Ascorbic acid biosensor using ascorbate oxidase immobilized on alkylamine glass beads

A biosensor for ascorbic acid based on enzyme kinetics of ascorbate oxidase (E.C.1.10.3.3.) was developed. The enzyme was extracted fromCucurbita maxima, or jerimun and immobilized by covalent bounding, using glutaradehyde as a bifunctional agent, on alkylamine glass beads, with and without enzyme active site protection. A low-cost, home-made oxygen electrode was applied as a transducer. The system has sensitivity from 62.5 up to 500 μM of ascorbic acid with satisfactory operation for more than 2 mo.     

Test films for test-determinations on the base of reagents, immobilized in gelatinous gel

Gelatinous solidified layers of the photographic film were used for the immobilization of analytical reagents for detection and determination of reductants and primary aromatic amines. It was shown, that the films with immobilized iron(III)-Dipy or iron(III)-Phen complexes as test films for reductants and films with immobilized aldehydes (vanillin, p-dimethylaminobenzaldehyde) as the test films for primary aromatic amines can be used. The improving of reagents immobilization in the presence of sodium dodecylsulfate micelles was obtained. Metrological characteristics of visual detection and photometric determination using test films were evaluated on the basis of statistical approach and investigation of detection probability distribution in the concentration range of unreliable reaction. The suggested test films for the determination of ascorbic acid, analgin (dipyrone), novocaine and streptocide in drugs were examined successfully.     

Fabrication of dissolved O2 metric uric acid biosensor using uricase epoxy resin biocomposite membrane

Uricase purified from 20-day-old leaves of cowpea was immobilized on to epoxy resin membrane with 80% retention of initial activity of free enzyme and a conjugation yield of 0.056 mg/cm². The uricase epoxy resin bioconjugate membrane was mounted over the sensing part of the combined electrode of ‘Aqualytic’ dissolved O₂ (DO) meter to construct a uric acid biosensor. The biosensor measures the depletion of dissolved O₂ during the oxidation of uric acid by immobilized uricase, which is directly proportional to uric acid concentration. The biosensor showed optimum response within 10-12 s at a pH 8.5 and 35 °C. A linear relationship was found between uric acid concentration from 0.025 to 0.1 mM and O₂ (mg/l) consumed. The biosensor was employed for measurement of uric acid in serum. The mean value of uric acid in serum was 4.92 mg/dl in apparently healthy males and 3.11 mg/dl in apparently healthy females. The mean analytic recoveries of added uric acid in reaction mixture (8.9 and 9.8 mg/dl) were 93.6 ± 2.34 and 87.18 ± 3.17% respectively. The within and between batch CVs were <6.5 and <5.0%, respectively. The serum uric acid values obtained by present method and standard enzymic colorimetric method, showed a good correlation (r = 0.996) and regression equation being y = 0.984x + 0.0674. Among the various metabolites tested only, glucose (11%), urea (38%), NaCl (25%) and cholesterol (13%) and ascorbic acid (56%) caused decrease, while, MgSO₄ and CaCl₂ had no effect on immobilized enzyme. The enzyme electrode showed only 32% decrease during its use for 100 times over a period of 60 days at 4 °C.     

Potentiometric determination of glucose by enzymatic oxidation in a flow system

A potentiometric determination is described for glucose based on oxidation by 1,4-benzoquinone with immobilized glucose oxidase as catalyst in an enzyme reactor. The electrode is preceded by an analytical dialysis unit to remove proteins. The ratio of quinone to hydroquinone was measured with a flow-through gold electrode. Another gold electrode preceded the enzyme reactor to correct for serum components (e.g. ascorbic acid) which can also reduce quinone. The operating range is 0.04–10 × 10^-3 M β-D-glucose. The dialysis proceeds with a linear dependence on glucose concentration, and the dialysis ratio can be adjusted by changing the buffer flow rate.     

Determination of ascorbic acid based on a peroxidase oscillator reaction

This paper reports a new method for determination of ascorbic acid based on a peroxidase-oxidase (PO) biochemical oscillator reaction. The oscillation period changes linearly with concentration of ascorbic acid in the range 0.175–17.5 μM with a correlation coefficient of 0.9982 (n=6) based on oxygen measurement with an oxygen electrode. The results compared well with those obtained by an AOAC procedure.     

On‐Line Monolithic Enzyme Reactor Fabricated by Sol‐Gel Process for Elimination of Ascorbic Acid While Monitoring Dopamine

Catecholamine, a well‐known neurotransmitter, is released to restore heart function after the occurrence of cardiac ischemia. Endogenous ascorbic acid interferes considerably with the monitoring of neurotransmitters such as catecholamine and glutamate. In this work we have successfully developed a nanoliter volume monolithic enzyme reactor and integrated it with a carbon film electrode for monitoring dopamine in order to scavenge such electroactive interferents as ascorbic acid. A monolithic silica support prepared by the sol‐gel process has a large through‐pore structure and a nanoporous surface. The low back pressure resulting from the large though‐pore structure makes it possible to immobilize the enzyme and introduce fluid into the flow monitoring system by using a microsyringe pump. Ascorbate oxidase (AOx) was immobilized on the nanoporous surface of a monolithic matrix by physical adsorption and used for converting the ascorbic acid into its electrochemically inert form, dehydroascorbic acid, while monitoring dopamine. We showed that more than 99.8% of 100 μM of ascorbic acid could be converted to its oxidized form in the monolithic enzyme reactor. We succeeded in monitoring dopamine at a concentration of less than 100 nM in the presence of 50 μM of ascorbic acid. Therefore, this work demonstrated that a monolithic silica support offers excellent potential in regards to realizing a highly selective enzymatic reactor for biosensors.     

A simple and selective sensor for the determination of ascorbic Acid in vitamin C tablets based on paptode.

A method for the determination of ascorbic acid in vitamin C tablets based on a very simple paptode design on TLC strips is described. This procedure is based on the reduction of iron(III) with ascorbic acid and the formation of a colorful red complex with immobilized 2,2'-dipyridyl (dipy) on TLC strips. The linear range of the system was 20-200 ppm with a detection limit of 1 ppm and a relative standard deviation of 1.5% (n = 28). The parameters, such as pH, concentration of iron(III), concentration of dipy and the volume of dipy per 1 cm(2) of TLC strips, were optimized. The proposed sensor was successfully applied for the determination of ascorbic acid in vitamin C tablets.     

Reagentless Glucose Biosensor Based on the Direct Electrochemistry of Glucose Oxidase on Carbon Nanotube‐Modified Electrodes

The direct electrochemistry of glucose oxidase (GOD) was revealed at a carbon nanotube (CNT)‐modified glassy carbon electrode, where the enzyme was immobilized with a chitosan film containing gold nanoparticles. The immobilized GOD displays a pair of redox peaks in pH 7.4 phosphate buffer solutions (PBS) with the formal potential of about ?455 mV (vs. Ag/AgCl) and shows a surface‐controlled electrode process. Bioactivity remains good, along with effective catalysis of the reduction of oxygen. In the presence of dissolved oxygen, the reduction peak current decreased gradually with the addition of glucose, which could be used for reagentless detection of glucose with a linear range from 0.04 to 1.0 mM. The proposed glucose biosensor exhibited high sensitivity, good stability and reproducibility, and was also insensitive to common interferences such as ascorbic and uric acid. The excellent performance of the reagentless biosensor is attributed to the effective enhancement of electron transfer between enzyme and electrode surface by CNTs, and the biocompatible environment that the chitosan film containing gold nanoparticles provides for immobilized GOD.     

Microdialysis sampling and monitoring of uric acid in vivo by a chemiluminescence reaction and an enzyme on immobilized chitosan support membrane

A new detection system based on microdialysis sampling and chemiluminescence (CL) reaction was developed for in vivo monitoring of uric acid (UA) with high sensitivity, selectivity and accuracy. The uric acid is indirectly monitored by CL detection of enzymatic reaction product formation (H₂O₂), catalyzed by Uricase. A microprobe was modified and coated with immobilized enzyme through a Streptavidin-biotin mediated linker by using a chitosan support membrane, polyurethane trapped ferrocene film is employed to protect the probe surface and diminish the interference from reductant molecules, which often are present in the blood (e.g. ascorbic acid). The earlier mentioned probe and the constructed sensor can detect uric acid in the range of 0.01-1 mM with detection limit (3σ) of 5 μM. Finally, the system is used to monitor uric acid (UA) variation through an acute myocardial infarction (AMI) model. Following AMI-induced oxidative stress, the UA level decreases continuously, thus suggesting that UA plays a protective role as a substitute antioxidant. Furthermore, the in vivo monitoring results show good agreement with those obtained by a standard method, and the procedure is recommended for in vivo and real time monitoring of UA. In addition, the proposed method can be more accurate since the UA may be potentially oxidized by in vitro exposure to oxygen in the presence of a catalyst.     

Ascorbic Acid and Salicylic Acid Mitigate NaCl Stress in Caralluma tuberculata Calli

Plants exposed to salt stress undergo biochemical and morphological changes even at cellular level. Such changes also include activation of antioxidant enzymes to scavenge reactive oxygen species, while morphological changes are determined as deformation of membranes and organelles. Present investigation substantiates this phenomenon for Caralluma tuberculata calli when exposed to NaCl stress at different concentrations. Elevated levels of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) in NaCl-stressed calli dwindled upon application of non-enzymatic antioxidants; ascorbic acid (AA) and salicylic acid (SA). Many fold increased enzymes concentrations trimmed down even below as present in the control calli. Electron microscopic images accentuated several cellular changes upon NaCl stress such as plasmolysed plasma membrane, disruption of nuclear membrane, increased numbers of nucleoli, alteration in shape and lamellar membrane system in plastid, and increased number of plastoglobuli. The cells retrieved their normal structure upon exposure to non-enzymatic antioxidants. The results of the present experiments conclude that NaCl aggravate oxidative molecules that eventually alleviate antioxidant enzymatic system. Furthermore, the salt stress knocked down by applying ascorbic acid and salicylic acid manifested by normal enzyme level and restoration of cellular structure.     

Electrodeposited Cu‐Au Alloy Nanoparticles for Uric Acid Electrochemical Biosensor with Quick Response

A uric acid (UA) electrochemical biosensor based on the Cu‐Au alloy nanoparticles (NPs) and uricase was developed. The electrodeposition technique of Cu‐Au alloy NPs was selected to be a convenient potentiostatic method at –0.8 V in a single solution containing both Au(III) and Cu²⁺. Cyclic voltammetry and scanning electron microscopy proved the successful deposition of Cu‐Au alloy NPs. EIS demonstrated the good conductivity of Cu‐Au alloy NPs. The enzyme was immobilized on the surface of Cu‐Au alloy NPs modified electrode by casting with chitosan solution. The ultimate biosensor showed linear amperometric response towards UA in the concentration range of 3.0 to 26.0 μM with a detection limit of 0.8 μM. The main feature of the biosensor was its short response time, which was attributed to the good conductivity of Cu‐Au alloy NPs. Furthermore, the biosensor could avoid the interference of ascorbic acid and oxygen.     

Amperometric L-glutamate sensor using a novel L-glutamate oxidase from Streptomyces platensis NTU 3304

Streptomyces platensis NTU 3304, isolated from soil samples, produces extracellular L-glutamate oxidase in liquid culture. Strains of this species have never been reported to be able to produce this enzyme. The purified enzyme was immobilized onto a cellulose triacetate membrane which was held at an oxygen electrode. The sensor was specific to L-glutamate in accordance with the properties of the novel L-glutamate oxidase. The time required for each assay in batch operation was less than 3 min. A linear relationship is observed between the decrease in dissolved oxygen and the concentration of L-glutamate between 20 and 140 mg l^?1 (ca. 0.12 and 0.84 mM). The sensor retained 95% of its original activity after 400 assays over a period of 3 weeks. The sensor was applied to determine the concentration of L-glutamate in broth samples during L-glutamic acid fermentation. Good correlations were achieved between results obtained with the sensor and by enzymatic analysis using glutamate dehydrogenase.     

An amperometric uric acid biosensor based on chitosan-carbon nanotubes electrospun nanofiber on silver nanoparticles

A novel amperometric uric acid biosensor was fabricated by immobilizing uricase on an electrospun nanocomposite of chitosan-carbon nanotubes nanofiber (Chi–CNTsNF) covering an electrodeposited layer of silver nanoparticles (AgNPs) on a gold electrode (uricase/Chi–CNTsNF/AgNPs/Au). The uric acid response was determined at an optimum applied potential of ?0.35 V vs Ag/AgCl in a flow-injection system based on the change of the reduction current for dissolved oxygen during oxidation of uric acid by the immobilized uricase. The response was directly proportional to the uric acid concentration. Under the optimum conditions, the fabricated uric acid biosensor had a very wide linear range, 1.0–400 μmol L^?1, with a very low limit of detection of 1.0 μmol L^?1 (s/n?=?3). The operational stability of the uricase/Chi–CNTsNF/AgNPs/Au biosensor (up to 205 injections) was excellent and the storage life was more than six weeks. A low Michaelis–Menten constant of 0.21 mmol L^?1 indicated that the immobilized uricase had high affinity for uric acid. The presence of potential common interfering substances, for example ascorbic acid, glucose, and lactic acid, had negligible effects on the performance of the biosensor. When used for analysis of uric acid in serum samples, the results agreed well with those obtained by use of the standard enzymatic colorimetric method (P?>?0.05).

Ascorbic acid biosensor based on laccase immobilized on an electrode modified with a self-assembled monolayer and coated with functionalized quantum dots

Laccase was immobilized on an electrode modified with a cysteine self-assembled monolayer and coated with functionalized quantum dots. The immobilized laccase is capable of directly transferring an electron. Immobilized laccase retained its activity to oxidize ascorbic acid (AA), and the apparent Michaelis–Menten constant was found to be 0.47 mM. The modified electrode was used to determine AA in the 10 to 140 μM concentration range, with linear response curve and a detection limit of 1.4 μM (s/n?=?3).     

Preparation and application of a new glucose sensor based on iodide ion selective electrode

An electrode for glucose has been prepared by using an iodide selective electrode with the glucose oxidase enzyme. The iodide selective electrode used was prepared from 10% TDMAI and PVC according our previous study. The enzyme was immobilized on the iodide electrode by holding it at pH 7 phosphate buffer for 10 min at room temperature. The H₂O₂ formed from the reaction of glucose was determined from the decrease of iodide concentration that was present in the reaction cell. The iodide concentration was followed from the change of potential of iodide selective electrode. The potential change was linear in the 4×10⁻⁴ to 4×10⁻³ M glucose concentration (75-650 mg glucose/100ml blood) range. The slope of the linear portion was about 79 mV per decade change in glucose concentration. Glucose contents of some blood samples were determined with the new electrode and consistency was obtained with a colorimetric method. The effects of pH, iodide concentration, the amount of enzyme immobilized and the operating temperature were studied. No interference of ascorbic acid, uric acid, iron(III) and Cu(II) was observed. Since the iodide electrode used was not an AgI-Ag₂S electrode, there was no interference of common ions such as chloride present in biological fluids. The slope of the electrode did not change for about 65 days when used 3 times a day.     

Low‐Potential Detection of Glucose with a Biosensor Based on the Immobilization of Glucose Oxidase on Polymer/Manganese Oxide Layered Nanocomposite

A nanocomposite with poly(diallyldimethylammonium), PDDA, intercalated between manganese oxide layers is constructed on a graphite electrode surface through one‐step electrodeposition and used to adsorb glucose oxidase (GOD). The immobilized GOD displays a pair of stable and quasireversible redox peaks with a formal potential of ?468 mV in pH 7.0 buffer solutions and exhibits excellent electrocatalysis to the reduction of oxygen. In the presence of dissolved oxygen, the reduction peak current decreased gradually with the addition of glucose, indicating that the immobilized GOD kept its bioactivity. Thus a reagentless biosensor for glucose at a low detection potential was established. The linear concentration range is from 0.02 to 2.78 mM with a detection limit of 9.8 μM. The proposed glucose biosensor was insensitive to common interferences such as ascorbic and uric acids etc.     

Salmonella typhi determination using voltammetric amplification of nanoparticles: A highly sensitive strategy for metalloimmunoassay based on a copper-enhanced gold label

A highly sensitive electrochemical amplification immunoassay for Salmonella typhi (S. typhi) determination has been developed for the first time by using a copper-enhanced gold nanoparticle label coupled with anodic stripping voltammetry. Monoclonal antibodies for S. typhi were first immobilized on polystyrene microwells and then captured by S. typhi bacteria. After an immunoreaction occurred, a polyclonal, antibody-colloidal gold conjugate was added to bind to the S. typhi bacteria. Next, a copper-enhancer solution containing ascorbic acid and copper (II) sulfate was added into the polystyrene microwells. The ascorbic acid was employed to reduce the copper (II) ions to copper (0), which was subsequently deposited onto the gold nanoparticle tags. After the copper was dissolved in nitric acid, the released copper ions were detected by anodic stripping voltammetry. The amount of deposited copper was related to the amount of gold nanoparticle tag present, which was controlled by the amount S. typhi attached to the polyclonal antibody-colloidal gold conjugate. Therefore, the anodic stripping peak current was linearly dependent on the S. typhi concentration over concentration range of 1.30 × 10² cfu/mL to 2.6 × 10³ cfu/mL in a logarithmic plot, with a detection limit as low as 98.9 cfu/mL. The influences of the relevant experimental variables, such as the concentration of copper and the reaction time of S. typhi with antibody, were investigated. We also successfully applied this method to determine the presence of S. typhi in human serum. Our results are a step towards developing more sensitive and reliable nanoparticle immunoassays.     

Development of a bienzyme reactor sensor system for the determination of ornithine

A bienzyme reactor sensor system with amperometric detection was developed for the determination of ornithine. The system based on the immobilized enzymes (ornithine carbamyl transferase and pyruvate oxidase) consisted of a buffer tank, a peristaltic pump, an enzyme reactor, an oxygen electrode and a recorder. Then, 0.1 M MOPS buffer, containing pyruvic acid (0.5 mM) and carbamyl phosphate (0.5 mM), was continuously transferred into the system at 35 °C. Phosphate ion was formed enzymatically by transformation of ornithine in the presence of carbamyl phosphate. Pyruvate oxidase is activated by the presence of phosphate. Therefore, ornithine was determined from the oxygen consumed upon oxidation of pyruvic acid catalyzed by pyruvate oxidase in the presence of phosphate ion. The limit of detection was 0.05 mM and the response was linear to 3 mM (R²=0.9905). The variation coefficients were 4.9 (n=15) and 3.9% (n=15) for 1.1 and 3.0 mM standard ornithine, respectively. Good comparative results (R²=0.9238) were observed between ornithine contents in prawn muscle determined by the proposed system and by the HPLC. One assay was completed within 4 min. The immobilized enzymes were stable for 2 months at 4 °C and more than 150 samples could be continually determined using this enzyme reactor.     

Immobilization of monoamine oxidase on eggshell membrane and its application in designing an amperometric biosensor for dopamine

An amperometric biosensor for dopamine is described. It is based on the enzyme monoamine oxidase immobilized on a glutaraldehyde-activated eggshell membrane that was deposited on a glassy carbon electrode. The Michaelis-Menten constant (Km) is 0.087 mM. Optimum pH and temperature conditions were obtained at pH 7.0 and 37 °C, respectively. The sensor showed a detection limit of 20 μM, a linear range from 50 μM to 250 μM, and a storage stability of ~25 days. In order to further improve the performance, a Nafion coating was applied on the electrode surface which gave favorable results with respect to shelf life of the enzyme (~40 days), the limit of detection, and the selectivity over ascorbic acid and uric acid.     

Interaction of some antioxidants with Belousov-Zhabotinsky reaction based on catechol-BrO 3 − -Mn2+-H2SO4 system

Temporal evolution of a new Mn(II) catalyzed Belousov-Zhabotinsky (BZ) chemical oscillator with catechol (1.2-dihydroxybenzene) as organic substrate is reported within narrow range of concentrations of initial reagents at 30°C. After optimizing the oscillation parameters the system was perturbed with the antioxidants like ascorbic acid and inosine. It is found that ascorbic acid acts as co-substrate within certain concentration limit, whereas inosine acts as a quencher of oscillations. Addition of ascorbic acid to the BZ system decreases induction time thus acting synergistically to help the reaction to enter quickly into the oscillatory regime. A good linear dependence of induction time on the concentration of ascorbic acid (R ² = 0.9948) and inosine (R ² = 0.955) is reported. Inosine has been found to increase the induction time and quench the oscillations. It is mentioned that the magnitude of induction time decreases to a greater extent with ascorbic acid as compared to the magnitude of its increase with the same concentration of inosine. This is pointing to the fact that ascorbic acid is stronger antioxidant than inosine as depicted by their interaction with catechol-based BZ chemical oscillator. Temporal evolution of the BZ reaction with the injection of antioxidants at different stages of reaction is also reported.