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).

Enzyme biosensor systems based on porous silicon photoluminescence for detection of glucose, urea and heavy metals.

A phenomenon of changes in photoluminescence of porous silicon at variations in medium pH is proposed to be used as a basis for the biosensor system development. The method of conversion of a biochemical signal into an optical one is applied for direct determination of glucose and urea as well as for inhibitory analysis of heavy metal ions. Changes in the quantum yield of porous silicon photoluminescence occur at varying pH of the tested solution due to the enzyme-substrate reaction. When creating the biosensor systems, the enzymes urease and glucose oxidase (GOD) were used as a bioselective material; their optimal concentrations were experimentally determined. It was shown that the photoluminescence intensity of porous silicon increased by 1.7 times when increasing glucose concentration in the GOD-containing reaction medium from 0 to 3.0mM, and decreased by 1.45 times at the same increase in the urea concentration in the urease-containing reaction medium. The calibration curves of dependence of the biosensor system responses on the substrate concentrations are presented. It is shown that the presence of heavy metal ions (Cu(2+), Pb(2+), and Cd(2+)) in the tested solution causes an inhibition of the enzymatic reactions catalyzed by glucose oxidase and urease, which results in a restoration of the photoluminescence quantum yield of porous silicon. It is proposed to use this effect for the inhibitory analysis of heavy metal ions.

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.

Stimulation of human olfactory receptor 17-40 with odorants probed by surface plasmon resonance.

We report here the results of human olfactory receptor (OR) 17-40 stimulation with some odorants probed by means of the double-channel surface plasmon resonance platform NanoSPR-6. OR 17-40 tagged with N-terminal cmyc sequence was heterologously co-expressed with Galpha(olf) protein in yeast, and receptor-carrying nanosomes were prepared from yeast membrane fraction. Then, receptors were specifically captured via anti-cmyc antibody attached to the gold-coated substrate in orientated or random way. Measurement of odorants effects were carried out in the presence of GTP-gamma-S in differential mode in order to compensate bulk changes of refractive index. For the first time, biosensing efficiency of olfactory films was discussed in terms of their thickness and Galpha(olf) accessibility to GTP-gamma-S. Bell-shaped response profile with two maxima (near 1 nM and near 1 microM) was established for helional, which is documented as highly specific agonist of OR 17-40. Unrelated odorant heptanal used as control, did not evoke significant variations of differential signal.

Sensitivity and specificity improvement of an ion sensitive field effect transistors-based biosensor for potato glycoalkaloids detection.

Butyryl cholinesterase of different origin along with variations of the time of enzyme immobilization on the potentiometric transducer surface is offered to control the ion sensitive field effect transistor (ISFET)-based biosensor sensitivity. Because butyryl cholinesterase has been already used to develop the sensors for heavy metals, organophosphorus/carbamate pesticides, and steroidal glycoalkaloids analysis, the present study has been focused on the investigation and adjustment of the ISFET-based biosensor specificity exclusively to the glycoalkaloids. Utilization of ethylendiaminetetracetate (a complexon of heavy metal ions) and phosphotriesterase (a highly efficient catalyst for the hydrolysis of organophosphorus compounds) enabled the highly specific determination of glycoalkaloids at the background of lead and mercury (up to 500 microM of ions concentration) and paraoxon (up to 100 microM of pesticide concentration). The developed biosensor has been validated for glycoalkaloids detection in potato varieties cultivated in Ukraine, and the results obtained are compared to those measured by the methods of HPLC and TLC.

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.

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.

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.

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.