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Pesticides are largely used at worldwide level to improve food production, fulfilling the needs of the global population which is increasing year by year. Although pesticides are beneficial for crop production, their extensive use has serious consequences for the pollution of the produced food as well as for soil and groundwaters. Indeed, it is reported that 50% of sprayed pesticides reach different destinations other than their target species, including soil, surface waters, and groundwaters. For this reason, we developed a flower-like origami paper-based device for pesticides detection in aerosol phase for precision agriculture. In detail, the paper-based electrochemical platform detects paraoxon, 2,4-dichlorophenoxyacetic acid, and glyphosate at ppb levels by measuring their inhibitory activity towards three different enzymes namely butyrylcholinesterase, alkaline phosphatase, and peroxidase enzyme, respectively. This integrated electrochemical device is composed of three office paper-based screen-printed electrodes and filter paper-based pads loaded with enzymes and enzymatic substrates. The pesticide detection is carried out by measuring through chronoamperometric technique the initial and residual enzymatic activity by using a smartphone-assisted potentiostat and evaluating the percentage of inhibition, proportional to the amount of aerosolized pesticides. This paper-based device was able to detect the three classes of pesticides in aerosol phase with limits of detection equal to 30 ppb, 10 ppb, and 2 ppb, respectively for 2,4-D, glyphosate, and paraoxon., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Coronavirus disease 2019 (COVID-19) has been recognized as a global pandemic outbreak, opening the most severe socio-economic crisis since World War II. Different scientific activities have been emerged in this global scenario, including the development of innovative analytical tools to measure nucleic acid, antibodies, and antigens in the nasopharyngeal swab, serum, and saliva for prompt identification of COVID-19 patients and to evaluate the immune response to the vaccine. The detection of SARS-CoV-2 in saliva remains a challenge for the lack of sufficient sensitivity. To address this issue, we developed a novel paper-based immunoassay using magnetic beads to support the immunological chain and 96-well wax-printed paper plate as a platform for color visualization by using a smartphone combined with Spotxel free-charge app. To assess the reliability of the measurement of SARS-CoV-2 in saliva, untreated saliva was used as a specimen and the calibration curve demonstrated a dynamic range up to 10 μg/mL, with a detection limit equal to 0.1 μg/mL. The effectiveness of this sustainable analytical tool in saliva was evaluated by comparing the data with the nasopharyngeal swab specimens sampled by the same patients and tested with Real-Time PCR reference method, founding 100% of agreement, even in the case of high Cycle Threshold (CT) numbers (low viral load). Furthermore, the positive saliva samples were characterized by the next-generation sequencing method, demonstrating the capability to detect the Delta variant, which is actually (July 2021) the most relevant variant of concern., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Botulinum neurotoxins (BoNTs) produced by soil bacterium Clostridium botulinum are cause of botulism and listed as biohazard agents, thus rapid screening assays are needed for taking the correct countermeasures in a timely fashion. The gold standard method relies on the mouse lethality assay with a lengthy analysis time, i.e., 2-5 days, hindering the prompt management of food safety and medical diagnosis. Herein, we propose the first paper-based antibody-free sensor for reliable and rapid detection of BoNT/A and BoNT/C, exploiting their cleavage capability toward a synthetic peptide able to mimic the natural substrate SNAP-25. The peptide is labelled with the electroactive molecule methylene blue and immobilized on the paper-based electrode modified with gold nanoparticles. Because BoNT/A and BoNT/C can cleave the peptide with the removal of methylene blue from electrode surface, the presence of these neurotoxins in the sample leads to a signal decrease proportional to BoNT amount. The biosensor developed with the selected peptide and combined with smartphone assisted potentiostat is able to detect both BoNT/A and BoNT/C with a linearity up to 1 nM and a detection limit equal to 10 pM. The applicability of this biosensor was evaluated with spiked samples of orange juice, obtaining recovery values equal to 104 ± 6% and 98 ± 9% for 1 nM and 0.5 nM of BoNT/A, respectively., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The diffusion of novel SARS-CoV-2 coronavirus over the world generated COVID-19 pandemic event as reported by World Health Organization on March 2020. The huge issue is the high infectivity and the absence of vaccine and customised drugs allowing for hard management of this outbreak, thus a rapid and on site analysis is a need to contain the spread of COVID-19. Herein, we developed an electrochemical immunoassay for rapid and smart detection of SARS-CoV-2 coronavirus in saliva. The electrochemical assay was conceived for Spike (S) protein or Nucleocapsid (N) protein detection using magnetic beads as support of immunological chain and secondary antibody with alkaline phosphatase as immunological label. The enzymatic by-product 1-naphtol was detected using screen-printed electrodes modified with carbon black nanomaterial. The analytical features of the electrochemical immunoassay were evaluated using the standard solution of S and N protein in buffer solution and untreated saliva with a detection limit equal to 19 ng/mL and 8 ng/mL in untreated saliva, respectively for S and N protein. Its effectiveness was assessed using cultured virus in biosafety level 3 and in saliva clinical samples comparing the data using the nasopharyngeal swab specimens tested with Real-Time PCR. The agreement of the data, the low detection limit achieved, the rapid analysis (30 min), the miniaturization, and portability of the instrument combined with the easiness to use and no-invasive sampling, confer to this analytical tool high potentiality for market entry as the first highly sensitive electrochemical immunoassay for SARS-CoV-2 detection in untreated saliva., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The development of portable and user-friendly sensing platforms is a hot topic in the field of analytical chemistry. Among others, electroanalytical approaches exhibit a high amenability for reaching this purpose, i.e. the commercial strips for diabetes care are an obvious success. However, providing fully-integrated and reagent-free methods is always a leitmotiv. In this work, we evaluated the use of a disposable pipette tip, opportunely configured to demonstrate the first example of an electrochemical biosystem in a pipette tip, namely bio-lab-on-a-tip. The combination of a pipette tip, wire electrodes, enzyme, and cotton wool filter, allows the fabrication of a novel electroanalytical platform that does not need expertise-required tasks. To demonstrate the feasibility of this novel method, glucose is detected in beverages by means of chronoamperometry. The experimental setup, entirely built inside the pipette tip, is able to 1) block impurities/interferences from matrix, 2) load/release reagents for the bio-assay, 3) reduce the operating task to zero, and 4) perform electrochemical detection. With optimized experimental parameters, the bio-lab-on-a-tip is able to detect glucose linearly up to 10 mM with a detection limit of 170 μM. The effectiveness of the platform was confirmed by testing commercial beverages, e.g. Coca-Cola and Coca-Cola Zero, with high accuracy. In addition, the shelf-life of the novel device was evaluated, highlighting the role of cotton wool filter for providing a suitable environment for glucose oxidase stability. The novel concept can be easily generalized for further applications in the field of non-invasive clinical diagnostics and in-situ environmental monitoring., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the overall scenario of precision medicine, we propose a novel paper-based lab-on-a-chip to deliver a cost-effective and easy to use sensing tool for customized administration of drugs in Alzheimer's disease. Among several drugs, we designed the device for evaluating the efficacy of compounds (e.g. physostigmine, rivastigmine, donepezil) which are able to inhibit in a reversible way the cholinesterase enzyme. Because cholinesterase activity is peculiar to each patient, the administration of customized amount of the drug can improve the treatment efficacy and the quality of patient life, avoiding side effects due to the overdosage. In detail, we exploited Vivid™ Plasma Separation membrane to threat the whole blood sample, filter paper to load the reagents needed for the measurement, and office paper to print electrodes able to measure the butyrylcholinesterase activity, delivering a reagent free analytical tool. The calibration curve of butyrylcholinesterase obtained in blood sample provided linearity between 2 and 12 U/mL, with sensitivity of 0.050 ± 0.004 μA mL/U. The physostigmine, rivastigmine, and donepezil inhibition activities toward the butyrylcholinesterase enzyme were also measured in blood sample with linearity up to respectively 0.5 μM, 25 μM, 30 μM, and detection limits of 0.009 μM, 0.4 μM, 0.3 μM. These results demonstrate the capability of paper-based origami sensors as point of care devices to customize the drug administration in Alzheimer's disease., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pancreatic ductal adenocarcinoma is the predominant neoplastic disease of the pancreas and it represents the fourth most frequent cause of death in cancer-related disease, with only 8% of survivors after 5-year to the diagnosis. The main issues of this type of cancer rely on fast progress (i.e. 14 months from T1 to a T4 stage), nonspecific symptoms with delay in diagnosis, and the absence of effective screening strategies. To address the lack of early diagnosis, we report a cost-effective paper-based biosensor for the detection of miRNA-492, which is recognised as a biomarker for pancreatic ductal adenocarcinoma. To design a miniaturised, sensitive, and robust paper-based platform, an electrochemical sensor was screen-printed on office paper previously wax-patterned via wax-printing technique. The paper-based sensor was then engineered with a novel and highly specific peptide nucleic acid (PNA) as the recognition element. The formation of PNA/miRNA-492 adduct was evaluated by monitoring the interaction between the positively charged ruthenium (III) hexamine with uncharged PNA and/or negatively charged PNA/miRNA-492 duplex by differential pulse voltammetry. The paper-based biosensor provided a linear range up to 100 nM, with a LOD of 6 nM. Excellent selectivity towards one- and two-base mismatches (1MM, 2MM) or scrambled (SCR) sequences was highlighted and the applicability for biomedical analyses was demonstrated, measuring miRNA-492 in undiluted serum samples., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A novel amperometric algae-based biosensor was developed for the detection of photosynthetic herbicides in river water. The green photosynthetic algae Chlamydomonas reinhardtii was immobilized on carbon black modified screen-printed electrodes, exploiting carbon black as smart nanomaterial to monitor changes in algae oxygen evolution during the photosynthetic process. The decrease of oxygen evolution, occurring in the presence of herbicides, results in a decrease of current signals by means of amperometric measurements, in an analyte concentration dependent manner. Atrazine as case study herbicide was detected in a concentration range of 0.1 and 50 μM, with a linear range from 0.1 to 5 μM and a detection limit of 1 nM. No interference was observed in presence of 100 ppb arsenic, 20 ppb copper, 5 ppb cadmium, 10 ppb lead, 10 ppb bisphenol A, and 1 ppb paraoxon, tested as safety limits. A ~25% matrix effect and satisfactory recovery values of 107 ± 10% and 96 ± 8% were obtained in river water for 3 and 5 μM of atrazine, respectively. Stability studies were also performed obtaining a high working stability up to 10 h and repeatability with an RSD of 1.1% (n = 12), as well as a good storage stability up to 3 weeks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Advances in cutting-edge technologies including nanotechnology, microfluidics, electronic engineering, and material science have boosted a new era in the design of robust and sensitive biosensors. In recent years, carbon black has been re-discovered in the design of electrochemical (bio)sensors thanks to its interesting electroanalytical properties, absence of treatment requirement, cost-effectiveness (c.a. 1 €/Kg), and easiness in the preparation of stable dispersions. Herein, we present an overview of the literature on carbon black-based electrochemical (bio)sensors, highlighting current trends and possible challenges to this rapidly developing area, with a special focus on the fabrication of carbon black-based electrodes in the realisation of sensors and biosensors (e.g. enzymatic, immunosensors, and DNA-based)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Biosensor development exploiting various transduction principles is characterized by a strong competition to reach high detectability, portability and robustness. Nevertheless, a literature-based comparison is not possible, as different conditions are employed in each paper. Herein, we aim at evaluating which measurement, photons or electrons, yields better biosensor performance. Upon outlining an update in recent achievements to boost analytical performance, amperometry and chemiluminescence (CL)-based biosensors are directly compared employing the same biospecific reagents and analytical formats. Horseradish peroxidase (HRP) and hydrogen peroxide concentrations were directly measured, while glucose and mouse IgG were detected employing an enzyme paper-based biosensor and an immunosensor, respectively. Detectability was down to picomoles of hydrogen peroxide (4 pmol for CL and 210 pmol for amperometry) and zeptomoles of HRP (45 zmol for CL and 20 zmol for amperometry); IgG was detected down to 12 fM (CL) and 120 fM (amperometry), while glucose down to 17 μM (CL) and 40 μM (amperometry). Results showed that amperometric and CL biosensors offered similar detectability and analytical performance, with some peculiarities that suggest complementary application fields. As they generally provided slightly higher detectability and wider dynamic ranges, CL-based biosensors appear more suitable for point-of-care testing of clinical biomarkers, where detectability is crucial. Nevertheless, as high detectability in CL biosensors usually requires longer acquisition times, their rapidity will allocate electrochemical biosensors in real-time monitoring and wearable biosensors. The analytical challenge demonstrated that these biosensors have competitive and similar performance, and between photons and electrons the competition is still open., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the last decades, there is a growing search for analytical strategies to ensure clinical analysis without the need of laboratory set-up and skilled personnel. Indeed, user-friendly and low-cost devices are highly valued in the era of sustainability for their capability to be applied in low-resource contexts, such as developing countries. To address this issue, herein we report a 96-well paper-based and laboratory setup-free optical platform for the detection of butyrylcholinesterase enzyme (BChE) activity in human serum. We used chromatographic paper to realize a novel analytical tool exploiting its porous structure for reagentless synthesize Prussian Blue Nanoparticles (the sensing element), as well to load all the reagents required for the measurement. The principle of BChE activity detection relies on the reaction between the enzymatic product thiocholine and Prussian Blue, giving the Prussian White with subsequently Prussian Blue's fading, detected by a common office scanner supported by ImageJ software. Using this novel paper-based optical platform, BChE activity was linearly detected in the 2-15 U/mL range with a detection limit down to 0.8 U/mL. The accuracy was successfully demonstrated by recovery study with spiked serum and by comparing the data with the gold standard method., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The synthesis and employment of volatile toxic compounds as chemical weapons with a large-scale destructive power has introduced a new insidious threat over the last century. In this framework, the development of wearable sensing tools represents a critical point within the security field, in order to provide early alarm systems. Herein, a novel wearable electrochemical biosensor was developed for the rapid and on-site detection of mustard agents. Since a chemical attack is typically carried out by spraying these volatile agents into air, the sensor was designed in order to be able to measure mustard agents directly in the aerosol phase, further than in the liquid phase. The electrodes were screen-printed onto a filter paper support, which allowed to harness the porosity of paper to pre-load all the needed reagents into the cellulose network, and hence to realise an origami-like and reagent-free device. Mustard agent detection was carried out by monitoring their inhibitory effects toward the choline oxidase enzyme, through the amperometric measurement of the enzymatic by-product hydrogen peroxide. A carbon black/Prussian blue nanocomposite was used as a bulk-modifier of the conductive graphite ink constituting the working electrode, allowing for the electrocatalysis of the hydrogen peroxide reduction. After having verified the detecting capability toward a mustard agent simulant, the applicability of the resulting origami-like biosensor was demonstrated for the rapid and real-time detection of real sulfur mustard, obtaining limits of detection equal to 1 mM and 0.019 g·min/m 3 for liquid and aerosol phase, respectively., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Herein, we report an impedimetric DNA-based aptamer sensor for a single-step detection of B. anthracis spore simulant (B. cereus spore). Specifically, we designed a miniaturized label-free aptasensor for B. cereus spores based on a gold screen-printed electrode functionalized with B. cereus spores-binding aptamer (BAS-6R). Several parameters were optimized to fabricate the aptasensor such as the concentration of DNA aptamer solution (0.5 µM), the time (48 h), the temperature (4 °C), and the pH (7.5) for aptamer immobilization on the working electrode surface. Once the aptasensor was developed, it was tested against B. cereus spores 14579 evaluating the effect of incubation time and MgCl 2 concentration. Under the optimized conditions (incubation time equal to 3 h and absence of MgCl 2 ), B. cereus spores 14579 were detected with a linear range between 10 4 CFU/ml and 5 × 10 6 CFU/ml and a detection limit of 3 × 10 3 CFU/ml. Furthermore, the study of selectivity toward B. cereus 11778, B. subtilis, Legionella pneumophila, and Salmonella Typhimurium has demonstrated the capability of this sensor to detect B. cereus spores, proving the suitability of the DNA-based sensing element combined with a portable instrument for a label-free measurement on site of B. anthracis spore simulant., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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 ± 1% and 88 ± 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Paper-based microfluidic devices are gaining large popularity because of their uncontested advantages of simplicity, cost-effectiveness, limited necessity of laboratory infrastructure and skilled personnel. Moreover, these devices require only small volumes of reagents and samples, provide rapid analysis, and are portable and disposable. Their combination with electrochemical detection offers additional benefits of high sensitivity, selectivity, simplicity of instrumentation, portability, and low cost of the total system. Herein, we present the first example of an integrated paper-based screen-printed electrochemical biosensor device able to quantify nerve agents. The principle of this approach is based on dual electrochemical measurements, in parallel, of butyrylcholinesterase (BChE) enzyme activity towards butyrylthiocholine with and without exposure to contaminated samples. The sensitivity of this device is largely improved using a carbon black/Prussian Blue nanocomposite as a working electrode modifier. The proposed device allows an entirely reagent-free analysis. A strip of a nitrocellulose membrane, that contains the substrate, is integrated with a paper-based test area that holds a screen-printed electrode and BChE. Paraoxon, chosen as nerve agent simulant, is linearly detected down to 3µg/L. The use of extremely affordable manufacturing techniques provides a rapid, sensitive, reproducible, and inexpensive tool for in situ assessment of nerve agent contamination. This represents a powerful approach for use by non-specialists, that can be easily broadened to other (bio)systems., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A reliable and cost-effective electrochemical method for the detection of deoxynivalenol (DON) in cereals and cereal-based food samples based on the use of a novel anti-DON Fab fragment is presented. The analytical system employed, Enzyme-Linked-Immunomagnetic-Electrochemical (ELIME) assay, is based on the use of immunomagnetic beads (IMBs) coupled with eight magnetized screen-printed electrodes (8-mScPEs) as electrochemical transducers. Using standard solutions of DON, a working range between 100 and 4500 ng/ml was obtained with an EC(50) of 380 ng/ml. The ELIME assay was employed to evaluate the cross-reactivity of the Fab fragment towards different trichothecenes revealing a good selectivity towards DON over other trichothecenes with the exception of 3-Ac-DON. The sensor was then applied to cereals and cereal-based food samples (wheat, breakfast cereal and baby-food) and a wide range of sample treatment procedures was tested. Within-laboratory precision (9-24% repeatability for breakfast cereals and 10-33% for baby-food) and recovery data (82-110% for breakfast cereals and 97-108% for baby-food) were calculated by analyzing blank breakfast cereals and baby-foods fortified with DON, demonstrating that the proposed method has the capability for use as a screening assay for DON in such products., (Copyright 2010 Elsevier B.V. All rights reserved.)

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., (2010 Elsevier B.V. All rights reserved.)

A novel biosensor assay format for aflatoxin based on acetylcholinesterase (AChE) inhibition by aflatoxin B(1) (AFB(1)) is proposed. The AChE was present in solution and an amperometric choline oxidase biosensor was used for monitoring its residual activity. To create the biosensor, the choline oxidase was immobilized by cross-linking onto screen-printed electrodes modified with Prussian Blue (PB) and these were used to detect the H(2)O(2) at low potential (-0.05V versus a screen-printed internal silver pseudoreference electrode). For the development of the AFB(1) assay, several parameters such as AChE and substrate concentration, the methanol effect, and pH were evaluated and optimized. The linear working range was assessed to be 10-60ppb. Concentrations as low as 2ppb, which correspond to the legal limit of AFB(1) in food for humans, were detected after a pre-concentration step. The suitability of the method was evaluated using commercial olive oil samples. A recovery equal to 78+/-9% for 10ppb of AFB(1) in olive oil samples was obtained.

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.

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.

Glucose biosensors based on the use of planar screen-printed electrodes modified with an electrochemical mediator and with glucose oxidase have been optimised for their application in the continuous glucose monitoring in diabetic patients. A full study of their operative stability and temperature dependence has been accomplished, thus giving useful information for in vivo applications. The effect of dissolved oxygen concentration in the working solution was also studied in order to evaluate its effect on the linearity of the sensors. Glucose monitoring performed with serum samples was performed to evaluate the effect of matrix components on operative stability and demonstrated an efficient behaviour for 72 h of continuous monitoring. Finally, these studies led to a sensor capable of detecting glucose at concentrations as low as 0.04 mM and with a good linearity up to 2.0 mM (at 37 degrees C) with an operative stability of ca. 72 h, thus demonstrating the possible application of these sensors for continuous glucose monitoring in conjunction with a microdialysis probe. Moreover, preliminary in vivo experiments for ca. 20 h have demonstrated the feasibility of this system.

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.

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.

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.

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.

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.

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.