Hybridization detection of enzyme-labeled DNA at electrically heated electrodes

In this report we describe an electrochemical DNA hybridization sensor approach, in which signal amplification is achieved using heated electrodes together with an enzyme as DNA-label. On the surface of the heatable low temperature co-fired ceramic (LTCC) gold electrode, an immobilized thiolated capture probe was hybridized with a biotinylated target using alkaline phosphatase (SA-ALP) as reporter molecule. The enzyme label converted the redox-inactive substrate 1-naphthyl phosphate (NAP) into the redox-active 1-naphthol voltammetrically determined at the modified gold LTCC electrode. During the measurement only the electrode was heated leaving the bulk solution at ambient temperature. Elevated temperature during detection led to increased enzyme activity and enhanced analytical signals for DNA hybridization detection. The limit of detection at 53 °C electrode temperature was 1.2 nmol/L.     

DNA hybridization "turns on" electro-catalysis at gold electrodes

The efficiency of electro-catalysis occurring at DNA-modified gold electrodes is highly dependently on the density of DNA monolayers, as a result, DNA hybridization can "turn on" electro-catalysis by increasing the DNA surface density.     

Carbon-nanotube-modified glassy carbon electrodes for amplified label-free electrochemical detection of DNA hybridization

The preparation and attractive performance of carbon-nanotube modified glassy-carbon (CNT/GC) electrodes for improved detection of purines, nucleic acids, and DNA hybridization are described. The surface-confined multiwall carbon-nanotube (MWCNT) facilitates the adsorptive accumulation of the guanine nucleobase and greatly enhances its oxidation signal. The advantages of CNT/GC electrodes are illustrated from comparison to the common unmodified glassy carbon, carbon paste and graphite pencil electrodes. The dramatic amplification of the guanine signal has been combined with a label-free electrical detection of DNA hybridization. Factors influencing the enhancement of the guanine signal are assessed and optimized. The performance characteristics of the amplified label-free electrochemical detection of DNA hybridization are reported in connection to measurements of nucleic-acid segments related to the breast-cancer BRCA1 gene.     

DNA hybridization at microbeads with cathodic stripping voltammetric detection

In electrochemical DNA hybridization sensors generally a single-stranded probe DNA was immobilized at the electrode followed by hybridization with the target DNA and electrochemical detection of the hybridization event at the same electrode. In this type of experiments nonspecific adsorption of DNA at the electrode caused serious difficulties especially in the case of the analysis of long target DNAs. We propose a new technology in which DNA is hybridized at a surface H and the hybridization is detected at the detection electrode (DE). This technology significantly extends the choice of hybridization surfaces and DEs. Here we use paramagnetic Dynabeads Oligo(dT)(25) (DBT) as a transportable reactive surface H and a hanging mercury drop electrode as DE. We describe a label-free detection of DNA and RNA (selectively captured at DBT) based on the determination of adenines (at ppb levels, by cathodic stripping voltammetry) released from the nucleic acids by acid treatment. The DNA and RNA nonspecific adsorption at DBT is negligible, making thus possible to detect the hybridization event with a great specificity and sensitivity. Specific detection of the hybridization of polyribonucleotides, mRNA, oligodeoxynucleotides, and a DNA PCR product (226 base pairs) is demonstrated. New possibilities in the development of the DNA hybridization sensors opened by the proposed technology, including utilization of catalytic signals in nucleic acid determination at mercury (e.g. signals of osmium complexes covalently bound to DNA) and solid DEs (e.g. using enzyme-labeled antibodies against chemically modified DNAs) are discussed.     

The detection of DNA deamination by electrocatalysis at DNA-modified electrodes

The method of electrocatalysis based on using a methylene blue (MB) as an electrochemical indicator and ferricyanide ions [Fe(CN)6]3- as an electron acceptor was applied in screening DNA for lesions caused by deamination of nucleobases. The damaged DNA was modeled by short 18-mer oligonucleotides containing the different number of mismatched target bases (uracil instead of cytosine residues). The hybridization capacity of these oligomers with complementary probes (immobilized on gold electrodes or free) was investigated by both electrochemical methods and UV spectroscopy. We have shown that the amplitude of the reduction signal corresponding to ferricyanide ions considerably increases in the presence of MB. This electrocatalytic effect allowed us to detect the changes in electrochemical properties of DNA caused by dU.dG mismatches. Using differential pulse voltammetry and cyclic voltammetry, we showed that the electron transport from the electrode through the double-stranded DNA to MB and then to ferricyanide ions is suppressed by the mismatches in duplex structure. According to UV-monitored melting data, single or multiple wobble dU.dG base pairs destabilize 18-mer DNA duplex by 9-27 degrees C.     

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.     

Electrochemical detection of DNA hybridization using methylene blue and electro-deposited zirconia thin films on gold electrodes

A novel electrochemical DNA biosensor based on methylene blue (MB) and zirconia (ZrO₂) thin films modified gold electrode for DNA hybridization detection is presented. Zirconia thin films were electrodynamically deposited onto the bare gold electrode in an aqueous electrolyte of ZrOCl₂ and KCl by cycling the potential between −1.1 and +0.7 V (versus Ag/AgCl) at a scan rate of 20 mV s⁻¹. Oligonucleotide probes with phosphate group at the 5′ end were attached onto the zirconia thin films because zirconia is affinity for phosphoric group. The surface density of the immobilized DNA molecules at the zirconia interface was investigated by fluorescence spectroscopy method. Hybridization was induced by exposure of the ssDNA-containing Au electrode to complementary ssDNA in solution. The decreases in the peak currents of MB, an electroactive label, were observed upon hybridization of probe with the target. The cathodic peak current (i_p) of MB after hybridization with the target DNA was linearly related to the logarithmic value of the target DNA concentration ranging from 2.25×10⁻¹⁰ to 2.25×10⁻⁸ mol l⁻¹. A detection limit of 1.0×10⁻¹⁰ mol l⁻¹ of oligonucleotides can be estimated.     

Carbon nanotube-enhanced electrochemical DNA biosensor for DNA hybridization detection

A novel and sensitive electrochemical DNA biosensor based on multi-walled carbon nanotubes functionalized with a carboxylic acid group (MWNTs-COOH) for covalent DNA immobilization and enhanced hybridization detection is described. The MWNTs-COOH-modified glassy carbon electrode (GCE) was fabricated and oligonucleotides with the 5'-amino group were covalently bonded to the carboxyl group of carbon nanotubes. The hybridization reaction on the electrode was monitored by differential pulse voltammetry (DPV) analysis using an electroactive intercalator daunomycin as an indicator. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics dramatically increased DNA attachment quantity and complementary DNA detection sensitivity. This is the first application of carbon nanotubes to the fabrication of an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization.     

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.     

Magnetically-induced solid-state electrochemical detection of DNA hybridization

A magnetic triggering of a solid-state electrical transduction of DNA hybridization is described. Positioning of an external magnet below the thick-film electrode attracts the DNA/particle network and enables the solid-state electrochemical stripping detection of the silver tracer. TEM imaging indicates that the hybridization event results in a three-dimensional aggregate structure in which duplex segments link the metal nanoparticles and magnetic spheres, and that most of this assembly is covered with the silver precipitate. This leads to a direct contact of the metal tag with the surface (in connection to the magnetic collection) and enables the solid-state electrochemical transduction (without prior dissolution and subsequent electrodeposition of the metal), using oxidative dissolution of the silver tracer. No such aggregates (and hence magnetic "collection") are observed in the presence of noncomplementary DNA, that is, without the linking hybrid. The new method couples high sensitivity of silver-amplified assays with effective discrimination against excess of closely related nucleotide sequences (including single-base imperfections). Such direct electrical detection of DNA/metal-particle assemblies can bring new capabilities to the detection of DNA hybridization, and could be applied to other bioaffinity assays.     

Nanoparticle based enhancement of electrochemical DNA hybridization signal using nanoporous electrodes

A novel nanoparticle-based enhanced methodology for the detection of ssDNA using nanoporous alumina filter membranes, containing pores of 200 nm in diameter, is reported. The blockage of the pores due to the hybridization is detected by measuring the decrease in the differential pulse voltammetric response of the [Fe(CN)(6)](4-/3-) redox indicator and using screen-printed carbon electrodes as transducing platform. Furthermore, 20 nm gold nanoparticle (AuNPs) tags are used in order to increase the sensitivity of the assay. The enhancement mechanism of DNA detection is due to an additional blocking effect induced by hybridization reaction by bringing AuNPs inside the pores. The developed methodology can be extended to other biosensing systems with interest not only for DNA but also for proteins and cells. The developed nanochannel/nanoparticle biosensing system would have enormous potential in future miniaturized designs adapted to mass production technologies such as screen-printing technology.     

Solid composite electrodes for DNA enrichment and detection

New plastic composite electrodes with appliance in medical diagnostic are described. The new electrode material offers the possibility of specific electrical enrichment and electrochemical analysis of nucleic acid sequences. To facilitate selective enrichment of target nucleic acids, specific probe oligonucleotides were attached covalently to free carboxyl groups of conducting polycarbonate/carbon fiber electrodes. Complementary oligonucleotides were enriched from analyte solutions by electric field supported methods. The analysis of the PCR product shows the efficiency and selectivity of the electrical enrichment. We have also shown that inexpensive and robust solid electrodes made of polycarbonate and conductive carbon powder are suitable for electrochemical examination of nucleic acids. The combination of electrochemical enrichment of DNA and subsequent electrochemical detection is a promising approach towards an inexpensive molecular diagnosis kit.     

Rapid hybridization at high salt concentration and detection of bacterial DNA using fluorescence polarization

The effects of NaCl concentration and temperature on the rate of hybridization of complementary single-stranded DNA (24-mers) were investigated. The single label of fluorescein was used for the probe DNA. The time courses of fluorescence polarization for the probe DNA were monitored. It was shown that detection of a specific DNA sequence (24-mer) was possible in less than 10 min using fluorescence polarization under the optimized conditions of 0.8 M NaCl at 46 degrees C in TE buffer. The effects of base-pair mismatches on DNA hybridization in the presence of NaCl or MgCl(2) were also investigated, and the specificity was considered by comparing the hybridization rate of the fluorescein-labeled probe. Determination of a specific DNA sequence was also possible in TE buffer containing 0.2 M MgCl(2). Moreover, in the presence of 0.2 M MgCl(2), there were no undesirable effects on hybridization and the presence of a single base pair mismatch could be identified. Rapid and specific determination of the DNA of enterohemorrhagic Escherichia coli, methicillin resistant Staphylococcus aureus and Legionella pneumophila, which had been multiplied by the asymmetric PCR, was performed under the optimized conditions for hybridization. It was confirmed that the conditions were also applicable to the hybridization between the probes and the amplified products of the actual bacterial genes. The combination of fluorescence polarization with the asymmetric PCR was quite effective. Moreover, the nested and asymmetric PCR product of bacterial gene could be detected effectively. The DNA detection method could also be used even if the specificity of the DNA amplification was not perfect and some unexpected bands were mixed with the target band during electrophoresis.     

Impedimetric genosensors for the detection of DNA hybridization

Impedance spectroscopy is proposed as the transduction principle for detecting the hybridization of DNA complementary strands. In our experiments, different DNA oligonucleotides were used as model gene substances. The gene probe is first immobilized on a graphite-epoxy composite working electrode based genosensor. Detection principle is based on changes of impedance spectra of a redox marker, the ferro/ferricyanide couple, after hybridization with target DNA. Resistance offered to the electrochemical reaction serves as the working signal, allowing for an unlabelled gene assay.      

Dendrimer-based biosensor for chemiluminescent detection of DNA hybridization

We report on a highly sensitive chemiluminescent (CL) biosensor for the sequenc-specific detection of DNA using a novel bio barcode DNA probe modified with gold nanoparticles that were covered with a dendrimer. The modified probe is composed of gold nanoparticles, a dendrimer, the CL reagent, and the DNA. The capture probe DNA was immobilized on magnetic beads covered with gold. It first hybridizes with the target DNA and then with one terminal end of the signal DNA on the barcoded DNA probe. CL was generated by adding H₂O₂ and Co(II) ions as the catalyst. The immobilization of dendrimer onto the gold nanoparticles can significantly enhance sensitivity and gives a detection limit of 6 fmol L^-1 of target DNA.

Liquid crystal-based detection of DNA hybridization using surface immobilized single-stranded DNA

The authors have investigated (a) the self-assembly of single-stranded DNA (ssDNA) on glass surfaces, and (b) the interaction of DNA with liquid crystals (LCs) on solid surfaces. The results suggest that ssDNA (compared to dsDNA) on the solid interface causes particularly different orientations in LCs. The LC molecules assume a uniform homeotropic orientation on the surface with a typical surface ssDNA coverage of ~2.4 × 10¹² molecules per square cm. Once complementary DNA is hybridized on the surface, the homotropic orientation of the LCs becomes disrupted. This orientation transition can be visually observed by using a crossed polarizer. The findings were exploiting to design an assay for target DNA (= analyte DNA) that has an ~0.1 nM detection limit. The assay is highly selective and can easily differentiate target DNA from single-base mismatch and non-complementary DNA. In our perception, it represents a powerful, label-free and portable DNA detection scheme.

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Graphical abstract Schematic illustration of the mechanism for orientation behavior of a liquid crystal film supported on different surfaces. The homeotropic orientation of LC molecules was induced by ssDNA with appropriate surface coverage and was disrupted by ssDNA with lower or higher surface coverage or P1/T1 complex. 5CB: 4-Cyano-4′-pentylbiphenyl. TEA: Triethoxysilylbutyraldehyde.

     

Sensitive voltammetric detection of DNA damage at carbon electrodes using DNA repair enzymes and an electroactive osmium marker

This paper presents a new approach to electrochemical sensing of DNA damage, using osmium DNA markers and voltammetric detection at the pyrolytic graphite electrode. The technique is based on enzymatic digestion of DNA with a DNA repair enzyme exonuclease III (exoIII), followed by single-strand (ss) selective DNA modification by a complex of osmium tetroxide with 2,2'-bipyridine. In double-stranded DNA possessing free 3'-ends, the exoIII creates ss regions that can accommodate the electroactive osmium marker. Intensity of the marker signal measured at the pyrolytic graphite electrode responded well to the extent of DNA damage. The technique was successfully applied for the detection of (1) single-strand breaks (ssb) introduced in plasmid DNA by deoxyribonuclease I, and (2) apurinic sites generated in chromosomal calf thymus DNA upon treatment with the alkylating agent dimethyl sulfate. The apurinic sites were converted into the ssb by DNA repair endonuclease activity of the exoIII enzyme. We show that the presented technique is capable of detection of one lesion per approximately 10(5) nucleotides in supercoiled plasmid DNA.     

DNA hybridization detection with water-soluble conjugated polymers and chromophore-labeled single-stranded DNA

A sensor is provided that detects single-stranded deoxyribonucleic acid (ssDNA) with a specific base sequence. The ssDNA sequence sensor comprises an aqueous solution containing a cationic water-soluble conjugated polymer [in this case, poly(9,9-bis(6'-N,N,N-trimethylammonium)-hexyl)-fluorene phenylene), 1] with a ssDNA labeled with a dye (in this case, fluorescein). The emission of light from the sensor solution with the wavelength characteristic of the probe oligonucleotide indicates the presence of ssDNA with a specific base sequence complementary to that of the probe ssDNA-fluorescein. Maximum energy transfer from 1 to the signaling chromophore occurs when the ratio of polymer chains to DNA strands is approximately 1:1. Energy transfer from 1 results in a fluorescein emission that is more intense than that observed by direct excitation of the chromophore. Furthermore, the decrease in energy transfer upon addition of electrolyte indicates that electrostatic forces dominate the interactions between 1 and DNA.     

DNA hybridization chain reaction and DNA supersandwich self-assembly for ultrasensitive detection

The fabrication of sensitive sensors with high selectivity is highly desirable for the detection of some important biomarkers,such as nucleic acids,proteins,small molecules and ions.DNA hybridization chain reaction(HCR) and DNA supersandwich self-assembly(SSA) are two prevalent enzyme-free signal amplification strategies to improve sensitivity of the sensors.In this review,we firstly describe the characteristics about DNA HCR and DNA SSA,and then summarize the advances in the one-dimensional DNA nanostructures assisted by HCR and SSA.This review has been divided into three parts according to the two signal amplification methods and highlights recent progress in these two strategies to improve the detection sensitivity of proteins,nucleic acids,small molecules and ions.