Immunomagnetic bead-based bioassay for the voltammetric analysis of the breast cancer biomarker HER2-ECD and tumour cells using quantum dots as detection labels.

An electrochemical magnetic immunosensing strategy was developed for the determination of HER2-ECD, a breast cancer biomarker, and breast cancer cells in human serum. A sandwich assay was performed on carboxylic acid-functionalized magnetic beads (MBs) using a screen-printed carbon electrode (SPCE) as transducer surface. The affinity process was detected using electroactive labels; core/shell streptavidin-modified CdSe@ZnS Quantum Dots (QDs). Cd2+ ions, released from the QDs, were determined by differential pulse anodic stripping voltammetry (DPASV). An assay time of 90 min, with an actual hands-on time of about 20 min, a linear range between 0.50-50 ng·mL-1 of HER2-ECD and a limit of detection of 0.29 ng·mL-1 were achieved. Analysis of live breast cancer cells was also performed using the optimized assay. Breast cancer cell lines SK-BR-3 (a HER2-positive cell line), MDA-MB-231 (a HER2-negative cell line) and MCF-7 (a cell line with low HER2 expression) were tested. The selectivity of the assay towards SK-BR-3 cells was confirmed. A concentration-dependent signal that was 12.5× higher than the signal obtained for the HER2-negative cells (MDA-MB-231) and a limit of detection of 2 cells·mL-1 was obtained. Graphical abstractSchematic representation of the electrochemical immunomagnetic assay for the determination of the breast cancer biomarker HER2-ECD and cancer cells using magnetic beads (MBs), a screen-printed carbon electrode (SPCE) as transducer surface and quantum dots (QD) as electroactive labels.

Detection of the peanut allergen Ara h 6 in foodstuffs using a voltammetric biosensing approach.

A voltammetric biosensor for Ara h 6 (a peanut allergen) detection in food samples was developed. Gold nanoparticle-modified screen-printed carbon electrodes were used to develop a sandwich-type immunoassay using two-monoclonal antibodies. The antibody-antigen interaction was detected through the electrochemical detection of enzymatically deposited silver. The immunosensor presented a linear range between 1 and 100 ng/ml, as well as high precision (inter-day RSD ≤9.8%) and accuracy (recoveries ≥96.7%). The detection and quantification limits were 0.27 and 0.88 ng/ml, respectively. It was possible to detect small levels of Ara h 6 in complex food matrices.

Seafood product safety: A hybrid graphene/gold-based electrochemical immunosensor for fish allergen analysis.

Seafood product labels with accurate allergen contents can avoid and/or minimize allergic reactions. Therefore, an electrochemical immunosensor for the analysis of ß-parvalbumin (ß-PV, a major fish allergen) was developed. Screen-printed carbon electrodes were nanostructured with reduced graphene oxide and gold nanoparticles. The platform was characterized by scanning electron microscopy and elemental analysis. In a sandwich-type assay (∼75 min), the antigen-antibody interaction was detected by chronoamperometry using horseradish peroxidase and TMB-H2O2. A linear range of 25-3000 ng/mL, a sensitivity of 2.99 µA.mL/ng, and a limit of detection of 9.9 ng/mL (corresponding to 0.40 ng in the analysed aliquot) were obtained. The selectivity and possible interferences were assessed by analysing several other food allergens and a marine toxin. The sensor was applied to the analysis of 17 commercial foods and the effect of culinary processing (e.g., grilled, canned, smoked) on the ß-PV concentration was assessed. Traces of ß-PV were successfully quantified and ELISA was used to assess the results.

Voltammetric immunosensor based on oxidized carbon nanotubes/MnFe2O4 hybrid nanoplatform for amplified detection of celery (Apium graveolens).

Celery is a food allergen that must be included in the ingredient list of commercial food products in the European Union. This is a challenge for the food industry because of potential cross-contamination and undeclared ingredients because of their low concentrations. So, the food industry requires expedited high-performance analytical methods. The development, validation and application of a magnetic nanomaterial-based voltammetric immunosensor is reported to quantify a major celery allergen (Api g 1), achieving a low limit of detection (32 pg·mL-1, in a 40-µL sample). The applicability of the biosensor was evaluated by analysing twenty food products and the lowest Api g 1 content (1.1 ± 0.9 mg·kg-1) was quantified in a cooked sample. The selectivity of the method and the interference of similar fresh products (e.g., parsley, basil) were evaluated. This portable and easy-to-use biosensor can be a fit-for-purpose solution to tackle a major problem for the food industry.

An electrochemical deamidated gliadin antibody immunosensor for celiac disease clinical diagnosis.

The first electrochemical immunosensor (EI) for the detection of antibodies against deamidated gliadin peptides (DGP) is described here. A disposable nanohybrid screen-printed carbon electrode modified with DGP was employed as the transducer's sensing surface. Real serum samples were successfully assayed and the results were corroborated with an ELISA kit. The presented EI is a promising analytical tool for celiac disease diagnosis.

Fe3O4@Au Core-Shell Magnetic Nanoparticles for the Rapid Analysis of E. coli O157:H7 in an Electrochemical Immunoassay.

Escherichia coli (E. coli) O157:H7 is a pathogenic bacterium that causes serious toxic effects in the human gastrointestinal tract. In this paper, a method for its effective analytical control in a milk sample was developed. To perform rapid (1 h) and accurate analysis, monodisperse Fe3O4@Au magnetic nanoparticles were synthesized and used in an electrochemical sandwich-type magnetic immunoassay. Screen-printed carbon electrodes (SPCE) were used as transducers, and electrochemical detection was performed by chronoamperometry using a secondary horseradish peroxidase-labeled antibody and 3,3',5,5'-tetramethylbenzidine. This magnetic assay was used to determine the E. coli O157:H7 strain in the linear range from 20 to 2 × 106 CFU/mL, with a limit of detection of 20 CFU/mL. The selectivity of the assay was tested using Listeria monocytogenes p60 protein, and the applicability of the assay was assessed by analyzing a commercial milk sample, demonstrating the usefulness of the synthesized nanoparticles in the developed magnetic immunoassay.

Tracking Arachis hypogaea Allergen in Pre-Packaged Foodstuff: A Nanodiamond-Based Electrochemical Biosensing Approach.

The present work reports a nanodiamond-based voltammetric immunosensing platform for the analysis of a food allergen (Ara h 1) present in peanuts (Arachis hypogaea). The possibility of the usage of nanodiamonds (d = 11.2 ± 0.9 nm) on screen-printed carbon electrodes (SPCE/ND) in a single-use two-monoclonal antibody sandwich assay was studied. An enhanced electroactive area (~18%) was obtained and the biomolecule binding ability was improved when the 3D carbon-based nanomaterial was used. The antibody-antigen interaction was recognized through the combination of alkaline phosphatase with 3-indoxyl phosphate and silver ions. Linear Sweep Voltammetry (LSV) was applied for fast signal acquisition and scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) support the voltammetric approach and confirm the presence of silver particles on the electrode surface. The proposed immunosensor provided a low limit of detection (0.78 ng·mL−1) and highly precise (RSD < 7.5%) and accurate results. Quantification of Ara h 1 in commercial foodstuffs (e.g., crackers, cookies, protein bars) that refer to the presence of peanuts (even traces) on the product label was successfully achieved. The obtained data were in accordance with recovery results (peanut addition, %) and the foodstuff label. Products with the preventive indication "may contain traces" revealed the presence of peanuts lower than 0.1% (m/m). The method's results were validated by comparison with an enzyme-linked immunosorbent assay. This allows confident information about the presence of allergens (even at trace levels) that leads to profitable conditions for both industry and consumers.

Food allergen control: Tropomyosin analysis through electrochemical immunosensing.

Regulations of the EU obliges the indication of the presence of allergens on food labels. This work reports the development of an electrochemical immunosensor to determine tropomyosin (TPM) - a major shellfish allergen - prevailing in the muscles of crustacean species. Two linear ranges between the signal and TPM concentration were obtained: between 2.5 and 20 ng mL-1 and between 30 and 200 ng mL-1, with a lowest limit of detection of 0.47 ng mL-1. The selectivity of the optimized immunoassay, tested with other food allergens (e.g., Cyp c 1, a fish allergen), assures the effective detection of TPM, enabling successful control of foodstuff labelling. Several (12) foods, containing high and low TPM concentrations and TPM-free samples, were analysed using the sensor. A conventional ELISA kit and recovery assays were used to evaluate the accuracy of the results.

Tracking a Major Egg Allergen to Assess Commercial Food Label Compliance: Towards a Simple and Fast Immunosensing Device.

An amperometric immunosensor was developed for the analysis of the major egg-white allergen ovotransferrin (Gal d 3) in commercial food products because the (accidental) intake, skin contact with, and/or inhalation of eggs can lead to severe disorders in allergic individuals. Employing a sandwich-type immunosensing strategy, screen-printed carbon electrodes (SPCE) were biomodified with anti-Gal d 3 (capture) antibodies, and the allergen's detection was achieved with anti-Gal d 3 antibodies labelled with horseradish peroxidase (HRP). The 3,3',5,5'-tetramethylbenzidine (TMB)/H2O2 reaction with HRP was used to obtain the electrochemical (amperometric) signal. An attractive assay time of 30 min and a remarkable analytical performance was achieved. The quantification range was established between 55 and 1000 ng·mL−1, with a limit of detection of 16 ng·mL−1. The developed method demonstrated good precision (Vx0 = 5.5%) and provided precise results (CV < 6%). The sensor also detected extremely low amounts (down to 0.010%) of egg. The analysis of seven raw and/or cooked egg and egg-white samples indicated that food processing influences the amount of allergen. Furthermore, to assure the compliance of product labelling with EU legislation, 25 commercial food ingredients/products were analysed. The accuracy of the results was confirmed through an ELISA assay. The stability of the ready-to-use sensing surface for 20 days allows a reduction of the reagents' volumes and cost.

Voltammetric Immunosensor to Track a Major Peanut Allergen (Ara h 1) in Food Products Employing Quantum Dot Labels.

Tracking unreported allergens in commercial foods can avoid acute allergic reactions. A 2-step electrochemical immunosensor was developed for the analysis of the peanut allergen Ara h 1 in a 1-h assay (<15 min hands-on time). Bare screen-printed carbon electrodes (SPCE) were used as transducers and monoclonal capture and detection antibodies were applied in a sandwich-type immunoassay. The short assay time was achieved by previously combining the target analyte and the detection antibody. Core/shell CdSe@ZnS Quantum Dots were used as electroactive label for the detection of the immunological interaction by differential pulse anodic stripping voltammetry. A linear range between 25 and 1000 ng·mL-1 (LOD = 3.5 ng·mL-1), an adequate precision of the method (Vx0 ≈ 6%), and a sensitivity of 23.0 nA·mL·ng-1·cm-2 were achieved. The immunosensor was able to detect Ara h 1 in a spiked allergen-free product down to 0.05% (m/m) of peanut. Commercial organic farming cookies and cereal and protein bars were tested to track and quantify Ara h 1. The results were validated by comparison with an ELISA kit.

Electrochemical Immunosensor for the Simultaneous Determination of Two Main Peanut Allergenic Proteins (Ara h 1 and Ara h 6) in Food Matrices.

Efficiently detecting peanut traces in food products can prevent severe allergic reactions and serious health implications. This work presents the development of an electrochemical dual immunosensor for the simultaneous analysis of two major peanut allergens, Ara h 1 and Ara h 6, in food matrices. A sandwich immunoassay was performed on a dual working screen-printed carbon electrode using monoclonal antibodies. The antibody-antigen interaction was detected by linear sweep voltammetry through the oxidation of enzymatically deposited silver, which was formed by using detection antibodies labeled with alkaline phosphatase and a 3-indoxyl phosphate/silver nitrate mixture as the enzymatic substrate. The assay time was 2 h 20 min, with a hands-on time of 30 min, and precise results and low limits of detection were obtained (Ara h 1: 5.2 ng·mL-1; Ara h 6: 0.017 ng·mL-1). The selectivity of the method was confirmed through the analysis of other food allergens and ingredients (e.g., hazelnut, soybean and lupin). The dual sensor was successfully applied to the analysis of several food products and was able to quantify the presence of peanuts down to 0.05% (w/w). The accuracy of the results was confirmed through recovery studies and by comparison with an enzyme-linked immunosorbent assay. Tracking food allergens is of utmost importance and can be performed using the present biosensor in a suitable and practical way.

Molecularly imprinted polymer-based electrochemical sensors for environmental analysis.

The ever-increasing presence of contaminants in environmental waters is an alarming issue, not only because of their harmful effects in the environment but also because of their risk to human health. Pharmaceuticals and pesticides, among other compounds of daily use, such as personal care products or plasticisers, are being released into water bodies. This release mainly occurs through wastewater since the treatments applied in many wastewater treatment plants are not able to completely remove these substances. Therefore, the analysis of these contaminants is essential but this is difficult due to the great variety of contaminating substances. Facing this analytical challenge, electrochemical sensing based on molecularly imprinted polymers (MIPs) has become an interesting field for environmental monitoring. Benefiting from their superior chemical and physical stability, low-cost production, high selectivity and rapid response, MIPs combined with miniaturized electrochemical transducers offer the possibility to detect target analytes in-situ. In most reports, the construction of these sensors include nanomaterials to improve their analytical characteristics, especially their sensitivity. Moreover, these sensors have been successfully applied in real water samples without the need of laborious pre-treatment steps. This review provides a general overview of electrochemical MIP-based sensors that have been reported for the detection of pharmaceuticals, pesticides, heavy metals and other contaminants in water samples in the past decade. Special attention is given to the construction of the sensors, including different functional monomers, sensing platforms and materials employed to achieve the best sensitivity. Additionally, several parameters, such as the limit of detection, the linear concentration range and the type of water samples that were analysed are compiled.

Quantum dots as nanolabels for breast cancer biomarker HER2-ECD analysis in human serum.

Early detection of cancer increases the possibility for an adequate and successful treatment of the disease. Therefore, in this work, a disposable electrochemical immunosensor for the front-line detection of the ExtraCellular Domain of the Human Epidermal growth factor Receptor 2 (HER2-ECD), a breast cancer biomarker, in a simple and efficient manner is presented. Bare screen-printed carbon electrodes were selected as the transducer onto which a sandwich immunoassay was developed. The affinity process was detected through the use of an electroactive label, core/shell CdSe@ZnS Quantum Dots, by differential pulse anodic stripping voltammetry in a total time assay of 2 h, with an actual hands-on time of less than 30 min. The proposed immunosensor responded linearly to HER2-ECD concentration within a wide range (10-150 ng/mL), showing acceptable precision and a limit of detection (2.1 ng/mL, corresponding to a detected amount (sample volume = 40 µL) of 1.18 fmol) which is about 7 times lower than the established cut-off value (15 ng/mL). The usefulness of the developed methodology was tested through the analysis of spiked human serum samples. The reliability of the presented biosensor for the selective screening of HER2-ECD was confirmed by analysing another breast cancer biomarker (CA15-3) and several human serum proteins.

Screen-Printed Electrode-Based Sensors for Food Spoilage Control: Bacteria and Biogenic Amines Detection.

Food spoilage is caused by the development of microorganisms, biogenic amines, and other harmful substances, which, when consumed, can lead to different health problems. Foodborne diseases can be avoided by assessing the safety and freshness of food along the production and supply chains. The routine methods for food analysis usually involve long analysis times and complex instrumentation and are performed in centralized laboratories. In this context, sensors based on screen-printed electrodes (SPEs) have gained increasing importance because of their advantageous characteristics, such as ease of use and portability, which allow fast analysis in point-of-need scenarios. This review provides a comprehensive overview of SPE-based sensors for the evaluation of food safety and freshness, focusing on the determination of bacteria and biogenic amines. After discussing the characteristics of SPEs as transducers, the main bacteria, and biogenic amines responsible for important and common foodborne diseases are described. Then, SPE-based sensors for the analysis of these bacteria and biogenic amines in food samples are discussed, comparing several parameters, such as limit of detection, analysis time, and sample type.

Disposable electrochemical immunosensor for analysis of cystatin C, a CKD biomarker.

Specific monitoring of cystatin C (CysC) levels in biological fluids is critical for diagnosis, treatment and mechanistic understanding of a spectrum of diseases, particularly chronic kidney disease (CKD). Despite evidences that CysC correlates with the high risk and/or progression of CKD, its use in clinical practice is still scarce. In this context, we report the development of a simple and sensitive immunosensor for the detection of CysC. The biosensor combines the technology of cost-effective screen-printed electrodes with the high specificity of a sandwich immunoassay. Optimized conditions showed that the sensor operates in a linear range between 10 and 100 ng mL-1, with a detection limit and a sensitivity of 6.0 ng mL-1 and 6.4 ±â€¯0.3 µA ng mL-1 cm-2, respectively. Moreover, the sensor provided precise results (RSD ≤ 6.2%) and the quantification of CysC in CKD serum samples revealed to be in agreement with the values obtained by a particle-enhanced nephelometric immunoassay. In this light, the proposed immunosensor qualifies for clinical application, constituting a step forward in the development of fast, sensitive and cost-effective diagnostic tools that can improve the current medical care settings of CKD patients.

Improving the extraction of Ara h 6 (a peanut allergen) from a chocolate-based matrix for immunosensing detection: Influence of time, temperature and additives.

The extraction of Ara h 6 (a peanut allergen) from a complex chocolate-based food matrix was optimized by testing different temperatures, extraction times, and the influence of additives (NaCl and skimmed milk powder) in a total of 36 different conditions. Analyses were carried out using an electrochemical immunosensor. Three conditions were selected since they allowed the extraction of the highest levels of Ara h 6. These extractions were performed using 2g of sample and 20ml of Tris-HNO3 (pH=8) containing: a) 0.1M NaCl and 2g of skimmed milk powder at 21°C for 60min; b) 1M NaCl and 1g of skimmed milk powder at 21°C for 60min; and c) 2g of skimmed milk powder at 60°C for 60min. Recoveries were similar or higher than 94.7%. This work highlights the importance to adjust extraction procedures regarding the target analyte and food matrix components.

Electroanalytical determination of paroxetine in pharmaceuticals.

Electroanalytical methods based on square-wave adsorptive-stripping voltammetry (SWAdSV) and flow-injection analysis with SWAdSV detection (FIA-SWAdSV) were developed for the determination of paroxetine (PRX). The methods were based on the reduction of PRX at a mercury drop electrode at -1.55V versus Ag/AgCl, in a borate buffer of pH 8.8, and the possibility of accumulating the compound at the electrode surface. Because the presence of dissolved oxygen did not interfere significantly with the analysis, it was also possible to determine PRX using FIA-SWAdSV. This method enables analysis of up to 120 samples per hour at reduced costs. Both methods developed were validated and successfully applied to the quantification of PRX in pharmaceutical products.

Electroanalytical study of the antidepressant sertraline.

A flow injection square wave cathodic stripping voltammetric method has been developed for the determination of sertraline in a pharmaceutical preparation. The method shows linearity between peak current intensity and sertraline concentration for the interval between 0.20 x 10(-6) and 1.20 x 10(-6) mol L(-1). Limits of detection and quantification were found to be 1.5 x 10(-7) and 5.0 x 10 (-7) mol L(-1), respectively. Up to 70 samples per hour can be analysed with a good precision (R.S.D. = 2.5%). The proposed method was successfully applied to the determination of sertraline in a commercial product. In the voltammetric determination of sertraline in flow, a high sample rate is obtained at reduced costs, opening the possibility to compete with the chromatographic methods generally used for this analysis.

Bioelectrochemically-assisted reductive dechlorination of 1,2-dichloroethane by a Dehalococcoides-enriched microbial culture.

The aim of this study was to verify the possibility to use a polarized graphite electrode as an electron donor for the reductive dechlorination of 1,2-dichloroethane, an ubiquitous groundwater contaminant. The rate of 1,2-DCA dechlorination almost linearly increased by decreasing the set cathode potential over a broad range of set cathode potentials (i.e., from -300 mV to -900 mV vs. the standard hydrogen electrode). This process was primarily dependent on electrolytic H2 generation. On the other hand, reductive dechlorination proceeded (although quite slowly) with a very high Coulombic efficiency (near 70%) at a set cathode potential of -300 mV, where no H2 production occurred. Under this condition, reductive dechlorination was likely driven by direct electron uptake from the surface of the polarized electrode. Taken as a whole, this study further extends the range of chlorinated contaminants which can be treated with bioelectrochemical systems.

Detection of Ara h 1 (a major peanut allergen) in food using an electrochemical gold nanoparticle-coated screen-printed immunosensor.

A gold nanoparticle-coated screen-printed carbon electrode was used as the transducer in the development of an electrochemical immunosensor for Ara h 1 (a major peanut allergen) detection in food samples. Gold nanoparticles (average diameter = 32 nm) were electrochemically generated on the surface of screen-printed carbon electrodes. Two monoclonal antibodies were used in a sandwich-type immunoassay and the antibody-antigen interaction was electrochemically detected through stripping analysis of enzymatically (using alkaline phosphatase) deposited silver. The total time of the optimized immunoassay was 3h 50 min. The developed immunosensor allowed the quantification of Ara h 1 between 12.6 and 2000 ng/ml, with a limit of detection of 3.8 ng/ml, and provided precise (RSD <8.7%) and accurate (recovery >96.6%) results. The immunosensor was successfully applied to the analysis of complex food matrices (cookies and chocolate), being able to detect Ara h 1 in samples containing 0.1% of peanut.