Urea potentiometric biosensor based on urease immobilized on chitosan membranes

Potentiometric biosensors based on urease (E.C. 3.5.1.5.) immobilized on chitosan membranes coupled to all-solid-state nonactin ammonium ion selective electrodes are described. The enzyme was immobilized on the chitosan membranes by four procedures: (A) adsorption; (B) adsorption followed by reticulation with dilute aqueous glutaraldehyde solution; (C) activation with glutaraldehyde followed by contact with the enzyme solution; and (D) activation with glutaraldehyde, contact with the enzyme solution and reduction of the Schiff base with sodium borohydride. The response characteristics of the biosensors obtained with these enzymatic membranes were determined and compared. The biosensor with best response characteristics, obtained by procedure (B), showed the following characteristics of response to urea: (i) linearity in the 10(-4) to 10(-2) M range; (ii) slope of up to 56 mV per decade; (iii) response time between 30 s and 2 min; and (iv) lifetime of 2 months. This biosensor was tested in the determination of urea in blood serum samples.     

Urea Biosensors Based on PVC Membrane Ion-Selective Electrodes

Abstract

This paper presents a general method of enzyme immobilization at the surface of ion selective membranes. Covalent binding of enzymes directly on the electrode surface is a very effective method that results in stable enzymatic membranes. As an example the construction of enzymatic sensors for urea determination based on ammonium and hydrogen carbonate ion selective electrodes is presented. The optimum working conditions for these biosensors were found. Bioelectrodes based on an ammonium sensor show very good analytical parameters: dynamic stability - over 2 months without decrease of sensitivity, response time - shorter then 20 s. high sensitivity, determination range from 0.3 to 70 mM. In the contrast to the ammonium ion based biosensors, those constructed on the basis of anion selective electrodes have worse analytical parameters. It is mainly due to poor selectivity and instability of an applied ion selective electrode. In spite of this, both types of urea biosensors were used for measurements in the differential potentiometry mode. The application of such system increased the sensitivity of urea determination.     

Array of potentiometric sensors for simultaneous analysis of urea and potassium

Urea biosensors based on urease immobilized by crosslinking with BSA and glutharaldehyde coupled to ammonium ion-selective electrodes were included in arrays together with potassium, sodium and ammonium PVC membrane ion-selective electrodes. Multivariate calibration models based on PCR and PLS2 were built and tested for the simultaneous determination of urea and potassium. The results show that it is possible to obtain PCR and PLS2 calibration models for simultaneous determination of these two species, based on a very small set of calibration samples (nine samples). Coupling of biosensors with ion-selective electrodes in arrays of sensors raises a few problems related to the limited stability of response and unidirectional cross-talk of the biosensors, and this matter was also subjected to investigation in this work. Up to three identical urea biosensors were included in the arrays, and the data analysis procedure allowed the assessment of the relative performance of the sensors. The results show that at least two urea biosensors should be included in the array to improve urea determination. The prediction errors of the concentration of urea and potassium in the blood serum samples analyzed with this array and a PLS2 calibration model, based on nine calibration samples, were lower than 10 and 5%, respectively.     

Improved Performance of the Potentiometric Biosensor for the Determination of Creatinine

Abstract

Contemporary methods of analyzing creatinine engage chemicals harmful to the environment and generate large volumes of waste disposals. By introducing a membrane‐based potentiometric biosensor with immobilized creatinine deaminase, the measurements can be performed by miniaturized portable devices that are easy to handle and allow rapid analysis with minimum consumption of chemicals. Thus, the enzymatic creatinine biosensors were revisited and optimized with respect to repeatability, sensitivity, limit of detection (LOD), and response time. A detection limit of 0.3 µM and a sensitivity of 58.78±0.03 mV (23.5°C) were obtained in tris buffer at pH=7.4 after introduction of shielding of all electronics and software filtering. Measurements performed by flow injection analysis (FIA) showed that the response time could be lowered to approximately 30 sec using sample volumes of 30 µl. Interferences were corrected for by application of the Nicolsky‐Eisenman equation, thus allowing determination of creatinine in matrices resembling those of clinical measurements. Investigations of sandwich structures showed that the sensitivity decreased as a function of the number of membranes on top of the immobilized layer of active creatinine deaminase. It was thus shown that the sensitivity depends on the distance of diffusion of species from the sample solution through the membranes to the enzyme.     

Novel Anion Exchangers for Electrodes with Improved Selectivity to Divalent Anions

It has been found that replacing of several long‐chain alkyl substituents at the nitrogen atom of lipophilic quaternary ammonium salts (QAS) by methyls results in a dramatic increase of the potentiometric selectivity of ion‐selective electrodes (ISE) with QAS‐based plasticized PVC membranes to some divalent anions against the monovalent ones. The discussed effect of QAS cation nature on the potentiometric selectivity is also partly retained for ISE with neutral carrier‐based membranes doped with QAS to provide anion permselectivity. This opens up new possibilities to control the potentiometric selectivity of ISE for divalent anions by the appropriate selection of the anion exchanger.     

Synthesis and characterization of covalently immobilized bis-crown ether based potassium ionophore

The synthesis of a novel covalently immobilized crown ether based potassium ionophore is presented. Apart from previously proposed methods for the preparation of PVC linked ionophores based on the chemical modification of functionalized PVC polymers, the hereby proposed procedure involves the direct copolymerization of a suitable derivative of the bis-crown ether type potassium ionophore (BME 44) and vinyl chloride monomer. The analytical performance of the potentiometric ion selective electrodes incorporating the PVC bound ionophore were optimized and determined. Compared with electrodes based on other bis-crown ether type immobilized potassium selective ionophores a slightly improved logK(K, Na)(Pot) and a longer lifetime was found. Spectral imaging and chronoamperometry were used to study the mobility of different bis-crown ether derivatives in plasticized PVC membranes.     

Potentiometric Biosensor System Based on Recombinant Urease and Creatinine Deiminase for Urea and Creatinine Determination in Blood Dialysate and Serum

A biosensor system for simultaneous determination of creatinine and urea in blood serum and dialysate samples was developed. It consisted of creatinine and urea biosensors based on a potentiometric transducers with two identical pH‐sensitive field‐effect transistors. In creatinine biosensor, creatinine deiminase immobilized via photopolymerization in PVA/SbQ polymer on one transistor served as a biorecognition element, while bovine serum albumin in PVA/SbQ polymer placed on the second transistor was used for reference. The urea biosensor was created in the same way but recombinant urease was used instead of creatinine deiminase. The linear ranges of creatinine and urea measurement were 0.02–2 mM and 0.5–15 mM, correspondingly, which allowed simultaneous determination of the metabolites. Response time of the biosensor system was 2–3 min; RSD of responses did not exceeded 5 %. The biosensors demonstrated absence of non‐selective response towards components of blood dialysate and serum. Urea and creatinine concentrations were determined in 20 samples of blood dialysate and serum. The results correlated well with traditional methods of analysis. Creatinine and urea biosensors were stable during five months of storage (during this time the responses decreased by about 10 %). The proposed biosensor system can be effectively used for analysis of serum samples and for hemodialysis control.     

Recent trends in potentiometric sensor arrays--a review

Nowadays there exists a large variety of ion sensors based on polymeric or solid-state membranes that can be used in a sensor array format in many analytical applications. This review aims at providing a critical overview of the distinct approaches that were developed to build and use potentiometric sensor arrays based on different transduction principles, such as classical ion-selective electrodes (ISEs) with polymer or solid-state membranes, solid-contact electrodes (SCE) including coated wire electrodes (CWE), ion-sensitive field-effect transistors (ISFETs) and light addressable potentiometric sensors (LAPS). Analysing latest publications on potentiometric sensor arrays development and applications certain problems are outlined and trends are discussed.     

Creatinine biosensor based on ammonium ion selective electrode and its application in flow-injection analysis

A new, highly sensitive, fast responding and stable potentiometric biosensor for creatinine determination is developed. The biosensor is based on an ammonium ion-selective electrode. Creatinine deiminase (EC 3.5.4.21) is chemically immobilized on the surface of the polymeric ion-sensitive membrane in the form of monomolecular layer using a simple, one-step carbodiimide covalent attachment method. The resulting enzyme electrodes are useful for measurement under flow injection analysis (FIA) conditions. The biosensors exhibit excellent operational and storage stability. The enzyme electrodes retain over 70% of initial sensitivity after ten weeks of work under FIA conditions. The storage stability at 4 °C is longer than half a year without loss of sensitivity. Under optimized conditions near 30 samples per hour can be analyzed and the determination range (0.02-20.0 mmol l⁻¹) fully covers creatinine concentrations important from clinical and biomedical point of view. The simple biosensor/FIA system has been successfully used for determination of creatinine in urine, serum and posthemodialysate samples.     

Comparison of Multi‐walled Carbon Nanotubes and Poly(3‐octylthiophene) as Ion‐to‐Electron Transducers in All‐Solid‐State Potassium Ion‐Selective Electrodes

Multi‐walled carbon nanotubes (MWCNTs) were compared with poly(3‐octylthiophene) (POT) as ion‐to‐electron transducer in all‐solid‐state potassium ion‐selective electrodes with valinomycin‐based ion‐selective membranes. MWCNTs and POT were mixed with the other components of the potassium ion‐selective membrane cocktail (valinomycin, KTpClPB, o‐NPOE, PVC, THF) which was then applied on a glassy carbon (GC) substrate to prepare single‐piece ion‐selective electrodes (SPISEs). Results from potentiometric and impedance measurements showed that the MWCNT‐based electrodes have a more reproducuible standard potential and a lower overall impedance than the electrodes based on POT. Both types of electrodes showed similar sensitivity to potassium ions and no redox sensitivity.     

Visible and FTIR Microscopic Observation of Bisthiourea Ionophore Aggregates in Ion‐Selective Electrode Membranes

Since conventional response models for ionophore‐based ISEs are based on the assumption of a homogeneous membrane phase, they cannot accurately predict the response of membranes containing self‐aggregating ionophores. However, meaningful conclusions about the relationship between ionophore structure and potentiometric responses can only be drawn if ionophore aggregation is properly recognized. This study demonstrates that dark field visible microscopy and FTIR microspectroscopy are valuable tools for the observation of such ionophore self‐aggregation and, thereby, the development of new ionophore‐based ISEs. Sulfate selective electrodes with solvent polymeric membranes containing bisthiourea ionophores that differ only by peripheral nonpolar substituents were shown to exhibit very different interferences from the sample pH. On one hand, optimized electrodes based on an ionophore with a phenyl substituent on each thiourea group ( 1 ) do not respond to pH at all and function well as sulfate‐selective electrodes. On the other hand, membranes containing a more lipophilic ionophore with two additional hexyl‐substituted adamantyl groups ( 2 ) exhibit severe pH interference at pH values as low as pH 5. The observation of membranes containing ionophore 2 with dark field visible microscopy and FTIR microspectroscopy shows supramolecular aggregation, and explains the startling difference between the potentiometric responses of the two types of electrodes.     

Functional layers for chemical sensors based on conducting polypyrrole

Functional thin layers based on polypyrrole were used in electrochemical sensors as mixed conducting interfaces between ion‐selective membranes and the wiring. In particular, new types of ion‐selective electrodes for potentiometric measurement of pH value and concentration of sulfate ions in solutions were developed. The resulting electrodes do not need any inner liquid junction. First determinations of the sensor parameters sensitivity, selectivity and long term stability indicate a good performance of the prepared sensors. The results imply that interfaces, containing polypyrrole, could be an interesting basis for the construction of a new type of all‐solid‐state ion‐selective electrodes.     

Quantitative Analysis of Active Pharmaceutical Ingredients (APIs) Using a Potentiometric Electronic Tongue in a SIA Flow System

An advanced potentiometric electronic tongue and Sequential Injection Analysis (SIA) measurement system was applied for the quantitative analysis of mixtures containing three active pharmaceutical ingredients (APIs): acetaminophen, ascorbic acid and acetylsalicylic acid, in the presence of various amounts of caffeine as interferent. The flow‐through sensor array was composed of miniaturized classical ion‐selective electrodes based on plasticized PVC membranes containing only ion exchangers. Partial Least Squares (PLS) analysis of the steady‐state sensor array responses, measured in API mixtures prepared by the SIA system permitted a correct quantitative analysis of acetylsalicylic acid and ascorbic acid. Further optimization using multiway PLS fed by dynamic responses without additional feature extraction did not improve significantly the resolution of acetaminophen. Lastly, the chemometric treatment, involving the extraction of dynamic components of the transient response employing the Wavelet transform, the removal of less‐significant coefficients by means of Causal Index pruning and training of an Artificial Neural Network (ANN) with the selected coefficients, allowed the simultaneous determination of all the three studied APIs, while counterbalancing any interference due to caffeine.     

Miniaturized module with biosensors for potentiometric determination of urea

We describe the construction of a miniaturized module which allows carrying out potentiometric urea determination with the usage of biosensors. The module was fabricated using new hybrid technologies developed in our group which combine ceramic and polymeric materials. Its simplicity and easy way of preparation makes the system very useful for analytical measurements in a flow mode. Application of urease-modified polymer membranes deposited on silver screen-printed electrodes in the module allows to determine urea concentration in clinical range. Moreover, it is a very promising construction for other applications in that other enzymes can be immobilized and various bioanalytes can be determined using this module.     

An improved electrode for the assay of urea in blood

An improved urea enzyme electrode is applied for the determination of urea in blood samples. The electrode is based on the enzymatic hydrolysis of urea, and potentiometric detection of the ammonium ion produced. A silicone rubber-based nonactin ammonium ion-selective electrode serves as the sensor. The selectivity coefficients of this electrode were 6.5 for NH₄⁺/K⁺; 750 for NH₄⁺/Na⁺, and much higher for other cations. The reaction layer of the electrode was made of urease enzyme chemically immobilized on polyacrylic gel. The prepared gel was stable at 4° for over four months. The electrodes retained their activity for over one month. A three-electrode system, which allowed dilution to a constant interference level, was applied to avoid interfering effects in blood samples. Analyses of blood sera showed good agreement with a standard spectrophotometric method. Routine clinical assays of blood urea are feasible.     

Potentiometric Electronic Tongue for Taste Assessment of Ibuprofen Based Pharmaceuticals

The flavor aspect of pharmaceutical formulations is very important in terms of their acceptability for the patient. This work is aimed at using of a novel promising type of electronic tongue (ET), consisting of a potentiometric sensor array, for the discrimination and assessment the taste of commercial pharmaceuticals based on ibuprofen (Ibuflam 4 %). The sensor array was formed by six ion‐selective membranes (ISMs) based on both specific and non‐specific active components. Analysis of potentiometric signal outputs including statistical data processing utilizing the principal component analysis (PCA) method allowed evaluating the role of individual active components of the ISMs in the sense of taste control of the pharmaceutical preparation Ibuflam 4 % before and after flavoring. The sensing array can be used to identify differences in flavor within individual batches from the same producer, since ISMs provide a different response to all components present in the analyzed pharmaceutical. The ET contains ISMs which are able to recognize the bitter taste in the framework of the batches of ibuprofen based pharmaceuticals. To recognize bitter taste, it was essential to include ISMs, providing a response to a particular type of taste, namely, specific and non‐specific ISMs. The experimental findings show that taste‐sensing potentiometric sensor system can be a good alternative to the human panel and is able to detect the slight changes in taste.     

Simulations of Redox Mediation within Bioactive Hydrogels of Amperometric Biosensors

Highly hydrated bioactive hydrogels containing immobilized oxidoreductase enzymes and immobilized redox mediators were simulated as the biorecognition layer of amperometric biosensors. The linear dynamic range of the amperometric response of mediated biosensors increases and moves to higher concentration brackets with an increase in the concentration of mediator. This informs the design of biosensors that target the same analyte but possesses several independently addressable electrodes modified with hydrogels that contain different concentrations of mediator. Increases in enzyme concentration increase the linear dynamic range but does not alter the sensitivity of amperometric biosensors. Both sensitivity and linear dynamic range of mediated amperometric enzyme biosensors may be “tuned” by varying the concentrations of the enzyme and the mediator. Simulations effectively guide the initial domains of study of complex systems such as implantable biosensors.     

Integration of microfabricated needle-type glucose sensor devices with a novel thin-film Ag/AgCl electrode and plasma-polymerized thin film: mass production techniques

We developed an integrated array of needle-type biosensors employing a novel process of fabrication, comprising conventional semiconductor fabrication and micromachining technology. Amperometric sensing electrodes with plasma-polymerized films and a thin-film Ag/AgCl reference electrode were directly integrated on a glass substrate with thin-film process, e.g., sputtering. An enzyme was immobilized on the electrode via the plasma-polymerized film, which was deposited directly on the substrate using a dry process. The novel thin-film Ag/AgCl reference electrode showed stable potentials in concentrated chloride solutions for a long period. The plasma-polymerized film is considered to play an important role as an interfacial design between the sensing electrode and the immobilized enzyme considering that the film is extremely thin, adheres well to the substrate (electrode) and has a highly cross-linked network structure and functional groups, such as amino groups. The results showed increments of the sensor signal, probably because the plasma-polymerized film allowed a large amount of enzyme to be immobilized. The greatest advantage is that the process can permit the mass production of high-quality biosensors at a low cost.     

Hybrid Electronic Tongue as a Tool for the Monitoring of Wine Fermentation and Storage Process

Hybrid electronic tongue based on potentiometric and voltammetric sensors was applied for the monitoring of wine production process. The sensor array formed by miniaturized ion‐selective electrodes and glassy carbon electrodes provided the analysis of the progress and correctness of wine fermentation and storage process, detection of the presence of disturbing factors and evaluation of the quality of final product. The efficiency of the proposed approach was compared with the monitoring of wine production carried out using standard reference methods. The results indicated that hybrid electronic tongue could be used as simple and reliable analytical tool dedicated to qualitative and quantitative assessment of wine production.     

Glucose and urea biosensors based on all solid-state PVC-NH2 membrane electrodes

Miniaturized urea and glucose sensors prepared by immobilization of glucose oxidase or urease directly onto all solid-state contact PVC-NH₂ membrane ammonium and hydrogen ion selective electrodes are described. The resulting biosensing membranes function equivalently to normal PVC membranes in terms of potentiometric response properties. The most important features of the glucose and urea sensors were high sensitivity, long life-time, easily built at a low cost, micro-construction and short response time. The characteristics of the glucose and urea sensors were examined in several buffer solutions at different concentrations and pH values. The influence of immobilization conditions on the dynamic response properties and life-time of the electrodes was studied. Under optimal conditions, the urea electrode showed a linear response between 5×10⁻² and 5×10⁻⁴ M urea, while the glucose electrode showed a linear response between 5×10⁻² and 1×10⁻⁴ M glucose.