Multilayer electrodes: Fully electroactive cyt c on gold as a part of a DNA/protein architecture (original) (raw)
Related papers
Interactions between cytochrome c and DNA strands self-assembled at gold electrode
International Journal of Molecular Sciences, 2007
In this work, we reported the investigation on the interaction between DNA strands self-assembled at gold electrodes and an electron transfer protein, cytochrome c. We observed that cytochrome c exhibited well-defined electrochemistry in both double-stranded and single-stranded DNA films. This suggested that the electron transfer reaction of cytochrome c arose possibly due to the electron hopping along DNA strands rather than wiring along the double helix. We also compared the heterogeneous electron transfer rate of cytochrome c with that of a ruthenium complex, which further confirmed this mechanism.
Procedia Engineering, 2012
A novel third generation biosensor was developed based on one-shot adsorption of chemically modified cytochrome c (cyt c) onto bare gold electrodes. The introduction of short-chain thiol derivatives (mercaptopropionic acid, MPA) on the lysine residues of cyt c enabled the very fast formation (<5 min) of an electroactive biological self-assembled monolayer (SAM) exhibiting a quasi-reversible electrochemical behavior and a fast direct electron transfer (ET). The high value estimated for the heterogeneous ET rate constant, k s = 1600 s-1 , indicates that short anchors might facilitate the ET via an efficient orientation of the heme pocket. In comparison, no direct ET was observed in the case of native and long-anchor modified (mercaptoundecanoic acid, MUA) cyt c adsorbed on gold. The so-made amperometric biosensor enabled real-time and non-invasive detection of extracellular H 2 O 2 released by unicellular aquatic microorganisms Chlamydomonas reinhardtii as a consequence of cadmium-induced oxidative stress. Motivation and results Over the last two decades amperometric biosensors based on the enzymatically-catalyzed reduction of H 2 O 2 have attracted a wide interest due to their accurate sensitivity and specificity. In the case of the so-called third generation biosensors which rely on direct ET the ability to achieve an efficient electrical communication underlies resolving orientation and distance issues between the electrode surface and the protein redox center. 1 It is known that cyt c exhibits a very low ET once in contact to solid bare electrodes which results in a poor electrochemical behavior. 2 Thus, a number of studies have focused on modifying the gold electrode surface in order to generate a suitable molecular environment that prevents from cyt c denaturing and enhances the ET rate constant. Here we report a chemically modified cyt c that forms adsorbed monolayer onto bare gold and exhibits very fast ET rate constant. The introduction of thiol derivatives-via chemical coupling on cyt c lysine residues-that act as anchor molecules on gold prevents protein denaturing and enables rational "tuning" of the ET rate with the chain length. As expected for native cyt c no faradaic peak developed on the capacitive current whereas well defined peaks are visible for cyt c-MPA which corresponds to the electrochemical oxidation and reduction of heme group (Fig. 1). However, cyt c-MUA that exhibits a longer anchor-spacer shows no direct ET albeit its electroactivity is restored as soon as small gold nanoparticles act as electron shuttle. In contrast, the cyt c-MPA layer shows a quasi-reversible electrochemical behavior indicating that fast interfacial ET mechanism is predominant due to a favorable protein orientation on gold. The voltammetric monitoring of MPA-cyt c chemisorption on bare gold emphasizes the rapidity of the electroactive protein layer formation with the maximum peak intensity reached within 5 minutes (20 cycles at = 0.05 V s-1), as shown in Figure 2A. Moreover, the analysis of the peaks intensity shows a reversible electrochemical behaviour with a value of Ia/Ic = 0.96. The calculation of the E FWHM = 86 mV which is below the theoretical value of 90.6 mV defines a nerstian monoelectronic process strongly adsorbed on the electrode surface. 3 The surface coverage () of electroactive MPA-cyt c on gold was estimated to be 4x10-12 mol cm-2 which corresponds roughly to 25 % of the theoretical value reported by Nakano et al. 4 for a fully packed cyt c surface coverage. It is also observable that the formal potential E°' =-48 mV (vs Ag/AgCl) is negative-shifted as compared to native cyt c in solution, as expected for a covalently immobilized protein. 5 Furthermore, cyclicvoltammetry was performed at different scan rates in order to study the ET mechanism taking place at a MPA-cyt c modified electrode. The voltammograms in Figure 2B show the influence of the scan rate both on the peak current intensity and the peak-to-peak separation. As depicted in Figure 2C the anodic and cathodic peaks currents are linearly proportional to the scan rate in the range between 0.01 and 0.6 V s-1 , which is expected for a surface-controlled electrochemical process. An estimated value of the heterogeneous ET rate constant, k s , has been calculated from the analysis of peak-to-peak separations fusing Laviron's method. Considering a charge transfer coefficient = 0.5 a high value of k s = 1600 s-1 was calculated, which is in good agreement with those reported for cyt c covalently attached onto a SAM of mercaptobutyric acid 6 or a mixed SAM of MPA/mercaptoethanol. 7
Fabrication of DNA monolayers on gold substrates and guiding of DNA with electric field
2003
We report electrically controlled selective coating of gold electrodes with mixed monolayers of oligonucleotides and alkanethiol passivation molecules. Gold nanoparticles are used as labels for visualization and voltage between the electrodes is applied for guiding the oligonucleotides. We discuss the efficiency of the guiding and monolayer preparation procedure.
Layer-by-layer assembly of electro-active gold nanoparticle/cytochrome c multilayers
Biosensors and Bioelectronics, 2009
In this study we have tested the use of modified gold nanoparticles (GNPs) for introduction into cytochrome c (CytC) multilayer assemblies, constructed by the layer-by-layer deposition technique. For this purpose gold nanoparticles of different size are synthesized and the GNPs modified with mercaptopropionic acid (MPA). The particles are characterized by UV-vis-spectroscopy and transmission electron microscopy (TEM). The modified particles have been studied with respect to their binding capability on a CytC layer by SPR. The negatively charged particles exhibit rather fast adsorption kinetics on CytC. We can also show that CytC binds to MPA-modified gold nanoparticles.
Electrical Probes of DNA-Binding Proteins
Methods in enzymology, 2017
A DNA electrochemistry platform has been developed to probe proteins bound to DNA electrically. Here gold electrodes are modified with thiol-modified DNA, and DNA charge transport chemistry is used to probe DNA binding and enzymatic reaction both with redox-silent and redox-active proteins. For redox-active proteins, the electrochemistry permits the determination of redox potentials in the DNA-bound form, where comparisons to DNA-free potentials can be made using graphite electrodes without DNA modification. Importantly, electrochemistry on the DNA-modified electrodes facilitates reaction under aqueous, physiological conditions with a sensitive electrical measurement of binding and activity.
Electrochemical probe for the monitoring of DNA–protein interactions
Biosensors and Bioelectronics, 2010
Self-assembly of thiol-terminated oligonucleotides on gold substrates provides a convenient way for DNA-functionalized surfaces. Here we describe the development of an electrochemical assay for the detection of DNA-protein interactions based on the modification of the electrochemical response of methylene blue (MB) intercalated in the DNA strands. Using a functionalized electrode with double stranded DNA carrying T3 RNA polymerase binding sequence, we show a substantial attenuation of the current upon the DNA-protein interaction. Moreover, a Langmuir binding isotherm for T3 RNA polymerase (T3 Pol) gives a dissociation constant K D equal to 0.46 ± 0.23 M. Such value is 100 times lower than the calculated K D for the non-specific interaction of bovine serum albumin (BSA) with T3 Pol promoter. In addition, the use of the T7 RNA polymerase (T7 Pol) promoter instead of the T3 Pol promoter induces an increase of K D from 0.46 M to more than 25 M. Accordingly, this strong decrease in the affinity of T3 Pol towards an off-target DNA promoter reveals an electrochemical sequence-specific discrimination of DNA-protein interactions. In conclusion, our results show that the developed electrochemical test allows the monitoring of DNA-protein interactions with high specificity and with an in situ protein detection threshold at a nanomolar range.
Bioconjugate Chemistry, 2008
The base pair stack of DNA has been demonstrated as a medium for long range charge transport chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to structural perturbations in the base pair stack as occur with lesions, single base mismatches, and protein binding. We have exploited this sensitivity for the development of reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers. Here we discuss the characteristic features, applications, and advantages of DNA-mediated electrochemistry.
Oligonucleotide-modified electrodes for fast electron transfer to cytochrome c
Electrochemistry Communications, 1999
Gold electrodes were modified with short ds-oligonucleotides via thiol binding to form a thin and stable surface layer. The modification was characterised by impedance measurements and used as a promoter for fast electron transfer to cytochrome c. The protein was investigated both immobilised and in solution showing reversible electrochemical behaviour in each case. The modification proved to have a good adsorption capability for the redox protein which was also found to be reversible. In the immobilised state at the electrode cytochrome c reacted with superoxide radicals in solution, exemplified by cyclic voltammetric measurements.