Biomedical subjects
Publications and source records attributed to Ciara K O'Sullivan.
We report the first use of an electrochemical aptasensor for selective potassium recognition, based on a conformational change, affording an electric signal transduced electrochemically by square wave voltammetry or electrochemical impedance spectroscopy.
A bifunctional derivative of the thrombin-binding aptamer with a redox-active Fc moiety and a thiol group at the termini of the aptamer strand was synthesized. The ferrocene-labeled aptamer thiol was self-assembled through S-Au bonding on a polycrystalline gold electrode surface and the surface was blocked with 2-mercaptoethanol to form a mixed monolayer. By use of a fluorescent molecular beacon, the effect of counterions on quadruplex formation was established. The aptamer-modified electrode was characterized electrochemically by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The modified electrode showed a voltammetric signal due to a one-step redox reaction of the surface-confined ferrocenyl moiety of the aptamer immobilized on the electrode surface in 10 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer of pH 8.0. An increase in the DPV current signal was evident after blocking with 2-mercaptoethanol, effectively removing aptamer nonspecifically absorbed rather than bound to electrode surface or due to the formation of the aptamer-thrombin affinity interaction. The impedance measurement, in agreement with the differential pulse voltammetry (DPV), showed decreased Faradaic resistances in the same sequence. The "signal-on" upon thrombin association could be attributed to a change in conformation from random coil-like configuration on the probe-modified film to the quadruplex structure. The DPV of the modified electrode showed a linear response of the Fc oxidation signal to the increase in the thrombin concentration in the range between 5.0 and 35.0 nM with a linear correlation of r = 0.9988 and a detection limit of 0.5 nM. The molecular beacon aptasensor was amenable to full regeneration by simply unfolding the aptamer in 1.0 M HCl, and could be regenerated 25 times with no loss in electrochemical signal upon subsequent thrombin binding.
A novel impedimetric aptasensor using a mixed self-assembled monolayer composed of thiol-modified thrombin binding aptamer and 2-mercaptoethanol on a gold electrode is reported. The changes of interfacial features of the electrode were probed in the presence of the reversible redox couple, Fe(CN)6(3-/4-), using impedance measurements. The electrode surface was partially blocked due to the self-assembly of aptamer or the formation of the aptamer-thrombin complex, resulting in an increase of the interfacial electron-transfer resistance detected by electrochemical impedance spectroscopy or cyclic voltammetry. The aptasensor was regenerated by breaking the complex formed between the aptamer and thrombin using 2.0 M NaCl solution, and the immobilized aptamer subsequently was used for repeated detection of thrombin. The aptamer-functionalized electrode showed a linear response of the charge-transfer resistance to the increase of thrombin concentration in the range of 5.0-35.0 nM and the thrombin was easily detectable to a concentration of 2.0 nM.
Immense effort has been placed on the realization of immunoassays exploiting displacement of a suboptimum target, due to the ease of use and applicability to immunochromatographic strips and immunosensors. Most of the efforts reported to date focus on the use of a suboptimal target that is displaceable by the target toward which the antibody has higher affinity. Limited success has been achieved due to difficulty in obtaining suboptimal targets to which the antibody has enough affinity to bind while at the same time having lower levels of affinity in comparison to the target to facilitate displacement. Aptamers are synthetic oligonucleotides specifically selected to bind a certain target. Thanks to their high affinity and sensitivity, aptamers appear as alternative candidates to antibodies for analytical devices and several enzyme-linked aptamer assays and aptasensors have been reported. Aptamers, in contrast to antibodies, require the formation of a three-dimensional structure for target binding and can thus be anticipated to have a much higher affinity for binding its target rather than a modified form of the target (e.g., enzyme-labeled target). This phenomenon can be exploited for the development of a displacement assay, using enzyme-labeled target as a suboptimal displaceable molecule. Here, we report the first demonstration of the exploitation of an aptamer in an extremely rapid and highly sensitive displacement assay. Surface plasmon resonance studies demonstrated the thrombin-binding aptamer to have a lower affinity for enzyme-labeled thrombin than unmodified thrombin, with respective K(D) of 1.1 x 10(-8) and 2.9 x 10(-9) M. The assay is extremely rapid, requiring only 10 min for completion, and exhibits a detection limit lower than that obtainable with competitive enzyme-linked aptamer assays and comparable to that of hybrid aptamer-antibody assays. Optimal storage conditions for precoated microtiter plates (consisting of coated aptamer and captured labeled target) were elucidated, and the results demonstrated their amenability to long-term storage, facilitating commercially viable displacement enzyme-linked aptamer assays that simply require sample addition, with a total assay time, including color development, of 30 min.
Explore the source record for details and available documents.
Aptamers are synthetic oligonucleotides specifically selected to bind a certain target. Thanks to their high affinity and sensitivity, aptamers appear as alternative candidates to antibodies for analytical devices and several assays have been reported. However, and contrary to what happens with DNA probes, the aptamers' ability to bind their targets depends on folding and 3-D structure, which may be affected by the incubation conditions and buffer composition. In this report, a systematic evaluation of the parameters with potential effect on the ELAA (Enzyme Linked Aptamer Assay) performance has been carried out. Additionally, diverse ELAA and mixed ELISA/ELAA formats exploiting the thrombin-binding aptamer have been optimized and their efficiencies compared. ELAA results have been confirmed using nuclear magnetic resonance, electrophoresis, and surface plasmon resonance. Our results indicate that parameters such as immobilization strategy, incubation time/temperature, and buffer composition should be optimized for each aptamer as they affect folding and, thus, binding efficiency. Among the studied assays, the mixed ELISA/ELAA sandwich formats showed the lowest limit of detection observed (<1 nM thrombin), while a competition ELAA appeared as the best assay in terms of high sensitivity (1.8 nM) and short assay time (1 h, 30 min). The elucidation of optimal parameters for assay performance reported here clearly indicates that aptamers are unique structures. Formation of the 3-D structures required for target binding is influenced by variable parameters, and unlike DNA/antibody based assays, there are no general recommendations, with each assay requiring individual optimization of parameters.
The current work describes the optimisation of a screen-printed electrode (SPE) system for measurement of a variety of seafood toxins, such as okadaic acid, brevetoxin, domoic acid and tetrodotoxin. A disposable screen-printed carbon electrode coupled with amperometric detection of p-aminophenol at +300 mV vs. Ag/AgCl, produced by the label, alkaline phosphatase, was used for signal measurement. ELISA was primarily used to develop all toxin systems, prior to transferring to SPE. The sensors incorporate a relevant range for toxin detection, by which humans become ill, with detection limits achieved at SPE to the order of ng ml (-1) (ppb) or lower in some cases. The SPE system is simple and cost-effective due to their disposable nature, and analysis time is complete in 30 min. In addition, analyses can be achieved outside of a laboratory environment allowing for in-field measurements. Recovery experiments on selected toxins using the relevant working ranges highlighted the functionality of these systems yielding a +/-10% deviation for the true value.
Aptamers are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. They are isolated from combinatorial libraries of synthetic nucleic acid by an iterative process of adsorption, recovery and reamplification. Aptamers, first reported in 1990, are attracting interest in the areas of therapeutics and diagnostics and offer themselves as ideal candidates for use as biocomponents in biosensors (aptasensors), possessing many advantages over state of the art affinity sensors. The properties of aptamers, their applicability to biosensor technology, current research and future prospects are addressed in this short review.