Annika Lindgren

Biosensors based on novel peroxidases with improved properties in direct and mediated electron transfer

Native horseradish peroxidase (HRP) on graphite has revealed approximately 50% of the active enzyme molecules to be in direct electron transfer (ET) contact with the electrode surface. Some novel plant peroxidases from tobacco, peanut and sweet potato were kinetically characterised on graphite in order to find promising candidates for biosensor applications and to understand the nature of the direct ET in the case of plant peroxidases. From measurements of the mediated and mediatorless currents of hydrogen peroxide reduction at the peroxidase-modified rotating disk electrodes (RDE), it was concluded that the fraction of enzyme molecules in direct ET varies substantially for the different plant peroxidases. It was observed that the anionic peroxidases (from sweet potato and tobacco) demonstrated a higher percentage of molecules in direct ET than the cationic ones (HRP and peanut peroxidase). The peroxidases with a high degree of glycosylation demonstrated a lower percentage of molecules in direct ET. It could, thus, be concluded that glycosylation of the peroxidases hinders direct ET and that a net negative charge on the peroxidase (low pI value) is beneficial for direct ET. Especially noticeable are the values obtained for sweet potato peroxidase (SPP), revealing both a high percentage in direct ET and a high rate constant of direct ET. The peroxidase electrodes were used for determination of hydrogen peroxide in RDE mode (mediatorless). SPP gave the lowest detection limit (40 nM) followed by HRP and peanut peroxidase.

Amperometric detection of phenols using peroxidase-modified graphite electrodes

Abstract This report describes the development of the new concept utilising various peroxidases for determination of phenolic compounds in flow injection (FI) mode. A solid graphite electrode was modified with peroxidase (EC 1.11.1.X) and used in an amperometric detection system. At the peroxidase electrode phenolic compounds can be oxidised in the presence of hydrogen peroxide (H2O2). The phenoxy radicals formed during the enzymatic oxidation can be reduced back electrochemically at −50 m V vs. Ag AgCl . This reduction current is proportional to the concentration of the phenolic compound. Different methods of coupling horseradish peroxidase (HRP, EC 1.11.1.7) were tested, including adsorption and covalent immobilisation. It was found that the best electrodes gave a detection limit for p-cresol of 0.4 μM (using 60 μM H2O2 in the flow carrier). Peroxidases of different biological origins were adsorbed to graphite electrodes and the selectivity of these electrodes was studied for eight phenolic compounds. All the peroxidase electrodes in the experiment, except the chloroperoxidase electrode, were sensitive to all the phenolic compounds tested.

Direct electron transfer catalysed by recombinant forms of horseradish peroxidase: insight into the mechanism

Abstract The paper presents the first results on recombinant horseradish peroxidase (HRP) electrochemistry obtained on graphite with a rotating disk electrode system. Recombinant HRP demonstrates a higher percentage of properly oriented molecules than the native enzyme. The first important conclusion based on the recombinant HRP electrochemistry is that glycosylation hinders direct electron transfer (ET). The single-point mutants with limited activity toward phenolic substrates, viz. Asn70Val and Asn70Asp showed no changes in the registered current upon the addition of p -cresol, catechol, p -aminophenol and guaiacol and, thus, in this particular case mediated ET was not more advantageous than direct ET. The rate constants for direct ET were comparable for all mutants tested in this study demonstrating that direct ET does not depend on the enzymes ability or inability to oxidise phenolic substrates. The results obtained in this study demonstrate the true mediatorless nature of enzyme-catalysed direct ET.

Direct electron transfer of cellobiose dehydrogenase from various biological origins at gold and graphite electrodes

Direct electron transfer was observed for cellobiose dehydrogenases (CDH) from three different fungi, viz. CDH from Phanerochaete chrysosporium, Sclerotium rolfsii and Humicola insolens, in the presence of cellobiose when the enzymes were adsorbed on graphite electrodes. The redox wave of the heme cofactor of CDH could be demonstrated on thiol modified gold electrodes using cyclic voltammetry for Phanerochaete CDH and Humicola CDH; however, the electrocatalytic current for cellobiose oxidation could only be registered for Phanerochaete CDH.

Comparison of rotating disk and wall-jet electrode systems for studying the kinetics of direct and mediated electron transfer for horseradish peroxidase on a graphite electrode

Abstract This paper presents a comparison between the results obtained for the kinetics of direct and mediated electroreduction of hydrogen peroxide at horseradish peroxidase (HRP) modified graphite electrodes, used either as a rotating disk electrode (RDE) or as the working electrode in a wall-jet flow through cell. The advantages of using a wall-jet flow system compared with a RDE system for kinetic investigations are that there is no need to account for substrate consumption, especially in the case of desorption of enzyme, and also when studying product-inhibited enzymes. The comparison reveals that identical results can be obtained using either technique. The ratio of HRP molecules on the graphite surface in direct electron transfer contact was determined as 48±4%, and the turnover number of the heterogeneous electron transfer between adsorbed HRP and the graphite electrode was determined as 1.9±0.3 s −1 .

Direct heterogeneous electron transfer of recombinant horseradish peroxidases on gold

Clean polycrystalline gold electrodes were modified with native glycosylated horseradish peroxidases (HRP) or two different recombinant (carbohydrate free) HRPs; recombinant wild-type HRP (rec-HRP) and recombinant HRP containing a six histidine-tag at the C-terminus of the polypeptide chain (rec-HRP-His), respectively. Only the electrodes modified with the recombinant HRPs exhibited high current responses to H2O2 due to relatively rapid direct electron transfer (ET) between recombinant HRP and gold. The absence of a carbohydrate shell on rec-HRP and the additionally existing histidine-tag on rec-HRP-His improved the electrode sensitivity to H2O2 by more than 100 times if compared with the response observed at gold modified with native HRP. Rotating disk electrode experiments indicated that the heterogeneous electron transfer rates are equal to 4.7 and 7.5 s-1 for direct electron transfer between the gold electrode and rec-HRP or rec-HRP-His, respectively.

Bioelectrochemical characterisation of cellobiose dehydrogenase modified graphite electrodes: ionic strength and pH dependences

Abstract The bioelectrocatalysis of cellobiose and lactose was studied at graphite electrodes modified with adsorbed cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium . At a low concentration of substrate, there was an optimum in the bioelectrocatalytic current around pH 4.7, whereas at high substrate concentrations, the catalytic current was decreasing continuously with increasing pH. An increase of the ionic strength of the buffer solution by the addition of 80 mM of NaCl resulted in a 50% increase of the catalytic current. Slow sweep cyclic voltammograms were used to extract the formal potential of the heme domain of the enzyme in the presence of the substrate, cellobiose, at different pH values. The formal potential of the heme decreased with increasing pH. Based on the results in this work, it was confirmed that there was a direct, non-mediated, electron transfer between the heme of CDH and the graphite electrode. Experimental results revealed that the enzyme was bound strongly to the surface of the graphite electrode and that this graphite–enzyme interaction did not result in denatured or a strongly changed structure of the heme domain of CDH on the electrode surface. The mechanism of the bioelectrocatalysis at graphite electrodes modified with CDH is discussed, focusing on the rate limiting steps of the electrocatalytic process.

Development of a cellobiose dehydrogenase modified electrode for amperometric detection of diphenols

A new amperometric biosensor based on cellobiose dehydrogenase (CDH) was created for the detection of ortho- and para-diphenolic compounds. The developed electrode efficiently discriminates between diphenolic and monophenolic compounds. The analyte, a diphenolic compound, is oxidised on the surface of a graphite electrode at an applied potential of +300 mV vs. Ag/AgCl. The diphenol is then regenerated by the adsorbed CDH in the presence of cellobiose, thus allowing an amplified response signal. Different parameters of the CDH–electrode system were optimised, e.g., applied potential, immobilisation time, flow rate, substrate concentration and storage conditions. Using the optimised parameters the sensitivity and detection limits for various diphenolic compounds were evaluated, resulting in detection limits below 5 nM for most of the compounds tested. The highest sensitivity recorded was obtained for dopamine, 3.6 A l mol–1 cm–2. The operational stability of the electrodes was high: during 2 h of continuous operation only a 1–2% decrease in response signal was observed.

Flow injection analysis of phenolic compounds with carbon paste electrodes modified with tyrosinase purchased from different companies

Abstract Tyrosinase-modified carbon paste electrodes were prepared using lyophilised powder of the enzyme purchased from different companies. The selectivity of these electrodes for nine phenolic compounds, including six substituted catechols, has been studied. The signals obtained for catechol were always higher than those found for other phenolic compounds. Cyclic voltammetry and flow injection measurements indicated that the response of the tyrosinase-modified carbon paste electrodes was limited by the rate of the enzymatic oxidation of catechols. Different approaches of paste electrode preparation have been studied and compared. Direct mixing of enzyme into the graphite powder doped with the osmium based mediator, resulted in the highest sensitivity for the studied substrates. However, substrate selectivity was found to be dependent on the source of enzyme used for electrode preparation.

Optimisation of a heterogeneous non-competitive flow immunoassay comparing fluorescein, peroxidase and alkaline phosphatase as labels

Off- and on-line strategies for a non-competitive heterogeneous flow immunoassay were developed comparing three different labels. The samples, containing the model compounds digoxin or digoxigenin, were either pre-incubated off-line or on-line in a mixing coil with excess of labelled anti-digoxigenin Fab-fragments. The excess of Fab-fragments was then separated from the digoxin bound Fab-fragments by passing the sample through a column with immobilised digoxin. The off-line immunochemical detection system is suitable for sensitive high through-put screening of the analytes, whereas the on-line system is more suitable for coupling as a post-column detection unit to liquid chromatography. The digoxin and digoxigenin content in the sample were quantified using fluorescein (F) and enzyme (peroxidase (POD), alkaline phosphatase (AP)) labelled Fab-fragments. The fluorescein label was directly measured with the fluorescence detector, whereas a fluorescent enzyme product was measured in the two enzyme based systems, using 3-(p-hydroxyphenyl)-propionic acid (HPPA) and hydrogen peroxide for POD and, and 4-methylumbelliferyl phosphate (4-MUP) for AP. The highest sensitivity and lowest limit of detection (LOD) was obtained with the Fab-POD system with LODs for digoxin and digoxigenin in the off- and on-line configurations of 0.025 and 0.01 nM, respectively. The sample through-put for the off- and on-line systems were 43 and 32 samples per hour, respectively.