Emmanuel I. Iwuoha

Organic phase enzyme electrodes : kinetics and analytical applications

Abstract The suitability of organic media for enzymatic reactions has led to tremendous advancement in biosynthesis and, more recently, biosensing. Organic phase enzyme-based biosensors (OPEE), i.e. biosensors that operate in organic media, combine the high selectivity and specificity of enzymes with the ability to control their reactivity by altering some physiochemical properties of the reaction media, such as solvent polarity and degree of hydration. Classical enzymology predicts that hydrophobic organic solvents, rather than hydrophilic solvents, are suitable for enzyme activity, on account of the latters ability to strip the enzyme of essential water of hydration necessary for the flexibility and polarity of the active site micro-environment. We have demonstrated with solvents, such as acetonitrile, acetone, butan-2-ol, chloroform, hexane and tetrahydrofuran, that organic-phase amperometric biosensors can be developed to exhibit excellent reactivities in both hydrophilic and hydrophobic organic solvents by controlling the degree of hydration of the biosensing environment. Amperometric biosensors containing tyrosinase, glucose oxidase (GOx), horseradish peroxidase (HRP), and more recently chemically modified HRP, have been constructed and successfully applied to assays in organic solvents. Several enzyme immobilization matrices have been used in our laboratory for the fabrication of sensors. Poly(estersulphonic acid)-entrapped tyrosinase and HRP electrodes have been found to be very effective biosensors in a variety of organic solvents for determining phenols, organic peroxides and pesticide compounds. The Os-polymers, [Os(bpy)2(PVP)nCl]Cl [bpy = 2,2′-bipyridyl, PVP = poly(4-vinylpyridine); n = 10, 20 or 25] and [Os(byp)2(PVI)10Cl]Cl [PVI = poly(4-vinylimidazole)] have been used with bifunctional cross-linking agents, such as glutaraldehyde or polyethylene glycol, to form reagentless biosensors with GOx, tyrosinase and HRP (native and modified forms of HRP). These biosensors, based on the electrostatic complexation of the enzyme and the positively charged Os-polymer, have been found to be very stable in organic phase. GOx sensors that employ soluble mediators, such as, ferrocenemonocarboxylic acid, exhibited better catalytic efficiency, k cat ′ K M ′ in the organic phase than in the aqueous phase. In our studies we have demonstrated that the reactivities of amperometric organic phase biosensors follow the Michaelis-Menten kinetic paradigm. However, kinetic analyses have shown that the values of the sensor biocatalytic parameters, including Imax, kcat′ and KM′ depend on solvent media. Under certain conditions, k cat ′ K M ′ is the second order rate constant through ΔG=/ −RT ln [ k cat ′ K M ′ )hlkBT], where T, h, and kB are the absolute temperature, Plancks constant and Boltzmanns constant, respectively. Thus, k cat ′ K M ′ is a measure of the activation of the biosensor redox-catalytic reaction in different solvents. This parameter has been evaluated at different temperatures in both aqueous and organic media. The ΔG=/ values (the difference in Gibbs energy between transition and ground state of sensor reaction for a given solvent and substrate) in organic and aqueous media have been used to evaluate the role of the solvent on the stabilization of the transition state on the electrocatalytic reaction. Organic phase HRP biosensors have also been applied in our laboratory for the determination of thiourea, ethylenethiourea and other thio-compounds (which are the parent compounds for some pesticides). The thio-compounds act as inhibitors. Their determination is based on the change in the electrocatalytic current of peroxide reduction by the biosensor, which accompanies the addition of the organic sulphides. Tyrosinase electrodes have been applied as detectors in reversed-phase HPLC for the detection of the eight phenols that are normally found in cigarette filters. Native HRP and bis-succinimide-modified-HRP electrodes have also been applied in the amperometric determination of organic peroxides, the aim being to produce a more sensitive, reproducible and stable organic phase HRP electrode.

Drug metabolism biosensors: electrochemical reactivities of cytochrome P450cam immobilised in synthetic vesicular systems.

Biosensors containing cytochrome P450cam in a didodecyldimethylammonium bromide vesicular system were prepared by cross-linking onto a glassy carbon electrode (GCE) with glutaraldehyde in the presence of bovine serum albumin. Cyclic voltammetric responses of the sensor in air-free buffer solution showed that the sensor exhibited reversible electrochemistry due to direct electron exchange between the haem Fe(3+/2+) redox system and the GCE surface. In air-saturated solution containing camphor, the biosensor gave an irreversible electrocatalytic current which is compatible with the monooxygenation of the substrate. Steady state amperometric experiments with camphor, adamantanone and fenchone were performed with a biosensor prepared by cross-linking P450cam with glutaraldehyde onto a Pt disc electrode. The sensor was characterised by fast amperometric responses, attaining steady-state in about 20 s in a cobalt sepulchrate mediated electrochemical system. The kinetic parameters of the biosensor were analysed using the electrochemical Michaelis Menten equation. The estimated apparent Michaelis-Menten constant, Km, values for the biosensors were in the range of 1.41-3.9 mM.

Development of an “electrically wired” amperometric immunosensor for the determination of biotin based on a non-diffusional redox osmium polymer film containing an antibody to the enzyme label horseradish peroxidase

Abstract An amperometric immunosensor was developed based on a non-diffusional redox polymer used for transferring electrons between the electrode surface and the antigen (horseradish peroxidase; HRP) bound to the anti-HRP antibody on the sensing surface. The redox polymer [Os(bpy)2(PVP)10Cl]Cl (bpy = bipyridyl, PVP = poly-4-vinylpyridine) was co-immobilised with the antibody on the electrode surface by crosslinking the antibody with glutaraldehyde to form a combined sensing and electron transfer system. The resulting sensing film exhibits the classical features of a kinetically fast redox couple strongly bound to an electrode surface. The properties of the sensor depend on the content of osmium polymer, the extent of crosslinking and the loading of the antibody in the film. The detection limit for HRP of the sensor was found to be 0.01 μg ml−1, which is one order of magnitude lower than that for a traditional ELISA experiment. By employing an antibiotin antibody with an immuno-competitive approach, the sensor exhibited excellent performance for the detection of the hapten biotin.

Reactivities of amperometric organic phase peroxidase-modified electrodes in the presence and absence of thiourea and ethylenethiourea as inhibitors

Abstract The reactivities of amperometric horseradish peroxidase (HRP) electrodes in acetonitrile, methanol and acetone were studied by cyclic voltammetry. 1,1-Dimethylferrocene (DMFc) was used as a soluble electron transfer mediator. The responses of the HRP electrode to butanone peroxide (BTP) as a substrate, and thiourea (THU) and ethylenethiourea (ETU) as inhibitors, were measured. Michaelis-Menten analyses of the calibration curves obtained for BTP, THU and ETU in the solvents were carried out to determine the apparent Michaelis-Menten constant (K′m), the limiting current (Imax) and the apparent inhibition constant (K′i). The catalytic efficiency of the biosensor was determined as the ratio of the peak current controlled by the enzyme catalytic reduction of BTP to that controlled by the diffusion of DMFc to the electrode surface ( I k I d ). The values of the Michaelis-Menten kinetic parameters and I k I d were used to assess the performance of the peroxide sensor in the presence and absence of inhibitors. Our results indicate a decrease in sensor performance with increasing solvent hydrophilicity (methanol > acetonitrile > acetone). This has been attributed to the ability of the hydrophilic solvents to desorb essential water of hydration from the enzyme active site. This removal of water decreases the rate of enzymatic reaction and subsequently the catalytic performance of the sensor.

Solvent effects on the reactivities of an amperometric glucose sensor

Reactivities of organic phase biosensors containing 5.1 pmol cm−2 glucose oxidase (GOx) on glassy carbon (GC) or Pt electrode surfaces (0.071 cm2 in area) were evaluated in acetonitrile, acetone, butan-2-ol, tetrahydrofuran and 0.1 M phosphate buffer(pH 7.0). Each of the organic media contained 10% v/v of water. Ferrocenemonocarboxylic acid was used as a soluble electron transfer mediator for the detection of glucose in these solvents. Tafel analyses of the cyclic voltammograms (CVs) of the electrocatalytic reaction gave Tafel slopes of between 103 and 129 mV decade−1, which are in good agreement with the theoretical value of 118 mV decade−1. Constant-potential amperometric studies on GOx-modified rotating Pt disc electrodes (RDEs) were carried out at 0.45 V, a potential dictated by the limiting catalytic currents IK of the CV experiments. The apparent turnover rate constant k′cat of GOx in the biosensor and its catalytic efficiency k′catK′m were estimated from the results of the RDE experiments. Changing from the aqueous buffer to organic media produced a drastic decrease in k′cat, which is more than two orders of magnitude lower in butan-2-ol. This sensor characteristic is related to the lower solvent-dependent diffusibility of glucose in the sensor for the organic systems vis-a-vis phosphate buffer. The normalized catalytic efficiencies, (k′cat/K′m)org solv/(k′mcat/K′m)buffer show an enhancement of biosensor efficiency on changing from phosphate buffer to polar organic solvents. The k′catK′m values are indicators of the degree of activation of the biosensors electrocatalytic reaction. Greater stabilization of the transition state of the electroenzymatic process by organic phases relative to phosphate buffer was ascertained from the normalized catalytic efficiency. The enhanced catalytic efficiency of the organic phase sensors is attributed solely to the activation of the catalytic reaction of GOx and β-d-glucose.

Investigation of the effects of polar organic solvents on the activity of tyrosinase entrapped in a poly(ester-sulphonic acid) polymer

Abstract The behaviour of an amperometric organic-phase phenol sensor was investigated in acetonitrile, acetone and tetrahydrofuran reaction media containing 20% v/v water. The biosensor consisted of tyrosinase immobilised on a Pt or glassy carbon electrode in a poly(estersulphonic acid) polymer matrix. Analyses of the results of the cyclic voltammograms of the enzyme electrode in the presence of 100 μM phenol show that the sensor is effective in the three solvent media. The normalised catalytic currents ( I k, normalised ) of the sensor in the three solvents are 35·5, 26·0 and 8·02, for acetone, acetonitrile and tetrahydrofuran, respectively. I k, normalised is the ratio of the cyclic voltammetric reduction current at -150 mV in the presence of 100 μM phenol to the background current at the same potential. The steady-state amperometric results show that the sensor exhibits fast response to phenol, and the kinetics of the responses agree with the Michaelis-Menten model in all solvent media. The I max , K m app values show that the phenol sensor is about five times more sensitive in acetone and acetonitrile than in tetrahydrofuran. Diffusion coefficient value of phenol ( D phenol ) computed from potential step studies on a 25 μm Pt microelectrode are 9·85, 8·80 and 6·34 x 10 −6 cm2 s −1 , for acetone, acetonitrile and tetrahydrofuran, respectively. The similarity in the D phenol values suggests that the flux of the analyte to the similarity surface does not alone determine the sensitivity of the tyrosinase sensor.

Effects of acetonitrile on horseradish peroxidase (HRP)-anti HRP antibody interaction.

The effects of the water-miscible organic solvent acetonitrile on the enzymatic activity of horseradish peroxidase (HRP) and on HRP-anti-HRP binding have been investigated. Results showed that both the catalytic activity of HRP and the binding ability of the antibody were affected on increasing the concentration of the organic solvent. The activity of HRP varied with the organic composition of the solvent, indicating that the conformation of the enzyme was affected. The binding ability of the antibody also decreased significantly with an increase of the organic composition of the solvent, and in absolute acetonitrile, the activity of the antibody is about 500 times lower than that in aqueous medium. Binding reversibility experiments indicated that the antibody was not irreversibly damaged in solutions with acetonitrile composition greater than 80% and below 40%; however, an irreversible decrease in the binding was observed in solutions with an acetonitrile composition between 40 and 80%. The reduction in the binding ability is probably due to the irreversible conformation changes in the antibody.

Characterisation of electrosynthetic l-dopa-melanin films by electrochemical and spectroelectrochemical techniques

Abstract Melanin was electrochemically synthesised by the oxidation of l -dopa in aqueous medium. The intermediates of the oxidation process were characterised by cyclic voltammetry and spectroelectrochemistry. The final product of melanin oxidation exhibited properties of a heterogeneous system containing a mixture of quinones and other oxidation intermediates of l -dopa. The charge transport properties of the redox polymeric compound was studied by chronocoulometric techniques using phosphate, perchlorate and sulphonate as electrolytes. The melanin electron transport coefficient ( D e 1/2 C es ) estimated for melanin in the various electrolytes are in good agreement with what is reported for other redox polymers. UV spectroelectrochemical reactivities of the melanogenesis process was used to identify some of the intermediate products. The excellent charge transport characteristics of melanin and its redox characteristics suggest that it could be used as an efficient immobilisation matrix for biomolecules in constructing reagentless amperometric biosensors.

Polymer-Based Amperometric Biosensors

Electrochemical detection of analytes can give rise to very elegant methods in analytical chemistry. Such electroanalytical applications are characterized by excellent sensitivity, good selectivity toward the analyte, and simplicity in the design and use of instrumentation. However, the limited number of electrode/conductor surfaces available and the problem of electrode instability caused by passivation of electrode and slow electrode kinetics mean that there is a restricted number of analytes suitable for electrochemical detection. To circumvent this and other related problems, electrode surfaces can be modified to change chemical and physical properties of the electrode material. The added impetus of these chemically modified electrodes (CMEs) is tailoring the electrode surface for improved quantitative and qualitative detection of a particular analyte. This is due to the high selectivity of the electrode surface for catalytic, ion-exchange or complexation reactions, improvements in permselectivity, or high specificity due to incorporating various biocomponents.

Amperometric determination of butanone peroxide and hydroxylamine via direct electron transfer at a horseradish peroxidase-modified platinum electrode

An amperometric organic-phase peroxide biosensor was constructed by immobilizing horseradish peroxidase (HRP) entrapped within poly(ester-sulfonic acid) Eastman AQ-55D polymer matrix on a platinum electrode surface. The enzyme electrode functioned by direct electron transfer between the HRP redox centre and the platinum surface. This mediator-free biosensor was applied to the determination of butanone peroxide and hydroxylamine, which are HRP substrate and inhibitor, respectively. Fast sensor responses were obtained in 30 s for both the substrate and the inhibitor. The biosensor showed higher catalytic efficiency in acetonitrile (1.1 µA l mmol–1) than in methanol (0.9 µA l mmol–1). However, the sensor exhibited greater percentage inhibition of HRP activity in the less catalytically efficient methanol medium (516% inhibition 1 mmol–1) than in acetonitrile (345% inhibition l mmol–1). This reversal in the performance of the sensor towards hydroxylamine in the organic solvents is attributed to the reaction media polarity combined with the butanone peroxide-induced conformational change of the enzyme active site.