Aptamers are artificial nucleic acid ligands, specifically generated against certain targets, such as amino acids, drugs, proteins or other molecules. In nature they exist as a nucleic acid based genetic regulatory element called a riboswitch. For generation of artificial ligands, they are isolated from combinatorial libraries of synthetic nucleic acid by exponential enrichment, via an in vitro iterative process of adsorption, recovery and reamplification known as systematic evolution of ligands by exponential enrichment (SELEX). Thanks to their unique characteristics and chemical structure, aptamers offer themselves as ideal candidates for use in analytical devices and techniques. Recent progress in the aptamer selection and incorporation of aptamers into molecular beacon structures will ensure the application of aptamers for functional and quantitative proteomics and high-throughput screening for drug discovery, as well as in various analytical applications. The properties of aptamers as well as recent developments in improved, time-efficient methods for their selection and stabilization are outlined. The use of these powerful molecular tools for analysis and the advantages they offer over existing affinity biocomponents are discussed. Finally the evolving use of aptamers in specific analytical applications such as chromatography, ELISA-type assays, biosensors and affinity PCR as well as current avenues of research and future perspectives conclude this review.
We demonstrate a highly sensitive nano aptasensor for anthrax toxin through the detection of its polypeptide entity, protective antigen (PA toxin) using a PA toxin ssDNA aptamer functionalized single-walled carbon nanotubes (SWNTs) device. The aptamer was developed in-house by capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX) and had a dissociation constant (K d ) of 112 nM. The aptasensor displayed a wide dynamic range spanning up to 800 nM with a detection limit of 1nM. The sensitivity was 0.11 per nM and it was reusable six times. The aptasensor was also highly selective for PA toxin with no interference from human and bovine serum albumin, demonstrating it as a potential tool for rapid and point-of-care diagnosis for anthrax.
An electrochemical molecular beacon aptasensor for fast and sensitive assay of thrombin is developed and characterized. A bifunctional derivative of the thrombin-binding aptamer, 15-base long with a redox-active ferrocene moiety and a pendant hexanethiol linker group at the termini of the aptamer strand was immobilized on Au electrode surface. The electrochemical behavior of the aptasensor was studied using cyclic voltammetry (CV), chronoamperometry (CA), chronopotentiometry (CP), square-wave voltammetry (SWV) and chronopotentiometric stripping analysis (PSA) techniques. Taken together, these experiments support the switch "on" mechanism of the system when the thrombin binds to the immobilized aptamer. The variation of response to the concentration of thrombin, the target protein, was evaluated by square-wave voltammetry and chronopotentiometric stripping analysis in HEPES buffer solution (pH 8.0, 0.01 M). The aptasensor showed a specific response to thrombin in the range 1.0 to 35 nM with a detection limit of subnanomolar level concentrations. The reaction time, reproducibility, specificity and stability of the aptasensor were also studied.
Using the technique of liquid crystal templating a series of high surface area mesoporous platinum microelectrodes was fabricated. The underpotential deposition of metal ions at such electrodes was found to be similar to that at conventional platinum electrodes. The phenomena of underpotential deposition, in combination with the intrinsic properties of mesoporous microelectrodes (i.e. a high surface area and efficient mass transport) was exploited for the purpose of anodic stripping voltammetry. In particular the underpotential deposition of Ag(+), Pb(2+) and Cu(2+) ions was investigated and it was found that mesoporous microelectrodes were able to quantify the concentration of ions in solution down to the ppb range. The overall behaviour of the mesoporous electrodes was found to be superior to that of conventional microelectrodes and the effects of interference by surfactants were minimal.
Self-assembled monolayers (SAM) were obtained on gold electrodes using thioctic esters of benzo [1,3]dioxinol. These SAMs present a group that can be electroactivated selectively and was used for the directed, reagentless, covalent patterning of proteins. The advantage of this moiety is that it allows electroactivation at low potentials increasing selectivity and reliability. In this study, the efficiency of this patterning system is examined. Cyclic voltammetry (CV) was used to confirm the electroactive nature of SAM modified electrodes, showing fast and complete electrochemical deprotection with one scan. The enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) were patterned on the SAM-modified electrode through Schiffs base formation after electrochemical deprotection, confirming the selective nature of the electroactive substrate. Amperometric response was measured after the GOx immobilization showing high selective response. Real time monitoring is shown by immobilization of HRP on the SAM modified surface using electrochemical surface plasmon resonance (ESPR) after electrochemical deprotection, again showing high selective response when compared to the protected control.
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