Electrochemical immunosensor detection of antigliadin antibodies from real human serum.

The determination of antigliadin antibodies from human serum samples is of vital importance for the diagnosis of an autoimmune disease such as celiac disease. An electrochemical immunosensor that mimics traditional ELISA type architecture has been constructed for the detection of antigliadin antibodies with control over the orientation and packing of gliadin antigen molecules on the surface of gold electrodes. The orientation of the antigen on the surface has been achieved using a carboxylic-ended bipodal alkanethiol that is covalently linked with amino groups of the antigen protein. The bipodal thiol presents a long poly(ethyleneglycol)-modified chain that acts as an excellent non-specific adsorption barrier. The bipodal nature of the thiol ensured a good spacing and hence good diffusion properties of electroactive species through the self-assembled monolayer, which is vital for the efficiency of the constructed electrochemical immunosensor. The electrochemical immunosensor was characterized using surface plasmon resonance as well as electrochemical impedance spectroscopy. Amperometric evaluation of the sensor with polyclonal antigliadin antibodies showed stable and reproducible low limits of detection (46 ng/mL; % RSD = 8.2, n = 5). The behaviour and performance of the electrochemical immunosensor with more complex matrixes such as reference serum solutions and real patient samples was evaluated and compared with commercial ELISA kits demonstrating an excellent degree of correlation in thirty minutes total assay time; the electrochemical immunosensor not only delivers a positive or negative result, it allows the estimation of semi-quantitative antibody contents based on the comparison against clinical reference solutions.

Copper UPD as non-specific adsorption barrier in electrochemical displacement immunosensors.

Non-specific adsorption events are responsible to a large extent for the lack of reliability and applicability of electrochemical immunosensors. In the particular case of displacement-based immunosensors, as an approach to achieve reagentless, labelless and easy to use immunosensors, the hindering effect of then non-specific adsorption is amplified when the system presents a low affinity constant between biorecognition element and target. The application of Copper UPD as non-specific adsorption barrier in combination with the use of self-assembled monolayers (SAM) to provide efficient binding of biomolecules to the immunosensor electrode surface is shown to be a very promising mechanism to construct protein resistant surfaces with no harming effects on the electrochemical transducing mechanism. The electrochemical immunodetection of TCA (2,4,6-Trichloroanisole) has been chosen as example for a real case study. A monoclonal antibody to detect the target TCA and an appropriate sub-optimum antigen were used. In addition to a rational strategy for displacement immunosensor development, the decrease of non-specific adsorption phenomena by introducing Copper UPD is reported here. With such strategy an electrochemical displacement immunosensor with a limit of detection of 200ppb and response time of 10min is achieved.

Towards a fast-responding, label-free electrochemical DNA biosensor.

DNA sensors and sensor arrays (biochips) have become an important tool in molecular biology and biotechnology in recent years. For low-throughput, easy-to-use devices it is desirable that they be of low cost, reagentless, and label-free. Displacement sensors with electrochemical detection offer these advantages, and therefore the development of such a detection principle is show in this work. An HRP-labeled oligonucleotide was sub-optimally pre-hybridized with a capture probe and was displaced upon introduction of the fully complementary probe target, producing a decrease in signal that was proportional to the sample concentration. This detection scheme has been demonstrated colorimetrically and electrochemically, obtaining a total signal displacement of 55% only 5 min after introduction of the sample.

Reagentless biosensors based on self-deposited redox polyelectrolyte-oxidoreductases architectures.

Reagentless fructose and alcohol biosensors have been produced with a versatile enzyme immobilisation technique which mimics natural interactions and flexibility of living systems. The electrode architecture is built up on electrostatic interactions by the sequential adsorption of redox polyelectrolytes and redox enzymes giving rise to the efficient transformation of substrate fluxes into electrocatalytic currents. All investigated multilayer structures were self-deposited on 3-mercapto-1-propanesulfonic acid monolayers self-assembled on gold electrodes. Fructose dehydrogenase, horseradish peroxidase (HRP) and the couple HRP-alcohol oxidase were electrochemically connected with a cationic poly[(vinylpyridine)Os(bpy)2Cl] redox polymer (RP) interface in a layer-by-layer self-deposited architecture. The dependence of the distance on the electrochemical response of this interface was also studied showing a clear decrease in the Faradaic current when the distance to the electrode surface was increased. The sensitivities obtained for each biosensor were 19.3, 58.1 and 10.6 mA M(-1) cm(-1) for fructose, H2O2 and methanol, respectively. The sensitivity values can be easily controlled by a rational deposition and manipulation of the charge in the catalytic layers. The electrostatic assembly of the electrochemical interface and the catalytic layers resulted in integrated biochemical systems in which mass transfer diffusion and heterogeneous catalytic and electron transfer steps are efficiently coupled and can be easily manipulated.

Electrochemical immunosensor for the detection of atrazine.

An amperometric immunosensor for the detection of the herbicide atrazine has been developed. A redox polymer PVPOs(bpy)2Cl was co-immobilized with the specific antibody on the surface of the electrode by crosslinking with PEGDGE to form an electron-conducting hydrogel. In a competitive assay the occurrence of the antibody-antigen reaction on the surface of the sensing film was detected through the 'electrical wiring' of the redox centres of antigen-labelled horseradish peroxidase and the electrode surface in the presence of H2O2 at 0.1 V (vsAg/AgCl).

A new type of hydrophilic carbon paste electrodes for biosensor manufacturing: binder paste electrodes.

The effect of two types of carbon pastes and two osmium-based redox mediators on the response of amperometric enzyme electrodes for glucose was examined. A hydrophobic mediator and a hydrophilic cationic mediator were prepared and mixed in a paste that contained either mineral oil as the pasting liquid, or a polycationic electrolyte without oil. It was found that the current densities were increased by a factor of 25 when the oil-based paste was replaced by the hydrophilic one (binder paste, BP) and five- to six-fold when the hydrophilic mediator was used in place of the hydrophobic. The linear range for the glucose oxidase electrodes was extended to concentrations higher than 60 mM. The glucose electrodes were preliminary optimized and their half-life time reached more than 12 h when operated continuously under vigorous stirring when the 'pasting' polyelectrolyte was crosslinked. At a working potential of 400 mV versus the Ag/AgCl saturated electrode, the saturating current densities per geometric surface area were 1.2 mA/cm2 +/- 0.2 (n = 7). These 'binder paste electrodes' are the first reported bulk modified electrodes without hydrophobic pasting liquid or cover membranes, and present an interesting research and application tool.

Electron transfer via redox hydrogels between electrodes and enzymes.

The molecular "wiring" of redox enzymes provides the basis for amperometric enzyme and affinity sensors. The key features of the useful redox polymers are hydrophilicity and flexibility.

Preliminary investigations of an amperometric oligosaccharide dehydrogenase-based electrode for the detection of glucose and some other low molecular weight saccharides.

Biosensors for the determination of sugars were constructed using oligosaccharide dehydrogenase (ODH) and soluble phenazine methosulfate (PMS) or an osmium-based three-dimensional redox hydrogel. In the latter case the enzyme and poly(1-vinylimidazole) complexed with osmium (4,4'-dimethylbpy)2Cl were cross-linked with poly(ethylene glycol) diglycidyl ether. Both electrode configurations showed similar sensitivities for glucose in the range between 8 and 21 muAmM-1 cm-2. The responses for 10 mono and oligosaccharides were studied. There was no response for fructose. In the concentration range 0.1-20 mM the relative sensitivities were determined for arabinose (96%), xylose (3%), mannose (50%), galactose (11%), glucose (100%), maltose (24%), lactose (12%), cellobiose (34%) and maltotriose (10%).

Design, characterization, and one-point in vivo calibration of a subcutaneously implanted glucose electrode.

A 0.29-mm-diameter flexible electrode designed for subcutaneous in vivo amperometric monitoring of glucose is described. The electrode was designed to allow "one-point" in vivo calibration, i.e., to have zero output current at zero glucose concentration, even in the presence of other electroreactive species of serum or blood. A valid zero point, along with a measurement of the glucose concentration in a withdrawn sample of blood at which the current is known, defined the sensitivity in the linear response range. The electrode was four-layered, with the layers serially deposited within a 0.125-mm recess upon the tip of a polyimide-insulated 0.25-mm gold wire. The recessed structure reduced the sensitivity to movement and allowed, through control of the depth of the recess, control of the transport of glucose and thus the range of linearity. The recess contained the four polymeric layers, with a total mass less than 5 micrograms and no leachable components. The bottom glucose concentration-to-current transducing layer consisted of the enzyme "wiring" redox polymer poly[(vinylimidazole)Os(bipyridine)2Cl]+ , complexed with recombinant glucose oxidase and cross-linked with poly(ethylene glycol) diglycidyl ether, to form an electron-conducting hydrogel. The layer was overcoated with an electrically insulating layer of polyaziridine-cross-linked poly(allylamine), on which an immobilized interference-eliminating horseradish peroxidase based film was deposited. An outer biocompatible layer was formed by photo-cross-linking derivatized poly(ethylene oxide). The current output of a typical electrode at 10 mM glucose and at 37 degrees C was 35 nA, the apparent Km was 20 mM, and the 10-90% response time was approximately 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)

L-alpha-glycerophosphate and L-lactate electrodes based on the electrochemical "wiring" of oxidases.

The title electrodes were constructed by coimmobilizing the respective FAD oxidases on solid electrode surfaces with a poly(vinyl pyridine) polymer which was N-derivatized with bromoethylamine and Os(bpy)2Cl2. The redox-polymer-enzyme hydrogels were cross-linked on the electrode surface using poly(ethylene glycol) diglycidyl ether. As in the case of glucose oxidase, the redox polymer acts as an electron relaying "wire" transferring electrons directly from the enzymes' FADH2 centers to the electrode. This transfer competes with the natural process of reoxidation of FADH2 by molecular oxygen. The variation of the response of these electrodes with the atmosphere (N2 or air), pH, and substrate concentration was determined. The pH profile of the electrocatalytic current differs from that of the activity of the free enzymes, exhibiting a broader maximum, shifted to higher pH values. The observed sensitivities and linear ranges are respectively 2 x 10(-2) A M-1 cm-2 and 2.7 mM for L-alpha-glycerophosphate, and 0.3 A M-1 cm-2 and 0.2 mM for L-lactate that may be compared to 2 x 10(-2) A M-1 cm-2 and 10 mM for glucose. The 0-90% response time for all electrodes is 1 s or less.