A mediator-free phenol biosensor based on immobilizing tyrosinase to ZnO nanoparticles

A mediator-free phenol biosensor was developed. The low-isoelectric point tyrosinase was adsorbed on the surface of high-isoelectric point ZnO nanoparticles (nano-ZnO) facilitated by the electrostatic interactions and then immobilized on the glassy carbon electrode via the film forming by chitosan. It was found that the nano-ZnO matrix provided an advantageous microenvironment in terms of its favorable isoelectric point for tyrosinase loading and the immobilized tyrosinase retaining its activity to a large extent. Moreover, there is no need to use any other electron mediators. Phenolic compounds were determined by the direct reduction of biocatalytically generated quinone species at -200mV (vs. saturated calomel electrode). The parameters of the fabrication process and the various experimental variables for the enzyme electrode were optimized. The resulting biosensor can reach 95% of steady-state current within 10s, and the sensitivity was as high as 182microAmmol(-1)L. The linear range for phenol determination was from 1.5x10(-7) to 6.5x10(-5)molL(-1) with a detection limit of 5.0x 10(-8)molL(-1) obtained at a signal/noise ratio of 3. In addition, the apparent Michaelis-Menten constant (K(m)(app)) and the stability of the enzyme electrode were estimated. The performance of the developed biosensor was compared with that of biosensors based on other immobilization matrices.     

Silica sol-gel composite film as an encapsulation matrix for the construction of an amperometric tyrosinase-based biosensor

An amperometric tyrosinase enzyme electrode for the determination of phenols was developed by a simple and effective immobilization method using sol-gel techniques. A grafting copolymer was introduced into sol-gel solution and the composition of the resultant organic-inorganic composite material was optimized, the tyrosinase retained its activity in the sol-gel thin film and its response to several phenol compounds was determined at 0 mV vs. Ag/AgCl (sat. KCl). The dependences of the current response on pH, oxygen level and temperature were studied, and the stability of the biosensor was also evaluated. The sensitivity of the biosensor for catechol, phenol and p-cresol was 59.6, 23.1 and 39.4 microA/mM, respectively. The enzyme electrode maintained 73% of its original activity after intermittent use for three weeks when storing in a dry state at 4 degrees C.     

Versatile method of cholinesterase immobilisation via affinity bonds using Concanavalin A applied to the construction of a screen-printed biosensor

Development of new and more reliable methods to immobilise biomolecules has emerged rapidly due to a continuous need for more stable, sensitive and reliable biosensors. This paper reports a new method of acetylcholine-esterase (AChE) immobilisation based on the high affinity interaction between the glycoproteic enzyme and Concanavalin A (Con A). In order to establish the nature of the link formed between the glycoenzyme, lectin and support, three different configurations are presented. The optimum immobilisation procedure was further used for biosensor manufacturing. The non-specific adsorption is around 3% and the chemical cross-linking of the proteins is avoided. The optimised method allows loading of the working electrode surface with different amounts of enzyme ranging from 0.3 to 3.3 mIU with a good operational stability. The sensor showed a linear response range to acetylthiocholine substrate between 10 and 110 micromol l(-1) with a sensitivity of 3.6 mA l mol(-1). The applicability of the method to the detection of organophosphorus insecticides resulted in a detection limit of 10(-8) mol l(-1) for chlorpyriphos.     

Amperometric tyrosinase biosensor based on Fe3O4 nanoparticles-chitosan nanocomposite

A novel tyrosinase biosensor based on Fe(3)O(4) nanoparticles-chitosan nanocomposite has been developed for the detection of phenolic compounds. The large surface area of Fe(3)O(4) nanoparticles and the porous morphology of chitosan led to a high loading of enzyme and the entrapped enzyme could retain its bioactivity. The tyrosinase-Fe(3)O(4) nanoparticle-chitosan bionanocomposite film was characterized with atomic force microscopy and AC impedance spectra. The prepared biosensor was used to determine phenolic compounds by amperometric detection of the biocatalytically liberated quinone at -0.2V vs. saturated calomel electrode (SCE). The different parameters, including working potential, pH of supporting electrolyte and temperature that governs the analytical performance of the biosensor have been studied in detail and optimized. The biosensor was applied to detect catechol with a linear range of 8.3 x 10(-8) to 7.0 x 10(-5)mol L(-1), and the detection limit of 2.5 x 10(-8)mol L(-1). The tyrosinase biosensor exhibits good repeatability and stability. Such new tyrosinase biosensor shows great promise for rapid, simple, and cost-effective analysis of phenolic contaminants in environmental samples. The proposed strategy can be extended for the development of other enzyme-based biosensors.     

Development of tyrosinase biosensor based on quantum dots/chitosan nanocomposite for detection of phenolic compounds

A sensitive and simple amperometric biosensor for phenols was developed based on the immobilization of tyrosinase into CdS quantum dots/chitosan nanocomposite matrix. The nanocomposite film with porous nanostructure, excellent hydrophilicity and biocompatibility resulted in high enzyme loading, and the tyrosinase (Tyr) immobilized in this novel matrix retained its activity to a large extent. The CdS quantum dots/chitosan nanocomposite film was characterized by scanning electron microscopy and electrochemical impedance spectroscopy, and the parameters of the various experimental variables for the biosensor were optimized. Under the optimal conditions, the designed biosensor displayed a wide linear response to catechol over a concentration range of 1.0 × 10⁻⁹ to 2.0 × 10⁻⁵ M with a high sensitivity of 561 ± 9.7 mA M⁻¹ and a low detection limit down to 0.3 nM at a signal-to-noise ratio of 3. The CdS quantum dots/chitosan nanocomposites could provide a novel matrix for enzyme immobilization to promote the development of biosensing and biocatalysis.     

Development of a micro-planar amperometric bile acid biosensor for urinalysis

The determination of bile acid concentration in urine is useful for the screening and diagnosis of various hepatobiliary diseases. Currently, there is no concise method to determine bile acid concentration in urine. This study describes a bile acid biosensor fabricated by electrochemical technique for urinalysis. The micro-planar electrodes employed for the study consisted of a working electrode (platinum), a counter electrode (platinum) and a reference electrode (silver/silver chloride (Ag/AgCl)). The sensor chip was coated with Nafion using a spin-coater in order to both eliminate many interference species in urine and achieve long-term stability of the reference electrode. Nafion coating allowed the sensor chip to prevent the electrode reaction from interference species in urine, because it is charged negative strongly (Nafion contains sulfonic acid group). Three enzymes (bile acid sulfate sulfatase: BSS, beta-hydroxysteroid dehydrogenase: beta-HSD, and NADH oxidase: NHO) were immobilized by glutaraldehyde (GA: cross-linker) onto the sensor chip, because the immobilization of enzymes by GA is simple and commonly carried out. The sensor chip was able to detect bile acid in buffer solution. The optimum enzyme ratio immobilized onto the sensor chip was BSS:beta-HSD:NHO=4:4:20 U/1 chip. There was a relationship between the concentration of bile acid and the response current value. The dynamic range of the sensor chip was 2-100 microM for bile acid. Additionally, bile acid in the urine specimen could be detected using this bile acid biosensor. We present a simple and rapid bile acid biosensor with high sensitivity and high reproducibility.     

Amperometric glucose biosensor based on gold-deposited polyvinylferrocene film on Pt electrode

The preparations and performances of the novel amperometric biosensors for glucose based on immobilized glucose oxidase (GOD) on modified Pt electrodes are described. Two types of modified electrodes for the enzyme immobilization were used in this study, polyvinylferrocene (PVF) coated Pt electrode and gold deposited PVF coated Pt electrode. A simple method for the immobilization of GOD enzyme on the modified electrodes was described. The enzyme electrodes developed in this study were called as PVF-GOD enzyme electrode and PVF-Au-GOD enzyme electrode, respectively. The amperometric responses of the enzyme electrodes were measured at constant potential, which was due to the electrooxidation of enzymatically produced H2O2. The electrocatalytic effects of the polymer, PVF, and the gold particles towards the electrooxidation of the enzymatically generated H2O2 offers sensitive and selective monitoring of glucose. The biosensor based on PVF-Au-GOD electrode has 6.6 times larger maximum current, 3.8 times higher sensitivity and 1.6 times larger linear working portion than those of the biosensor based on PVF-GOD electrode. The effects of the applied potential, the thickness of the polymeric film, the amount of the immobilized enzyme, pH, the amount of the deposited Au, temperature and substrate concentration on the responses of the biosensors were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. Finally the effects of interferents, stability of the biosensors and applicability to serum analysis of the biosensor were also investigated.     

Hydrophobic salt-modified Nafion for enzyme immobilization and stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells. In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes. In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion. Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells. This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.     

Nanographene-based tyrosinase biosensor for rapid detection of bisphenol A

Hydrophilic nanographene (NGP) prepared by ball milling of graphite was used as the support to construct a novel tyrosinase biosensor for determination of bisphenol A (BPA). The performances of the nanographene-based tyrosinase biosensor were systematically compared with those of multiwall carbon nanotubes (MWNTs) modified tyrosinase biosensors. The results indicated that the nanographene-based tyrosinase biosensor provided significant advantages over MWNTs-based tyrosinase biosensor in term of response, repeatability, background current and limit of detection (LOD), which could be attributed to its larger specific surface area and unique hierarchical tyrosinase-NGP nanostructures. The nanographene-based tyrosinase biosensor displayed superior analytical performance over a linear range from 100 nmol L(-1) to 2000 nmol L(-1), with LOD of 33 nmol L(-1) and sensitivity of 3108.4 mA cm(-2)M(-1). The biosensor was further used for detecting BPA (leaching from different vessels) in tap water, and the accuracy of the results was validated by high performance liquid chromatography (HPLC). The nanographene-based tyrosinase biosensor proved to be a promising and reliable tool for rapid detection of BPA leached from polycarbonate plastic products and for on-site rapid analysis of emergency pollution affairs of BPA.     

Amperometric glucose biosensor based on layer-by-layer assembly of multilayer films composed of chitosan, gold nanoparticles and glucose oxidase modified Pt electrode

A new strategy for fabricating glucose biosensor was presented by layer-by-layer assembled chitosan (CS)/gold nanoparticles (GNp)/glucose oxidase (GOD) multilayer films modified Pt electrode. First, a cleaned Pt electrode was immersed in poly(allylamine) (PAA), and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/PAA/Pt). Second, the GOD/GNp/PAA/Pt electrode was immersed in CS, and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/CS/GOD/GNp/PAA/Pt). Third, different layers of multilayer films modified Pt electrodes were assembled by repeating the second process. Film assembling and characterization were studied by quart crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The results confirmed that the assembling process of multilayer films was simple to operate, the immobilized GOD displayed an excellent catalytic property to glucose, and GNp in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. The amperometric response of the biosensors uniformly increased from one to six layers of multilayer films, and then reached saturation after the seven layers. Among the resulting biosensors, the biosensor based on the six layers of multilayer films was best. It showed a wide linear range of 0.5-16 mM, with a detection limit of 7.0 microM estimated at a signal-to-noise ratio of 3, fast response time (within 8s). Moreover, it exhibited good reproducibility, long-term stability and interference free. This method can be used for constructing other thin films, which is a universal immobilization method for biosensor fabrication.     

Cytochrome P450 (CYP) enzymes and the development of CYP biosensors

Cytochrome P450s (CYPs) are a large family of heme-containing monooxygenase enzymes involved in the first-pass metabolism of drugs and foreign chemicals in the body. CYP reactions, therefore, are of high interest to the pharmaceutical industry, where lead compounds in drug development are screened for CYP activity. CYP reactions in vivo require the cofactor NADPH as the source of electrons and an additional enzyme, cytochrome P450 reductase (CPR), as the electron transfer partner; consequently, any laboratory or industrial use of CYPs is limited by the need to supply NADPH and CPR. However, immobilizing CYPs on an electrode can eliminate the need for NADPH and CPR provided the enzyme can accept electrons directly from the electrode. The immobilized CYP can then act as a biosensor for the detection of CYP activity with potential substrates, albeit only if the immobilized enzyme is electroactive. The quest to create electroactive CYPs has led to many different immobilization strategies encompassing different electrode materials and surface modifications. This review focuses on different immobilization strategies that have been used to create CYP biosensors, with particular emphasis on mammalian drug-metabolizing CYPs and characterization of CYP electrodes. Traditional immobilization methods such as adsorption to thin films or encapsulation in polymers and gels remain robust strategies for creating CYP biosensors; however, the incorporation of novel materials such as gold nanoparticles or quantum dots and the use of microfabrication are proving advantageous for the creation of highly sensitive and portable CYP biosensors.     

Fabrication of a carbon fiber paper as the electrode and its application toward developing a sensitive unmediated amperometric biosensor

Carbon fiber paper (CFP), a material frequently used as the diffusion layer in fuel cells, was found recently to exhibit a potential as an electrode for the development of sensitive, unmediated biosensors. After nitrogen plasma treatment, the CFP exhibited a quasi-reversible behavior to the redox couple (e.g., ferricyanide) with an electron transfer rate constant of 7.2 × 10(-3)cms(-1). This rate constant is approximately double that of a Pt-electrode and is much higher than that of many carbon-based electrodes. The unmediated CFP-based tyrosinase biosensor fabricated for this study exhibited an optimal working potential and operating pH value of -0.2V and 6.5, respectively. Compared to other unmediated tyrosinase biosensors, the CFP-based tyrosinase biosensor offers a high sensitivity for the monitoring of phenolic compounds (17.8, 7.1, 5.2 and 3.7 μA μM(-1)cm(-2) for catechol, phenol, bisphenol and 3-aminophenol, respectively). The lowest detection limit for catechol, phenol, bisphenol and 3-aminophenol was 2, 5, 5 and 12 nM, respectively. Furthermore, this biosensor exhibited a good repeatability, a fast response time (around 10s), and a wide linear dynamic range of detection for phenolic compounds.     

Development of amperometric biosensor for glucose based on a novel attractive enzyme immobilization matrix: calcium carbonate nanoparticles

Calcium carbonate nanoparticles (nano-CaCO3) may be a promising material for enzyme immobilization owing to their high biocompatibility, large specific surface area and their aggregation properties. This attractive material was exploited for the mild immobilization of glucose oxidase (GOD) in order to develop glucose amperometric biosensor. The GOD/nano-CaCO3-based sensor exhibited a marked improvement in thermal stability compared to other glucose biosensors based on inorganic host matrixes. Amperometric detection of glucose was evaluated by holding the modified electrode at 0.60 V (versus SCE) in order to oxidize the hydrogen peroxide generated by the enzymatic reaction. The biosensor exhibited a rapid response (6s), a low detection limit (0.1 microM), a wide linear range of 0.001-12 mM, a high sensitivity (58.1 mAcm-2M-1), as well as a good operational and storage stability. In addition, optimization of the biosensor construction, the effects of the applied potential as well as common interfering compounds on the amperometric response of the sensor were investigated and discussed herein.     

Immobilization and direct electrochemistry of glucose oxidase on a tetragonal pyramid-shaped porous ZnO nanostructure for a glucose biosensor

A tetragonal pyramid-shaped porous ZnO (TPSP-ZnO) nanostructure is used for the immobilization, direct electrochemistry and biosensing of proteins. The prepared ZnO has a large surface area and good biocompatibility. Using glucose oxidase (GOD) as a model, this shaped ZnO is tested for immobilization of proteins and the construction of electrochemical biosensors with good electrochemical performances. The interaction between GOD and TPSP-ZnO is examined by using AFM, N(2) adsorption isotherms and electrochemical methods. The immobilized GOD at a TPSP-ZnO-modified glassy carbon electrode shows a good direct electrochemical behavior, which depends on the properties of the TPSP-ZnO. Based on a decrease of the electrocatalytic response of the reduced form of GOD to dissolved oxygen, the proposed biosensor exhibits a linear response to glucose concentrations ranging from 0.05 to 8.2mM with a detection limit of 0.01mM at an applied potential of -0.50V which has better biosensing properties than those from other morphological ZnO nanoparticles. The biosensor shows good stability, reproducibility, low interferences and can diagnose diabetes very fast and sensitively. Such the TPSP-ZnO nanostructure provides a good matrix for protein immobilization and biosensor preparation.     

Label-free detection of bacterial RNA using polydiacetylene-based biochip

A novel acetylcholinesterase (AChE)/choline oxidase (ChOx) bienzyme amperometric acetylcholine biosensor based on gold nanoparticles (AuNPs) and multi-walled carbon nanotubes (MWCNTs) has been successfully developed by self-assembly process in combination of sol-gel technique. A thiolated aqueous silica sol containing MWCNTs and ChOx was first dropped on the surface of a cleaned Pt electrode, and then AuNPs were assembled with the thiolated sol-gel network. Finally, the alternate deposition of poly (diallyldimethylammonium chloride) (PDDA) and AChE was repeated to assemble different layers of PDDA-AChE on the electrode for optimizing AChE loading. Among the resulting biosensors, the biosensor based on two layers of PDDA-AChE multilayer films showed the best performance. It exhibited a wide linear range, high sensitivity and fast amperometric response, which were 0.005-0.4mM, 3.395 μA/mM, and within 15s, respectively. The biosensor showed long-term stability and acceptable reproducibility. More importantly, this study could provide a simple and effective multienzyme immobilization platform for meeting the demand of the effective immobilization enzyme on the electrode surface.     

Development of DNA electrochemical biosensor based on covalent immobilization of probe DNA by direct coupling of sol-gel and self-assembly technologies

A new procedure for fabricating deoxyribonucleic acid (DNA) electrochemical biosensor was developed based on covalent immobilization of target single-stranded DNA (ssDNA) on Au electrode that had been functionalized by direct coupling of sol-gel and self-assembled technologies. Two siloxanes, 3-mercaptopropyltrimethoxysiloxane (MPTMS) and 3-glycidoxypropyltrimethoxysiloxane (GPTMS) were used as precursors to prepare functionally self-assembly sol-gel film on Au electrode. The thiol group of MPTMS allowed assembly of MPTMS sol-gel on gold electrode surface. Through co-condensation between silanols, GPTMS sol-gel with epoxide groups interconnected into MPTMS sol-gel and enabled covalent immobilization of target NH(2)-ssDNA through epoxide/amine coupling reaction. The concentration of MPTMS and GPTMS influenced the performance of the resulting biosensor due to competitive sol-gel process. The linear range of the developed biosensor for determination of complementary ssDNA was from 2.51 x 10(-9) to 5.02 x 10(-7)M with a detection limit of 8.57 x 10(-10)M. The fabricated biosensor possessed good selectivity and could be regenerated. The covalent immobilization of target ssDNA on self-assembled sol-gel matrix could serve as a versatile platform for DNA immobilization and fabrication of biosensors.     

Biosensor for the determination of phenols based on cross-linked enzyme crystals (CLEC) of laccase

Cross-linked enzyme crystals (CLECs) are a versatile form of biocatalyst that can also be used for biosensor application. Laccase from Trametes versicolor (E.C.1.10.3.2) was crystallized, cross-linked and lyophilized with beta-cyclodextrin. The CLEC laccase was found to be highly active towards phenols like 2-amino phenol, guaiacol, catechol, pyrogallol, catechin and ABTS (non-phenolic). The CLEC laccase was embedded in 30% polyvinylpropylidone (PVP) gel and mounted into an electrode to make the sensor. The biosensor was used to detect the phenols in 50-1000 micromol concentration level. Phenols with lower molecular weight such as 2-amino phenol, catechol and pyrogallol gave a short response time where as the higher molecular weight substrates like catechin and ABTS had comparatively a long response time. The optimum pH of the analyte was 5.5-6.0 when catechol was used as substrate. The CLEC laccase retained good activity for over 3 months.     

Determination of phenolic compounds by a polyphenol oxidase amperometric biosensor and artificial neural network analysis

The determination of phenolic compounds is significant given its toxicity, even at very low concentration levels. Amperometric determination of phenols is a simple technique available. Direct oxidation of phenols can be used, but another possibility is the use of polyphenol oxidase (tyrosinase) enzyme biosensors that oxidises the phenolic compounds into their corresponding quinones. Reduction of the resulting quinones accomplishes the amplification of the amperometric signal, as long as the result of the reduction process is the corresponding cathecol, this being able to be oxidised again by the polyphenol oxidase immobilized on the surface of the biosensor. In this communication, simultaneous determination of different phenols was carried out combining biosensor measurements with chemometric tools, in what is known as electronic tongue. The departure information used was the overlapped reduction voltammogram generated with the amperometric biosensor based on polyphenol oxidase. Artificial Neural Networks (ANN) were used for extraction and quantification of each compound. Phenol, cathecol and m-cresol formed the three-analyte study case resolved in this work. Good prediction ability was attained, and so, the separate quantification of these three phenols was accomplished.     

Highly ordered mesoporous carbons as electrode material for the construction of electrochemical dehydrogenase- and oxidase-based biosensors

In this work, the excellent catalytic activity of highly ordered mesoporous carbons (OMCs) to the electrooxidation of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H(2)O(2)) was described for the construction of electrochemical alcohol dehydrogenase (ADH) and glucose oxidase (GOD)-based biosensors. The high density of edge-plane-like defective sites and high specific surface area of OMCs could be responsible for the electrocatalytic behavior at OMCs modified glassy carbon electrode (OMCs/GE), which induced a substantial decrease in the overpotential of NADH and H(2)O(2) oxidation reaction compared to carbon nanotubes modified glassy carbon electrode (CNTs/GE). Such ability of OMCs permits effective low-potential amperometric biosensing of ethanol and glucose, respectively, at Nafion/ADH-OMCs/GE and Nafion/GOD-OMCs/GE. Especially, as an amperometric glucose biosensor, Nafion/GOD-OMCs/GE showed large determination range (500-15,000mumoll(-1)), high sensitivity (0.053nAmumol(-1)), fast (9+/-1s) and stable response (amperometric response retained 90% of the initial activity after 10h stirring of 2mmoll(-1) glucose solution) to glucose as well as the effective discrimination to the possible interferences, which may make it to readily satisfy the need for the routine clinical diagnosis of diabetes. By comparing the electrochemical performance of OMCs with that of CNTs as electrode material for the construction of ADH- and GOD-biosensors in this work, we reveal that OMCs could be a favorable and promising carbon electrode material for constructing other electrochemical dehydrogenase- and oxidase-based biosensors, which may have wide potential applications in biocatalysis, bioelectronics and biofuel cells.     

Highly sensitive amperometric biosensors for phenols based on polyaniline-ionic liquid-carbon nanofiber composite

A novel polyaniline-ionic liquid-carbon nanofiber (PANI-IL-CNF) composite was greenly prepared by in situ one-step electropolymerization of aniline in the presence of IL and CNF for fabrication of amperometric biosensors. The scanning electron micrographs confirmed that the PANI uniformly grew along with the structure of CNF and the PANI-IL-CNF composite film showed a fibrillar morphology with the diameter of around 95 nm. A phenol biosensor was constructed by immobilizing tyrosinase on the surface of the composite modified glassy carbon electrode via the cross-linking step with glutaraldehyde. The biosensor exhibited a wide linear response to catechol ranging from 4.0 x 10(-10) to 2.1 x 10(-6)M with a high sensitivity of 296+/-4 AM(-1)cm(-2), a limit of detection down to 0.1 nM at the signal to noise ratio of 3 and applied potential of -0.05 V. According to the Arrhenius equation, the activation energy for enzymatic reaction was calculated to be 38.8 kJmol(-1) using catechol as the substrate. The apparent Michaelis-Menten constants of the enzyme electrode were estimated to be 1.44, 1.33, 1.16, 0.65 microM for catechol, p-cresol, phenol, m-cresol, respectively. The functionalization of CNF with PANI in IL provided good biocompatible platform for biosensing and biocatalysis.