Amperometric determination of lysine using a lysine oxidase biosensor based on rigid-conducting composites.

In this study, amperometric biosensors based on rigid conducting composites are developed for the determination of lysine. These lysine biosensors consist of chemically immobilized lysine oxidase membranes attached to either graphite-methacrylate or peroxidase-modified graphite-methacrylate electrodes. The enzymatic degradation of lysine releases hydrogen peroxide, which is the basis of the amperometric detection. The direct oxidation of hydrogen peroxide is monitored at +1000 mV with a graphite-methacrylate electrode, while with the peroxidase-modified electrode reductive detection is performed. In addition, for the peroxidase-modified biocomposite electrode, both direct electron transfer and hydroquinone-mediated detection are studied. For the lysine biosensor based on the hydroquinone-mediated peroxidase biocomposite, the linear range is up to 1.6 x 10(-4) M, the sensitivity 11300 microA/M, the repeatability 1.8%, the detection limit 8.2 x 10(-7) M and the response time t95% is 42 s. The proposed biosensors are used to determine lysine in pharmaceutical samples. Results are consistent with those obtained with the standard method.

Determination of lysine in pharmaceutical samples containing endogenous ammonium ions by using a lysine oxidase biosensor based on an all-solid-state potentiometric ammonium electrode.

A new potentiometric method is proposed to determine lysine in pharmaceutical samples. This method is based on a lysine biosensor consisting of a chemically immobilized lysine oxidase membrane attached to an all-solid-state ammonium electrode. Lysine is degraded in the sensor to release ammonium, which is detected by means of the ammonium electrode. The presence of endogenous ammonium in the samples interferes with these determinations, since the response measured corresponds to the sum of the ammonium generated enzymatically and that present in the sample. This is a general drawback for all biosensors based on the detection of ammonium. Study of samples containing both lysine and ammonium showed that concentration ranges exist in which a near-logarithmic relationship between potentials measured and lysine concentrations is found. Therefore, within these ranges, lysine can be determined by using the standard addition method, with the subsequent data treatment involving an iterative linearization procedure. Results obtained with the proposed potentiometric method are consistent with those given by the standard method for amino acid analysis.

Development of a biparametric bioanalyser for creatinine and urea. Validation of the determination of biochemical parameters associated with hemodialysis.

The construction and evaluation of an automated urea and creatinine biparametric biosystem using flow injection analysis (FIA) are described. The biosystem uses enzyme reactions that hydrolyse urea and creatinine producing ammonium ions. The enzymes used were creatinine deiminase and urease, which are immobilized covalently in flow reactors. The reactor with creatinine deiminase has the enzyme immobilized on controlled-pore glass beads, whereas urease is immobilized on a nylon open tubular reactor. Detection is realised with a flow-through ammonium ion-selective electrode with an inner solid-state contact (graphite-epoxy composite). Ammonium ions are separated from alkali ion interferents through a gas-diffusion cell. The bioanalyser is fully automated using software and electronics developed ex profeso in our laboratories. The analyser was validated off-line by measuring urea and creatinine from discrete effluent samples from hemodialysis equipment. Results agreed with concurrent analyses realised using hospital laboratory methods. There were no significant differences between the two sets of results at the 95% confidence level. Finally, the biparametric bioanalyser was validated on-line by measuring creatinine and urea levels in artificial kidney effluents. These measurements were useful in the determination of key biochemical parameters of clinical interest such as the mass of urea and creatinine extracted from the patient as well as the initial concentration of creatinine and urea in blood plasma. When the results of the bioanalyser were compared with those yielded by the usual methods, they showed no significant differences at the 95% confidence level when determining the mass of the analytes extracted by the hemodialyser or when determining the urea concentration in blood plasma. However, when measuring the creatinine concentration in blood plasma using the developed bioanalyser, significant differences appeared.

Flow injection immunoanalysis based on a magnetoimmunosensor system.

A new immunosensor integrated to a flow system has been developed. It is based on magnetic immunoparticles immobilized on a solid-state transducer using a magnetic field. The described technique renews the immunoparticles reproducibly for each analysis allowing a good measurement precision. The developed experimental approach permits the implementation of an automated immunoassay that is quick (analytical cycle < 30 min) and sensitive in the micromolar concentration range. The system was applied to the determination of rabbit immunoglobulin G as an analyte model.

Development of electrochemical immunosensing systems with renewable surfaces.

The repeated use of immunochemically modified solid phases in electrochemical immunosensor analysis is the driving interest of this work. Two new strategies have been developed. One of these strategies is aimed at the development of a manual methodology. It comprises the construction of amperometric immunosensors based on rigid biocomposites. These biocomposites are formed by a conducting polymer composite matrix that acts as a reservoir of an immobilized immunologic material. The surface of the biocomposite can be renewed by a simple polishing procedure. The second strategy involves the design of an automatic methodology. It features an immunochemical analytical system using flow injection techniques. The potentiometric detection uses a solid phase formed by immunologic reagents immobilized in magnetic particles. These particles are fixed to the sensor with the use of a magnetic field. The renewal of the reactive surface is achieved by the release and activation of the restraining magnetic field and the manipulation of the flow. The analytical properties of these immunosensors were evaluated measuring RIgG using a competitive technique and measuring GaRIgG with a sandwich methodology. The labelling enzymes of the immunoconjugates were peroxidase in amperometric measurements and urease in potentiometric measurements.

Amperometric immunosensors based on rigid conducting immunocomposites.

Novel polishable immunosensors based on rigid biocomposite materials have been constructed. These biocomposites contain graphite powder, rabbit IgG, and methacrylate or epoxy resins. This material acts as a reservoir for the biological molecules and as a transducer at the same time. In order to study the potential analytical properties of this new type of material, a competitive binding assay was developed to determine the RIgG present in a sample with the aid of goat anti-rabbit IgG labeled with alkaline phosphatase. Using phenyl phosphate as a substrate, the phenol produced by the enzymatic reaction was amperometrically detected at 800 mV (vs Ag/AgC1). The surface of the immunosensor can be regenerated by simply polishing, obtaining fresh immunocomposite ready to be used in a new competitive assay.

Validation of an automatic urea analyser used in the continuous monitoring of hemodialysis parameters.

The validation of an automatic urea analyser used in the monitoring of hemodialysis processes is reported. The analyser can indirectly determine dialysis parameters as dialysis delivery (KT/V) and protein catabolism (PCRn). These parameters are useful for the prescription and optimization of hemodialysis. The analyser, based on a previously-reported flow-injection analytical biosystem, was connected on-line to the effluent of a dialysis machine during several hemodialysis sessions. The urea concentration data were continuously processed and dialysis parameters were obtained in quasi real time by means of the integration of an adjusted time-dependent exponential function. These values were compared with those obtained by applying the methods traditionally employed in hospital laboratories. The evaluation comprised 24 data sets from several patients of different gender and age. No significant differences were found between the KT/V and PCRn results obtained with the usual method and those results produced by the analyser proposed here.

On-line monitoring of urea in effluent liquid during haemodialysis.

An analytical system specially built for on-line urea monitoring is reported. Measurements are carried out in the effluent of a haemodialysis machine. The measuring system employs the dialyser inflow stream as a carrier solution channel in a continuous fashion. The analyser periodically samples the outflow stream of the dialyser by means of an automatic injection valve. The analyser features a bioreactor consisting of immobilized urease and a gas-diffusion module. It is through this module that the urea is converted to ammonia gas which is transferred to another carrier channel, this transports the ammonium ion to a tubular, all-solid-state, ion-sensitive electrode. A timer controls the transport, injection, the measuring and the recording subsystems. The analyser has been used during actual haemodialysis sessions. Urea clearances were also measured in batch, using conventional spectrophotometric clinical equipment. The correlation between both methodologies was sufficient to confirm the usefulness of the developed on-line analyser to monitor the optimal length of haemodialysis sessions.

Covalent binding of urease on ammonium-selective potentiometric membranes.

As part of the development of disposable urea bioselective probes, the covalent binding of urease on ammonium-selective potentiometric membranes has been assessed. Nonactin/bis(1-butylpentyl)adipate/poly(vinylchloride) (PVC) membranes, directly applied to an internal solid contact (conductive epoxy-graphite composite), has been used as a support for covalent immobilization of urease. Two types of all-solid-state construction process have been assayed: thin layers of cellulose acetate (CA) were coated on the PVC ammonium-selective membranes (type 1) and blends of PVC and CA at various ratios were used as ammonium-selective membrane matrices (type 2). Urease was covalently attached to CA via aldehyde groups. These groups were created on the polysaccharide with sodium periodate to which the enzyme was immobilized through a spacer (hexamethylenediamine). The viability of both types of probe for the determination of ammonium ions was assessed after each step of the activation process. Results indicated that type 2 potentiometric probes are altered after the treatment with sodium periodate. Good results were obtained with type 1 probes. Their dynamic concentration range of response to urea was from 2 x 10(-5) to 0.01 M with a sensibility of 50 mV/decade.

Chloride determination in serum by a flow-injection analysis precipitation pseudo-titration technique using a flow-through all-solid-state silver electrode.

A simply constructed, tubular, all-solid-state, flow-through silver electrode for flow-injection analysis (F.I.A.) is described. Use of a single line manifold accommodating the silver electrode, with a low level of silver ion (5 x 10(-4) M) in the carrier stream, is a useful method to determine chloride in serum, by means of a precipitation pseudo-titration F.I.A. technique. The sampling frequency is about 60/h.