Mediated amperometric determination of xylose and glucose with an immobilized aldose dehydrogenase electrode.

An enzyme electrode was constructed for amperometric determination of xylose and glucose. The electrode is based on the PQQ-dependent membrane-bound aldose dehydrogenase (ALDH) from Gluconobacter oxydans. ALDH was covalently immobilized on a graphite electrode. Immobilized dimethylferrocene, soluble ferrocene carboxylic acid and phenazine methosulphate were used as electron transfer mediators. When xylose was measured electrochemically using an electrode modified with ALDH and dimethylferrocene, the linear measurement range extended to 100 mM. For glucose measurement the linear measurement range was about one-tenth of that for xylose. The electrode showed fairly good stability; 50% of the original electrode response was still obtained after 5 days of intermittent use. The effect of possible leakage of adsorbed mediator was determined by measuring the response of an electrode with soluble mediator as a function of time. The reproducibility of the electrode was good, the standard deviation of the electrode response in ten measurements with the same electrode being only 2.7%.     

Electrode and cuvette for rapid continuous CO2 tension and pH measurement in blood

An electrode and cuvette system has been developed for the continuous and rapid measurement of either blood CO2 tension or pH. The CO2 electrode consists of a 1.5-mm-diameter flat-tip glass pH electrode covered by a film of carbonic anhydrase solution, over which a 25-micron-thick dimethyl silicone membrane is attached. Porous ceramic filled with 20% polyacrylamide, equilibrated with a salt solution, serves as a salt bridge between a Ag-AgCl reference electrode and the pH electrode surface. The electrode is housed in a four-port cuvette assembly. Blood from a vessel of interest is delivered to the cuvette by means of an occlusive roller pump. The cuvette maintains the electrode and blood at a constant temperature and directs a continuous jet of blood against the electrode surface. The cuvette also allows for easy and frequent calibration of the electrode with either gas or liquid standards. The 90% response time of the CO2 electrode is 3.0 s for liquids and 1.3 s for gases. Removal of the dimethyl silicone membrane and carbonic anhydrase film yields a pH electrode that can continuously measure blood pH with a 90% response time of 1.6 s.     

Design and response characteristics of an enzyme electrode for measurement of penicillin in fermentation broth

A principle for the construction of an autoclavable enzyme electrode is presented. Some characteristics of a penicillin electrode constructed according to this principle are given. The response time is ˜1 min. The response to increasing concentrations of penicillin is linear in buffered samples but logarithmic in unbuffered samples. The reason for the linearity is discussed. A local pH decrease in the enzyme, which is a is a consequence of the enzymatic reaction, reduces the buffering capacity within the electrode and thus increases the sensitivity. It is suggested that this increased sensitivity eliminates the logarithmic response predicted by the Nernst equation for a pH-based enzyme electrode.     

Selectivity enhancement of an Escherichia coli bacterial electrode using enzyme and transport inhibitors

The feasibility of using specific enzyme and transport inhibitors to minimize the glutamine response of a potentiometric microbial sensor is demonstrated. The glutamine response of a bacterial electrode prepared with Escherichia coli as the biocatalyst in conjunction with an ammonia gas-sensing electrode was greatly reduced by treating the electrode with the enzyme inhibitor 6-diazo-5-oxo-L-norleucine (DONL) and the transport inhibitor gamma-L-glutamylhydrazide. Each inhibitor effectively decreased glutamine response to a level sufficiently low to be considered negligible in clinical studies. Although the sensor ultimately recovered from the effects of a single exposure to an inhibitor, continuous exposure at an optimum concentration maintained a low response to glutamine. Furthermore, the treatment of the sensor with both inhibitors simultaneously resulted in a negligible response to glutamine of <1 mV, indicating that both inhibitors are necessary for optimum inhibition of glutamine response. This approach is promising as a means of enhancing the selectivity of microbial sensors.     

A simple, sensitive, and accurate alcohol electrode

The construction and performance of an enzyme electrode is described which specifically detects lower primary aliphatic alcohols in aqueous solutions. The electrode consists of a commercial Clark-type oxygen electrode on which alcohol oxidase (E.C. 1.1.3.13) and catalase were immobilized. The decrease in electrode current is linearly proportional to ethanol concentrations between 1 and 25 ppm. The response of the electrode remains constant during 400 assays over a period of two weeks. The response time is between 1 and 25 min. Assembly of the electrode takes less than 1 h.     

The role of polypyrrole as charge transfer mediator and immobilization matrix for d-fructose dehydrogenase in a fructose sensor

A fructose dehydrogenase (FDH) modified electrode is produced by the electroadsorption of a layer of FDH on a platinum electrode followed by the electropolymerization of a polypyrrole (PPy) film around and over the enzyme. This immobilizes and stabilizes the enzyme as well as providing an electron transfer pathway to the electrode. The amperometric response to fructose and the enzymatic activity are measured as a function of PPy film thickness. The electrode is shown to have a maximum response at a PPy thickness of approximately the thickness of the enzyme layer. A measure of the electrode efficiency is also obtained, this is the amperometric response to fructose as a percentage of that expected on the basis of the enzyme activity. The functioning of the electrode is also dependent on the counter-ion used for PPy polymerization. This is shown to be mainly related to the nucleation and growth of the PPy film in the interfacial region.     

Glucose biosensor prepared by glucose oxidase encapsulated sol-gel and carbon-nanotube-modified basal plane pyrolytic graphite electrode

A new glucose biosensor has been fabricated by immobilizing glucose oxidase into a sol-gel composite at the surface of a basal plane pyrolytic graphite (bppg) electrode modified with multiwall carbon nanotube. First, the bppg electrode is subjected to abrasive immobilization of carbon nanotubes by gently rubbing the electrode surface on a filter paper supporting the carbon nanotubes. Second, the electrode surface is covered with a thin film of a sol-gel composite containing encapsulated glucose oxidase. The carbon nanotubes offer excellent electrocatalytic activity toward reduction and oxidation of hydrogen peroxide liberated in the enzymatic reaction between glucose oxidase and glucose, enabling sensitive determination of glucose. The amperometric detection of glucose is carried out at 0.3 V (vs saturated calomel electrode) in 0.05 M phosphate buffer solution (pH 7.4) with linear response range of 0.2-20 mM glucose, sensitivity of 196 nA/mM, and detection limit of 50 microM (S/N=3). The response time of the electrode is < 5s when it is stored dried at 4 degrees C, the sensor showed almost no change in the analytical performance after operation for 3 weeks. The present carbon nanotube sol-gel biocomposite glucose oxidase sensor showed excellent properties for the sensitive determination of glucose with good reproducibility, remarkable stability, and rapid response and in comparison to bulk modified composite biosensors the amounts of enzyme and carbon nanotube needed for electrode fabrication are dramatically decreased.     

Model-based optimization of a conductive matrix enzyme electrode

A mathematical model has been developed to describe the mechanism for internal mass transfer and enzyme reaction kinetics of an amperometric conductive matrix enzyme electrode. The model is simplified and solved analytically to arrive at a representation for the response slope in the linear range as well as for the response time. This is the first time that the response time of an enzyme electrode is described by a mathematical model. Simulations give information on how the design parameters influence the performance of the electrode for a glucose oxidase catalyzed sensing reaction process. Based on this information, several designs were constructed and tested showing suitable agreement with theoretical predictions. Finally, an optimized electrode was designed and validated.     

Carbonate ion-selective electrode with reduced interference from salicylate

A carbonate ion-selective electrode for determination of total carbon dioxide species such as carbon dioxide, bicarbonate and carbonate with reduced interference from salicylate is described. Derivatives of trifluoroacetophenone were used as neutral carriers for carbonate. A polymer-free liquid carbonate-selective membrane with a cellophane outer membrane was found to give a carbonate-selective electrode with a negligible response to salicylate. The electrical contact was obtained by insertion of a silver/silver chloride electrode directly into the liquid membrane. The electrode does not require any aqueous filling solution and is therefore maintenance-free. The response to carbon dioxide species was found to be highly reproducible with a response time of 1-2 min at total carbon dioxide concentrations in the range from 5 to 50 mM. The lifetime of the electrode was at least 3 months. The electrode is regarded as very promising for clinical analysis of carbon dioxide species in body fluids such as plasma and serum.     

Mercaptoethylpyrazine promoted electrochemistry of redox protein and amperometric biosensing of uric acid

Electrochemistry of microperoxidase-11 (MPx-11) anchored on the mixed self-assembled monolayer (SAM) of 2-(2-mercaptoethylpyrazine) (PET) and 4,4'-dithiodibutyric acid (DTB) on gold (Au) electrode and the biosensing of uric acid (UA) is described. MPx-11 has been covalently anchored on the mixed SAM of PET and DTB on Au electrode. MPx-11 on the mixed self-assembly exhibits reversible redox response characteristic of a surface confined species. The heterocyclic ring of PET promotes the electron transfer between the electrode and the redox protein. The apparent standard rate constant kapps obtained for the redox reaction of MPx-11 on the mixed monolayer is approximately 2.15 times higher than that on the single monolayer of DTB modified electrode. MPx-11 efficiently mediates the electrocatalytic reduction of H2O2. MPx-11 electrode is highly sensitive to H2O2 and it shows linear response for a wide concentration range. The electrocatalytic activity of the MPx-11 electrode is combined with the enzymatic activity of uricase (UOx) to fabricate uric acid biosensor. The bienzyme assembly is highly sensitive towards UA and it could detect UA as low as 2 microM at the potential of -0.1 V. The biosensor shows linear response with a sensitivity of 3.4+/-0.08 nA cm(-2) microM(-1). Ascorbate (AA) and paracetamol (PA) do not significantly interfere in the amperometric sensing of UA.     

Enzyme electrode for phenol

An enzyme electrode is described for quantitative determination of phenol at micromolar concentrations. Immobilized phenol hydroxylase is attached to the surface of a Clark oxygen electrode. The Maximum rate of oxygen consumption is linearly dependent on phenol concentration over the 0.5–50μM range. The electrode can be used for at least 150 assays without an activity loss. Readout is very rapid—within 30 sec of sample addition. The electrode response is independent of pH between pH 6.5 and 9.5. The response increases linearly with temperature in the interval 10–40°C. It is necessary to incubate the enzyme electrode in a buffer containing NADPH for a few minutes before the addition of sample. This is to make the electrode response independent of the diffusion rate of this cosubstrate. This and other diffusional effects on the performance of the phenol electrode are discussed.     

A polyvinylchloride-membrane based anion selective electrode for continuous registration of delta pH (interior alkaline) with salicylate as the indicator probe

An anion sensitive electrode has been constructed with the use of the lipid soluble cation benzyl-dimethyl-hexadecylammonium analogous to the procedure described for tetraphenylphosphonium-sensitive electrodes [Shinbo, T., Kamo, N., Kurihara, K. and Kobatake, Y. (1978) Arch. Biochem. Biophys. 187, 414-422]. The anion electrode responds to salicylate concentrations above 400 microM with a Nernstian sensitivity. Less lipid soluble anions like chloride and phosphate do not interfere. Below 400 microM salicylate the response of the electrode decreases gradually so that the sensitivity of the electrode is less than 10 mV per decade change at concentrations of the anion of 50 microM. A computer program has been developed to fit the electrode response curve with a polynomal function of the fourth power. Additional software-allows calculation of changes in the concentration of the salicylate anion, also under conditions where the sensitivity of the electrode for the anion is not constant. In this way the electrode can be used to measure changes in salicylate concentration that occur in a suspension of bacteria when, upon energization, a pH gradient is generated. 31P nuclear magnetic resonance measurements demonstrated that the pH gradient measured with the salicylate-sensitive electrode in the phototrophic bacterium Rhodopseudomonas sphaeroides is quantitatively correct. The response time of the electrode decreases from 1 min at 20 microM salicylate to 10 s at concentrations greater than or equal to 200 microM.     

The calibration and use of a calcium ion-specific electrode for kinetic studies of mitochondrial calcium transport.

A calcium ion-specific electrode has been used to study calcium transport by isolated,hepatic mitochondria. The methodology used requires only a sensitive pH meter operated in the millivolt mode with the electrode. Free calcium ion concentrations may be followed continuously. Using incubation conditions which cause release of intramitochondrial calcium, the calcium electrode system may also be used to determine total. intramitochondrial calcium. Techniques for the calibration of the electrode response are discussed. Free calcium ion concentrations have been calculated from total calcium concentrations and the association constants for the binding species present in the assay medium. The observation that the electrode response is linear to submicromolar concentrations allows calculation of a linear least-squares fit of millivolt reading to computed free calcium ion concentration. A computer program written in BASIC for these computations is included in Appendix material. The half-maximal rate constant for mitochondrial calcium uptake has been found to occur at a free calcium ion concentration of 6.5 μm. The interaction or Hill coefficient for the process is 2.3, indicating positive cooperativity.     

An enzyme electrode for specific determination of L-lysine: A real-time control sensor

Lysine decarboxylase (L-lysine carboxylyase, E.C.4.1.1.18) is immobolized on a carbon dioxide gas-sensing electrode, by copolymerization with gelatin using the bifuncitional agent glutaraldehyde. The enzyme electrodes thus prepared are used in a continuous flow system to measure the concentration of L-lysine in a mixture of amino acids. The measuring time for each sample is about 3 min, including response and rinsing times. The electrode response is linear between 0.01-1 g/L and has a high specificity for L-lysine. The enzyme electrode response to lysine at concentrations below 0.5 g/L is stable on repeated use for at least 500 assays.     

Construction and characterization of a multi-layer enzyme electrode: Covalent binding of quinoprotein glucose dehydrogenase onto gold electrodes

Multilayers of quinoprotein glucose dehydrogenase were assembled onto modified gold electrodes. As a primary modifier the bifunctional 3,3′-dithiodipropionic acid bis(N-hydroxysuccinimide ester) was chemisorbed. Glucose dehydrogenase was covalently bound to this activated electrode in a stepwise procedure. In the presence of glucose, the electrode functions as a sensor for electron acceptors. Catalytic current, as observed for p-aminophenol, was used to characterize electrode performance. The dependence of the electrode response on the number of enzyme layers showed that the transition from a kinetic to a diffusion-limited sensor is reached at 6–7 enzyme layers. The response of multilayer electrode is stable over a broad range of pH and ionic strength of the bulk solution. It also shows good stability: after 2 months, 75% of its original activity remained.     

Internal standard method for the measurement of choline and acetylcholine by capillary electrophoresis with electrochemical detection

An internal standard method has been developed for the determination of the neurotransmitter acetylcholine and/or its metabolic precursor choline. This approach couples the high separation efficiency of capillary electrophoresis with the sensitivity and selectivity of electrochemical detection at an enzyme-modified electrode. Indirect electrochemical detection is accomplished at a 25 microm platinum electrode modified by cross-linking the enzymes choline oxidase and acetylcholinesterase with glutaraldehyde. Although in this simple form of electrode fabrication there is a gradual loss of response from the electrochemical detector with time, accurate quantitation is achieved by the addition of butyrylcholine, which is also a substrate for acetylcholinesterase, as an internal standard. A linear response is achieved between 0 and 125 microM with a limit of detection of 2 microM (25 fmol). The utility of this method was demonstrated by monitoring the kinetics of choline uptake in synaptosomal preparations.     

Urate oxidase electrode based on dissolved oxygen probe for urine uric acid determination

A biosensor for the specific determination of uric acid in urine was developed using urate oxidase (EC 1.7.3.3) in combination with a dissolved oxygen probe. Urate oxidase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated teflon membrane to serve as enzyme electrode. The electrode response was maximum when 50 mM glycine buffer was used at pH 9.2 and 35 degrees C. The enzyme electrode response depends linearly on uric acid concentration between 5-40 microM with a response time of 5 min. The enzyme electrode is stable for more than 2 weeks and during this period over 35 assays were performed.     

Quantitative measurements of NO reaction kinetics with a Clark-type electrode.

Nitric oxide (NO) plays important physiological roles in the body. Knowledge regarding the kinetics of NO catabolism is important for understanding the biological functions of NO. Clark-type NO electrodes have been frequently employed in measuring the kinetics of NO reactions; however, the slow response time of these electrodes can cause measurement errors and limit the application of the electrode in measurements of fast NO reactions. In this study, a simplified diffusion model is given for describing the response process of the NO electrode to the change of NO concentration. The least-square method is used in fitting the currents calculated from the diffusion equation to the experimental curves for determining the diffusion parameters and rate constants. The calculated currents are in excellent accordance with the experimental curves for different NO reaction kinetics. It has been demonstrated that when using an NO electrode with a response time of approximately 6 s to measure fast NO reactions with a half-life of approximately 1s, the response currents of the electrode have large differences compared to the curve of actual NO concentration in the solution; however, the rate constant of NO decay can still be accurately determined by computer simulations with the simplified diffusion model. Theoretical analysis shows that an NO electrode with a response time of 6 s (D/L2=0.06 s-1) and the lowest detection limit of 1 nM NO can be used in measuring kinetics of extremely rapid NO reactions with a half-life below 10 ms.     

Electrochemically amplified detection for lipopolysaccharide using ferrocenylboronic acid

A novel electrochemical technique for lipopolysaccharide (LPS) detection has been developed using a combination of ferrocenylboronic acid derivatives and an enzyme-modified electrode. The enzyme-modified electrode was constructed from a gold electrode modified with a bovine serum albumin membrane containing diaphorase. Ferrocenylboronic acid derivatives are oxidized on the electrode, and then regenerated by a diaphorase-catalyzed reaction in the presence of NADH. The consumption/regeneration cycle for ferrocenylboronic acid derivatives resulted in a chemically amplified current response. The current response for ferrocenylboronic acid derivatives decreased in association with its complexation with glycosyl units of LPS, and this current decrease caused by LPS was also amplified by the recycling process. On the other hand, the addition of a monosaccharide such as D-mannose or D-galactose induced no response at the same LPS concentration. The enzyme membrane immobilized on the electrode plays an important role in selectivity as well as chemical amplification. In addition, the enzyme-modified electrode exhibited a rapid response of 5 min for LPS, which is much faster than the currently used method. The detection limit of LPS from Escherichia coli O127:B8 was as low as 50 ng ml-1.     

Characterization and electrocatalytic properties of Prussian blue electrochemically deposited on nano-Au/PAMAM dendrimer-modified gold electrode

Gold electrode was modified with 3-mercaptopropionic acid (MPA) and further reacted with poly(amidoamine) (PAMAM) dendrimer (generation 4.0) then attached the nano-Au to obtain films on which Prussian blue (PB) was electrochemically deposited to afford much wider pH adaptive range, much better electrochemical stability and excellent electrochemical response. The microstructure and electrochemical behavior of Au/MPA/PAMAM/nano-Au/PB electrode were investigated by scanning electron microscopy (SEM) and cyclic voltammetry. The electrochemical response of the Au/MPA/PAMAM/nano-Au/PB-modified electrode for the electrocatalytic reduction of hydrogen peroxide was investigated, and it was found that the sensitivity as well as the corresponding detection limits were improved as compared to the voltammetric response of a Au/PB-modified electrode and Au/MPA/PAMAM/PB electrode. Based on this, a new electrochemical sensor for determination of hydrogen peroxide has been developed.