Electrochemical detection of osmium tetroxide-labeled PCR-products by means of protective strands

This communication reports about how single-stranded 136 base polymerase chain reaction (PCR) products labeled with electrochemically active osmium tetroxide bipyridine can be detected voltammetrically by hybridization with probe strands immobilized on gold electrodes. These electroactive ssDNA targets have been obtained by means of Lambda Exonuclease treatment of the double-stranded PCR products followed by hybridization of the remaining single strands with short protective strands and covalent labeling with osmium tetroxide bipyridine. Square-wave voltammetric signals of these osmium labels have been obtained only upon hybridization with the immobilized probe strands. An optimal 50 °C hybridization temperature has been found with a saturation of the probe layer at 30 min hybridization time and 7.5 nmol/l target concentration. The blank capture probe layer alone did not yield any signal. Unprotected strands produced almost no interference. Such double-selective switch-on electrochemical hybridization assays hold great promise for the specific detection of PCR products.     

Determination for Enterobacter cloacae based on a europium ternary complex labeled DNA probe

The fast detection and accurate diagnosis of the prevalent pathogenic bacteria is very important for the treatment of disease. Nowadays, fluorescence techniques are important tools for diagnosis. A two-probe tandem DNA hybridization assay was designed for the detection of Enterobacter cloacae based on time-resolved fluorescence. In this work, the authors synthesized a novel europium ternary complex Eu(TTA)₃(5-NH₂-phen) with intense luminescence, high fluorescence quantum yield and long lifetime before. We developed a method based on this europium complex for the specific detection of original extracted DNA from E. cloacae. In the hybridization assay format, the reporter probe was labeled with Eu(TTA)₃(5-NH₂-phen) on the 5′-terminus, and the capture probe capture probe was covalent immobilized on the surface of the glutaraldehyde treated glass slides. The original extracted DNA of samples was directly used without any DNA purification and amplification. The detection was conducted by monitoring the fluorescence intensity from the glass surface after DNA hybridization. The detection limit of the DNA was 5 × 10⁻¹⁰ mol L⁻¹. The results of the present work proved that this new approach was easy to operate with high sensitivity and specificity. It could be conducted as a powerful tool for the detection of pathogen microorganisms in the environment.     

Electrochemical Detection of Asymmetric PCR Products by Labeling with Osmium Tetroxide

Single stranded DNA‐targets from asymmetric polymerase chain reaction (PCR) of a sequence of the gram positive, spore forming bacterium Clostridium acetobutylicum were detected by square‐wave voltammetry after labeling with osmium tetroxide bipyridine and hybridization with DNA capture probes immobilized on gold electrodes. The asymmetric PCR, performed with a 10‐fold excess of the forward‐primer, was used without any further purification for hybridization with protective strands and covalent labeling with osmium tetroxide bipyridine. Square‐wave voltammetric signals of 20 nmol/L targets were significantly higher at 50 °C compared with 23 °C hybridization temperature. A fully noncomplementary protective strand yielded thoroughly modified targets unable for further hybridization. Coupling this with thermal discrimination opens new opportunities for sequence specific DNA detection.     

Nanostructured rough gold electrodes as platforms to enhance the sensitivity of electrochemical genosensors

An electrochemical DNA genosensor constructed by using rough gold as electrode support is reported in this work. The electrode surface nanopatterning was accomplished by repetitive square-wave perturbing potential (RSWPP). A synthetic 25-mer DNA capture probe, modified at the 5′ end with a hexaalkylthiol, able to hybridize with a specific sequence of lacZ gene from the Enterobacteriaceae bacterial family was assembled to the rough gold surface. A 25 bases synthetic sequence fully complementary to the thiolated DNA capture probe and a 326 bases fragment of lacZ containing a fully matched sequence with the capture probe, which was amplified by a specific asymmetric polymerase chain reaction (aPCR), were employed as target sequences. The hybridization event was electrochemically monitored by using two different indicators, hexaammineruthenium (III) chloride showing an electrostatic DNA binding mode, and pentaamineruthenium-[3-(2-phenanthren-9-yl-vinyl)-pyridine] (in brief RuL) which binds to double stranded DNA (dsDNA) following an intercalative mechanism. After optimization of the different variables involved in the hybridization and detection reactions, detection limits of 5.30 pg μL⁻¹ and 10 pg μL⁻¹ were obtained for the 25-mer synthetic target DNA and the aPCR amplicon, respectively. A RSD value of 6% was obtained for measurements carried out with 3 different genosensors prepared in the same manner.     

Biocompatible core-shell nanoparticle-based surface-enhanced Raman scattering probes for detection of DNA related to HIV gene using silica-coated magnetic nanoparticles as separation tools

A novel, highly selective DNA hybridization assay has been developed based on surface-enhanced Raman scattering (SERS) for DNA sequences related to HIV. This strategy employs the Ag/SiO₂ core-shell nanoparticle-based Raman tags and the amino group modified silica-coated magnetic nanoparticles as immobilization matrix and separation tool. The hybridization reaction was performed between Raman tags functionalized with 3′-amino-labeled oligonucleotides as detection probes and the amino group modified silica-coated magnetic nanoparticles functionalized with 5′-amino-labeled oligonucleotides as capture probes. The Raman spectra of Raman tags can be used to monitor the presence of target oligonucleotides. The utilization of silica-coated magnetic nanoparticles not only avoided time-consuming washing, but also amplified the signal of hybridization assay. Additionally, the results of control experiments show that no or very low signal would be obtained if the hybridization assay is conducted in the presence of DNA sequences other than complementary oligonucleotides related to HIV gene such as non-complementary oligonucleotides, four bases mismatch oligonucleotides, two bases mismatch oligonucleotides and even single base mismatch oligonucleotides. It was demonstrated that the method developed in this work has high selectivity and sensitivity for DNA detection related to HIV gene.     

Quantitative detection of well-based DNA array using switchable lanthanide luminescence

In this report a novel wash-free method for multiplexed DNA detection is demonstrated employing target specific probe pairs and switchable lanthanide luminescence technology on a solid-phase array. Four oligonucleotide capture probes, conjugated at 3′ to non-luminescent lanthanide ion carrier chelate, were immobilized as a small array on the bottom of a microtiter plate well onto which a mix of corresponding detection probes, conjugated at 5′ to a light absorbing antenna ligand, were added. In the presence of complementary target nucleic acid both the spotted capture probe and the liquid-phase detection probe hybridize adjacently on the target. Consequently the two non-luminescent label molecules self-assemble and form a luminescent mixed lanthanide chelate complex. Lanthanide luminescence is thereafter measured without a wash step from the spots by scanning in time-resolved mode. The homogeneous solid-phase array-based method resulted in quantitative detection of synthetic target oligonucleotides with 0.32 nM and 0.60 nM detection limits in a single target and multiplexed assay, respectively, corresponding to 3× SD of the background. Also qualitative detection of PCR-amplified target from Escherichia coli is described.     

Electrochemical detection of modified maize gene sequences by multiplexed labeling with osmium tetroxide bipyridine

In this report we demonstrate an approach for the electrochemical detection of four sequences from maize and genetically modified (GM) maize by means of square-wave voltammetry (SWV). After multiplexed labeling with osmium tetroxide bipyridine ([OsO₄(bipy)]), the target oligonucleotides are hybridized with a complementary DNA capture probe immobilized on gold electrodes. The multiplexed labeling was performed by mixing the four target strands with the respective oligonucleotides 80% homologous to the central target recognition sequences in order to protect the latter from binding of [OsO₄(bipy)] to its thymine or cytosine residues. All components were added to the same solution. No significant decreases in SWV hybridization signals were observed after such multiplexed labeling of up to four target strands in the same reaction batch. Obtained voltammetric signals were significantly higher at 50 °C compared to 25 °C hybridization temperature and very low response was observed for non-complementary strands. Multiplexed labeling with osmium tetroxide bipyridine holds great promise for the development of simple and effective voltammetric detection protocols for GM organisms.     

Sensitive and simple detection of Escherichia coli strain based on time-resolved fluorescence DNA hybridization assay

A two-probe tandem DNA hybridization assay based on time-resolved fluorescence was employed to detect Escherichia coli strain. The amino modified capture probe was covalently immobilized on the common glass slide surface. The Eu(TTA)₃(5-NH₂-phen) with the characteristics of long lifetime and intense luminescence was labeled with reporter probe. The original extracted DNA samples without the purification and amplification process were directly used in the hybridization assay. The concentration of capture probe, hybridization temperature, hybridization and washing time were optimized. The detection limit is about 1.49 × 10³ CFU mL⁻¹E. coli cells, which is comparable to the value of most microbiology methods. The proposed method has the advantages of easy operation, satisfactory sensitivity and specificity, which can provide a promising technique for monitoring the microorganisms.     

Tris(2,2′-bipyridyl)cobalt(III)-doped silica nanoparticle DNA probe for the electrochemical detection of DNA hybridization

A new and sensitive electrochemical DNA hybridization detection assay, using tris(2,2′-bipyridyl)cobalt(III) [Co(bpy)₃³⁺]-doped silica nanoparticles as the oligonucleotide (ODN) labeling tag, and based on voltammetric detection of Co(bpy)₃³⁺ inside silica nanoparticles, is described. Electro-active Co(bpy)₃³⁺ is not possible for directly linking with DNA, it is doped into the silica nanoparticles in the process of nanoparticles synthesis for DNA labeling with trimethoxysilylpropydiethylenetriamine (DETA) and glutaraldehyde as linking agents. The Co(bpy)₃³⁺ labeled DNA probe is used to hybridize with target DNA immobilized on the surface of glassy carbon electrode. Only the complementary sequence DNA (cDNA) could form a double-stranded DNA (dsDNA) with the DNA probe labeled with Co(bpy)₃³⁺ and give an obvious electrochemical response. A three-base mismatch sequence and non-complementary sequence had negligible response. Due to the large number of Co(bpy)₃³⁺ molecules inside silica nanoparticles linked to oligonucleotide DNA probe, the assay showed a high sensitivity. It allows the detection at levels as low as 2.0×10⁻¹⁰ mol l⁻¹ of the target oligonucleotides.     

Enzyme-amplified electrochemical hybridization assay based on PNA, LNA and DNA probe-modified micro-magnetic beads

In the present study, we investigated the properties of PNA and LNA capture probes in the development of an electrochemical hybridization assay. Streptavidin-coated paramagnetic micro-beads were used as a solid phase to immobilize biotinylated DNA, PNA and LNA capture probes, respectively. The target sequence was then recognized via hybridization with the capture probe. After labeling the biotinylated hybrid with a streptavidin–enzyme conjugate, the electrochemical detection of the enzymatic product was performed onto the surface of a disposable electrode. The assay was applied to the analytical detection of biotinylated DNA as well as RNA sequences. Detection limits, calculated considering the slope of the linear portion of the calibration curve in the range 0–2 nM were found to be 152, 118 and 91 pM, coupled with a reproducibility of the analysis equal to 5, 9 and 6%, calculated as RSD%, for DNA, PNA and LNA probes respectively, using the DNA target. In the case of the RNA target, the detection limits were found to be 51, 60 and 78 pM for DNA, PNA and LNA probes respectively.     

A DNA biosensor based on the electrocatalytic oxidation of amine by a threading intercalator

An electrochemical biosensor for the detection of DNA based a peptide nucleic acid (PNA) capture probe (CP) modified indium tin oxide electrode (ITO) is described in this report. After hybridization, a threading intercalator, N,N′-bis[(3-propyl)-imidazole]-1,4,5,8-naphthalene diimide (PIND) imidazole complexed with Ru(bpy)₂Cl (PIND-Ru, bpy = 2,2′-bipyridine), was introduced to the biosensor. PIND-Ru selectively intercalated to double-stranded DNA (ds-DNA) and became immobilized on the biosensor surface. Voltammetric tests showed highly stable and reversible electrochemical oxidation/reduction processes and the peak currents can directly be utilized for DNA quantification. When the tests were conducted in an amine-containing medium, Tris-HCl buffer for example, a remarkable improvement in the voltammetric response and noticeable enhancements of voltammetric and amperometric sensitivities were observed due to the electrocatalytic activity of the [Ru(bpy)₂Cl] redox moieties. Electrocatalytic current was observed when as little as 3.0 attomoles of DNA was present in the sample solution.     

Human Serum Albumin−Gold Nanoparticle Based Impedimetric Sensor for Sensitive Detection of miRNA-200c

Here, human serum albumin conjugated gold nanoparticles (HSA−AuNPs) were synthesized by a simple route to develop an impedimetric sensor for miRNA-200c detection based on a selective oligo-hybridization process without any labeling. The synthetic DNA capture probe for miRNA-200c was decorated onto the HSA−AuNPs modified pencil graphite electrodes. Impedimetric signals were monitored after the hybridization process between the DNA probe and target miRNA-200c. HSA−AuNPs adsorption time, incubation time of the capture probe and hybridization time-temperature were optimized. The proposed miRNA-200c biosensor demonstrated proper sensitivity and selectivity, low detection limit (1.13 fM), good reproducibility and simple direct detection of miRNA-200c in serum.     

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.     

Electrochemical Detection of DNA Hybridization Based on the Probe Labeled with Carbon‐Nanotubes Loaded with Silver Nanoparticles

A sensitive electrochemical method for the detection of DNA hybridization based on the probe labeled with multiwall carbon‐nanotubes (MWNTs) loaded with silver nanoparticles (Ag‐MWNTs) has been developed. MWNTs were electroless‐plated with a large number of silver nanoparticles to form Ag‐MWNTs. Probe single strand DNA (ss‐DNA) with a thiol group at the 3′‐terminal labeled with Ag‐MWNTs by self‐assembled monolayer (SAM) technique was employed as an electrochemical probe. Target ss‐DNA with a thiol group was immobilized on a gold electrode by SAM technique and then hybridized with the electrochemical probe. Binding events were monitored by differential pulse voltammetric (DPV) signal of silver nanoparticles. The signal difference permitted to distinguish the match of two perfectly complementary DNA strands from the near perfect match where just three base pairs were mismatched. There was a linear relation between the peak current at +120 mV (vs. SCE) and complementary target ss‐DNA concentration over the range from 3.1×10^?14 to 1.0×10^?11 mol/L with a detection limit of 10 fmol/L of complementary target ss‐DNA. The proposed method has been successfully applied to detection of the DNA sequence related to cystic fibrosis. This work demonstrated that the MWNTs loaded with silver nanoparticles offers a great promising approach for sensitive detection of DNA hybridization.     

Electrochemical analysis of microRNAs with hybridization chain reaction-based triple signal amplification

Selective and sensitive detection of trace microRNA is important for early diagnosis of diseases due to its expression level related to diseases. Herein, a triple signal amplification strategy is developed for trace microRNA-21 (miRNA-21) detection by combining with target-triggered cyclic strand displacement reaction (TCSDR), hybridization chain reaction (HCR) and enzyme catalytic amplification. Four DNA hairpins (H1, H2, H3, H4) are employed to form an ultralong double-strand DNA (dsDNA) structure, which is initiated by target miRNA-21. As H3 and H4 are labeled with horseradish peroxidase (HRP), numerous HRPs are loaded on the long dsDNA, producing significantly enhanced electrocatalytic signals in the hydrogen peroxide (H₂O₂) and 3,3′,5,5′-tetramethylbenzidine (TMB) reaction strategy. Compared with single signal amplification, the triple signal amplification strategy shows higher electrochemical response, wider dynamic range and lower detection limit for miRNA-21 detection with excellent selectivity, reproducibility and stability. Taking advantage of the triple signal amplification strategy, the proposed electrochemical biosensor can detect miRNA-21 in 10 HeLa cell lysates, suggesting that it is a promising method for fruitful assay in clinical diagnosis.     

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.     

Nanostructured Screen Printed Graphite Electrode for the Development of a Novel Electrochemical Genosensor

The aim of this work is the preparation of DNA‐sensing architectures based on gold nanoparticles (AuNPs) in conjunction with an enzyme‐amplified detection to improve the analytical properties of genosensor. In order to assess the utility of study as DNA‐sensing devices, a thiolated DNA capture probe sequence was immobilized on the gold nanoparticle modified surface. After labeling of the biotinylated hybrid with a streptavidin‐alkaline phosphatase conjugate, the electrochemical detection of the enzymatic product was performed on the surface of a disposable electrode. Two different enzymatic substrates to detect the hybridization event were studied. In the first case, the enzyme catalyzed the hydrolysis of α‐naphthyl phosphate; the product is electroactive and has been detected by means of differential pulse voltammetry (DPV). In the second one, the enzyme catalyzed the precipitation of an insoluble and insulating product on the sensing interface. In this case, the electrochemical transduction of the hybridization process was performed by electrochemical impedance spectroscopy (EIS).     

Development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus

We describe a DNA microarray system using a bipolar integrated circuit photodiode array (PDA) chip as a new platform for DNA analysis. The PDA chip comprises an 8 × 6 array of photodiodes each with a diameter of 600 μm. Each photodiode element acts both as a support for an immobilizing probe DNA and as a two-dimensional photodetector. The usefulness of the PDA microarray platform is demonstrated by the detection of high-risk subtypes of human papilloma virus (HPV). The polymerase chain reaction (PCR)-amplified biotinylated HPV target DNA was hybridized with the immobilized probe DNA on the photodiode surface, and the chip was incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles bound to the biotin of the HPV target DNA precipitates silver metal particles at the chip surfaces, which block light irradiated from above. The resulting drop in output voltage depends on the amount of target DNA present in the sample solution, which allows the specific detection and the quantitative analysis of the complementary target DNA. The PDA chip showed high relative signal ratios of HPV probe DNA hybridized with complementary target DNA, indicating an excellent capability in discriminating HPV subtypes. The detection limit for the HPV target DNA analysis improved from 1.2 nM to 30 pM by changing the silver development time from 5 to 10 min. Moreover, the enhanced silver development promoted by the gold nanoparticles could be applied to a broader range of target DNA concentration by controlling the silver development time. Figure An optical image of the PDA chip and target DNA detection through silver enhancement Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Electrochemical biosensor based on nanogold-modified poly-eriochrome black T film for BCR/ABL fusion gene assay by using hairpin LNA probe

A novel electrochemical biosensor is described for detection of breakpoint cluster region gene and a cellular abl (BCR/ABL) fusion gene in chronic myelogenous leukemia (CML) by using thiolated-hairpin locked nucleic acids (LNA) as the capture probe. The hairpin LNA probe was immobilized on the nanogold (NG)/poly-eriochrome black T (EBT) film-modified glassy carbon electrode (GCE). The immobilized LNA probe could selectively hybridize with its target DNA on LNA/NG/EBT/GCE surface. The immobilization and hybridization of the LNA probe were characterized with cyclic voltammetry and electrochemical impedance spectroscopy. The hybridization of the immobilized LNA probe with the target DNA was detected by differential pulse voltammetry with the electroactive methylene blue as an indicator. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization, and a high sensitivity. This novel electrochemical biosensor has been used for assay of PCR real sample with satisfactory result.     

电化学DNA生物传感器*

对特异DNA序列的检测在基因相关疾病的诊断、军事反恐和环境监测等方面均具有非常重要的意义,DNA传感器的研究就是为了满足对特异DNA序列的快速、便捷、高灵敏度和高选择性检测的需要。近年来涌现出了多种传感策略,根据检测方法的不同可以大致分为光学传感器、电化学传感器、声学传感器等。由于电化学检测方法本身所具有的灵敏、快速、低成本和低能耗等特点,电化学DNA传感器已成为一个非常活跃的研究领域并在近几年中得到了快速发展。本文概括了近年来在DNA传感器的重要分支——电化学DNA传感器领域内的一些重要进展,主要包括DNA探针在传感界面上的固定方法和各种电化学DNA杂交信号的检测方法。