Creatinine sensitive biosensor based on ISFETs and creatinine deiminase immobilised in BSA membrane

A creatinine sensitive biosensor based on ion sensitive field-effect transistors (ISFETs) with immobilised creatinine deiminase (CD) is developed. CD is immobilised on the transducer surface by classical cross-linking with bovine serum albumin (BSA) in a glutaraldehyde (GA) vapour. The linear dynamic ranges of biosensors are between 0 and 5 mM creatinine concentration, and the sensor sensitivity depends on the sample buffer concentration. Minimal detection limit for creatinine determination in model solution with 144 mM NaCl and 5% BSA, pH 7.4, is about 10 muM. Biosensor responses are reproducible and stable during continuous work at least for 8 h, and the relative standard deviation of sensor response is approximately 3% (n=48, for creatinine concentrations of 0.2 and 0.6 mM). An investigation about storage stability of creatinine sensitive ENFETs kept in dry at 4-6 degrees C shows that biosensors demonstrate an excellent storage stability for at least 6 months and more. Moreover, creatinine sensitive enzymatic field-effect transistors (ENFETs), demonstrating very good performances, are very selective and specific and well suitable for hemodialysis monitoring.     

Novel Multiplexed Biosensor System for the Determination of Lactate and Pyruvate in Blood Serum

Multiplexing is one of the main current trends in biosensors, especially important for clinical diagnosis. However, simultaneous determination of several substances in one sample is often difficult due to different performance and working conditions of separate biosensors. This work was aimed at the development of a multiplexed biosensor system for the determination of lactate and pyruvate concentrations in liquid samples (i. e., blood serum). The system consisted of two amperometric biosensors based on lactate oxidase and pyruvate oxidase, which worked simultaneously in a single measuring cell. Conditions for the biosensor system work were selected. Linear range of lactate determination was 5–1000 μM, pyruvate – 10–5000 μM. Steady‐state response time was 30 s and 50 s for the lactate and pyruvate biosensors, respectively. After 2 weeks of storage biosensor responses decreased to 95 % (lactate biosensor) or 82 % (pyruvate biosensor) of the initial value. A scheme of analysis of the concentrations of lactate and pyruvate in human blood serum was proposed. The lactate and pyruvate concentrations as well as their ratio in human blood serum samples were determined and compared with the control method. The proposed biosensor system is suitable for the rapid detection of lactate, pyruvate and their ratio and can be used for clinical diagnosis, e. g., evaluation of the reasons of lactic acidosis and prognosis of patient's recovery.     

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.     

Characterization of biosensors based on membranes containing a conducting polymer

A new type of biosensor based on the coupling of an enzyme to an ion-selective membrane containing a conducting polymer is evaluated. The results obtained with the enzyme field- effect transistor (ENFET) and the ion-selective electrode (ISE) for the determination of creatinine and urea are compared. The presence of the conducting polymer significantly lowers the detection limit for creatinine by one decade to 10^?7 and 10^?4 M for the ENFET and ISE, respectively. The determination of urea in urine and serum with the ENFET was carried out, and the results correlated well with those obtained by spectrophotometry.     

Formation and dichroism of poly(vinyl alcohol)–iodine complex in photocurable film

The formation of polyiodine complexes was investigated in a photocurable poly(vinyl alcohol) modified N‐methyl‐4(4′‐formylstyryl)pyridinium methosulfate acetal (PVA‐SbQ), which is a photofunctional group that causes photodimerization. PVA‐SbQ films with polyiodine complexes were prepared to be photocured, iodinated and soaked in a boric acid solution. The formation of PVA–polyiodine complexes was studied during iodinating and while in the boric acid treatment through UV–vis absorption spectrometry, resonance Raman spectrometry and IR absorption spectrometry. As a result, polyiodines were formed in the photocurable PVA‐SbQ films, and the formation of PVA–polyiodine complexes was enhanced by boric acid treatment. It was found that the SbQ‐ratio of PVA‐SbQ affects the formation of PVA–polyiodine complexes. The photocrosslinking by the dimerization of SbQ groups helps to form the PVA–I₅⁻ complex during the boric acid treatment. Based on this effect, we demonstrated a unique recording method by the PVA–polyiodine complex formation. The PVA‐SbQ film cured by the irradiation of the liner polarized light showed that dichroism of the PVA–polyiodine complexes formed after iodination. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparison of Single Layer and Bilayer Biosensors Based on Crosslinking of Penicillinase for Potentiometric Detection of Penicillin in Milk and Antibiotics

The use of bilayers for fabrication of biosensors is advantageous for increasing enzyme loading. Substantial improvement in sensitivity is often achieved through immobilisation of the enzyme in both layers. In particular, the use of cross linking agents such as glutaraldehyde (GLA), bovine serum albumin (BSA) and polyvinyl alcohol (PVA) are beneficial for enhancing enzyme stability and, hence, for fabricating stable biosensors. The successful fabrication of a single layer BSA‐GLA‐P’nase biosensor for potentiometric detection of penicillin is described. Subsequently, the three crosslinking agents were employed with two polymers, polypyrrole (PPy) and polytyramine (PTy), together with penicillinase (P’nase) for fabrication of PPy‐NO₃‐P’nase/BSA‐PVA‐P’nase and PTy‐NO₃‐P’nase/BSA‐GLA‐P’nase bilayer biosensors. The analytical performances of the bilayer biosensors were then compared with the single layer BSA‐GLA‐P’nase biosensor for the determination of penicillin in milk and amoxycillin tablets. While the determination of penicillin in milk was somewhat problematic, its determination in amoxicillin tablets proved to be successful, with recoveries of 102±15 % obtained with the PPy‐NO₃‐P’nase/BSA‐PVA‐P’nase biosensor, 100±19 % with PTy‐NO₃‐P’nase/BSA‐GLA‐P’nase biosensor and 103±5 % with BSA‐GLA‐P’nase biosensor. Notably, the results of the latter agreed favourably with those obtained through a reference titrimetric method.     

Urea biosensor based on amperometric pH-sensing with hematein as a pH-sensitive redox mediator

The natural dye hematein in water solution was used as a pH-sensitive redox-active mediator for amperometric pH-sensing. The electrochemical characteristics were studied using cyclic voltammetry and chronoamperometry. Several types of urea biosensors were constructed with urease on the surface of platinum and graphite composite electrodes or in the bulk of the graphite composite. They were used for the amperometric urea determination at a working potential of 0 mV (versus SCE) using 0.5 mM hematein. Detection limits and response linearity was in the micromolar range depending on the biosensor type, concentration and pH of buffers used. An interference study of various cations, anions, and substances, which may be present in real samples demonstrated good selectivity for the determination of urea. The biosensors showed good operational (>3 h) and storage (>3 months) stability. The results of urea determination in blood and urine obtained by biosensor correlated well with those obtained by a spectrophotometric reference method.     

Enzymatic Biosensor Based on the ISFET and Photopolymeric Membrane for the Determinaion of Urea

An ISFET based enzymatic biosensor was developed for the determination of urea. Immobilization of urease was accomplished by the use of liquid mixture which contained vinylpyrrolidone, oligouretane metacrylate and oligocarbonate metacrylate and which can form a polymer under the influence of ultraviolet. The biosensor has the following characteristics: the linear field of responses is in the range of 0.05–20 mM, curve slope – 38 mV/pC, and response time 5–10 min. The increase of the temperature from 28 to 41 °C leads to 15% increase in the intensity of the response of the biosensor. The maximum response is observed at pH 6.0–6.5. At the increase of the NaCl concentration in solution up to 300 mM the biosensor response drops off and achieves half of its initial level. NH₄Cl causes a stronger inhibition of enzyme activity comparing to NaCl. The results obtained with the developed biosensor correlate with the data of standard calorimetric methods. The intensity of the biosensor response decreases gradually during 40 days up to 80% of the initial level. The biosensors prepared with a fresh membrane or membrane preserved during 46 days at 2 °C gave similar responses in solution with an equal concentration of a substrate. It is concluded that the developed enzymatic biosensor is perspective for its clinical application for the determination of urea in blood and that the proposed method to prepare a selective biological membrane may be in a simple way included in integral technology of the semiconductor transducer manufacturing.     

The Use of Poly(Vinyl Alcohol) to Cross‐link Penicillinase for the Fabrication of a Penicillin Potentiometric Biosensor

Chemically cross‐linked PVA films are permeable matrices for the fabrication of biosensors. PVA provides an attractive immobilisation method as it preserves the enzymatic activity. Penicillinase (P’nase) was cross‐linked with poly(vinyl alcohol) (PVA) and bovine serum albumin (BSA). The optimum conditions for the of BSA‐PVA‐P’nase film were: 2.5 % w/v PVA, 0.006 % w/v BSA, 2.4 mM penicillin (Pen) and 16 U/mL P’nase. The minimum detectable concentration was 1.7 µM. The linear concentration range obtained for the BSA‐PVA film was 7.5–283 µM. The BSA‐PVA P’nase biosensor detected penicillin in amoxycillin with an average percentage recovery of 97±12 %. Higher penicillin concentrations (10–20 ppm) were detected more successfully than lower concentrations (≤5 ppm). These results indicate that further work is required to enable the successful detection of lower penicillin concentrations such as 5 ppm.     

Potentiometric determination of dialysate urea nitrogen

An enzymatically modified ammonium ion-selective electrode has been applied for the determination of urea in spent dialysate. The biosensor has been used in a simple flow-injection analysis (FIA) system. The system enables one to perform over 25 dialysate urea nitrogen (DUN) determinations per hour. The interferences from other components of posthemodialysis fluid were eliminated by simultaneous measurements with non-modified enzymatically ion-selective electrode. It is possible to use both the sensors in a simplified differential potentiometric system. The results of DUN determination using the biosensor/FIA system and a conventional method of urea determination were comparable. The presented analytical system can potentially find wider biomedical application in the monitoring of hemodialysis progress.     

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.     

Effective and Functional Surface Design for Biosensing Applications Based on a Novel Conducting Polymer and PMMA/Clay Nanocomposite

Surface functionalization plays a crucial role in the design of biosensors. For this purpose, a novel functional monomer, 6‐(4,7‐bis(2,3‐dihydrothieno[3,4‐b][1,4]dioxin‐5‐yl)‐2H‐benzo[d][1,2,3]triazol‐2‐yl)hexan‐1‐amine (BEDOA‐6), was designed and synthesized. Poly(BEDOA‐6) was utilized as an immobilization matrix for glucose oxidase biosensor construction. Moreover, polymethylmethacrylate (PMMA) layered silicate nanocomposites were prepared by in situ suspension polymerization. Conducting polymer surface was modified with PMMA/clay nanocomposite material and a glucose biosensor was developed. In addition, XPS and SEM were utilized to characterize the surface properties. The biosensor shows a wide linear range between 2.8 µM and 1.2 mM to glucose with a low detection limit of 1.99 µM. Finally, the biosensor was tested on serum samples containing actual human blood. The results were in well‐agreement with a reference method.     

Simultaneous Determination of Creatine and Creatinine using Monocrystalline Diamond Paste–Based Amperometric Biosensors

Abstract

In order to determine creatine and creatinine, amperometric diamond paste biosensors were proposed. A bienzymatic biosensor based on creatinase and sarcosine oxidase was used for the assay of creatine and a trienzymatic biosensor based on creatinase, sarcosine oxidase, and creatininase was proposed for the assay of creatinine. The linear concentration ranges are of fmol/L magnitude order, with very low limits of detection. The biosensors proved to be highly reliable for determination of creatine and creatinine as raw materials in pharmaceutical formulations as well as in serum samples.     

Dynamics of cyclodimerization and viscoelasticity of photo‐crosslinkable PVA

We investigated the interfacial properties of poly(vinyl alcohol) carrying UV‐crosslinkable pendant quaternized stilbazole (styrylpyridinium), PVA‐SbQ. The extent and dynamics of PVA‐SbQ cyclodimerization reactions and crosslinking induced by UV irradiation were monitored in situ and in real time by quartz crystal microgravimetry (QCM). Sensograms reflecting time‐dependent changes in density and viscoelasticity of crosslinking films followed a Boltzmann sigmoidal model, depending on precursor film composition and irradiation power. The shifts in QCM frequency and energy dissipation upon PVA‐SbQ cyclodimerization correlated with three photo‐crosslinking phases involving soft‐to‐rigid transitions, namely, induction (initiation), main crosslinking (interaction), and termination. PVA‐SbQ films crosslinked to different degree were used as protein carriers and a slower release profile was determined for the films that underwent more extensive crosslinking. Overall, this study demonstrates for the first time the dynamics of PVA‐SbQ crosslinking and its impact in system viscoelasticity and protein release. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2015 , 53, 345–355     

Spectrophotometric bioanalytical flow-injection system for control of hemodialysis treatment

A spectrophotometric flow-injection analysis (FIA) system for monitoring clinical hemodialysis is demonstrated. The role of a dialysate urea detector incorporated in this bioanalytical system is played by an optical flow-through biosensor based on Prussian Blue film with chemically linked urease forming a monomolecular layer of the enzyme. This pH-enzyme optode-FIA system is useful for the selective determination of post-dialysate urea in the range of concentration corresponding to its level in real clinical samples (2-16 mmol l(-1)). This bioanalytical system allows the analysis of about 15 samples of spent dialysate per hour. The operational and storage stabilities of the applied biosensor are longer than 2 weeks and 2 months, respectively. Clinical evaluation of the bioanalytical system was performed.     

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.     

Thick film biosensors for metabolites in undiluted whole blood and plasma samples

The new electrochemical thick film biosensors from Roche Diagnostics are presented. Following considerations about the principal requirements that biosensors have to fulfil to be useful for diagnostic purposes, the basic design of these thick film biosensors is shown. In this paper, the new generation of biosensors for glucose, lactate and urea are presented, as well as data from a new biosensor for creatinine. All biosensors are designed for multiple use, at minimum 500 samples or 1 week in-use (depending on type of enzyme used), for determinations in undiluted whole blood or plasma, with extra electrodes to compensate for interferences. The sensors are integrated in a disposable cassette requiring 38 microtl sample volume. The analytical ranges of the sensors scope well with the normal and pathological concentrations of metabolites in human blood, e.g. for glucose 0.5-40.0 mmol/L. Both biosensors and interference-compensating electrodes are developed to have a cycle time of 90 s maximum. Method comparison diagrams show excellent correlation of results obtained by biosensors compared to results achieved by reference methods. In addition, the possibility of urea and creatinine determinations in diluted urine is presented.     

Chronocoulometric determination of urea in human serum using an inkjet printed biosensor

A biosensor for the determination of urea in human serum was fabricated using a combination of inkjet printed polyaniline nanoparticles and inkjet printed urease enzyme deposited sequentially onto screen-printed carbon paste electrodes. Chronocoulometry was used to measure the decomposition of urea via the doping of ammonium at the polyaniline-modified electrode surface at -0.3 V vs. Ag/AgCl. Ammonium could be measured in the range from 0.1 to 100 mM. Urea could be measured by the sensor in the range of 2-12 mM (r(2)=0.98). The enzyme biosensor was correlated against a spectrophotometric assay for urea in 15 normal human serum samples which yielded a correlation coefficient of 0.85. Bland-Altman plots showed that in the range of 5.8-6.6 mM urea, the developed sensor had an average positive experimental bias of 0.12 mM (<2% RSD) over the reference method.     

Nitrogen‐Doped Carbon Hollow Spheres for Immobilization,Direct Electrochemistry,and Biosensing of Protein

Nitrogen‐doped carbon hollow spheres (NCHS) were designed for the immobilization and biosensing of proteins. Chitosan was first functionalized with glutaraldehyde to form cross‐linked chitosan with free ? CHO groups (GCS). The as‐prepared GCS was used for dispersion of nitrogen‐doped carbon hollow spheres. Using glucose oxidase (GOD) as a model, the NCHS was tested for immobilization of redox proteins and the design of electrochemical biosensors. GOD molecules immobilized in the nanocomposites showed direct electrochemistry with a formal potential of ?0.448 V and well electrochemical performance. The proposed biosensor exhibited a linear response to glucose concentrations ranging from 3.7 µM to 18.0 mM with a detection limit of 1.2 µM and a sensitivity of 11.85 µA mM^?1. This biosensor was also applied to detect glucose in human serum samples, accomplishing good recovery in the range of 92–105 %. The nanocomposites provided a good matrix for protein immobilization and biosensor fabrication.