Nucleobase modification as redox DNA labelling for electrochemical detection

Basic aspects of DNA electrochemistry with a strong focus on the use of modified nucleobases as redox probes for electrochemical bioanalysis are reviewed. Intrinsic electrochemical properties of nucleobases in combination with artificial redox-active nucleobase modifications are frequently applied in this field. Synthetic approaches (both chemical and enzymatic) to base-modified nucleic acids are briefly summarized and their applications in redox labelling are discussed. Finally, analytical applications including DNA hybridization, primer extension, PCR, SNP typing, DNA damage and DNA-protein interaction analysis are presented (critical review, 91 references).     

Polymer-DNA hybrids as electrochemical probes for the detection of DNA

The syntheses of several norbornene block copolymers containing oligonucleotide and ferrocenyl side chains and their use in the electrochemical detection of DNA are described. Two kinds of DNA-containing block copolymers with either ferrocenyl or dibromoferrocenyl groups were prepared via ring-opening metathesis polymerization (ROMP). Based on these two distinct ferrocene derivatives, a triblock copolymer labeling strategy was developed. With this strategy, the identity of DNA target can be determined by the E1/2s of the ferrocenyl moieties and the ratio of peak currents. These polymers exhibit predictable and tailorable electrochemical properties, high DNA duplex stability, and unusually sharp melting transitions, which are highly desirable characteristics for DNA detection applications. Significantly, single-base mismatches could be easily detected using two distinct block copolymers as dual-channel detection probes in an electrochemical DNA detection format.     

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.     

Effect of redox label tether length and flexibility on sensor performance of displacement-based electrochemical DNA sensors

This article summarizes the sensor performance of four electrochemical DNA sensors that exploit the recently developed displacement-replacement sensing motif. In the absence of the target, the capture probe is partially hybridized to the signaling probe at the distal end, positioning the redox label, methylene blue (MB), away from the electrode. In the presence of the target, the MB-modified signaling probe is released; one type of probe is capable of assuming a stem-loop probe (SLP) conformation, whereas the other type adopts a linear probe (LP) conformation. Independent of the sensor architecture, all four sensors showed “signal-on” sensor behavior. Unlike the previous report, here we focused on elucidating the effect of the redox label tether length and flexibility on sensor sensitivity, specificity, selectivity, and reusability. For both SLP and LP sensors, the limit of detection was 10 pM for sensors fabricated using a signaling probe with three extra thymine (T3) bases linked to the MB label. A limit of detection of 100 pM was determined for sensors fabricated using a signaling probe with five extra thymine (T5) bases. The linear dynamic range was between 10 pM and 100 nM for the T3 sensors, and between 100 pM and 100 nM for the T5 sensors. When compared to the LP sensors, the SLP sensors showed higher signal enhancement in the presence of the full-complement target. More importantly, the SLP-T5 sensor was found to be highly specific; it is capable of discriminating between the full complement and single-base mismatch targets even when employed in undiluted blood serum. Overall, these results highlight the advantages of using oligo-T(s) as a tunable linker to control flexibility of the tethered redox label, so as to achieve the desired sensor response.     

Dosimetric gelator probes and their application as sensors

Supramolecular gels formed by the self-assembly of organic molecules are useful in many areas from materials to medicine. Of the different applications, exploitation of gels for the visual detection of analytes is a fairly recent trend in gel chemistry. Most of the gel-based sensors rely on non-covalent interactions between the gelator molecules and the added chemical analytes and therefore, often suffer from less selectivity and long response time. In this context, dosimetric gelator probes are superior to other gel-based sensors with high selectivity and fast response time. Unlike non-covalent binding sites, dosimetric gelators typically contain a reaction centre and undergo a specific chemical reaction selective to an analyte resulting in either formation or rupturing of covalent bonds. In this review, we provide an up-to-date report of various reaction-based gel systems applied for the sensing of analytes. We elaborately discuss the concept, design principles, self-assembly properties, and reaction mechanisms of such gelators. We also highlight the limitations, challenges, and the necessity of further exploration of dosimetric gels in this domain.     

Organic radical-ion salts as selective electrochemical sensors

A series of semiconducting organic radical-ion salts has been studied with regard to the potential use of such compounds in solid-state ion-selective electrodes. Improvements in working activity ranges and selectivities were obtained in comparison with previously investigated salts of TCNQ; excellent calibration graphs were obtained for lead(II), copper([I) and tctraphenylarsonium ions.     

Unusual metals as electrode materials for electrochemical sensors

Although noble metals are still widely used in electroanalysis, a plethora of different nonconventional metals is now enriching the panorama of materials acting as the electrochemical transducer in sensing systems. In particular, Ti, Cu, Co, Fe, Mo, Ta, W, Rh, Bi, Sb, Te and Pb are discussed here in view of their peculiar physicochemical properties and of the interesting electrocatalytic activities ascribable to these elements and to the relevant metal oxide ultrathin films that spontaneously form at the electrode–solution interface. This behaviour, exploitable in electroanalysis for the detection of a number on analytes, is often accompanied by low price and high resistance to corrosion and to abrasion characterising these materials. These peculiarities encourage the possible use of the cited metals in a wide number of analytical frames, ranging from process control to bioimplantable sensing systems.     

Films of lutetium bisphthalocyanine nanowires as electrochemical sensors

Lutetium bisphthalocyanine (LuPc(2)) nanowires have been successfully obtained by electrophoretic deposition (EPD). The influence of the deposition conditions and annealing in the structure of the films has been studied by AFM, SEM, X-ray diffraction (XRD), UV-vis absorption, and near-infrared (NIR). The electrochemical properties of the EDP films immersed in different electrolytic solutions (KCl, MgCl(2), KClO(4), HCl, and NaOH) indicate that anions diffuse inside the film to maintain the electroneutrality and the kinetics follows the Randles-Sevcik equation. The stability of the response increases strongly upon annealing due to the improvement of the adhesion of the sensitive material to the substrate. The EPD films have been successfully used to detect caffeic acid (an antioxidant of interest in the food industry). The anodic peak associated with the oxidation of caffeic acid appears at 0.54 V and is linearly dependent on the caffeic acid concentration in the 6 × 10(-5) M to 5 × 10(-4) M range with a detection limit of 3.12 × 10(-5) M. The electrochemical behavior of the annealed LuPc(2) EPD films is similar to that observed using Langmuir-Blodgett (LB) nanostructured films. However, the different molecular organization of the molecules inside the film causes differences in the shape and position of the peaks. Although LuPc(2) sensors prepared with both EPD and LB techniques provide stable and reproducible responses, the use of EPD is preferred for real sensing applications because of its lower cost, shorter preparation time, and longer lifetime.     

Extending the dynamic range of electrochemical sensors using multiple modified electrodes

Multiple electrodes, combined with a chemometric strategy to calibrate the measurement response, have been used for the determination of an analyte across a broader dynamic range than is possible with a single electrode. The model system used for the detection of copper comprised electrodes modified with a self-assembled monolayer. The electrodes were modified with the copper-complexing species (3-mercaptopropionic acid, thioctic acid, and the peptides cysteine and Gly-Gly-His) and copper was determined over concentrations ranging from nanomolar to millimolar using voltammetric analysis. We have demonstrated that by combining the calibration functions from the four electrodes a better estimate (i.e. with smaller variance) of the concentration of the analyte is obtained. Measurement uncertainty is expressed for independently prepared electrodes, which allows the possibility of commercial production and factory calibration. The principles of using multiple electrodes modified with recognition elements with different affinities for the target analyte to extend the dynamic range of sensors is a general one that could be applied to other analytes.     

Porous Cu-NiO modified glass carbon electrode enhanced nonenzymatic glucose electrochemical sensors

Porous Cu-NiO nanocomposites were successfully prepared by calcination of the Cu-Ni(OH)(2) precursor at 400 °C for 2 h. During the process of calcination, Ar was used to deaerate O(2). The structure and morphology of Cu-NiO were characterized by X-ray diffraction spectrum (XRD), energy dispersive X-ray analyses (EDX), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Using porous Cu-NiO nanocomposites, a simple non-enzymatic amperometric sensor has been fabricated (Cu-NiO/GCE) and evaluated by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and typical amperometric method. When applied to detect glucose by the amperometric method, Cu-NiO/GCE produced an ultrahigh sensitivity of 171.8 μA mM(-1), with a low detection limit of 0.5 μM (S/N = 3). What's more, interference from common co-existing species, such as UA, AA, and fructose can be avoided at the sensor. Results in this study imply that porous Cu-NiO nanocomposites are promising nanomaterials for the enzyme-free determination of glucose.     

Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl transferase. Application in electrochemical DNA hybridization and protein-DNA binding assays

A simple approach to DNA tail-labelling using terminal deoxynucleotidyl transferase and modified deoxynucleoside triphosphates is presented. Amino- and nitrophenyl-modified dNTPs were found to be good substrates for this enzyme giving 3'-end stretches of different lengths depending on the nucleotide and concentration. 3-Nitrophenyl-7-deazaG was selected as the most useful label because its dNTP was efficiently incorporated by the transferase to form long tail-labels at any oligonucleotide. Accumulation of many nitrophenyl tags per oligonucleotide resulted in a considerable enhancement of voltammetric signals due to the nitro group reduction, thus improving the sensitivity of electrochemical detection of the tail-labelled probes. We demonstrate a perfect discrimination between complementary and non-complementary target DNAs sequences by tail-labelled hybridization probes as well as the ability of tumour suppressor p53 protein to recognize a specific binding site within tail-labelled DNA substrates, making the methodology useful in electrochemical DNA hybridization and DNA-protein interaction assays.     

Inkjet-printed electrochemical sensors

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Rubber-based substrates modified with carbon nanotubes inks to build flexible electrochemical sensors

The development of a solid-contact potentiometric sensor based on conducting rubbers using a carbon nanotubes ink is described here. Commercial rubbers are turned into conductive ones by a simple and versatile method, i.e. painting an aqueous dispersion of single-walled carbon nanotubes on the polymer surface. On this substrate, both the working ion-selective electrode and the reference electrode are built in order to form an integrated potentiometric cell. As a proof-of-principle, selective potassium electrodes are fully characterized giving comparable performances to conventional electrodes (sensitivity, selectivity, stability, linear range, limit of detection and reproducibility). As an application of the rubber-based electrodes, a bracelet was constructed to measure potassium levels in artificial sweat. Since rubbers are ubiquitous in our quotidian life, this approach offers great promise for the generation of chemical information through daily objects.     

Gold chloride electrodes as electrochemical sensors for liquid chromatography

A fast, simple, inexpensive and reproducible method for the fabrication of a gold chloride electrode is presented. The response of this electrode is highly stable and it leads to improved detection for various redox species. Experiments demonstrating the utility of the gold chloride electrode as a sensitive sensor for liquid chromatography are described.     

Nanomaterial-based electrochemical sensors and optical probes for detection and imaging of peroxynitrite: a review

Peroxynitrite (PON for short) is a powerful nitrating, nitrosating and oxidative agent for cellular constituents. In vivo, PON is formed through the diffusion-controlled reaction between superoxide radical (O₂ ^?- ) and nitric oxide (?NO). This critical review (with 67 refs.) covers the state of the art in nanomaterial-based (a) detection and imaging of PON inside cells and (b) monitoring of cellular events such as cellular oxidative burst by using optical or electrochemical methods. It starts with the formation, fate and pathophysiology of PON in vivo. The next part summarizes nanomaterial based electrochemical microsensors featuring nanofilms and nanostructured electrodes, nanospheres, 3D nanostructures and graphene-supported catalysts. A following chapter covers techniques based on optical nanoprobes, starting with nanomaterials used in optical detection of PON (including quantum dots, carbon dots, fluorescent organic polymer dots, rare earth nanocrystals including upconversion nanoparticles, iron oxide nanoparticles, gold nanoparticles, and fluorophore-modified nanoporous silicon). This is followed by subsections on strategies for optical detection of PON (including color changes, fluorescence quenching, activation and recovery), and on schemes for optimized spatial and temporal resolution, for improving sensitivity, selectivity, and (photo)stability. We then address critical issues related to biocompatibility, pharmacokinetics, give a number of representative practical applications and discuss challenges related to PON detection. The review concludes with a discussion of latest developments and future perspectives.

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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.     

Simultaneous electrochemical immunoassay of three liver cancer biomarkers using distinguishable redox probes as signal tags and gold nanoparticles coated carbon nanotubes as signal enhancers

A novel strategy for simultaneous determination of three liver cancer biomarkers based on three redox labels with distinct voltammetric peaks was described. Gold nanoparticles coated carbon nanotubes were used as carriers to immobilize redox probes labeled antibodies and to amplify the signals.     

Biomembrane mimetic electrochemical sensors

Biomimetic approaches to the assembling of regular layers of electrochemical sensors make it possible to extend their performance because of the tuning shape and charge of the lipid analogs and implementation of artificial receptors in the lipid layers. The role of artificial components in modification of the properties of the surface layers and application of the nanopore devices are of particular interest. In review, recent trends in assembling modified lipid membranes and their artificial analogs with nanopores have been considered with particular emphasis on their use in electrochemical sensors.     

Quadruplex-based molecular beacons as tunable DNA probes

Molecular beacons (MBs) are fluorescent nucleic acid probes with a hairpin-shaped structure in which the 5' and 3' ends are self-complementary. Due to a change in their emissive properties upon recognition with complementary sequences, MBs allow the diagnosis of single-stranded DNA or RNA with high mismatch discrimination, in vitro and in vivo. Whereas the stems of MB hairpins usually rely on the formation of a Watson-Crick duplex, we demonstrate in this report that the preceding structure can be replaced by a G-quadruplex motif (G4). Intramolecular quadruplexes may still be formed with a central loop composed of 12 to 21 bases, therefore extending the sequence repertoire of quadruplex formation. G4-MB can efficiently be used for oligonucleotide discrimination: in the presence of a complementary sequence, the central loop hybridizes and forms a duplex that causes opening of the quadruplex stem. The corresponding G4-MB unfolding can be detected by a change in its fluorescence emission. We discuss the thermodynamic and kinetic opportunities that are provided by using G4-MB instead of traditional MB. In particular, the intrinsic feature of the quadruplex motif facilitates the design of functional molecular beacons by independently varying the concentration of monovalent or divalent cations in the medium.