Development of electrochemical biosensors based on sol-gel enzyme encapsulation and protective polymer membranes

Protective polymer coatings have been used to enhance the retention of enzymes in sol-gel films as immobilisation phases in electrochemical biosensors. Carbon film electrodes were electrochemically modified with poly(neutral red) (PNR). These electrodes were coated with oxysilane sol-gels incorporating glucose oxidase and an outer coating of carboxylated PVC (CPVC) or polyurethane (PU), with and without Aliquat-336 or isopropyl myristate (IPM) plasticizer, was applied. The biosensors were characterised electrochemically using cyclic voltammetry and amperometry, electrochemical impedance spectroscopy and scanning electron microscopy. Impedance spectra showed that the electrode surface is most active when the sol-gel–GOx layer is not covered with a membrane. However, membranes without plasticizer extend the lifetime of the biosensor to more than 2 months when PU is used as an outer membrane. The linear range of the biosensors was found to be 0.05–0.50 mM of glucose and the biosensor with PU outer membrane exhibited higher sensitivity (ca.117 nA mM⁻¹) in the region of linear response than that with CPVC. The biosensors were applied to glucose measurement in natural samples of commercial orange juice.     

Bio-smart materials: kinetics of immobilized enzymes in p(HEMA)/p(pyrrole) hydrogels in amperometric biosensors

Various strategies are being pursued to confer the highly specific molecular recognition properties of bioactive molecules to the transducer action of inherently conductive polymers. We have successfully integrated inherently conductive polypyrrole within electrode-supported, UV cross-linked hydroxyethyl methacrylate (HEMA)-based hydrogels. These electroactive composites were used as matrixes for the physical immobilization of several oxidase enzymes to fabricate clinically important biosensors. Measurements were made of the amperometric responses via H₂O₂ oxidation for each biosensor. Apparent Michaelis constants, K_m(app), for glucose oxidase immobilized in p(HEMA) membranes and in p(HEMA)/p(Pyrrole) composite membranes were 13.8 and 43.7 mM respectively compared to 33 mM in solution. The inclusion of polypyrrole in the hydrogel network increased the thermal stability of the immobilized enzyme at 60°C by 30% and 40% compared to p(HEMA) membranes and solution phase respectively. The composite also yielded larger I_max values (19 μA/cm⁻²) for glucose biosensors compared to similar glucose biosensors fabricated without the conducting polymer (15 μA). K_m(app) values for cholesterol oxidase immobilized in the same composite films were ca. three orders of magnitude higher than the K_m for the soluble enzyme. The polypyrrole component is shown to reduce diffusive transport but to confer thermal stability to these biosensors.     

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 of Lactose Biosensor Based on β‐Galactosidase and Glucose Oxidase Immobilized into Gelatin

In this article, we describe the preparation of a new lactose biosensor based on electrode coating with β‐galactosidase and glucose oxidase immobilized gelatin. For this purpose, β‐galactosidase and glucose oxidase enzymes were immobilized onto gelatin by crosslinking with glutaraldehyde. Properties of the immobilized β‐galactosidase and glucose oxidase enzymes electrode have been studied. The effects of glutaraldehyde concentration, temperature and pH variations and reusability were among the subjects analyzed. Lactose biosensors were subjected to continuous repeated use in order to observe reusability and shelf life; where standard lactose and milk samples were used as substrate solutions. Continuous reuse experiments showed that most of the lactose biosensors activities were retained even after the 10th use in a period of 30 days.     

Modified gas-permeable silicone rubber membranes for covalent immobilisation of enzymes and their use in biosensor development

Novel enzyme membranes are introduced. Modified polymeric gas-permeable layers were developed enabling biological components which have available reactive groups (-NH2, -OH, -SH, -COOH) to couple covalently on to their surfaces. Therefore, gas-permeable two component room temperature vulcanizing (2K-RTV) silicone rubber was modified using additional cross-linking agents. Triethoxysilanes with functional groups on their side chains such as epoxy or amino groups were used. A special attribute of the resulting gas-permeable membranes is that their formation and modification occur simultaneously during one reaction step. IR spectroscopy was used to observe the changes in the polymeric structure due to the reaction with the additional cross-linking agents. Sensors equipped with these layers are suitable to measure dissolved gases such as O2, CO2 and NH3 consumed or produced by enzymes converting their substrates. Determination of glucose, a well investigated enzymatic detection process, was chosen to demonstrate the applicability of the enzyme immobilisation. Glucose oxidase was immobilised on the membranes and glucose was detected by amperometric measurement of oxygen consumption. It is expected that this immobilisation method will also be useful for miniaturised planar biosensors.     

Application of Amperometric Enzyme Biosensors for Wine and Must Analysis

The work presents application of amperometric biosensors based on platinum printed electrodes and immobilized enzymes alcohol oxidase, glucose oxidase and lactate oxidase for wine analysis. Created devices demonstrate wide dynamic range of work, good stability and high selectivity to the substrate, and are successfully applied for 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 and chromatography method is shown. Created biosensors can be used as a basis of commercial device for express analysis of ethanol, glucose and lactate in wine and must during its fermentation.     

Layer-by-layer construction of protein architectures through avidin–biotin and lectin–sugar interactions for biosensor applications

In this review, the preparation and properties of protein architectures constructed by layer-by-layer (LbL) deposition through avidin–biotin and concanavalin A (Con A)–sugar interactions are discussed in relation to their use for optical and electrochemical biosensors. LbL films can be constructed through the alternate deposition of avidin and biotin-labeled enzymes on the surfaces of optical probes and electrodes. The enzymes retain their catalytic activity, resulting in the formation of optical and electrochemical biosensors. Alternatively, Con A can be used to construct enzyme-containing LbL films and microcapsules using sugar-labeled enzymes. Some enzymes such as glucose oxidase and horseradish peroxidase can be used for this purpose without labeling with sugar, because these enzymes contain intrinsic hydrocarbon chains on their molecular surfaces. The Con A/enzyme LbL architectures were successfully used to develop biosensors sensitive to specific substrates of the enzyme. In addition, Con A-based films can be used for the optical and electrochemical detection of sugars.     

New Immobilized Enzyme Membranes for Tailor-Made Biosensors

Abstract

Commercially available polyamide preactivated membranes for immunodiagnostic use were found suitable for the preparation of immobilized enzyme membranes adaptable to biosensors. Membranes with immobilized glucose oxidese as model enzyme, tested with a microprocessor-based device involving an enzymatic electrode, gave excellent results. The extremely simple and fast procedure allows anyone to prepare such bioactive membranes easily, possibly from his own enzyme preparation and within a few minutes set up a specific probe from commercially available sensors.     

Electrochemical Oxidation of Glucose Using Mutant Glucose Oxidase from Directed Protein Evolution for Biosensor and Biofuel Cell Applications

In this study, electrochemical characterisation of glucose oxidation has been carried out in solution and using enzyme polymer electrodes prepared by mutant glucose oxidase (B11-GOx) obtained from directed protein evolution and wild-type enzymes. Higher glucose oxidation currents were obtained from B11-GOx both in solution and polymer electrodes compared to wt-GOx. This demonstrates an improved electrocatalytic activity towards electrochemical oxidation of glucose from the mutant enzyme. The enzyme electrode with B11-GOx also showed a faster electron transfer indicating a better electronic interaction with the polymer mediator. These encouraging results have shown a promising application of enzymes developed by directed evolution tailored for the applications of biosensors and biofuel cells.     

Macromolecule encapsulation in diazoresin-based hollow polyelectrolyte microcapsules

A stable enzyme encapsulation technique based on the conversion of weak interactions between diazo resin/poly(styrene sulfonate) to covalent bonds was explored. Photosensitive diazoresin-based polyelectrolyte microcapsules were prepared via layer-by-layer electrostatic self-assembly of poly(styrene sulfonate) and diazoresin on MnCO(3) templates. UV-vis and zeta-potential measurements confirmed the alternate deposition of {PSS/DAR} multilayers on the micrometer-sized dissolvable templates. The DAR-based microcapsules were demonstrated to be permeable to enzymes prior to UV irradiation, while the permeability of the multilayer wall was changed substantially after photo-cross-linking. Encapsulated molecules were stably entrapped after UV irradiation, as shown by confocal microscopy and atomic force microscopy images. Activity assays revealed that encapsulated glucose oxidase possessed 52.8% of the catalytic activity exhibited by the same amount of free enzyme, proving the preservation of native conformation and accessibility of substrate. This encapsulation technique is promising for many biomedical and biotechnological applications, particularly enzyme biosensors, which require stable immobilization of functional components while allowing sufficient transport rates for substrate molecules.     

Fiberoptic biosensors based on chemiluminescent reactions

The chemiluminescence of luminol in the presence of H₂O₂ has been exploited to develop fiberoptic biosensors associated with flow injection analysis systems. A chlorophenol sensor was developed based on the ability of certain halophenols to enhance the peroxidase-catalyzed luminol chemiluminescence. Horseradish peroxidase immobilized on a collagen membrane was used. Ten chlorophenols have been tested with this chemiluminescent-based sensor. The lower detection limit was obtained with 4-chloro-3-methylphenol and was equal to 0.01 μM. Electrochemiluminescent-based fiberoptic biosensors for glucose and lactate were also developed using glucose oxidase or lactate oxidase immobilized on polyamide membranes. In the presence of oxidase-generated H₂O₂, the light emission was triggered electrochemically by means of a glassy carbon electrode polarized at +425 mV vs a platinum pseudo-reference electrode. The detection limits for glucose and lactate were 150 and 60 pmol, respectively, and the dynamic ranges were linear from 150 pmol to 600 nmol and from 60 pmol to 60 nmol, respectively.     

Electrochemical Biosensors Based on Enzymatic Reactors Filled by Various Types of Silica and Amalgam Powders for Measurements in Flow Systems

Two enzymatic biosensors with amalgam powder reactors and twelve enzymatic biosensors with various silica powder reactors were fabricated and tested in this work. The enzymatic reactors based on silver amalgam powder provide high sensitivity and they are convenient for faster flow rates. Experiments with six silica materials showed that mesoporous silica SBA‐15 was the best one in terms of covering by enzymes, sensitivity and lifetime of biosensors. The current response of the SBA15‐glucose oxidase (GOx) sensor started to decrease only after 200 day testing and after 406 days the peak current was still 35.9 % of the initial value. The used amalgam tubular detector in a flow system allowed working at highly negative potentials, which significantly increased the sensitivity of determinations. Statistical results of parallel measurement of model solutions with the fabricated biosensors show their high accuracy (RSD=0.28–1.81 %) and sensitivity (6.2–14.3 µmol L^?1). The proposed SBA15‐GOx biosensor was successfully used for determination of glucose in commercial honey.     

Renewable dehydrogenase-based interfaces for bioelectronic applications

Bioelectronic interfaces that establish electrical communication between redox enzymes and electrodes have potential applications as biosensors, biocatalytic reactors, and biological fuel cells. However, these interfaces contain labile components, including enzymes and cofactors, which have limited lifetimes and must be replaced periodically to allow long-term operation. Current methods to fabricate bioelectronic interfaces do not allow facile replacement of these components, thus limiting the useful lifetime of the interfaces. In this paper we describe a versatile new fabrication approach that binds the enzymes and cofactors using reversible ionic interactions. This approach allows the interface to be removed via a simple pH change and then replaced to fully regenerate the biocatalytic activity. The positively charged polyelectrolyte poly(ethylenimine) was used to ionically bond a dehydrogenase enzyme and its cofactor to a gold electrode that was functionalized with 3-mercaptopropionic acid and the electron mediator toluidine blue O. By reducing the pH, the surface-bound 3-mercaptopropionic acid was protonated, disrupting the ionic bonds and releasing the enzyme-modified polyelectrolyte. After neutralization, fresh enzyme and cofactor were bound, regenerating the bioelectronic interface. Cyclic voltammetry, chronoamperometry, constant potential amperometry, electrochemical impedance spectroscopy, and Fourier transform infrared spectroscopy analyses were used to characterize the bioelectronic interfaces. For the two enzymes tested (secondary alcohol dehydrogenase and sorbitol dehydrogenase) and their respective cofactors (beta-nicotinamide adenine dinucleotide phosphate and beta-nicotinamide adenine dinucleotide), the reconstituted interface exhibited a surface coverage, an electron-transfer coefficient, and a turnover rate similar to those of the original interface.     

Development of Novel Glucose and Pyruvate Biosensors at Poly(Neutral Red) Modified Carbon Film Electrodes. Application to Natural Samples

Amperometric biosensors based on the corresponding oxidase enzyme with poly(neutral red) redox mediator have been developed for the determination of glucose and pyruvate. The enzymes have been immobilized on top of poly(neutral red) modified carbon film electrodes with glutaraldehyde as the cross‐linking agent. The biosensors were characterized by cyclic voltammetry and by electrochemical impedance spectroscopy. The glucose biosensor exhibited a linear response in the range 90 μM to 1.8 mM with a detection limit of 22 μM and the pyruvate biosensor in the range 90 to 600 μM with a detection limit of 34 μM. The relative standard deviations were found to be 2.1% (n=3) and 2.8% (n=4) respectively. The interference effects of various compounds were also studied. The glucose content of several types of wine and the amount of pyruvate in onion and garlic were determined and the results were compared with those obtained by standard spectrophotometric methods.     

Characterization of natural chitosan membranes from the carapace of the soldier crab Mictyris brevidactylus and its application to immobilize glucose oxidase in amperometric flow-injection biosensing system

This study investigated characteristics of a chitosan membrane from the carapace of the soldier crab Mictyris brevidactylus intended to construct an amperometric biosensor. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used in this study to characterize these chitosan membranes intended for constructing enzymatic biosensors. Chitosan membranes suffering various durations (>10 min) of deacetylation had small charge-transfer resistances (<7.88 kohms) but large double-layer capacitances (>0.55 microF). They were found in EIS where both the solution resistance and Warburg impedance upon electrode interface were almost independent of the durations and degree of deacetylation. The degree of deacetylation and the thickness of chitosan membranes were also determined. Membrane thickness was slightly dependent with the duration but degree of deacetylation was slightly dependent on the duration. Chitosan membranes with various thicknesses suffered various durations of deacetylation, but this did not influence their electrochemical characteristics. The chitinous membrane was covalently immobilized with glucose oxidase (EC 1.3.4.3) and then attached onto the platinum electrode of a homemade amperometric flow cell. Sensor signal was linearly related to glucose concentration (r=0.999 for glucose up to 1.0 mM). The system was sensitive (S/N>5 for 10 microM glucose) and reproducible (CV<1.3% for 50 microM glucose, n=5).     

Electrochemical Kinetic Characterization of Redox Mediated Glucose Oxidase Reactions: A Simplified Approach

One of the main drawbacks affecting first‐generation electrochemical biosensors in the analysis of real matrices is the interference of electroactive species present in the sample under investigation. Several approaches have been attempted to overcome this problem in the past ten years but the best results were achieved by using mediated based electrochemical biosensors. Despite this, the kinetic of the redox mediators‐enzymatic proteins interaction has not been studied deeply enough. In this work we have developed a theoretical‐methodological approach for the characterization of the kinetic of interaction between redox enzymes and substrates and/or redox mediators. Particularly, the interaction of glucose oxidase (GOx) with several commercially available redox mediators has been studied by means of amperometry and cyclic voltammetry. The main kinetic parameters for different mediators were exploited and discussed with the aim of finding the best mediator for a glucose biosensor to be used on real samples.     

Pervaporation and properties of chitosan-poly(acrylic acid) complex membranes

Polyelectrolyte complex membranes between chitosan as a cationic polyelectrolyte and poly(acrylic acid) as an anionic species were prepared by blending two polymer solutions in different ratio. Characterization of chitosan-poly(acrylic acid) complex membrane was investigated by Fourier transform-infrared (FT-IR), wide angle X-ray diffractometer, dielectric analyzer. Their mechanical properties were studied by universal testing machine. The swelling of polyelectrolyte membranes was studied. Thermal properties of polyelectrolyte membranes from chitosan and poly(acrylic acid) by varying blend ratios showed a shift in transition temperatures of polyelectrolyte complexes. Polyelectrolyte complex membranes from chitosan and poly(acrylic acid) had pH sensitive characteristics as determined by FT-IR studies and swelling behaviors. Pervaporation performances were investigated with various organic mixtures; water-ethanol, water-isopropanol, methanol-methyl t-butyl ether mixtures. An increase of poly(acrylic acid) content in the polyelectrolyte complex membranes affected the swelling behavior and pervaporation performance of water-ethanol mixture. Permeation flux decreased and the water concentration in the permeate was close to 100% upon increasing the feed alcohol concentration.     

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.     

New CNT/poly(brilliant green) and CNT/poly(3,4-ethylenedioxythiophene) based electrochemical enzyme biosensors

A combination of the electroactive polymer poly(brilliant green) (PBG) or conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) with carbon nanotubes to obtain CNT/PBG and CNT/PEDOT modified carbon film electrodes (CFE) has been investigated as a new biosensor platform, incorporating the enzymes glucose oxidase (GOx) as test enzyme, alcohol oxidase (AlcOx) or alcohol dehydrogenase (AlcDH). The sensing parameters were optimized for all biosensors based on CNT/PBG/CFE, CNT/PEDOT/CFE platforms. Under optimized conditions, both GOx biosensors exhibited very similar sensitivities, while in the case of AlcOx and AlcDH biosensors, AlcOx/CNT/PBG/CFE was found to give a higher sensitivity and lower detection limit. The influence of dissolved O₂ on oxidase-biosensor performance was investigated and was shown to be different for each enzyme. Comparisons were made with similar reported biosensors, showing the advantages of the new biosensors, and excellent selectivity against potential interferents was successfully demonstrated. Finally, alcohol biosensors were successfully used for the determination of ethanol in alcoholic beverages.     

An origami paper device for complete elimination of interferents in enzymatic electrochemical biosensors

Many efforts have been made to prevent interferences in enzymatic electrochemical biosensors by permselective membranes or mediators with low redox potential. However, it is difficult to completely eliminate interferents without compromised sensitivity by these traditional procedures. We propose here a method based on an origami paper device that separates the electrochemical reactions of interferents and substrates for complete depletion of interferents and precise analysis of substrates. Interferents such as ascorbate, urate and paracetamol were completely consumed by a simple electrolysis step, while substrates were quantitatively analyzed by coulometry. With GOx as a model enzyme, an interference-free and calibration-free coulometric glucose biosensor has been demonstrated successfully. The proposed origami paper device provides a facile and easy-controlled approach to eliminate the electroactive interferents completely for enzymatic electrochemical biosensors.