Bioconjugates of mercaptocarboxylic acids functionalized AuNP and superoxide dismutase for superoxide electrochemical monitoring.

The use of gold nanoparticles/superoxide dismutase (AuNP/SOD) bioconjugates is described as building blocks in SOD biosensor development for the quantification of superoxide in cell culture media. AuNP functionalization with 11-mercaptoundecanoic acid (MUA) and 4-mercaptobenzoic acid (MBA) (AuNPMUA and AuNPMBA) was used to improve SOD immobilization through EDC/NHS coupling using their -COOH terminus, leading to the formation of more stable bioconjugates. AuNP and AuNP/SOD bioconjugates were characterized by SEM to determine their size and morphology, UV-Vis for optical properties, FT-IR, and Raman spectroscopies for chemical functional group analysis and EDX for elemental analysis. Electrochemical methods were used to characterize the Au/AuNP-modified electrodes. For the optimization of the biosensor architecture, different AuNP/enzyme bioconjugates were prepared by varying the amount of both enzyme and AuNP, as well as their incubation time. Finally, the biosensors incorporating the bioconjugates were characterized by fixed potential amperometry and voltammetric analysis in order to establish the enzymatic mechanism and to elucidate the best biosensor architecture for monitoring superoxide in cell culture media. The best sensitivity value for superoxide detection corresponded to 41.2 nA µM cm-2, achieved by a biosensor based on AuNPMBA/SOD bioconjugates monitored through fixed potential amperometry at 0.3 V vs. Ag/AgCl, with a limit of detection of 1.0 µM, and overall very good operational stability, maintaining 91% of the initial sensitivity after 30 days. Finally, the optimized biosensor was employed for the quantification of successive additions of superoxide in cell culture media, with excellent recovery values.

An antibody-based amperometric biosensor for 20S proteasome activity and inhibitor screening.

The 20S proteasome enzyme complex is involved in the proteolytic degradation of misfolded and oxidatively damaged proteins and is a focus of medical research for the development of compounds with pharmaceutical properties, which are active in cancer cells and/or neurodegenerative diseases. The present study aims to develop a biosensor for investigating the 20S proteasome activity and inhibition by means of electrochemical methods. The 20S proteasome is best immobilized at the electrode surface through bio-affinity interactions with antibodies that target different subunits on the 20S proteasome, enabling the investigation of the effect of an enzyme's orientation on biosensor response. The enzymatic activity is analyzed by fixed potential amperometry with the highest sensitivity of 24 µA cm-2 mM-1 and a LOD of 0.4 µM. The detection principle involves the oxidation of an electroactive probe that is released from the enzyme's substrates upon proteolysis. The most sensitive biosensor is then used to study the multicatalytic activity of the 20S proteasome, i.e. the caspase-, trypsin- and chymotrypsin-like activity, by analyzing the biosensor's sensitivity towards different substrates. The behavior of the immobilized 20S proteasome is investigated as a function of substrate concentration. The kinetic parameters are derived and compared with those obtained when the enzyme was free in solution, with K0.5 values being one to two orders of magnitude lower in the present case. Two 20S inhibitors, epoxomicin and bortezomib, are investigated by analyzing their influence on the 20S biosensor response. The proposed analytical method for proteasome activity and inhibitor screening has the main advantage of being cost-effective compared to the ones typically employed.

Immobilized Antibodies on Mercaptophenylboronic Acid Monolayers for Dual-Strategy Detection of 20S Proteasome.

A dual strategy for the electrochemical detection for 20S proteasome (20S) is proposed, based on the oriented immobilization of a capture monoclonal antibody (Abß) on a self-assembled monolayer of 4-mercaptophenylboronic acid (4-MPBA) on gold electrodes, which led to the Au/4-MPBA/Abß immunosensor. The methodology comprises the correlation of 20S concentration with (i) its proteolytic activity toward the Z-LLE-AMC substrate, using the Au/4-MPBA/Abß/20S, and (ii) the enzymatic activity of an alkaline phosphatase (AlkP) from the AlkP-labeled secondary antibody (Abcore-AlkP), which involves the conversion of aminophenylphosphate to the electroactive aminophenol using Au/4-MPBA/Abß/20S/Abcore-AlkP. The step-by-step construction of the immunosensor and the interactions at its surface were evaluated by surface plasmon resonance and gravimetric analysis with quartz crystal microbalance, showing a high affinity between both antibodies and 20S. Morphological analysis by scanning electron microscopy demonstrated a pattern of parallel lines upon immobilization of Abß on 4-MPBA and morphological changes to a well-organized granular structure upon binding of 20S. A voltametric and impedimetric characterization was performed after each step in the immunosensor construction. The two detection strategies were evaluated. It was shown that the immunosensor responds linearly with 20S concentration in the range between 5 and 100 µg mL-1, which corresponds to proteasome levels in serum in the case of diverse pathological situations, and LoD values of 1.4 and 0.2 µg mL-1 were calculated for the detection strategies. The immunosensor was applied to the detection of 20S in serum samples with recovery values ranging from 101 to 103%.

Hybrid Nanocomposite Platform, Based on Carbon Nanotubes and Poly(Methylene Blue) Redox Polymer Synthesized in Ethaline Deep Eutectic Solvent for Electrochemical Determination of 5-Aminosalicylic Acid.

A novel hybrid composite of conductive poly(methylene blue) (PMB) and carbon nanotubes (CNT) was prepared for the detection of 5-aminosalicylic acid (5-ASA). Electrosynthesis of PMB with glassy carbon electrode (GCE) or with carbon nanotube modified GCE was done in ethaline deep eutectic solvent of choline chloride mixed with ethylene glycol and a 10% v/v aqueous solution. Different sensor architectures were evaluated in a broad range of pH values in a Britton-Robinson (BR) buffer using electrochemical techniques, chronoamperometry (CA), and differential pulse voltammetry (DPV), to determine the optimum sensor configuration for 5-ASA sensing. Under optimal conditions, the best analytical performance was obtained with CNT/PMBDES/GCE in 0.04 M BR buffer pH 7.0 in the range 5-100 µM 5-ASA using the DPV method, with an excellent sensitivity of 9.84 µA cm-2 µM-1 (4.9 % RSD, n = 5) and a detection limit (LOD) (3σ/slope) of 7.7 nM, outclassing most similar sensors found in the literature. The sensitivity of the same sensor obtained in CA (1.33 µA cm-2 µM-1) under optimal conditions (pH 7.0, Eapp = +0.40 V) was lower than that obtained by DPV. Simultaneous detection of 5-ASA and its analogue, acetaminophen (APAP), was successfully realized, showing a catalytic effect towards the electro-oxidation of both analytes, lowering their oxidation overpotential, and enhancing the oxidation peak currents and peak-to-peak separation as compared with the unmodified electrode. The proposed method is simple, sensitive, easy to apply, and economical for routine analysis.

Photodynamic therapy efficacy enhanced by dynamics: the role of charge transfer and photostability in the selection of photosensitizers.

Progress in the photodynamic therapy (PDT) of cancer should benefit from a rationale to predict the most efficient of a series of photosensitizers that strongly absorb light in the phototherapeutic window (650-800 nm) and efficiently generate reactive oxygen species (ROS = singlet oxygen and oxygen-centered radicals). We show that the ratios between the triplet photosensitizer-O2 interaction rate constant (kD) and the photosensitizer decomposition rate constant (kd), kD/kd, determine the relative photodynamic activities of photosensitizers against various cancer cells. The same efficacy trend is observed in vivo with DBA/2 mice bearing S91 melanoma tumors. The PDT efficacy intimately depends on the dynamics of photosensitizer-oxygen interactions: charge transfer to molecular oxygen with generation of both singlet oxygen and superoxide ion (high kD) must be tempered by photostability (low kd). These properties depend on the oxidation potential of the photosensitizer and are suitably combined in a new fluorinated sulfonamide bacteriochlorin, motivated by the rationale.

Novel cells integrated biosensor based on superoxide dismutase on electrospun fiber scaffolds for the electrochemical screening of cellular stress.

A novel electrochemical biosensor was developed to monitor fibroblast cells stress levels for the first time in situ under external stimuli based on the recognition of superoxide anion released upon cell damage. The biosensor comprised metallized polycaprolactone electrospun fibers covered with zinc oxide for improved cell adhesion and signal transduction, whilst stable bioconjugates of mercaptobenzoic acid-functionalized gold nanoparticles/superoxide dismutase were employed as recognition bioelements. Biosensors were first tested and optimized for in situ generated superoxide detection by fixed potential amperometry at +0.3 V, with minimal interferences from electroactive species in cell culture media. L929 fibroblast cells were then implanted on the optimized biosensor surface and the biosensor morphologically characterized by scanning electron microscopy (SEM) and fluorescence microscopy, which illustrated the network-type pattern of fibroblasts adjacent to the fiber scaffold. Fibroblast stress was induced by zymosan and monitored at the cells integrated biosensor using fixed potential amperometry (CA) with a sensitivity of 26 nA cm-2 µg mL-1 zymosan and electrochemical impedance spectroscopy (EIS), with similar sensitivity of the biosensor considering the Rs and Z' parameters of around 0.13 Ω cm2 µg-1 mL and high correlation factors R2 of 0.9994. The obtained results underline the applicability of the here developed biosensor for the electrochemical screening of the fibroblast cells stress. The concept in using low-cost biocompatible polymeric fibers as versatile scaffolds for both enzyme immobilization and cell adhesion, opens a new path in developing biosensors for the in-situ investigation of a variety of cellular events.

Disposable superoxide dismutase biosensors based on gold covered polycaprolactone fibers for the detection of superoxide in cell culture media.

A novel and disposable biosensor based on superoxide dismutase (SOD) immobilized on gold metallized polycaprolactone electrospun polymeric fibers (PCl/Au) has been developed for the determination of superoxide (O2•-) in cell culture media. SOD biosensors were constructed employing three immobilization methods: cross-linking with EDC/NHS at a cysteine self-assembled monolayer (PCl/Au/SODCYS), biopolymer encapsulation with chitosan (PCl/Au/SODCHI) and cross-linking with glutaraldehyde (PCl/Au/SODGA). Scanning electron microscopy was performed at the three different biosensors to evaluate their surface morphologies. Biosensors were employed for the electrochemical detection of superoxide by fixed potential amperometry at different applied potentials, with two distinct enzymatic mechanisms being proposed: i) the reduction of the enzymatically generated peroxide, at -0.3 V, for which the PCl/Au/SODCHI biosensor presented the highest value of sensitivity of 40.1 µA mM-1 cm-2, and ii) the regeneration of the enzyme catalytic copper centre, at +0.3 V, for which the PCl/Au/SODCYS biosensor had the highest sensitivity value of 16.1 µA mM-1 cm-2. The proposed recognition mechanisms were further confirmed by cyclic voltammetric measurements, which enabled also to determine the amount of immobilized electroactive SOD, with highest value corresponding to the PCl/Au/SODCYS biosensor. The biosensors with best analytical performance, PCl/Au/SODCYS and PCl/Au/SODCHI, were further investigated for stability and selectivity, with best results for the PCl/Au/SODCYS, chosen for superoxide monitoring in cell culture media. The study is promising for future application of PCl/Au/SODCYS for the on-line superoxide monitoring of superoxide in cell cultures, grown directly on the biosensor itself.

PC-12 Cell Line as a Neuronal Cell Model for Biosensing Applications.

PC-12 cells have been widely used as a neuronal line study model in many biosensing devices, mainly due to the neurogenic characteristics acquired after differentiation, such as high level of secreted neurotransmitter, neuron morphology characterized by neurite outgrowth, and expression of ion and neurotransmitter receptors. For understanding the pathophysiology processes involved in brain disorders, PC-12 cell line is extensively assessed in neuroscience research, including studies on neurotoxicity, neuroprotection, or neurosecretion. Various analytical technologies have been developed to investigate physicochemical processes and the biosensors based on optical and electrochemical techniques, among others, have been at the forefront of this development. This article summarizes the application of different biosensors in PC-12 cell cultures and presents the modern approaches employed in neuronal networks biosensing.

Methylene blue and neutral red electropolymerisation on AuQCM and on modified AuQCM electrodes: an electrochemical and gravimetric study.

The phenazine monomers neutral red (NR) and methylene blue (MB) have been electropolymerised on different quartz crystal microbalance (QCM) substrates: MB at AuQCM and nanostructured ultrathin sputtered carbon AuQCM (AuQCM/C), and NR on AuQCM and on layer-by-layer films of hyaluronic acid with myoglobin deposited on AuQCM (AuQCM-{HA/Mb}(6)). The surface of the electrode substrates was characterised by atomic force microscopy (AFM), and the frequency changes during potential cycling electropolymerisation of the monomer were monitored by the QCM. The study investigates how the monomer chemical structure together with the electrode morphology and surface structure can influence the electropolymerisation process and the electrochemical properties of the phenazine-modified electrodes. Differences between MB and NR polymerisation, as well as between the different substrates were found. The electrochemical properties of the PNR-modified electrodes were analysed by cyclic voltammetry and electrochemical impedance spectroscopy and compared with the unmodified AuQCM. The results are valuable for future applications of modified AuQCM as substrates for electroactive polymer film deposition and applications in redox-mediated electrochemical sensors and biosensors.

Direct Immobilization of Biomolecules through Magnetic Forces on Ni Electrodes via Ni Nanoparticles: Applications in Electrochemical Biosensors.

The present work describes a new simple procedure for the direct immobilization of biomolecules on Ni electrodes using magnetic Ni nanoparticles (NiNPs) as biomolecule carriers. Ni electrodes were fabricated by electroplating, and NiNPs were chemically synthesized. The chemical composition, crystallinity, and granular size of Ni electrodes, NiNP, and NiNP-modified Ni electrodes (NiNP/Ni) were determined by X-ray diffraction, scanning electron microscopy, and X-ray photoelectron spectroscopy (XPS). The electrochemical characterization of Ni electrodes by cyclic voltammetry and electrochemical impedance spectroscopy confirmed the existence of nickel oxides, hydroxides, and oxohydroxide films at the surface of Ni. Magnetic characterization and micromagnetic simulations were performed in order to prove that the magnetic force is responsible for the immobilization process. Further, Ni electrodes were employed as amperometric sensors for the detection of hydrogen peroxide because it is an important performance indicator for a material to be applied in biosensing. The working principle for magnetic immobilization of the enzyme-functionalized NiNP, without the use of external magnetic sources, was demonstrated for glucose oxidase (GOx) as a model enzyme. XPS results enabled to identify the presence of GOx attached to the NiNP (GOx-NiNP) on Ni electrodes. Finally, glucose detection and quantification were evaluated with the newly developed GOx-NiNP/Ni biosensor by amperometry at different potentials, and control experiments at different electrode materials in the presence and absence of NiNP demonstrated their importance in the biosensor architecture.

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.

Electrochemical sensors and biosensors based on redox polymer/carbon nanotube modified electrodes: a review.

The aim of this review is to present the contributions to the development of electrochemical sensors and biosensors based on polyphenazine or polytriphenylmethane redox polymers together with carbon nanotubes (CNT) during recent years. Phenazine polymers have been widely used in analytical applications due to their inherent charge transport properties and electrocatalytic effects. At the same time, since the first report on a CNT-based sensor, their application in the electroanalytical chemistry field has demonstrated that the unique structure and properties of CNT are ideal for the design of electrochemical (bio)sensors. We describe here that the specific combination of phenazine/triphenylmethane polymers with CNT leads to an improved performance of the resulting sensing devices, because of their complementary electrical, electrochemical and mechanical properties, and also due to synergistic effects. The preparation of polymer/CNT modified electrodes will be presented together with their electrochemical and surface characterization, with emphasis on the contribution of each component on the overall properties of the modified electrodes. Their importance in analytical chemistry is demonstrated by the numerous applications based on polymer/CNT-driven electrocatalytic effects, and their analytical performance as (bio) sensors is discussed.

Synthesis, structure, and spectral and electrochemical properties of chromium(III) tris-(8-hydroxyquinolinate).

The kinetically inert chromium(III) tris-(8-hydroxyquinolinate), Crq3, has been synthesized, crystallized from 90% methanol-water, and characterized by MALDI-TOF mass spectrometry, thermogravimetry, FTIR, NMR spectroscopy, and X-ray powder diffraction. It is formed as a methanol solvate, but the solvent can be removed by heating. Large paramagnetic shifts and spectral broadening in (1)H NMR spectra indicate electron delocalization between the metal and the ligand. DFT calculations show it is present as the meridional isomer, with the HOMO largely based on one of the metal 3d orbitals and the LUMO essentially localized on the ligands. Cyclic voltammetry (CV) in acetonitrile solutions shows four oxidation peaks and two, less intense reduction waves on the first scan. The HOMO energy determined from the first oxidation peak is fairly close to that obtained by DFT, in agreement with this being mainly metal based. Although the number of peaks decreases on subsequent CV scans, the complex shows markedly enhanced electrochemical stability compared with aluminium(III) tris-(8-hydroxyquinolinate). Solution UV/visible absorption and solid diffuse reflectance spectra have a weak, long wavelength band, assigned to the metal based d-d transition, in addition to the normal, ligand based bands seen in metal quinolates. The energy of the lowest energy band is identical to the HOMO-LUMO separation obtained by cyclic voltammetry, in agreement with the above description. The compound is only weakly luminescent, in contrast to many other metal quinolates, due to the lowest energy transition being metal rather than ligand based. The potential of this compound as an electron transporting/hole blocking layer in optoelectronic devices is indicated.

Simple electrochemical sensor for caffeine based on carbon and Nafion-modified carbon electrodes.

A simple, economic, highly sensitive and highly selective method for the detection of caffeine has been developed at bare and Nafion-modified glassy carbon electrodes (GCE). The electrochemical behaviour of caffeine was examined in electrolyte solutions of phosphate buffer saline, sodium perchlorate, and in choline chloride plus oxalic acid, using analytical determinations by fixed potential amperometry, phosphate buffer saline being the best. Modifications of the GCE surface with poly(3,4-ethylenedioxythiophene) (PEDOT), Nafion, and multi-walled carbon nanotubes were tested in order to evaluate possible sensor performance enhancements, Nafion giving the most satisfactory results. The effect of interfering compounds usually found in samples containing caffeine was examined at GCE without and with Nafion coating, to exclude interferences, and the sensors were successfully applied to determine the caffeine content in commercial beverages and drugs.

A new self-assembled layer-by-layer glucose biosensor based on chitosan biopolymer entrapped enzyme with nitrogen doped graphene.

The layer-by-layer (LbL) technique has been used for the construction of a new enzyme biosensor. Multilayer films containing glucose oxidase, GOx, and nitrogen-doped graphene (NG) dispersed in the biocompatible positively-charged polymer chitosan (chit(+)(NG+GOx)), together with the negatively charged polymer poly(styrene sulfonate), PSS(-), were assembled by alternately immersing a gold electrode substrate in chit(+)(NG+GOx) and PSS(-) solutions. Gravimetric monitoring during LbL assembly by an electrochemical quartz microbalance enabled investigation of the adsorption mechanism and deposited mass for each monolayer. Cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the LbL modified electrodes, in order to establish the contribution of each monolayer to the overall electrochemical properties of the biosensor. The importance of NG in the biosensor architecture was evaluated by undertaking a comparative study without NG in the chit layer. The GOx biosensor's analytical properties were evaluated by fixed potential chronoamperometry and compared with similar reported biosensors. The biosensor operates at a low potential of -0.2V vs., Ag/AgCl, exhibiting a high sensitivity of 10.5 µA cm(-2) mM(-1), and a detection limit of 64 µM. This study shows a simple approach in developing new biosensor architectures, combining the advantages of nitrogen-doped graphene with the LbL technique for enzyme immobilization.

A new modified conducting carbon composite electrode as sensor for ascorbate and biosensor for glucose.

A new carbon-based conducting composite has been developed as electrochemical sensor and biosensor for the amperometric detection of ascorbate and glucose. Electrocatalytic oxidation of ascorbate has been done successfully at unmodified cellulose acetate-graphite composite electrodes, the sensor being highly sensitive, selective and with a low detection limit at 0.0 V vs. SCE and was successfully applied for ascorbate determination in commercial fruit juice samples. An interference free glucose biosensor has also been developed, based on the immobilisation of glucose oxidase by cross-linking with glutaraldehyde on poly (neutral red) modified composite electrodes. The biosensor exhibits a higher sensitivity of 31.5+/-1.7 microA cm(-2) mM(-1) than other carbon-composite-based glucose biosensors, a detection limit of 20.3 microM and a very short response time.

Development and characterization of a new conducting carbon composite electrode.

A new conducting composite flexible material prepared from cellulose acetate (CA) polymer and graphite has been developed and used for the fabrication of electrodes, which were then characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Scanning electron microscopy (SEM) was used to provide information concerning the morphology of the composite electrode surface. The potential window, background currents and capacitance were evaluated by cyclic voltammetry in the pH range from 4.6 to 8.2. The voltammetry of model electroactive species demonstrates a close to reversible electrochemical behaviour, under linear diffusion control. The electroactive area of the composite electrodes increases after appropriate electrode polishing and electrochemical pre-treatment. The electrodes were used as substrate for the electropolymerisation of the phenazine dye neutral red, for future use as redox mediator in electrochemical biosensors. The composite electrodes were also successfully used for the amperometric detection of ascorbate at 0.0 V vs. SCE, and applied to the measurement of ascorbate in Vitamin C tablets; the sensor exhibits high sensitivity and a low detection limit of 7.7 microM. Perspectives for use as a versatile, mechanically flexible and robust composite electrode of easily adaptable dimensions are indicated.

An alcohol oxidase biosensor using PNR redox mediator at carbon film electrodes.

A new amperometric biosensor for ethanol monitoring has been developed and optimised. The biosensor uses poly(neutral red) (PNR), as redox mediator, which is electropolymerised on carbon film electrodes and alcohol oxidase (AlcOx) from Hansenula polymorpha as recognition element, immobilised by cross-linking with glutaraldehyde (GA) in the presence of bovine serum albumin (BSA) as carrier protein. Optimisation of variables affecting the system was performed and, for chronoamperometric measurements, a potential of -0.300 V versus saturated calomel electrode was chosen in 0.1M sodium phosphate buffer saline at pH 7.5. The optimised biosensor showed a good sensitivity of 171.8+/-14.8nAmM(-1) and the corresponding detection limit (signal-to-noise-ratio=3) of 29.7+/-1.5 microM. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 57.6% of its initial sensitivity remaining after 3 weeks (the sensor was used two to three times per week). No significant interferences were found from compounds usually present in wine. The biosensor was used for the determination of ethanol in Portuguese red and white wines.

Design and application of a flow cell for carbon-film based electrochemical enzyme biosensors.

A flow cell has been designed for use with an electrochemical enzyme biosensor, based on low-cost carbon-film electrodes. Three types of mediators were used: cobalt and copper hexacyanoferrates and poly(neutral red) (PNR), covered with glucose oxidase (GOx) immobilised by cross-linking with glutaraldehyde in the presence of bovine serum albumin or inside a oxysilane sol-gel network. Mixtures of sol-gel precursors were made from 3-aminopropyl-triethoxysilane (APTOS) together with methyltrimethoxysilane (MTMOS), methyltriethoxysilane (MTEOS), tetraethyloxysilane (TEOS) or 3-glycidoxypropyl-trimethoxysilane (GOPMOS), and the best chosen for encapsulation. Optimisation in batch mode, using amperometric detection at fixed potential, showed the PNR-GOx modified carbon-film electrodes to be best for flow analysis for both glutaraldehyde and sol-gel enzyme immobilisation. Both types of enzyme electrode were tested under flow conditions and the reproducibility and stability of the biosensors were evaluated. The biosensors were used for fermentation monitoring of glucose in grape must and interference studies were also performed.