Covalent immobilization of oligonucleotides on p-aminophenyl-modified carbon screen-printed electrodes for viral DNA sensing

DNA-sensing platforms were prepared by covalently attaching oligonucleotide capture probes onto p-aminophenyl-functionalized carbon surfaces and applied to the determination of an amplified herpes virus DNA sequence in an electrochemical hybridization assay.     

Coupling of a Reagentless Electrochemical DNA Biosensor with Conducting Polymer Film and Nanocomposite as Matrices for the Detection of the HIV DNA Sequences

Abstract

This paper describes a reagentless electrochemical DNA biosensor applied to the detection of human immunodeficiency virus (HIV) sequences based on electrochemical impedance spectroscopy (EIS). The novel DNA biosensor has been elaborated by means of an opposite‐charged adsorption Au‐Ag nanocomposite to a conductive polymer polypyrrole (PPy) modified platinum electrode (Pt) and self‐assembly the mercapto oligonucleotide probes onto the surface of modified electrode via the nanocomposite. The duplex formation was detected by measuring the electrochemical impedance signal of nucleic acids in phosphate buffer solution (PBS). Such response is based on the concomitant conductivity changes of the PPy film and nanocomposite. The reagentless scheme has been characterised using 21‐mer synthetic oligonucleotides as models: parameters affecting the hybridization assay were explored and optimized. The detection limit is 5.0×10^?10 M of target oligonucleotides at 3σ. The potential for development of reagentless DNA hybridization analysis in the clinical diagnosis is being pursued.     

Effect of oligonucleotide probes substituted by deoxyinosines on the specificity of SNP detection on the DNA microarray

One of the main factors that can affect the quality of microarray results is the microarray hybridization specificity. The key factor that affects hybridization specificity is the design of the probes. In this paper, we described a novel oligonucleotide probe containing deoxyinosines aimed at improving DNA hybridization specificity. We compared different probes to determine the distance between deoxyinosine base and SNPs site and the number of deoxyinosine bases. The new probe sequences contained two set of deoxyinosines (each set had two deoxyinosines), in which the interval between SNP site and each set of deoxyinosines was two bases. The new probes could obtain the highest hybridization specificity. The experimental results showed that probes containing deoxyinosines hybridized effectively to the perfectly matched target and improved the hybridization specificity of DNA microarray. By including a simple washing step after hybridization, these probes could distinguish matched targets from single‐base‐mismatched sequences perfectly. For the probes containing deoxyinosines, the fluorescence intensity of a match sequence was more than eight times stronger than that of a mismatch. However, the intensity ratio was only 1.3 times or less for the probes without deoxyinosines. Finally, using hybridization of the PCR product microarrays, we successfully genotyped SNP of 140 samples using these new labeled probes. Our results show that this is a useful new strategy for modifying oligonucleotide probes for use in DNA microarray analysis.     

基于纳米金胶标记DNA探针的电化学DNA传感器研究

以纳米金胶为标记物,将其标记于人工合成的5-端巯基修饰的寡聚核苷酸片段上,制成了具有电化学活性的金胶标记DNA电化学探针;在一定条件下,使其与固定在玻碳电极表面的靶序列进行杂交反应,利用ssDNA与其互补链杂交的高度序列选择性和极强的分子识别能力,以及纳米金胶的电化学活性,实现对特定序列DNA片段的电化学检测以及对DNA碱基突变的识别.     

微囊藻属DNA电化学传感器的研制

利用自组装法将巯基修饰的DNA探针与6-巯基-1-己醇(MCH)固定到金电极表面,制备了微囊藻属特定DNA传感器,将该传感器与完全互补的微囊藻DNA序列、完全不互补序列,以及单碱基错配序列进行杂交,以Hoechst 33258为杂交指示剂,应用循环伏安法和线性扫描伏安法研究了该传感器对目标DNA的电化学检测行为.研究表明,当与完全互补DNA杂交后,Hoechst 33258氧化信号有明显的增强.实验对自组装时间、MCH浸泡时间及杂交液离子浓度进行了优化.结果表明,当自组装时间为90 min,MCH浸泡时间为1 h,杂交溶液中NaCl浓度为0.3 mol/L时,电化学信号最好.目标DNA的氧化峰电流值与其浓度在1×10~(-8) ～1×10~(-6) mol/L范围内呈良好的线性关系,检出限为8.1×10~(-9) mol/L.     

Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes

New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive detection of transgenic maize at percentages lower than 1%.     

Amplified label-free electrical detection of DNA hybridization

A new protocol is described for amplifying label-free electrochemical measurements of DNA hybridization based on the enhanced accumulation of purine nucleobases in the presence of copper ions . Such electrical DNA assays involve hybridization of the target to inosine-substituted oligonucleotide probes (captured on magnetic beads), acidic dipurinization of the hybrid DNA, and adsorptive chronopotentiometric stripping measurements of the free nucleobases in the presence of copper ions. Both amplified adenine and guanine peaks can be used for detecting the DNA hybridization. The dramatic signal amplification advantage of this type of detection has been combined with efficient magnetic removal of non-complementary DNA, use of microliter sample volumes and disposable transducers. Factors influencing the signal enhancement were assessed and optimized. A detection limit of 40 fmol (250 pg) was obtained with 10 min hybridization and 5 min adsorptive-accumulation times. The advantages of this procedure were demonstrated by its application in the detection of DNA segments related to the BRCA1 breast cancer gene. The copper enhancement holds great promise not only for the detection of DNA hybridization, but also for trace measurement of nucleic acids.     

Dual enzyme electrochemical coding for detecting DNA hybridization

Enzyme-based hybridization assays for the simultaneous electrochemical measurements of two DNA targets are described. Two encoding enzymes, alkaline phosphatase and beta-galactosidase, are used to differentiate the signals of two DNA targets in connection to chronopotentiometric measurements of their electroactive phenol and alpha-naphthol products. These products yield well-defined and resolved peaks at +0.31 V (alpha-naphthol) and +0.63 V (phenol) at the graphite working electrode (vs. Ag/AgCl reference). The position and size of these peaks reflect the identity and level of the corresponding target. The dual target detection capability is coupled to the amplification feature of enzyme tags (to yield fmol detection limits) and with an efficient magnetic removal of non-hybridized nucleic acids. Proper attention is given to the choice of the substrates (for attaining well resolved peaks), to the activity of the enzymes (for obtaining similar sensitivities), and to the selection of the enzymes (for minimizing cross interferences). The new bioassay is illustrated for the simultaneous detection of two DNA sequences related to the BCRA1 breast-cancer gene in a single sample in connection to magnetic beads bearing the corresponding oligonucleotide probes. Prospects for electrochemical coding of multiple DNA targets are discussed.     

DNA bioprints: simple nonisotopic DNA fingerprints with biotinylated probes

The discovery of multilocal DNA fingerprinting represented a revolution in criminal identification and paternity testing. However, for routine use in clinical laboratories, the standard DNA fingerprint methodology is too complex. We have been successful in the development of a simplified DNA nonisotopic fingerprinting system using biotin-labeled probes which we have called DNA bioprints. To achieve this we explored three main technical features: utilization of biotinylated nonradioactive probes as a simpler substitute for 32P-labeled probes, utilization of oligonucleotide probes as a simpler substitute for recombinant probes, and direct hybridization in the dried agarose gel as a simpler substitute for Southern blots. In this article we review our results in the development of DNA bioprints.     

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。     

Fabrication of DNA microarrays on nanoengineered polymeric ultrathin film prepared by self-assembly of polyelectrolyte multilayers

Microarray-based technology is in need of flexible and cost-effective chemistry for fabrication of oligonucleotide microarrays. We have developed a novel method for the fabrication of oligonucleotide microarrays with unmodified oligonucleotide probes on nanoengineered three-dimensional thin films that are deposited on glass slides by consecutive layer-to-layer adsorption of polyelectrolytes. Unmodified oligonucleotide probes were spotted and immobilized on these multilayered polyelectrolyte thin films (PET) by electrostatic adsorption and entrapment on the porous structure of the PET film. The PET provides higher probe binding capacity and thus higher hybridization signal than that of the traditional two-dimensional aminosilane and poly-L-lysine coated slides. Immobilized probe densities of 3.4 x 10(12)/cm2 were observed for microarray spots on PET with unmodified 50-mer oligonucleotide probes, which is comparable to the immobilized probe densities of alkyamine-modified 50-mer probes end-tethered on an aldehyde-functionalized slide. The study of hybridization efficiency showed that 90% of immobilized probes on PET film are accessible to target DNA to form duplex format in hybridization. The DNA microarray fabricated on PET film has wider dynamic range (about 3 orders of magnitude) and lower detection limit (0.5 nM) than the conventional amino- and aldehyde-functionalized slides. Oligonucleotide microarrays fabricated on these PET-coated slides also had consistent spot morphology. In addition, discrimination of single nucleotide polymorphism of 16S rRNA genes was achieved with the PET-based oligonucleotide microarrays. The PET microarrays constructed by our self-assembly process is cost-effective, versatile, and well suited for immobilizing many types of biological active molecules so that a wide variety of microarray formats can be developed.     

Imaging of DNA hybridization on microscopic polypyrrole patterns using scanning electrochemical microscopy (SECM): the HRP bio-catalyzed oxidation of 4-chloro-1-naphthol

We illustrate in this paper the successful combination of the direct and feedback mode of scanning electrochemical microscopy (SECM) for the writing of oligonucleotide patterns on thin gold films alongside the imaging of DNA hybridization. The patterning process was achieved using the direct mode of SECM, where the electrical field established between the SECM tip and the gold interface was used to drive the local deposition of micrometre sized polypyrrole spots to which a 15(mer) oligonucleotide (ODN) strand was linked covalently. Imaging of the deposited polypyrrole-ODNs was achieved by means of the feedback mode of SECM using Ru(NH(3))(6)(3+) as the mediator. The detection of the hybridization reaction of the ODN probes with their biotinylated complementary strands using SECM was possible after subsequent reactions with streptavidin and biotinylated horseradish peroxidase (HRP). The HRP-biocatalyzed oxidation of 4-chloro-1-naphthol (1) in the presence of H(2)O(2), and the precipitation of the insoluble product 4-chloro-1-naphthon (2) on the hybridized areas on the gold film caused a local alteration of conductivity. Such a change in conductivity was sensitively detected by the SECM tip and allowed imaging of DNA arrays in a fast and straightforward way.     

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.     

Cadmium sulfide nanocluster-based electrochemical stripping detection of DNA hybridization

A novel, sensitive electrochemical DNA hybridization detection assay, using cadmium sulfide (CdS) nanoclusters as the oligonucleotide labeling tag, is described. The assay relies on the hybridization of the target DNA with the CdS nanocluster oligonucleotide DNA probe, followed by the dissolution of the CdS nanoclusters anchored on the hybrids and the indirect determination of the dissolved cadmium ions by sensitive anodic stripping voltammetry (ASV) at a mercury-coated glassy carbon electrode (GCE). The results showed that only a complementary sequence could form a double-stranded dsDNA-CdS with the DNA probe and give an obvious electrochemical response. A three-base mismatch sequence and non-complementary sequence had negligible response. The combination of the large number of cadmium ions released from each dsDNA hybrid with the remarkable sensitivity of the electrochemical stripping analysis for cadmium at mercury-film GCE allows detection at levels as low as 0.2 pmol L(-1) of the complementary sequence of DNA.     

DNA hybridization electrochemical biosensor using a functionalized polythiophene

A simple and label-free electrochemical sensor for recognition of the DNA sensor event was prepared by electrochemical polymerization of 4-hydroxyphenyl thiophene-3-carboxylate. Poly(4-hydroxyphenyl thiophene-3-carboxylate) (PHPT) was synthesized electrochemically onto glassy carbon electrode and characterized by cyclic voltammetry, FTIR and AFM measurements. An ODN-probe was physisorbed onto PHPT film and tested on hybridization with complementary ODN segments. A biological recognition can be monitored by comparison with electrochemical signal (cyclic voltammogram) of single and double strand state oligonucleotide. The oxidation current of double strand state oligonucleotide is lower than that of single strand, that is corresponding to the decrease of electroactivity of PHPT with the increase of stiffness of polymer structure. Physisorbed ODN-probe and its hybridization were observed morphologically onto ITO electrodes using AFM. The sensitivity of the electrochemical sensor is 0.02 μA/nmol, detection limit is 1.49 nmol and it has good selectivity.     

DNA biosensor based on the electrochemiluminescence of Ru(bpy)3(2+) with DNA-binding intercalators

This paper reports a novel detection method for DNA hybridization based on the electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) with a DNA-binding intercalator as a reductant of Ru(bpy)(3)(3+). Some ECL-inducible intercalators have been screened in this study using electrochemical methods combined with a chemiluminescent technique. The double-stranded DNA intercalated by doxorubicin, daunorubicin, or 4',6-diamidino-2-phenylindole (DAPI) shows a good ECL with Ru(bpy)(3)(2+) at +1.19 V (versus Ag/AgCl), while the non-intercalated single-stranded DNA does not. In order to stabilize the self-assembled DNA molecules during ECL reaction, we constructed the ECL DNA biosensor separating the ECL working electrode with an immobilized DNA probe. A gold electrode array on a plastic plate was assembled with a thru-hole array where oligonucleotide probes were immobilized in the side wall of thru-hole array. The fabricated ECL DNA biosensor was used to detect several pathogens using ECL technique. A good specificity of single point mutations for hepatitis disease was obtained by using the DAPI-intercalated Ru(bpy)(3)(2+) ECL.     

Comparison of Different Strategies on DNA Chip Fabrication and DNA‐Sensing: Optical and Electrochemical Approaches

New strategies for the construction of DNA chips and the detection of DNA hybridization will be discussed in this review. The focus will be on the use of polypyrrole as a linker between a substrate and oligonucleotide probes. The modification step is based on the electrochemical copolymerization of pyrrole and oligonucleotides bearing a pyrrole group on its 5′ end. This strategy was employed for the immobilization of oligonucleotides on millimeter‐sized electrodes, microelectrode arrays, as well as for the local structuring of homogeneous gold surfaces. Our approaches for the localized patterning of gold surfaces will be also discussed. Localized immobilization was achieved by using an electrospotting technique, where a micropipette served as an electrochemical cell where spot sizes with 800 μm diameters were fabricated. The use of a microcell using a Teflon covered metal needle with a cavity of 100 μm resulted in immobilized probe spots of 300 μm. Scanning electrochemical microscopy (SECM) was also used, and surface modifications of 100 μm were obtained depending on the experimental conditions. Different detection methods were employed for the reading of the hybridization event: fluorescence imaging, surface plasmon resonance imaging (SPRI), photocurrent measurements, and voltamperometric measurements using intercalators. Their advantages concerning the various immobilization strategies will also be discussed.     

Electrochemical DNA Hybridization Assay: Enzyme‐Labeled Detection of Mutation in p53 Gene

This paper discusses a new electrochemical DNA hybridization sensing approach based on the detection of a linked enzyme label. In this method we employ enzyme that is attached to a tethered ssDNA oligomer on the surface and the target analyte is a complementary ssDNA oligomer that does not require any pre‐treatment. The advantage of using of enzyme label is in its amplification of the registration of the hybridization event due to the catalytic reaction facilitated in the process. One particular novelty is associated with the use of enzymes that directly communicate with the electrode surface thus allowing for minimizing the need of additional reagents in the assay. The electrochemical assay was demonstrated when using mixed self‐assembled monolayers from thiolated oligonucleotide and 6‐mercapto 1‐hexanol on gold surfaces. Horseradish peroxidase (HRP) is attached to the surface tethered oligonucleotide using streptavidin‐biotin chemistry, and the enzyme successfully established direct electron transfer (DET) with the electrode or mediated electron transfer (MET) using a mediator. Hybridization results in increasing the angle of contact between electrode and DNA and also the stiffness of the ds DNA, which results in displacing the enzyme away from the electrode surface, and thereby reducing the occurrence of direct electron transfer between the enzyme and the electrode. The cyclic voltammetry showed a clear distinction in response between the complete complementary sequence and the two‐base mismatch sequence. Ellipsometric measurements show that the thickness of the thiol modified oligonucleotide on gold surfaces changes before and after hybridization for the complementary sequence, where as a minimal change in thickness was observed for the noncomplementary sequence. The model target analyte in this study was TP53 gene where a specific mutation is a marker for a list of cancers. Mutations of the TP53 gene have been demonstrated in tumors of the colon, breast, lung, ovary, bladder, and many other organs. Analysis of p53 mutations may provide useful information for the diagnosis, prognosis and therapy of cancer.     

Lead Sulfide Nanoparticle as Oligonucleotides Labels for Electrochemical Stripping Detection of DNA Hybridization

We report a method for the detection of DNA hybridization in connection to lead sulfide (PbS) nanoparticle tags and electrochemical stripping measurement of the lead. A kind of lead sulfide nanoparticle with free carboxyl groups on its surface was synthesized in aqueous solution. The nanoparticle was used as a marker to label a sequence‐known oligonucleotide, which was then employed as a DNA probe for identifying a target ssDNA immobilized on a PPy modified electrode based on a specific hybridization reaction. The hybridization events were monitored by the oxidation dissolution of the lead sulfide anchored on the hybrids and the indirect determination of the lead ions by anodic stripping voltammetry (ASV). The detection limit is 0.3 pmol L^?1 of target oligonucleotides. The PbS nanoparticle combining its easy conjugation to the DNA molecule with the highly sensitive stripping voltammetry detection of lead shows its promising application in the electrochemical DNA hybridization analysis assay.     

Electrochemical transduction of DNA hybridization at modified electrodes by using an electroactive pyridoacridone intercalator

A synthetic redox probe structurally related to natural pyridoacridones was designed and electrochemically characterised. These heterocycles behave as DNA intercalators due to their extended planar structure that promotes stacking in between nucleic acid base pairs. Electrochemical characterization by cyclic voltammetry revealed a quasi-reversible electrochemical behaviour occurring at a mild negative potential in aqueous solution. The study of the mechanism showed that the iminoquinone redox moiety acts similarly to quinone involving a two-electron reduction coupled with proton transfer. The easily accessible potential region with respect to aqueous electro-inactive window makes the pyridoacridone ring suitable for the indirect electrochemical detection of chemically unlabelled DNA. Its usefulness as electrochemical hybridization indicator was assessed on immobilised DNA and compared to doxorubicin. The voltamperometric response of the intercalator acts as an indicator of the presence of double-stranded DNA at the electrode surface and allows the selective transduction of immobilised oligonucleotide hybridization at both macro- and microscale electrodes.