Biosensors Based on Nano-Gold/Zeolite-Modified Ion Selective Field-Effect Transistors for Creatinine Detection.

The combination of advantages of using zeolites and gold nanoparticles were aimed to be used for the first time to improve the characteristic properties of ion selective field-effect transistor (ISFET)-based creatinine biosensors. The biosensors with covalently cross-linked creatinine deiminase using glutaraldehyde (GA) were used as a control group, and the effect of different types of zeolites on biosensor responses was investigated in detail by using silicalite, zeolite beta (BEA), nano-sized zeolite beta (Nano BEA) and zeolite BEA including gold nanoparticle (BEA-Gold). The presence of gold nanoparticles was investigated by ICP, STEM-EDX and XPS analysis. The chosen zeolite types allowed investigating the effect of aluminium in the zeolite framework, particle size and the presence of gold nanoparticles in the zeolitic framework.After the synthesis of different types of zeolites in powder form, bare biosensor surfaces were modified by drop-coating of zeolites and creatinine deiminase (CD) was adsorbed on this layer. The sensitivities of the obtained biosensors to 1 mM creatinine decreased in the order of BEA-Gold > BEA > Nano BEA > Silicalite > GA. The highest sensitivity belongs to BEA-Gold, having threefold increase compared to GA, which can be attributed to the presence of gold nanoparticle causing favourable microenvironment for CD to avoid denaturation as well as increased surface area. BEA zeolites, having aluminium in their framework, regardless of particle size, gave higher responses than silicalite, which has no aluminium in its structure. These results suggest that ISFET biosensor responses to creatinine can be tailored and enhanced upon carefully controlled alteration of zeolite parameters used to modify electrode surfaces.

Creatinine Deiminase Adsorption onto Silicalite-Modified pH-FET for Creation of New Creatinine-Sensitive Biosensor.

In the work, silicalite particles were used for the surface modification of pH-sensitive field-effect transistors (pH-FETs) with the purpose of developing new creatinine-sensitive biosensor. Creatinine deiminase (CD) adsorbed on the surface of silicalite-coated pH-FET served as a bioselective membrane. The biosensor based on CD immobilized in glutaraldehyde vapor (GA) was taken as control. The creatinine-sensitive biosensor obtained by adsorption on silicalite was shown to have better analytical characteristics (two- to threefold increased sensitivity to creatinine, three- to fourfold lesser response and recovery times, a decrease of the detection limit of creatinine determination to 5 µM, etc.).Additionally, the biosensor based on CD adsorbed on silicalite (Sil/CD) was characterized by high signal reproducibility (relative standard deviation (RSD) for creatinine measurement = 2.6 %) and stability during storage (over 13 months). It was experimentally confirmed that the proposed biosensor was not sensitive either to high concentrations of sodium chloride or to the macromolecular protein fractions and can be used for direct quantitative analysis of creatinine in the blood serum.It was concluded that the method of CD adsorption on silicalite is well-suited for the creation of creatinine-sensitive biosensor with improved working characteristics.

Development of Silicalite/Glucose Oxidase-Based Biosensor and Its Application for Glucose Determination in Juices and Nectars.

The application of silicalite for improvement of enzyme adsorption on new stainless steel electrodes is reported. Glucose oxidase (GOx) was immobilized by two methods: cross-linking by glutaraldehyde (GOx-GA) and cross-linking by glutaraldehyde along with GOx adsorption on silicalite-modified electrode (SME) (GOx-SME-GA). The GOx-SME-GA biosensors were characterized by a four- to fivefold higher sensitivity than GOx-GA biosensor. It was concluded that silicalite together with GA sufficiently enhances enzyme adhesion on stainless steel electrodes. The developed GOx-SME-GA biosensors were characterized by good reproducibility of biosensor preparation (relative standard deviation (RSD)-18 %), improved signal reproducibility (RSD of glucose determination was 7 %), and good storage stability (29 % loss of activity after 18-day storage). A series of fruit juices and nectars was analyzed using GOx-SME-GA biosensor for determination of glucose concentration. The obtained results showed good correlation with the data of high-performance liquid chromatography (HPLC) (R = 0.99).

Thin-film amperometric multibiosensor for simultaneous determination of lactate and glucose in wine.

An amperometric multi-biosensor based on lactate and glucose oxidases has been developed for determination of lactate and glucose in wine. Gold thin-film amperometric electrodes were used as multi-transducers. Analytical characteristics of the multi-biosensor developed were studied. The minimum detectable concentration was 5×10(-6) mol/l for both glucose and lactate. High reproducibility and storage stability of the multi-biosensor are demonstrated in this paper. Lactate and glucose were determined in wine, and a good correlation was obtained with concentrations determined using high-performance liquid chromatography (correlation coefficient for glucose R(2)=0.998, for lactate R(2)=0.718).

Highly sensitive electrochemical biosensor for bisphenol A detection based on a diazonium-functionalized boron-doped diamond electrode modified with a multi-walled carbon nanotube-tyrosinase hybrid film.

A highly sensitive electrochemical biosensor for the detection of Bisphenol A (BPA) in water has been developed by immobilizing tyrosinase onto a diazonium-functionalized boron doped diamond electrode (BDD) modified with multi-walled carbon nanotubes (MWCNTs). The fabricated biosensor exhibits excellent electroactivity towards o-quinone, a product of this enzymatic reaction of BPA oxidation catalyzed by tyrosinase. The developed BPA biosensor displays a large linear range from 0.01 nM to 100 nM, with a detection limit (LOD) of 10 pM. The feasibility of the proposed biosensor has been demonstrated on BPA spiked water river samples. Therefore, it could be a promising and reliable analytical tool for on-site monitoring of BPA in waste water.

Nano- and microsized zeolites as a perspective material for potentiometric biosensors creation.

A number of potentiometric biosensors based on coimmobilization of enzymes with different types of zeolite on pH-ion-sensitive field-effect transistor (ISFET) have been developed. Their working characteristics have been determined and compared. It was shown that clinoptilolite and zeolite Beta polymorph A (BEA) are more promising for creating biosensors than zeolite A. Changing the concentration of zeolite BEA in membranes, it is possible to extend the biosensor linear measurement range. The two-layer method of deposition of the enzyme with clinoptilolite was found to provide a significant increase in the biosensor sensitivity to substrates, whereas thermal modification of the zeolite BEA crystals can improve analytical characteristics of potentiometric biosensors for detection of toxic substances. These results show that it is possible to regulate the ISFET characteristics for different enzyme-based biosensors by tailoring the electrode surfaces via different zeolites. This makes zeolites strong candidates for integration into biosensors as ISFET modifiers.

Application of silicalite-modified electrode for the development of sucrose biosensor with improved characteristics.

The application of silicalite for improvement of working characteristics of conductometric enzyme biosensors for determination of sucrose was studied in this research. Biosensors based on different types of silicalite-modified electrodes were studied and compared according to their analytical characteristics. Polyethylenimine/glutaraldehyde/silicalite-modified biosensors showed higher sensitivity compared with others type of biosensors. Moreover, the polyethylenimine/glutaraldehyde/silicalite sucrose biosensors were characterized by high selectivity and signal reproducibility (relative standard deviation (RSD) = 2.78% for glucose measurements and RSD = 3.2% for sucrose measurements). Proposed biosensors were used for determination of sucrose in different samples of beverages. The obtained results had good correlation with results obtained by HPLC. Thus, polyethylenimine/glutaraldehyde/silicalite-modified biosensors have shown perspective characteristics for the development of effective conductometric enzyme biosensors.

Sensitive impedimetric biosensor for direct detection of diazinon based on lipases.

Two novel impedimetric biosensors for highly sensitive and rapid quantitative detection of diazinon in aqueous medium were developed using two types of lipase, from Candida Rugosa (microbial source) (CRL) and from porcine pancreas (animal source) (PPL) immobilized on functionalized gold electrode. Lipase is characterized to specifically catalyze the hydrolysis of ester functions leading to the transformation of diazinon into diethyl phosphorothioic acid (DETP) and 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP). The developed biosensors both presented a wide range of linearity up to 50 µM with a detection limit of 10 nM for Candida Rugosa biosensor and 0.1 µM for porcine pancreas biosensor. A comparative study was carried out between the two biosensors and results showed higher efficiency of Candida Rugosa sensor. Moreover, it presented good accuracy and reproducibility, had very good storage and multiple use stability for 25 days when stored at 4°C.

Study of zeolite influence on analytical characteristics of urea biosensor based on ion-selective field-effect transistors.

A possibility of the creation of potentiometric biosensor by adsorption of enzyme urease on zeolite was investigated. Several variants of zeolites (nano beta, calcinated nano beta, silicalite, and nano L) were chosen for experiments. The surface of pH-sensitive field-effect transistors was modified with particles of zeolites, and then the enzyme was adsorbed. As a control, we used the method of enzyme immobilization in glutaraldehyde vapour (without zeolites). It was shown that all used zeolites can serve as adsorbents (with different effectiveness). The biosensors obtained by urease adsorption on zeolites were characterized by good analytical parameters (signal reproducibility, linear range, detection limit and the minimal drift factor of a baseline). In this work, it was shown that modification of the surface of pH-sensitive field-effect transistors with zeolites can improve some characteristics of biosensors.

Application of amperometric biosensors for analysis of ethanol, glucose, and lactate in wine.

This article presents the application of amperometric biosensors based on platinum printed electrodes SensLab and immobilized enzymes, alcohol oxidase, glucose oxidase, and lactate oxidase, for wine analysis. Created devices demonstrate linear response to ethanol, glucose, and lactate within the concentration range 0.3-20 mM, 0.04-2.5 mM, and 0.008-1 mM, respectively. No decrease in ethanol and glucose biosensor activity is revealed during 2 months after fabrication, and the operational stability of the lactate biosensor is sufficient only during 4 days. Developed biosensors showed high selectivity to the substrate and are successfully applied to the analysis of such complex mixtures as wine and must. Good correlation of the results of analysis of different wines and must obtained by amperometric biosensors with immobilized oxidases and traditional methods is shown. Created biosensors can be used as a basis of a commercial device for express analysis of ethanol, glucose, and lactate in wine and must during its fermentation. Application of such devices for quality control in foodstuff industry can have great economical effect because determination by biosensors is less expensive, labor-intensive, and lengthy than traditional methods of analysis.

Conductometric Microbiosensors for Environmental Monitoring.

This review presents the principles of conductometric measurements in ionic media and the equivalent electrical circuits of different designs for conductometric measurements. These types of measurements were first applied for monitoring biocatalytic reactions. The use of conductometric microtransducers is then presented and detailed in the case of pollutant detection for environmental monitoring. Conductometric biosensors have advantages over other types of transducers: they can be produced through inexpensive thinfilm standard technology, no reference electrode is needed and differential mode measurements allow cancellation of a lot of interferences. The specifications obtained for the detection of different pesticides, herbicides and heavy metal ions, based on enzyme inhibition, are presented as well as those obtained for the detection of formaldehyde, 4- chlorophenol, nitrate and proteins as markers of dissolved organic carbon based on enzymatic microbiosensors.

Enzyme biosensors based on ion-selective field-effect transistors.

The key theoretical principles of the work on ion-selective field-effect transistor connected with their application in bioanalytical practice, some specifics of modern microtechnologies for their creation, and measurement schemes with set-ups are discussed. The achievements in the creation of enzyme biosensors based on ion-selective field-effect transistors and prospects for their application are described in detail.

Conductometric nitrate biosensor based on methyl viologen/Nafion/nitrate reductase interdigitated electrodes.

A highly sensitive, fast and stable conductometric enzyme biosensor for determination of nitrate in water is reported for the first time. The biosensor electrodes were modified by methyl viologen mediator mixed with nitrate reductase (NR) from Aspergillus niger by cross-linking with glutaraldehyde in the presence of bovine serum albumin and Nafion((R)) cation-exchange polymer. The process parameters for the fabrication of the enzyme electrode and various experimental variables such as pH, the enzyme loading and time of immobilization in glutaralaldehyde vapor were investigated with regard to their influence on sensitivity, limit of detection, dynamic range and operational and storage stability. The biosensor can reach 95% of steady-state conductance value in about 15s. Linear calibration in the range of 0.02 and 0.25 mM with detection limits of 0.005 mM nitrate was obtained with a signal-to-noise ratio of 3. When stored in 5 mM phosphate buffer (pH 7.5) at 4 degrees C, the sensor showed good stability over 2 weeks.

Analysis of the potato glycoalkaloids by using of enzyme biosensor based on pH-ISFETs.

The applicability of an enzyme biosensor based on pH-ISFETs for direct determination of total glycoalkaloids content in real potato samples, without any pre-treatment, is shown. The results of determination of the total glycoalkaloids concentrations in potato samples from different experimental varieties obtained by the biosensor are well correlated with the analogous data obtained by the HPLC method with standard complex sample pre-treatment procedure. The detection of total glycoalkaloids content by biosensors is reproducible, the relative standard deviation was around 3%. The dependence of total glycoalkaloids content on various parts of the potato tuber and their size, different growing area has been shown using the biosensor developed. The method based on biosensors is cheap, easy to operate and requires a shorter analysis time than the one needed using traditional methods for glycoalkaloids determination. The biosensor can operate directly on potato juice, or even directly on a suspension of potato or plant material. It can provide a way to save time and costs, with a possibility of taking rapid assessment of total glycoalkaloids content in a wide variety of potato cultivars. Furthermore the operational and storage stability of this biosensor are quite good with a drift lower than 1% per day and response being stable for more than 3 months.

Conductometric tyrosinase biosensor for the detection of diuron, atrazine and its main metabolites.

The determination of diuron, atrazine, desisopropylatrazine (DIA) and desethylatrazine (DEA) were investigated using conductometric tyrosinase biosensor. Tyrosinase was immobilised on the biosensor sensitive part by allowing it to mix with bovine serum albumin (BSA) and then cross-linking in saturated glutaraldehyde (GA) vapour for 30min. The determination of pollutants in a solution was performed by comparison of the output signal (i.e percentage of the enzymatic activity) of the biosensor before and after contact with pollutants. The measurement of the enzymatic activity was performed using 4-chlorophenol, phenol and catechol substrates and response times ranging from 1 to 5min were observed. A 4-chlorophenol substrate was used to detect pesticides. A 30min contact time of the biosensor in the pollutant solution was used. Under the experimental conditions employed, detection limits for diuron and atrazine were about 1ppb and dynamic range of 2.3-2330 and 2.15-2150ppb were obtained for diuron and atrazine, respectively. A relative standard deviation (n=3) of the output signal was estimated to be 5% and a slight drift of 1.5muSh(-1) was observed. The 90% of the enzyme activity was still maintained after 23 days of storage in a buffer solution at 4 degrees C.

Biosensors based on enzyme field-effect transistors for determination of some substrates and inhibitors.

This paper is a review of the authors' publications concerning the development of biosensors based on enzyme field-effect transistors (ENFETs) for direct substrates or inhibitors analysis. Such biosensors were designed by using immobilised enzymes and ion-selective field-effect transistors (ISFETs). Highly specific, sensitive, simple, fast and cheap determination of different substances renders them as promising tools in medicine, biotechnology, environmental control, agriculture and the food industry. The biosensors based on ENFETs and direct enzyme analysis for determination of concentrations of different substrates (glucose, urea, penicillin, formaldehyde, creatinine, etc.) have been developed and their laboratory prototypes were fabricated. Improvement of the analytical characteristics of such biosensors may be achieved by using a differential mode of measurement, working solutions with different buffer concentrations and specific agents, negatively or positively charged additional membranes, or genetically modified enzymes. These approaches allow one to decrease the effect of the buffer capacity influence on the sensor response in an aim to increase the sensitivity of the biosensors and to extend their dynamic ranges. Biosensors for the determination of concentrations of different toxic substances (organophosphorous pesticides, heavy metal ions, hypochlorite, glycoalkaloids, etc.) were designed on the basis of reversible and/or irreversible enzyme inhibition effect(s). The conception of an enzymatic multibiosensor for the determination of different toxic substances based on the enzyme inhibition effect is also described. We will discuss the respective advantages and disadvantages of biosensors based on the ENFETs developed and also demonstrate their practical application.

Development and optimisation of biosensors based on pH-sensitive field effect transistors and cholinesterases for sensitive detection of solanaceous glycoalkaloids.

Highly sensitive biosensors based on pH-sensitive field effect transistors and cholinesterases for detection of solanaceous glycoalkaloids have been developed, characterised and optimised. The main analytical characteristics of the biosensors developed have been studied under different conditions and an optimal experimental protocol for glycoalkaloids determination in model solution has been proposed. Using such a biosensor and an enzyme reversible inhibition effect, the total potato glycoalkaloids content can be determined within the range of 0.2-100 microM depending on the type of alkaloid, with lowest detection limits of 0.2 microM for alpha-chaconine, 0.5 microM for alpha-solanine and 1 microM for solanidine. The dynamic ranges for the compounds examined show that such biosensors are suitable for a quantitative detection of glycoalkaloids in real potato samples. High reproducibility, operational and storage stability of the biosensor developed have been shown.