Amperometric biosensor for the determination of creatine

An amperometric biosensor for the determination of creatine was developed. The carbon rod electrode surface was coated with sarcosine oxidase (SOX) and creatine amidinohydrolase by cross-linking under glutaraldehyde vapour. The SOX from Arthrobacter sp. 1–1 N was purified and previously used for creation of a creatine biosensor. The natural SOX electron acceptor, oxygen, was replaced by an redox mediating system, which allowed amperometric detection of an analytical signal at +400-mV potential. The response time of the biosensor was less than 1 min. The biosensor showed a linear dependence of the signal vs. creatine concentration at physiological creatine concentration levels. The optimal pH in 0.1 M tris(hydroxymethyl)aminomethane (Tris)–HCl buffer was found to be at pH 8.0. The half-life of the biosensor was 8 days in 0.1 M Tris–HCl buffer (pH 8.0) at 20 °C. Principal scheme of consecutively followed catalytic reactions used to design a biosensor for the determination of creatine     

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).     

Amperometric bienzyme electrode for l-carnitine

An electrochemical sensor with two soluble enzymes allows assay of 0.1–10 mM, l- carnitine with an accuracy of 2%. The assay takes about 2 min. The first enzyme, carnitine dehydrogenase, catalyzes the oxidation of l-carnitine by NAD⁺. The NADH formed is oxidized by hexacyanoferrate(III) in a reaction catalyzed by diaphorase, and the hexacyanoferrate(II) produced is oxidized by electrolysis at 0.3 V. The sensor can be used for several days.     

Enantioselective screen-printed amperometric biosensor for the determination of D-amino acids

D-amino acids are generally considered to be important markers of bacterial contamination of food products. A screen-printed amperometric biosensor for the detection of D-amino acids has been constructed by the immobilization of D-amino acid oxidase on a graphite working electrode of a screen-printed strip modified with Prussian Blue and Nafion layers. Enzyme immobilization was then carried out by cross-linking of a mixture of the enzyme and bovine serum albumin with glutaraldehyde. As a result of the mediator addition and because of the multi-layer construction of the biosensor, including a polymer layer to avoid the interferences, the limit of the detection of the developed biosensor was two orders of magnitude improved in comparison to other screen-printed biosensors, as far as the determination of amino acids is concerned. Additional modification of the graphite electrode with carbon nanotubes led to a significant enhancement of the signal magnitude. A fast linear response of the developed biosensor was subsequently observed in static measurements for D-alanine in the concentration range from 5 to 200 microM. Excellent enantioselectivity towards D-amino acids was discovered. During the experiment, D-amino acids were detected in fruit juices and some milk samples. The complex matrix of natural milk samples had no influence on the response of the biosensor. The results were in good agreement with those obtained by capillary electrophoresis measurements.     

A screen-printed disposable biosensor for selective determination of putrescine

We have developed screen-printed carbon electrodes for the determination of putrescine (Put) via the enzyme monoamine oxidase that was immobilized on the surface of the electrode by cross-linking it with bovine serum albumin using glutaraldehyde. A mixture of 5 % of tetrathiofulvalene (TTF) and carbon ink was used for the fabrication of the screen-printed working electrode. Put was amperometrically detected by measurement of the current due to the oxidation of the mediator TTF. The use of TTF lowers the working potential to +250 mV (vs. a screen-printed Ag/AgCl reference electrode). Response is linear in the range from 16 to 101 μM, and the detection limit is 17.2?±?4.6 μM, with a reproducibility of 9.6 % (n?=?4) in terms of relative standard deviation. The effects of potentially interfering biogenic amines such as cadaverine, histamine, spermine, spermidine and tryptamine were also evaluated. The biosensor was successfully applied to the determination of Put in zucchini and anchovies.

Tyrosinase/laccase bienzyme biosensor for amperometric determination of phenolic compounds

Tyrosinase/laccase bienzyme biosensor for amperometric determination of phenolic compounds was constructed. Enzymes were immobilized in titania gel matrix. The obtained biosensor was successfully used for determination of 2,6-dimethoxyphenol, 4-tertbutylcatechol, 4-methylcatechol, 3-chlorophenol and catechol. The highest sensitivity and the widest linear range were noticed for catechol, 234 mA L mol^− 1 and 2.0 × 10^− 7–3.2 × 10^− 5 mol/L, respectively. Detection limit for catechol, at signal-to-noise ratio of 3 was 1.3 × 10^− 7 mol/L.     

Amperometric biosensor for the rapid determination of salicylate in whole blood

A method for the determination of salicylate in whole blood is described. The assay uses salicylate hydroxylase to convert salicylate to catechol in the presence of NADH and molecular oxygen. The formation of catechol is monitored amperometrically by oxidation at +300 mV vs.Ag/AgCl and the size of the oxidation current is related to the concentration of salicylate in the sample. The reagents are incorporated into the working electrode of a disposable strip, allowing measurements to be made on a drop of blood within 1 min. The functional range of the assay can be extended to the equivalent of 7.2 mM plasma salicylate by incorporating benzoate as a component of the reaction system. The method has the advantages of simplicity and speed compared with standard procedures, and should prove especially useful in suspected overdose situations.     

Amperometric biosensors based on nafion coated screen-printed electrodes for the determination of cholinesterase inhibitors

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.     

Amperometric choline biosensor fabricated through electrostatic assembly of bienzyme/polyelectrolyte hybrid layers on carbon nanotubes

We report a flow injection amperometric choline biosensor based on the electrostatic assembly of the choline oxidase (ChO) enzyme and a bienzyme of ChO and horseradish peroxidase (HRP) onto multi-wall carbon nanotubes (MWCNT) modified glassy carbon (GC) electrodes. These choline biosensors were fabricated by immobilization of enzymes on the negatively charged MWCNT surface through alternately assembling a cationic poly(diallydimethylammonium chloride) (PDDA) layer and an enzyme layer. Using this layer-by-layer assembling approach, a bioactive nanocomposite film of PDDA/ChO/PDDA/HRP/PDDA/CNT (ChO/HRP/CNT) and PDDA/ChO/PDDA/CNT (ChO/CNT) was fabricated on the GC surface. Owing to the electrocatalytic effect of carbon nanotubes, the measurement of faradic responses resulting from enzymatic reactions has been realized at low potential with acceptable sensitivity. The ChO/HRP/CNT biosensor is more sensitive than the ChO/CNT one. Experimental parameters affecting the sensitivity of biosensors, e.g., applied potential, flow rate, etc., were optimized and potential interference was examined. The response time for this choline biosensor is fast (few seconds). The linear range of detection for the choline biosensor is from 5.0 x 10(-5) to 5.0 x 10(-3) M and the detection limit is about 1.0 x 10(-5) M.     

Amperometric determination of sulfite using screen-printed electrodes modified with metallic nanoparticles

We report on the amperometric determination of sulfite using screen-printed carbon electrodes (SPCEs) modified with gold and silver nanoparticles that were deposited on the electrode to improve the capabilities of detection. The electrode is fairly selective and responds to sulfite with an oxidation current (at 300 mV and pH 6) in the 9.80 to 83.33 μM concentration range. The precision in terms of repeatability and reproducibility is 14.4 % and 10.7 % in the case of SPCEs modified by gold nanoparticles. The method was applied to the determination of sulfite in drinking water, pickle juice and vinegar. Recoveries ranged from 96 % to 104 %.

Amperometric biosensor for oxalate determination in urine using sequential injection analysis

An amperometric flow biosensor for oxalate determination in urine samples after enzymatic reaction with oxalate oxidase immobilized on a modified magnetic solid is described. The solid was magnetically retained on the electrode surface of an electrode modified with Fe (III)-tris-(2-thiopyridone) borate placed into a sequential injection system preceding the amperometric detector. The variables involved in the system such as flow rate, aspired volumes (modified magnetic suspension and sample) and reaction coil length were evaluated using a Taguchi parameter design. Under optimal conditions, the calibration curve of oxalate was linear between 3.0-50.0 mg·L^-1, with a limit of detection of 1.0 mg·L^-1. The repeatability for a 30.0 mg·L^-1 oxalate solution was 0.7%. The method was validated by comparing the obtained results to those provided by the spectrophotometric method; no significant differences were observed.     

Amperometric glyceride biosensor

Glycerol dehydrogenase (GDH) and lipase have been used for the amperometric determination of glycerol and triglycerides on modified carbon electrodes. Carbon electrodes were modified with adsorbed Meldola Blue, Nile Blue or Toluidine Blue O. Electrochemical oxidation of NADH was realized at 0V vs saturated Ag/AgCl electrode. NADH was produced by the catalytic oxidation of glycerol in the presence of glycerol dehydrogenase immobilized on the surface of an electrode. GDH was adsorbed on the electrode, entrapped in gelatin, immobilized in polylysine gel, or trapped in two types of organic salts. Sensitivity of the electrodes vary from 2 to 9 nA/mM glycerol with steady state achieved in a time of between 20 s and 8 min, depending on the method of immobilization. Triglycerides were determined after a 5 min pre-incubation period in a mixture of lipases with different specificity.     

Amperometric biosensor based on tyrosinase immobilized in hydrotalcite-like compounds film for the determination of polyphenols

A tyrosinase (Tyr) biosensor has been constructed by immobilizing tyrosinase on the surface of Mg–Al–CO₃ hydrotalcite-like compound film (HTLc) modified glassy carbon electrode (GCE) for the determination of polyphenols. The negatively charged tyrosinase was adsorbed firmly on the surface of a positively charged HTLc/GCE by electrostatic interactions and retained its activity to a great degree. The modified electrode was characterized by cyclic voltammetry and AC impedance spectra. Polyphenols were determined by a direct reduction of biocatalytically generated quinone species. The different parameters, including pH, temperature, and enzyme loading were investigated and optimized. Under the optimum conditions, Tyr/HTLc electrode gave a linear response range of 3–300, 0.888–444, and 0.066–396 μM with a detection limit (S/N = 3) of 0.1, 0.05, and 0.003 μM for catechol, caffeic acid, and quercetin, respectively. In addition, the repeatability and stability of the enzyme electrode were estimated. Total polyphenol contents of real samples were also determined to study the potential applicability of the Tyr/HTLc/GCE biosensor.     

Amperometric sensors based on tyrosinase-modified screen-printed arrays

This paper describes the design, development and characteristics of a tyrosinase (polyphenol oxidase) modified amperometric screen-printed biosensor array, with the enzyme cross-linked in a redox-hydrogel namely the PVI₁₃-dmeOs polymer. Two types of Au-screen-printed four-channel electrode arrays, differing in design and insulating layer, were compared and investigated. Au-, graphite-coated-Au- and Carbopack C-coated-Au-surfaces, serving as the basis for tyrosinase immobilisation, were investigated and the performances of the different arrays were evaluated and compared in terms of their electrocatalytic characteristics, as well as operational- and storage stability using catechol as model substrate. It was found that the Carbopack C-coated array was the best choice for tyrosinase immobilisation procedure mainly due to a higher mechanical stability of the deposited enzyme layer, combined with good sensitivity and stability for up to 6 months of use. In the batch mode the biosensors responded linearly to catechol up to 30 μM with limits of detection from 0.14 μM. Parameters from cyclic voltammograms indicated that the reversibility of the direct electrochemical reaction for catechol on the three types of electrode surfaces (no tyrosinase modification) was not the limiting factor for the construction and performance of tyrosinase biosensors.     

Amperometric biosensor for xanthine with supramolecular architecture

Xanthine oxidase modified with 1-adamantanyl residues was supramolecularly immobilized on Au electrodes coated with Au nanoparticles coated with a perthiolated beta-cyclodextrin polymer; the analytical response of the electrode toward xanthine was evaluated.     

Development of a cytochrome c-based screen-printed biosensor for the determination of the antioxidant capacity of orange juices

This paper describes the development of an amperometric cytochrome c (cyt c)-based biosensor and its later application to the quantification of the scavenging capacity of antioxidants. The enzymatic biosensor was constructed by covalently co-immobilizing both cyt c and XOD on a mercaptoundecanol/mercaptoundecanoic acid (MU/MUA) mixed self-assembled monolayer (SAM)-modified screen-printed gold electrode. The applicability of this method was shown by analyzing the antioxidant capacity of pure substances, such as ascorbic acid and Trolox, and natural sources of antioxidants, particularly 5 orange juices.     

A bienzyme electrode for tyrosine-containing peptides determination

A bienzyme electrode for monitoring biologically important peptides containing tyrosine has been established on the basis of mushroom tyrosinase and quinoprotein glucose dehydrogenase (GDH). Tyrosine residues bound in the peptide chain are oxidized by tyrosinase resulting in consumption of oxygen. Subsequently, the dopaquinone residues are reduced in the GDH catalysed reaction which is driven by an excess of glucose. This reaction cycle leads to a considerable increase of sensitivity. Both enzymes were entraped in poly(vinyl)alcohol matrix and placed on the surface of a Clark-type oxygen electrode (the working platinum electrode was potentiostated at −600 mV) (vs. Ag/AgCl reference electrode) between a polypropylene and cellulose membrane. The bienzyme-modified Clark-type oxygen electrode has a lower detection limit of 0.2 μM for the opioid peptides Tyr-D-Ser-Gly-Phe-Leu-Thr, Leu-enkephalin, Tyr-D-Ala-Gly-Phe-D-Leu and morphiceptin. The dependence of response of the electrode on the length of peptide chain and position of tyrosine residue is discussed. The new electrode has been applied to the quality control of tyrosine containing peptides from pharmaceutical formulations and from peptide synthesis.     

Amperometric determination of pesticides using a biosensor based on a polishable graphie-epoxy biocomposite

The determination of organophosphorus and carbamate pesticides was carried out using an amperometric transducer based on a robust, polishable and easily mechinable biocomposite. The biocomposite material contains graphite powder, a non-conducting epoxy resin and acetylcholinesterase. The enzyme retains its bioactivity in the rigid epoxy-graphite matric. Measurements were carried out with acetylhiocholine as a substrate. Thiocholine produced by enzymatic hydrolysis was oxidized electrochemically at 70 mV (vs. Ag/AgCl in pH 7.0 buffered solution with 0.1 M phosphate and 0.1 m KCl). The decrease rate of substrate steady-state current after the addition of pesticide was used for evaluation. The method of construction allows for the repetitive use of the electrode. Simple polishing procedures are used to regenerate the bioactive transducer surface.     

Amperometric biosensor based on D-aminoacid oxidase for the R-perindopril assay

A new amperometric biosensor based on D-aminoacid oxidase is described for the assay of R-perindopril. R-perindopril can be determined in the 400–¶20 nmol/L concentration range; the detection limit is ¶10 nmol/L. The selectivity was checked with S-perindopril, D- and L-proline, and polyvinylpyrrolidone. The main interfering species was D-proline. An automated system for the assay of R-perindopril based on the concept of flow injection with an amperometric biosensor (based on D-aminoacid oxidase) as detector is also described. The system is suitable for the on-line monitoring of R-perindopril at a sampling rate of 72 samples/h, in the linear range: 100 nmol/L –20 nmol/L with an RSD better than 0.09% (n = 10).     

Amperometric biosensor based on D-aminoacid oxidase for the R-perindopril assay

A new amperometric biosensor based on D-aminoacid oxidase is described for the assay of R-perindopril. R-perindopril can be determined in the 400-20 nmol/L concentration range; the detection limit is 10 nmol/L. The selectivity was checked with S-perindopril, D- and L-proline, and polyvinylpyrrolidone. The main interfering species was D-proline. An automated system for the assay of R-perindopril based on the concept of flow injection with an amperometric biosensor (based on D-aminoacid oxidase) as detector is also described. The system is suitable for the on-line monitoring of R-perindopril at a sampling rate of 72 samples/h, in the linear range: 100 nmol/L -20 nmol/L with an RSD better than 0.09% (n = 10).