Abdel-Nasser Kawde

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.

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.

Pencil-based renewable biosensor for label-free electrochemical detection of DNA hybridization

Abstract The characteristics and advantages of a renewable DNA hybridization biosensor based on a pencil electrode transducer are described. The surface of this biosensor can be renewed rapidly, by a simple mechanical extrusion, hence, obviating the need for an additional regeneration step. The sensor, thus, responds rapidly to the ‘switching’ between target and non-complementary oligonucleotide solutions, with the use of fresh surfaces erasing memory effects. The intrinsic redox activity of the target DNA is employed for detecting the duplex formation. Relevant experimental parameters were examined and optimized. The selectivity of the new device was demonstrated for the detection of a single-point mutation in the BRCA1 breast cancer gene. Such low-cost, renewable graphite transducers provide an attractive alternative to conventional carbon electrodes used for transducing DNA hybridization. By eliminating the needs for a regeneration step and an external indicator, the device offers a greatly simplified operation and holds promise for decentralized genetic testing.

Renewable pencil electrodes for highly sensitive stripping potentiometric measurements of DNA and RNA

Renewable graphite pencil electrodes are demonstrated to be excellent materials for adsorptive stripping measurements of trace nucleic acids. While displaying an attractive stripping performance, comparable to that of conventional carbon paste electrodes, the pencil electrode offers a convenient (mechanical) renewal, with each stripping potentiogram recorded at a fresh surface. Various pencil lead materials and lengths have been examined and experimental variables of the pretreatment and measurement procedures have been explored and optimized. The extremely low detection limits (e.g., 3 micrograms l-1 tRNA with 10 min accumulation) are coupled to a good surface-to-surface reproducibility (RSD of 6.4% for 14 repetitive measurements of 1 mg l-1 ssDNA).

Gold nanoparticle-modified graphite pencil electrode for the high-sensitivity detection of hydrazine.

A novel gold nanoparticle-modified graphite pencil electrode (AuNP-GPE) is prepared just by immersing a bare GPE in AuNP solution, followed by heating for 15 min. The bare and modified GPEs are characterized by FE-SEM imaging and cyclic voltammetry. The AuNP-GPEs showed excellent electrocatalytic activities with respect to hydrazine oxidation, with good reproducibility. To reduce the quantification and detection limits, and increase the hydrazine sensitivity, the pH and square wave voltammetry parameters are optimized. A square wave voltammetry study as a function of the hydrazine concentration showed that the AuNP-GPE detectors quantification limit was 100 nmol L(-1) hydrazine, much lower than the value obtained using amperometry (10 µmol L(-1)). The limits of detection (at 3σ) for hydrazine sensing at AuNP-GPEs using square wave voltammetry and amperometry were 42 nmol L(-1) and 3.07 µmol L(-1). Finally, the modified electrode was used to determine the hydrazine concentration in drinking water, and satisfactory results are obtained. This simple, rapid, low-cost method for fabricating a modified electrode is an attractive approach to the development of new sensors.

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.

Graphite pencil electrodes as electrochemical sensors for environmental analysis: a review of features, developments, and applications

Graphite pencil electrodes (GPEs) are carbon-based electrodes that are recognized by their low cost, simplicity, commercial availability, ease of modification and disposability. GPEs are attractive substrates for electrochemical sensing because of their unique feature of “disposability” compared to other commonly used carbon-based electrodes. Mechanically rigid GPEs are easy to modify and miniaturize. The sensitivity and selectivity of GPE toward certain analytes can be enhanced by applying different modification materials. The primary focus of this review article is to highlight the applications of GPEs in the analysis of inorganic and organic pollutants in different environmental matrices. This review gives a brief overview of the various types of inorganic and organic pollutants and their impact on the environment. The key features of modified GPEs that enhance their electrocatalytic activity toward detection of certain target analytes are critically appraised. In the end, we summarize the current status, weaknesses and future prospects of GPE based sensors for environmental analysis.

Visual Detection of Single-Base Mismatches in DNA Using Hairpin Oligonucleotide with Double-Target DNA Binding Sequences and Gold Nanoparticles

We describe a hairpin oligonucleotide (HO) with double-target DNA binding sequences in the loop and 11-base in the stem for visual detection of single-base mismatches (SBM) in DNA with highly specificity. The thiol-modified HO was immobilized on gold nanoparticle (Au-NP) surface through a self-assembling process. The strategy of detecting SBM depends on the unique molecular recognition properties of HO to the perfect-matched DNA and SBM to generate different quantities of duplex DNA on the Au-NP surface, which are captured on the test zone of lateral flow test strip via the DNA hybridization reaction between the duplex DNA and preimmobilized DNA probe. Accumulation of Au-NPs produces the characteristic red bands, enabling visual detection of SBM. It was found that the ability of HO to differentiate perfect-matched DNA and SBM was increased dramatically by incorporating double-target DNA binding sequences in the loop of HO. The signal ratio between perfect-matched DNA and SBM was up to 28, which is much higher than that of conventional HO or molecular beacon. The approach was applied to detect the mutation sites, Arg142Cys and Gly529Ile, of transglutaminase 1 gene in autosomal recessive congenital ichthyosis. The results presented here show that the new HO is a potential molecular recognition probe for the future development of nucleic acid-based biosensors and bioassays. The approach can be used for point-of-care diagnosis of genetic diseases and detecting infectious agents or warning against bio-warfare agents.

A novel, fast and cost effective graphene-modified graphite pencil electrode for trace quantification of L-tyrosine

A simple and novel method for detecting L-tyrosine in urine was introduced using a graphene-modified graphite pencil electrode (GR-modified GPE). Graphene oxide (GO) was directly reduced using cyclic voltammetry (CV) on the surface of the GPE. The synthesized GO was characterized by FTIR and Raman spectroscopy. The morphology of the electrode surface was characterized by field emission-scanning electron microscopy (FE-SEM) and the electrochemical properties were characterized by CV, electrochemical impedance spectroscopy (EIS), and square wave voltammetry (SWV). The graphene layer on the GPE dramatically enhanced the electroactive surface area and electrochemical oxidation of L-tyrosine. A satisfactory linear response was obtained in the square wave voltammogram from 0.8 μM to 60 μM, with a regression constant (R2) of 0.9995. The modified electrode yielded a low L-tyrosine limit of detection of 0.07 μM. The present modification process is completed within 5 min compared to other reported time-consuming methods. The modified electrode surface was free from interfering species and successfully applied to the determination of L-tyrosine in human urine. The low cost and easy to modify electrode displayed excellent sensitivity, selectivity, and reproducibility, a low limit of detection and a wide linear response range.