共查询到20条相似文献,搜索用时 46 毫秒
1.
Anh TM Dzyadevych SV Soldatkin AP Duc Chien N Jaffrezic-Renault N Chovelon JM 《Talanta》2002,56(4):627-634
An enzyme biosensor for the determination of 4-chlorophenol in water solutions based on potentiometric pH-sensitive field-effect transistors as semiconductor transducer and tyrosinase immobilised in saturated glutaraldehyde vapours as biorecognition element has been described for the first time. The main analytical characteristics were studied under different conditions, as well as the possibility to optimise these working parameters. Different factors, such as pH of immobilisation, the enzyme loading and time of immobilisation in glutaralaldehyde vapours were investigated with regard to the influence on sensitivity, limit of detection, dynamic range, and operational and storage stability. The best result gives a limit of detection close to 20 ppm and a dynamic range from 25 to 1000 ppm with sensitivity 2 mV mM(-1). The operational stability was not less-than15 h and the R.S.D. were approximately 3% for intra-sensors responses and approximately 7% for inter-sensors responses. The storage stability was >15 days. 相似文献
2.
Karolina Labus Katarzyna Szymańska Jolanta Bryjak Andrzej B. Jarzębski 《Chemical Papers》2015,69(8):1058-1066
Tyrosinase from Agaricus bisporus was immobilised covalently on mesostructured siliceous foam (MCF) and three mesoporous silicas of SBA-15 type of different pore sizes, regarded as the reference, to reveal that MCF was the superior enzyme support. All the carriers were functionalised using 3-aminopropyltrimethoxysilane and the enzyme was attached covalently via glutaraldehyde or by simple adsorption and it was also cross-linked with glutaraldehyde in selected samples. The experiments indicated that only tyrosinase attached covalently was highly active and that postimmobilisation cross-linking slightly reduced its activity with no improvement in stability. MCF-bound tyrosinase was the best biocatalyst with monophenolase and diphenolase activities of 3627 U mL?1 and 53040 U mL?1 of carrier sediment, respectively. Inactivation studies at 55°C showed that MCF-bound tyrosinase was 20 times more stable than the native enzyme, whereas for typical SBA-15 it was only 12 times. A comparative study with other, non-siliceous enzyme supports indicated that aminated MCF appeared to be the carrier of choice for the covalent attachment of tyrosinase. 相似文献
3.
Developing a biosensor which is capable of simultaneously monitoring l-Dopa levels in multiple samples besides requiring small reaction volume is of great value. The present study describes the detection of l-Dopa using tyrosinase enzyme extracted from Amorphophallus campanulatus and immobilized on the surface of the microplate wells. Among the different approaches used for immobilizing tyrosinase onto the microplate wells, glutaraldehyde treatment was found to be most effective. Besides enzyme activity, ESEM–EDS (environmental scanning electron microscope–energy dispersive system) and Atomic Force Microscopy (AFM) were also carried out to confirm the immobilization of tyrosinase enzyme onto the microplate well surface. This immobilized biocomponent was then integrated with an optical transducer for l-Dopa detection and it showed good reproducibility. The sensing property of the system was studied by measuring the initial rate of dopachrome formation at 475 nm. The calibration plot gave a linear range of detection from 10–1000 μM and the detection limit was calculated to be 3 μM. The immobilized biocomponent was stable for 41 days and was reused up to nine times. Spiked samples (blood plasma) were also analyzed using this biocomponent. This microplate based biosensor thus provides a convenient system for detection of multiple samples in a single run. 相似文献
4.
《Electrophoresis》2017,38(3-4):486-493
A new method for screening tyrosinase inhibitors from traditional Chinese medicines (TCMs) was successfully developed by capillary electrophoresis with reliable online immobilized enzyme microreactor (IMER). In addition, molecular docking study has been used for supporting inhibition interaction between enzyme and inhibitors. The IMER of tyrosinase was constructed at the outlet of the capillary by using glutaraldehyde as cross‐linker. The parameters including enzyme reaction, separation of the substrate and product, and the performance of immobilized tyrosinase were investigated systematically. Because of using short‐end injection procedure, the product and substrate were effectively separated within 2 min. The immobilized tyrosinase could remain 80% active for 30 days at 4°C. The Michaelis–Menten constant of tyrosinase was determined as 1.78 mM. Kojic acid, a known tyrosinase inhibitor, was used as a model compound for the validation of the inhibitors screening method. The half‐maximal inhibitory concentration of kojic acid was 5.55 μM. The method was successfully applied for screening tyrosinase inhibitors from 15 compounds of TCM. Four compounds including quercetin, kaempferol, bavachinin, and bakuchiol were found having inhibitory potentials. The results obtained in this work were supported by molecular docking study. 相似文献
5.
Amperometric biosensing of carbamate and organophosphate pesticides utilizing screen-printed tyrosinase-modified electrodes 总被引:1,自引:0,他引:1
A tyrosinase (Tyr) screen-printed biosensor based on the electroreduction of enzymatically generated quinoid products was electrochemically characterized and optimized for determination of carbamates and organophosphorus pesticides. A composite electrode prepared by screen-printing a cobalt (II) phthalocyanine (CoPc) modified cellulose-graphite composite on a polycarbonate support was employed as electrochemical transducer. The Tyr biosensor was prepared by immobilization of enzyme on the composite electrode surface by cross-linking with glutaraldehyde and bovine serum albumin. Parameters affecting the biosensor response such as response time, enzyme loading, concentration and pH of the buffer solution were optimized utilizing catechol as substrate. The maximum response for o-quinone enzymatically generated was obtained after 2 min of reaction. A good reproducibility and high operational stability were found for Tyr biosensor (60 units) at 50 mM phosphate buffer, pH 6.50. Under these conditions, the useful lifetime of biosensor was 10 days. After 15 days, the biosensor could be used with 20% of the initial value. Inhibition studies on the o-quinone steady-state current (at −0.20 V versus Ag/AgCl) were performed to investigate the inhibition kinetics of the pesticides in the enzymatic activity of mushroom tyrosinase. The results shown that the methyl parathion and carbofuran can lead to competitive inhibition process of the enzyme, while diazinon and carbaryl act as mixed inhibitors. Linear relationships were found for methyl parathion (6-100 ppb), diazinon (19-50 ppb), carbofuran (5-90 ppb) and carbaryl (10-50 ppb). Analysis of natural river water samples spiked with 30 ppb of each pesticide showed recoveries between 92.50% and 98.50% and relative standard deviations of 2%. 相似文献
6.
Jolanta Kochana Katarzyna Hnida Grzegorz Sulka Paweł Knihnicki Joanna Kozak Agnieszka Gilowska 《Chemical Papers》2015,69(8):1130-1135
Polypyrrole nanowires (PPyNWs) were fabricated and examined as a structural component of amperometric biosensor matrix. An enzyme, tyrosinase (TYR), was immobilized onto PPyNWs using glutaraldehyde (GA). Matrix composite morphology was investigated using scanning electron microscopy. Electrochemical behavior of the prepared PPyNWs/GA/TYR biosensor towards catechol was studied and the assessment of its analytical characteristics was carried out taking into account linear range, sensitivity, repeatability, reproducibility and operational stability. 相似文献
7.
Recent research performed in our laboratory (using a butyrylcholinesterase + choline oxidase enzyme electrode) suggested the validity of the biosensor approach using enzyme inhibition OPEEs (i.e. enzyme electrodes working in organic phase) in the case of organophosphorus and carbamate pesticides, which are poorly soluble in aqueous solutions. Since these pesticides are generally much more soluble in chloroform than in water, the present research aimed at analysing this class of pesticides using a tyrosinase inhibition OPEE operating in water-saturated chloroform medium. The tyrosinase biosensor was assembled using an oxygen amperometric transducer coupled to the tyrosinase enzyme, immobilized in kappa-carrageenan gel. Lastly a detailed comparison between the inhibition monoenzymatic tyrosinase and inhibition bienzymatic (butyrylcholinesterase + choline oxidase) OPEEs was performed and discussed in this work. 相似文献
8.
Tyrosinase from a plant source Amorphophallus companulatus was immobilized on eggshell membrane using glutaraldehyde. Among the three different approaches used for immobilization, activation of eggshell membrane by glutaraldehyde followed by enzyme adsorption on activated support could stabilize the enzyme tyrosinase and was found to be effective. Km and Vmax values for dopamine hydrochloride calculated from Lineweaver-Burk plot were 0.67 mM and 0.08 mM min−1, respectively. Studies on effect of pH showed retention of more than 90% activity over a pH range 5.0-6.5. Membrane bound enzyme exhibited consistent activity in the temperature range 20-45 °C. Shelf life of immobilized tyrosinase system was found to be more than 6 months when stored in phosphate buffer at 4 °C. An electrochemical biosensor for dopamine was developed by mounting the tyrosinase immobilized eggshell membrane on the surface of glassy carbon electrode. Dopamine concentrations were determined by the direct reduction of biocatalytically liberated quinone species at −0.19 V versus Ag/AgCl (3 M KCl). Linearity was observed within the range of 50-250 μM with a detection limit of 25 μM. 相似文献
9.
Tyrosinase was immobilized on Chitosan (CTS) beads to produce 3,4‐dihydroxy‐L‐phenylalanine (L ‐DOPA) from L ‐tyrosine. Epichlorohydrin (ECH), ethylene glycol diglycidyl ether (EGDE), and glutaraldehyde (GLU) were used as coupling agents, respectively. Ultraviolet/visible measurements on CTS films showed that the reaction intermediate (L ‐dopaquinone) attacked the amino groups on CTS, so the amine residues on chitosan were capped by acetic acid anhydride (Ac) or formaldehyde (Fm) to avoid the deactivation of the immobilized tyrosinase. The pH and temperature of the maximal rate to produce L‐DOPA were investigated. GLU (coupling agent) and Ac (capping agent) were selected for practical utility. A 7.5% (w/v) concentration of GLU was found to attain maximal activity of the immobilized enzyme. The thermal stability of tyrosinase immobilized on CTS‐GLU‐Ac, and after treatment with sodium borohydride, was enhanced to a great extent. The L ‐DOPA converting efficiency in the environmental conditions of this study decreased from 45.1% to 39.9% (between 1st and 30th batch). This immobilized tyrosinase can be used practically in the production of L‐DOPA from L‐tyrosine. 相似文献
10.
D. Mruga Dr. O. Soldatkin K. Paliienko A. Topcheva Dr. N. Krisanova D. Kucherenko Prof. Dr. T. Borisova Dr. S. Dzyadevych Dr. A. Soldatkin 《Electroanalysis》2021,33(5):1299-1307
Today, the concentration of glutamate in the patient′s blood is an important indicator in medical diagnostics; therefore, it is necessary to have a simple, accurate, and fast tool to obtain the data. Here, a recently developed amperometric glutamate-sensitive biosensor was optimized to improve its characteristics. The platinum disk electrode was used as a transducer. As a bioselective element we used the enzyme glutamate oxidase, covalently crosslinked with bovine serum albumin by glutaraldehyde. Circumstances of enzyme immobilization on the transducer‘s surface were optimized (enzyme and glutaraldehyde concentrations and immobilization duration). To test the ability of this biosensor to work in biological environments containing complex biological substances, the influence of the working solution was investigated (concentration of the working buffer, its temperature, presence of the protein in the analyzed sample). The linear range of biosensor was from 5 to 600 μM of glutamate and the sensitivity was 150–200 nA/mM. Measurements of glutamate concentrations in the blood plasma were performed by biosensor and glutamate dehydrogenase assay. The linear correlation between the methods was found in a range of 50.4–182.5 μM (R2=0.99). Thus, it has been shown that the developed biosensor makes it possible to measure the concentration of glutamate in blood plasma. 相似文献
11.
Mignani A Luciano G Lanteri S Leardi R Scavetta E Tonelli D 《Analytica chimica acta》2007,599(1):36-40
An amperometric glucose biosensor was developed using an anionic clay matrix of hydrotalcitic nature (Ni/Al-NO3 HT) as enzyme support, which was electrochemically synthesized at −0.90 V versus SCE, using a rotating disk Pt electrode to assure homogeneity of the electrodeposition suspension. The biorecognition element was glucose oxidase (GOx) immobilized on HT during the electrosynthesis, which was followed by cross-linking with glutaraldehyde vapours to avoid the enzyme release.The performances of the biosensor, in terms of sensitivity to glucose calculated from the slope of the calibration curve, are dependent on parameters related to the electrodeposition.An experimental design was applied to detect the optimal conditions of electrosynthesis in order to optimize the glucose biosensor performance. The factors taken into account were enzyme concentration and Ni/Al molar ratio. A full factorial design was performed to study linear interactions between factors and their quadratic effects and the optimal setup was evaluated by the isoresponse curves. The significant factors were enzyme concentration (linear and quadratic terms) and the interaction between enzyme concentration and Ni/Al molar ratio. Under the optimized electrodeposition conditions, the reproducibility of the biosensor fabrication was very good, being the RSD of the sensitivity about 5%. 相似文献
12.
Carvalho Gabriela M. J. Alves Tito Lívio M. Freire Denise M. G. 《Applied biochemistry and biotechnology》2000,84(1-9):791-800
The production of l-DOPA using l-tyrosine as substrate, the enzyme tyrosinase (EC 1.14.18.1) as biocatalyst, and l-ascorbate as reducing agent for the o-quinones produced by the enzymatic oxidation of the substrates was studied. Tyrosinase immobilization was investigated on
different supports and chemical agents: chitin flakes activated with hexamethylenediamine and glutaraldehyde as crosslinking
agent, chitosan gel beads, chitosan gel beads in the presence of glutaraldehyde, chitosan gel beads in the presence of polyvinyl
pyrrolidone, and chitosan flakes using glutaraldehyde as crosslinking agent. The last support was considered the best using
as performance indexes the following set of immobilization parameters: efficiency (90.52%), yield (11.65%), retention (12.87%),
and instability factor (0.00). The conditions of immobilization on chitosan flakes were optimized using a two-level full factorial
experimental design. The independent variables were enzyme-support contact time (t), glutaraldehyde concentration (G), and the amount of enzyme units initially offered (U
C). The response variable was the total units of enzymatic activity shown by the immobilized enzyme (U
IMO). The optimal conditions were t=24 h, G=2% (v/v), and U
C=163.7 U. Under these conditions the total units of enzymatic activity shown by the immobilized enzyme (U
IMO) was 23.3 U and the rate of l-DOPA production rate was 53.97 mg/(L·h). 相似文献
13.
Nahid Soltani Firooz Reza Panahi Babak Mokhtarani Farshad Yazdani 《Cellulose (London, England)》2017,24(3):1407-1416
Tyrosinase is used to eliminate phenolic compounds from wastewater. Therefore, its immobilization is important to enhance catalytic efficiency. Papery materials are of particular interest for use as support for enzyme immobilization since the porous microstructure of fiber networks in papers can provide a suitable reaction environment, especially in flow-type catalytic reactions. However, immobilization of protein onto papery structure needs chemical modifications in severe conditions. To overcome this challenge, a cellulosic paper was directly amine-functionalized in moderate conditions and used for tyrosinase immobilization. The support was pretreated with HCl (0.5 N) solution and then sequentially immersed in ethylenediamine (EDA), glutaraldehyde solution (2% v/v) and the crude enzyme. In comparison with the untreated one, the immobilized enzyme on the EDA-treated support offered a 3.7-fold increase in activity. The FTIR spectra as well as EDX analysis proved the presence of amine groups in the cellulosic paper and also covalent immobilization of tyrosinase on the modified support. When considering the effect of pH on the activity at 25 °C, a maximum relative activity of 134% at pH 6 was revealed. Similarly, evaluating the effect of temperature on the activity at pH 7 displayed a maximum relative activity of 152% at 35 °C. The immobilized enzyme was suitable for use for more than four cycles to degrade a phenolic compound at severe pH and temperature conditions. Additionally, the immobilized enzyme was active after treatment of the surface at different pHs and temperatures for 105 min. The chemically modified cellulosic paper can be used as a support for enzyme immobilization. 相似文献
14.
Sensitive amperometric biosensors for phenols compounds, based on tyrosinase (polyphenoloxidase, PPO) immobilized on a Pt electrode in an electropolymerized poly-amphiphilic pyrrole matrix or cross-linked with glutaraldehyde, were constructed and compared. Steady-state amperometric measurements, performed at -50 mV vs. SCE in aqueous phosphate buffer containing LiClO(4) 0.1 M (pH 7) as well as in a chloroform solution containing 0.1 M C(6)H(5)CH(2)N(CH(3))(3)Cl, were used in order to compare the electroanalytical and kinetic parameters of the investigated amperometric biosensors in aqueous and nonaqueous media. It was established that the polypyrrole matrix has a higher efficiency for enzyme retention resulting in higher bioelectrode sensitivity, both in aqueous buffer (690 microA M(-1)) and in chloroform (149 microA M(-1)). 相似文献
15.
《Analytical letters》2012,45(8):1399-1409
Abstract A rapid procedure for determining phenols in olive oils, based on an organic-phase enzyme electrode, is described. Direct assays are performed in chloroform solutions which support the tyrosinase activity. This class (phenol)-specific enzyme strongly adheres to the surface of the graphite transducer. The resulting wall-jet detector offers effective flow injection operation, with a detection limit of 4×10?7 M (0.8 ng) phenol and sample frequency of 60 h?1. Applicability to olive-oil samples of different origins is illustrated. 相似文献
16.
Screen-printed electrodes coated with the nafion layer have been investigated for cholinesterase biosensor design. The butyrylcholinesterase (ChE) from horse serum was immobilised onto the nafion layer by cross-linking with glutaraldehyde vapours. The biosensors obtained showed better long-term stability and lower working potential in comparison to those obtained with no nafion coating. The sensitivity of a biosensor toward organophosphate pesticides is not affected by the nafion coating. The detection limits were found to be 3.5x10(-7) M for trichlorfon and 1.5x10(-7) M for coumaphos. 相似文献
17.
Campanella L Dragone R Lelo D Martini E Tomassetti M 《Analytical and bioanalytical chemistry》2006,384(4):915-921
Several triazine pesticides, such as atrazine, are much more soluble in several organic solvents, such as chloroform, than
in water. Our recent research was aimed at analyzing this class of pesticides using tyrosinase OPEE (organic phase enzyme
electrodes), exploiting their inhibiting action on the tyrosinase enzyme when operating in water-saturated chloroform medium.
In this work we studied the response of a tyrosinase inhibition enzyme sensor to several triazinic (simazine, propazine, terbuthylazine)
and benzotriazinic (azinphos-ethyl and azinphos-methyl) pesticides (LOD=0.5×10−9 mol l−1). Recovery trials were also performed in vegetal matrixes (corn, barley, lentils). Lastly, the effect of the solvent (chloroform
or water) on the inhibition process was investigated via Hill’s equation and the diffusion of analyte from the solvent to
the enzyme membrane. 相似文献
18.
Amperometric screen-printed biosensor arrays with co-immobilised oxidoreductases and cholinesterases
Amperometric screen-printed biosensor arrays for detection of pesticides (organophosphates and carbamates) and phenols have been developed. Cholinesterases (AChE and BChE), tyrosinase (TYR), peroxidases (SBP, soybean and HRP, horseradish) and cellobiose dehydrogenase (CDH) were combined on the same array consisting of one Ag/AgCl reference electrode surrounded by eight radially distributed working electrodes of either carbon or platinum. Mainly cross-linking with glutaraldehyde was employed for enzyme immobilisation. The substrates for the enzymes were acetylthiocholine for cholinesterases (ChEs), cellobiose for CDH and hydrogen peroxide for peroxidases. Hydrogen peroxide was generated in the presence of glucose by co-immobilised glucose oxidase (GOx). All measurements were performed in an electrochemical steady state system specially constructed for eight channel screen-printed electrode arrays. The achieved relative standard deviation values calculated for different enzyme substrates (10 measurements) were typically below 7% and one assay was completed within less than 10 min. The detection limits for pesticides and phenols were in the nanomolar and micromolar ranges, respectively. The developed biosensor array was evaluated on wastewater samples. To simplify interpretation of results, the measured data were treated with multivariate analysis-principal component analysis (PCA). 相似文献
19.
The development and optimization of an analytical method using enzymatic biosensors able to operate in organic solvents [organic phase enzyme electrodes (OPEEs)] for the determination of the water content in food fats (butter, margarine) or pharmaceutical or cosmetic ointments is described. The method is based on the increase in enzymatic activity which is related to the increase in the percentage water content in the organic phase into which the biosensor is dipped. The enzymes used to assemble the biosensors were tyrosinase or catalase, the substrates were phenol or p-cresol and tert-butyl hydroperoxide, respectively, and the organic solvents were acetonitrile or dioxane. A gas diffusion amperometric electrode for oxygen measurement was used as electrochemical transducer. The results were compared with those obtained applying the Karl Fischer method to the same food or drug matrices. The correlations among the two methods proved satisfactory, as the difference in the computed values of water content was never higher than 7%. Also, the precision of measurements was acceptable (RSD < 6%) in all the analyses of real matrices. 相似文献
20.
壳聚糖固定化血管紧张素转化酶及其性质 总被引:3,自引:0,他引:3
以壳聚糖微球为载体, 戊二醛为交联剂固定化血管紧张素转化酶, 研究了酶固定化的最优条件和固定化酶的性质. 结果表明, 在戊二醛质量分数为2.5%、给酶量为8 mg/mL时, 固定化酶的比活性最大, 为0.085 U/g. 固定化酶在40~50 ℃, pH在7~9之间有最大活性, 其米氏常数Km为2.39 mmol/L. 同时, 固定化酶具有良好的稳定性, 可重复利用. 相似文献