Modified Gold Electrode Surface Research Articles

DNA Sensor for Recognition of Native Yeast DNA Sequence with Methylene Blue as an Electrochemical Hybridization Indicator

An electrochemical DNA sensor based on the recognition of single-stranded yeast DNA (ssDNA) immobilized on gold electrode to its complementary ssDNA (cDNA) is presented for hybridization detection. The yeast single-stranded DNA was covalently bound on a self-assembled 3-mercaptopropionic acid monolayer by using water soluble N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) as a linker. The self-assembled monolayer prevented the nonspecific adsorption of long DNA chain on the electrode surface. The covalently immobilized ssDNA could selectively hybridize with cDNA in solution to form double-stranded DNA (dsDNA) on the surface. The increase of peak currents of methylene blue (MB), an electroactive indicator, upon the hybridization of immobilized ssDNA with cDNA in the solution was observed, which was used to monitor the recognition of yeast DNA sequence. The interaction of immobilized ssDNA and dsDNA with MB also resulted in a transition of electrode process from diffusion-controlled to surface-controlled process. The adsorption constants of methylene blue on ssDNA and dsDNA modified gold electrode surface were found to be (3.3±0.3)×103 M−1 and (6.6±0.4)×103 M−1, respectively, indicating a higher affinity of dsDNA to MB.

Sensitivity enhancement of DNA sensors by nanogold surface modification

A novel amplified microgravimetric gene sensing system was developed using quartz crystal microbalance modified by gold nanoparticles anchored on its 1,6-hexanedithiol modified gold electrode surface, and ultrasensitive detection of DNA hybridization was accomplished at the level of at least 2×10 −16 M .

Synthesis of 5-(ω-sulfhydrylalkyl)salicylaldehydes as precursors for the preparation of alkanethiol-modified metal salens

Using multistep syntheses, we obtained two alkanethiol-modified salicylaldehydes, namely 5-(2-sulfhydrylethyl)salicylaldehyde and 5-(6-sulfhydrylhexyl)salicylaldehyde. These compounds are precursors for the preparation of alkanethiol-substituted metal salens, which can potentially be used to form surface-modified gold electrodes.

Synthesis and Electrochemical Behaviour of Vinylferrocene-Propylene Sulfide-Graft Copolymers

Poly(vinylferrocene)–graft–poly(propylene sulfide) copolymers have been synthesized and were used to form surface-modified gold electrodes (SMEs) by self-assembly and drop-evaporation techniques. The electrochemical behaviours and stabilities of the polymer films in the SMEs in 1 M H2SO4 media were investigated by cyclic voltammetry. The SMEs exhibited the expected electrochemical response and it was confirmed that the electrontransfer reactions occurred on the surface of the electrode. Repeated cycling of new SMEs indicated that films containing sulfur groups were more robust than similar SMEs based on simple poly(vinylferrocene). The stability of the SMEs increased with increasing proportions of propylene sulfide in the graft polymers.

Cytochrome c′ from Chromatium vinosum on gold electrodes

Cytochrome c′ from Chromatium vinosum (CVCP) was immobilized at a surface-modified gold electrode. Characterization of the CVCP electrode showed a quasi-reversible, diffusionless electrochemical redox behavior of the surface adsorbed protein with a formal potential of −128±5 mV vs. Ag/AgCl. The heterogeneous electron transfer rate constant of adsorbed CVCP was determined to be about 50 s −1. Different immobilization strategies were compared. The interaction of the immobilized CVCP with nitric oxide (NO) was investigated and applied for a primary amperometric detection of NO in solution.

Interfacial Recognition of Sugars by Boronic Acid-Carrying Self-Assembled Monolayer

Disulfides that carried two boronic acid groups at their ends were prepared by coupling of 3-aminophenylboronic acid with dithiodialiphatic acid (DTAA; number of methylene groups: 1, 2, and 3) using a water-soluble carbodiimide. The compounds obtained formed a self-assembled monolayer (SAM) on both silver and gold colloids and on gold electrodes as proven by surface-enhanced Raman spectroscopy (SERS) and cyclic voltammetry (CV). Small sugars (fructose, glucose, mannose, and galactose) and polysaccharide (mannan) were bound to the boronic acid SAM on the gold colloid as detected by the changes in absorption of the colloid at 522 nm. The affinity of small sugars for the phenylboronic acid group was in the order fructose ≫ mannose > galactose > glucose, and these results could be attributed to the regioselective binding of the phenylboronic acid group in the SAM to hydroxyl groups of the sugars. The binding of sugars or sugar-carrying protein (ovalbumin) to the surface-modified gold electrode was also shown by the increase in potential difference in the cyclic voltammograms. The effect of the distance between the sulfur atom and the phenylboronic acid group in the SAM on the recognition of sugars is also discussed.

Superoxide electrode based on covalently immobilized cytochrome c: modelling studies

We have recently described an optimised electrode for the detection of enzymatic and cellular superoxide (O 2 •−) production based on cytochrome c immobilized covalently at a surface-modified gold electrode and applied this system to the study of free radical production by activated human glioblastoma cells. In this paper we report the development of a mathematical model for the O 2 •− electrode responding to enzymically produced O 2 •− which should enable the determination of absolute concentrations of O 2 •− in biological systems when this electrode is employed for direct, real-time monitoring of free radical release and interactions.

Redox thermodynamics of the native and alkaline forms of eukaryotic and bacterial class I cytochromes c.

The reduction potentials of beef heart cytochrome c and cytochromes c2 from Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Rhodobacter capsulatus were measured through direct electrochemistry at a surface-modified gold electrode as a function of temperature in nonisothermal experiments carried out at neutral and alkaline pH values. The thermodynamic parameters for protein reduction (DeltaS degrees rc and DeltaH degrees rc) were determined for the native and alkaline conformers. Enthalpy and entropy terms underlying species-dependent differences in E degrees and pH- and temperature-induced E degrees changes for a given cytochrome were analyzed. The difference of about +0.1 V in E degrees between cytochromes c2 and the eukaryotic species can be separated into an enthalpic term (-DeltaDeltaH degrees rc/F) of +0.130 V and an entropic term (TDeltaDeltaS degrees rc/F) of -0.040 V. Hence, the higher potential of the bacterial species appears to be determined entirely by a greater enthalpic stabilization of the reduced state. Analogously, the much lower potential of the alkaline conformer(s) as compared to the native species is by far enthalpic in origin for both protein families, and is largely determined by the substitution of Met for Lys in axial heme ligation. Instead, the biphasic E degrees /temperature profile for the native cytochromes is due to a difference in reduction entropy between the conformers at low and high temperatures. Temperature-dependent 1H NMR experiments suggest that the temperature-induced transition also involves a change in orientation of the axial methionine ligand with respect to the heme plane.

Preparation of gold surfaces with biospecific affinity for NAD(H)-dependent lactate dehydrogenase

Gold surfaces with biospecific affinity for NAD(H)-dependent lactate dehydrogenase have been prepared by covalent attachment of several triazine dyes to chemisorbed functionalized alkanethiol self-assembled monolayers. The dyes which work as coenzyme analoga build a site-specific affinity complex with the enzyme by binding through its NAD +-binding pocket. This immobilization method implies a biospecific recognition of the enzyme which is expected to yield lactate dehydrogenase self-assembled monolayers in which the enzyme is oriented facing its NAD +-binding pocket (and therefore its active site) towards the metal surface. Self-assembled monolayers with Cibacron Blue F3G-A as anchored ligand showed always the highest affinity for lactate dehydrogenase. However, the biospecific affinity of the ligand-anchored monolayer was strongly dependent on the nature of the alkanethiol underlayer used to bind the triazine dye. Higher enzymatic surface coverages were obtained with mixed self-assembled monolayers providing a low amount of ligand (less than a 10% of a densely packed alkanethiol monolayer) bound to the metal surface through a long and flexible spacer. Lactate dehydrogenase- modified gold electrode surfaces catalyzed the electro-oxidation of lactate only when the biological cofactor (NAD +) was present in the reaction mixture, thus suggesting that the NAD +-binding pocket used to anchor the enzyme to the monolayer is not involved in the enzymatic reaction.

Electrochemical investigations of the intermolecular electron transfer between cytochrome c and NADPH-cytochrome P450-reductase

The electron exchange reaction of cytochrome c with NADPH-cytochrome P450 reductase was investigated electrochemically. For this purpose the electrochemical behavior of cytochrome c in solution, adsorbed or covalently immobilized on a modified gold electrode surface was studied. In the case when cytochrome c was in solution or only electrostatically adsorbed on the electrode surface, fast electron transfer was observed with NADPH-cytochrome P450 reductase. When cytochrome c was covalently bound to the electrode surface, in spite of quasi-reversible electron exchange with the electrode, no electron transfer was observed with NADPH-cytochrome P450 reductase. These results suggest that electrostatically adsorbed cytochme c on the modified electrode surface has some mobility that allows re-orientation as required to interact both with the electrode and with the NADPH-cytochrome P450 reductase. In contrast, covalent binding of cytochrome c leads to permanent orientation towards the electrode surface and thus blocks the cytochrome c electron accepting site from the reductase.

Self-assembled monolayers with biospecific affinity for lactate dehydrogenase for the electroenzymatic oxidation of lactate

Surface modified gold electrodes with high biospecific affinity for NAD(H)-dependent lactate dehydrogenase have been prepared by covalent attachment of several traizine dyes to stepwise functionalized mixed alkanethiol self-assembled monolayers. The biospecific affinity of such ligand-anchored monolayers to bind submonolayer amounts of enzyme was demonstrated from the course of the protein adsorption events monitored by surface plasmon resonance. Electroenzymatic activity measurements of lactate dehydrogenase modified surfaces for the reaction of lactate oxidation, carried out ‘ex situ’ at different stages of protein layer growth, allowed the optimization of the preparative procedure to yield reproducible enzymatic electrodes with a low amount of unspecifically bound protein. A short adsorption time, as well as a high concentration of enzyme in the solution used for protein layer growth, led to lactate dehydrogenase-modified gold electrode surfaces with a high electroenzymatic activity arising mainly from biospecifically bound species. The lowest amount of unspecifically adsorbed protein was found for ligand-anchored monolayers prepared from mixed alkanethiol underlayers with an excess of positively charged groups. The lack of electroenzymatic activity shown by lactate dehydrogenase modified electrodes in the absence of soluble coenzyme (NAD +) indicates that none of the investigated ligand-anchored monolayers could provide an efficient electronic pathway from the metal to the active site of the enzyme. Therefore, the monolayers acted just as an anchoring system for lactate dehydrogenase.

Surface-modified gold electrodes with biospecific affinity for lactate dehydrogenase based on Cibacron Blue 3FG-A self-assembled monolayers

Abstract Surface-modified gold electrodes with biospecific affinity for NAD(H)-dependent lactate dehydrogenase (LDH) have been prepared from mixed self-assembled monolayers bearing the coenzyme-analogous, Cibacron Blue 3FG-A, as anchored pendant group. Enzymatic monolayers are formed by formation of a complex upon the site-specific binding of Cibacron Blue to the enzyme through its NAD + -binding pocket. In such monolayers the enzyme may be expected to lie oriented on the electrode surface with its NAD + -binding pocket (and therefore its active site) connected directly to the metal through a spacer. However, since Cibacron Blue is not electroactive within the range of potentials used to electro-oxidize lactate, the ligand monolayer was found not to be involved in the electroenzymatic reaction, but worked only as an anchoring system for the enzyme. LDH-modified electrodes were able to electro-oxidize lactate at small overpotentials only in the presence of soluble coenzyme, NAD + . The electroenzymatic activity of LDH-modified electrodes depended on the amount of enzyme immobilized and on the concentration of lactate in the reaction mixture, with a Michaelis constant K M = 10 mM for lactate. The overall electroenzymatic activity of LDH-modified electrodes was found to arise from both, biospecifically and unspecifically, adsorbed enzyme. Whereas the amperometric response due to the biospecifically adsorbed species dropped off following the first shot of lactate, that due to unspecifically adsorbed enzyme remained stable after several electro-oxidation runs and only disappeared after inactivation of the enzyme.

Correlation of the Structural Decomposition and Performance of Pyridinethiolate Surface Modifiers at Gold Electrodes for the Facilitation of Cytochrome c Heterogeneous Electron-Transfer Reactions

This paper describes the results of an electrochemical and spectroscopic (infrared reflection and X-ray photoelectron spectroscopies) investigation of the modified gold electrode surfaces prepared from dilute ethanolic solutions of 4-mercaptopyridine (PySH) and 4,4‘-dipyridyl disulfide (PySS). Both precursors have been used extensively as facilitators for the electron transfer of redox proteins like cytochrome c (cyt c). During the course of an investigation of the interfacial architectures formed from the two different precursors, a previously unreported structural instability in the adlayers was discovered. This instability manifests itself as a decrease in the ability of the modified surfaces to facilitate the electron transfer of cyt c that correlates with an increase of the immersion time in the precursor solutions. Results are presented that delineate the decrease in facilitator performance and probe the structural changes resulting in the decrease in performance. Together, the electrochemical and surface spectroscopic findings reveal that the modified surfaces spontaneously decompose to yield an adlayer composed largely of adsorbed atomic and oligomeric sulfur, an adlayer that we found to be ineffective in the facilitation of the electron transfer reaction of cyt c. The implications of these findings on the use of this type of modifier to studies of electron transfer reactions of redox proteins and to issues of the general structural stability of organosulfur-based monolayers are briefly discussed.

Parathyroid hormone induces superoxide anion burst in the osteoclast: evidence for the direct instantaneous activation of the osteoclast by the hormone.

We have shown that superoxide anion (O2-) production by the osteoclast can be used as an index of the osteoclast activity since the agents that inhibit and stimulate the osteoclast also diminish and stimulate O2- production respectively. Therefore, we have investigated the mechanism of parathyroid hormone (PTH)-mediated stimulation of osteoclast function in terms of its effect on O2- generation. The determination of O2- generation was carried out by employing cytochrome c immobilised on a surface-modified gold electrode. The basal level of free radical production by the osteoblast-like cells (ROS 17/2.8) was 10(4)-fold lower than by osteoclasts cultured on bone. PTH had no acute effect on free radical production by the osteoblasts. The exposure of the osteoclasts cultured on bone to PTH led to a dramatic and immediate stimulation of O2- generation which was unaffected by the presence of ROS 17/2.8 cells. The osteoclasts co-cultured with ROS 17/2.8 cells and exposed to PTH for 3 h were also found to produce greater stimulation of O2- than the osteoclasts exposed to PTH alone. A competitive leukotriene D4 antagonist REV 5901, which also inhibits 5-lipoxygenase, did not block O2- generation by osteoclasts cultured alone or in the presence of osteoblasts. Therefore, we conclude that PTH directly stimulates osteoclasts to produce O2-; this may be the main mode of activation of the osteoclasts, although an osteoblast-mediated effect of the hormone cannot be ruled out.

Macroscopically rapid electron transfer of cytochrome c522 with high thermostability at bare and surface-modified gold electrodes

Macroscopically rapid electron transfer of cytochrome c522 with high thermostability at bare and surface-modified gold electrodes

Effect of calcitonin, elevated calcium and extracellular matrices on superoxide anion production by rat osteoclasts.

The electrochemical determination of superoxide anion generation by isolated mammalian osteoclasts was carried out by employing cytochrome c, immobilized at a surface-modified gold electrode. Osteoclasts cultured on bone generated 15-fold more superoxide anions than cells plated on plastic. Superoxide anion production by osteoclasts cultured on bone was almost completely abolished by salmon calcitonin, and partially abolished following exposure to elevated extracellular calcium. We therefore conclude that osteoclast activity is influenced substantially by the extracellular matrix.

Direct electron transfer bioelectronic interfaces: application to clinical analysis

Bioelectronic interfaces based on direct electron transfer to proteins and enzymes immobilised at functional electrode surfaces are currently under development and the potential of two such systems for application to clinical measurement will be outlined. The first is the detection of free radical production via direct electrochemistry of cytochrome c immobilised covalently at modified gold electrodes. The redox protein cytochrome c has been immobilised covalently to gold electrodes surface-modified with N-acetyl cysteine via carbodiimide condensation. The electrodes thus produced were used to measure directly the enzymatic and cellular production of the superoxide anion radical (O 2 −). The superoxide radical reduced the immobilised cytochrome c which was immediately re-oxidised by the surface-modified gold electrode poised at a potential of +25 mV ( vs Ag/AgCl). The electron transfer rate constant (k et) of this process was 3.4 ± 1.2 s −1. The rate of current generation was directly proportional to the rate of O 2 − production. The essentially reagentless system produced was designed to be applied ultimately to continuous monitoring of free radical activity in vivo since there is evidence that oxygen-derived free radical species act as mediators which cause and perpetuate inflammation in disease states, including rheumatoid arthritis and neurodegenerative disorders. The second systems are pseudo-homogeneous immunoassays based on direct electron transfer to horseradish peroxidase. Horseradish peroxidase enzyme electrodes based on activated carbon (HRP-ACE) have been constructed by simple passive adsorption. Direct electron transfer between the electrodes and HRP resulted in the electroenzymatic reduction of hydrogen peroxide at potentials lower than +480 mV ( vs Ag/AgCl) and a current sensitivity for peroxide of 637 nA μM −1 cm −2 at +50 mV ( vs Ag/AgCl). A linear response to hydrogen peroxide measurement was obtained over the range 0.2–150 μM. Kinetic analysis of the HRP-ACE system gave a heterogeneous rate constant (k′ ME) of 5.3 × 10 −3 cm s −1 and an enzyme turnover number (k′ cat,E) of 2.8 × 10 −2 cm s −1. The HRP-ACE electrode showed excellent storage stability in phosphate buffered saline (pH 7.4) at 4°C, with a calculated half-life of 235 days. The potential for electrodes, based on the HRP/ACE interaction, to be applied to clinical measurement in model no wash competitive and non-competitive (‘pseudo-homogeneous’) immunoassays is described.

Direct Voltammetric Behavior of Rs. rubrum Cytochrome c′ at a 2-Mercaptosuccinate-modified Gold Electrode

Abstract The direct electrochemical redox behavior of cytochrome c′ was first observed at a surface-modified gold electrode by 2-mercaptosuccinate. Cytochrome c′ showed quasi-reversible redox responses at the electrode, and the heterogeneous rate constant for the redox reaction was estimated to be the order of 10−4 cm/s.

Redox-induced conformational changes in myoglobin and hemoglobin: electrochemistry and ultraviolet-visible and Fourier transform infrared difference spectroscopy at surface-modified gold electrodes in an ultra-thin-layer spectroelectrochemical cell.

Using suitable surface-modified electrodes, we have developed an electrochemical system which allows a reversible heterogeneous electron transfer at high (approximately 5 mM) protein concentrations between the electrode and myoglobin or hemoglobin in an optically transparent thin-layer electrochemical (OTTLE) cell. With this cell, which is transparent from 190 to 10,000 nm, we have been able to obtain electrochemically-induced Fourier-transform infrared (FTIR) difference spectra of both proteins. Clean protein difference spectra between the redox states were obtained because of the absence of redox mediators in the protein solution. The reduced-minus-oxidized difference spectra are characteristic for each protein and arise from redox-sensitive heme modes as well as from polypeptide backbone and amino acid side chain conformational changes concomitant with the redox transition. The amplitudes of the difference bands, however, are small as compared to the total amide I absorbance, and correspond to approximately 1% (4%) of the reduced-minus-oxidized difference absorbance in the Soret region of myoglobin (hemoglobin) and to less than 0.1% of the total amide I absorbance. Some of the bands in the 1560-1490-cm-1 spectral regions could be assigned to side-chain vibrational modes of aromatic amino acids. In the conformationally sensitive spectral region between 1680 and 1630 cm-1, bands could be attributed to peptide C = O modes because of their small (2-5 cm-1) shift in 2H2O. A similar assignment could be achieved for amide II modes because of their strong shift in 2H2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrochemical sensors for direct reagentless measurement of superoxide production by human neutrophils.

Electrochemical sensors based on immobilised cytochrome c or superoxide dismutase for the measurement of superoxide radical production by stimulated neutrophils are described. Cytochrome c was immobilised covalently at a surface-modified gold electrode and by passive adsorption to novel platinised activated carbon electrodes (PACE). The reoxidation of cytochrome c at the electrode surface upon reduction by superoxide was monitored using both xanthine/xanthine oxidase and stimulated neutrophils as sources of the free radical. In addition, bovine Cu/Zn superoxide dismutase was immobilised to PACE by passive adsorption and superoxide, generated by xanthine/xanthine oxidase, detected by oxidation of hydrogen peroxide produced by the enzymic dismutation of the superoxide radical. A biopsy needle probe electrode based on cytochrome c immobilised at PACE and suitable for continuous monitoring of free radical production was constructed and characterised.