Walter M. de Azevedo

Glucose Biosensor Using Glucose Oxidase Immobilized in Polyaniline

A biosensor for glucose utilizing kinetics of glucose oxidase (EC 1.1.3.4.) was developed. The enzyme was immobilized on polyaniline by covalent bonding, using glutaraldehyde as a bifunctional agent. The system showed a linear response up to 2.2 mM of glucose with a response time of 2.5–4.0 min. In addition, the immobilized enzyme had a higher activity between pH 6.5 and 7.5. The system retained 50% of its activity after 30 d of daily use. The optical absorption spectra of the polyaniline/glucose oxidase electrode after glucose had been added to the buffer solution showed that the absorption band around 800 nm had changed considerably when glucose was allowed to react with the electrode. This optical variation makes polyaniline a very promising polymer for use as a support in optical sensor for clinical application.

Polyvinyl alcohol-glutaraldehyde network as a support for protein immobilisation

Polyvinyl alcohol-glutaraldehyde (PVA-glut) network was synthesised in bead and disc forms and used for protein immobilisation. Xanthine oxidase, a-amylase and amyloglucosidase were covalently fixed on the beads yielding preparations with specific activities retention of 72.3%, 1.6% and 1.4%, respectively. Km of xanthine oxidase PVA-glut beads (24 ± 4 µM) was slightly higher than that estimated for the soluble enzyme (16 ± 2 mM). Antigens from Schistosoma mansoni were covalently fixed onto PVA-glut discs and ELISA was carried out. The relationship between the amount of fixed antigen and the results obtained by ELISA showed a hyperbolic curve and the saturation of antigen-antibody complex was achieved at the plateau.

Increasing glucose determination range by flow injection analysis (FIA) using glucose oxidase immobilised on polyaniline

A Flow Injection Analysis (FIA) for glucose using glucose oxidase (E.C.1.1.3.4) was developed. The enzyme was immobilised on polyaniline chemically synthetized on internal surface of silicon tube (2.0 mm of diameter and length from 1 to 5 meters), using glutaraldehyde as a bifunctional agent. The system was able to measure glucose at levels from 5 to 500mM with a response time of 2min using 250μl of sample. The system has been operated satisfactorily for one month with more than 300 assays with loss of 60% of activity.

A Conductimetric System Based on Polyaniline for Determination of Ammonia in Fertilizers

Abstract A simple conductimetric system to determine ammonia concentration using a sensor based on a conductor polymer was developed. The sensitive element to ammonia is a thin polyaniline film deposited by chemical synthesis in an acrylic substrate prepared before hand with two graphite electrodes. The conductance of the polyaniline film decreases when exposed to the ammonia gas and this variation can be related to the ammonia concentration. To determine ammonia in fertilizer samples a system consisting of a measurement cell, a conductivity meter and a strip chart recorder was used. The results were compared with those obtained by three different laboratories employing a Kjeldahl method and are in good agreement. The detection range of the system was 0.6 to 3.7 μg.mL−1 with a response time of 4 minutes. The relative standard deviation of the proposed method was about 5%.

Polyaniline–dacron composite as solid phase in enzyme linked immunosorbent assay for Yersinia pestis antibody detection

Polyaniline (PANI) was chemically synthesized on a dacron disk surface and an antigen (F1 fraction) obtained from Yersinia pestis was covalently fixed onto this composite via glutaraldehyde. The enzyme linked immunosorbent assay (ELISA) or rapid ELISA procedure detected immunoglobulin G (IgG) anti-F1 fraction in human serum employing this derivative. The appropriate conditions for carrying out the test were established as an antigen concentration of 2 microg/PANI-dacron disk, peroxidase labeled goat anti-human IgG conjugate diluted 4000 times, and a serum dilution of 1:100. The PANI-dacron disks showed greater antigen retention than conventional poly(vinyl chloride) plates and less antibody unspecific adsorption.

The use of polyvinyl alcohol glutaraldehyde antigen coated discs for laser induced fluorescence detection of plague

Abstract F1-antigen purified from Yersinia pestis was covalently linked to polyvinyl alcohol (PVA) glutaraldehyde cross-linked discs synthesized by acid catalysis. This derivative was incubated with fluorescein labeled antibody against F1-antigen and excited at 4880 A by either an argon laser or a dye laser. The fluorescence was detected at 5200 A. The appearance of the transition at 5200 A was indicative of positive complex formation, since PVA-glutaraldehyde does not fluoresce at this wavelength. The observed fluorescence was compared with samples treated with patient sera. A positive response was indicated by a decrease in the fluorescence at 5200 A. An additional series of experiments was carried out by first treating the F1-antigen-PVA-glutaraldehyde disc with patient sera, followed by fluorescein labeled goat anti-human IgG conjugate. A positive response was indicated by an increase in the observed fluorescence. Both approaches were equally capable of detecting antibodies in patient sera. However, the first technique was more rapid. When these data are compared against the standard passive hemagglutination assay presently used in Brazil, it has been found to have far greater sensitivity for the detection of plague.

Magnetic polysiloxane-polyvinyl alcohol composite as solid-phase in chemiluminescent assays

A polysiloxane and polyvinyl alcohol interpenetrating polymer network was synthesised and its ferromagnetic derivative was used as solid support for antigen covalent immobilisation in chemiluminescent assays. Only 0.625 μg of either Trypanosoma cruzi or Schistosoma mansoni antigens immobilized onto the magnetic particles (2.5 mg) were sufficient to detect the correspondent human IgG within a nanogram scale.

The use of polyvinyl alcohol glutaraldehyde as solid-phase in ELISA for plague

Discs of polyvinyl alcohol cross-linked with glutaraldehyde were synthesized under acid catalysis (H2SO4). Then, the antigen F1 purified from Yersinia pestis was covalently linked to this modified polymer. Afterwards, an enzyme-linked immunosorbent assay (ELISA) was established for the diagnosis of plague in rabbit and human. The best conditions for the method were achieved by using 1.3 micrograms of F1 prepared in 0.067 M phosphate buffer, pH 7.2, containing 1 M NaCl (PBS); anti-IgG peroxidase conjugate diluted 6,000 times and as a blocking agent 3% w/v skim milk in PBS. The titration of positive rabbit serum according to this procedure detected antibody concentrations up to 1:12,800 times. The present method, the conventional ELISA and passive haemagglutination assay are compared.

Lanthanide-Organic Gels as a Multifunctional Supramolecular Smart Platform

A multifunctional smart supramolecular platform based on a lanthanide-organic hydrogel is presented. This platform, which provides unique biocompatibility and tunable optical properties, is synthesized by a simple, fast, and reproducible eco-friendly microwave-assisted route. Photoluminescent properties enable the production of coated light-emitting diodes (LED), unique luminescent barcodes dependent on the excitation wavelength and thin-films for use in tamper seals. Moreover, piroxicam entrapped in hydrogel acts as a transdermal drug release device efficient in inhibiting edemas as compared to a commercial reference.

Polyvinyl alcohol-glutaraldehyde as solid-phase in Elisa for Schistosomiasis

Soluble adult Schistosoma mansoni antigen preparation (SWAP) was covalently fixed onto polyvinyl alcohol-glutaraldehyde discs and an enzyme linked-immunosorbent assay (ELISA) was set up. The best conditions for the assay were established and it was found that small amount of antigen such as 1.5 micrograms was required. A comparison between this procedure and the conventional ELISA was proceeded. A reliable method of antigen immobilization was achieved and the low prices of the employed reagents are economically attractive.