Thiol-modified Gold Electrodes Research Articles

An electrochemical biosensor for the detection of Pb2+ based on G-quadruplex DNA and gold nanoparticles

We present a novel simple strategy for the detection of Pb2+ based on G-quadruplex DNA and gold nanoparticles. First, gold nanoparticles were chemically adsorbed onto the surface of a thiol-modified gold electrode. Subsequently, the substrate DNA1 was adsorbed onto the surfaces of the gold nanoparticles via thiol-gold bonds, so that the complementary guanine-rich DNA2 could be hybridized to the gold electrode in sequence. [Ru(NH3)6]3+ (RuHex), which can be electrostatically adsorbed onto the anionic phosphate of DNA, served as an electrochemical probe. The presence of Pb2+ can induce DNA2 to form a stable G-quadruplex and fall off the gold electrode. The amount of RuHex remaining on the electrode surface was determined by electrochemical chronocoulometry (CC). The prepared biosensor showed high sensitivity for Pb2+ with a linear range with respect to ln(cPb2+) from 0.01 to 200nM and a low detection limit of 0.0042nM under optimal conditions. Because of the high selectivity of the Pb2+-specific DNA2, the designed biosensor also showed low false-positive signal rates with other metal ions in real-world examples. Therefore, this strategy has the potential for practical application in environmental monitoring. Graphical abstract ᅟ.

Improved peroxide biosensor based on Horseradish Peroxidase/Carbon Nanotube on a thiol-modified gold electrode.

A new 3-dimensional (3D) network of crosslinked Horseradish Peroxidase/Carbon Nanotube (HRP/CNT) on a thiol-modified Au surface has been described in order to build up the effective electrical wiring of the enzyme units with the electrode. The synthesized 3D HRP/CNT network has been characterized with cyclic voltammetry and amperometry which results the establishment of direct electron transfer between the redox active unit of HRP and the Au surface. Electrochemical measurements reveal that the high biological activity and stability is exhibited by the immobilized HRP and a quasi-reversible redox peak of the redox centre of HRP was observed at about -0.355 and -0.275V vs. Ag/AgCl. The electron transfer rate constant, KS and electron transfer co-efficient α were found as 0.57s-1 and 0.42, respectively. Excellent electrocatalytic activity for the reduction of H2O2 was exhibited by the developed biosensor. The proposed biosensor modified with HRP/CNT 3D network displays a broader linear range and a lower detection limit for H2O2 determination. The linear range is from 1.0×10-7 to 1.2×10-4M with a detection limit of 2.2.0×10-8M at 3σ. The Michaelies-Menten constant Kapp M value is estimated to be 0.19mM. Moreover, this biosensor exhibits very high sensitivity, good reproducibility and long-time stability.

Insights into Interprotein Electron Transfer of Human Cytochrome c Variants Arranged in Multilayer Architectures by Means of an Artificial Silica Nanoparticle Matrix.

The redox behavior of proteins plays a crucial part in the design of bioelectronic systems. We have demonstrated several functional systems exploiting the electron exchange properties of the redox protein cytochrome c (cyt c) in combination with enzymes and photoactive proteins. The operation is based on an effective reaction at modified electrodes but also to a large extent on the capability of self-exchange between cyt c molecules in a surface-fixed state. In this context, different variants of human cyt c have been examined here with respect to an altered heterogeneous electron transfer (ET) rate in a monolayer on electrodes as well as an enhanced self-exchange rate while being incorporated in multilayer architectures. For this purpose, mutants of the wild-type (WT) protein have been prepared to change the chemical nature of the surface contact area near the heme edge. The structural integrity of the variants has been verified by NMR and UV–vis measurements. It is shown that the single-point mutations can significantly influence the heterogeneous ET rate at thiol-modified gold electrodes and that electroactive protein/silica nanoparticle multilayers can be constructed with all forms of human cyt c prepared. The kinetic behavior of electron exchange for the mutant proteins in comparison with that of the WT has been found altered in some multilayer arrangements. Higher self-exchange rates have been found for K79A. The results demonstrate that the position of the introduced change in the charge situation of cyt c has a profound influence on the exchange behavior. In addition, the behavior of the cyt c variants in assembled multilayers is found to be rather similar to the situation of cyt c self-exchange in solution verified by NMR.

Integrating Enzymes into Cytochrome c –Based Multilayer Architectures – General Considerations and Example with Fructose Dehydrogenase

The creation of artificial electron transfer (ET) chains based on the defined arrangement of enzymes and redox proteins on electrode surfaces represents an interesting field in bioelectronics1. One approach exploits the self exchange properties of cytochrome c (cyt c) to build up new sensing electrodes2. In this study we have investigated the ET reaction of the flavin-dependent enzyme fructose dehydrogenase (FDH) with the redox protein cyt c. Two different pH optima are detected for the reaction of both proteins in solution - at acidic and neutral pH. When one reaction partner - cyt c - is immobilized on a thiol-modified gold electrode and FDH is in solution ET proceeds efficiently at neutral pH which can be concluded from well-shaped catalytic currents obtained in cyclic voltammetry. In addition, a defined dependence on the substrate concentration has been found. In acidic media the reaction can also be verified but appears to be less efficient. Furthermore, it can be demonstrated that both partners can be assembled in a stable multilayer architecture, using the biopolymer DNA as a negatively charged polyelectrolyte building block. The defined layered deposition of DNA and a mixture of FDH and cyt c is confirmed by surface plasmon resonance (SPR) measurements. Prepared on electrodes, substantial catalytic currents are recorded upon addition of fructose. The response can be enhanced by the number of layers deposited on the surface. This shows that an artificial signal chain can be constructed through multiple protein layers. FDH is not the only enzyme which can be immobilized and connected to the electrode by means of the redox protein cyt c. Cellobiose dehydrogenase, sulfite oxidase or billirubin oxidase are other examples3,4. This may indicate that cyt c based multilayers can have some general applicability when some preconditions are fullfilled. 1. Feifel et al , in Biosensors Based on Aptamers and Enzymes, Springer 2014, 140, 253-298 2. Lisdat et al., Chemical Communications 3 2009, 274-283. 3. Feifel et al., Angew. Chemie 126(22) 2014 5782-86 4 Dronov et al. JACS 2008 130(4) 1122.

Layered Protein Assemblies on Electrodes Exploiting Multistep Electron Transfer Chains for Dual Analyte Detection

In recent years there is considerable progress in constructing artificial signal chains which follow natural examples. This can result in sensitive systems which are turned on by the presence of a certain analyte. One approach is based on the construction of protein arrangements using the redox protein cytochrome c (cyt c) [1]. Here electron transfer through several layers of the protein on electrodes has been achieved. The defined arrangement of the biomolecule is an important precondition for a functional system. The layer-by-layer adsorption technique has been shown to be advantageous with this respect. Different building blocks have been applied including polyanilline derivatives, DNA and nanoparticles [2,3]. Furthermore, first examples for the construction of bi-protein assemblies with catalytic properties have been demonstrated, e.g. with sulfite oxidase [4].In this study it is first demonstrated that bi-protein assemblies using cyt c and the enzyme cellobiose dehydrogenase (CDH) can be constructed in a layered design using modified silica nanoparticles as building block [5]. It is found, that glycosylation of the enzyme significantly influences the layered assembly and the electron transfer chain from the substrate to the electrode. Conditions have been found, so that several catalytically active protein layers can be formed on the electrode surface, thus allowing the adjustment of the electrode response to lactose by the numbers of deposited layers.Based on these results a tri-protein architecture has been created on a thiol-modified gold electrode. Again the layer-by-layer deposition method is used, but additionally the multi-copper enzyme Laccase has been immobilised within this structure. The formation of the entity with cyt c, Lac, CDH, and SiNPs is first confirmed by quartz crystal microbalance (QCM) experiments, and then investigated electrochemically by cyclic voltammetry (CV).Within this architecture the two enzymes are connected to the electrode via cyt c. Since the activity of the enzymes is controlled by the delivery or withdrawal of electrons, the redox state of cyt c can be used for switching the activity of the biocatalysts. Since electron transfer throughout the whole layered entity is feasible, Lac and CDH in different distances to the electrode can be addressed. The switchable reaction cascades for Lac and CDH are functioning in an non-separated matrix without disturbing the reaction of the other biocatalyst. Whereas the CDH allows lactose detection, the activity of laccase is influenced by the oxygen content of the solution.The approach represents an advance in mimicking of biological electron transfer cascades by fixing protein-protein reactions to electrodes in the immobilized state without the need of any mediating shuttle molecule..[1 ] F. Lisdat, R. Dronov, H. Möhwald, F. W. Scheller, D. G. Kurth, Chemical Communications3 (2009) 274[2] D. Sarauli; J. Tanne; D. Schäfer; I. W. Schubart; F. Lisdat, Electrochemistry Communications11 (12) (2009) 2288[3] S. C. Feifel, F. Lisdat, Journal of Nanobiotechnology(2011) 9:59[4] R.V. Dronov, D.G. Kurth, H. Möhwald, R. Spricigo, S. Leimkühler, U. Wollenberger, K. V. Rajagopalan, F. W. Scheller, F. Lisdat, JACS130 (4) (2008) 1122[5] S. C. Feifel, A. Kapp, R. Ludwig, L. Gorton, F. Lisdat, RSC Advances 3 (2013) 3428

Investigation of the pH-Dependent Electron Transfer Mechanism of Ascomycetous Class II Cellobiose Dehydrogenases on Electrodes

Cellobiose dehydrogenase (CDH) is capable of direct electron transfer (DET) on various carbon and thiol-modified gold electrodes. As a result, these systems have been utilized as biocatalyst in biosensors and biofuel cell anodes. Class I CDHs, from basidiomycetous fungi, are highly specific to cellulose or lactose, and DET is only observed at pH values below 5.5. To extend the applicability of CDH-based electrodes, the catalytic properties and the behavior on electrode surfaces of ascomycetous class II CDHs from Chaetomium attrobrunneum, Corynascus thermophilus, Dichomera saubinetii, Hypoxylon haematostroma, Neurospora crassa, and Stachybotrys bisbyi were investigated. We found that class II CDHs have diverse properties but generally show a lower substrate specificity than class I CDHs by converting also glucose and maltose. Intramolecular electron transfer (IET) and DET at neutral and alkaline pH were observed and elucidated by steady-state kinetics, pre-steady-state kinetics, and electrochemical measurements. The CDHs ability to interact with the electron acceptor cytochrome c and to communicate with electrode surfaces through DET at various pH conditions was used to classify the investigated enzymes. In combination with stopped-flow measurements, a model for the kinetics of the pH-dependent IET is developed. The efficient glucose turnover at neutral/alkaline pH makes some of these new CDHs potential candidates for glucose biosensors and biofuel cell anodes.

Functionalized Palladium Nanoparticles for Hydrogen Peroxide Biosensor

We present a comparison between two biosensors for hydrogen peroxide (H2O2) detection. The first biosensor was developed by the immobilization of Horseradish Peroxidase (HRP) enzyme on thiol-modified gold electrode. The second biosensor was developed by the immobilization of cysteamine functionalizing palladium nanoparticles on modified gold surface. The amino groups can be activated with glutaraldehyde for horseradish peroxidase immobilization. The detection of hydrogen peroxide was successfully observed in PBS for both biosensors using the cyclic voltammetry and the chronoamperometry techniques. The results show that the limit detection depends on the large surface-to-volume ratio attained with palladium nanoparticles. The second biosensor presents a better detection limit of 7.5 μM in comparison with the first one which is equal to 75 μM.

A study of glycoprotein–lectin interactions using quartz crystal microbalance

A study of biospecific interactions between lectins and glycoproteins using a quartz crystal microbalance biosensor with dissipation monitoring (QCM-D) was reported. Four lectins were covalently immobilised on the thiol-modified gold electrode of the QCM chips in order to obtain sensing surfaces. The frequency shift served as analytical signal and the dissipation shift provided additional information about the viscoelastic properties of the glycoprotein–lectin complex formed on the surface of the QCM chip. The working conditions of the assay were optimised. The interaction between different lectins and glycoproteins was characterised by specific frequency shifts and each glycoprotein displayed its own unique lectin-binding pattern. This lectin pattern can serve as a finger print for the discrimination between various glycoproteins. The biosensor enabled quantitative determination of glycoproteins in the concentration range of 50 μg mL −1 to 1 mg mL −1 with good linearity and R.S.D. of less than 6.0%. An additional advantage of the proposed biosensor was the possibility to re-use the same lectin surfaces during a long period of time (2 month) without changes in analytical response. This was experimentally achieved by the application of a proper regeneration solution (10 mM glycine–HCl, pH 2.5). The lectin-based quartz crystal microbalance technique is suitable both for rapid screening and for quantitative assay of serum glycoproteins.

Superoxide Biosensing with Engineered Cytochrome c

Several mutation positions have been chosen for introducing positively charged lysines in human cytochrome c (cyt c) with the aim of increasing the reaction rate with superoxide radicals (SO) and thus, the sensitivity of an electrochemical cyt c based SO biosensor. The impact of the mutations on structural and redox properties as well as on the reaction rate with SO are verified. Four mutants show a higher reaction rate with the radical compared to the wild type. These mutants are used for the construction of SO sensors based on thiol-modified gold electrodes and covalently fixed proteins. The E66K mutant electrode has a clearly higher sensitivity in comparison to the wildtype based sensor.

Cytochrome c Mutants for Superoxide Biosensors

The effect of introducing positive charges (lysines) in human cytochrome c (cyt c) on the redox properties and reaction rates of cyt c with superoxide radicals was studied. The mutated forms of this electron-transfer protein are used as sensorial recognition elements for the amperometric detection of the reactive oxygen radical. The proteins were prepared by site-directed mutagenesis focusing on amino acids near the heme edge. The 11 mutants of human cyt c expressed in the course of this research have been characterized by UV-vis spectroscopy, circular dichroism, and NMR spectroscopy to verify overall structure integrity as well as axial coordination of the heme iron. The mutants are investigated voltammetrically using promoter-modified gold electrodes with respect to redox activity and formal redox potential. The rate constants for the reaction with superoxide have been determined spectrophotometrically. Four mutants show a higher reaction rate with the radical as compared to the wild type. These mutants are used for the construction of superoxide sensors based on thiol-modified gold electrodes and covalently fixed proteins. We found that the E66K mutant-based electrode has a clearly higher sensitivity in comparison with the wild-type-based sensor while retaining the high selectivity and showing a good storage stability.

Interference from Trace Copper in Electrochemical Investigations Employing Carboxylic Acid Terminated Thiol Modified Gold Electrodes

AbstractUnexpectedly, electrochemistry at variable chain length carboxylic acid terminated alkylthiol self‐assembled monolayers (SAMs) on gold electrodes gives rise to a Faradaic process in buffered aqueous electrolyte solution. In particular, the three‐carbon chain length, 3‐mercaptopropionic acid (MPA), exhibits a chemically reversible process with a mid‐point potential of 175 mV vs. Ag/AgCl under conditions of cyclic voltammetry. This process is associated with the presence of trace (parts per billion) amounts of copper(II) ions present in the chemical reagents used to prepare the aqueous electrolyte and also from the gold electrode itself. The carboxylic acid moiety on the SAM concentrates Cu2+ ions by coordination and this surface confined layer is then reduced. Methods to minimize the interference of Cu2+ ions at carboxylic acid terminated SAM are discussed and caution with respect to the interpretation of protein electrochemistry is recommended when using carboxylic acid functionalized SAMs to provide biocompatible electrochemical transduction surfaces, unless a metal free environment can be obtained.

Diffuse Layer Properties of Thiol-Modified Gold Electrodes Probed by Direct Force Measurements

The diffuse layer properties of modified gold electrodes under potentiostatic control have been determined by direct force measurements. These measurements have been performed with a colloidal probe consisting of a silica particle attached to the cantilever of an atomic force microscope. The gold electrodes were modified by self-assembled monolayers (SAMs) of different thickness. Additionally, the terminating functional groups of the monolayer have been varied. The interaction force profiles have been fit to the full solutions of the nonlinear Poisson-Boltzmann equation. An accurate quantitative description of the force profiles has been obtained by taking charge regulation between the surfaces into account. The diffuse layer potentials obtained from these fits were studied in dependence of the potential applied to the gold electrode. The capacitance of the SAM and the potential of zero charge (pzc) have been determined for various SAMs of different thickness and surface termination. The values obtained by our direct force measurements are in agreement with the ones reported by classical electrochemical techniques. The capacitance of the SAM depends primarily on the thickness of the monolayer and its crystalline structure. Pronounced differences in the pzc for the different functional groups have been found. These changes are related to the dipole moment of the functional group terminating the SAM. Our data are in agreement with ion adsorption, but this effect seems to be less pronounced than for bare gold electrodes.

Direct heterogeneous electron transfer reactions of fungal laccases at bare and thiol-modified gold electrodes

Mediatorless (direct) electron transfer between bare and thiol-modified gold electrodes and fungal laccases from different sources has been demonstrated. The electrochemical activity of the enzymes from basidiomycetes Trametes hirsuta, Trametes ochracea, and Cerrena maxima under aerobic and anaerobic conditions can clearly be observed using cyclic voltammetry and spectroelectrochemistry. Bioelectroreduction of oxygen by T. hirsuta laccase immobilized on amino-thiophenol-modified gold electrodes, starting at +625 mV vs. NHE, is demonstrated and differences in bioelectrocatalysis of the enzyme immobilized on bare and thiol-modified electrodes are shown. It was found that hydrogen peroxide was one of the products of oxygen electroreduction on gold electrodes modified with fungal laccases, whereas no significant peroxide formation was observed for T. hirsuta laccase immobilised on thiol-modified gold electrodes. Thus, a hypothesis about two different mechanisms of oxygen electroreduction by fungal laccases adsorbed on bare and thiol-modified electrodes is proposed.

QCM Study of the Aggregation of Starburst PAMAM Dendrimers on the Surface of Bare and Thiol-Modified Gold Electrodes

The aggregation of starburst PAMAM (poly(amidoamine)) dendrimers (bearing either 64 carboxylic or amine peripheral groups) from micromolar aqueous neutral solutions on the surface of bare and thiol (HS(CH2)2COOH or HS(CH2)2NH3Cl) modified gold electrodes has been studied using the QCM technique. As expected, the electrostatic interaction between protonated amine groups and carboxylate functional groups has been shown to promote much larger surface coverage values than those obtained for naked clean gold substrates. Computer fitting of the obtained titration curves to Langmuir and Frumkin isotherm models suggests that the main effect of the surface attached thiol is not an increased overall adsorption energy but a favorable lateral interaction between the adsorbed dendrimer molecules.

Measurement of the Rate of Adsorption of Electroactive Cytochrome c to Modified Gold Electrodes by Electrochemical Impedance Spectroscopy

The electrochemistry of a system containing a surface-confined electroactive species is characterized by electrochemical impedance spectroscopy, and the results are displayed on a Cole−Cole plot. The rate of adsorption of electroactive horse-heart cytochrome c to a carboxythiol-modified gold electrode is experimentally monitored by continuously measuring the change in the magnitude of the pseudocapacitance, which is directly proportional to the amount of adsorbed protein. The time dependence of the adsorption of cytochrome c to the thiol-modified gold electrode shows a significant period of diffusion-controlled behavior. Limitations on the frequency of measurement are outlined in relationship to the magnitude of the standard rate constant of the electron-transfer reaction.

Direct electron transfer of cellobiose dehydrogenase from various biological origins at gold and graphite electrodes

Direct electron transfer was observed for cellobiose dehydrogenases (CDH) from three different fungi, viz. CDH from Phanerochaete chrysosporium, Sclerotium rolfsii and Humicola insolens, in the presence of cellobiose when the enzymes were adsorbed on graphite electrodes. The redox wave of the heme cofactor of CDH could be demonstrated on thiol modified gold electrodes using cyclic voltammetry for Phanerochaete CDH and Humicola CDH; however, the electrocatalytic current for cellobiose oxidation could only be registered for Phanerochaete CDH.

Direct electron transfer between the heme of cellobiose dehydrogenase and thiol modified gold electrodes

Abstract Cellobiose dehydrogenase (CDH) is an extracellular fungal enzyme with two domains, one containing flavin adenine dinucleotide (FAD) and one containing heme. The electrochemistry of CDH, as well as its cleaved FAD- and heme-subunits, was studied using a membrane electrode, i.e. the enzyme was trapped under a permselective membrane on a cystamine or 3-mercaptopropionic acid modified gold electrode. Direct un-mediated electron transfer (ET) between the heme of CDH and thiol modified gold electrodes was demonstrated using cyclic voltammetry. At low sweep rate (10 mV s −1 ) and low pH (pH 4.3) up-hill ET from heme to FAD in CDH was observed. The formal potential of the heme in CDH and in the cleaved heme-subunit was found to be the same and equal to −41 mV versus Ag ∣ AgCl at pH 5.1. The dependence of the formal potential on the pH (in the pH range 3.6–6.0) indicates the presence of one redox-linked ionisable functional group. Entropy and enthalpy changes were determined in variable temperature experiments as follows, Δ S °′=−194±14 J mol −1 K −1 and Δ H °′=−74±6 kJ mol −1 . The electrocatalytic behaviour of the CDH electrodes was demonstrated by addition of the enzyme substrate, cellobiose. The catalytic current was shown to decrease upon increased pH, in accordance with previous kinetic data in solution. The model of electron transport from the substrate (cellobiose) to FAD, and then through the heme domain to the electrode was confirmed in the experiments.

The influence of surfactants on the electron-transfer reaction at self-assembled thiol monolayers modifying a gold electrode

Monolayer films of thiols with different terminal groups (CH3, COOH and NH2) were prepared on gold electrodes using a self-assembling procedure. Cyclic voltammetry was used to investigate the electron-transfer reaction of potassium ferricyanide at the thiol modified gold electrode in the presence of surfactants. It was found that surfactants show some effects on the electron-transfer reaction at the thiol modified electrode. Cationic surfactants can improve the reversibility of a redox reaction at the thiol modified gold electrodes, while anionic surfactants cause a little inhibition of electron transfer of the redox at both methyl and carboxyl terminal thiol modified gold electrodes. Surfactants can interact with thiol monolayers in different ways, changing the structure and properties of the monolayer film, and can further affect electron transfer at the modified gold electrode.

A reagentless amperometric hydrogen peroxide biosensor based on covalently binding horseradish peroxidase and thionine using a thiol-modified gold electrode

Abstract A new approach to construct a reagentless hydrogen peroxide biosensor is described. Horseradish peroxidase and thionine are covalently bound to a cysteamine-assembled gold electrode using glutaraldehyde as a bifunctional reagent. Thionine immobilised in this way can shuttle electrons between the electrode and the redox activity center of the enzyme. The sensor was highly sensitive to hydrogen peroxide with a detection limit of 8.0×10 −7 mol l −1 and a response time of less than 4 s. The effects of the applied potential and the pH values of the buffer solution on the response of the sensor were investigated for optimum analytical performance.

The distribution of standard rate constants for electron transfer between thiol-modified gold electrodes and adsorbed cytochrome c

The magnitude of the total rate constant of an electrochemical reaction at small overpotentials is shown to depend on the exponentials of the distance between the electron donor and acceptor, the reorganization energy, and the formal potential of the reaction. When a population distribution of one or more of these three parameters exists, the effect on the measured standard rate constant can be illuminated by using small-amplitude a.c. electrochemical impedance spectroscopy. Assuming a Gaussian distribution of the logarithm of the standard rate constants, the results from the system of cytochrome c molecules adsorbed on thiol-modified gold electrodes yield a standard deviation of approximately 0.5 for log k°/ k avg°. The peak broadening of reversible voltammograms is related to a comparable distribution of formal potentials.