Impedimetric aptasensor for tobramycin detection in human serum.

An RNA aptamer is proposed as a recognition element for the detection of tobramycin in human serum. A displacement assay was developed using faradaic-electrochemical impedance spectroscopy (F-EIS) as a detection technique. Two modified aptamers, a partially (ATA) and a fully O-methylated aptamer (FATA) were evaluated and compared. The affinity constant, K(D), for both aptamers was estimated by F-EIS resulting virtually identical within the experimental error. The selectivity towards other aminoglycosides was also studied. The analytical characteristics were evaluated in aqueous solution using both aptamers and FATA was selected for human serum experiments. Using a 1:0.5 dilution of the serum, a linear range between 3 µM and 72.1 µM was obtained, which included the therapeutic range of the antibiotic.

SPR sensing of small molecules with modified RNA aptamers: detection of neomycin B.

We have studied how the modification of the RNA aptamer evolved against neomycin B at 2' position of ribose with a methyl group influences the affinity of the interaction. Using surface plasmon resonance (SPR) and faradaic impedance spectroscopy (FIS) an affinity constant in the muM range was calculated. The results showed that the modification of the aptamer does not significantly alter the affinity of the aptamer for the antibiotic. This finding opens up the possibility of designing modified RNA aptamers resistant to endonucleases without variation of the analytical features. In addition to this, we propose a competitive assay for the detection of neomycin B using SPR as a transduction technique. A range of quantification between 10 nM and 100 microM was obtained, which shows the feasibility of detecting small molecules using aptamers with high sensitivity.

PCR-coupled electrochemical sensing of Legionella pneumophila.

Human infections with Legionella pneumophila represent a public health problem. Current culture assays for surveillance and control of L. pneumophila in water are time-consuming and limited by the sensitivity, especially when samples also contain microorganisms that inhibit Legionella growth. In this work, an electrochemical method, different from real-time polymerase chain reaction (PCR) approaches, for semiquantitative evaluation of L. pneumophila is presented. A PCR assay targeting the 16S-rRNA gene of L. pneumophila giving rise to a 95-mer amplicon was established. Amplicons were hybridized to a biotin-labeled reporter sequence and then to a thiolated stem-loop structure immobilized onto gold electrodes as a reporter molecule with 1-naphthyl phosphate as a substrate. 1-Naphthol enzymatically generated was determined by differential pulse voltammetry (DPV). For a constant number of amplification cycles, results show that the voltammetric signal is related to the number of copies in the sample thus achieving a useful semiquantitative estimation of L. pneumophila. After 40 cycles of PCR amplification this methodology has a limit of detection of 10 genomes, allowing the reliable detection of 10(2) genomes of L. pneumophila as well as distinguishing 10(3) and 10(4) genomes of the pathogen, values related to corrective actions in water systems in buildings, in accordance with the legislation currently in force.

Hairpin-DNA probe for enzyme-amplified electrochemical detection of Legionella pneumophila.

An electrochemical genosensor for the detection of nucleic acid sequences specific of Legionella pneumophila is reported. An immobilized thiolated hairpin probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the signaling probe, and streptavidin-alkaline phosphatase as reporter molecule. The activity of the immobilized enzyme was voltammetrically determined by measuring the amount of 1-naphthol generated after 2 min of enzymatic dephosphorylation of 1-naphthyl phosphate. The sensor allows discrimination between L. pneumophila and L. longbeachae with high sensitivity under identical assay conditions (no changes in stringency). A limit of detection of 340 pM L. pneumophila DNA, and a linear relationship between the analytical signal and the logarithm of the target concentration to 2 muM were obtained. Experimental results show the superior sensitivity and selectivity of the hairpin-based assay when compared with analogous sandwich-type assays using linear capture probes.

Computational predictions and experimental affinity distributions for a homovanillic acid molecularly imprinted polymer.

Density Functional Theory calculations have been used to select, among a set of chemicals traditionally used in the formulation of non-covalent molecularly imprinted polymers (MIPs), the best functional monomer and porogenic solvent for the construction of a recognition element for the dopamine metabolite homovanillic acid (HVA). Theoretical predictions were confirmed through batch binding assays and voltammetric detection. The computational method predicts that trifluoromethacrylic acid and toluene are the monomer and solvent rendering the highest stabilization energy for the pre-polymerization adducts. HVA-MIP prepared using this formulation gives rise to a binding isotherm that is accurately modelled by the Freundlich isotherm. The binding properties of this polymer were estimated using affinity distribution analysis. An apparent number of sites of 13 micromol g(-1) with an average affinity constant of 2 x 10(4) M(-1) was obtained in the concentration window studied.

Amplified label-free electrocatalytic detection of DNA in the presence of calcium ions.

A new label-free electrocatalytic method for the detection of DNA, is presented, in which DNA is the catalyst. The method takes advantage of the catalytic properties of the electrooxidised adenines within DNA toward the oxidation of NADH. This catalytic event results in an enhancement in the oxidation current of the electrooxidised adenines within DNA. Further improvement in this analytical signal is achieved in the presence of Ca(2+) ions. Parameters affecting the electrocatalytic current, such as pH or concentration of Ca(2+) ions have been investigated and optimised. Finally, the analytical features of the developed method are obtained. This method constitutes a more sensitive and reproducible alternative to other methods that use the oxidation current of the electrooxidised adenines without coupling to any catalytic event. A limit of detection of 33 fmol deoxyadenylic acid icosanucleotide (dA)(20), is obtained without labels.

Computational approach to the rational design of molecularly imprinted polymers for voltammetric sensing of homovanillic acid.

A methodology based on density functional theory calculations for the design of molecularly imprinted polymers (MIPs) is described. The method allows the rational choice of the most suitable monomer and polymerization solvent among a set of chemicals traditionally used in MIP formulations for the molecular imprinting of a given template. It is based on the comparison of the stabilization energies of the prepolymerization adducts between the template and different functional monomers. The effect of the polymerization solvent is included using the polarizable continuum model. A voltammetric sensor for homovanillic acid was constructed using different MIPs as recognition element, confirming that the solvent (toluene) and functional monomer (methacrylic acid) selected according to the theoretical predictions lead to the most efficient molecular recognition sensing phase. With the voltammetric sensor prepared using the MIP designed according to the theoretical predictions, a linear response for concentrations of homovanillic acid between 5 x 10(-8) and 1 x 10(-5) M can be obtained. The limit of detection is 7 x 10(-9) M. The selectivity obtained for homovanillic acid over other structurally related compounds buttresses the validity of this strategy of design.

Flavin adenine dinucleotide as precursor for NADH electrocatalyst.

The generation of a new electrocatalytic system for NADH after oxidizing flavin adenine dinucleotide (FAD) is shown. The oxidation is performed in alkaline medium until +1.4 V (Ag/AgCl) at graphite electrodes. The catalytic activity is ascribed to the electrooxidized moiety of FAD and not to quinone surface groups. A comparison between this catalyst and that attributed to poly(FAD) (Karyakin, A. A.; Ivanova Y. N.; Revunova, K. V.; Karyakina, E. E. Anal. Chem. 2004, 76, 2004-2009.) is presented. It is concluded that the surface quinone groups generated during the strong anodization of the electrode in acidic medium at 2-2.5 V and not the poly(FAD) are responsible for the catalytic activity described in the above mentioned work.

5-Hydroxytryptophan as a precursor of a catalyst for the oxidation of NADH.

Following oxidation of 5-hydroxytryptophan (5-HTPP) at a pyrolytic graphite electrode at pH 7.5, two quasi-reversible redox couples emerge at -0.170 and +0.032 V, respectively, due to oxidation products strongly adsorbed to the electrode surface. These redox processes have been electrochemically and kinetically characterized in terms of the dependence of the formal potential (E degrees ') with pH, variation of the current density with scan rate, operational stability, and electron-transfer rate constant (k(s)). The wave centered at +0.032 V could mediate the oxidation of NADH, exhibiting a strong and persistent electrocatalytic response. A quinone-imine structure has been proposed as the electrocatalytically active species. The kinetics of the reaction between the mediator and NADH has been characterized via rotating disk electrode voltammetry, and it has been found that the rate constant for the reaction is dependent on the solution concentration of NADH. 5-HTPP modified electrodes could be employed in the amperometric detection of NADH with a limit of detection in the nanomolar range. Moreover, 5-HTPP modified electrodes retain their electrocatalytic activity for at least one week. The potential application of these electrodes to amperometric biosensor is demonstrated.

Current strategies for electrochemical detection of DNA with solid electrodes.

A review of current strategies aimed at detecting nucleic acids (NA) using NA-modified solid electrodes reveals the versatility and potential of electrochemical detection in this field. What emerged at the beginning of 90s as a very promising detection system in DNA technology is now resulting in the first commercial devices. Many aspects of the experimental design, for example surface immobilisation and detection schemes, are outlined and evaluated. Although most approaches use hybridisation as the recognition reaction, those not based on hybridisation are also included. As is finally shown, great advances have been achieved, although further developments are required if electrochemical devices are to be suitable for routine measurement.

Voltammetric response of diclofenac-molecularly imprinted film modified carbon electrodes.

A voltammetric sensor for the determination of diclofenac was developed, based on the molecular recognition of the analyte by molecularly imprinted methacrylate-ethyleneglycol dimethacrylate co-polymers. Pre-polymerisation solutions were deposited onto the surface of a glassy carbon electrode and a polymer film was obtained after spin coating control of thickness and in situ thermal polymerisation. After the template extraction from the resultant film, re-binding of diclofenac is performed from acetonitrile solutions containing the analyte. The amount of bonded diclofenac was then evaluated by differential pulse voltammetry in different electrolytes. The best results were obtained in 0.025 M citrate solution pH 6 containing 10% of acetonitrile. This medium favours the release of diclofenac from the polymer binding sites. In this way, the voltammetric transduction of the molecular recognition event is achieved. Voltammetric selectivity measurements revealed negligible interferences from diclofenac family of anti-inflammatory drugs, such as niflumic or meclofenamic acids.

Modified carbon paste electrodes for flow injection amperometric determination of isocitrate dehydrogenase activity in serum.

A carbon paste electrode modified with the adsorbed products of the electrochemical oxidation of adenosine triphosphate is described. The electrode was applied to the amperometric electrocatalytic detection of the reduced form of both nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate. The catalytic oxidation current shows a linear dependence on the concentration of the reduced form of nicotinamide adenine dinucleotide up to 1x10(-4)M, with a detection limit of 5x10(-9)M. Modified carbon paste electrodes were coated with an electrogenerated film of nonconducting poly(o-phenylenediamine) to obtain a stable amperometric response for at least 150h. In addition to static measurements, determination of both reduced cofactors was carried out in a flow injection analysis system with a thin-layer amperometric detection cell. The electrocatalytic monitoring of reduced nicotinamide adenine dinucleotide phosphate was applied to flow injection measurement of isocitrate dehydrogenase activity in serum. The results were in good agreement with those for the standard spectrophotometric test kit. The proposed method consumed less time and reagents and provided better precision than the standard method.

Voltammetric determination of underivatized oligonucleotides on graphite electrodes based on their oxidation products.

A new electrochemical method to determine underivatized oligonucleotides is developed. The electro-oxidation of the adenine moieties of adsorbed oligonucleotides at elevated potentials on pyrolytic graphite electrodes (PGE) in neutral or alkaline media gives rise to electroactive products strongly adsorbed on the electrode surface. The extent of the redox processes of these products, with formal potential close to 0 V (vs Ag /AgCl) at pH 10, correlates well with the amount of parent oligonucleotide. Various electrochemical techniques have been compared and applied to the detection of specific DNA sequences and synthetic homopolynucleotides. Detection limits of 2 and 10 ng for (dA)20 and a 21-mer sequence of HIV-1, respectively, have been achieved using sample volumes of 10 microL. Moreover, the adsorbed oxidized oligonucleotide shows electrocatalytic activity toward the oxidation of NADH. The capability of the new method to detect DNA hybridization is discussed.