A novel combined thermometric and amperometric biosensor for lactose determination based on immobilised cellobiose dehydrogenase

A novel method for lactose determination in milk is proposed. It is based on oxidation of lactose by cellobiose dehydrogenase (CDH) from the basidiomycete Phanerochaete chrysosporium, immobilised in an enzyme reactor. The reactor was prepared by cross-linking CDH onto aminopropyl-silanised controlled pore glass (CPG) beads using glutaraldehyde. The combined biosensor worked in flow injection analysis (FIA) mode and was developed for simultaneous monitoring of the thermometric signal associated with the enzymatic oxidation of lactose using p-benzoquinone as electron acceptor and the electrochemically generated current associated with the oxidation of the hydroquinone formed. A highly reproducible linear response for lactose was obtained between 0.05 mM and 30 mM. For a set of more than 500 samples an R.S.D. of less than 10% was achieved. The assay time was ca. 2 min per sample. The sensor was applied for the determination of lactose in dairy milk samples (milk with a fat content of 1.5% or 3% and also "lactose free" milk). No sample preparation except dilution with buffer was needed. The proposed method is rapid, suitable for repeated use and allows the possibility to compare results from two different detection methods, thus providing a built-in quality assurance. Some differences in the response observed between the methods indicate that the dual approach can be useful in mechanistic studies of redox enzymes. In addition, a dual system opens up interesting possibilities for studies of enzyme properties and mechanisms.     

An amperometric biosensor for polyphenolic compounds in red wine

In the present work, a biosensor was developed with Laccase Coriolus Versicolor as the biological reconnaissance element immobilized on derivatized polyethersulphone membranes and applied to a Pt-Ag, AgCl US electrode base. Its application to several polyphenols usually found in red wine (caffeic acid, gallic acid, catechin, rutin, trans-resveratrol, quercetin and malvidin) was tested. It was observed that an amperometric response was obtained for catechin at +100 mV (versus Ag, AgCl) and caffeic acid at -50 mV in acetate buffer solutions (pH 4.5) having 12% ethanol. At pH 3.5 and +100 mV the biosensor was sensitive to both substrates and their response was additive. A limit of detection of 1.0 x 10(-6) M, linearity ranging from 2.0 to 14.0 x 10(-6) M, high sensitivity (0.0566 mAM(-1)) and reproducibility (R.S.D. <10%) were achieved for equimolar mixed solutions of catechin and caffeic acid. Under the same experimental conditions the other polyphenols tested individually did not yield any biosensor response. The application of the biosensor to red wine samples required a previous solid phase extraction for polyphenols enrichment. In fact, attempts to apply the biosensor in red wine using the "standard addition" methodology showed that large interferences occurred, as was to be expected. Reduction currents of -0.33 +/- 0.03 nA were obtained when the biosensor was used with the wine extract at +100 mV. This current could be ascribed to catechin and caffeic acid, although some interference by other polyphenols at the matrix level seemed to persist. The present biosensor showed promising applications for the wine analysis in future.     

An amperometric enzyme biosensor for real‐time measurements of cellobiohydrolase activity on insoluble cellulose

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi‐crystalline and amorphous, can be monitored directly and in real‐time by an enzyme‐modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross‐linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH‐catalyzed reaction with cellobiose, was recorded under constant‐potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH‐biosensors showed high sensitivity (87.7 µA mM^?1 cm^?2), low detection limit (25 nM), and fast response time (t_95% ～ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH‐biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β‐anomer of cello‐oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real‐time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2012; 109: 3199–3204. © 2012 Wiley Periodicals, Inc.     

An enzyme electrode for amperometric measurement of D-amino acid

A carbon paste enzyme electrode has been developed for measurement of D-amino acids that employs a fatty acid modified FAD to prevent leaching of this essential cofactor to the surrounding aqueous environment and which serves as an enzyme stabilizing agent. The lower limit of detection is at least 10(-4) M and the electrode has a linear range of 10(-4) to 3 x 10(-3) M and a response time of 180 s. Twenty L-amino acids were tested and none of them elicited responses when electrodes were exposed to 0.5 mM concentration increases over a baseline level. On the other hand, some response was observed when exposed to 18 of 20 D-amino acids varying from 2 to 200% of the corresponding D-alanine response. Electrodes were shown to have longevities of over 30 days while maintaining 85% of their original sensitivity. Electrodes showed activity over a pH of 6.2-11.7 with a maximum at 9.2 and over temperatures of 10-47 degrees C with a maximum at 37 degrees C.     

An amperometric uric acid biosensor based on modified Ir-C electrode

The level of uric acid (UA) has a high relationship with gout, hyperuricemia and Lesch-Nyan syndrome. The determination of UA is an important indicator for clinics and diagnoses of kidney failure. An amperometric UA biosensor based on an Ir-modified carbon (Ir-C) working electrode with immobilizing uricase (EC 1.7.3.3) was developed by thick film screen printing technique. This is the first time to report the utilization of an uricase/Ir-C electrode for the determination of UA by using chronoamperometric (CA) method. The high selectivity of UA biosensor was achieved due to the reduction of H(2)O(2) oxidation potential based on Ir-C electrode. Using uricase/Ir-C as the sensing electrode, the interference from the electroactive biological species, such as ascorbic acid (AA) and UA (might be directly oxidized on the sensing electrode) was slight at the sensing potential of 0.25 V (versus Ag/AgCl). UA was detected amperometrically based on uricase/Ir-C electrode with a sensitivity of 16.60 microAmM(-1) over the concentration range of 0.1-0.8 mMUA, which was within the normal range in blood. The detection limit of UA biosensor was 0.01 mM (S/N=6.18) in pH 7 phosphate buffer solution (PBS) at 37 degrees C. The effects of pH, temperature, and enzymatic loading on the sensing characteristics of the UA biosensor were also investigated in this study.     

An amperometric glucose biosensor prototype fabricated by thermal inkjet printing

The prototype of an amperometric glucose biosensor was realized by thermal inkjet printing using biological and electronic water-based inks, containing a glucose oxidase (GOD) from Aspergillus niger and the conducting polymer blend poly(3,4-ethylenedioxythiophene/polystyrene sulfonic acid) (PEDOT/PSS), respectively. The biosensor was fabricated microdepositing PEDOT/PSS and GOD, in sequence, on ITO-glass, by a commercial inkjet printer, with the help of a commercial software. High density microdots matrices were so-realized, with a calculated resolution of about 221 x 221 dpi (dot per inch). By means of a rapid and easy assay it was demonstrated that no activity loss occurred upon the printing of GOD, despite of the use of a thermal printhead. The device was encapsulated in a semipermeable membrane of cellulose acetate, applied by dip-coating, in order to prevent dissolution of the enzyme and/or PEDOT/PSS in water. The preliminary response of the electrode was measured in an aqueous glucose solution in the presence of ferrocenemethanol (FeMeOH) as a mediator, and resulted linear up to 60 mM in glucose. The best sensitivity value achieved was 6.43 microAM(-1) cm(-2) (447 nAM(-1) U(-1) cm(-2)). The characteristics of the device, and the possible performance improvements have been analyzed and discussed. The reported findings indicate that inkjet printing could be a viable instrument for the easy construction of a working biosensor via direct digital design using biological and conductive polymer based inks. Such an approach may be seen as an example of "biopolytronics".     

An interference free amperometric biosensor for the detection of biogenic amines in food products

In this work is reported the development and application of an amperometric biosensor for the determination of total biogenic amines content by using the commercial diamino oxidase (DAO from Porcine kidney E.C. 1.4.3.6) as the biocomponent, entrapped by glutaraldehyde onto an electrosynthesized bilayer film. In order to minimize both the fouling and the interference caused by the direct electrochemical oxidation of both the analytes (i.e., biogenic amines) and the common interferents usually present in food products the performances of Pt and Au electrodes and of several electroproduced anti-interferents mono- and bi-layer films were tested. In spite of a very low activity of the commercial DAO, the biosensor displayed a high response sensitivity in flow experiments, short response time, a good linear response and low detection limits. The excellent anti-interference characteristics allowed the use of the biosensor in screening analysis of food products.     

A rapid assay for pectinesterase activity which can be used as a prescreen for pectinesterase inhibitors.

A rapid assay for inhibition of pectinesterase has been developed to facilitate large scale screening for possible inhibitors. The assay gives quantitative results which can be confirmed by other assay techniques. The assay uses an agar plate in which pectin and an indicator have been incorporated. The hydrolysis of pectin to pectinic acid by pectinesterase causes a lowering of the pH of the medium and produces a color change. The area of the color change produced by the hydrolysis of pectin is directly proportional to the activity of the pectinesterase.     

An amperometric biosensor fabricated from electro-co-deposition of sodium alginate and horseradish peroxidase

A convenient and effective strategy for fabrication of hydrogen peroxide biosensor based on sodium alginate (SA) and polyvinyl butyral (PVB) as matrices was reported in this paper. The horseradish peroxidase (HRP) and SA were electro-co-deposited onto the surface of gold electrode, and the HRP–SA/Au electrode was further coated with PVB. The interaction between HRP and SA was characterized by UV–vis absorption spectroscopy, and the fabricating process of biosensor was characterized by electrochemical impedance spectroscopy (EIS). The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. Experimental conditions were investigated which influence the performance of the biosensor, such as pH, and applied potential. The biosensor showed a linear response to H₂O₂ over a concentration range from 7.0 × 10⁻⁶ to 4.1 × 10⁻³ M with a detection limit of 1.8 × 10⁻⁶ M based on a signal-to-noise ratio of 3 under optimum conditions. The value of HRP in the composite was evaluated to be 1.38 mM. The biosensor obtained from this study possesses high sensitivity, good reproducibility, and long-term stability.     

A dual enzyme amperometric biosensor for monitoring organophosphorous pesticides

An amperometric biosensor consisting of two enzyme membranes, one a potato layer rich in acid phosphatase and the other immobilized glucose oxidase membrane, when operated in conjunction with a Clark type dissolved O 2 elec-trode, detected the pesticide, Paraoxon, at 1 g/ml. The advantage of this biosensor is that the inhibition of acid phosphatase by the pesticide is reversible and thereby eliminates the problem of enzyme inactivation and the necessity for its reactivation which is not efficient.     

A nylon membrane based amperometric biosensor for polyphenol determination

A nylon membrane based amperometric biosensor employing banana fruit polyphenol oxidase (PPO) is presented for polyphenol detection. Nylon membrane was first activated and then coupled with chitosan. PPO was covalently attached to this membrane through glutaraldehyde coupling. The membrane bioconjugate was characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) study and then mounted onto Au electrode using parafilm to construct a working electrode. Once assembled along with Ag/AgCl as reference and Pt as auxiliary electrode, the biosensor gave optimum response within 15 s at pH 7.5 and 30 °C, when polarized at +0.4 V. The response (in mA) was directly proportional to polyphenol concentration in the range 0.2–400 μM. The lower detection limit of the biosensor was 0.2 μM. The biosensor was employed for determination of polyphenols in tea, beverages and water samples. The enzyme electrode showed 25% decrease in initial activity after 150 reuses over 6 months, when stored at 4 °C.     

Transglycosylation of cellobiose by partially purified Trichoderma viride cellulase.

A commercial cellulase from Trichoderma viride was fractionated into three fractions, F1, F2, and F3, in order to investigate transglycosylation activities. Among these fractions, F3, which demonstrated highly hydrolytic activity toward p-nitrophenyl beta-D-glucopyranoside and Avicel, most effectively catalyzed the transglycosylation of cellobiose and converted cellobiose into beta-Glc-(1-->6)-beta-glc-(1-->4)-Glc and beta-Glc-(1-->6)-beta-Glc-(1-->6)-beta-Glc(1-->4)-Glc. The F3 fraction contained the enzyme to catalyze beta-glucosyl transfer toward only the C-6 position of the sugar acceptor, and thus it is expected to be of use for syntheses of functional oligosaccharides.     

A chemically mediated amperometric biosensor for monitoring eubacterial respiration

The development of a novel biosensor system for measuring the respiratory activity of whole eubacterial cells is described. The biosensor incorporates a physically immobilized layer of cells held in intimate contact with an amperometric transducing electrode and uses a chemical mediator, potassium ferricyanide, to divert electrons from the respiratory system of the bacteria to the poised electrode. The current thus produced is proportional to the level of respiratory activity of the immobilized bacterial cells and can be monitored by a computer interface system. The paper outlines the principles of the biosensor and describes the results of a screen of potentially useful eubacteria. Also described are the effects of physical parameters on the sensor and a strategy for the long term preservation of the biosensor by freeze-drying.     

An amperometric uric acid biosensor based on multiwalled carbon nanotube-gold nanoparticle composite

An amperometric uric acid biosensor was fabricated by immobilizing uricase (EC 1.7.3.3) onto gold nanoparticle (AuNP)/multiwalled carbon nanotube (MWCNT) layer deposited on Au electrode via carbodiimide linkage. Determination of uric acid was performed by oxidation of enzymically generated H₂O₂ at 0.4 V. The sensor showed optimal response within 7 s at 40 °C in 50 mM Tris–HCl buffer (pH 7.5). The linear working range of the biosensor was 0.01–0.8 mM. The limit of detection (LOD) was 0.01 mM. The sensor measured uric acid levels in serum of healthy individuals and persons suffering from gout. The analytical recoveries of the added uric acid, 10 and 20 mg L^–1, were 98.0% and 96.5%, respectively. Within- and between-batch coefficients of variation were less than 5.6% and less than 4.7%, respectively. A good correlation (r = 0.998) was obtained between serum uric acid values by the standard enzymic colorimetric method and the current method. A number of serum substances had practically no interference. The sensor was used in more than 200 assays and had a storage life of 120 days at 4 °C.     

Development of a micro-planar amperometric bile acid biosensor for urinalysis

The determination of bile acid concentration in urine is useful for the screening and diagnosis of various hepatobiliary diseases. Currently, there is no concise method to determine bile acid concentration in urine. This study describes a bile acid biosensor fabricated by electrochemical technique for urinalysis. The micro-planar electrodes employed for the study consisted of a working electrode (platinum), a counter electrode (platinum) and a reference electrode (silver/silver chloride (Ag/AgCl)). The sensor chip was coated with Nafion using a spin-coater in order to both eliminate many interference species in urine and achieve long-term stability of the reference electrode. Nafion coating allowed the sensor chip to prevent the electrode reaction from interference species in urine, because it is charged negative strongly (Nafion contains sulfonic acid group). Three enzymes (bile acid sulfate sulfatase: BSS, beta-hydroxysteroid dehydrogenase: beta-HSD, and NADH oxidase: NHO) were immobilized by glutaraldehyde (GA: cross-linker) onto the sensor chip, because the immobilization of enzymes by GA is simple and commonly carried out. The sensor chip was able to detect bile acid in buffer solution. The optimum enzyme ratio immobilized onto the sensor chip was BSS:beta-HSD:NHO=4:4:20 U/1 chip. There was a relationship between the concentration of bile acid and the response current value. The dynamic range of the sensor chip was 2-100 microM for bile acid. Additionally, bile acid in the urine specimen could be detected using this bile acid biosensor. We present a simple and rapid bile acid biosensor with high sensitivity and high reproducibility.     

An amperometric microbial biosensor development based on Candida tropicalis yeast cells for sensitive determination of ethanol

Different branchs of industry need to use ethanol in their production and some progress and not only the industry also to determine ethanol sensitively, accurately, fast and economical is very important. For the sensitive determination of ethanol a new amperometric biosensor based on Candida tropicalis cells, which contains alcohol oxidase enzyme, immobilized in gelatin by using glutaraldehyde was developed. In the study, before the microbial biosensor construction C. tropicalis cells were activated and cultured in a culture medium. By using gelatine and glutaraldehyde (0.1%) C. tropicalis cells obtained in logarithmic phase were immobilized and fixed on a pretreated teflon membrane of a dissolved oxygen probe. Ethanol determination is based on the assay of the differences on the respiration activity of the cells on the oxygenmeter in the absence and the presence of ethanol. The microbial biosensor response was depend linearly on ethanol concentration between 0.5 and 7.5 mM with 2 min response time. In the optimization studies of the microbial biosensor the most suitable microorganism amount were found as 4.42 mg cm(-2) and also phosphate buffer (pH:7.5; 50 mM) and 30 degrees C were obtained as the optimum working conditions. In the characterization studies of the microbial biosensor some parameters such as substrate specificity, interference effects of some substances on the biosensor response, operational and storage stability were carried out.     

Development and study of an amperometric biosensor for the in vitro measurement of low concentration of putrescine in blood

An amperometric biosensor was developed for the in vitro determination of putrescine in blood samples because elevated level of putrescine in blood can be a diagnostic indicator of certain kinds of cancer. The electrochemical transducer consisted of a flat form, three electrode amperometric micro-cell fabricated with thin film photolithography on flexible Kapton substrate. An immobilized putrescine oxidase (PUO) layer provided the biocatalytic oxidation of the putrescine, while the generated hydrogen peroxide was detected on the platinum-working electrode. An electropolymerized poly(m-phenylenediamine) (pPDA) size-exclusion layer was used to protect the working electrode from fouling and to prevent signal generation by common electroactive interferents present in blood. The preparation of the biocatalytic enzyme- and outer protective layers was optimized for improved sensitivity and response time. A detection limit of 50 nM was achieved in pH-adjusted whole blood samples, which is below pathological levels.     

Ascorbate oxidase based amperometric biosensor for organophosphorous pesticide monitoring.

An amperometric principle based biosensor containing tissues of cucumber, rich in ascorbic acid oxidase, was used for the detection of organophosphorous (OP) pesticide ethyl paraoxon, which inhibits the activity of ascorbic acid oxidase. The optimal concentration of ascorbic acid used as substrate was found to be 5.67 mM. The biosensor response was found to reach steady state within 2 min. A measurable inhibition (> 10%) was obtained with 10 min incubation of the enzyme electrode with different concentrations of the pesticide. There was a linear relationship between the percentage of inhibition of the enzyme substrate reaction and the pesticide (ethyl paraoxon) concentration in the range 1-10 ppm with a regression value 0.9942.