Giuseppe Palleschi

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.

Low Doses of Pristine and Oxidized Single-Wall Carbon Nanotubes Affect Mammalian Embryonic Development

Several in vitro and in vivo studies suggest local and systemic effects following exposure to carbon nanotubes. No data are available, however, on their possible embryotoxicity in mammals. In this study, we tested the effect of pristine and oxidized single-wall carbon nanotubes (SWCNTs) on the development of the mouse embryo. To this end, SWCNTs (from 10 ng to 30 μg/mouse) were administered to female mice soon after implantation (postcoital day 5.5); 10 days later, animals were sacrificed, and uteri, placentas, and fetuses examined. A high percentage of early miscarriages and fetal malformations was observed in females exposed to oxidized SWCNTs, while lower percentages were found in animals exposed to the pristine material. The lowest effective dose was 100 ng/mouse. Extensive vascular lesions and increased production of reactive oxygen species (ROS) were detected in placentas of malformed but not of normally developed fetuses. Increased ROS levels were likewise detected in malformed fetuses. No increased ROS production or evident morphological alterations were observed in maternal tissues. No fetal and placental abnormalities were ever observed in control animals. In parallel, SWCNT embryotoxicity was evaluated using the embryonic stem cell test (EST), a validated in vitro assay developed for predicting embryotoxicity of soluble chemical compounds, but never applied in full to nanoparticles. The EST predicted the in vivo data, identifying oxidized SWCNTs as the more toxic compound.

Evaluation of the use of free and immobilised acetylcholinesterase for paraoxon detection with an amperometric choline oxidase based biosensor

Abstract Aspects related to the use of free and immobilised acetylcholinesterase (AChE) for the development of an amperometric biosensor for organophosphorus (OP) insecticides based on enzyme inhibition have been studied. Procedures based on free and immobilised AChE were previously studied from many authors but the measurement of the residual enzyme activity has been directly carried out in the same sample where incubation of AChE with OP has been performed. With AChE immobilised on a nylon net and not assembled on the sensor it is possible to perform incubation and measurement steps separately: the incubation step is performed in the sample containing the inhibitor, the measurement step is performed in a standard solution containing the specific substrate in the buffer. With this two-step procedure, a Pt electrode, only covered with a thin layer of cross-linked choline oxidase and without any membrane, has also been used in batch conditions obtaining interesting results in terms of sensitivity, precision and limit of detection.

Enzyme electrodes with improved mechanical and analytical characteristics obtained by binding enzymes to nylon nets

Abstract Enzyme electrodes obtained by binding enzymes on nylon net show improved mechanical and analytical characteristics. The chemical binding process is mild so that the final membrane retains a high enzyme activity. The resulting membrane also has high mechanical strength and can be assembled on the electrode surface several times without damage. Response times without nylon net are excellent. Examples with single and dual enzymes coupled with an oxygen or ammonia electrode are discussed.

Prussian Blue and enzyme bulk-modified screen-printed electrodes for hydrogen peroxide and glucose determination with improved storage and operational stability

The addition to the carbon ink, a major component of a screen-printed electrode (SPE), of an aliquot (10%) of Prussian Blue (PB)-modified glassy carbon (PB-GC) particles, resulted in an interference free “ready to use” amperometric H 2O2 sensor (Vapp =− 50 mV) with a LOD of 3 × 10 −7 mol/l and a sensitivity of 135 mA (mol l −1 cm 2 ). A storage stability of up to 8 months and an operational stability of 3 days has been achieved making these sensors suitable for mass-production. Glucose and lysine biosensors have been assembled immobilizing glucose oxidase (Gox) and lysine oxidase (LyOx) with glutaraldehyde and Nafion ® onto the PB bulk-modified electrodes. A LOD of 4 × 10 −6 mol/l for glucose and 5 × 10 −6 mol/l for lysine with a linear range up to 0.5 and 0.7 mmol/l, respectively, have been observed. A ready to use glucose biosensor was then developed mixing PB-modified glassy carbon (7.5%), carbon ink (87.5%) and glucose oxidase (GOx, 5%) and using the mixture for the printing step of a SPE working electrode. A LOD of 3× 10 −5 mol/l and a linearity range up to 6 × 10 −3 mol/l of glucose have been achieved, together with a storage stability up to 20 weeks (at RT) and an operational stability of 1 day with 11 calibration curves performed.

Monoclonal antibody based electrochemical immunosensor for the determination of ochratoxin A in wheat

Competitive electrochemical enzyme-linked immunosorbent assays based on disposable screen-printed electrodes have been developed for quantitative determination of ochratoxin A (OTA). The assays were carried out using monoclonal antibodies in the direct and indirect format. OTA working range, I(50) and detection limits were 0.05-2.5 and 0.1-7.5mugL(-1), 0.35 (+/-0.04) mugL(-1) and 0.9 (+/-0.1) mugL(-1), 60 and 100mugL(-1) in the direct and indirect assay format, respectively. The immunosensor in the direct format was selected for the determination of OTA in wheat. Samples were extracted with aqueous acetonitrile and the extract analyzed directly by the assay without clean-up. The I(50) in real samples was 0.2mugL(-1) corresponding to 1.6mug/kg in the wheat sample with a detection limit of 0.4mug/kg (calculated as blank signal -3sigma). Within- and between-assay variability were less than 5 and 10%, respectively. A good correlation (r=0.9992) was found by comparative analysis of naturally contaminated wheat samples using this assay and an HPLC/immunoaffinity clean-up method based on the AOAC Official Method 2000.03 for the determination of OTA in barley.

Determination of organophosphorus insecticides with a choline electrochemical biosensor

Abstract Organophosphorus insecticides have been determined with an amperometric hydrogen peroxide based choline biosensor. This class of pesticides inhibits cholinesterase enzymes which in the presence of their substrates produce choline. The decrease in activity of these enzymes is monitored by the choline sensor and is correlated to the concentration of pesticide present in solution. Pesticides such as paraoxon, parathion, methyl paraoxon and methyl parathion have been coupled with acetylcholine and butyrylcholine esterases. Results showed that although the enzyme activity of acetylcholinesterase (AChE) was higher than butyrylcholinesterase (BuChE), the lower specificity of BuChE resulted in a higher enzyme inhibition. The detection limit was less than 1 ppb with an incubation time of 120 min and 2 ppb when the incubation time was 30 min. This method has been applied to the analysis of pesticides in water samples. Results, compared with those obtained with a different analytical procedure (liquid/liquid extraction and GLC determination), demonstrated that our method is a good analytical choice to measure the total anticholinesterase activity in water samples.

3,3', 5,5'-tetramethylbenzidine as electrochemical substrate for horseradish peroxidase based enzyme immunoassays. A comparative study

The use of 3,3′,5,5′-tetramethylbenzidine (TMB) as an electrochemical substrate for horseradish peroxidase (HRP) was investigated. HRP activity has been detected using flow injection analysis at a glassy carbon working electrode polarised at +100 mV versus Ag/AgCl in 0.1 mol l–1 citrate–phosphate buffer (pH 5.0). The optimum concentrations were 2 × 10–4 mol l–1 TMB and 10–3 mol l–1 H2O2. The detection limit obtained after 15 min of incubation was 8.5 × 10–14 mol l–1 HRP with the amperometric method. This limit was lower than that obtained using hydroquinone as HRP substrate and comparable to that with the p-aminophenyl phosphate–alkaline phosphatase system. Better performance was achieved with amperometric than spectrophotometric detection using TMB in a competitive ELISA for rabbit immunoglobulin G as a model analyte.

Amperometric ammonium ion and urea determination with enzyme-based probes

Amperometric enzyme probes for ammonium and urea have been assembled and evaluated using immobilized glutamate dehydrogenase and urease enzymes coupled with platinum electrodes. Analytical parameters such as pH, buffer, temperature, probe life-time, enzyme immobilization, cofactor concentration and response time have been optimized. Ammonium was detected in the range 10(-5)-3 x 10(-4) mol l-1. Better reproducibility and stability were achieved using the enzyme GLDH type III and NADH at a concentration of 10(-3) mol l-1. Urea has been determined in the range 10(-5)-3 x 10(-4) mol l(-1) using the enzyme urease first in solution and then immobilized on nylon net. The analysis was based on an amperometric measurement which gives a linear relationship between current and analyte concentration. This considerably improved the sensitivity of the analysis when compared with the potentiometric-based procedures. Moreover, this method does not suffer from the potassium ion interference which affects the potentiometric nonactin-based NH+4 electrodes. Analysis of ammonium and urea were carried out in standard solutions and in saliva samples. Results compared with a spectrophotometric reference procedure correlated well.

Determination of Organophosphorus and Carbamic Pesticides with a Choline and Acetylcholine Electrochemical Biosensor

Abstract Pesticides as paraoxon and aldicarb have been determined with an amperometric hydrogen peroxide based choline sensor. These pesticides inhibit the enzyme acetylcholinesterase which in presence of its substrate, acetylcholine, produces choline. When these pesticides are in presence of acetylcholinesterase, the activity of this enzyme decreases; this causes a decrease of choline production which is monitored by a choline sensor and correlated to the concentration of pesticide in solution. Two different procedures were followed: one with both the enzymes acetylcholinesterase and choline oxidase immobilized, the second one with the acetylcholinesterase in solution and the choline oxidase immobilized. Parameters as pH, buffer, enzyme concentration, substrate concentration and reaction and incubation times were optimized. Results showed that these compounds can be detected in the range 10 – 100 ppb. The use of the enzyme in solution gave the best results with a detection limit of 2 ppb pesticide.