脱氧核糖核酸在硬脂酸铝离子膜上固定杂交及BAR基因片段检测

将石墨粉、固体石蜡和硬脂酸按一定比例混合制得表面富含羧基的碳糊电极,然后在电极表面组装荷正电的铝离子膜。在硬脂酸铝离子膜上进行DNA探针的固定和与目标基因的杂交。以亚甲蓝为杂交指示剂,用循环伏安法优化了DNA的固定和杂交条件。应用该电化学生物传感器以微分脉冲伏安法对转基因玉米外源BAR基因片段进行了检测,结果令人满意。     

基于聚硫堇膜对DNA杂交的直接电化学检测

In this work, the application of a conducting polymer, poly（thionine）, modified electrode as matrix to DNA immobilization as well as transducer to label-free DNA hybridization detection was introduced. The electropolymerization of thionine onto electrode surface was carried out by a simple two-step method, which involved a preanodization of glassy carbon electrode at a constant positive potential in thionine solution following cyclic voltammetry scans in the solution. Electrochemical detection was performed by differential pulse voltammetry in the electroactivity potential domain of poly（thionine）. The resulting poly（thionine） modified electrode showed a good stability and electroactivity in aqueous media during a near neutral pH range. Additionally, the pendant amino groups on the poly（thionine） chains enabled poly（thionine） modified electrode to immobilize phosphate group terminated DNA probe via covalent linkage. Hybridization process induced a clear decrease in poly（thionine） redox current, which was corresponding to the decrease in poly（thionine） electroactivity after double stranded DNA was formed on the polymer film. The detection limit of this electrochemical DNA hybridization sensor was 1.0 × 10^-10mol/L. Compared with complementary sequence, the hybridization signal values of 1-base mismatched and 3-base mismatched samples were 63.9% and 9.2%, respectively.     

Methylene blue as an electrochemical discriminator of single- and double-stranded oligonucleotides immobilised on gold substrates.

Self-assembly of thiol-terminated oligonucleotides on gold substrates provides a convenient and versatile route to DNA-functionalised surfaces. Here we show that the square-wave voltammetric peak position of methylene blue complexed to thiol-terminated single-stranded oligonucleotides immobilised on gold electrodes differs from that of methylene blue complexed to thiol-terminated double-stranded oligonucleotides immobilised on gold electrodes. The peak potential of methylene blue at the single-stranded oligonucleotide array was consistently found to occur at potentials ca. 10-15 mV more positive than that at double-stranded oligonucleotide arrays, the precise difference being dependent on the direction of the voltammetry. This voltammetric behaviour mirrors that found for methylene blue bound to freely diffusing single- and double-stranded calf thymus DNA and suggests that the immobilised oligonucleotides retain the methylene blue binding properties of their freely diffusing counterparts. Thus methylene blue provides a simple electrochemical indicator for the status of oligonucleotide-functionalised gold surfaces.     

Electrochemical Determination of the p53 Tumor Suppressor Gene Using a Gold Nanoparticle-Graphene Nanocomposite Modified Glassy Carbon Electrode

A novel sensitive and simple electrochemical DNA sensor is reported for the determination of p53 tumor suppressor gene. A gold nanoparticle/graphene nanocomposite-modified glassy carbon electrode was prepared and methylene blue was used as the hybridization redox indicator. Scanning electron microscopic and electrochemical characterization demonstrated that the gold nanoparticles and graphene were present on the electrode. The resulting sensor provided suitable electrochemical response to the p53 tumor suppressor gene with a linear dynamic range from 0.1 to 1000?nM. The limit of detection was 0.012?nM. The sensor was able to differentiate a complete complementary DNA sequence, single-base mismatched DNA sequence, and a three-base mismatched DNA sequence. The precision of the device was satisfactory, with a relative standard deviation of 4.1% for 11 measurements. The combination of gold nanoparticles and a graphene nanocomposite provided enhanced capabilities for the determination of DNA for clinical applications.     

Electrochemistry of microparticles of tris (2,2′-bipyridine) ruthenium(II) hexafluorophosphate

The electrochemical behaviour of tris(2,2′-bipyridine)ruthenium(II) hexafluorophosphate (Ru(II)) microparticles, immobilised on a graphite electrode and adjacent to an aqueous electrolyte solution, has been studied by cyclic voltammetry and an in situ spectroelectrochemical technique. The solid Ru(II) complex exhibits one reversible redox couple with a formal potential (E_f) of 1.1 V versus Ag¦AgCl. The continuous cyclic voltammetric experiments showed that the Ru(II) microparticles are stable during the electrochemical conversions. The in situ spectroelectrochemical study showed that the absorbance at 463 nm decreased due to the oxidation of Ru(II) to Ru(III). Upon reduction, the growth of absorbance at 463 nm was observed due to the formation of Ru(II) complex and this process was reversible.     

Electrochemical properties of Doyle catalyst immobilized on carbon paste in the presence of DNA

The electrochemical behaviour of Doyle catalyst, dirhodium(II) tetrakis [methyl-2-oxopyrrolidine-5(S)-carboxylate] (Rh2(5S-MEPY)4), immobilised in graphite powder was evaluated preparing the carbon paste electrode, as well as its electrochemical properties in the presence (DCDE) and absence (DCE) of DNA. In both cases, one redox couple at 0.35 V vs. SCE in 0.5 mol l(-1) KCl solution at pH 7 and 10 mV s(-1) was observed. The resolution of the peak current in the voltammetric studies and other electrochemical properties were improved when the Doyle catalyst was immobilised in the presence of DNA. The estimated rate constants were of 17 and 26 s(-1) for a scan rate of 1 V s(-1) for DCE and DCDE, respectively. Furthermore, the interaction between rhodium carboxylates and electrolytes become more evident, suggesting a good hydrophilic and conductor character of this biopolymer.     

An Electrochemical DNA Sensor Based on Electrodepositing Aluminum Ion Films on Stearic Acid‐Modified Carbon Paste Electrode and Its Application for the Detection of Specific Sequences Related to Bar Gene and CP4 Epsps Gene

An electrochemical DNA biosensor was fabricated by immobilizing DNA probe on aluminum ion films that were electrodeposited on the surface of the stearic acid‐modified carbon paste electrode (CPE). DNA immobilization and hybridization were characterized with cyclic voltammetry (CV) by using methylene blue (MB) as indicator. MB has a couple of well‐defined voltammetric redox peaks at the CPE. The currents of redox peaks of MB decreased after depositing aluminum ion films on the CPE (Al(III)/CPE) and increased dramatically after immobilizing DNA probe (ssDNA/Al(III)/CPE). Hybridization of DNA probe led to a marked decrease of the peak currents of MB, which can be used to detect the target single‐stranded DNA. The conditions for the preparation of Al(III)/CPE, and DNA immobilization and hybridization were optimized. The specific sequences related to bar transgene in the transgenic corn and the PCR amplification of CP4 epsps gene from the sample of transgenic roundup ready soybean were detected by differential pulse voltammetry (DPV) with this new electrochemical DNA biosensor. The difference between the peak currents of MB at ssDNA/Al(III)/CPE and that at hybridization DNA modified electrode (dsDNA/Al(III)/CPE) was applied to determine the specific sequence related to the target bar gene with the dynamic range comprised between 1.0×10^?7 mol/L to 1.0×10^?4 mol/L. A detection limit of 2.25×10^?8 mol/L of oligonucleotides can be estimated.     

Protein adsorption on nanoporous TiO2 films: a novel approach to studying photoinduced protein/electrode transfer reactions

We have investigated the use of nanoporous TiO2 films as substrates for protein immobilisation. Such films are of interest due to their high surface area, optical transparency, electrochemical activity and ease of fabrication. These films moreover allow detailed spectroscopic study of protein/electrode electron transfer processes. We find that protein immobilisation on such films may be readily achieved from aqueous solutions at 4 degrees C with a high binding stability and no detectable protein denaturation. The nanoporous structure of the film greatly enhances the active surface area available for protein binding (by a factor of up to 850 for an 8 microns thick film). We demonstrate that the redox state of proteins such as immobilised cytochrome-c (Cyt-c) and haemoglobin (Hb) may be modulated by the application of an electrical bias potential to the TiO2 film, without the addition of electron transfer mediators. The binding of Cyt-c on the TiO2 films is investigated as a function of film thickness, protein concentration, protein surface charge and ionic strength. We demonstrate the potential use of immobilised Hb on such TiO2 films for the detection of dissolved CO in aqueous solutions. We further show that protein/electrode electron transfer may be initiated by UV bandgap excitation of the TiO2 electrode. Both photooxidation and photoreduction of the immobilised proteins can be achieved. By employing pulsed UV laser excitation, the interfacial electron transfer kinetics can be monitored by transient optical spectroscopy, providing a novel probe of protein/electrode electron transfer kinetics. We conclude that nanoporous TiO2 films may be useful both for basic studies of protein/electrode interactions and for the development of novel bioanalytical devices such as biosensors.     

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.     

基于PAMAM/聚2,6-吡啶二甲酸膜DNA电化学生物传感器的制备及对禽病毒基因检测的研究

以铂电极上聚合的2,6-吡啶二甲酸(PDC)膜组装G5.0树状高分子(PAMAM)固定ssDNA探针, 制备了一种新型的DNA电化学生物传感器. 用[Fe(CN)6]3－/4－作氧化还原指示剂, 以电化学交流阻抗和循环伏安技术对探针ssDNA的固定和杂交进行了表征. 实验表明, 当ssDNA在复合膜上固定及与其互补序列杂交后, 电极表面的传递电阻(Ret)依次增大. 因此, 可以利用Ret的明显差异, 以此固定探针的修饰电极, 对互补序列DNA进行无标记交流阻抗检测. 基于该生物传感器结合交流阻抗技术对禽病毒基因进行检测, 在优化实验条件下, 靶基因ssDNA-2在2.0×10－11～1.0×10－8 mol•L－1线性范围内, 其浓度与电极表面的电子传递电阻(Ret)之间呈良好的线性关系, 检测限为3.6×10－12 mol•L－1. 表明该方法为病毒灵敏地检测提供了一个有益的传感平台.     

Simple diazonium chemistry to develop specific gene sensing platforms

A simple strategy for covalent immobilizing DNA sequences, based on the formation of stable diazonized conducting platforms, is described. The electrochemical reduction of 4-nitrobenzenediazonium salt onto screen-printed carbon electrodes (SPCE) in aqueous media gives rise to terminal grafted amino groups. The presence of primary aromatic amines allows the formation of diazonium cations capable to react with the amines present at the DNA capture probe. As a comparison a second strategy based on the binding of aminated DNA capture probes to the developed diazonized conducting platforms through a crosslinking agent was also employed. The resulting DNA sensing platforms were characterized by cyclic voltammetry, electrochemical impedance spectroscopy and spectroscopic ellipsometry. The hybridization event with the complementary sequence was detected using hexaamineruthenium (III) chloride as electrochemical indicator. Finally, they were applied to the analysis of a 145-bp sequence from the human gene MRP3, reaching a detection limit of 210 pg μL⁻¹.     

Detection of short oligonucleotide sequences of hepatitis B virus using electrochemical DNA hybridisation biosensor

A novel, sensitive and selective electrochemical hybridisation biosensor was developed for the detection of the hepatitis B virus (HBV) using a manganese(II) complex as electrochemical indicator and a DNA probe-modified carbon paste electrode as the biosensor (DNA/CPE). The results showed that this complex could be accumulated electrochemically the immobilised dsDNA layer rather than in the single-stranded DNA (ssDNA) layer. On the basis of this, the manganese complex was used as an electrochemical hybridisation indicator for the detection of oligonucleotides related to HBV. The hybridisation event was evaluated on the basis of the difference between the reduction signals of the manganese(II) complex with the probe DNA prior to and post hybridisation with a target sequence using a differential pulse mode. Several factors affecting the immobilisation and hybridisation of oligonucleotides as well as the indicator’s accumulation were investigated. Experiments with a non-complementary and mismatch sequences demonstrated the good selectivity of the biosensor. Using this approach, the HBV target oligonucleotide’s sequence could be quantified over arange from 0.22 ng L^?1 to 5.40 ng L^?1, with a linear correlation coefficient of 0.9994 and the limit of detection of 0.07 ng L^?1.     

Label-free oligonucleotide detection method based on a new L-cysteine-dihydroartemisinin complex electroactive indicator

A novel base-mismatched oligonucleotide assay method based on label-free electrochemical biosensor was developed, in which the L-cysteine (Cys)-dihydroartemisinin (DHA) complex was used as a new electroactive indicator. In DNA sensor, Cys-DHA complex was initially formed on electrode surface by cathodic scanning, and target oligonucleotide was conjugated with Cys-terminated DHA indicator through electrostatic interaction under optimal pH. The subsequent sequence assay was responsive to hybridization recognition, which target oligonucleotide was captured by the surface-anchored DNA/Cys-DHA probe. The electrochemical signals of biosensor before and after hybridization were compared basing the measurements of semi-derivative linear scan voltammetry (SDLSV) and electrochemical impedance spectroscopy (EIS). On the basis of signal amplification of electroactive indicator and specific recognition of DNA probe, five target oligonucleotides with different mismatched bases were assayed, and a detection limit reached 0.3 nM. Furthermore, atomic force microscopy (AFM) was used to visually characterize specific recognition spots of biosensor at nanoscale. This study demonstrated a new electroactive molecule-based, biomolecule-involved electroactive indicator and its application in recognition and detection of complementary and base-mismatched oligonucleotide.     

Cyclic voltammetry of echinomycin and its interaction with double-stranded and single-stranded DNA adsorbed at the electrode.

Interactions of echinomycin (Echi) with DNA was studied by cyclic voltammetry (CV) with hanging mercury drop electrode (HMDE). Echinomycin was electrochemically active, yielding several signals. Interaction of Echi with dsDNA attached to a hanging mercury drop electrode resulted in high Echi signals, suggesting a strong binding of Echi to dsDNA by bis-intercalation at the electrode surface. Under the same conditions, interaction of Echi with ssDNA produced almost no Echi signal. This behavior is in agreement with a strong binding of Echi to dsDNA and a very weak binding of Echi to ssDNA observed earlier in solution. Echi, thus, appears to be a good candidate for redox indicator in electrochemical DNA hybridization sensors.     

以乙二胺为手臂分子制备的DNA修饰电极及其伏安性能

Carboxyl was formed on the surface of glassy carbon electrode(GCE) by electrochemical oxidation. Ethylenediamine(En) was used as the arm molecule to link carboxyl with dsDNA using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) as the activators to prepare dsDNA modified electrode(dsDNA/En/GCE). It was shown that dsDNA couM be covalently immobilized on the surface of GCE. ssDNA modified electrode(ssDNA/En/GCE) was obtained via the thermal denaturation of dsDNA/En/GCE. The dsDNA/En/GCE and ssDNA/En/GCE were characterized by voltammetry with methylene blue(MB) as the indicator. The results indicated that the currents of the redox peaks of MB at ssDNA/En/GCE were larger than those at dsDNA/En/GCE, and the currents of the redox peaks at En/GCE were the smallest. The peak-currents of MB at the DNA modified electrode had good reproducibility after multi-denaturation and hybridization cycles.     

Oligonucleotide-modified electrodes for fast electron transfer to cytochrome c

Gold electrodes were modified with short ds-oligonucleotides via thiol binding to form a thin and stable surface layer. The modification was characterised by impedance measurements and used as a promoter for fast electron transfer to cytochrome c. The protein was investigated both immobilised and in solution showing reversible electrochemical behaviour in each case. The modification proved to have a good adsorption capability for the redox protein which was also found to be reversible. In the immobilised state at the electrode cytochrome c reacted with superoxide radicals in solution, exemplified by cyclic voltammetric measurements.     

A generic platform for the addressable functionalisation of electrode surfaces through self-induced "electroclick"

A novel and general strategy for the immobilisation of functional objects onto electrodes is described. The concept is based on the addition of two pendant ethynyl groups onto a bis(pyridyl)amine derivative, which acts as a molecular platform. This platform is pre-functionalised with an N(3)-tagged object of interest by Huisgen cycloaddition to one of the ethynyl groups in biphasic conditions. Hence, when complexed by Cu(II) , this molecular-object holder can be immobilised, by a "self-induced electroclick", through the second ethynyl group onto N(3)-alkanethiol self-assembled monolayers on a gold electrode. Two different functional groups, a redox innocent ((CH(2))(3)-Ph) and an electrochemical probe (ferrocene), were immobilised by following this strategy. The in situ electrochemical grafting showed, for both systems, that the kinetics of immobilisation is fast. The voltammetric characterisation of the surface-tagged functionalised copper complexes indicated that a good surface coverage was achieved and that a moderately fast electron-transfer reaction occurs. Remarkably, in the case of the redox-active ferrocenyl-immobilised system, the electrochemical response highlighted the involvement of the copper ion of the platform in the kinetics of the electron transfer to the ferrocene moiety. This platform is a promising candidate for applications in surface addressing in areas as diverse as biology and materials.     

Designs of Enterobacteriaceae Lac Z Gene DNA Gold Screen Printed Biosensors

This paper describes specific electrochemical enterobacteriaceae lac Z gene DNA sensors based on immobilization of a thiolated 25 base single stranded probe onto disposable screen printed gold electrodes (gold SPEs). Two configurations have been evaluated. In the first one, the capture probe was attached to the electrode surface through its ? SH moiety, while mercaptohexanol (MCH) was used as spacer for the displacement of nonspecifically adsorbed oligonucleotide molecules. The hybridization event between the probe and target DNA sequences was detected at ?0.20 V by square‐wave voltammetry (SWV), using methylene blue (MB) as electrochemical indicator. The second genosensor configuration involved modification of gold high temperature SPEs with a 3,3′‐dithiodipropionic acid di(N‐succinimidyl ester) (DTSP) self‐assembled monolayer (SAM). Moreover, 2‐aminoethanol was used as blocking agent, and further modification with avidin allowed binding of the biotinylated enterobacteriaceae lac Z gene DNA probe. An enzyme amplified detection scheme was applied, based on the coupling of streptavidin‐peroxidase to the biotinylated complementary target, after the hybridization process, and immobilization of tetrathiafulvalene (TTF) as redox mediator atop the modified electrode. The amperometric response obtained at ?0.15 V after the addition of hydrogen peroxide was used to detect the hybridization process. Experimental variables concerning sensors composition and electrochemical transduction were evaluated in both cases. A better precision and reproducibility in the fabrication process, as well as a higher sensitivity were achieved using the biotinylated probe‐based sensor configuration. A limit of detection of 0.002 ng/μL was obtained without any preconcentration step.     

Voltammetric Studies on the Recognition of a Copper Complex to Single‐ and Double‐Stranded DNA and Its Application in Gene Biosensor

A mixed‐ligands copper complex [Cu(phendione)(DAP)]SO₄ (phendione=1,10‐phenanthroline‐5,6‐dione, DAP=2,3‐diaminophenazine) was synthesized. Cyclic voltammetry showed that the complex underwent an obvious decrease of redox peak currents and positive shift of formal potential after interaction with double‐stranded DNA (dsDNA), suggesting that the copper complex behaved as a typical metallointercalator for dsDNA, The recognition properties of the copper complex to single‐stranded DNA (ssDNA) and dsDNA were assessed using surface‐based electrochemical methods and the results suggested that the complex had obviously different redox signals at ssDNA and dsDNA modified electrodes. The copper complex was further used as an electroactive indicator for the detection of cauliflower mosaic virus (CaMV) 35S promoter gene.     

电化学石英晶体微天平研究界面电场对DNA杂交的影响

采用电化学石英晶体微天平, 现场监测不同界面电场下完全匹配的靶标DNA和不完全匹配的靶标DNA分别与寡聚核苷酸探针分子杂交的过程. 结果表明, 电极表面荷正电时DNA表观杂交效率比电极表面荷负电时高, 但假阳性比较显著; 而电极表面荷负电时能有效地抑制错配杂交. 探讨了引入界面电场后探针分子取向和微观作用力对DNA杂交的影响.