Origami multiple paper-based electrochemical biosensors for pesticide detection.

Herein, we propose the first three-dimensional origami paper-based device for the detection of several classes of pesticides by combining different enzyme-inhibition biosensors. This device was developed by integrating two different office paper-based screen-printed electrodes and multiple filter paper-based pads to load enzymes and enzymatic substrates. The versatile analysis of different pesticides was carried by folding and unfolding the filter paper-based structure, without any addition of reagents and any sample treatment (i.e. dilution, filtration, pH adjustment). The paper-based platform was employed to detect paraoxon, 2,4-dichlorophenoxyacetic acid, and atrazine by exploiting the capability of these different types of pesticides (i.e. organophosphorus insecticides, phenoxy-acid herbicides, and triazine herbicide) to inhibit butyrylcholinesterase, alkaline phosphatase, and tyrosinase, respectively. The degree of inhibition correlating to the quantity of pesticides was evaluated by chronoamperometrically monitoring the enzymatic activity in the absence and in the presence of pesticides by using a portable potentiostat. To improve the sensitivity, the paper-based electrodes were modified with carbon black alone in the case of platforms for 2,4-dichlorophenoxyacetic acid and atrazine detection, or decorated with Prussian blue nanoparticles for the detection of paraoxon. The paper-based device was applied for the detection of paraoxon, 2,4-dichlorophenoxyacetic acid, and atrazine at ppb level in both standard solutions and river water sample. The accuracy of this origami multiple paper-based electrochemical biosensor was evaluated in river water by recovery studies, obtaining satisfactory values (e.g. for paraoxon 90 ±â€¯1% and 88 ±â€¯2%, for 10 and 20 ppb, respectively). The proposed three-dimensional origami paper device allows for rapid, cost-effective and accurate pesticide detection in surface water as a result of combining filter and office papers, screen-printing, wax-printing and nanomaterial technology.

Recent advances in biosensors based on enzyme inhibition.

Enzyme inhibitors like drugs and pollutants are closely correlated to human and environmental health, thus their monitoring is of paramount importance in analytical chemistry. Enzymatic biosensors represent cost-effective, miniaturized and easy to use devices; particularly biosensors based on enzyme inhibition are useful analytical tools for fast screening and monitoring of inhibitors. The present review will highlight the research carried out in the last 9 years (2006-2014) on biosensors based on enzyme inhibition. We underpin the recent advances focused on the investigation in new theoretical approachs and in the evaluation of biosensor performances for reversible and irreversible inhibitors. The use of nanomaterials and microfluidic systems as well as the applications of the various biosensors in real samples is critically reviewed, demonstrating that such biosensors allow the development of useful devices for a fast and reliable alarm system.

A three-dimensional carbon nanotube network for water treatment.

The bulk synthesis of freestanding carbon nanotube (CNT) frameworks is developed through a sulfur-addition strategy during an ambient-pressure chemical vapour deposition process, with ferrocene used as the catalyst precursor. This approach enhances the CNTs' length and contorted morphology, which are the key features leading to the formation of the synthesized porous networks. We demonstrate that such a three-dimensional structure selectively uptakes from water a mass of toxic organic solvent (i.e. o-dichlorobenzene) about 3.5 times higher than that absorbed by individual CNTs. In addition, owing to the presence of highly defective nanostructures constituting them, our samples exhibit an oil-absorption capacity higher than that reported in the literature for similar CNT sponges.

Towards the development of a single-step immunosensor based on an electrochemical screen-printed electrode strip coupled with immunomagnetic beads.

This work investigates the behaviour of two alternative systems that model the crucial event involved in any ELISA test, i.e. the molecular recognition between an antigen and its specific antibody on a solid phase, and its measurement. Each approach is devised with the goal of making possible a single-step, separation and wash-free amperometric magneto-immunosensor. Magnetic particles (MBs) are used as support for the immobilization of rabbit IgGs that are recognized by the specific anti-rabbit IgG-HRP. The assay protocol is based on the use of a series of small "reservoirs" containing phosphate buffer, hydroquinone, anti-rabbit IgG-HRP and an appropriate amount of MB-rabbit IgG. After a brief incubation, the content of each "reservoir" is transferred to one of the wells of a 8-well magnetized-screen-printed electrode strip. The resulting MB-IgG-anti-IgG-HRP chain, is then concentrated on the working electrode surface for electrochemical measurement. Two different approaches to monitor this immunological reaction are investigated. The first one is based on the enzyme-channeling principle (ECP) and involves the use of a second enzyme, glucose oxidase (GOD), immobilized on the working electrode previously modified with Prussian Blue. Since the H(2)O(2) produced by GOD is the co-substrate of the HRP enzyme, glucose is added into the well and the current, generated by the residual H(2)O(2), is measured. The second, more direct, approach is performed without exploiting ECP (no GOD enzyme), by adding H(2)O(2) into the well and measuring the current generated by the HRP product on a pristine screen-printed electrode. Both approaches yielded a typical sigmoidal binding curve, illustrating the discrimination between the signal produced by the immuno-bound HRP concentrated on the electrode surface, and the background signal due to HRP in the bulk solution.

Acetylcholinesterase biosensor based on single-walled carbon nanotubes--Co phtalocyanine for organophosphorus pesticides detection.

A simple and reliable technique has been developed for the construction of an amperometric acetylcholinesterase biosensor based on screen-printed carbon electrodes. For the first time, one-step modification using single-walled carbon nanotubes and Co phtalocyanine has been proposed to decrease the working potential and to increase the signal of thiocholine oxidation. The biosensor developed made it possible to detect 5-50 ppb of paraoxon and 2-50 ppb of malaoxon with detection limits of 3 and 2 ppb, respectively (incubation 15 min). The biosensor showed high reproducibility when measurements of the substrate and inhibitor were performed (R.S.D. about 1% and 2.5%, respectively). The reliability of the inhibition measurements was confirmed by testing spiked samples of sparkling and tape waters.

An acetylcholinesterase biosensor for determination of low concentrations of Paraoxon and Dichlorvos.

The characterization of an economic and ease-to-use carbon paste acetylcholinesterase (AChE) based biosensor to determine the concentration of pesticides Paraoxon and Dichlorvos is discussed. AChE hydrolyses acetylthiocholine (ATCh) in thiocoline (TC) and acetic acid (AA). When AChE is immobilized into a paste carbon working electrode kept at +410 mV vs. Ag/AgCl electrode, the enzyme reaction rate using acetylthiocholine chloride (ATCl) as substrate is monitored as a current intensity. Because Paraoxon and Dichlorvos inhibit the AChE reaction, the decrease of the current intensity, at fixed ATCl concentration, is a measure of their concentration. Linear calibration curves for Paraoxon and Dichlorvos determination have been obtained. The detection limits resulted to be 0.86 ppb and 4.2 ppb for Paraoxon and Dichlorvos, respectively, while the extension of the linear range was up 23 ppb for the former pesticide and up to 33 ppb for the latter. Because the inhibited enzyme can be reactivated when immediately treated with an oxime, the biosensor reactivation has been studied when 1,1'-trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were used. TMB-4 resulted more effective. The comparison with the behavior of similar AChE based biosensors is also presented.

A thionine-modified carbon paste amperometric biosensor for catechol and bisphenol A determination.

A thionine-modified carbon paste electrode for catechol and Bisphenol A (BPA) detection is presented. Graphite powder was modified by adsorbing thionine as electrochemical mediator. The electrochemical response of the modified carbon paste electrode (CPE) was determined before electrode modification with tyrosinase. Then, tyrosinase was added in order to assemble a biosensor. Once established the best operative conditions, an interelectrode reproducibility around 7% was obtained and the resulting biosensor showed improved sensitivities and (S=139.6+/-1.1 nA/microM for catechol and S=85.4+/-1.5 nA/microM for BPA) in comparison with the biosensor constructed without thionine (S=104.4+/-0.5 nA/microM for catechol and S=51.1+/-0.6 nA/microM for BPA) and low detection limits (0.15 microM for both the electrodes and analytes). Also the comparison with the results reported in the literature showed higher sensitivity and lower detection limit for our biosensor. Moreover the functioning of the thionine-tyrosinase CPE was validated following a biodegradation process of water polluted by BPA and comparing the time changes of BPA concentration inferred by the biosensor calibration curve and those determined by means of HPLC measurements.

Cardiac autonomic regulation after lung exposure to carbon nanotubes.

The ultrafine (UF) component of airborne pollution may impair cardiovascular autonomic control, a high-risk condition for cardiovascular adverse events. Since engineered nanoparticles, such as single-walled carbon nanotubes (SWCNTs) share physicochemical properties with UF, they might have similar adverse effects. Aim of the study was to evaluate arterial baroreflex function (BRF) at baseline, 24 h after the first instillation, immediately before the second one, and 2 weeks later, in adult Wystar-Kyoto conscious rats undergoing two intratracheal instillations of SWCNT (eight rats) or phosphate buffer saline (PBS) (five rats) at 2-week interval. During each session, 30-min continuous recording of arterial pressure and pulse interval was performed by a telemetered catheter implanted in the abdominal aorta of the rats. BRF was studied by the sequence technique. SWCNTs dispersed in PBS (1 mg/ml) were administered immediately after sonication (1 microg/g body weight). A significant decrease in the number of baroreflex sequences (from 498 +/- 27.1 at baseline to 287 +/- 40.2 at the recording performed after 4 weeks; P < 0.05) was observed in SWCNT-instilled rats, whereas no significant change was detected in controls. These data suggest that SWCNTs may alter the BRF, thus affecting the autonomic cardiovascular control regulation.

A bienzyme electrochemical probe for flow injection analysis of okadaic acid based on protein phosphatase-2A inhibition: an optimization study.

A bienzyme electrochemical probe has been assembled and used to monitor the inhibition of the enzyme protein phosphatase-2A (PP2A) by okadaic acid (OA), taking advantage of the particular characteristics of a biochemical pathway in which PP2A is involved. This enzyme has significant activity toward glycogen phosphorylase a (PHOS a), which in turn catalyzes the conversion of glycogen to glucose-1-phosphate (G-1-P). In addition, PP2A is strongly inhibited by OA and its derivatives. Due to this combination of properties, PP2A was employed to develop an assay system involving a preliminary phase of off-line enzymatic incubations (OA/PP2A, PP2A/PHOS a, PHOS a/glycogen+phosphate). This off-line step was followed by the electrochemical detection of H2O2, which is the final product of two sequential enzymatic reactions: G-1-P with alkaline phosphatase (AP) producing glucose, then glucose with glucose oxidase (GOD) producing hydrogen peroxide. These two enzymes were coimmobilized on a nylon net membrane that was placed over an H2O2 platinum probe inserted into a flow injection analysis (FIA) system. During a first phase of the study, all analytical parameters were optimized. During a subsequent phase, the inhibition of PP2A enzyme by OA was evaluated. The calibration of the system shows a working range for detection of OA between 30 and 250 pg ml(-1). The total analysis time is the sum of 50 min for the off-line enzymatic incubations and 4 min for the biosensor response.

Construction, assembling and application of a trehalase-GOD enzyme electrode system.

Trehalose is a disaccharide important in foods, serving as a glucose source in many and also as an additive in the food preparation. Because of its peculiar physico-chemical properties it plays an important role as preservative in drying and deep-freezing treatments. A new biosensor for trehalose determination has been realized by means of a flow system, based on a reactor in which the trehalase enzyme catalyses its hydrolysis into two alpha,d-glucose molecules, and a GOD (glucose oxidase) amperometric biosensor is employed for the glucose determination. The optimum operative conditions have been laid out and a particular attention has been paid to the immobilization procedure of the two enzymes. The electrode used is of the SPE (screen-printed electrode) type and has been activated with the Prussian Blue (PB) and then assembled using GOD immobilized with Nafion. The reactor has been prepared with the trehalase enzyme chemically immobilized on an Immunodyne ABC membrane. As demonstration of its utility, the biosensor has been tested on a real sample of Boletus edulis mushroom.

Enzymatic determination of BPA by means of tyrosinase immobilized on different carbon carriers.

Different tyrosinase carbon paste modified electrodes to determine bisphenol A (BPA) concentration in aqueous solutions have been constructed. Variables examined were in the carbon paste composition and in particular: (i) the immobilized enzyme amount; (ii) the carbon type (powder, single or multi-walled nanotubes); (iii) the nature of the pasting oil (mineral oil, hexadecane and dodecane). For each biosensor type the amperometric response was evaluated with reference to the linear range and sensitivity. Constant reference has been made to the amperometric signals obtained, under the same experimental conditions, towards the catechol, a specific phenolic substrate for tyrosinase. The most efficient biosensors were those constructed by using the following composition for the carbon paste: 10% of tyrosinase, 45% of single wall carbon nanotubes (SWCN) and 45% of mineral oil. This biosensor formulation displayed the following electrochemical characteristics: a sensitivity equal to 138 microA/mM, LOD of 0.02 microM (based on three times the S/N ratio), linear range of 0.1-12 microM and response time of 6 min. This experimental work represents a first attempt at construction of a new carbon nanotube-tyrosinase based biosensor able to determine the concentration of BPA, one of the most ubiquitous and hazardous endocrine disruptors which can pollute the drinking and surface water, as well as many products of the food chain.

Electrochemical immunosensor array using a 96-well screen-printed microplate for aflatoxin B1 detection.

A novel analytical immunosensor array, based on a microtiter plate coupled to a multichannel electrochemical detection (MED) system using the intermittent pulse amperometry (IPA) technique, is proposed for the detection of aflatoxin B1 (AFB1). In the present work, the electrochemical behaviour and electroanalytical performance of the thick-film carbon sensors (also designated as screen-printed electrodes) incorporated in the multichannel electrochemical plate were first evaluated. Then the 96-well screen-printed microplate was modified in accord with a competitive indirect enzyme-linked immunoassay (ELISA) format for aflatoxin B1 detection. The measurements were performed using both spectrophotometric and electrochemical procedures and the results of the calibration curves, detection limit (LOD), sensitivity and reproducibility of the respective assay systems were evaluated. The immunoassay was then applied for analysis of corn samples spiked with AFB1 before and after the extraction treatment, in order to study the extraction efficiency and the matrix effect, respectively. These studies have shown that using this system, AFB1 can be measured at a level of 30 pg/mL and with a working range between 0.05 and 2 ng/mL. Good recoveries (103+/-8%) were obtained, demonstrating the suitability of the proposed assay for accurate determination of the AFB1 concentration in corn samples. The specificity of the assay was assessed by studying the cross-reactivity of PAb relative to AFB1. The results indicated that the PAb could readily distinguish AFB1 from other aflatoxins, with the exception for AFG1.

A disposable immunosensor for detection of 17beta-estradiol in non-extracted bovine serum.

This paper reports the assembly of a disposable immunosensor based on the direct competitive enzyme-linked immunosorbent assay (ELISA), for simple and fast measurement of 17beta-estradiol (17beta-E2) in bovine serum, using screen-printed electrodes (SPEs) and a Palm-Sens portable electrochemical detector. The immunosensor strip was assembled immobilising, by passive adsorption, anti-rabbit IgG onto the surface of the working SPE electrode. After the interaction between anti-rabbit IgG and rabbit anti-17beta-E2 polyclonal antibodies (PAb), the competition was performed using 17beta-estradiol-alkaline phosphatase conjugate (17beta-E2-AP) synthesised in our laboratory. The enzymatic substrate used for signal generation was 1-naphthylphosphate and its conversion to an electroactive product (1-naphthol) was measured using differential pulse voltammetry (DPV). To develop a prototype for field measurements, the entire competitive protocol has been optimised directly in a blank non-extracted bovine serum. According to the new EU criteria established by the Commission Decision 2002/657/EC for qualitative and quantitative screening methods, the detection capability (CCbeta), was determined. The CCbeta value resulted below the action limit (40 pg mL(-1)) fixed for 17beta-E2) Spiked and real samples were analysed using the electrochemical immunostrips obtaining precision values (relative standard deviation, R.S.D.%) ranging from 8.6 to 17.0% and a recovery (R%) from 88.2 to 120.0%. Results obtained on real samples were confirmed by liquid chromatography coupled on-line with tandem mass spectrometry (LC-MS/MS) using an atmospheric pressure chemical ionisation (APCI) source and a heated nebulizer (HN) interface; this is the method currently used to confirm illegal hormone administration for regulatory purposes. The disposable immunosensor appears suitable as a screening tool for field analysis of bovine serum estradiol.

An electrochemical immunosensor for aflatoxin M1 determination in milk using screen-printed electrodes.

The production and assembling of disposable electrochemical AFM1 immunosensors, which can combine the high selectivity of immunoanalysis with the ease of the electrochemical probes, has been carried out. Firstly immunoassay parameters such as amounts of antibody and labelled antigen, buffer and pH, length of time and temperature of each steps (precoating, coating, binding and competition steps) were evaluated and optimised in order to set up a spectrophotometric enzyme-linked immunosorbent assay (ELISA) procedure. This assay exhibited a working range between 30 and 160 ppt in a direct competitive format. Then electrochemical immunosensors were fabricated by immobilising the antibodies directly on the surface of screen-printed electrodes (SPEs), and allowing the competition to occur between free AFM1 and that conjugated with peroxidase (HRP) enzyme. The electrochemical technique chosen was the chronoamperometry, performed at -100 mV. Furthermore, studies of interference and matrix effects have been performed to evaluate the suitability of the developed immunosensors for the analysis of aflatoxin M1 directly in milk. Results have shown that using screen-printed electrodes aflatoxin M1 can be measured with a detection limit of 25 ppt and with a working range between 30 and 160 ppt. A comparison between the spectrophotometric and electrochemical procedure showed that a better detection limit and shorter analysis time could be achieved using electrochemical detection.

Acetylcholinesterase sensor based on screen-printed carbon electrode modified with prussian blue.

Acetylcholinesterase (ChE) sensor based on Prussian blue (PB) modified electrode was developed and tested for the detection of organophosphorus and carbamic pesticides. The signal of the sensor was generated in PB mediated oxidation of thiocholine recorded at +200 mv in DC mode. ChE from electric eel was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA) on the surface of screen-printed carbon electrode covered with PB and Nafion. The content of the surface layer (specific enzyme activity, Nafion and BSA amounts) was optimized to establish high and reliable response toward the substrate and ChE inhibitors. The ChE/PB sensor makes it possible to detect Aldicarb, Paraoxon and Parathion-Methyl with limits of detection 30, 10 and 5 ppb, respectively (incubation 10 min). The feasibility of practical application of the ChE/PB sensor developed for the monitoring of degradation of the pesticides in wine fermentation was shown. To diminish matrix interferences, the electrolysis of the grape juice with Al anode and evaporation of ethanol were suggested, however the procedures decrease the sensitivity of pesticide detection and stability of the sample tested.

A novel continuous subcutaneous lactate monitoring system.

A novel continuous lactate monitoring system has been developed modifying the GlucoDay portable medical device (A. Menarini Diagnostics), already present in the European market, and used to continuously measure glucose levels. Lactate oxidase based biosensors have been developed immobilising the enzyme on nylon net and placing it on a Pt electrode. The biosensor was connected to the portable device provided with a micro-pump and coupled to a microdialysis system. It is capable to record subcutaneous lactate every 3 min. In vitro analytical results confirmed that the sensors respond linearly in the interval of concentration between 0.1 and 10 mmol/L, covering the whole physiological range. During prolonged monitoring periods, the response of the biosensors remained stable, showing a limited drift of 8%, within 60 h. Stability tests are still on route. However, preliminary results have shown a shelf life of about 10 months. In vivo experiments performed on healthy rabbits have demonstrated the good accuracy and reproducibility of the system. A correlation coefficient equal to 0.9547 (N=80) was found, which represents a good correlation between the GlucoDay and the laboratory reference analyser. A 16 h in vivo monitoring on a healthy volunteer has been also performed.

Novel planar glucose biosensors for continuous monitoring use.

Novel planar glucose biosensors to be used for continuous monitoring have been developed. The electrodes are produced with the "screen printing" technique, and present a high degree of reproducibility together with a low cost and the possibility of mass production. Prior to enzyme immobilisation, electrodes are chemically modified with ferric hexacyanoferrate (Prussian Blue). This allows the detection of the hydrogen peroxide produced by the enzymatic reaction catalysed by GOD, at low applied potential (ca. 0.0 V versus Ag/AgCl), highly limiting any electrochemical interferences. The layer of Prussian Blue (PB) showed a high stability at the working conditions (pH 7.4) and also after 1 year of storage dry at RT, no loss of activity was observed. The assembled glucose biosensors, showed high sensitivity towards glucose together with a long-term operational and storage stability. In a continuous flow system, with all the analytical parameters optimised, the glucose biosensors detected glucose concentration as low as 0.025 mM with a linear range up to 1.0mM. These probes were also tested over 50-60 h in a continuous flow mode to evaluate their operational stability. A 0.5 mM concentration of glucose was continuously fluxed into a biosensor wall-jet cell and the current due to the hydrogen peroxide reduction was continuously monitored. After 50-60 h, the drift of the signal observed was around 30%. Because of their high stability, these sensors suggest the possibility of using such biosensors, in conjunction with a microdialysis probe, for a continuous monitoring of glucose for clinical purposes.

Disposable immunosensor for the determination of domoic acid in shellfish.

The construction of an electrochemical immunosensor coupled to differential pulse voltammetry (DPV) for the detection of domoic acid (DA), a neurotoxic aminoacid responsible for the human syndrome known as "Amnesic Shellfish Poisoning" (ASP), is proposed here. The method involves the use of disposable screen-printed electrodes (SPEs) for the immunosensor development based on a "competitive indirect test". Domoic acid conjugated to bovine serum albumin (BSA-DA) was coated onto the working electrode of the SPE, followed by incubation with sample (or standard toxin) and anti-DA antibody. An anti-goat IgG-alkaline phosphatase (AP) conjugate was used for signal generation. A spectrophotometric enzyme-linked immunosorbent assay (ELISA) was used in a preliminary phase of development, prior to transferring the assay to the SPEs. Results showed a detection limit equal to 5 ng/ml of toxin. The electrochemical system is simple and cost-effective due to the disposable nature of the SPEs, and the analysis time is 150 min, shorter than that for the spectrophotometric method. The suitability of the assay for DA quantification in mussels was also evaluated. Samples were spiked with DA before and after the sample treatment to study the extraction efficiency and the matrix effect, respectively. After treatment, samples were analysed using a 1:250 v/v dilution in PBS-M (phosphate saline buffer pH 7.4 + CH3OH 10%) to minimise the matrix effect and allow for the detection of 20 microg/g of DA in mussel tissue. This represents the maximum acceptable limit defined by the Food and Drug Administration [Compliance Programme 7303.842. Guidance Levels, Table 3, p. 248, http://www.fda.org]. The optimised ELISA systems were then used, in parallel with a conventional HPLC method, to detect and confirm DA in shellfish extract in order to verify the performance of the electrochemical system. Very good recoveries were obtained, demonstrating the suitability of the proposed assay for accurate determination of the DA concentration in mussel samples.

A microdialysis technique for continuous subcutaneous glucose monitoring in diabetic patients (part 1).

The performances and the stability of a novel subcutaneous glucose monitoring system have been evaluated. GlucoDay (A. Menarini I.F.R. S.r.l, Florence Italy) is a portable instrument provided with a micro-pump and a biosensor coupled to a microdialysis system capable of recording the subcutaneous glucose level every 3 min. Long and short term stability of the biosensor are discussed and the results of some critical in vitro and in vivo (on rabbits) experiments are reported. A linear response up to 30 mM has been found for in vivo glucose concentration. The sensitivity referred to blood glucose is better than 0.1 mM and the zero current is typically below the equivalent of 0.1 mM. In the accuracy study a mean bias of 2.7 mg/dl and a correlation coefficient equal to 0.9697 have been found. At room temperature, an excellent membrane stability assures good performances up to 6 months from the first use.

Prussian Blue based screen printed biosensors with improved characteristics of long-term lifetime and pH stability.

The promising advantages of Prussian Blue (PB) as catalyst and of the thick film screen printing technology have been combined to assemble sensors with improved characteristics for the amperometric determination of H(2)O(2). PB-modified screen printed electrodes were applied to detect H(2)O(2) at an applied potential of -0.05 V versus the internal screen printed Ag pseudoreference electrode, showing a detection limit of 10(-7) mol l(-1), a linearity range from 10(-7) to 5x10(-5) mol l(-1), a sensitivity of 234 microA mmol l(-1) cm(-2), and a high selectivity. Improved stability at alkaline pH values was also observed, which made possible their use with enzymes having an optimum basic pH. Then, the immobilisation of a single enzyme (glucose oxidase (GOD) or choline oxidase (ChOX)) or of two enzymes, acetylcholinesterase (AchE) coimmobilised with ChOX, has been performed on the surface of PB modified screen-printed electrodes (SPEs) using glutaraldehyde and Nafion. ChOX has been selected as an example of enzyme working at alkaline pH. The choline biosensors showed a detection limit of 5x10(-7) mol l(-1), a wide linearity range (5x10(-7)-10(-4) mol l(-1)), a high selectivity and a remarkable long term stability of 9 months at 4 degrees C, and at least 4 weeks at room temperature. Similar analytical characteristics and stability were observed with the acetylcholine biosensors.