Detection of DNA immobilized on bare gold electrodes and gold nanoparticle-modified electrodes via electrogenerated chemiluminescence using a ruthenium complex as a tag

Electrogenerated chemiluminescence (ECL) for DNA hybridization detection is demonstrated based on DNA that was self-assembled onto a bare gold electrode and onto a gold nanoparticles modified gold electrode. A ruthenium complex served as an ECL tag. Gold nanoparticles were self-assembled on a gold electrode associated with a 1,6-hexanedithiol monolayer. The surface density of single stranded DNA (ssDNA) on the gold nanoparticle modified gold electrode was 4.8?×?10¹⁴ molecules per square centimeter which was 12-fold higher than that on the bare gold electrode. Hybridization was induced by exposure of the target ssDNA gold electrode to the solution of ECL probe consisting of complementary ssDNA tagged with ruthenium complex. The detection limit of target ssDNA on a gold nanoparticle modified gold electrode (6.7?×?10^?12 mol L^?1) is much lower than that on a bare gold electrode (1.2?×?10^?10 mol L^?1). The method has been applied to the detection of the DNA sequence related to cystic fibrosis. This work demonstrates that employment of gold nanoparticles self-assembled on a gold electrode is a promising strategy for the enhancement of the sensitivity of ECL detection of DNA.     

Immobilization of cytochrome c on cysteamine-modified gold electrodes with EDC as coupling agent

Cyclic voltammetry has been applied for the characterization of cross-linked horse heart cytochrome c (HHC) on cysteamine-modified gold electrodes. The cross-linking, i.e. amide bond formation, between the proteins was achieved by using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as coupling reagent. The optimal conditions for the formation of the HHC film were determined by varying the HHC concentration. In addition the reproducibility, stability and the influence of the scan rate upon these films were investigated with cyclic voltammetry. The protein film stability in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution was tested by UV/vis absorption spectroscopy.     

Transparent gold as a platform for adsorbed protein spectroelectrochemistry: investigation of cytochrome c and azurin

The majority of protein spectroelectrochemical methods utilize a diffusing, chemical mediator to exchange electrons between the electrode and the protein. In such methods, electrochemical potential control is limited by mediator choice and its ability to interact with the protein of interest. We report an approach for unmediated, protein spectroelectrochemistry that overcomes this limitation by adsorbing protein directly to thiol self-assembled monolayer (SAM) modified, thin (10 nm), semitransparent gold. The viability of the method is demonstrated with two diverse and important redox proteins: cytochrome c and azurin. Fast, reversible electrochemical signals comparable to those previously reported for these proteins on ordinary disk gold electrodes were observed. Although the quantity of protein in a submonolayer adsorbed at an electrode is expected to be insufficient for detection of UV-vis absorption bands based on bulk extinction coefficients, excellent spectra were detected for each of the proteins in the adsorbed state. Furthermore, AFM imaging confirmed that only a single layer of protein was adsorbed to the electrode. We hypothesize that interaction of the relatively broad gold surface plasmon with the proteins' electronic transitions results in surface signal enhancement of the molecular transitions of between 8 and 112 times, allowing detection of the proteins at much lower than expected concentrations. Since many other proteins are known to interact with gold SAMs and the technical requirements for implementation of these experiments are simple, this approach is expected to be very generally applicable to exploring mechanisms of redox proteins and enzymes as well as development of sensors and other redox protein based applications.     

Electrochemical characterization of polyelectrolyte/gold nanoparticle multilayers self-assembled on gold electrodes

Polyelectrolyte/gold nanoparticle multilayers composed of poly(l-lysine) (pLys) and mercaptosuccinic acid (MSA) stabilized gold nanoparticles (Au NPs) were built up using the electrostatic layer-by-layer self-assembly technique upon a gold electrode modified with a first layer of MSA. The assemblies were characterized using UV-vis absorption spectroscopy, cyclic and square-wave voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy. Charge transport through the multilayer was studied experimentally as well as theoretically by using two different redox pairs [Fe(CN)(6)](3-/4-) and [Ru(NH(3))(6)](3+/2+). This paper reports a large sensitivity to the charge of the outermost layer for the permeability of these assemblies to the probe ions. With the former redox pair, dramatic changes in the impedance response were obtained for thin multilayers each time a new layer was deposited. In the latter case, the multilayer behaves as a conductor exhibiting a strikingly lower impedance response, the electric current being enhanced as more layers are added for Au NP terminated multilayers. These results are interpreted quite satisfactorily by means of a capillary membrane model that encompasses the wide variety of behaviors observed. It is concluded that nonlinear slow diffusion through defects (pinholes) in the multilayer is the governing mechanism for the [Fe(CN)(6)](3-/4-) species, whereas electron transfer through the Au NPs is the dominant mechanism in the case of the [Ru(NH(3))(6)](3+/2+) pair.     

Evaluation of endothelial cell adhesion onto different protein/gold electrodes by EIS

To study cell attachment to biomaterials, several proteins such as fibronectin, collagen IV, heparin, immunoglobulin G, and albumin have been deposited onto polystyrene adsorbed on a self-assembled monolayer (silane or thiol) on glass or gold, respectively. The different steps of this multilayer assembly have been characterized by electrochemical impedance spectroscopy (EIS). These data are compared to those of adhesion rate, viability percentage, and cytoskeleton labeling for a better understanding of the cell adhesion process to each protein. All the proteins are endothelial cell adhering biomolecules but not with the same features. A linear relationship has been established between adhesion rate and resistance of the endothelial cell/protein interface for all negatively charged proteins.     

DNA nanosensors based on the use of single gold nanowire electrodes and Methylene Blue as an intercalator

The authors describe an electrochemical DNA nanosensor based on the use of single gold nanowire electrodes (AuNWEs). The probe DNA is immobilized on the AuNWE via Au-S bonds that are formed between thiol-terminated DNA and the gold surface. Single AuNWEs were prepared by an improved laser-assisted pulling method and hydrofluoric acid etching. The nanoelectrodes were characterized by cyclic voltammetry and COMSOL simulation. Square wave voltammetry was used to monitor the DNA hybridization event between probe DNA and target DNA by using Methylene Blue (MB) as an intercalator of dsDNA. Under optimal conditions, the peak current for MB (best measured at a potential of ?0.2 V vs. Ag/AgCl) increases linearly with the logarithm of the analyte concentration in the 1.0 f. to 10 nM range, with a 0.48 fmM detection limit at an S/N ratio of 3. The assay is highly selective, reproducible and stable. Considering the small overall dimensions and high sensitivity, this nanoelectrode potentially can be applied to in-vivo sensing of DNA inside living cells

Open image in new window

Graphical abstract Schematic presentation of an electrochemical DNA nanosensor using single gold nanowire electrodes and based on the interaction of thiol-terminated DNA and gold surface. It was used to detect complementary DNA with high selectivity and sensitivity.

     

DNA-modified electrodes; part 4: optimization of covalent immobilization of DNA on self-assembled monolayers

The covalent immobilization of DNA onto self-assembled monolayer (SAM) modified gold electrodes (SAM/Au) was studied by X-ray photoelectron spectrometry and electrochemical method so as to optimize its covalent immobilization on SAMs. Three types of SAMs with hydroxyl, amino, and carboxyl terminal groups, respectively, were examined. Results obtained by both X-ray photoelectron spectrometry and cyclic voltammetry show that the largest covalent immobilization amount of dsDNA could be gained on hydroxyl-terminated SAM/Au. The ratio of amount of dsDNA immobilized on hydroxyl-terminated SAMs to that on carboxyl-terminated SAMs and to that on amino-terminated SAMs is (3-3.5): (1-1.5): 1. The dsDNA immobilized covalently on hydroxyl-terminated SAMs accounts for 82.8-87.6% of its total surface amount (including small amount of dsDNA adsorbed). So the hydroxyl-terminated SAM is a good substrate for the covalent immobilization of dsDNA on gold surfaces.     

Electrochemiluminescence of CdTe quantum dots as labels at nanoporous gold leaf electrodes for ultrasensitive DNA analysis

A new electrochemiluminescence (ECL) DNA assay is developed using quantum dots (QDs) as DNA labels. When nanoporous gold leaf (NPGL) electrodes are used, sensitivity of the ECL assay is remarkably increased due to ultra-thin nanopores. In this assay, target DNA (t-DNA) is hybridized with capture DNA (c-DNA) bound on the NPGL electrode, which is fabricated by conjugating amino-modified c-DNA to thioglycolic acid (TGA) modified at the activated NPGL electrode. Following that, amino-modified probe DNA is hybridized with the t-DNA, yielding sandwich hybrids on the NPGL electrode. Then, mercaptopropionic acid-capped CdTe QDs are labeled to the amino group end of the sandwich hybrids. Finally, in the presence of S₂O₈²⁻ as coreactant, ECL emission of the QD-labeled DNA hybrids on the NPGL electrode is measured by scanning the potential from 0 to −2 V to record the curve of ECL intensity versus potential. The maximum ECL intensity (I_m,ECL) on the curve is proportional to t-DNA concentration with a linear range of 5 × 10⁻¹⁵ to 1 × 10⁻¹¹ mol/L. The ECL DNA assay can be used to determine DNA corresponding to mRNA in cell extracts in this study.     

Surface characterization of a cross‐linked cytochrome c film on cysteamine‐modified gold electrodes

The aim of the present paper is to characterize a cross‐linked horse heart cytochrome c (HHC) film on cysteamine‐modified gold electrodes. The HHC film was deposited using 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide (EDC) as a coupling agent. Attenuated total reflection infrared (ATR‐IR) spectroscopic analyses were performed to characterize the newly formed surface on a qualitative and conformational level. The film thickness was measured using a noncontact optical surface profiler, while quantitative data and information on the heterogeneity of the film were obtained by means of synchrotron radiation X‐ray micro fluorescence (SR micro‐XRF). Results indicate that, in addition to electrochemical studies, spectroscopic analysis methods are essential to gain insight in the effect of immobilization strategies on protein conformations. The latter is of relevance in the development and optimization of biosensors. Copyright © 2009 John Wiley & Sons, Ltd.     

Electrochemical studies of cytochrome c on gold electrodes with promotor of humic acid and 4-aminothiophenol

p-Aminothiophenol (PATP) and humic acids (HA or HAs) were applied jointly as the electron transfer accelerants of redox reactions of cytochrome c (Cyt c) on gold electrodes. The electrochemical properties of the modified electrodes were studied by field emission scanning electron microscope, ultraviolet-visible spectroscopy, electrochemical impedance spectroscopy, Raman spectroscopy and cyclic voltammetry. The immobilized Cyt c displayed a couple of stable and well-defined redox peaks with a formal potential of −0.101 V (vs. SCE) in pH 7.0 phosphate buffer solution. Cyt c adsorption is in the form of a monolayer with average surface coverage of 5.28 pmol cm⁻². The electron transfer rate constant was calculated to be 2.14 s⁻¹. It indicate that the HA film acted as a good adsorption matrix for Cyt c and an excellent accelerant for the redox of Cyt c. The Cyt c-HA modified gold electrode showed a new couple of well-marked redox peaks when 2,4-dichlorophenol was added to the test solution.     

Electric detection of target DNA by fabricating gold nanowire bridges on planar nanogap electrodes

For the electrical detection of target DNA (partial avian influenza virus/H1N1/HA sequence) prepared via asymmetric PCR, we fabricated DNA-templated conducting gold nanowire bridges on planar nanogap electrodes using positively charged gold nanoparticles.     

Photocurrent response after enzymatic treatment of DNA duplexes immobilized on gold electrodes: electrochemical discrimination of 5-methylcytosine modification in DNA

We demonstrate a photoelectrochemical approach to the detection of the methylation status of cytosine bases in DNA. We prepared anthraquinone (AQ) photosensitizer-tethered oligodeoxynucleotide (ODN) duplexes bearing 5-methylcytosine (mC) or the corresponding cytosine (C) at a restriction site of the ODN strand immobilized on gold electrodes, and measured their photocurrent responses arising from hole transport after enzymatic digestion. Treatment with HapII or HhaI of the duplexes bearing normal C led to strand cleavage, and the photosensitizer unit was eliminated from the ODN strand immobilized on the gold electrode, exclusively reducing the photocurrent density. With a similar treatment, the duplexes bearing mC showed higher photocurrent responses arising from hole transport through the duplex. This significant difference in the photocurrent response between mC and normal C residues in DNA on the gold electrodes is potentially applicable to the detection of mC modification in DNA.     

DNA as a template for synthesis of fluorescent gold nanoclusters

Water-soluble blue-emitting gold nanoclusters have been synthesized using dsDNA as a template without any additional reducing agent. The features of the formed nanoclusters have been revealed by fluorescence and electronic absorption spectroscopy as well as transmission electron microscopy. The prepared gold nanoclusters have been highly stable at physiological pH without any further modification.     

Effect of size and protein environment on electrochemical properties of gold nanoparticles on carbon electrodes

We studied the electrochemical properties of gold nanoparticles (GNPs) and their complexes with proteins using square-wave voltammetry. Effect of the nanoparticle size and detection procedure was explored upon the oxidation of GNPs on a glassy carbon electrode (GCE). For pre-characterized GNPs of 13, 35 and 78 nm diameter, the oxidation peak potential was + 0.98, + 1.03 and + 1.06 V vs. Ag/AgCl, respectively. The conjugation of GNPs with four different proteins was verified by UV–Vis spectroscopy and atomic force microscopy indicated the formation of protein shells around GNPs. This process hampered the oxidation of GNPs on bare GCE causing pronounced decrease in the current response by an average factor of 72. GCE modification with carbon nanotubes weakly influenced the sensitivity of GNP detection but resulted in a 14.5-fold signal increase averaged for all GNP–protein complexes. The acidic dissolution and electrodeposition of GNPs or their complexes adsorbed on GCE allowed superior signal amplification directly proportional to nanoparticle size. The results are useful for the optimization of voltammetric analysis of GNP–protein complexes and can be extended to the characterization of other metal nanostructures and their complexes with biological components.     

DNA sensor based on an Escherichia coli lac Z gene probe immobilization at self-assembled monolayers-modified gold electrodes

A novel approach to construct an electrochemical DNA sensor based on immobilization of a 25 base single-stranded probe, specific to E. coli lac Z gene, onto a gold disk electrode is described. The capture probe is covalently attached using a self-assembled monolayer of 3,3′-dithiodipropionic acid di(N-succinimidyl ester) (DTSP) and mercaptohexanol (MCH) as spacer. Hybridization of the immobilized probe with the target DNA at the electrode surface was monitored by square wave voltammetry (SWV), using methylene blue (MB) as electrochemical indicator. Variables involved in the sensor performance, such as the DTSP concentration in the modification solution, the self-assembled monolayers (SAM) formation time, the DNA probe drying time atop the electrode surface and the amount of probe immobilized, were optimized.

A good stability of the single- and double-stranded oligonucleotides immobilized on the DTSP-modified electrode was demonstrated, and a target DNA detection limit of 45 nM was achieved without signal amplification. Hybridization specificity was checked with non-complementary and mismatch oligonucleotides. A single-base mismatch oligonucleotide gave a hybridization response only 7 ± 3%, higher than the signal obtained for the capture probe before hybridization. The possibility of reusing the electrochemical genosensor was also tested.     

The adsorption of cytochromes on a modified surface of gold electrodes

The adsorption of cytochromes b ₅ and c on the surface of gold electrodes, including the surface modified with cysteine, was studied. The quartz crystal microbalance method with parallel dissipation energy measurements, microcontact printing, and atomic-force microscopy were used to show that the special features of the structure and morphology of two-component cytochrome b ₅ and c films were determined by the nature of the proteins themselves and the influence of the modifying “sublayer.” The largest changes in the weight of films and dissipation energy were observed in the adsorption of cytochrome b ₅ on a cytochrome c film deposited on a cysteine sublayer. Atomic-force microscopy measurements showed that strong interaction between cytochrome c and b ₅ molecules on the surface of gold modified with cysteine could be related to the formation of the corresponding protein complex.     

Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes

An electrochemical DNA biosensor based on the recognition of single stranded DNA (ssDNA) by hybridization detection with immobilized complementary DNA oligonucleotides is presented. DNA and oligonucleotides were covalently attached through free amines on the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamino)propyl-N′-ethylcarbodiimide hydrochloride (EDC) onto a carboxylate terminated alkanethiol self-assembled monolayers (SAM) preformed on a gold electrode (AuE). Differential pulse voltammetry (DPV) was used to investigate the surface coverage and molecular orientation of the immobilized DNA molecules. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE, mismatched hybrid-modified AuE, and the probe-modified AuE which indicates the MB signal is determined by the extent of exposed bases. Control experiments were performed using a non-complementary DNA sequence. The effect of the DNA target concentration on the hybridization signal was also studied. The interaction of MB with inosine substituted probes was investigated. Performance characteristics of the sensor are described.     

Electrocatalytic activity of DNA on electrodes as an indication of hybridisation

Electron transfer between metal electrodes and ferro/ferricyanide is completely suppressed at low ionic concentration. We describe here a new phenomenon related to this reaction: an immobilisation of thiolated single-stranded DNA on gold electrodes retains this activity at low ionic strength up to the level corresponding to the high ionic strength. In contrast, a hybridisation of the complementary DNA with the thiolated single-stranded DNA followed by a binding onto the electrodes, attenuated the electrocatalytic effect. These effects can be used for discrimination between single-stranded DNA and double-stranded DNA and for semi-quantitative measurement of complementary DNA in a sample.     

基于多壁碳纳米管和纳米金修饰电极测定特殊序列DNA的电化学生物传感器的制备

基于多壁碳纳米管和纳米金复合膜修饰电极制备了特殊序列的靶DNA的电化学生物传感器.该传感器以六氨基合钌为杂交指示剂,用差示脉冲伏安法进行检测DNA杂化,其响应信号与靶DNA浓度在1.0×10<'-12>～1.0×10<'-7>mol/L范围内呈线性关系,检测限达3.5×10<'-13>mol/L.该传感器能区分单碱基错配的靶DNA.     

Electrochemical detection of DNA hybridization using methylene blue and electro-deposited zirconia thin films on gold electrodes

A novel electrochemical DNA biosensor based on methylene blue (MB) and zirconia (ZrO₂) thin films modified gold electrode for DNA hybridization detection is presented. Zirconia thin films were electrodynamically deposited onto the bare gold electrode in an aqueous electrolyte of ZrOCl₂ and KCl by cycling the potential between −1.1 and +0.7 V (versus Ag/AgCl) at a scan rate of 20 mV s⁻¹. Oligonucleotide probes with phosphate group at the 5′ end were attached onto the zirconia thin films because zirconia is affinity for phosphoric group. The surface density of the immobilized DNA molecules at the zirconia interface was investigated by fluorescence spectroscopy method. Hybridization was induced by exposure of the ssDNA-containing Au electrode to complementary ssDNA in solution. The decreases in the peak currents of MB, an electroactive label, were observed upon hybridization of probe with the target. The cathodic peak current (i_p) of MB after hybridization with the target DNA was linearly related to the logarithmic value of the target DNA concentration ranging from 2.25×10⁻¹⁰ to 2.25×10⁻⁸ mol l⁻¹. A detection limit of 1.0×10⁻¹⁰ mol l⁻¹ of oligonucleotides can be estimated.