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.     

E-DNA sensors for convenient, label-free electrochemical detection of hybridization

We review the development of reagentless, electrochemical sensors for the sequence-specific detection of nucleic acids that are based on the target-induced folding or unfolding of electrode-bound oligonucleotides. These devices, which are sometimes termed E-DNA sensors, are comprised of an oligonucleotide probe modified on one terminus with a redox reporter and attached to an electrode at the other. Hybridization of this probe DNA to a target oligonucleotide influences the rate at which the redox reporter collides with the electrode, leading to a detectable change in redox current. Because all sensing elements of this method are strongly linked to the interrogating electrode, E-DNA sensors are label-free, operationally convenient and readily reusable. As E-DNA signaling is predicated on a binding-specific change in the dynamics of the probe DNA (rather than simply monitoring the adsorption of a target to the sensor surface) and because electroactive contaminants (interferents) are relatively rare, this class of sensors is notably resistant to false positives arising from the non-specific adsorption of interferents, and performs well even when challenged directly with blood serum, soil and other complex sample matrices. We review the history of and recent advances in this promising DNA and RNA detection approach.     

Redox cycling amplified electrochemical detection of DNA hybridization: application to pathogen E. coli bacterial RNA

An electrochemical genosensor in which signal amplification is achieved using p-aminophenol (p-AP) redox cycling by nicotinamide adenine dinucleotide (NADH) is presented. An immobilized thiolated capture probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the detection probe, and streptavidin-alkaline phosphatase as reporter enzyme. The phosphatase liberates the electrochemical mediator p-AP from its electrically inactive phosphate derivative. This generated p-AP is electrooxidized at an Au electrode modified self-assembled monolayer to p-quinone imine (p-QI). In the presence of NADH, p-QI is reduced back to p-AP, which can be re-oxidized on the electrode and produce amplified signal. A detection limit of 1 pM DNA target is offered by this simple one-electrode, one-enzyme format redox cycling strategy. The redox cycling design is applied successfully to the monitoring of the 16S rRNA of E. coli pathogenic bacteria, and provides a detection limit of 250 CFU μL⁻¹.     

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.     

Magnetic bead-based label-free electrochemical detection of DNA hybridization.

Magnetic bead capture has been used for eliminating non-specific adsorption effects hampering label-free detection of DNA hybridization based on stripping potentiometric measurements of the target guanine at graphite electrodes. In particular, the efficient magnetic separation has been extremely useful for discriminating against unwanted constituents, including a large excess of co-existing mismatched and non-complementary oligomers, chromosomal DNA, RNA and proteins. The new protocol involves the attachment of biotinylated oligonucleotide probes onto streptavidin-coated magnetic beads, followed by the hybridization event, dissociation of the DNA hybrid from the beads, and potentiometric stripping measurements at a renewable graphite pencil electrode. Such coupling of magnetic hybridization surfaces with renewable graphite electrode transducers and label-free electrical detection results in a greatly simplified protocol and offers great promise for centralized and decentralized genetic testing. A new magnetic carbon-paste transducer, combining the solution-phase magnetic separation with an instantaneous magnetic collection of the bead-captured hybrid, is also described. The characterization, optimization and advantages of the genomagnetic label-free electrical protocol are illustrated below for assays of DNA sequences related to the breast-cancer BRCA1 gene.     

Label-free electrochemical detection of DNA hybridization on gold electrode

A label-free electrochemical detection protocol for DNA hybridization is reported for the first time by using a gold electrode (AuE). The oxidation signal of guanine was monitored at +0.73 V by using square wave voltammetry (SWV) on self-assembled l-cysteine monolayer (SAM) modified AuE. The electrochemical determination of hybridization between an inosine substituted capture probe and native target DNA was also accomplished. 6-mer adenine probe was covalently attached to SAM via its amino link at 5^′ end. Then, 6-mer thymine-tag of the capture probe was hybridized with the adenine probe, thus left the rest of the oligonucleotide available for hybridization with the target. The dependence of the guanine signal upon the concentration of the target was observed. Probe modified AuE was also challenged with non-complementary and mismatch containing oligonucletides. Label-free detection of hybridization on AuE is greatly advantageous over the existing carbon and mercury electrode materials, because of its potential applicability to microfabrication techniques. Performance characteristics of the genosensor are described, along with future prospects.     

Rapid DNA electrochemical biosensing platform for label-free potentiometric detection of DNA hybridization

This paper described a novel electrochemical DNA biosensor for rapid specific detection of nucleic acids based on the sulfonated polyaniline (SPAN) nanofibre and cysteamine-capped gold nanoparticle (CA-G_NP) layer-by-layer films. A precursor film of 3-mercaptopropionic acid (MPA) was firstly self-assembled on the Au electrode surface. CA-G_NP was covalently deposited on the Au/MPA electrode to obtain a stable substrate. SPAN nanofibre and CA-G_NP were alternately layer-by-layer assembled on the stable substrate by electrostatic force. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)₆]^3−/4− as the redox indicator. The (CA-G_NP/SPAN)_n films showed satisfactory ability of electron transfer and excellent redox activity in neutral media. Negatively charged probe ssDNA was immobilized on the outer layer of the multilayer film (CA-G_NP) through electrostatic affinity. Chronopotentiometry and electrochemical impedance spectroscopy were employed to obtain the direct electrochemical readout for probe ssDNA immobilization and hybridization using [Fe(CN)₆]^3−/4− in solution as the mediator. While electrochemical impedance spectroscopy led to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provided the total resistance at the interfaces of the modified electrodes. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports was found. Chronopotentiometry was suggested as a rapid transduction means (a few seconds). Based on the (CA-G_NP/SPAN)_n films, the target DNA with 20-base could be detected up to 2.13 × 10⁻¹³ mol/L, and the feasibility for the detection of base-mismatched DNA was also demonstrated.     

Tin oxide nanoparticles-polymer modified single-use sensors for electrochemical monitoring of label-free DNA hybridization

In this study, SnO₂ nanoparticles (SNPs)-poly(vinylferrocenium) (PVF⁺) modified single-use graphite electrodes were developed for electrochemical monitoring of DNA hybridization. The surfaces of polymer modified and polymer-SNP modified pencil graphite electrodes (PGEs) were firstly characterized by using SEM analysis. The electrochemical behaviours of these electrodes were also investigated using the differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques. The polymer-SNP modified PGEs were then tested for the electrochemical sensing of DNA based on the changes at the guanine oxidation signals. Experimental parameters, such as; different modifications in DNA oligonucleotides, DNA probe concentrations were examined to obtain more sensitive and selective electrochemical signals for nucleic acid hybridization. After optimization studies, DNA hybridization was investigated in the case of complementary of hepatitis B virus (HBV) probe, mismatch (MM), and noncomplementary (NC) sequences.     

Washing-free electrochemical DNA detection using double-stranded probes and competitive hybridization reaction

A new electrochemical DNA detection method using double-stranded probes and competitive hybridization reaction offers highly selective discrimination of single base mismatch without post-hybridization washing.     

New supramolecular interactions for electrochemical sensors development: different cucurbit[8]uril sensing platform designs

Three different strategies for cucurbit[8]uril immobilization on a glassy carbon electrode have been assayed. The electrochemical properties of the resulting modified electrodes in solutions containing neutral, positively and negatively charged potential cucurbit[8]uril guests were investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The comparison of the electrochemical behaviour exhibited by the unmodified electrodes against various probes, with respect to that of each modified electrode, resulted in an appropriate method to choose among different strategies for the development of electrochemical sensors. These sensors are based on the incorporation of the cucurbit[8]uril molecular selection properties that depend on the chemical characteristics of the potential analytes. Furthermore, atomic force microscopy was employed for the characterization of the different surfaces developed.     

Comparison between electrochemical and optoelectrochemical impedance measurements for detection of DNA hybridization

The principles of the electrochemical and optoelectrochemical impedance measurements on bare electrolyte/dielectric/semiconductor structures are described. The analysis of the experimental curves allows access to several indications concerning the electrical behavior of such structures. The application of these techniques to follow the electrical behavior of structures modified with two biological systems was investigated. The antibody/antigen recognition did not change the surface charge and, therefore, did not affect the impedance curves with respect to the applied potential. By contrast, the hybridization of two complementary DNA strands on the surface of the structure induced a variation of flat band potential of the semiconductor leading to a shift of impedance curves along the potential axis. This means that it is possible to detect directly the DNA hybridization without the use of labeled probes. The use of light allows the surface to be probed locally. In the future, the application of this technique for direct detection of hybridization on DNA chips should be possible.     

Preparation of silver nanoparticles on cellulose nanocrystals and the application in electrochemical detection of DNA hybridization

Synthesis of Ag nanopaticles was carried out with carboxylated cellulose nanocrystals as the scaffolds by reducing metallic cations using NaBH₄. Ag particles with a size less than 10 nm were readily prepared and dispersed well. The carboxyl and hydroxyl groups of carboxylated cellulose nanocrystals supplied a coordination effect to adsorb metallic cations and Ag nanoparticles, which prevent the aggregation of nanoparticles. The carboxylated cellulose nanocrystals carrying Ag nanoparticles were used as labels for electrical detection of DNA hybridization.     

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.     

Particle-based detection of DNA hybridization using electrochemical stripping measurements of an iron tracer

Two particle-based procedures for monitoring DNA hybridization based on electrochemical stripping detection of an iron tracer are described. The first protocol involves probes labeled with gold-coated iron core-shell nanoparticles, while the second route relies on detecting the iron content of magnetic-sphere tags. In both cases, the captured iron-containing particles are dissolved following the hybridization, and the released iron is quantified by cathodic-stripping voltammetry in the presence of the 1-nitroso-2-naphthol ligand and a bromate catalyst. Both protocols offer high sensitivity, a well-defined concentration dependence, and minimal contributions from non-complementary nucleic acids. The iron-containing particle signal amplifiers thus represent a very useful addition to the arsenal of metal tracers employed in electrical bioassays.     

Screen-printed electrochemical hybridization biosensor for the detection of DNA sequences from the Escherichia coli pathogen

An electrochemical biosensor for the specific detection of short DNA sequences from the E. coli pathogen is described. This hybridization device relies on the immobilization of a 25-mer oligonucleotide probe, from the E. coli lacZ gene, onto a screen-printed carbon electrode. Chronopotentiometric detection of the Co(bpy)₃⁺³ indicator is used for monitoring the hybridization event. Numerous variables of the assay protocol, including those of the probe immobilization step, the hybridization event, and the indicator association/detection, are characterized and optimized. Hybridization times of 2- and 30-min are sufficient for detecting 300- and 50 ng/mL, respectively, of the E. coli DNA target. Applicability to analysis of untreated environmental water samples is illustrated. Such single-use electrochemical sensors hold great promise for decentralized environmental and food testing for the E. coli pathogen.     

Nanomaterial-based electrochemical sensors for detection of glucose and insulin

Journal of Solid State Electrochemistry - Electrochemical sensors for the detection of specific biomolecules have attracted a lot of interest over the recent years due to their high sensitivity,...     

Chemical control of electrode functionalization for detection of DNA hybridization by electrochemical impedance spectroscopy

We report sensitive label-free detection of DNA oligonucleotide sequences using ac impedance measurements. The surface attachment chemistry is critical, and using mixed self-assembled monolayers on a gold electrode results in much better performance than homogeneous self-assembled monolayers. Contrary to expectations, binding of the target sequence reduces rather than increases the charge transfer resistance. Similar behavior is observed on indium tin oxide electrodes, and we ascribe it to the hydrophilicity and rigidity of the DNA duplex that cause it to reside further from the electrode surface and facilitate the approach of negatively charged redox moieties to the interface.     

New flavin and deazaflavin oligonucleotide conjugates for the amperometric detection of DNA hybridization

The functionalization of an oligonucleotide by flavin and deazaflavin derivatives allowed an amperometric determination of the hybridization process through the disappearance of the electroactivity of the free oligonucleotide and the appearance of a new electrochemical signal characteristic of the resulting duplex.     

Improved electrochemical performances of polyaniline nanotubes-poly-L-lysine composite for label-free impedance detection of DNA hybridization

A sensitive label-free DNA hybridization biosensing platform was fabricated based on the synergistic effect of polyaniline nanotubes (PANI_nt) and poly-L-lysine (pLys). The composite of pLys and PANI_nt was coated onto the carbon paste electrode (CPE) to form a uniform and very stable nanocomposite membrane. The pLys in the composite film not only acts as a membrane to retain good electron transfer capability of PANI_nt even at physiological pH, but also possesses fine biocompatibility for bio-analytes. DNA probes with negatively charged phosphate groups were readily linked to the positively charged pLys surface due to the strong electrostatic affinity. The synergistic effect of PANI_nt and pLys could significantly enhance the sensitivity of DNA hybridization recognition. The phosphinothricin acetyltransferase (PAT) gene fragment from transgenic corn and the polymerase chain reaction amplification of the terminator of nopaline synthase gene from the real sample of a kind of transgenic soybean were detected by this DNA electrochemical biosensor via label-free impedance method. This stable composite gives convenient permselectivity properties as a transducer material for the design of modern electrochemical impedance biosensor using [Fe(CN)₆]^3?/4? as an indicator.     

Significance of nanomaterials in electrochemical sensors for nitrate detection: A review

Nanomaterial-enabled electrochemical sensors are designed as an economical, efficient, and user-friendly analytical tool for on-site and routine nitrate analysis over a wide range of environmental samples. The remarkable advances and tunable attributes of nanomaterials have greatly improved the analytical performance of electrochemical nitrate sensors. In this review, a comprehensive elucidation of the recent advances in nanomaterial-based electrochemical nitrate sensors is presented. The review firstly provides a general introduction, followed by typical electrochemical sensing methods. The next two sections detail various nanomaterials, including graphene derivatives, carbon nanotubes/fibers, metal/bimetal/metal oxide nanoparticles, and conducting polymers for modifying electrodes in enzymatic and non-enzymatic electrochemical nitrate sensors. Finally, the perspectives and current challenges in achieving real-world applications of nanomaterial-based electrochemical nitrate sensors are outlined.