Functionalization of a poly(amidoamine) dendrimer with ferrocenyls and its application to the construction of a reagentless enzyme electrode

Poly(amidoamine) dendrimers having various degrees of modification with the redox-active ferrocenyls were prepared by controlling the molar ratio of ferrocenecarboxaldehyde to amine groups of dendrimers. By alternate layer-by-layer depositions of partial ferrocenyl-tethered dendrimers (Fc-D) with periodate-oxidized glucose oxidase (GOx) on a Au surface, an electrochemically and enzymatically active multilayered assembly of enzyme was constructed. The resulting GOx/Fc-D multilayer-associated electrodes were electrochemically analyzed, and the surface concentration of ferrocenyl groups, active enzyme coverage, and sensitivity were estimated. A 32% dendrimer modification level of surface amines to ferrocenyls was found to be an optimum in terms of enzyme-dendrimer network formation, electrochemical interconnectivity of ferrocenyls, and electrode sensitivity. With the prepared Fc(32%)-tethered dendrimers, mono- and multilayered GOx/Fc-D electrodes were constructed, and their electrochemical and catalytic properties were characterized. The bioelectrocatalytic signals from the multilayered GOx/Fc-D electrodes were shown to be directly correlated to the number of deposited bilayers. From this result, it seems that the electrode sensitivity is directly controllable, and the multilayer-forming strategy with partial ferrocenyl-tethered dendrimers is useful for the construction of reagentless biosensors.     

Multilayered assembly of dendrimers with enzymes on gold: thickness-controlled biosensing interface

A new approach to construct a multilayered enzyme film on the Au surface for use as a biosensing interface is described. The film was prepared by alternate layer-by-layer depositions of G4 poly(amidoamine) dendrimers and periodate-oxidized glucose oxidase (GOx). The cyclic voltammograms obtained from the Au electrodes modified with the GOx/dendrimer multilayers revealed that bioelectrocatalytic response is directly correlated to the number of deposited bilayers, that is, to the amount of active enzyme immobilized on the Au electrode surface. From the analysis of voltammetric signals, the coverage of active enzyme per GOx/dendrimer bilayer during the multilayer-forming steps was estimated, which demonstrates that the multilayer is constructed in a spatially ordered manner. Also, with the ellipsometric measurements, a linear increment of the film thickness was registered, supporting the formation of the proposed multilayered structure. The E5D5 electrode showed the sensitivity of 14.7 microA x mM(-1) glucose x cm(-2) and remained stable over 20 days under day-by-day calibrations. The proposed method is simple and would be applicable to the constructions of thickness- and sensitivity-controllable biosensing interfaces composed of multienzymes as well as a single enzyme.     

Composite carbon paste biosensor for phenolic derivatives based on in situ electrogenerated polypyrrole binder

Amperometric biosensors based on new composite carbon paste (CPE) electrodes have been designed for the determination of phenolic compounds. The composite CPEs were prepared by in situ generation of polypyrrole (PPy) within a paste containing the enzyme polyphenol oxidase (PPO). The best paste composition (enzyme/pyrrole monomer/carbon particles/Nujol) was determined for a model enzyme, glucose oxidase, according to the enzymatic activity of the resulting electrodes and to the enzyme leakage from the paste during storage in phosphate buffer. The in situ electrogenerated PPy enables improvement in enzyme immobilization within the paste since practically no enzyme was lost in solution after 72 h of immersion. Moreover, the enzyme activity remains particularly stable under storage since the biocomposite structure maintains 80% of its activity after 1-month storage. Following the optimization of the paste composition, PPO-based carbon paste biosensors were prepared and presented excellent analytical properties toward catechol detection with a sensitivity of 4.7 A M(-1) cm(-2) and a response time lower than 20 s. The resulting biosensors were finally applied to the determination of epicatechin and ferulic acid as flavonol and polyphenol model, respectively.     

ENFET glucose biosensor produced with dendrimer encapsulated Pt nanoparticles

The biosensors based on ENFETs for direct glucose concentration analysis have been fabricated by introducing dendrimer encapsulated Pt nanoparticles and glucose oxidase (GOx) via a layer-by-layer self-assembly method. The free amine groups located on each poly(amidoamine) dendrimer molecule were exploited to immobilize enzyme through covalent attachment. Depending on metal nanoparticles within dendrimers and biocompatibility of dendrimers, the fabricated glucose sensitive ENFET shows obviously enhanced sensitivity and extended lifetime compared with the conventional ones. The fabricated sensor has a linear range of 0.25–2.0 mM, and a detection limit of ca. 0.15 mM. The influence of buffer concentration, ionic strength and pH was discussed. The biosensor also has good stability, which response could be used for detecting of glucose samples at intervals for at least 1 month when it stored in dry state at 4 °C.     

Optimal environment for glucose oxidase in perfluorosulfonated ionomer membranes: improvement of first-generation biosensors

An optimal environment for glucose oxidase (GOx) in Nafion membranes is achieved using an advanced immobilization protocol based on a nonaqueous immobilization route. Exposure of glucose oxidase to water-organic mixtures with a high (85-95%) content of the organic solvent resulted in stabilization of the enzyme by a membrane-forming polyelectrolyte. Such an optimal environment leads to the highest enzyme specific activity in the resulting membrane, as desired for optimal use of the expensive oxidases. Casting solution containing glucose oxidase and Nafion is completely stable over 5 days in a refrigerator, providing almost absolute reproducibility of GOx-Nafion membranes. A glucose biosensor was prepared by casting the GOx-Nafion membranes over Prussian Blue-modified glassy carbon disk electrodes. The biosensor operated in the FIA mode allows the detection of glucose down to the 0.1 microM level, along with high sensitivity (0.05 A M(-1) cm(-2)), which is only 10 times lower than the sensitivity of the hydrogen peroxide transducer used. A comparison with the recently reported enzyme electrodes based on similar H2O2 transducers (transition metal hexacyanoferrates) shows that the proposed approach displays a dramatic (100-fold) improvement in sensitivity of the resulting biosensor. Combined with the attractive performance of a Prussian Blue-based hydrogen peroxide transducer, the proposed immobilization protocol provides a superior performance for first-generation glucose biosensors in term of sensitivity and detection limits.     

Kinetic and equilibrium binding analysis of protein-ligand interactions at poly(amidoamine) dendrimer monolayers

The interaction of streptavidin (SA) with a biotinylated surface has been of great interest in the development of an interfacial layer for protein immobilization based on self-assembled monolayers (SAMs) and polymeric layers. Here, we demonstrate the unique characteristics of protein-ligand interactions on dendrimer monolayers based on kinetic and equilibrium binding analyses. With amine-ended poly(amidoamine) dendrimers from the first (G1) to fourth (G4) generation, the formation of even, compact dendrimer monolayers on gold was confirmed using FT-IR spectroscopy and ellipsometry. For the SA-biotin interaction, quantitative analysis of bound SA using surface plasmon resonance showed that the saturation binding level of SA was fairly higher in all dendrimer layers when compared to other tested systems of 11-mercaptoundecylamine SAMs and a poly(L-lysine) layer. Kinetic studies revealed that the initial binding rate of SA up to the saturation level was 2-fold higher in all dendrimer layers than in the SAMs regardless of the surface density of functionalized biotin. Concurrently, the dendrimer layers led to much higher values of sticking probability, which is defined as the probability that the SA molecule adsorbs upon collision with a biotinylated surface, at a fixed SA coverage, and prolonged the significant levels around the maximum probability with increasing SA coverage. Plots of the saturation coverage of SA versus the SA concentration in solution showed that SA binding onto the biotinylated G1 and G3 layers fit to a Langmuir isotherm model. Taken together, faster binding of SA and highly ordered packing of the molecules seems to be achieved through typical properties of the dendrimer monolayers such as surface distribution of functionalized biotin, surface corrugation, and flexibility of highly branched larger dendrimers, which provides a guideline for the construction and analysis of an interfacial layer in biosensing applications.     

Integration of enzymes and electrodes: spectroscopic and electrochemical studies of chitosan-enzyme films

A new film-forming solution was developed for the efficient immobilization of enzymes on solid substrates. The solution consisted of a biopolymer, chitosan (CHIT), that was chemically modified with a permeability-controlling agent, Acetyl Yellow 9 (AY9), using glutaric dialdehyde (GDI) as a molecular tether. A model enzyme, glucose oxidase (GOx), was mixed with the CHIT-GDI-AY9 solution and cast on the surface of platinum electrodes to form robust CHIT-GDI-AY9-GOx films for glucose biosensing. UV-visible and infrared spectroscopies were used to determine the composition of the films. The optimized films contained on average 1 molecule of AY9/3 glucosamine units of chitosan and 25 free GDI tethers/1 molecule of GOx. The electrochemical assays of the films indicated both a very high efficiency of enzyme immobilization (approximately 99%) and large enzyme activity (60 units cm(-2)). The latter translated into a high sensitivity (42 mA M(-1) cm(-2)) of the Pt/CHIT-GDI-AY9-GOx biosensor toward glucose. The biosensor operated at 0.450 V, had a fast response time (t90% < or = 3 s), and was free of typical interferences, and its dynamic range covered 3 orders of magnitude of glucose concentrations. The lowest actually detectable concentration was 10 microM glucose. In addition, the biosensor displayed a practical shelf life and excellent operational stability, e.g. its response was stable during 24-h testing under continuous polarization and continuous flow of 5.0 mM glucose solution. The proposed approach to enzyme immobilization is simple, efficient, and cost-effective and should be of importance in the development of biosensors based on other enzymes that are more expensive than glucose oxidase.     

基于生物素-亲和素系统的酶固定化及纳米金增效的研究

应用石英晶体微天平(QCM)研究了基于生物素一亲和素系统的葡萄糖氧化酶的固定化,探讨了纳米金修饰QCM金基片对酶固定化的一系列过程的影响。在本实验条件下,利用生物素．亲和素系统能较好地固定化葡萄糖氧化酶,经过纳米金颗粒修饰的QCM基片对酶的吸附量比未经修饰的基片可提高1倍以上。     

Direct electrochemistry of laccase immobilized on au nanoparticles encapsulated-dendrimer bonded conducting polymer: application for a catechin sensor

The direct electrochemistry of laccase was promoted by Au nanoparticle (AuNP)-encapsulated dendrimers (Den), which was applied for the detection of catechin. To increase the electrical properties, AuNPs were captured in the interiors of the dendrimer (Den-AuNPs) as opposed to attachment at the periphery of dendrimer. To prepare Den-AuNPs, the Au(III) ions were first coordinated in the interior of dendrimer with nitrogen ligands and then reduced to form AuNPs. The size of AuNPs encapsulated within the interior of the dendrimer was determined to be 1.7 +/- 0.4 nm. AuNPs-encapsulated dendrimers were then used to covalently immobilize laccase (PDATT/ Den(AuNPs)/laccase) through the formation of amide bonds between carboxylic acid groups of the dendrimer and the amine groups of laccase. Each layer of the PDATT/Den(AuNPs)/laccase probe was characterized using CV, EIS, QCM, XPS, SEM, and TEM. The PDATT/Den(AuNPs)/laccase probe displayed a well-defined direct electron-transfer (DET) process of laccase. The quasi-reversible redox peak of the Cu redox center of the laccase molecule was observed at -0.03/+0.13 V vs Ag/AgCl, and the electron-transfer rate constant was determined to be 1.28 s (-1). A catechin biosensor based on the electrocatalytic process by direct electrochemistry of laccase was developed. The linear range and the detection limit in the catechin analysis were determined to be 0.1-10 and 0.05 +/- 0.003 microM, respectively. Interference effects from various phenolic and polyphenolic compounds were also studied, and the general applicability of the biosensor was evaluated by selective analysis of real samples of catechin.     

Enzyme specific activity in functionalized nanoporous supports

Here we reveal that enzyme specific activity can be increased substantially by changing the protein loading density (P(LD)) in functionalized nanoporous supports so that the enzyme immobilization efficiency (I(e), defined as the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in solution) can be much higher than 100%. A?net negatively charged glucose oxidase (GOX) and a net positively charged organophosphorus hydrolase (OPH) were entrapped spontaneously in NH(2)-?and HOOC-functionalized mesoporous silica (300??, FMS) respectively. The specific activity of GOX entrapped in FMS increased with decreasing P(LD). With decreasing P(LD), I(e) of GOX in FMS increased from<35% to>150%. Unlike GOX, OPH in HOOC-FMS showed increased specific activity with increasing P(LD). With increasing P(LD), the corresponding I(e) of OPH in FMS increased from 100% to>200%. A protein structure-based analysis of the protein surface charges directing the electrostatic interaction-based orientation of the protein molecules in FMS demonstrates that substrate access to GOX molecules in FMS is limited at high P(LD), consequently lowering the GOX specific activity. In contrast, substrate access to OPH molecules in FMS remains open at high P(LD) and may promote a more favorable confinement environment that enhances the OPH activity.     

GOX-functionalized nanodiamond films for electrochemical biosensor

The importance of nanodiamond in biological and technological applications has been recognized recently, and applied in drug delivery, biochip, sensors and biosensors. Under this investigation, nanodiamond (ND) and nitrogen doped nanodiamond (NND) were deposited on n-type silicon films, and later functionalized with enzyme Glucose oxidase (GOX). The GOX functionalized doped and undoped ND films were characterized using combination of several techniques; i.e. FTIR spectroscopy, Raman spectroscopy, atomic force microscopy (AFM) and electrochemical techniques. ND/GOX and NND/GOX thin films on n-type silicon have been found to provide sensitive glucose sensor. GOX has been chosen as a model enzyme system to functionalize with ND at molecular level to understand the glucose biosensor.     

A comparative study of enzyme immobilization strategies for multi-walled carbon nanotube glucose biosensors

This work addresses the comparison of different strategies for improving biosensor performance using nanomaterials. Glucose biosensors based on commonly applied enzyme immobilization approaches, including sol-gel encapsulation approaches and glutaraldehyde cross-linking strategies, were studied in the presence and absence of multi-walled carbon nanotubes (MWNTs). Although direct comparison of design parameters such as linear range and sensitivity is intuitive, this comparison alone is not an accurate indicator of biosensor efficacy, due to the wide range of electrodes and nanomaterials available for use in current biosensor designs. We proposed a comparative protocol which considers both the active area available for transduction following nanomaterial deposition and the sensitivity. Based on the protocol, when no nanomaterials were involved, TEOS/GOx biosensors exhibited the highest efficacy, followed by BSA/GA/GOx and TMOS/GOx biosensors. A novel biosensor containing carboxylated MWNTs modified with glucose oxidase and an overlying TMOS layer demonstrated optimum efficacy in terms of enhanced current density (18.3 ± 0.5 μA mM(-1) cm(-2)), linear range (0.0037-12 mM), detection limit (3.7 μM), coefficient of variation (2%), response time (less than 8 s), and stability/selectivity/reproducibility. H(2)O(2) response tests demonstrated that the most possible reason for the performance enhancement was an increased enzyme loading. This design is an excellent platform for versatile biosensing applications.     

A bioconjugated polyglycerol dendrimer with glucose sensing properties

In this work, the biological and electrochemical properties of glucose biosensor based on polyglycerol dendrimer (PGLD) is presented. Streptokinase (SK), glucose oxidase (GOx) and phosphorylcholine (PC) were immobilized onto PGLD to obtain a blood compatible bioconjugate with glucose sensing properties. The bioconjugated PGLD was entrapped in polyaniline nanotubes (PANINT's) through template electrochemical polymerization of aniline. PANINT's were used as electron mediator due to their high ability to promote electron-transfer reactions involving GOx. Platelet adhesion, fibrinolytic activity and protein adsorption were studied by in vitro experiments to examine the interaction of blood with PGLD biosensor. The PGLD biosensor exhibits a strong and stable amperometric response to glucose. The enzyme affinity for the substrate (K (M) (app) ) indicates that the enzyme activity was not significantly altered after the bioconjugation of GOx with PGLD dendrimer. The bioelectrochemical properties suggest that the bioconjugated PGLD developed in this work appears to be a good candidate for providing interfaces for implantable biosensors, especially oxidoreductase-based sensors.     

Spectroscopic investigations of poly(propyleneimine)dendrimers using the solvatochromic probe phenol blue and comparisons to poly(amidoamine) dendrimers.

The physical and chemical properties of PPI dendrimers' interior were investigated using the fluorescent, solvatochromic probe phenol blue. In aqueous solutions of each generation studied, two discrete dye populations were clearly observed. PPI dendrimers were shown to form a tight, nonpolar association with the vast majority of available dye, within the dendrimer interior, near the core. In the steady-state fluorescence emission spectra, a microenvironment of decreasing polarity in increasingly larger-generation PPI dendrimers (up to G3) was seen for the associated probe. Each of the remaining larger-generation dendrimers provided a microenvironment of essentially equal polarity. Fluorescence anisotropy values for phenol blue in the PPI dendrimers demonstrated the dye's sensitivity to the changing molecular volumes of the dendrimer generations. Model compounds that mimicked PPI's surface groups and branching moieties were used to better define the associated dye's location. The mimics further confirmed that phenol blue was associated inside the dendrimer, where it did not interact with the dendrimer surface groups. The comparison of amine-terminated PPI and PAMAM dendrimers clearly demonstrated the effects of their structural differences and the ability of phenol blue to have sensed those differences, including the initiator core length, branching unit length, and branching unit chemical composition.     

Potentiometric response characteristics of polycation-sensitive membrane electrodes toward poly(amidoamine) and poly(propylenimine) dendrimers

The potentiometric response characteristics of polycation-sensitive membrane electrodes toward two classes of polycationic dendrimers are examined. Using appropriately formulated polymer membrane electrodes composed of a dinonylnaphthalenesulfonate (DNNS) salt in a plasticized polyurethane matrix, it is shown that poly(amidoamine) (PAMAM) and poly(propylenimine) (PPI) dendrimers are readily detected at submicrogram per milliliter levels via a nonequilibrium response mechanism. The relationship between the total EMF response (at equilibrium) and the specific dendrimer structure is also examined. For both the PAMAM and PPI species, it is shown that the total EMF response does not change significantly with dendrimer generation number; however, the nonequilibrium analytically useful response curves are shifted to higher mass concentrations as the generation number is increased. The relative contributions of the terminal primary amines and the interior tertiary amines of the dendrimers to the observed EMF response are investigated by synthesis of various dendrimer derivatives (acetylated, quaternized, etc.). By comparing the total EMF responses for these derivatives as a function of sample pH, it is demonstrated that the lipophilic cation exchanger (DNNS) within the membrane phase can likely interact electrostatically with both protonated forms of the terminal primary amines and interior tertiary amines of the dendrimer structures. The practical application of the nonequilibrium potentiometric detection of dendrimers for monitoring their interaction with DNA is also demonstrated.     

Monolayer studies of sugar- and nucleoside-terminated dendrimers at the air–water interface

Sugar- and adenosine-terminated dendrimers, [1,2-o-Isopropylideneribosyl-(G₁-12acid), -(G₂-36acid)] and [Adenosyl-(G₁-12acid), -(G₂-36acid)], were synthesized using Newkome's dendrimer synthetic method. Langmuir and Langmuir–Blodgett (LB) monolayers of these dendrimers have been constructed and characterized at the air–water interface and on solid substrates by measuring surface pressure–molecular area (Π–A) isotherms, atomic force microscopy (AFM), ellipsometry and contact angle measurement. Π–A isotherms and AFM images showed that these dendrimers formed stable and homogeneous monolayers without aggregation on pure water surface. The first and second generation of sugar-terminated dendrimers show molecular areas of 647 and 1359 Ų, respectively. Ellipsometry measurement indicates that the thickness of both the first and the second generation of sugar-terminated dendrimers were about 10 Å. This reflects a flat orientation of both molecules at the air–water interface. On the other hand, the first generation of adenosine-terminated dendrimer shows an area of 105.6 Ų per molecule with a thickness of 16 Å, and for the second generation, the area was 738.4 Ų with a thickness of 27 Å. These results suggested that adenosine-terminated dendrimers maintain a spherical form at the air–water interface. It was found that small difference in the structure of thymine and uracil in the subphase critically affects the interaction of the molecules and conformation of the dendrimers at the interface.     

金属与非金属纳米颗粒增强葡萄糖生物传感器

为了提高葡萄糖传感器的灵敏度和抗干扰性,利用纳米增强效应,以Au、Ag、Pt、SiO2纳米颗粒及金属-无机复合纳米颗粒与聚乙烯醇缩丁醛(PVB)构成复合固定酶膜基质,采用溶胶-凝胶法固定葡萄糖氧化酶(GOD),组成葡萄糖生物传感器.研究表明,纳米颗粒可以大幅度地提高固定化酶的催化活性,增加电极的电流响应灵敏度,改进生物传感器的抗干扰性能,使信噪比提高了32倍.     

Immobilization method for the preparation of biosensors based on pH shift-induced deposition of biomolecule-containing polymer films.

Miniaturization of amperometric biosensors is crucially dependent on the availability of methods for the nonmanual immobilization of biological recognition elements on the transducer surface. From an aqueous polymer suspension, the precipitation of a polymer film with entrapped biological recognition elements is initiated by electrochemically induced oxidation of H20 at the electrode surface. Using the locally generated H+ gradient, acidic side chains of the polymer are titrated, leading to a change in the polymer solubility and hence to the controlled deposition of a polymer film. To investigate the properties and limitations of this immobilization technology, the specific features of a glucose biosensor based on polymer-entrapped glucose oxidase and amperometric detection of enzymatically generated H202 were investigated. Besides the reproducibility of the immobilization procedure, the sensitivity (14.59 mA cm(-2) M(-1) at pH 7), long-term stability (up to 5000 measurements in a sequential-injection analyzer), dependence on enzyme concentration, polymer thickness, and possibilities to fabricate multilayer sensor architectures were exploited. In addition, the miniaturization potential of this nonmanual immobilization technology was evaluated by investigating the modification of microband electrode arrays and cross talk between the neighboring microsensors.     

Nanofabrication of helical peptide-shelled dendrimers

This study describes nanofabrication of helical peptide-shelled dendrimers using a Langmuir monolayer technique. Poly(amido amine) dendrimers (G3) modified with poly(gamma-benzyl-L-glutamate) [number averaged degree of polymerization, n = 12, 17, and 34 (G3-PBLGs hereafter)] were newly prepared by graft polymerization of gamma-benzyl-L-glutamate-N-carboxy anhydride initiated with amino groups of the dendrimer surface. The hydrodynamic diameters of G3-PBLGs were determined to be 6.9 +/- 0.7, 8.2 +/- 1.0, and 11.9 +/- 1.7 nm for n = 12, 17, and 34, respectively, by means of dynamic light scattering. These values were consistent with the theoretical diameters of G3-PBLGs, which were calculated by considering the alpha-helical PBLG segment length. G3-PBLGs were found to form stable monomolecular films with high collapse pressures above 40 mN m-1 at the air-water interface. In addition, these monolayers could be successfully transferred onto various solid substrates. Circular dichroism and Fourier transfer infrared spectroscopies of the deposited G3-PBLGs monolayers showed that PBLG segments took an alpha-helical conformation over a wide range of surface pressure even on solid substrates as well as in bulk solutions. Monolayer thicknesses of these Langmuir-Blodgett films, estimated by x-ray photoelectron spectroscopy and atomic force microscopy, were compatible with the hydrodynamic diameters of G3-PBLGs.     

Biosensors based on membrane-bound enzymes immobilized in a 5-(octyldithio)-2-nitrobenzoic acid layer on gold electrodes

Gold electrodes were modified through chemisorption of 5-(octyldithio)-2-nitrobenzoic acid (ODTNB). ODTNB includes a long chain in a short-length thio acid, providing a heterogeneous-like alkanethiol layer after adsorption on gold electrodes. Membrane-bound enzymes, in particular D-fructose dehydrogenase (FDH), D-gluconate dehydrogenase (GADH), and L-lactic dehydrogenase (cytochrome b2) (Cyb2), were immobilized onto ODTNB-modified gold electrodes simply by adsorption. The short-length thio acid may provide electrostatic interactions with enzyme surface charges, while the alkanethiolate enables hydrophobic interaction with the largely lipophilic, membrane-bound enzymes. The immobilization of FDH, GADH, and Cyb2 onto ODTNB-modified gold surfaces has been studied with the quartz crystal microbalance (QCM). Spectrophotometric and electrochemical assays indicate that the immobilized enzyme retains its enzymatic activity after immobilization onto the ODTNB-modified gold surface. The amount of immobilized (and active) enzyme was estimated from QCM to be of the order of 2.5 x 10(-12)-5.3 x 10(-12) mol x cm(-2). A fructose biosensor was developed, making use of a gold surface modified with ODTNB and fructose dehydrogenase, employing hydroxymethylferrocene as a mediator in solution. Calibration curves exhibited a linear relation between the biosensor response and the substrate concentration up to 0.7 mM. Statistical analysis gave an excellent linear correlation (r = 0.9993) and a sensitivity of 6.1 mM(-1) fructose. The biosensor shows a significant stable catalytic current for at least 25 days.