Hybridization biosensor using di(2,2'-bipyridine)osmium (III) as electrochemical indicator for detection of polymerase chain reaction product of hepatitis B virus DNA

A novel hepatitis B virus (HBV) DNA biosensor was developed by immobilizing covalently single-stranded HBV DNA fragments to a gold electrode surface via carboxylate ester to link the 3(')-hydroxy end of the DNA with the carboxyl of the thioglycolic acid (TGA) monolayer. A short-stranded HBV DNA fragment (181bp) of known sequence was obtained and amplified by PCR. The surface hybridization of the immobilized single-stranded HBV DNA fragment with its complementary DNA fragment was evidenced by electrochemical methods using [Os(bpy)(2)Cl(2)](+) as a novel electroactive indicator. The formation of double-stranded HBV DNA on the gold electrode resulted in a great increase in the peak currents of [Os(bpy)(2)Cl(2)](+) in comparison with those obtained at a bare or single-stranded HBV DNA-modified electrode. The mismatching experiment indicated that the surface hybridization was specific. The difference between the responses of [Os(bpy)(2)Cl(2)](+) at single-stranded and double-stranded DNA/TGA gold electrodes suggested that the label-free hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. X-ray photoelectron spectrometry technique has been employed to characterize the immobilization of single-stranded HBV DNA on a gold surface.     

Fluorescein-labeled “arch-like” DNA probes for electrochemical detection of DNA on gold nanoparticle-modified gold electrodes

In the present study, a gold nanoparticle-modified gold electrode (nanogold electrode) was used to develop a novel fluorescein electrochemical DNA biosensor based on a target-induced conformational change. The nanogold electrode was obtained by electrodepositing gold nanoparticles onto a bare gold electrode. This modification not only immobilized probe oligonucleotides, but also adsorbed fluorescein onto the surface of the gold nanoparticles to form an “arch-like” structure. This article compares the electrochemical signal changes caused by the hybridization of “arch-like” DNA on nanogold electrode and linear DNA on bare gold electrode. The results showed that the adsorption effect of nanogold can enhance the sensitivity of the sensor. The linear range of target ssDNA is from 2.0 × 10⁻⁹ M to 2.0 × 10⁻⁸ M with a correlation coefficient of 0.9956 and detection limit (3σ) of 7.10 × 10⁻¹⁰ M. Additionally, the specificity and hybridization response of this simple sensor were investigated.     

Electrochemical properties of DNA-intercalating doxorubicin and methylene blue on n-hexadecyl mercaptan-doped 5'-thiol-labeled DNA-modified gold electrodes

Interactions between DNA-intercalating molecules, methylene blue (MB) and doxorubicin (DOX), and gold surface modified by various DNA species and n-hexadecyl mercaptan (HDM) were investigated by cyclic voltammetry (CV). Hydrophilic DOX was completely blocked by the HDM film from contacting the gold electrode whereas hydrophobic MB could readily partition into the film. Unlabeled single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) underwent non-specific adsorption on gold surface but the adsorbed DNA can be partially displaced by HDM. Thiol-labeled ssDNA and dsDNA adsorbed on gold surface via both thiol-gold linkage and non-specific interactions between DNA strands and gold. The non-specific interactions could be interrupted by the addition of HDM, forming a mixed monolayer containing both HDM and DNA attached to the gold surface at 5'-thiol termini. The presence of ssDNA and dsDNA in the monolayer facilitated the redox reaction of MB and DOX on the modified electrode. Both MB and DOX diffuse along the ssDNA in the ssDNA-containing monolayers, and they additionally intercalate into the dsDNA in the dsDNA-containing monolayers. No sufficient evidence is shown to indicate that an organized monolayer is formed by the thiol-labeled dsDNA on gold surface, and that the redox reactions of MB and DOX were carried out by electron transfer through DNA helix.     

Enhancement of DNA immobilization and hybridization on gold electrode modified by nanogold aggregates

Gold electrodes modified by nanogold aggregates (nanogold electrode) were obtained by the electrodeposition of gold nanoparticles onto planar gold electrode. The Electrochemical response of single-stranded DNA (ssDNA) probe immobilization and hybridization with target DNA was measured by cyclic voltammograms (CV) using methylene blue (MB) as an electroactive indicator. An improving method using long sequence target DNA, which greatly enhanced the response signal during hybridization, was studied. Nanogold electrodes could largely increase the immobilization amount of ssDNA probe. The hybridization amount of target DNA could be increased several times for the manifold nanogold electrodes. The detection limit of nanogold electrode for the complementary 16-mer oligonucleotide (target DNA1) and long sequence 55-mer oligonucleotide (target DNA2) could reach the concentration of 10(-9) mol/L and 10(-11) mol/L, respectively, which are far more sensitive than that of the planar electrode.     

Electrogenerated chemiluminescence of luminol in neutral and alkaline aqueous solutions on a silver nanoparticle self-assembled gold electrode.

The electrochemiluminescence (ECL) behaviour of luminol on a silver nanoparticle self-assembled gold electrode in neutral and alkaline solutions was investigated using conventional cyclic voltammetry (CV). The silver nanoparticle self-assembled gold electrode exhibited excellent ECL properties for the luminol ECL system. In neutral solutions, four ECL peaks (ECL-1-ECL-4) were observed at 0.73, 1.15, -0.46 and -1.35 V (vs. SCE), respectively. The intensities of these peaks were enhanced significantly compared with those on a bulk gold electrode and a gold nanoparticle self-assembled gold electrode. It was found that ECL-1 and ECL-2 on a silver nanoparticle-modified electrode were about 1000 and 1770 times stronger than those on a bare Au electrode and were about 17 and 15 times stronger than those on a gold nanoparticle-modified electrode, respectively. In alkaline solutions, four ECL peaks were also observed that were much stronger than those in neutral solutions, and ECL-1 and ECL-2 were enhanced by about three orders and one order of magnitude compared with those on a bare Au electrode and on a gold nanoparticle self-assembled electrode, respectively. Moreover, the silver nanoparticle-modified electrode exhibited good stability and reproducibility for luminol ECL. These peaks were found to depend on a number of factors, including silver nanoparticles on the surface of the modified electrode, potential scan direction, scan rate, scan range, the presence of O2 or N2, pH values, the concentrations of NaBr and luminol, and buffer solutions. The emitter of the ECL was confirmed as 3-aminophthalate by analysing the CL spectra. The surface state of the silver nanoparticle self-assembled electrode was characterized by scanning electron microscopy (SEM) and the interface property of the electrode was studied by electrochemical impedance spectroscopy (EIS). A mechanism for the formation of these ECL peaks is proposed. The results demonstrate that luminol has excellent ECL properties, such as strong ECL intensity and good reproducibility on a silver nanoparticle-modified gold electrode, in both neutral and alkaline solutions, which is of great potential in analytical applications.     

DNA point mutation detection based on DNA ligase reaction and nano-Au amplification: a piezoelectric approach

A novel piezoelectric method for DNA point mutation detection based on DNA ligase reaction and nano-Au-amplified DNA probes is proposed. A capture probe was designed with the potential point mutation site located at the 3' end and a thiol group at the 5' end to be immobilized on the gold electrode surface of quartz crystal microbalance (QCM). Successive hybridization with the target DNA and detection probe of nano-Au-labeled DNA forms a double-strand DNA (dsDNA). After the DNA ligase reaction and denaturing at an elevated temperature, the QCM frequency would revert to the original value for the target with single-base mismatch, whereas a reduced frequency response would be obtained for the case of the perfect match target. In this way, the purpose of point mutation discrimination could be achieved. The current approach is demonstrated with the identification of a single-base mutation in artificial codon CD17 of the beta-thalassemia gene, and the wild type and mutant type were discriminated successfully. The scanning electron microscope (SEM) image showing that plenty of gold nanoparticles remained on the electrode surface demonstrated that the nano-Au label served as an efficient signal amplification agent in QCM assay. A detection limit of 2.6 x 10(-9)mol/L of oligonucleotides was achieved. Owing to its ease of operation and low detection limit, it is expected that the proposed procedure may hold great promise in both research-based and clinical genomic assays.     

Development of an electrochemical DNA biosensor with a high sensitivity of fM by dendritic gold nanostructure modified electrode

In this paper, dendritic gold nanostructure (DenAu) modified electrode was obtained by direct electrodeposition of planar electrode into 2.8 mM HAuCl(4) and 0.1 M H(2)SO(4) solution under a very negative potential of -1.5 V. Scanning electron microscopy was used to characterize the growth evolution of DenAu with time. The whole DNA biosensor fabrication process based on the DenAu modified electrode was characterized by cyclic voltammetry and electrochemical impedance spectroscopy methods with the use of ferricyanide as an electrochemical redox indicator. The probe DNA immobilization and hybridization with target DNA on the modified electrode could be well distinguished by using methylene blue as an electrochemical hybridization indicator. The DenAu modified electrode could realize an ultra sensitivity of 1 fM toward complementary target DNA and a very wide dynamic detection range (from 1 fM to 1 nM).     

A sensitive electrochemiluminescent sensor chip based on the ssDNA-Ru(II) complex and aptamer for the determination of thrombin

In this work, an electrochemiluminescence (ECL) sensor chip for sensitive detection of thrombin (TB) was prepared using a screen-printed electrode (SPE) as a working electrode and an aptamer as a specific recognition moiety. To produce an ECL sensor chip, a layer of pL-Cys was immobilized on the surface of the SPE using the cyclic voltammetry scanning method. A layer of gold nanoparticles (AuNPs) was assembled through an Au–S bond and hairpin DNA was further immobilized on the electrode surface. Ru(bpy)₂(mcpbpy)²⁺, as a luminescent reagent, was covalently bound to single-stranded DNA (ssDNA) to prepare a luminescence probe ssDNA-Ru. The probe was hybridized with TB aptamer to form a capture probe. In the presence of TB, the TB aptamer in the capture probe bound to TB, causing the release of ssDNA-Ru that could bind to hairpin DNA on the electrode surface. The Ru(II) complex as a luminescent reagent was assembled onto the electrode, and pL-Cys was used as a co-reactant to enhance the ECL efficiency. The ECL signal of the sensor chip generated based on the above principles had a linear relationship with log TB concentration at the range 10 fM to1 nM, and the detection limit was 0.2 fM. Finally, TB detection using this method was verified using real blood samples. This work provides a new method using an aptamer as a foundation and SPE as a material for the detection of biological substances.     

Electrical recognition of label-free oligonucleotides upon streptavidin-modified electrode surfaces

For the purpose of developing a direct label-free electrochemical detection system, we have systematically investigated the electrochemical signatures of each step in the preparation procedure, from a bare gold electrode to the hybridization of label-free complementary DNA, for the streptavidin-modified electrode. For the purpose of this investigation, we obtained the following pertinent data; cyclic voltammogram measurements, electrochemical impedance spectra and square wave voltammogram measurements, in Fe(CN)₆ ³⁻/Fe(CN)₆ ⁴⁻ solution (which was utilized as the electron transfer redox mediator). The oligonucleotide molecules on the streptavidin-modified electrodes exhibited intrinsic redox activity in the ferrocyanide-mediated electrochemical measurements. Furthermore, the investigation of electrochemical electron transfer, according to the sequence of oligonucleotide molecules, was also undertaken. This work demonstrates that direct label-free oligonucleotide electrical recognition, based on biofunctional streptavidin-modified gold electrodes, could lead to the development of a new biosensor protocol for the expansion of rapid, cost-effective detection systems.     

A sensitive signal-on assay for MTase activity based on methylation-responsive hairpin-capture DNA probe

This work develops a simple, sensitive and signal-on electrochemical sensor for methyltransferase (MTase) activity analysis. The sensor is composed of a methylene blue-modi?ed "signaling DNA probe" and a "capture DNA probe" tethered methylation-responsive hairpin DNA (hairpin-capture DNA probe). The thiol- modified hairpin-capture DNA probe at 5' end was firstly self-assembled on gold electrode via Au-S bonding. Methylation-induced scission of hairpin-capture DNA probe would displace the hairpin section and remain the "capture DNA probe" section on the gold electrode. Subsequently, the remained "capture DNA probe" on the gold electrode can hybridize with the methylene blue-modi?ed "signaling DNA probe", mediating methylene blue onto the gold electrode surface to generate redox current. It was eT on state. The developed facile signal-on electrochemical sensing system showed a linear response to concentration of Dam MTase range from 0.1 to 1.0 U/mL. The detection limit of Dam MTase activity was determined to be 0.07 U/mL and the total detection time is 7h. The sensor also has the ability to provide information about the dynamics of methylation process. Furthermore, we demonstrated that this sensor could be utilized to screen inhibitors or drugs for Dam MTase.     

Electrochemical studies of kanamycin immobilization on self-assembled monolayer and interaction with DNA

The electrochemical characteristics of kanamycin onto self-assembled monolayer (SAM) modified gold electrode (SAM/Au) is investigated by cyclic voltammetry. In the potential range 0-0.6 V, Cu(II) yields a pair of stable redox waves at the bare gold electrode. E(pa) is located at 0.189 V and E(pc) at 0.254 V. In contrast, Cu(II) is reduced at a more positive potential and a decreasing current at the kanamycin SAM/Au electrode. Cu(II) and kanamycin can form a stoichiometry complex with chemical ratio of 2:1. The interaction of Cu(II)-kanamycin complex with calf thymus DNA is also studied in the solution. And the interactive mode between Cu(II)-kanamycin complex and DNA is verified by the fluorescence method. Binding constants (K) of the Cu(II)-kanamycin complex to DNA and binding site size (s) are calculated from voltammetric data and equal to 1.5 x 10(7) l/mol and 4 bp, respectively.     

A chronocoulometric aptasensor based on gold nanoparticles as a signal amplification strategy for detection of thrombin

A sensitive chronocoulometric aptasensor for the detection of thrombin has been developed based on gold nanoparticle amplification. The functional gold nanoparticles, loaded with link DNA (LDNA) and report DNA (RDNA), were immobilized on an electrode by thrombin aptamers performing as a recognition element and capture probe. LDNA was complementary to the thrombin aptamers and RDNA was noncomplementary, but could combine with [Ru(NH₃)₆]³⁺ (RuHex) cations. Electrochemical signals obtained by RuHex that bound quantitatively to the negatively charged phosphate backbone of DNA via electrostatic interactions were measured by chronocoulometry. In the presence of thrombin, the combination of thrombin and thrombin aptamers and the release of the functional gold nanoparticles could induce a significant decrease in chronocoulometric signal. The incorporation of gold nanoparticles in the chronocoulometric aptasensor significantly enhanced the sensitivity. The performance of the aptasensor was further increased by the optimization of the surface density of aptamers. Under optimum conditions, the chronocoulometric aptasensor exhibited a wide linear response range of 0.1–18.5 nM with a detection limit of 30 pM. The results demonstrated that this nanoparticle-based amplification strategy offers a simple and effective approach to detect thrombin.     

Analytical application of self assembled monolayers on gold electrodes: critical importance of surface pretreatment

Polycrystalline bare gold electrodes have been used as substrate for the preparation of self assembled monolayers (SAMs) of alkanethiols. Chemical cleaning and several mechanical polishing procedures of the electrode have been carefully tested in order to prepare monolayers of octadecyl-, mercaptans. By analyzing the cyclic voltammetric curves of hexacyanoferrate III and chlorpromazine and the electrochemical desorption of the coated monolayer, it appeared that the response at the bare electrodes is related to the cleaning procedure and that the structure of SAM coated gold electrodes is influenced by the polishing material used, i.e. diamond or alumina slurry. Electrochemical results have been confirmed by Auger analysis of the electrode surface.     

Electrochemical studies of DNA immobilization onto the azide-terminated monolayers and its interaction with taxol

A surface modification procedure for the creation of self-assembled monolayers (SAMs) that can be used as a scaffold for double-stranded DNA (dsDNA) incorporation onto the gold surfaces is described. The SAMs of an azidohexane thiol derivative were prepared on the Au electrode and then used for the immobilization of dsDNA. The electrochemical characteristics of dsDNA onto the SAM-modified gold electrode were investigated by cyclic voltammetry and electrochemical impedance spectroscopy, and the surface concentration of dsDNA onto the SAMs surface was estimated. The interaction of dsDNA with the anticancer drug, taxol (paclitaxel), was also studied on the surface of DNA/SAM/Au electrode. The observed decrease in the guanine oxidation peak current was used to monitor the interaction of taxol with DNA. The resulting Langmuir isotherm for taxol binding to DNA at the modified electrode was used to evaluate the binding constant of taxol-DNA. The results obtained supported the groove binding interaction of taxol with DNA. The modified electrode was used as a sensitive sensor for quantification of taxol in human serum sample.     

A gold nanoparticle-based chronocoulometric DNA sensor for amplified detection of DNA

We report a protocol for the amplified detection of target DNA by using a chronocoulometric DNA sensor (CDS). Electrochemistry is known to be rapid, sensitive and cost-effective; it thus offers a promising approach for DNA detection. Our CDS protocol is based on a 'sandwich' detection strategy, involving a capture probe DNA immobilized on a gold electrode and a reporter probe DNA loaded on gold nanoparticles (AuNPs). Each probe flanks one of two fragments of the target sequence. A single DNA hybridization event brings AuNPs, along with hundreds of reporter probes, in the proximity of the electrode. We then employ chronocoulometry to interrogate [Ru(NH3)6]3+ electrostatically bound to the captured DNA strands. This AuNP-amplified DNA sensor can selectively detect as low as femtomolar (zeptomoles) concentrations of DNA targets and conveniently analyze a breast cancer-associated BRCA-1 mutant DNA. The time range for the entire protocol is approximately 3 d, whereas the DNA sensing takes less than 2 h to complete.     

Electrochemical synthesis of gold nanostructure modified electrode and its development in electrochemical DNA biosensor

In this article, gold nanostructure modified electrodes were achieved by a simple one-step electrodeposition method. The morphologies of modified electrodes could be easily controlled by changing the pH of HAuCl₄ solution. The novel nanoflower-like particles with the nanoplates as the building blocks could be interestingly obtained at pH 5.0. The gold nanoflower modified electrodes were then used for the fabrication of electrochemical DNA biosensor. The DNA biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the use of ferricyanide as an electrochemical redox indicator. The DNA immobilization and hybridization on gold nanoflower modified electrode was studied with the use of [Ru(NH₃)₆]³⁺ as a hybridization indicator. The electrochemical DNA biosensor shows a good selectivity and sensitivity toward the detection of target DNA. A detection limit of 1 pM toward target DNA could be obtained.     

Quantum dots electrochemical aptasensor based on three-dimensionally ordered macroporous gold film for the detection of ATP

A sensitive electrochemical aptasensor was successfully fabricated for the detection of adenosine triphosphate (ATP) by combining three-dimensionally ordered macroporous (3DOM) gold film and quantum dots (QDs). The 3DOM gold film was electrochemically fabricated with an inverted opal template, making the active surface area of the electrode up to 9.52 times larger than that of a classical bare flat one. 5′-thiolated ATP-binding aptamer (ABA) was first assembled onto the 3DOM gold film via sulfur–gold affinity. Then, 5′-biotinated complementary strand (BCS) was immobilized via hybridization reaction to form the DNA/DNA duplex. Since the tertiary structure of the aptamer was stabilized in the presence of target ATP, the duplex can be denatured to liberate BCS. The reaction was monitored by electrochemical stripping analysis of dissolved QDs which were bound to the residual BCS through biotin-streptavidin system. The decrease of peak current was proportional to the amount of ATP. The unique interconnected structure in 3DOM gold film along with the "built-in" preconcentration remarkably improved the sensitivity. ATP detection with high selectivity, wide linear dynamic range of 4 orders of magnitude and high sensitivity down to 0.01 nm were achieved. The results demonstrated that the novel strategy was feasible for sensitive ATP assay and provided a promising model for the detection of small molecules.     

Development of immune cellular biosensing system for assessing chemicals on inducible nitric oxide synthase signaling activator

An immune cellular biosensing system has been constructed to assess immunomodulating effects of chemicals. Production of nitric oxide in the immune cellular biosensing system was used as readout of an immune cellular response for assessing the immunomodulating effects of chemicals. The macrophage-like cell line RAW264.7, which has signaling pathways of inducible nitric oxide synthase, was employed in the cellular biosensing system. The immune cellular biosensing system consisted of a Pt counter electrode, an Ag/AgCl reference electrode, and a gold electrode onto which a polyion complex layer was coated to allow adherence of the RAW264.7 cells. As the results of evaluating effects of a polyion complex layer on cell viabilities by using WST-8 assay, the polyion complex layer did not affect RAW264.7 cells. The polyion-coated gold electrode could measure NO without the drawback of electrochemical interference that occurs with differential pulse voltammetry. The detection limit of the immune cellular biosensing system was 4.2 nM released NO as measured by double potential step chronoamperometry. The potent immune activating abilities of lipopolysaccharide and interferon-gamma could be assessed by the cellular biosensing system; NO release from cells was detected within 600 ms.     

DNA microdevice for electrochemical detection of Escherichia coli 0157:H7 molecular markers

An electrochemical DNA sensor based on the hybridization recognition of a single-stranded DNA (ssDNA) probe immobilized onto a gold electrode to its complementary ssDNA is presented. The DNA probe is bound on gold surface electrode by using self-assembled monolayer (SAM) technology. An optimized mixed SAM with a blocking molecule preventing the nonspecific adsorption on the electrode surface has been prepared. In this paper, a DNA biosensor is designed by means of the immobilization of a single stranded DNA probe on an electrochemical transducer surface to recognize specifically Escherichia coli (E. coli) 0157:H7 complementary target DNA sequence via cyclic voltammetry experiments. The 21 mer DNA probe including a C6 alkanethiol group at the 5' phosphate end has been synthesized to form the SAM onto the gold surface through the gold sulfur bond. The goal of this paper has been to design, characterise and optimise an electrochemical DNA sensor. In order to investigate the oligonucleotide probe immobilization and the hybridization detection, experiments with different concentration of DNA and mismatch sequences have been performed. This microdevice has demonstrated the suitability of oligonucleotide Self-assembled monolayers (SAMs) on gold as immobilization method. The DNA probes deposited on gold surface have been functional and able to detect changes in bases sequence in a 21-mer oligonucleotide.     

Electrochemical quartz crystal microbalance studies on enzymatic specific activity and direct electrochemistry of immobilized glucose oxidase in the presence of sodium dodecyl benzene sulfonate and multiwalled carbon nanotubes

The electrochemical quartz crystal microbalance (EQCM) technique was utilized to monitor in situ the adsorption of glucose oxidase (GOD) and the mixture of GOD and sodium dodecyl benzene sulfonate (SDBS) onto Au electrodes with and without modification of multiwalled carbon nanotubes (MWCNTs) or SDBS/MWCNTs composite, and the relationship between enzymatic specific activity (ESA) and direct electrochemistry of the immobilized GOD was quantitatively evaluated for the first time. Compared with the bare gold electrode at which a little GOD was adsorbed and the direct electrochemistry of the adsorbed GOD was negligible, the amount and electroactivity of adsorbed GOD were greatly enhanced when the GOD was mixed with SDBS and then adsorbed onto the SDBS/MWCNTs modified Au electrode. However, the ESA of the adsorbed GOD was fiercely decreased to only 16.1% of the value obtained on the bare gold electrode, and the portion of adsorbed GOD showing electrochemical activity exhibited very low enzymatic activity, demonstrating that the electroactivity and ESA of immobilized GOD responded oppositely to the presence of MWCNTs and SDBS. The ESA results obtained from the EQCM method were well supported by conventional UV-vis spectrophotometry. The direct electrochemistry of redox proteins including enzymes as a function of their biological activities is an important concern in biotechnology, and this work may have presented a new and useful protocol to quantitatively evaluate both the electroactivity and ESA of trace immobilized enzymes, which is expected to find wider applications in biocatalysis and biosensing fields.