of the total electrochemically addressable electrode surface area, A(real). Values of A(real) up to 28 times that of the geometric electrode surface area, A(geo), were obtained. For diffusion-controlled reactions, overlapping diffusion zones between adjacent nanopores established limiting semi-infinite linear diffusion fields where the maximum current density was dependent on A(geo). The importance of measuring the surface area available for the immobilization was determined using the redox protein, cyt c. The area accessible to modification by a biological macromolecule, A(macro), such as cyt c was reduced by up to 40% compared to A(real), demonstrating that the confines of some nanopores were inaccessible to large macromolecules due to steric hindrances. Preliminary studies on the preparation of np-Au electrodes modified with osmium redox polymer hydrogels and Myrothecium verrucaria bilirubin oxidase (MvBOD) as a biocathode were performed; current densities of 500 μA cm(-2) were obtained in unstirred solutions.     

Amperometric hydrogen peroxide biosensor based on horseradish peroxidase-labeled nano-Au colloids immobilized on poly(2,6-pyridinedicarboxylic acid) layer by cysteamine.

A sensitive hydrogen peroxidase (H2O2) amperometric sensor based on horseradish peroxidase (HRP)-labeled nano-Au colloids has been proposed. Nano-Au colloids were immobilized by the thiol group of cysteamine, which was associated with the carboxyl groups of poly(2,6-pyridinedicarboxylic acid) (PPDA). With the aid of the hydroquinone, the sensor displayed excellent electrocatalytical response to the reduction of H2O2. Compared with the non-Au-colloid modified electrode, i.e., PPDA/HRP, the Au-colloid modified electrode exhibited better performance characteristics, including stability, reproducibility, sensitivity and accuracy. The biosensor shows a linear response to H2O2 in the range of 3.0 x 10(-7) - 2 x 10(-3) M. The detection limit was 1.0 x 10(-7) M.     

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.     

Transport effects in the oxygen reduction reaction on nanostructured, planar glassy carbon supported Pt/GC model electrodes

The role of transport and re-adsorption processes on the oxygen reduction reaction (ORR), and in particular on its selectivity was studied using nanostructured model electrodes consisting of arrays of Pt nanostructures of well-defined size and separation on a planar glassy carbon (GC) substrate. The electrochemical measurements were performed under controlled transport conditions in a double-disk electrode thin-layer flow-cell configuration; the model electrodes were fabricated by colloidal lithography techniques, yielding Pt nanostructures of well defined and controlled size and density (diameter: 140 or 85 nm, height: 20 or 10 nm, separation: from 1-2 to more than 10 diameters). The nanostructured model electrodes were characterized by scanning electron microscopy and electrochemical probing of the active surface area (via the hydrogen adsorption charge). The electrocatalytic measurements revealed a pronounced variation of the hydrogen peroxide yield, which increases by up to two orders of magnitude with increasing separation and decreasing size of the Pt nanostructures. Similar, though less pronounced effects were observed upon varying the electrolyte flow and thus the mass transport characteristics. These effects are discussed in a reaction model which includes (i) direct reduction to H(2)O on the Pt surface and (ii) additional H(2)O(2) formation and desorption on both Pt and carbon surfaces and subsequent partial re-adsorption and further reduction of the H(2)O(2) molecules on the Pt surface.     

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.     

Immobilization and electrochemical redox behavior of cytochrome c on fullerene film-modified electrodes

Two different fullerene film-modified electrodes were prepared and used for surface immobilization and electrochemical property investigation of horse heart cytochrome c (cyt c). Both a pristine fullerene film and fullerene-palladium (C(60)-Pd) polymer film-modified platinum, glassy carbon and indium-tin-oxide (ITO) electrodes were used. The immobilized cyt c was characterized by piezoelectric microgravimetry at a quartz crystal microbalance (QCM), UV-visible absorption, and X-ray photoelectron spectroscopy (XPS), as well as cyclic voltammetry (CV) techniques. The UV-visible spectral studies revealed a small blue shift of both the Soret and Q band of the heme moiety of cyt c, immobilized on the C(60)-Pd polymer film-modified ITO electrode, as compared to the bands of cyt c in solution suggesting that molecules of cyt c are densely packed onto the surface of the modified electrode. The CV studies revealed a quasi-reversible electrode behavior of the heme moiety indicating the occurrence of kinetically hindered electron transfer. A good agreement was found between the values of cyt c electrode surface coverage determined by piezoelectric microgravimetry and cyclic voltammetry. For piezoelectric microgravimetry, these values ranged from 0.5 x 10(-10) to 2.5 x 10(-10) mol cm(-2), depending upon the amount of cyt c present in solution and the time allowed for immobilization, which compared with a value of 3.6+/-0.4 x 10(-10) mol cm(-2) determined by CV. The possible mechanisms of cyt c immobilization on the C(60) film and C(60)-Pd film-modified electrodes are also discussed.     

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.