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Modified screen-printed electrodes for amperometric detection of H(2)O(2) and nicotinamide adenine dinucleotide (NADH) at low applied potential are presented in this paper. The sensors are obtained by modifying the working electrode surface with Prussian Blue, a well known electrochemical mediator for H(2)O(2) reduction. The coupling of this sensor with phenazine methosulfate (PMS) in the working solution gives the possibility of measuring both NAD(P)H and H(2)O(2). PMS reacts with NADH producing PMSH, which in the presence of oxygen, gives an equimolar amount of H(2)O(2). This allows the measurement of both analytes with similar sensitivity (357 mA mol(-1)L cm(-2) for H(2)O(2) and 336 mA mol(-1)L cm(-2) for NADH) and LOD (5x10(-7)mol L(-1) for H(2)O(2) and NADH) and opens the possibility of a whole series of biosensor applications. In this paper, results obtained with a variety of dehydrogenase enzymes (alcohol, malic, lactate, glucose, glycerol and glutamate) for the detection of enzymatic substrates or enzymatic activity are presented demonstrating the suitability of the proposed method for future biosensor applications.

In the present paper, a comparative study using Co-phthalocyanine and Prussian Blue-modified screen-printed electrodes, has been performed. Both the electrodes have demonstrated an easiness of preparation together with high sensitivity towards thicoholine (LOD=5 x 10(-7) and 5 x 10(-6) M for Co-phthalocyanine and Prussian Blue, respectively) with high potentialities for pesticide measurement. Prussian Blue-modified screen-printed electrodes were then selected for successive enzyme immobilization due to their higher operative stability demonstrated in previous works. AChE and BChE enzymes were used and inhibition effect of different pesticides was studied with both the enzymes. AChE-based biosensors have demonstrated a higher sensitivity towards aldicarb (50% inhibition with 50 ppb) and carbaryl (50% inhibition with 85 ppb) while BChE biosensors have shown a higher affinity towards paraoxon (50% inhibition with 4 ppb) and chlorpyrifos-methyl oxon (50% inhibition with 1 ppb). Real samples were also tested in order to evaluate the matrix effect and recovery values comprised between 79 and 123% were obtained.

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.

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 (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 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 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.

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.

Detection of phosphate using amperometry was investigated. The phosphomolybdate complex, formed by addition of nitric acid, ammonium molybdate and phosphate, was reduced at a carbon paste electrode polarised at +0.3V (versus Ag/AgCl). The major characteristics observed were simplicity of the equipment, a limited consumption of reagents and a low detection limit (0.3mumoll(-1)), with a linear range between 1 and 20mumoll(-1). The interference of silicate was completely eliminated using an appropriate concentration of nitric acid and ammonium molybdate. The amperometric detection of orthophosphate in seawater using the batch injection analysis (BIA) technique was reported. Moreover, a carbon paste microelectrode was constructed. Its use allows the analysis of small volume of samples with little dilution in supporting electrolyte. This method was applied to the determination of orthophosphate in cyanobacterial biofilms collected from Roman catacombs. There was a good statistical correlation between results obtained with the proposed method and the standard spectrophotometric method.

Cholinesterase sensors based on screen-printed electrodes modified with polyaniline, 7,7',8,8'-tetracyanoquinodimethane (TCNQ), and Prussian blue have been developed and tested for detection of anticholinesterase pesticides in aqueous solution and in spiked grape juice. The influence of enzyme source and detection mode on biosensor performance was explored. It was shown that modification of the electrodes results in significant improvement of their analytical characteristics for pesticide determination. Thus, the slopes of the calibration curves obtained with modified electrodes were increased twofold and the detection limits of the pesticides were reduced by factors of 1.6 to 1.8 in comparison with the use of unmodified transducers. The biosensors developed make it possible to detect down to 2 x 10(-8) mol L(-1) chloropyrifos-methyl, 5 x 10(-8) mol L(-1) coumaphos, and 8 x 10(-9) mol L(-1) carbofuran in aqueous solution and grape juice. The optimal conditions for grape juice pretreatment were determined to diminish interference from the sample matrix.

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.

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.

In this paper the production of antibodies against saxitoxin (STX) is described, as is the optimization and comparison of two competitive ELISA formats (direct and indirect) for the detection of this toxin. Tests were performed in a 96-well microplate using the toxin-specific polyclonal antibodies produced in our laboratory, obtained from rabbits immunized with saxitoxin-keyhole limpet hemocyanin (STX-KLH). In indirect ELISA format saxitoxin, conjugated to bovine serum albumin (STX-BSA) was coated onto the microtitre plate and incubated with standard toxin and anti-STX antibody. A goat anti-rabbit IgG Peroxidase conjugate was used to enable detection. In the direct ELISA format, STX standard, STX conjugate to horseradish peroxidase (STX-HRP), and enzyme substrate/chromogen solution were sequentially added to the microplate after antibody coating.Results showed the saxitoxin detection limit to be 3 and 10 pg mL(-1) for direct and indirect ELISA formats, respectively. The suitability of the assay for quantification of saxitoxin in mussels was also studied. Samples were spiked with saxitoxin before and after sample treatment to study the extraction efficiency and matrix effect, respectively. After treatment, samples were analysed at 1:1000 v/v dilution in PBS to minimize the matrix effect and to detect the regulatory limit of 40-80 micro g saxitoxin per 100 g mussels as stipulated by the Food and Drug Administration. The efficiency of extraction of saxitoxin was from 72 to 102%. These data were confirmed by liquid chromatography coupled with fluorimetric detection, the technique currently used for quantitative determination of toxins in seafood.

Electropolymerisation of nonconducting polymer, poly-(1,2-diaminobenzene) on the top of Prussian Blue (PB) modified electrode led to significant improvement of resulting hydrogen peroxide transducer selectivity and operational stability. The reported transducer retained 100% of response during 20 h under the continuous flow of 0.1 mM H(2)O(2), and thus improves the stability level in selective peroxide detection by one order of magnitude. The selectivity value of the PB-poly(1,2-DAB) based H(2)O(2) sensor in relation to ascorbate is approximately 600. No signals to acetaminophen and urate were investigated. PB-poly(1,2-diaminobenzene) modified electrode allows the detection of H(2)O(2) in the flow-injection mode down to 10(-7) M with the sensitivity 0.3 A M(-1) cm(-2), which is only two times lower compared to the uncovered PB based transducer.

This paper reports on the development and characterization of Prussian Blue-modified carbon paste electrodes. New methods of matrix modification with Prussian Blue are reported. Two different carbon pastes have been prepared, using mineral oil or solid paraffin, thus obtaining different types of sensors whose behavior toward the electrochemical reduction of hydrogen peroxide has been fully characterized. Results obtained with Prussian Blue-modified electrodes showed a long operational lifetime, an excellent stability in a wide range of pH (3-9), a high sensitivity, and a fast response time. In addition, the coupling of solid carbon paste modified with Prussian Blue and the enzymes glucose oxidase and choline oxidase led to the assembling of biosensors that showed an optimum working range at alkaline pH.

A simple and rapid flow system for the determination of lactulose in milk samples was developed. It is based on the hydrolysis of lactulose to galactose and fructose by the enzyme beta-galactosidase immobilised in a reactor. The amount of fructose produced was measured with an electrochemical biosensor based on the fructose dehydrogenase enzyme, K3[Fe(CN)6] as mediator and a platinum based electrochemical transducer. Parameters such as the enzyme immobilisation in the reactor and under the electrode surface, the lifetime of the beta-galactosidase reactor and of the dehydrogenase biosensor and the flow parameters were studied and optimised. Fructose was determined in the range 1 x 10(-6)-5 x 10(-3) mol l-1 with an RSD of about 2% and a detection limit of 5 x 10(-7) mol l-1. The use of a microdialysis probe as the sampling system permitted the direct measurement of lactulose in milk samples without pre-treatment in the range 1 x 10(-5)-5 x 10(-3) mol l-1. The sensitivity of the procedure allowed pasteurised, UHT and in-container sterilised milk to be distinguished.

A light, portable sampling device for the continuous collection and storage of subcutaneous fluid is described. It consists of a hollow-fibre probe for ultrafiltration, a long tube for sample storage and a vacuum tube as driving force. Introduction of a restriction in the flow path allows a constant pulse-free flow as low as 100 nl min-1. The contents of the storage tube reflect the metabolic events that have happened during the 24 h collection period. The ultrafiltrate fluid collected from the subcutaneous tissue is free from large molecules and can be continuously analysed. An application in combination with a bionsensor for the analysis of glucose is described, but the device also allows monitoring of other subcutaneous constituents such as ions, metabolites, drugs and their metabolites.

An enzyme-based flow-injection amperometric analysis system (FIA) for monitoring of uric acid and glucose is described. The oxidase and peroxidase enzymes are physically coimmobilised in a sandwich-type reactor and ferrocene serves as a mediator. The assays are based on the measurement of a reduction current resulting from the enzymatic reactions, at a glassy carbon electrode held at 0.00 mV (vs. Ag/AgCl). The high selectivity (ascorbic acid did not interfere) is coupled to high sensitivity (a detection limit of 30 and 60 nmol/l for uric acid and glucose, respectively; signal/noise = 3) and good stability (the enzymes remained active for more than 6 weeks at 30 degrees C). The usefulness of the assay in clinical chemistry is illustrated by the measurement of human serum uric acid and glucose concentration. The results obtained were in fairly good agreement with those obtained using conventional hospital laboratory methods.

Amperometric enzyme electrode probes have been constructed for the specific determination of L-lysine and used in batch and flow analysis. The enzyme lysine oxidase was immobilized on a preactivated polymer support which was placed on a platinum electrode. Additional blocking membranes conferred high stability, reproducibility and avoided electrochemical and enzyme interferences. Parameters including pH, temperature, storage and operational times were optimized. Lysine was determined in the range 10(-6)-2.10(-3)M with a detection limit of 5 x 10(-7)M. The Michaelis constant was 2 x 10(-3)M. This value was approximately two order of magnitudes higher than that reported in literature for the free enzyme. The response time of the probe was about 2 min in batch and flow analysis and 30 sec in flow injection analysis (FIA). The resulting probes were stable for more than three months with more than 300 analyses performed. The determination of lysine was carried out by both flow-through analysis and FIA. Analysis in feeds was carried out by acid hydrolysis to liberate lysine; then the solution was analyzed by the bioprobe and HPLC procedures. Results by the two methods correlated well.

Microdialysis probes have been tested to evaluate the influence of flow-rate, probe dimensions and temperature when used as sampling systems inserted subcutaneously for in vivo monitoring of glucose. The probe was coupled with a glucose biosensor obtained from a thin layer electrochemical cell generally used as detector for liquid chromatography. Glucose oxidase was immobilized on a nylon net membrane and it was placed over an acetate cellulose membrane into the cell in contact with the platinum anode.

Microdialysis coupled to a glucose biosensor led to a continuous monitor system in vivo for glucose. Several microdialysis probes were used to stabilize the biosensor response. In vivo experiments, especially when the probe was placed subcutaneously, showed that the sensitivity of the biosensor decreased continuously; various kinds of fibers with a molecular weight cut-off (MWCO) ranging from 6,000 to 20,000 were compared. A wall-jet flow cell as detector for glucose showed less interference when compared to a thin layer cell. Glucoday, a new commercial instrument based on this principle, is presented.

Serum cholinesterase activity and the dibucaine numbers have been determined by using a hydrogen peroxide electrode and the enzyme choline oxidase immobilized on a nylon net. The analysis procedure is extremely simple and very fast allowing 30 cholinesterase determinations per hour. Both cholinesterase activity and dibucaine number measurements could be performed in 5 min and by using serum samples of only 10 microliters. When used in sera the probe showed no interference from electroactive compounds present in blood, and also showed good stability and reproducibility. These features make this sensor appropriate for continuous extracorporeal circuit blood monitoring of succinylcholine during surgery.

Glucose, lactate and potassium ions have been continuously measured in whole blood by using two extracorporeal electrochemical biosensors and a new flow through potassium electrode. For experiments involving the "glucose clamp", lactate and potassium electrodes have been connected in series to an endocrine artificial pancreas "Betalike" properly modified. The same artificial pancreas has been used in experiments measuring the anaerobic threshold (AT) by continuous monitoring of lactate in athletes. In this case glucose and lactate sensors were connected in series for continuous measurement of both metabolites in blood in real time. The results are in agreement with the biochemical pathways involving such metabolites.

Glucose, lactate, and pyruvate can be continuously measured in whole blood by using three extracorporeal electrochemical biosensors. We fixed two newly developed sensors, for lactate and pyruvate, in series with an endocrine artificial pancreas ("Betalike") without affecting its performance. These new sensors for use with whole blood should be able to clarify the fate of the intermediary metabolites of glucose in diabetic patients receiving infusions of drugs or insulin.

We report the features of a sensor for determining L-lactate. An oxygen sensor, coupled to a nylon net with chemically bound L-lactate oxidase (EC 1.1.3.2), is inserted into an artificial pancreas (Biostator, Miles) "downstream" from the glucose sensor. We used the sensor to continuously monitor the L-lactate concentration in blood after a "glucose clamp" experiment with a diabetic patient. L-Lactate determinations in blood drawn from the patient every 15 min agreed well with results obtained by use of the L-lactate sensor.