Amperometric glucose biosensor with extended concentration range utilizing complexation effect of borate.

Borate buffer strongly decreases amperometric response of a glucose oxidase linked pO2 or H2O2 sensing electrode, extending substantially its linear calibration range. With increasing pH and concentration of the buffer the upper limit for glucose can be varied between 1 and 30 mmol l-1 glucose. The effect of borate ion is explained by the rapid complexation of glucose decreasing the equilibrium concentration of free beta-anomer, the specific substrate of glucose oxidase. The high loading of cross-linked enzyme inside the sensor membrane is necessary for the measurement to ensure an almost constant response factor (delta i per 1 mmol l-1) between pH 5 and 10. Analysis in stirred solution and in a flow-through system has been employed for the measurement of elevated glucose levels in heparinized human blood or plasma samples.     

Amperometric enzyme electrode for determination of theophylline in serum.

This paper describes an amperometric enzyme electrode for the rapid determination of theophylline in serum. The method is based on the catalysed oxidation of theophylline by the haem-containing enzyme theophylline oxidase. Results are presented for two approaches. First, ferrocene monocarboxylic acid was used as a mediator. The second-order rate constant was 1.1 x 10(3) 1 mol-1 s-1. Secondly, the organic conducting salt NMP.TCNQ was used to construct enzyme electrodes. These electrodes were employed for the rapid (60 s) measurement of theophylline in serum at a working potential of +100 mV versus Ag/AgCl. Linear calibration curves were obtained over the clinically relevant range (y = 0.13x + 0.22, n = 8). Caffeine, theobromine and 3-methylxanthine at levels up to 100 mg l-1 do not interfere and 1-methylxanthine shows cross-reactivity at concentrations greater than 50 mg l-1.     

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.     

Amperometric microbial biosensor for p-nitrophenol using Moraxella sp.-modified carbon paste electrode

An amperometric microbial biosensor for highly specific, sensitive and rapid quantitative determination of p-nitrophenol was developed. The biosensor takes advantage of the ability of Moraxella sp. to specifically degrade p-nitrophenol to hydroquinone, a more electroactive compound than p-nitrophenol. The electrochemical oxidation current of hydroquinone formed in biodegradation of p-nitrophenol was measured at Moraxella sp.-modified carbon paste electrode and correlated to p-phenol concentrations. The optimum response was realized by electrode constructed using 15 mg of dry cell weight per 1 g of carbon paste and operating at 0.3 V (versus Ag/AgCl reference) in pH 7.5, 20 mM sodium phosphate buffer. Operating at these optimum conditions the biosensor had excellent selectivity against phenol derivatives and was able to measure as low as 20 nM (2.78 ppb) p-nitrophenol with very good accuracy and reproducibility. The biosensor was stable for approximately 3 weeks when stored at 4 degrees C. The applicability of the biosensor to measure p-nitrophenol in lake water was demonstrated.     

Amperometric electrode for determination of urea using electrodeposited rhodium and immobilized urease

An amperometric biosensor was developed for determination of urea using electrodeposited rhodium on a polymer membrane and immobilized urease. The urease catalyzes the hydrolysis of urea to NH₄⁺ and HCO₃⁻ ions and the liberated ammonia is catalytically and electrochemically oxidized by rhodium present in the rhodinized membrane on the Pt working electrode. Three types of rhodinized polymer membranes were prepared by varying the number of electrodeposition cycles: membrane 1 with 10 deposition cycles, membrane 2 with 40 cycles and membrane 3 with 60 cycles. The morphologies of the rhodinized membranes were investigated by scanning electron microscopy and the results showed that the deposition of rhodium was like flowers with cornices-like centers. The influence of the amount of electrodeposited rhodium over the electrode sensitivity to different concentrations of ammonia was examined initially based on the cyclic voltammetric curves using the three rhodium modified electrodes. The obtained results convincingly show that electrode with rhodinized membrane 1, which contain the lowest amount of electrodeposited rhodium is the most active and sensitive regarding ammonia. It was found that the anodic oxidation peak of ammonia to nitrogen occurs at 0.60 V. In order to study the performance of urease amperometric sensor for the determination of urea, experiments at constant potential (0.60 V) were performed. The current–time experiments were carried out with urease rhodinized membrane 1 (10 cycles). The amperometric response increased linearly up to 1.75 mM urea. The detection limit was 0.05 mM. The urea biosensor exhibited a high sensitivity of 1.85 μA mM⁻¹ cm⁻² with a response time 15 s. The Michaelis–Menten constant K_m for the urea biosensor was calculated to be 6.5 mM, indicating that the immobilized enzyme featured a high affinity to urea. The urea sensor showed a good reproducibility and stability. Both components rhodium and urease contribute to the decreasing of the production cost of biosensor by avoiding the use of a second enzyme.     

Amperometric responses to acetylene, ethylene, and methane by a Clark-type oxygen electrode

Hydrogen-conditioned Clark electrodes exposed to successive acetylene samples displayed a decreasing curvilinear response. Slight electrode responses are also effected by saturated ethylene solutions. Thus, amperometric measurements of hydrogen or carbon monoxide in the aqueous environment by these electrodes are perturbated by the presence of acetylene and to a much lesser extent by ethylene. Methane-saturated solutions induced no electrode response.     

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.     

Amperometric flow-injection determination of sucrose with a mediated tri-enzyme electrode based on sucrose phosphorylase and electrocatalytic oxidation of NADH

A new sucrose electrode is described for the determination of sucrose without interference from glucose or fructose. The sucrose electrode is based on the tri-enzymatic system of sucrose phosphorylase, phosphoglucomutase and glucose-6-phosphate 1-dehydrogenase, where NAD(P)H is produced from the last enzymatic reaction and recycled into NAD(P)+ through its electrocatalytic oxidation by Os(4,4'-dimethyl-2,2-bypyridine)2(1,10-phenanthroline-5,6-dione). The electrodes were optimised with respect to the various construction parameters and carrier composition in a FIA system and their response as a function of the pH and flow-rate was examined. The electrodes were suitable for operation in a FIA system and the analysis of real samples showed good agreement with the reference method. Typical optimised electrodes showed detection limits of 1 mM sucrose, response time of 5 min, sensitivity 1.010 nA mM(-1), and current density of 8.38 microA cm(-2), using 200 mM PIPES pH 7.25 with 10 mM phosphate and 5 mM MgCl2 as carrier.     

Amperometric detection of bilirubin from a micro-sensing electrode with a synthetic bilirubin imprinted poly(MAA-co-EGDMA) film

Poly(methacrylic acid-co-ethyl glycol dimethylacrylate) (poly(MAA-co-EGDMA)) imprinted with alpha-bilirubin was shown to be able to bind alpha-bilirubin in our previous work. In this work, the corresponding imprinted polymer thin film was synthesized onto a thiol treated Au electrode by surface grafting polymerization. Bilirubin was able to be detected by an Au electrode, however, the electrode was not be able to discriminate bilirubin from the other matrix components if clinical samples were used. Therefore, the imprinted material was introduced so that the modified Au electrode could specifically detect bilirubin. Optimal potential was found to be 0.55 V and this was set for the rest of experiments. The imprinting factor of 3.16 was confirmed by comparing the signals from the MIP-Au and the NIP (non-imprinted polymer)-Au electrode. Calibration of the bilirubin concentration with respect to the current by the MIP-Au electrode was made within the range of 5mg/dl and a detection sensitivity of 0.644 microA/mg/dl (2.58 microA/cm(2)/mg/dl) was obtained. Furthermore, a linear correlation of the bilirubin concentration within 1.0mg/dl versus detection current was also achieved. Bilirubin was further detected by the MIP-Au electrode in the presence of fetal bovine serum (FBS). Repeated detection of bilirubin with at least three detection batches was performed and the reproducibility of the same piece of MIP-Au electrode was confirmed. The result was compared to those obtained from the serum and the solvent solution. The results indicated the feasibility of using the bilirubin imprinted poly(MAA-co-EGDMA) film as a sensing electrode for the clinical detection of bilirubin in serum.     

Amperometric biosensor based on coentrapment of enzyme and mediator by gold nanoparticles on indium-tin oxide electrode

A disposable pseudo-mediatorless amperometric biosensor has been fabricated for the determination of hydrogen peroxide (H2O2). In the current study, an indium-tin oxide (ITO) electrode was modified with thiol functional group by (3-mercaptopropyl)trimethoxysilane. The stable nano-Au-SH monolayer (AuS) was then prepared through covalent linking of gold nanoparticles and thiol groups on the surface of the ITO. The horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) were finally coentrapped by the colloidal gold nanoparticles. The immobilized TMB was used as an electron transfer mediator that displayed a surface-controlled electrode process at a scan rate of less than 50mV/s. The biosensor was characterized by photometric and electrochemical measurements. The results showed that the prepared AuS monolayer not only could steadily immobilize HRP but also could efficiently retain HRP bioactivity. Parameters affecting the performance of the biosensor, including the concentrations of the immobilized TMB and HRP, the pH value, and the reaction temperature, were optimized. Under the optimized experimental conditions, H(2)O(2) could be determined in a linear calibration range from 0.005 to 1.5mM with a correlation coefficient of 0.998 (n=14) and a detection limit of 1microM at a signal/noise ratio of 3. The proposed method provides a new alternative to develop low-cost biosensors by using ITO film electrodes from industrial mass production.     

Amperometric detection of insulin at renewable sol-gel derived carbon ceramic electrode modified with nickel powder and potassium octacyanomolybdate(IV)

A renewable three-dimensional chemically modified carbon ceramic electrode (CCE) containing nickel powder and K4[Mo(CN)8] was constructed by sol-gel technique. The electrochemical properties and stability of modified electrode was evaluated by cyclic voltammetry in pH range 4-10. The redox couple of [Mo(CN)8] (4-/3-) was shown both as a solute in electrolyte solution and as a component of a carbon based conducting composite electrode. The apparent electron transfer rate constant (ks) and transfer coefficient (alpha) were determined by cyclic voltammetry and they were about 17.1 and 0.57 s(-1), respectively. The catalytic activity of the modified CCE toward insulin oxidation was investigated at pH range of 3-8 by cyclic votammetry. The modified electrode showed excellent electrocatalytic activity toward insulin electroxidation at physiological pH value. The modified electrode was used for insulin detection chronoamperometrically at pH 7. Under optimized condition in amperometry method, the concentration calibration range, detection limit and sensitivity were 0.5-500 nM, 0.45 nM and 6140 nA/microM, respectively. Flow injection amperometric determination of insulin at pH 7.4, at this modified electrode yielded a calibration curve with the following characteristics, linear dynamic range 100-500 pM; sensitivity 8.1 nA/nM and detection limit 40 pM (based on S/N = 3). The inherent stability at wide pH range, high sensitivity, low detection limit, low cost and ease of preparation are of advantageous of this insulin sensor. This sensor indicates great promise for monitory insulin in chromatographic effluents.     

High hydrogen production rate of microbial electrolysis cell (MEC) with reduced electrode spacing

Practical applications of microbial electrolysis cells (MECs) require high hydrogen production rates and a compact reactor. These goals can be achieved by reducing electrode spacing but high surface area anodes are needed. The brush anode MEC with electrode spacing of 2 cm had a higher hydrogen production rate and energy efficiency than an MEC with a flat cathode and a 1-cm electrode spacing. The maximum hydrogen production rate with a 2 cm electrode spacing was 17.8 m(3)/m(3)d at an applied voltage of E(ap)=1 V. Reducing electrode spacing increased hydrogen production rates at the lower applied voltages, but not at the higher (>0.6 V) applied voltages. These results demonstrate that reducing electrode spacing can increase hydrogen production rate, but that the closest electrode spacing do not necessarily produce the highest possible hydrogen production rates.     

Amperometric glucose biosensors based on layer-by-layer assembly of chitosan and glucose oxidase on the Prussian blue-modified gold electrode

A glucose biosensor based on layer-by-layer (LBL) self-assembling of chitosan and glucose oxidase (GOD) on a Prussian blue film was developed. First, Prussian blue was deposited on a cleaned gold electrode then chitosan and GOD were assembled alternately to construct a multilayer film. The resulting amperometric glucose biosensor exhibited a fast response time (within 10 s) and a linear calibration range from 6 μM to 1.6 mM with a detection limit of 3.1 μM glucose (s/n = 3). With the low operating potential, the biosensor showed little interference to the possible interferents, including ascorbic acid, acetaminophen and uric acid, indicating an excellent selectivity.     

Reconstitution of apomyoglobin with extended biliverdins.

An analysis of the reconstitution of biliverdins with extended conformations and horse heart apomyoglobin was carried out. Biliverdins with the 5Z-syn, 10Z-syn, 15Z-anti and 5Z-anti, 10Z-syn, 15Z-anti conformations, as well as biliverdins with the Z,Z,Z, all-syn conformation recombined with apomyoglobin. In every case the P enantiomers were bound in excess to the M enantiomers, with exception of the 5-syn, 10-syn, 15-anti biliverdin where the M enantiomer bound preferentially to the protein. Biliverdins with an anti conformation at the C-10 meso bridge did not recombine with the protein. It was concluded that the presence of a syn conformation at the C-10 methine conferred to the biliverdin the necessary helicity to fit into the apomyoglobin heme pocket. This regioselectivity is of importance in view of the well known analogy between the ligand domains of myoglobin and the C-phycocyanins.     

Fluorescence decay studies of reduced nicotinamide adenine dinucleotide in solution and bound to liver alcohol dehydrogenase.

The monophoton counting technique was used to obtain the fluorescence decay kinetics of NADH (dihydronicotinamide adenine dinucleotide) bound to LADH (HORSE LIVER ALCOHOL DEHYDROGENAS). It was found that the fluorescence decay of the enzyme complex did not follow a single exponential decay law but that the data could be well described as a sum of two exponentials. The decay parameters of the enzyme complex do not depend on the degree of binding-site saturation. These results are interpreted in terms of a reversible excited-state reaction forming a nonfluorescent product. Fluorescence decay kinetics are also reported for NADH and related molecules in solution. The decay parameters, fluorescence emission maxima, and fluorescence intensities depend on solvent polarity and viscosity.     

Amperometric and impedimetric characterization of a glutamate biosensor based on Nafion and a methyl viologen modified glassy carbon electrode

An electrochemical biosensor based on a glassy carbon (GC) electrode chemically modified with the perfluorinated cation-exchange polymer Nafion and methyl viologen (MV) is described. The enzyme was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA), methyl viologen and Nafion. Operating variables such as the enzyme/BSA ratio, cross-linking time in glutaraldehyde vapor, methyl viologen and Nafion percentages were investigated with regard to their influence on the biosensor sensitivity by using glucose oxidase as the enzyme model due to its high stability and low cost. The glutamate biosensor was elaborated by using optimized parameters and its electrochemical properties were investigated by cyclic voltammetry, amperometry and by electrochemical impedance spectroscopy. The glutamate biosensor shows a detection limit of 20 microM and a linear range extended to 0.75 mM. Its selectivity was tested with 15 different amino acids, each with a concentration of 20 microM, 25 microM acetaminophen, 20 microM uric acid and 200 microM ascorbic acid. No amperometric response was observed for the interfering species. This good selectivity allows glutamate detection in biological media without previous separation of the analyte.     

Roles of zinc ion and reduced coenzyme in horse liver alcohol dehydrogenase catalysis. The mechanism of aldehyde activation.

1,4,5,6-Tetrahydronicotinamide adenine dinucleotide (H2NADH) has been investigated as a reduced coenzyme analog in the reaction between trans-4-N,N-dimethylaminocinnamaldehyde (I) (lambdamax 398 nm, epsilonmax 3.15 X 10-4 M-minus 1 cm-minus 1) and the horse liver alcohol dehydrogenase-NADH complex. These equilibrium binding and temperature-jump kinetic studies establish the following. (i) Substitution of H2NADH for NADH limits reaction to the reversible formation of a new chromophoric species, lambdamax 468 nm, epsilonmax 5.8 x 10-4 M-minus 1 cm-minus 1. This chromophore is demonstrated to be structurally analogous to the transient intermediate formed during the reaction of I with the enzyme-NADH complex [Dunn, M. F., and Hutchison, J. S. (1973), Biochemistry 12, 4882]. (ii) The process of intermediate formation with the enzyme-NADH complex is independent of pH over the range 6.13-10.54. Although studies were limited to the pH range 5.98-8.72, a similar pH independence appears to hold for the H2NADH system. (iii) Within the ternary complex, I is bound within van der Waal's contact distance of the coenzyme nicotinamide ring. (iv) Formation of the transient intermediate does not involve covalent modification of coenzyme. Based on these findings, we conclude that zinc ion has a Lewis acid function in facilitating the chemical activation of the aldehyde carbonyl for reduction, and that reduced coenzyme plays a noncovalent effector role in this substrate activating step.     

Amperometric glucose sensor based on coimmobilization of glucose oxidase and Poly(p-phenylenediamine) at a platinum microdisk electrode

A miniaturized glucose biosensor in which glucose oxidase (GOD) and poly(p-phenylenediamine) (poly-PPD) were coimmobilized at the surface of a platinum microdisk electrode was developed and used successfully for amperometric determination of glucose. The performance of sensors prepared at different monomer concentrations and polymerization potentials with different media was investigated in detail. It was found that similarly to poly(o-phenylenediamine) (poly-OPD), (poly-PPD) noticeably eliminated the electrochemical interference of ascorbic acid, uric acid, and l-cysteine. The amperometric response of glucose with the biosensor under optimal conditions exhibited a linear relationship in the range of 5.0 x 10(-5) to 3.0 x 10(-3) M with correlation coefficient 0.9995. According to the Michaelis-Menten equation, the apparent Michaelis constant for glucose and the maximum steady-state current density of the poly-PPD/GOD-modified microelectrode were 3.94 mM and 607.5 microA cm(-2), respectively. The current density of the sensor responding to glucose in the linear range can reach 160 microA cm(-2) mM(-1), which is far greater than that obtained using poly-OPD and poly(phenol) film. In addition, the stability of the sensor was examined over a 2-month period.