Electrochemical Quantitative Analysis of Nucleic Acids Using β‐Cyclodextrin Modified Gold Electrode

We present an electrochemical biosensor for the analysis of nucleic acids upon hybridization on the β‐cyclodextrin (β‐CD)‐modified gold electrode. The strategy is based on the following: The 5’‐ferrocene‐labeled single stranded capture probe DNA (5’‐fc‐ss‐DNA) was incorporated into the cavity of thiolated β‐CD which was immobilized on the surface of gold electrode. After hybridization of complementary target DNA, hybridized double stranded DNA (ds‐DNA) was released from the cavity of β‐CD. The difference of electrochemical properties on the modified gold electrode was characterized by cyclic voltametry and surface plasmon resonance. We successfully applied this method to the investigation of the sensor responses due to hybridization on various concentrations of applied target DNA. As a result, the label‐free electrochemical DNA sensor can detect the target DNA with a detection limit of 1.08 nM. Finally, our method does not require either hybridization indicators or other signalling molecules such as DNA intercalaters which most of electrochemical hybridization detection systems require.     

Femtomolar electrochemical detection of DNA targets using metal sulfide nanoparticles

A new electrochemical DNA sensor providing detection capabilities down to 100 attomol of target DNA has been developed. The method applies CdS, ZnS, and PbS nanoparticles conjugated with short DNA sequences which are immobilized via hybridization with complementary sequences on a gold surface. When the DNA target is added, it can be identified by ousting the existing hybridization between one of the DNA-nanoparticle conjugates and the surface DNA. The nanoparticles remaining at the surface are detected by stripping voltammetry. The setup is constructed to give a signal-off response with a build-in control signal as only one of two different metal sulfide signaling probes on the surface is removed by hybridization with the DNA target. The competition assay is, in principle, label-free since no labels are required for detection after addition of DNA target. The dissociation of PbS nanoparticles from the surface after addition of the DNA target has been imaged by fluid phase AFM.     

新型超支状液晶核酸传感器用于p53突变基因的检测

基于核酸杂交链式反应影响液晶取向的原理, 构建了一种新型的超支状液晶核酸传感器用于检测p53突变基因. 本文突破传统构建超支状分子的方式, 采用杂交链式反应方法, 以目标序列p53突变基因作为引发剂, 3种不同的发卡探针Hairpin A, Hairpin B和Hairpin C为单体, 在温和的条件下, 通过改变单体的浓度和反应时间自发杂交组装形成尺寸和分子量可控的超支状DNA(branched-like DNA, bDNA). 借助捕获探针将该超支状DNA连接到液晶传感基底表面, 观察液晶分子取向改变前后的光学信号, 实现了p53基因含249密码子突变序列的快速检测. 本方法有望为核酸诊断的发展提供一种新的方法和思路.     

Lable‐Free Electrochemical DNA Sensor Based on Gold Nanoparticles/Poly(neutral red) Modified Electrode

We present a new strategy for the label‐free electrochemical detection of DNA hybridization based on gold nanoparticles (AuNPs)/poly(neutral red) (PNR) modified electrode. Probe oligonucledotides with thiol groups at the 5‐end were covalently linked onto the surface of AuNPs/PNR modified electrode via S‐Au binding. The hybridization event was monitored by using differential pulse voltammetry (DPV) upon hybridization generates electrochemical changes at the PNR‐solution interface. A significant decrease in the peak current was observed upon hybridization of probe with complementary target ssDNA, whereas no obvious change was observed with noncomplementary target ssDNA. And the DNA sensor also showed a high selectivity for detecting one‐mismatched and three‐mismatched target ssDNA and a high sensitivity for detecting complementary target ssDNA, the detection limit is 4.2×10^?12 M for complementary target ssDNA. In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA‐hybridization conditions.     

Addressable electrode array device with IDA electrodes for high-throughput detection

An electrochemical device is proposed for high-throughput electrochemical detection that consists of 32 row and 32 column electrodes on a single glass substrate. The row and column electrodes are connected to interdigitated array (IDA) electrodes to form 1024 (32 × 32) addressable sensor points in the device. Electrochemical responses from each of the 1024 sensors were successfully acquired on the device within 1 min using redox cycling at individual IDA electrodes, which ensures application of the device to comprehensive, high-throughput electrochemical detection for enzyme-linked immunosorbent assay (ELISA), reporter gene assay for monitoring gene expressions, and DNA analysis.     

Label- and marker-free gene detection based on hybridization-induced conformational flexibility changes in a ferrocene-PNA conjugate probe

We report a strategy for label-free and marker-free gene detection transducing the hybridization event to an electrochemical signal based on the hybridization-induced conformational flexibility change in probe structure. The probe structure was designed to possess a ferrocene moiety as a reporter part and a cysteine moiety as an anchor part at each end of a peptide nucleic acid (PNA) as a recognition part. Electrochemical examination of probe-modified gold electrodes revealed that the ferrocene moiety was placed at the flexible end of the linear probe chain. Upon hybridization with a complementary target DNA, the resultant rigid duplex restricted the ferrocene motion to the electrode surface, causing a decrease in the observed current. The target DNA was detected with the detection limit of 1.44 x 10(-11) M. Thus the probe functioned as a 'self-reporting probe' and detection of the target DNA was demonstrated without the need for external indicators. Moreover, the sensor electrode was able repeatedly to detect the target DNA by the process of regeneration and could discriminate a mismatched DNA.     

基于纳米ZnO/壳聚糖复合膜DNA电化学传感器用于HIV基因的免标记检测

以室温固相合成法制备纳米ZnO,通过壳聚糖(CHIT)的成膜效应将纳米ZnO固定在玻碳电极(GCE)表面,制得的ZnO/CHIT/GCE电极成为DNA固定和杂交的良好平台。DNA的固定和杂交通过电化学交流阻抗进行表征。以电化学交流阻抗免标记法检测目标DNA,固定于电极表面的DNA探针与目标DNA杂交后使电极表面的电子传递电阻增大,以此作为检测信号可以高灵敏度地测定目标DNA。电化学阻抗谱检测人类免疫缺陷病毒(HIV)基因片段的线性范围为2.0×10-11~2.0×10-6mol/L,检出限为2.0×10-12mol/L。     

Indicator-based and indicator-free magnetic assays connected with disposable electrochemical nucleic acid sensor system

An indicator-based and indicator-free magnetic assays connected with a disposable pencil graphite electrode (PGE) were successfully developed, and also compared for the electrochemical detection of DNA hybridization. The oxidation signals of echinomycin (ECHI) and electroactive DNA bases, guanine and adenine, respectively were monitored in the presence of DNA hybridization by using differential pulse voltammetry (DPV) technique. The biotinylated probe was immobilized onto the magnetic beads (magnetic particles, microspheres) and hybridization with its complementary target at the surface of particles within the medium was exhibited successfully using electrochemical sensor system. For the selectivity studies, the results represent that both indicator-based and indicator-free magnetic assays provide a better discrimination for DNA hybridization compared to duplex with one-base or more mismatches. The detection limits (S/N = 3) of the magnetic assays based on indicator or indicator-free were found in nM concentration level of target using disposable sensor technology with good reproducibility. The characterization and advantages of both proposed magnetic assays connected with a disposable electrochemical sensor are also discussed and compared with those methods previously reported in the literature.     

Immobilization of DNA onto poly(dimethylsiloxane) surfaces and application to a microelectrochemical enzyme-amplified DNA hybridization assay

This paper describes immobilization of DNA onto the interior walls of poly(dimethylsiloxane) (PDMS) microsystems and its application to an enzyme-amplified electrochemical DNA assay. DNA immobilization was carried out by silanization of the PDMS surface with 3-mercaptopropyltrimethoxysilane to yield a thiol-terminated surface. 5'-acrylamide-modified DNA reacts with the pendant thiol groups to yield DNA-modified PDMS. Surface-immobilized DNA oligos serve as capture probes for target DNA. Biotin-labeled target DNA hybridizes to the PDMS-immobilized capture DNA, and subsequent introduction of alkaline phosphatase (AP) conjugated to streptavidin results in attachment of the enzyme to hybridized DNA. Electrochemical detection of DNA hybridization benefits from enzyme amplification. Specifically, AP converts electroinactive p-aminophenyl phosphate to electroactive p-aminophenol, which is detected using an indium tin oxide interdigitated array (IDA) electrode. The IDA electrode eliminates the need for a reference electrode and provides a steady-state current that is related to the concentration of hybridized DNA. At present, the limit of detection of the DNA target is 1 nM in a volume of 20 nL, which corresponds to 20 attomoles of DNA.     

Label‐Free and Label Based Electrochemical Detection of Hybridization by Using Methylene Blue and Peptide Nucleic Acid Probes at Chitosan Modified Carbon Paste Electrodes

A chitosan modified carbon paste electrode (ChiCPE) based DNA biosensor for the recognition of calf thymus double stranded DNA (dsDNA), single stranded DNA (ssDNA) and hybridization detection between complementary DNA oligonucleotides is presented. DNA and oligonucleotides were electrostatically attached by using chitosan onto CPE. The amino groups of chitosan formed a strong complex with the phosphate backbone of DNA. The immobilized probe could selectively hybridize with the target DNA to form hybrid on the CPE surface. The detection of hybridization was observed by using the label‐free and label based protocols. The oxidation signals of guanine and adenine greatly decreased when a hybrid was formed on the ChiCPE surface. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with target. The signals of MB were investigated at dsDNA modified ChiCPE and ssDNA modified ChiCPE and the increased peak currents were observed, in respect to the order of electrodes. The hybridization of peptide nucleic acid (PNA) probes with the DNA target sequences at ChiCPE was also investigated. Performance characteristics of the sensor were described, along with future prospects.     

Parallel Detection of Different DNA Sequences on One Gold Electrode

Motivated by the potential of electrochemical techniques to analyze hybridization events fast and in a simple and cost‐effective way we present here a detection system allowing a parallel electrochemical DNA analysis. For this purpose different probe DNA strands have been immobilized on one electrode. By the use of two different target DNA sequences, both marked with the redox active methylene blue, we can show that hybridization with the complementary probe sh“NA strands can occur without steric hindrance. Each target has been recognized down to 3nM with a very high specificity of the sensor. In addition, we can detect two different ssDNA targets labeled with different redox active molecules, methylene blue and ferrocene, on one sensor surface simultaneously.     

Label-Free DNA Biosensor for Electrochemical Detection of Short DNA Sequences Related to Human Papilloma Virus

Abstract

Highly sensitive label-free techniques of DNA determination are particularly interesting in relation to the present development of an electrochemical hybridization biosensor for the detection of short DNA fragments specific to the human papilloma virus (HPV). Unlabeled DNA probes have been immobilized by spontaneous coadsorption of thiolated single-stranded oligonucleotides (HS-ssDNA) onto the sensing surface of a screen-printed gold electrode (SPGE). The covalently immobilized single-stranded DNA probe (HS-ssDNA) could selectively hybridize with its complementary DNA (cDNA) in solution to form double-stranded DNA (dsDNA) on the surface. DNA is treated with acid (e.g., 0.5 M chloridric acid), and the acid-released purine bases are directly determined by square wave voltammetry (SWV).

Variables of the probe-immobilization and hybridization steps are optimized to offer convenient quantitation of HPV DNA target, in connection with a short hybridization time. Peak currents were found to increase in the following order: hybrid-modified SPGE, 11-base mismatched modified SPGE, 18-base mismatched SPGE, and the probe modified SPGE. Control experiments with noncomplementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. The effect of the target DNA concentration on the hybridization signal was also studied. Under optimal conditions, this sensor has a good calibration range with HPV DNA sequence detection limit of 2 pg · ml^?1 (S/N = 3).     

Amplified electrochemical DNA sensor using peroxidase-like DNAzyme

A novel electrochemical DNA sensor was developed here by using peroxidase-like G-quadruplex-based DNAzyme as a biocatalytic label. A hairpin structure including the G-quadruplex-based DNAzyme in a caged configuration and the target DNA probe were immobilized on Au-electrode surface. Upon hybridization with the target, the hairpin structure was opened, and the G-quadruplex-based DNAzyme was generated on the electrode surface, triggering the electrochemical oxidization of hydroquinone by H₂O₂, which provide a quantitative measure for the detection of the target DNA. The DNA target was analyzed with a detection limit of 0.6 nM. This method is simple and easy to design without direct conjugation of redox-active element.     

Amperometric detection of DNA hybridization on a gold surface depends on the orientation of oligonucleotide chains

We tested the possibility of amperometric detection of DNA hybridization on a gold surface influenced by the immobilization of oligonucleotide giving different orientations of single stranded DNA relative to the gold surface. The DNA sensor was fabricated by chemisorption of 18-mer oligonucleotide modified by a phosphorothioate group either at its 3' or both 3' and 5' terminal. After immobilization of oligonucleotide to the gold support, the sensor was immersed in 11-mercaptoundecanoic acid (MUA) solution. Further chemisorption of MUA resulted in approximately 10-fold increase of resistance of the organic layer. Addition of complementary oligonucleotide resulted in an increase of conductivity for DNA sensor oriented perpendicular to the gold support (DNA with one thiol group), while the conductance decreased for DNA sensor with single stranded DNA oriented parallel to the gold support (with DNA modified by thiol groups at both 3' and 5' terminals). Addition of non-complementary chain resulted a slight decrease or no change of sensor conductivity. The hybridization process at both types of DNA orientations is not cooperative and can be described by Langmuir isotherms. The hybridization event on gold support has been confirmed by mass detection using the quartz crystal microbalance technique.     

Electrochemical biosensor for detecting DNA sequences from the pathogenic protozoan Cryptosporidium parvum

An electrochemical hybridization biosensor was developed for the detection of short DNA fragments specific to the deadly waterborne pathogen Cryptosporidium. The sensor relies on the immobilization of a 38-mer oligonucleotide unique to the Cryptosporidium DNA onto the carbon-paste transducer, and employs a highly sensitive chronopotentiometric transduction mode for monitoring the hybridization event. Variables of the probe-immobilization, hybridization and indicator-detection steps are optimized to offer convenient quantitation of ng ml(-1) levels of the Cryptosporidium DNA target, in connection with short (3-15 min) hybridization times. The suitability for direct detection of the spiked Cryptosporidium DNA target in untreated drinking and river water samples is demonstrated. Similar performance characteristics are observed at DNA-coated microfabricated thick-film carbon strips. This and similar developments hold great promise for field screening of Cryptosporidium and other microorganisms in environmental samples.     

Electrochemical DNA Hybridization Detection Using DNA Cleavage

We report the new method for detection of DNA hybridization using enzymatic cleavage. The strategy is based on that S1 nuclease is able to specifically cleave only single strand DNA, but not double strand DNA. The capture probe DNA, thiolated single strand DNA labeled with electroactive ferrocene group, was immobilized on a gold electrode. After hybridization of target DNA of complementary and noncomplementary sequences, nonhybridized single strand DNA was cleaved using S1 nuclease. The difference of enzymatic cleavage on the modified gold electrode was characterized by cyclic voltammetry and differential pulse voltammetry. We successfully applied this method to the sequence‐selective discrimination between perfectly matched and mismatched target DNA including a single‐base mismatched target DNA. Our method does not require either hybridization indicators or other exogenous signaling molecules which most of the electrochemical hybridization detection systems require.     

Direct detection and discrimination of double-stranded oligonucleotide corresponding to hepatitis C virus genotype 3a using an electrochemical DNA biosensor based on peptide nucleic acid and double-stranded DNA hybridization

Development of an electrochemical DNA biosensor for the direct detection and discrimination of double-stranded oligonucleotide (dsDNA) corresponding to hepatitis C virus genotype 3a, without its denaturation, using a gold electrode is described. The electrochemical DNA sensor relies on the modification of the gold electrode with 6-mercapto-1-hexanol and a self-assembled monolayer of 14-mer peptide nucleic acid probe, related to the hepatitis C virus genotype 3a core/E1 region. The increase of differential pulse voltammetric responses of methylene blue, upon hybridization of the self-assembled probe with the target ds-DNA to form a triplex is the principle behind the detection and discrimination. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the developed DNA sensor responds selectively to the ds-DNA target. Diagnostic performance of the biosensor is described and the detection limit was found to be 1.8 × 10⁻¹² M in phosphate buffer solution, pH 7.0. The relative standard deviation of measurements of 100 pM of target ds-DNA performed with three independent probe-modified electrodes was 3.1%, indicating a remarkable reproducibility of the detection method.     

Detection of MicroRNAs using target-guided formation of conducting polymer nanowires in nanogaps

A nanogapped microelectrode-based biosensor array is fabricated for ultrasensitive electrical detection of microRNAs (miRNAs). After peptide nucleic acid (PNA) capture probes were immobilized in nanogaps of a pair of interdigitated microelectrodes and hybridization was performed with their complementary target miRNA, the deposition of conducting polymer nanowires, polyaniline (PAn) nanowires, is carried out by an enzymatically catalyzed method, where the electrostatic interaction between anionic phosphate groups in miRNA and cationic aniline molecules is exploited to guide the formation of the PAn nanowires onto the hybridized target miRNA. The conductance of the deposited PAn nanowires correlates directly to the amount of the hybridized miRNA. Under optimized conditions, the target miRNA can be quantified in a range from 10 fM to 20 pM with a detection limit of 5.0 fM. The biosensor array is applied to the direct detection of miRNA in total RNA extracted from cancer cell lines.     

Electrochemical microfluidic biosensor for the detection of nucleic acid sequences

A microfluidic biosensor with electrochemical detection for the quantification of nucleic acid sequences was developed. In contrast to most microbiosensors that are based on fluorescence for signal generation, it takes advantage of the simplicity and high sensitivity provided by an amperometric and coulorimetric detection system. An interdigitated ultramicroelectrode array (IDUA) was fabricated in a glass chip and integrated directly with microchannels made of poly(dimethylsiloxane) (PDMS). The assembly was packaged into a Plexiglas housing providing fluid and electrical connections. IDUAs were characterized amperometrically and using cyclic voltammetry with respect to static and dynamic responses for the presence of a reversible redox couple-potassium hexacyanoferrate (ii)/hexacyanoferrate (iii) (ferri/ferrocyanide). A combined concentration of 0.5 microM of ferro/ferricyanide was determined as lower limit of detection with a dynamic range of 5 orders of magnitude. Background signals were negligible and the IDUA responded in a highly reversible manner to the injection of various volumes and various concentrations of the electrochemical marker. For the detection of nucleic acid sequences, liposomes entrapping the electrochemical marker were tagged with a DNA probe, and superparamagnetic beads were coated with a second DNA probe. A single stranded DNA target sequence hybridized with both probes. The sandwich was captured in the microfluidic channel just upstream of the IDUA via a magnet located in the outside housing. Liposomes were lysed using a detergent and the amount of released ferro/ferricyanide was quantified while passing by the IDUA. Optimal location of the magnet with respect to the IDUA was investigated, the effect of dextran sulfate on the hybridization reaction was studied and the amount of magnetic beads used in the assay was optimized. A dose response curve using varying concentrations of target DNA molecules was carried out demonstrating a limit of detection at 1 fmol assay(-1) and a dynamic range between 1 and 50 fmol. The overall assay took 6 min to complete, plus 15-20 min of pre-incubation and required only a simple potentiostat for signal recording and interpretation.     

Fluorescence and Label Free Impedimetric DNA Detection on SnO2 Nanopillars

SnO₂ is a n‐type semiconductive oxide with attractive characteristics mainly based on easy elaboration and functionalization routes in addition to chemical robustness. We have fabricated SnO₂ nanopillars based DNA sensor to perform the label free (without any redox compound) impedimetric DNA detection. The non faradaic electrochemical impedance spectroscopic (EIS) behavior and more particularly the evolution of the polarization resistance the SnO₂ nanopillars has been thoroughly investigated upon the different steps of their functionalization process up to DNA hybridization. Similarly to our previous study on planar 2D SnO₂ surfaces, the DNA hybridization induces a systematic increase of the polarization resistance, the magnitude of which decreases with the target DNA concentration. This DNA concentration dependence matches the one obtained from epifluorescence intensity measurements. A common value of DNA detection limit, i.e. 2 nM, is found from both measurement techniques. Interestingly the 3D view intensity obtained by confocal scanning laser fluorescence microscopy confirms that the DNA molecules are mainly grafted along the SnO₂ nanopillars. Finally both impedance and fluorescence measurements obtained in the case of 1‐ and 2‐base mismatch hybridizations demonstrate the selectivity of the SnO₂ nanopillars based DNA sensor. These preliminary results open the way to further investigations on the influence of both the shape ratio and electrical properties of the SnO₂ nanopillars on the impedance variations related to DNA hybridization, notably in view of improving the sensor performances.