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The aim of the present research was to develop a highly selective and sensitive amperometric immunosensor for atrazine based on a “competition” assay procedure. An immunosensor device for the determination of triazinic pesticides based on two different competition procedures is therefore described. The immunosensor developed uses an amperometric el...
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... immunosensor was assembled ( Fig. 1) using an Immobilon membrane in which the antibody or the antigen was immobilized and which overlapped a cellulose acetate membrane (0.1 mm thick) placed on the lower end of the plastic cap of an amperometric elec- trode for H 2 O 2 . A nylon net and an O-ring were used to fix the Immobilon membrane to the head of the cap itself. The transducer used consisted of a classical amperometric electrode for hydrogen peroxide, provided with a plastic cap filled with a 0.1 M KCl solu- tion screwed onto the body of the electrode. A constant potential of +650 mV with respect to an Ag/AgCl/Cl − cathode was applied to the Pt anode. Horseradish peroxidase enzyme was used as a marker for immunocomplex ...
Citations
... The electrode was then washed with PB buffer 10 mM pH 7.4 to remove the nonbound atrazine. 52 DPV Measurements and Electrochemical Apparatus. The measurements were performed in a three-electrode electrochemical cell with a solution of FeCN 6 3−/4− 1.1 mM, 100 mM KCl in deionized water (R = 18.2 MΩ cm) in a potential range between [−0.4; +0.6] V. ...
In recent years, several efforts have been made to develop selective, sensitive, fast response, and miniaturized immunosensors with improved performance for the monitoring and screening of analytes in several matrices, significantly expanding the use of this technology in a broad range of applications. However, one of the main technical challenges in developing immunosensors is overcoming the complexity of binding antibodies (Abs) to the sensor surface. Most immobilizing approaches lead to a random orientation of Abs, resulting in lower binding site density and immunoaffinity. In this context, supramolecular chemistry has emerged as a suitable surface modification tool to achieve the preorganization of artificial receptors and to improve the functional properties of self-assembled monolayers. Herein, a supramolecular chemistry/nanotechnology-based platform was conceived to develop sensitive label-free electrochemical immunosensors, by using a resorcarene macrocycle as an artificial linker for the oriented antibody immobilization. To this aim, a water-soluble bifunctional resorc[4]arene architecture (RW) was rationally designed and synthesized to anchor gold-coated magnetic nanoparticles (Au@MNPs) and to maximize the amount of the active immobilized antibody (Ab) in the proper "end-on" orientation. The resulting supramolecular chemistry-modified nanoparticles, RW@Au@MNPs, were deposited onto graphite screen printed electrodes which were then employed to immobilize three different Abs. Furthermore, an immunosensor for atrazine (ATZ) analysis was realized and characterized by the differential pulse voltammetry technique to demonstrate the validity of the developed biosensing platform as a proof of concept for electrochemical immunosensors. The RW-based immunosensor improved AbATZ loading on Au@MNPs and sensitivity toward ATZ by almost 1.5 times compared to the random platform. Particularly, the electrochemical characterization of the developed immunosensor displays a linearity range toward ATZ within 0.05-1.5 ng/mL, a limit of detection of 0.011 ng/ml, and good reproducibility and stability. The immunosensor was tested by analyzing spiked fortified water samples with a mean recovery ranging from 95.7 to 108.4%. The overall good analytical performances of this immunodevice suggest its application for the screening and monitoring of ATZ in real matrices. Therefore, the results highlighted the successful application of the resorc[4]arene-based sensor design strategy for developing sensitive electrochemical immunosensors with improved analytical performance and simplifying the Ab immobilization procedure.
... Pesticides are considered not only a potential milk contaminant but also one of the major environmental pollutants due to their inevitable spread in water, soil, atmosphere, and hence passage into agricultural products (Ambrus and Yang 2016). Pesticide residues undergo degradation in soil but due to their high stability and low water solubility, they persist in the ecosystem and to exhibit long shelf life, i.e. organophosphates and organochlorines can be detected 15-20 years after their use (Campanella et al. 2011). Figure 4 displays the various pesticide classes and the most common pesticide of each class with their corresponding structures. ...
... The second method employing the SPEs is commercially available, displaying a wide dynamic range of 5.0 × 10 −8 -2.0 × 10 −5 M with a satisfactory sensitivity (LOD of 2.3 × 10 − 8 M) and 25 min analysis time. Even though the SPE biosensor was more sensitive and had a lower analysis time, both biosensors displayed major cross-reactivity with other triazines and minor with carbaryls (Campanella et al. 2011;Tomassetti et al. 2015). ...
Accidentally present contaminants or intentionally added adulterants in milk lead potentially to delivering not only unhealthy but seriously hazardous products. Thorough, fast and sensitive analytical tools are essential for monitoring of milk quality, and for screening of any objectionable contaminants. Biosensors represent an innovative, time-efficient and on-site solution to assess milk quality in addition to their specificity towards target analytes alongside high accuracy within such complex matrices. Most biosensors use antibodies, aptamers or enzymes as the bio-receptor and rely on optical, electrochemical or thermometric transduction to generate a signal. The simplest biosensors appear to be those based on a colorimetric assay, being simple and having a signal that can be detected visually. Electrochemical sensors are more specific and sensitive, though with more complicated designs, whereas thermometric sensors have not been thoroughly explored concerning biosensing contaminants in milk. This review discusses recent advances in the field of biosensors and analyzes the various methods of bio-recognition and transduction with regard to their advantages, limitations, and application to milk products. Additionally, challenges facing further development of these strategies to fulfil the increasing demand for fast and on-line milk quality control are also presented.
... Monitoring of agricultural pesticides has been studied in the field of development of amperometric biosensors, since these are devices that have high sensitivity and specificity, with the advantages of miniaturization (Mihos et al., 2014), easy transportation and reduced analysis time, being able to determine concentrations in scale of mgL -1 a μgL -1 (Zamora-Sequeira et al., 2019; Gil and De Melo, 2010). Enzymatic biosensors are assembled by immobilizing functionalized proteins that act specifically for a particular substrate (Yulaev et al., 2001;Campanella et al., 2011). Generally purified commercial enzymes are used in the assembly, which makes the device more expensive (Zamora-Sequeira et al., 2019). ...
... To overcome these issues, researchers have focused on the development of various kinds of biosensors [16,17], employing biological elements such as antibodies [18][19][20], aptamers [21,22], and enzymes [23][24][25]. However, these kinds of bioreceptors usually require complex purification and immobilization techniques that significantly increase the overall cost of the device [26]. ...
A novel mediatorless photo-bioelectrochemical sensor operated with a biofilm of the cyanobacterium Synechocystis PCC6803 wt. for herbicide detection with long term stability (>20 days) was successfully developed and tested. Photoanodic current generation was obtained in the absence of artificial mediators. The inhibitory effect on photocurrent of three commonly used herbicides (i.e., atrazine, diuron, and paraquat) was used as a means of measuring their concentrations in aqueous solution. The injection of atrazine and diuron into the algal medium caused an immediate photocurrent drop due to the inhibition of photosynthetic electron transport. The detected concentrations were suitable for environmental analysis, as revealed by a comparison with the freshwater quality benchmarks set by the Environmental Protection Agency of the United States (US EPA). In contrast, paraquat caused an initial increase (~2 h) of the photocurrent effect of about 200%, as this compound can act as a redox mediator between the cells and the anode. A relatively long-term stability of the biosensor was demonstrated, by keeping anodes colonized with cyanobacterial biofilm in the dark at 4 °C. After 22 days of storage, the performance in terms of the photocurrent was comparable with the freshly prepared biosensor. This result was confirmed by the measurement of chlorophyll content, which demonstrated preservation of the cyanobacterial biofilm. The capacity of this biosensor to recover after a cold season or other prolonged environmental stresses could be a key advantage in field applications, such as in water bodies and agriculture. This study is a step forward in the biotechnological development and implementation of storable mediatorless electrochemical biosensors for herbicide detection.
... Nevertheless, it also can be found in drinking water and in in various food products, which leads to environmental and human health problems. Recently, the ATZ detections by electroanalytical methods via various electrodes have been reported [21,22]. In the present work, we explore a new electrochemical platform for detection of atrazine in aqueous solution. ...
In this work, one dimensional self-ordered Nb2O5 nanotube arrays were grown from Nb substrate by anodization method and were modified with optimum amounts of chitosan (CS) and carboxyl-Fe3O4 nanoparticles. These nanocomposites were characterized by SEM, XRD, and XPS and used as electrode materials for the direct detection of atrazine traces in aqueous solution. The test signals were obtained from the interaction between anti-atrazine monoclonal antibody and atrazine and recorded using via differential pulse voltammetric (DPV) method in electrochemical system. These modified one-dimensional Nb2O5 nanotube arrays showed the linear detection ranges of 0.04 ng/ml–0.6 ng/ml and a detection limit of 0.018 ng/ml for atrazine under the optimized conditions. These above results indicate that these Nb2O5 nanotube arrays are promising candidates for electrochemical sensors.
... The latest methods involving gold nanoparticles, silver nanoparticles and graphene nanostructures claim ppb level detection limits for the pesticides [8,9,[27][28][29][30]. There are numerous biosensors, immunosensor methods and optical sensors available to detect the pesticides with enzyme activity [31][32][33][34][35][36][37][38]. ...
A pesticide residue in fruits & vegetables is one of the key issues affecting the export of rural products in
India. Pesticides exposure or intake of it causes majorly nervous system problems. The solutions to quantitate
them in field are rare and the pesticide residue detection in the parts per billion (ppb) ranges is challenging.
Except ELISA, none of the existing methods can detect pesticide residues as per European Union
regulations. We employed a new approach of concentrating field samples and used sodium polyacrylate
(SPA) as water absorbing material. The SPA beads concentrate the field samples and obtained sub ppb
range detection using an existing FPIA system and could improve overall sensitivity by 10-100 fold. Fluorescence
polarization Immunoassay (FPIA) using SPA beads can detect analysts in the ppb range, and
after concentration of sample the complete assay can be done in few seconds and can adopt with any existing
set of antigen-antibody. We have used three most commonly used pesticides 2,4-D, atrazine and
methyl parathion could attain 0.1, 0.5 and 3 ppb detection limit for 2,4-D, atrazine and methyl parathion
using this system. We also demonstrate a simple field ready device using sodium poly acrylate (SPA) and
immobilized reagents useful in a biochemical assay to enhance sensitivity to immobilize biochemicals is
reported for the first time.
... Moreover, labor-intensive manipulations are required to mix the solutions for the immunoreaction, separate the free antibody from the antigen-antibody complex (bound/free (B/F) separation) for selective assays, with low background signals. In enzyme-linked immunosorbent assay (ELISA)45, further sensitive detection can be achieved by using an enzyme-labeled secondary antibody and its substrate, but it needs a longer analysis time and cumbersome procedures. To overcome these drawbacks, immunoassay methods based on microfluidic devices have been developed recently6789101112131415. ...
In this article, we report a simple and rapid immunoassay based on microscale electrophoresis using a microchip equipped with a reagent-release cartridge at the sample reservoir. The cartridge consists of an outer tube, a paper filter retaining dried reagents and an antibody, and inner tubes to fix the paper filter. When a sample solution containing an antigen is introduced into the sample reservoir through the reagent-release cartridge, the reagents and the antibody retained on the filter dissolve immediately into the sample solution. The sample solution and reagents containing the antibody are then mixed, promoting rapid immunoreaction between the antigen and the antibody. After sample introduction into the reservoir, the antigen-antibody complex is rapidly separated from the free antibody by microchip zone electrophoresis. The immunoassays for bovine serum albumin and human IgG were easily performed merely by pouring the sample solution into the cartridge in the newly developed device followed by microchip electrophoresis, resulting in the rapid and clear separation of the immunocomplexes from the free antibodies. These results showed that the developed device was applicable to a rapid and simple immunoassay.
... The latest methods involving gold nanoparticles, silver nanoparticles and graphene nanostructures claim ppb level detection limits for the pesticides [8,9,[27][28][29][30]. There are numerous biosensors, immunosensor methods and optical sensors available to detect the pesticides with enzyme activity [31][32][33][34][35][36][37][38]. ...
A pesticide residue in fruits and vegetables is one of the key issues affecting the export of rural
products in India. Pesticides exposure or intake of it causes majorly nervous system problems,
which affects the health and may also lead to death. The solutions to quantitate them in field are
rare and the pesticide residue detection in the parts per billion (ppb) ranges is challenging.
Except ELISA, none of the existing methodscan detect pesticide residues as per EU regulations.
ELISA is one of the gold standards for quantitation of pesticides in the ppb range and none of the
optical techniques have detection range close to ELISA. We employed a new approach of
concentrating field samples and used SPA as water absorbing material. The SPA beads
concentrate the field samples and obtained sub ppb range detection using an existing FPIA system
and could improve overall sensitivity by 10–100 fold. We could reach pictogram level detection
limit, which is currently achieved by very advanced ELISA systems. Fluorescence polarization
Immuno Assay (FPIA) using SPA beads can detect analysts in the ppb range, and after
concentration of sample the complete assay can be done in few seconds and can adopt with any
existing set of antigen-antibody. We have used antibodies for the three most commonly used
pesticides 2, 4-D, atrazine and methyl parathion. The in-house FPIA device can perform assay for
any new pesticides introduced in market and not restricted to the current 3 pesticides in study.
FPIA is a competitive immunoassay method based on the increase in the polarization of the
fluorescence of a small fluorescent-labeled hapten (tracer/pesticide residue) when it is bound by
specific antibody. We could attain 0.1, 0.5 and 3 ppb detection limit for 2, 4-D, atrazine and
methyl parathion using this system. We also demonstrate a simple field ready device using sodium
poly acrylate (SPA) and immobilized reagents. Though sodium poly acrylate (SPA) is a commonly
used chemical for water absorption its use in a biochemical assay to enhance sensitivity to
immobilize biochemicals is reported for the first time.
Keywords: Sodium Polyacrylate, FPIA, 2, 4-D, Atrazine and Methyl Parathion.
... It is a known fact that immunosensors are the most selective biosensors, and our team, as well as other authors (Garcés-García, Morais, González-Martínez, Puchades, & Maquieira, 2004), has recently fabricated several immunosensors for pesticide determination (Tomassetti, Martini, & Campanella, 2012;Raman Suri, Boro, Nangia, & Gandhi, 2009;Rekha, Thakur, & Karanth, 2000). However this kind of immunosensor was able to operate only in aqueous solution and to test pesticides in aqueous matrices (Raman Suri et al., 2009;Tomassetti et al., 2012;Campanella, Eremin, Lelo, Martini, & Tomassetti, 2011). Therefore, when the problem arose of having to determine traces of pesticides in oily matrices, it was necessary to replace OPEEs (Organic Phase Enzyme Electrodes) with OPIEs (Organic Phase Immuno Electrodes). ...
Agent orange herbicides, organophosphate and triazinic pesticides
analysis in olive oil and industrial oil mill waste effluents using new
organic phase immunosensors
Elisabetta Martini, Giovanni Merola, Mauro Tomassetti, Luigi Campanella
... Our research group has worked on inhibition biosensors, in particular tyrosinase biosensors, designed to analyse triazine and benzotriazine compounds [19,20]. In recent years, our team has investigated the development of conventional immunosensors [21][22][23][24], or screen-printed methods [25,26] above all because they were found to be more selective for the different classes of pesticide. In this framework the aim of the present research was to compare the analytical features of a new SPR device with two conventional or screen-printed amperometric immunosensor devices. ...
... Test geometry: competition between atrazine and albumin-atrazine conjugated with biotin-avidin-peroxidase, both free in solution, for anti-atrazine immobilized in the membrane were the same as described in a previous papers [21,33]. Conventional and screen-printed immunosensors both used an amperometric electrode for hydrogen peroxide as transducer. ...
A detailed comparison was made of the analytical features of a new Surface Plasmon Resonance (SPR) immunodevice for triazine pesticide determination with those of two other amperometric (conventional and screen-printed) immunosensors and the advantages and disadvantages of the SPR method were thoroughly investigated. For conventional amperometric and screen-printed devices, “competitive” assays were used; conversely, the SPR transduction technique allowed a “direct” measurement format to be used. As far as the main analytical data are concerned, the SPR method does not seem to offer substantial advantages. Nevertheless the measurement time is much shorter and the measurement itself much easier to perform. Lastly several applications and recovery tests were carried out on bovine milk samples, before and after spiking, to check for triazine pesticides in the samples, obtaining satisfactory results.
