Stjepan Milardović

Glucose determination in blood samples using flow injection analysis and an amperometric biosensor based on glucose oxidase immobilized on hexacyanoferrate modified nickel electrode

Abstract A very simple amperometric enzyme-based glucose sensor is applied as detector in flow injection analysis (FIA) of diabetic patients blood glucose. The enzyme glucose oxidase (GOD) is immobilized by a glutaraldehyde/bovine serum albumin crosslinking procedure on the surface of an alkali nickel hexacyanoferrate thin film electrocrystallized on the nickel electrode. An alkali nickel hexacyanoferrate modified nickel electrode catalyzes the reduction of the hydrogen peroxide, formed by selective biocatalytic GOD action on glucose oxidation by oxygen present in sample solutions. The developed glucose sensor has shown good sensitivity at low negative operating potentials ( −300 to −100 mV vs. Ag AgCl ), sufficiently wide linear glucose concentration range (5 μM–2.9 mM), relatively long operating time (during three months of operation the initial sensitivity decreased only 30%) and no influence of the most common interferences present in human blood like proteins, ascorbic and uric acids.

Rapid Determination of Oxalate by an Amperometric Oxalate Oxidase‐Based Electrode

An enzymatic biosensor for determination of oxalate in different real food samples (spinach, sesame seed, tea leaves, and strawberries) as well as in human urine is described and evaluated. Amperometric oxalate biosensor is made from graphite (spetroscpic purity) electrode modified with chromium (III) heksacyanoferrate (II) film (CrHCF). Oxalate oxidase was immobilized using bovine serum albumin and glutaraldehyde cross-linking procedure. This enzyme biocatalyses the oxidation of oxalate to hydrogen peroxide and carbon dioxide. CrHCF film enables one to measure a current resulting in the reduction in the reduction of hydrogen peroxide at low potential (0 V vs. Hg/Hg_2Cl_2/3M KCl electrode), proportional to oxalate concentration. The electrocatalytic properties of CrHCF electrode were well pronounced in 0.05 M succinic buffer, pH 3.8, containing 0.1 M KCl and 5.4 mM EDTA. For determination of oxalate in some food matrices (tea leaves and strawberries) dialysis membrane as an outer coat was necessary to prevent electrochemical interferents. The sensitivity of biosensor was 31microA/(mM cm^2) in synthetic oxalic acid solutions. The biosensor has shown good linearity (R^2=0.9984) in the oxalate concentration range between 2.5 and 400 microM and its half-lifetime was longer than one month. The performance of the metod was compared with the reference enzymatic spectrophotometric method and a very good correlation was obtained between these results.

A novel method for flow injection analysis of total antioxidant capacity using enzymatically produced ABTS+ and biamperometric detector containing interdigitated electrode

Application of interdigitated array microelectrodes as electrochemical sensors for determination of antioxidant capacity is reported. Electrochemical measurements with interdigitated electrodes (IDE) were studied in both stationary solutions and the flow system. The method is based on biamperometric measurements using ABTS(*+)|ABTS redox couple in phosphate buffer solution, pH 7.40. During analysis, the ABTS radical cation was enzymatically produced by peroxidase in a tubular flow-through reactor. The performance of bioreactor was tested at different concentrations of immobilized enzyme, ABTS and hydrogen peroxide. The influence of flow rate on proper operation of the bioreactor was also studied. The results of antioxidant activity were determined using Trolox as a standard. The applied IDE detector accomplished good sensitivity of 0.3nA/microM of Trolox and offered linear range between 20 to 500microM of Trolox. The comparison of results (R(2)=0.9915) for antioxidant activity between spectroscopic and FIA biamperometric measurements by interdigitated electrodes confirmed the applicability of the proposed method for determination of antioxidant capacity.

A novel biamperometric biosensor for urinary oxalate determination using flow-injection analysis

A biosensor for determination of oxalate concentration in urine has been developed by immobilisation of oxalate oxidase and peroxidase on the surface of an interdigitated gold electrode. Enzyme immobilisation was performed using BSA and glutaraldehyde. Biamperometric measurements were made in flow conditions both in aqueous oxalate solutions (tested concentration range between 50 microM and 10 mM) and in real urine samples (tested measuring range between 5 and 100 microM). Optimal working conditions were examined for flow-injection analysis, and good correlation was achieved between added oxalate quantity and the one measured by biosensor in urine matrix (R(2)=0.9983). The influence of some interferences (ascorbic acid, uric acid, paracetamol, acetylsalicylic acid) was also studied using biamperometric measurement mode.

Chromogenic radical based optical sensor membrane for screening of antioxidant activity

Solid-state optical sensor membranes based on immobilised chromogenic radicals for the assessment of antioxidant activity have been studied. Two stable lipophilic chromogenic radicals, DPPH (2,2-diphenyl-1-picrylhydrazyl radical) and galvinoxyl radical, GV, (2,6-di-tert-butyl-alpha-(3,5-di-tert-butyl-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxy radical), were immobilised in plasticised PVC films and screened for suitability as indicators of antioxidative activity. The spectrophotometric characterisation of the polymer films containing immobilised free radicals was performed, and the response of the immobilised free radicals toward standard antioxidants was studied. It has been demonstrated that the immobilised radicals retain their reactivity towards antioxidants and the results suggest that the reactivity of immobilised radicals is comparable to standard solution-based DPPH assays. Polymer films containing immobilised DPPH radical respond to standard antioxidants in aqueous solutions by changing colour irreversibly from purple (absorption maximum at 520nm) to yellow. The initial slopes of the response curves to the phenolic antioxidant gallic acid, obtained in the 1-50mM concentration range, gave a linear calibration plot in a 1min exposure cuvette test. The polymer films were used to screen antioxidative activity of beverage and food samples known to contain antioxidants, such as black and green tea, coffee, red wine, fruit juice, olive oil and sunflower oil. It has been demonstrated that a rapid and simple qualitative screening test of untreated samples is possible using a test strip based on immobilised DPPH radical.

The study of some possible measurement errors in clinical blood electrolyte potentiometric (ISE) analysers

The understanding of the most important sources of error in potentiometric blood analyser which might contribute to better instruments measurement repeatability is very often marginalized in fabrications and daily operation of some commercial blood analysers. In this paper ISEs-potentiometric measurements were performed and validated in Clinical Institute of Laboratory Diagnosis of the Zagreb University School of Medicine and Clinical Hospital Centre, using a carefully designed and constructed fully automated (computerised) homemade ISE-based blood electrolyte analyser constructed with an in-line five-channel flow-through measuring cell. The influence of electrolyte concentration of the salt bridge is reported. Special attention has been paid to the reference electrode design, and constructions which can operate in open liquid junction and membrane restricted liquid junction modes are described.