Toonika Rinken

Dynamic model of amperometric biosensors. Characterisation of glucose biosensor output

An integrated model for the characterisation of the output signal course of oxidase-bound amperometric biosensors is presented and evaluated in the case of glucose biosensors. This model integrates two earlier proposed models, the model of oxygen transducer-based biosensors, allowing the prediction of steady state parameters from the transient response and the dynamic signal lag model, characterising the electrochemical diffusion-limited sensors. The integrated model allows the characterisation of the whole biosensor signal output, originating from the output curve itself with errors less than 3% and no need to determine the systems geometrical, diffusion and partition parameters.

Calibration of glucose biosensors by using pre-steady state kinetic data.

A new method for biosensor calibration and data processing, allowing the prediction of steady state parameters from the analysis of transient response curves (Rinken et al., 1996. Analytical Letters 29, 859), has been evaluated in the case of an oxygen sensor based two-substrate enzyme electrode for glucose determination. The electrochemical glucose biosensor was prepared by covering the surface of oxygen sensor with glucose oxidase (EC 1.1.3.4) immobilized in nylon mesh. This decreased the oxygen flow to the sensor in the presence of glucose and resulted in time-dependent decrease of the biosensor signal. Except the lag period of the response in the beginning of the assay, the oxygen consumption by the immobilized enzyme was described by an exponential function: [formula: see text] The parameter C, which corresponded to the steady-state output of the biosensor, was found to be the most suitable for glucose determination. The non-linear fitting for data of over 1000 independent experiments to the equation above always revealed correlation coefficients greater than 0.97. The calculation of the steady state parameter from the transient phase data makes the analysis fast and precise, especially for sensors with thick membranes, being convenient to use in the case of enzyme electrodes. The theoretical essence of the parameter C also gives valuable information for the optimal design of biosensors.

Characterization of glucose oxidase immobilization onto mica carrier by atomic force microscopy and kinetic studies

Glucose oxidase (E.C 1.1.3.4) immobilized onto activated surface of mica was analyzed by enzymatic kinetics and visualization with atomic force microscopy (AFM). The activity of the immobilized enzyme decreased with the decrease of concentration of gamma-aminopropyltrimethoxysilane used for the first step of activation of mica, while AFM analysis showed similar homogeneous filling of the surface with the enzyme. The comparison of enzyme activity with its surface filling revealed that there has to be additional vertical structures, which cannot be visualized by the methods of AFM. The simultaneous decrease of the silanizing agent and the concentration of the enzyme led to molecular resolution for the enzyme on the surface of mica. This allows to propose the described method also for analyzing other surfaces of solid materials with coupled biomolecules.

Biosensors for Biogenic Amines: The Present State of Art Mini-Review

The analysis of biogenic amines in fresh and processed food is of great importance not only for the potential risk these compounds have on human health, but also because these amines can perform as chemical indicators of food quality and enable the assessment of food processing conditions and/or microbial contamination. A good option for a rapid detection of biogenic amines is the application of biosensors, as these devices enable the obtainment of results in a few minutes without pretreatment of the analyzed material. Biosensors for biogenic amines comprise various combinations of different enzymes for selective biorecognition and signal transduction systems and are based on different signal mechanisms. The present review gives a condensed overview of the recent developments and issues in the construction of biosensors for the detection of most common biogenic amines found in food and other protein-containing material.

Determination of Kinetic Constants and Enzyme Activity from a Biosensor Transient Signal

Abstract The kinetic constants and activity of soluble and immobilized enzymes can be determined electrochemically with a biosensor by applying the dynamic integrated model, which includes the inertia of the registering device, for data management and calculations. This model of electrochemical diffusion-limited sensors allows the prediction of steady-state parameters for enzyme-catalyzed reactions from a biosensor transient response and reproducible calculation of real physico-chemical constants without any additional experimental data. This approach has been used for the estimation of activity parameters of soluble and immobilized on mica glucose oxidase and for the evaluation of immobilization efficiency.

Immobilisation and Kinetic Study of Tyrosinase for Biosensor Construction

ABSTRACT The catalytic properties and stability of soluble and immobilised in nylon-6,6 mesh tyrosinase were studied with the help of an oxygen sensor. A variety of methods were examined for the immobilisation of tyrosinase, although active immobilised enzyme was obtained only with the help of benzidine and dicyclohexylcarbodiimide. The immobilisation caused an increase in the Km value for catechol almost 2 times in comparison with that found for soluble enzyme (0.39 and 0.22 mM, respectively). The immobilised tyrosinase retained sufficient activity for several months. Due to its characteristic suicide inactivation induced by catechol, it is only of single use for analytical purposes. Obtained data were also used for evaluation of the model of oximeter-based biosensors. This model allows the calculation of steady-state parameters from transient state data, excluding the influence of accompanying side processes; for tyrosinase-bound biosensors it gave very reproducible results for automatic data processing.

Interference of the Simultaneous Presence of Different Biogenic Amines on the Response of an Amine Oxidase–Based Biosensor

Abstract The application of an amine oxidase–based biosensor, which was selective for several biogenic amines, was examined for the simultaneous detection of cadaverine, putrescine, and histamine. The biosensor response parameters in mixtures containing cadaverine or putrescine were 1.14 times less than the sum of corresponding parameters in single-substrate solutions. Histamine did not cause any change of the biosensor response in the presence of cadaverine and/or putrescine. This “screening” of biosensor signal parameters by putrescine and cadaverine should be considered during calibration of amine oxidase–based biosensors used for the detection of biogenic amines.

A Model of Oximeter - Based Enzyme Electrode

A model of an oximeter-based two-substrate enzyme electrode is presented. The simplifications of the general model lead to a solution to differential equations describing the influence of the rate of the enzyme reaction in combination with the diffusion phenomena on measurable oxygen flux. Both transient phase and steady state conditions for experimental data can be considered within the frames of the model and provide possibilities for the calibration of the biosensor. The optimal parameters for the biosensor calibration can be used in practical design of enzyme electrodes.

Rapid biosensing of Staphylococcus aureus bacteria in milk

Mastitis, mainly caused by different pathogens, is the most common disease in dairy cattle leading to the reduction of milk production and its quality. The development of analytical methods for rapid detection of the most common mastitis-causing microorganisms enabling timely treatment of sick and infected animals is particularly important. The routine detection of mastitis-causing bacteria commonly takes 2–3 days, too long to wait for starting the proper treatment. The proposed immunobiosensing system, based on specific binding of S. aureus in automatically renewable micro-columns and highly selective recognition of the bound bacteria by secondary antibodies, conjugated with a fluorescence marker, enabled the detection of S. aureus bacteria in milk within 17 minutes with a detection limit of 200 CFU ml−1.

Analyzing the biosensor signal in flows: Studies with glucose optrodes

Responses of enzymatic bio-optrodes in flow regime were studied and an original model was proposed with the aim of establishing a reliable method for a quick determination of biosensor signal parameters, applicable for biosensor calibration. A dual-optrode glucose biosensor, comprising of a glucose bio-optrode and a reference oxygen optrode, both placed into identical flow channels, was developed and used as a model system. The signal parameters of this biosensor at different substrate concentrations were not dependent on the speed of the probe flow and could be determined from the initial part of the biosensor transient phase signal, providing a valuable tool for rapid analysis. In addition, the model helped to design the biosensor system with reduced impact of enzyme inactivation to the system stability (20% decrease of the enzyme activity lead to only a 1% decrease of the slope of the calibration curve) and hence significantly prolong the effective lifetime of bio-optrodes.