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- Amperometric enzyme sensor for the rapid determination of histaminePublication . Torre, Ricarda; Costa Rama, Estefanía; LOPES, Paula; Nouws, Henri; Delerue-Matos, CristinaThe concentration of histamine, a biogenic amine, in fish is considered a fish spoilage indicator. Therefore, the development of a rapid-response and portable tool that allows its on-site analysis is very interesting since the consumption of high amounts of histamine causes scombroid poisoning. Thus, in this work a simple enzymatic sensor for the determination of histamine, based on a screen-printed carbon electrode and the enzyme diamine oxidase, was developed. The enzyme was immobilized on the electrode surface through a simple cross-linking procedure employing glutaraldehyde and bovine serum albumin. Chronoamperometry was used as the detection technique; the sensor showed a short response time (60 s, -0.3 V) and the measurements were performed by only using 40 μL of sample solution. The sensor provides an useful linear range (between 1 and 75 mg L-1 in fish extract), excellent reproducibility (RSD = 2.6%) and is reusable for up to 7 measurements. The feasibility of the sensor was tested performing histamine analysis in fish extracts achieving recovery values of 103%.
- Impact of magnetite nanoparticles on the syntrophic dechlorination of 1,2-dichloroethanePublication . Leitão, Patrícia; Aulenta, Federico; Rossetti, Simona; Nouws, Henri; Danko, Anthony S.In anaerobic environments microorganisms exchange electrons with community members and with soil and groundwater compounds. Interspecies electron transfer (IET) occurs by several mechanisms: diffusion of redox compounds or direct contact between cells. This latter mechanism may be facilitated by the presence of conductive nanoparticles (NP), possibly serving as electron conduits among microorganisms. Our study examined the effect of magnetite (Fe3O4) NP on the dechlorination of 1,2-dichloroethane (1,2-DCA) by a mixed-culture. The addition of NP (170 mg L− 1 total Fe) enhanced the acetate-driven reductive dechlorination of 1,2-DCA to harmless ethene (via reductive dihaloelimination) up to 3.3-times (2.3 μeq L− 1 d− 1 vs. 0.7 μeq L− 1 d− 1), while decreasing the lag time by 0.8 times (23 days) when compared to unamended (magnetite-free) microcosms. Dechlorination activity was correlated with the abundance of Dehalococcoides mccartyi, which accounted up to 50% of total bacteria as quantified by CARD-FISH analysis, pointing to a key role of this microorganism in the process. Given the widespread abundance of conductive minerals in the environment, the results of this study may provide new insights into the fate of 1,2-DCA and suggest new tools for its remediation by linking biogeochemical mechanisms.
- Disposable electrochemical immunosensor for analysis of cystatin C, a CKD biomarkerPublication . LOPES, Paula; Costa Rama, Estefanía; Beirão, Idalina; Nouws, Henri; Santos-Silva, Alice; Delerue-Matos, CristinaSpecific 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 ± 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.
- Tracking a Major Egg Allergen to Assess Commercial Food Label Compliance: Towards a Simple and Fast Immunosensing DevicePublication . Freitas, Maria; del Rio, Mariana; Nouws, Henri; Delerue-Matos, CristinaAn 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.
- Natural Magnetite Minerals Enhance 1,2-Dichloroethane Reductive DechlorinationPublication . Leitão, Patrícia; Tucci, Matteo; Cruz Viggi, Carolina; Nouws, Henri; Danko, Anthony S.; Aulenta, FedericoContamination of soil and groundwater by chlorinated solvents is an environmental issue of primary concern. Recently, electrically conductive iron particles have been proposed as a novel approach to accelerate anaerobic bioremediation processes. In fact, it was demonstrated that conductive particles facilitate the exchange of electrons between microorganisms via Direct Interspecies Electron Transfer (DIET) processes, thus enhancing the pollutant-degrading potential of the microbial community. However, the use of natural minerals in this context has not been reported so far. In this study, we applied, for the first time, natural magnetite and hematite to accelerate the reductive dechlorination of 1,2-dichloroethane by an enrichment culture in lab-scale anaerobic microcosms. After four feeding cycles, low magnetite-amended microcosms (13 mg/L) yielded the highest rate of 1,2-DCA reductive dechlorination and reduced methanogenic activity. By contrast, hematite did not display any apparent stimulatory effect. Surprisingly, in the presence of higher amounts of iron oxides, a weaker effect was obtained, probably because iron(III) present in the minerals competed for the electrons necessary for reductive dechlorination. For all microcosms, the concentration of the toxic byproduct vinyl chloride was negligible throughout the whole study. The SEM/EDS analysis confirmed the close interaction between the conductive iron oxide particles and the dechlorinating bacteria. This work opens the possibility of using natural conductive minerals for bioremediation applications as well as shedding light on the previously unrecognized role of such minerals in contaminated ecosystems
- Tracking Arachis hypogaea Allergen in Pre-Packaged Foodstuff: A Nanodiamond-Based Electrochemical Biosensing ApproachPublication . Freitas, Maria; Carvalho, André; Nouws, Henri; Delerue-Matos, CristinaThe 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.
- Improving the extraction of Ara h 6 (a peanut allergen) from a chocolate-based matrix for immunosensing detection: Influence of time, temperature and additivesPublication . Alves, Rita C.; Pimentel, Filipa B; Nouws, Henri P.A.; Silva, Túlio H B; Oliveira, M Beatriz P P; Delerue-Matos, CristinaThe 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.
- Removal of diclofenac and sulfamethoxazole from aqueous solutions and wastewaters using a three-dimensional electrochemical processPublication . Soares, Cristina; Correia-Sá, Luísa; Paíga, Paula; Barbosa, Carlos; Remor, Paula Verônica; Freitas, Olga; Moreira, Manuela M.; Nouws, Henri; Correia, Manuela; Ghanbari, Amir; Rodrigues, António J.; Oliveira, Carlos M.; Figueiredo, Sónia; Delerue-Matos, CristinaThe three-dimensional (3D) electrochemical treatment process was studied for the removal of two pharmaceuticals, diclofenac (anti-inflammatory) and sulfamethoxazole (antibiotic), in mono and bi-component systems. Adsorption and conventional two-dimensional electrochemical processes were initially studied and then combined to develop the 3D process. The influence of different operating parameters on the removal efficiency was studied: the distance between the cathode and the anode, the pharmaceutical and electrolyte (NaCl) concentrations, the pH, and the (carbon-based) adsorbent used as particulate electrode (biochar and commercial activated carbon, granulometry, and amount). The energy consumption and the electric energy per order were evaluated. The results demonstrate the efficiency of the 3D process for the removal of diclofenac and sulfamethoxazole from aqueous solutions, both for mono- and bi-component systems, achieving their complete removal respectively in 10 and 30 min, using a Mixed Metal Oxide anode (titanium-coated with RuO2-IrO2-TiO2), a stainless steel cathode, a biochar particulate electrode (1–2 mm), an initial pharmaceutical concentration of 10 mg/L, an inter-electrode distance of 7.5 cm, a pH value of 7 and a current density of 7 mA/cm2. The optimised 3D process was also successfully applied to a wastewater treatment plant effluent, but lower removal efficiencies were observed (after 30 min) for bi-component fortified samples; 49% for DCF and 86% for SMX, with energy consumptions of 1224 and 613 Wh/g and an electric energy per order of 19.1 and 8.77 kWh/m3 respectively. On the other hand, the pharmaceuticals were completely removed from the effluent when real concentrations (i.e. without their addition) were used
- Bioelectrochemical Dechlorination of 1,2-DCA with an AQDS-Functionalized Cathode Serving as Electron DonorPublication . Leitão, P.; Nouws, Hendrikus; Danko, A. S.; Aulenta, F.In the present study we describe a simple method to immobilize the redox mediator anthraquinone‐2,6‐disulfonate (AQDS) at the surface of graphite electrodes, by means of a commercial anion exchange membrane. Cyclic voltammetry experiments confirmed the efficacy of the immobilization protocol and the long‐term (over 70 days) electrochemical stability of the AQDS‐functionalized electrode. Potentiostatic (–300 mV vs. SHE) batch experiments proved the capability of the electrode in accelerating the bioelectrochemical reductive dechlorination of the groundwater contaminant 1,2‐dichloroethane (1,2‐DCA) to harmless ethene by a mixed microbial culture, by serving as electron donor in the process. Considering the reported broad range of anodic and cathodic reactions catalyzed by AQDS, the herein described functionalized electrode has a remarkable potential for application in the environmental and industrial sector.
- Preconcentration and sensitive determination of the anti-inflammatory drug diclofenac on a paper-based electroanalytical platformPublication . Costa-Rama, E.; Nouws, H.; Delerue-Matos, Cristina; Blanco-López, M.C.; Fernández-Abedul, M.T.This work describes the development of a paper-based platform for highly sensitive detection of diclofenac. The quantification of this anti-inflammatory drug is of importance in clinical (e.g. quality and therapeutic control) and environmental (e.g. emerging contaminant determination) areas. The easy-to-handle platform here described consists of a carbon-ink paper-based working electrode and two metallic wires, provided by a gold-plated standard connector, as reference and counter electrodes. The porous paper matrix enables the preconcentration of the sample, decoupling sample and detection solutions. Thus, relatively large sample volumes can be used, which significantly improves the sensitivity of the method. A wide dynamic range of four orders of magnitude, between 0.10 and 100 μM, was obtained for diclofenac determination. Due to the predominance of adsorption at the lowest concentrations, there were two linear concentration ranges: one comprised between 0.10 and 5.0 μM (with a slope of 0.85 μA μM-1) and the other between 5.0 and 100 μM (with a slope of 0.48 μA μM-1). A limit of detection of 70 nM was achieved with this simple device that provided accurate results with an RSD of ca. 5%. The platform was applied for diclofenac quantification in spiked tap water samples. The versatility of this design enabled the fabrication of a multiplexed platform containing eight electrochemical cells that work independently. The low cost, small size and simplicity of the device allow on-site analysis, which is very useful for environmental monitoring.