A Novel Catechol Oxidase Enzyme Electrode for the Specific Determination of Catechol

An enzyme electrode for the specific determination of catechol was developed by using catechol oxidase (EC 1.10.3.1) from eggplant (Solanum melangena L.) in combination with a dissolved oxygen probe. Optimization studies of the prepared catechol oxidase enzyme electrode established a phosphate buffer 50 mM at pH 7.0 and 35°C to provide the optimum conditions for affirmative electrode response. The enzyme electrode response depended linearly on a catechol concentration range of 5?10^-7-30?10^-5 M with a response time of 25 sec and substrate specificity of the catechol oxidase electrode of 100%. The biosensor retained its enzyme activity for at least 70 days.     

An ammonia-sensing air gap microelectrode

An ammonia-sensing air gap microelectrode has been designed on the basis of a neutral carrier pH-sensing inner electrode. This electrode has a tip diameter of 2 to 5 microns, has a simple design, is easy to fabricate, and has a long shelf life. Its response to ammonium is linear in the range 3 x 10(-5) to 10(-2) M and its response time (95%) is 10 to 15 s. The electrode was converted to a microsensor for urea by immobilization of urease within its tip. The linear response to urea ranged from 3 x 10(-4) to 10(-2) M and the response time was 15 to 20 s.     

Study and application of a new adenosine electrode with thymus tissue.

A new adenosine-selective membrane electrode using rabbit thymus tissue as catalyst is described. A typical response slope of 51.2 mV per concentration decade is observed over a linear range which extends from 3.16 x 10(-5) M to 5.62 x 10(-3) M. Detection limits of 2.99 x 10(-5) M have been established. Measured response times are 7 min. The coefficient of variation ranged from 1 to 5.62% (n = 7, m = 5). Fourteen compounds were specifically tested as possible interferents, but no significant response was observed. The standard recoveries of adenosine were from 95.3 to 104.0% (m = 5, n = 5), and the recoveries of adenosine in rabbit blood ranged from 94.0 to 108.4% (n = 3, m = 5) over the linear range. This tissue-based biosensor has excellent sensitivity and selectivity, and has additional advantages of simplicity and low cost. The biosensor can be used to measure directly the concentration of adenosine in body fluid samples without sample processing.     

A new film for the fabrication of an unmediated H2O2 biosensor

A novel and stable film made from polyethylene glycol (PEG) on pyrolytic graphite (PG) electrode was presented in this paper for incorporating horseradish peroxidase (HRP) to study the direct electrochemistry of the enzyme. In PEG film, HRP showed a thin-layer electrochemistry behavior. The apparent standard potential (E degrees ') was -0.379 V versus SCE at pH 7.2. Moreover, the PEG-HRP modified electrode exhibited excellent electrocatalytical response to the reduction of H2O2 with a calibration range between 2.0 x 10(-6) and 6.0 x 10(-4) M and a good linear relation from 2.0 x 10(-6) to 1.0 x 10(-4) M, on which an unmediated H2O2 biosensor was based. The detection limit of 6.7 x 10(-7) M was estimated when the signal-to-noise ratio was 3. The relative standard deviation (R.S.D.) was 4.7% for six successive determinations at a concentration of 4.0 x 10(-5) M. The apparent Michaelis-Menten constant (Km app) of the sensor was found to be 1.38 mM. Epinephrine, dopamine, and ascorbic acid did not interfere with the sensitive determination of H2O2.     

Real-time investigation of the interaction between primaquine phosphate and bovine serum albumin (BSA) by piezoelectric quartz crystal impedance analysis

Real-time investigation of the interaction between primaquine phosphate and bovine serum albumin by the piezoelectric quartz crystal impedance (PQCI) analysis was carried out for the first time. Three kinds of electrodes were investigated. Compared with bare gold (Au) electrode, the gold electrode self-assembled of nanogold colloids exhibits maintained biocompatibility, increased capacity and more bioactivity. Additionally, on the basis of the multi-dimensional information provided by the PQCI analysis, the real-time interaction information and the kinetics of the binding process was investigated and a response model was deduced. At 37 degrees C, the binding rate (k1), dissociation rate (k(-1)) and equilibrium constants (Ka) were 4.19x10(2) (mol l(-1))(-1) s(-1), 1.01x10(-3) s(-1) and 4.15x10(5) (mol l(-1))(-1) for the electrode modified by nanogold particles; 3.83x10(2) (mol l(-1))(-1) s(-1), 9.70x10(-4) s(-1) and 3.95x10(5) (mol l(-1))(-1) for the bare gold electrode, respectively.     

Highly selective oxalate-membrane electrode based on 2,2'-[1,4-butandiyle bis(nitrilo propylidine)]bis-1-naphtholato copper(II)

A new oxalate-selective electrode based on the complex 2,2'-[1,4-butandiyle bis(nitrilo propylidine)]bis-1-naphtholato copper(II) (CuL) as the membrane carrier was developed. The electrode exhibited a good Nernstian slope of -29.2+/-0.6 mV/decade (mean value+/-standard deviation, n=5) and a linear range of 5.0 x 10(-8) to 1.0 x 10(-1)M for oxalate. The limit of detection was 5.0 x 10(-8)M. This electrode represents a fast response time (i.e. 10-15s) and could be used for more than 3 months. The selectivity coefficients were determined by the fixed interference method (FIM) and could be used in the pH range of 2.0-7.0. It was employed as an indicator electrode for the determination of oxalate in water samples.     

Amperometric determination of choline released from rat submandibular gland acinar cells using a choline oxidase biosensor

A choline (CHO) biosensor based on the determination of H(2)O(2) generated at the electrode surface by the enzyme choline oxidase (CHOx) was developed. The biosensor consisted of CHOx retained onto a horseradish peroxidase (HRP) immobilized solid carbon paste electrode (sCPE). The HRPsCPE contained the molecule phenothiazine as redox mediator and CHOx was physically retained on the electrode surface using a dialysis membrane. Several parameters have been studied such as, mediator amount, influence of applied potential, etc. The CHO measurements were performed in 0.1 M phosphate buffer, pH 7.4. Amperometric detection of CHO was realized at an applied potential of 0.0 mV vs Ag/AgCl. The response is linear over the concentration range 5.0x10(-7)-7.0x10(-5) M, with a detection limit of 1.0x10(-7) M. This biosensor was used to detect choline released from phosphatidylcholine (PC) by phospholipase D (PLD) in isolated rat salivary gland cells stimulated by a purinergic agonist (ATP).     

Hyphomicrobium bacterial electrode for determination of monomethyl sulfate

A biosensor consisting of physically entrapped monomethyl sulfate (methyl sulfate) degrading bacterium, Hyphomicrobium MS 219, and a combined glass electrode has been developed for the determination of methyl sulfate. The response of the bacterial electrode is linear between 2.5 x 10(-2)M and 6.3 x 10(-1)M methyl sulfate with an effective response to concentrations as low as 10(-3)M and as high as 1M methyl sulfate. The probe has an average slope of 8 mV per concentration decade over the linear range. Response times vary from 5 min in the linear range to 30 min at the detection limit. The sensor has a lifetime of at least 1 week and shows high selectivity to methyl sulfate in the absence of other growth substrates.     

Direct electrochemistry of lactate dehydrogenase immobilized on silica sol-gel modified gold electrode and its application

The direct electrochemistry of lactate dehydrogenase (LDH) immobilized in silica sol-gel film on gold electrode was investigated, and an obvious cathodic peak at about -200 mV (versus SCE) was found for the first time. The LDH-modified electrode showed a surface controlled irreversible electrode process involving a one electron transfer reaction with the charge-transfer coefficient (alpha) of 0.79 and the apparent heterogeneous electron transfer rate constant (K(s)) of 3.2 s(-1). The activated voltammetric response and decreased charge-transfer resistance of Ru(NH(3))(6)(2+/3+) on the LDH-modified electrode provided further evidence. The surface morphologies of silica sol-gel and the LDH embedded in silica sol-gel film were characterized by SEM. A potential application of the LDH-modified electrode as a biosensor for determination of lactic acid was also investigated. The calibration range of lactic acid was from 2.0 x 10(-6) to 3.0 x 10(-5) mol L(-1) and the detection limit was 8.0 x 10(-7) mol L(-1) at a signal-to-noise ratio of 3. Finally, the effect of environmental pollutant resorcinol on the direct electrochemical behavior of LDH was studied. The experimental results of voltammetry indicated that the conformation of LDH molecule was altered by the interaction between LDH and resorcinol. The modified electrode can be applied as a biomarker to study the pollution effect in the environment.     

A new scheme of hybridization based on the Au(nano)-DNA modified glassy carbon electrode

DNA hybridization on the Au(nano)-DNA modified glassy carbon electrode (GCE) was investigated. The thiol modified probe oligonucleotides (SH-ssDNA) at the 5' phosphate end were assembled on the Au(nano)-DNA modified GCE surface. The electrochemical response of the probe immobilization and hybridization with target DNA was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. Gold nanoparticles can be dispersed effectively on the GCE surface in the presence of calf thymus DNA. Au(nano)-DNA modified GCE could greatly increase the active sites and enhance the response signal during immobilization and hybridization. The hybridization amount of target DNA could be greatly increased. The linear detection range of Au(nano)-DNA electrode for the complementary 21-mer oligonucleotide (cDNA) was achieved from 1.52 x 10(-10) to 4.05 x 10(-8) mol L(-1). The detection limit could reach the concentration of 10(-10) mol/L.     

Preparation of novel mercury-doped silver nanoparticles film glassy carbon electrode and its application for electrochemical biosensor

A novel mercury-doped silver nanoparticles film glassy carbon (Ag/MFGC) electrode was prepared in this study. Electrochemical behaviors of cysteine on the Ag/MFGC electrode were investigated by electrochemical impedance spectroscopy and cyclic voltammetry (CV). The results indicated that cysteine could be strongly adsorbed on the surface of the Ag/MFGC electrode to form a thin layer. The doped electrode could catalyze the electrode reaction process of cysteine, and the cysteine displayed a pair of well-defined and nearly reversible CV peaks at the electrode in an acetate buffer solution (pH 5.0). The Ag/MFGC electrode was used for determination of cysteine by differential pulse voltammetry. The linear range was between 4.0x10(-7) and 1.3x10(-5) mol/L, with a detection limit of 1.0x10(-7) mol/L and a signal-to-noise ratio of 3. The relative standard deviation was 2.4% for seven successive determinations of 1.0x10(-5) mol/L cysteine. The determinations of cysteine in synthetic samples and urinal samples were carried out and satisfactory results were obtained. Amperometric application of the Ag/MFGC electrode as biosensors is proposed.     

A piezoelectric quartz crystal impedance study on Cu(2+)-induced precipitation of bovine serum albumin in aqueous solution

Piezoelectric quartz crystal impedance (QCI) technique was used for monitoring the Cu(2+)-induced precipitation of bovine serum albumin onto the gold electrode. The critical precipitate concentration of Cu(2+) reflected by the significant decrease in the resonant frequency was estimated to be 9.98 x 10(-5) mol x l(-1), and the saturated adherence of the precipitate on the electrode occurred when the Cu(2+) concentration was greater than 9.79x10(-3) mol x l(-1). The frequency shift in air was about 85.5% of that in liquid, and the Deltaf(0)/DeltaR(1) ratio found in solution was 82.67 Hz Omega(-1), suggesting that the frequency response was predominated by the mass change due to precipitate adherence to the electrode surface. The response of the resonant frequency was analyzed using an equation Deltaf=a(0) + a(1) e(-t/tau(1)) + a(2) e(-t/tau(2)). The relationship between the total a(0) values and the Cu(2+) concentration was discussed.     

HRP/[Zn-Cr-ABTS] redox clay-based biosensor: design and optimization for cyanide detection

A novel inexpensive and simple amperometric biosensor, based on the immobilization of HRP into redox active [Zn-Cr-ABTS] layered double hydroxide, is applied to the determination of cyanide. The electrochemical transduction step corresponds to the reduction at 0.0 V of ABTS+* enzymatically formed in the presence of H2O2. The biosensor has a fast response to H2O2 (8s) with a linear range of 1.7 x 10(-9) to 2.1 x 10(-6) M and a sensitivity of 875 mA M(-1) cm(-2). The apparent Michaelis-Menten constant (KMapp) is 12 microM. The detection of cyanide is performed via its non competitive inhibiting action on the HRP/[Zn-Cr-ABTS] electrode. The concentration range of the linear response and the apparent inhibition constant (ki) are 5 x 10(-9) to 4 x 10(-8) and 1.4 x 10 (-7) M, respectively.     

Au-nanoparticles as an electrochemical sensing platform for aptamer-thrombin interaction

A novel electrochemical method for the detection of bioaffinity interactions based on a gold-nanoparticles sensing platform and on the usage of stripping voltammetry technique was developed. The oxidation of gold surface (resulted in gold oxide formation) upon polarization served as a basis for analytical response. As a model, thrombin-thrombin binding aptamer couple was chosen. The aptamer was immobilized on a screen-printed electrode modified with gold-nanoparticles by avidin-biotin technology. Cathodic peak area was found proportional to thrombin quantity specifically adsorbed onto electrode surface. Sigmoid calibration curve as is typical for immunoassay was obtained, with thrombin detection limit of 10(-9)M. Linear range corresponds from 10(-8) to 10(-5)M thrombin concentration or 2 x 10(-14) to 2 x 10(-11)mol/electrode (R=0.996). Binding of thrombin to an aptamer has also been detected using the ferricyanide/ferrocyanide redox couple as electrochemical indicator.     

Differential pulse anodic voltammetric determination of pantoprazole in pharmaceutical dosage forms and human plasma using glassy carbon electrode

Pantoprazole is used as an anti-ulcer drug through inhibition of H(+), K(+)-adenosine 5(')-triphosphatase in gastric parietal cells. It reduces the gastric acid secretion regardless of the nature of stimulation. The use of differential pulse voltammetry for the determination of pantoprazole in pharmaceutical dosage forms and human plasma using a glassy carbon electrode has been examined. The best voltammetric response was reached for a glassy carbon electrode in Britton-Robinson buffer solution of pH 5.0 submitted to a scan rate of 20.0 mVs(-1) and a pulse amplitude of 50.0 mV. This electroanalytical procedure was able to determine pantoprazole in the concentration range 6.0 x 10(-6)-8.0 x 10(-4)M. Precision and accuracy of the developed method was checked with recovery studies. The limit of detection and limit of quantitation were found to be 4.0 x 10(-7) and 9.0 x 10(-7)M, respectively. Rapidity, precision, and good selectivity were also found for the determination of pantoprazole in pharmaceutical dosage forms and human plasma. For comparative purposes high-performance liquid chromatography with a diode array and UV/VIS detection at 290.0 nm determination also was developed.     

The solubility of calcium oxalate in tissue culture media

The equilibrium parameters for calcium oxalate solubility in tissue culture media were investigated because of the current interest in oxalate toxicity. The calcium selective ion electrode methodology was evaluated and calcium concentrations from potentiometric calculations were verified by d-c argon plasma emission spectroscopy. The experimental K(sp)'s at 25 degrees C for Dulbecco's modified Eagle media and McCoys 5A media are equivalent to the literature K(sp) of 2.3 x 10(-9) for low ionic strength. The equilibrium concentration products, [Ca2+] [C2O2-(4)], are ten times higher than the K(sp)'s due to the high ionic strengths of tissue culture media. At 37 degrees C, addition of soluble oxalate at the 10(-3) to 10(-4) M level causes >50% precipitation of the oxalate resulting in equilibrium oxalate concentrations of less than 6 x 10(-5) M. This relatively inexpensive selective ion technique allows the determination of oxalate concentrations in equilibrium-saturated media which are substantially less than those calculated by the amount of soluble oxalate added to the media.     

Iron(II) phthalocyanine-modified carbon-paste electrode for potentiometric detection of ascorbic acid

A chemically modified electrode constructed by incorporating iron(II) phthalocyanine [Fe(II)Pc] into carbon-paste matrix was used as a sensitive potentiometric sensor for detection of ascorbic acid. The resulting electrode exhibits catalytic properties for the electrooxidation of ascorbic acid, and lowers the overpotential for the oxidation of this compound. The faster rate of electron transfer results in a near-Nernstian behavior of the modified electrode, and makes it a suitable potentiometric sensor for detection of ascorbic acid. A linear response in concentration range from 10(-6) to 10(-2) M (0.18--1800 microg ml(-1)) was obtained with a detection limit of 5 x 10(-7) M for the potentiometric detection of ascorbic acid. The modified electrode was used for the determination of ascorbic acid in vitamin preparations. The recovery was 97.2--102.4% for the vitamin added to the preparations with a relative standard deviation of less than 5%. The modified electrode exhibited a fast response time (<10 s),had good stability, and had an extended lifetime.     

Simultaneous voltammetric measurement of ascorbic acid, epinephrine and uric acid at a glassy carbon electrode modified with caffeic acid

A stable electroactive thin film of poly(caffeic acid) has been deposited on the surface of a glassy carbon electrode by potentiostatic technique in an aqueous solution containing caffeic acid. Poly(caffeic acid) was used as a modified electrode for the detection of ascorbic acid (AA), epinephrine (EP), uric acid (UA) and their mixture by cyclic voltammetry. This modified electrode exhibits potent and persistent electron-mediating behavior followed by well-separated oxidation peaks towards AA, EP and UA with activation overpotential. For the ternary mixture containing AA, EP and UA, the three compounds can well separate from each other at the scan rate of 20 mVs(-1) with a potential difference of 156, 132 and 288 mV between AA and EP, EP and UA and AA and UA, respectively, which was large enough to determine AA, EP and UA individually and simultaneously. The catalytic peak current obtained, was linearly dependent on the AA, EP and UA concentrations in the range of 2.0 x 10(-5) to 1.0 x 10(-3) mol l(-1), 2.0 x 10(-6) to 8.0 x 10(-5) mol l(-1) and 5.0 x 10(-6) to 3.0 x 10(-4) mol l(-1), and the detection limits for AA, EP and UA were 7.0 x 10(-6), 2.0 x 10(-7) and 6.0 x 10(-7) mol l(-1), respectively. The modified electrode shows good sensitivity, selectivity and stability, and has been applied to the determination of EP in practical injection samples and that of EP, UA and AA simultaneously with satisfactory results.     

A sensitive determination of estrogens with a Pt nano-clusters/multi-walled carbon nanotubes modified glassy carbon electrode

On the top of a multi-walled carbon nanotubes (MWNTs) modified glassy carbon electrode (MWNTs/GCE), Pt nanoclusters were electrochemically deposited, fabricating a Pt/MWNTs composite modified electrode, Pt/MWNTs/GCE. X-ray photoelectron spectroscopy, powder X-ray diffraction and field emission scanning electron microscope were used for the surface characterization of the electrode, and demonstrated the formation and distribution of Pt clusters of Pt nanoparticles of 8.4 nm in averaged size in the MWNTs matrix. The preliminary study found that this composite modified electrode has strong electrocatalytic activity toward the oxidation of estrogens involving estradiol, estrone and estriol. The voltammetric behavior of estrogens on this electrode was investigated by cyclic voltammetry, linear sweep voltammetry and square-wave voltammetry. In comparison with the MWNTs/GCE or a Pt nanoparticles modified GCE prepared in the similar way, this composite modified electrode exhibited much higher current sensitivity and catalytic activity. This electrode is also stable. The linear range of square-wave voltammetric determination was 5.0 x 10(-7)-1.5 x 10(-5)mol/L for estradiol, 2.0 x 10(-6)-5.0 x 10(-5)mol/L for estrone, and 1.0 x 10(-6)-7.5 x 10(-5)mol/L for estriol. Under an assumption that the concentration ratio of estradiol:estrone:estriol is 2:2:1, the real sample of blood serums was tested for the determination using this electrode. Satisfactory result was obtained with averaged recovery of 105%.     

Electrochemiluminescence determination of codeine or morphine with an organically modified silicate film immobilizing Ru(bpy)3(2+).

An ECL approach was developed for the determination of codeine or morphine based on tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)(3)(2+)) immobilized in organically modified silicates (ORMOSILs). Tetramethoxysilane (TMOS) and dimethyldimethoxysilane (DiMe-DiMOS) were selected as co-precursors for ORMOSILs, which were then immobilized on a surface of glassy carbon electrode (GCE) by a dip-coating process. Ru(bpy)(3)(2+) was immobilized in the ORMOSIL film via ion-association with poly(p-styrenesulphonate). The ORMOSIL-modified GCE presented good electrochemical and photochemical activities. In a flow system, the eluted codeine or morphine was oxidized on the modified GCE and reacted with immobilized Ru(bpy)(3)(2+) at a potential of +1.20 V (vs. Ag/AgCl). The modified electrode was used for the ECL determination of codeine or morphine and showed high sensitivity. The calibration curves were linear in the range 2 x 10(-8)-5 x 10(-5) mol/L for codeine and 1 x 10(-7)-3 x 10(-4) mol/L for morphine. The detection limit was 5 x 10(-9) mol/L for codeine and 3 x 10(-8) mol/L for morphine, at signal:noise ratio (S:N)=3. Both codeine and morphine showed reproducibility with RSD values <2.5% at 1.0 x 10(-6) mol/L. Furthermore, the modified electrode immobilized Ru(bpy)(3)(2+) was applied to the ECL determination of codeine or morphine in incitant samples.