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Highlights • A third-generation biosensor based on nitric oxide reductase (NOR) was developed. • A MWCNTs/BMIMBF 4 nanocomposite was optimized to entrap NOR. • PGE/[MWCNTs/BMIMBF 4 /NOR] direct electron transfer behavior was characterized. • High sensitivity was reached towards NO unmediated bioelectrocatalytic reduction. • The biosensor kept 79–116% of its initial response after one month. Abstract Nitric oxide (NO) has a crucial role in signaling and cellular physiology in humans. Herein, a novel third-generation biosensor based on the Marinobacter hydrocarbonoclasticus metalloenzyme (nitric oxide reductase (NOR)), responsible for the NO reduction in the denitrifying processes, was developed through the direct adsorption of a new nanocomposite (multiwalled carbon nanotubes (MWCNTs)/1 -n- butyl-3-methylimidazolium tetrafluoroborate (BMIMBF 4)/NOR) onto a pyrolytic graphite electrode (PGE) surface. The NOR direct electron transfer behavior (formal potential of -0.255 ± 0.003 V vs. Ag/AgCl) and electrocatalysis towards NO reduction (−0.68 ± 0.03 V vs. Ag/AgCl) of the PGE/[MWCNTs/BMIMBF 4 /NOR] biosensor were investigated in phosphate buffer at pH 6.0. Large enzyme loading (2.04 × 10−10 mol/cm2), acceptable electron transfer rate between NOR and the PGE surface (k s = 0.35 s-1), and high affinity for NO (K m = 2.17 μmol L-1) were observed with this biosensor composition. A linear response to NO concentration (0.23–4.76 μmol L-1) was perceived with high sensitivity (0.429 μA/μmolL-1), a detection limit of 0.07 μmol L-1, appropriate repeatability (9.1% relative standard deviations (RSD)), reproducibility (6.0–11% RSD) and 80–102% recoveries. The biosensor was stable during 1 month retaining 79–116% of its initial response. These data confirmed that NOR incorporated in the MWCNTs/BMIMBF 4 nanocomposite can efficiently maintain its bioactivity paving a new and effective way for NO biosensing. [ABSTRACT FROM AUTHOR]

In this work, a highly sensitive label-free immunosensor for detection of the main biomarker of Alzheimer’s disease (AD), amyloid beta 1–42 (Aβ (1–42)), is presented. A gold electrode was modified with a mercaptopropionic acid (MPA) self-assembled monolayer, electrodeposited gold nanoparticles (AuNPs) and a monoclonal antibody mAb DE2B4 to recognize Aβ; all the relevant experimental variables were optimized. Antibodies were functionalized through chemical modification (thiolation) to promote the antibody immobilization on the AuNPs surface with proper orientation which enabled the direct detection of Aβ(1–42). Scanning electron microscopy, square-wave voltammetry and electrochemical impedance spectroscopy were used to characterize the construction of the biosensor. Using the proposed immunosensor, Aβ(1–42) was specifically detected within the linear range of 10–1000 pg mL −1 with a 5.2 pg mL −1 and 17.4 pg mL −1 detection and quantification limit, respectively; recovery values for the tested spiking levels ranged from 90.3 to 93.6%. The immunosensor enables rapid, accurate, precise, reproducible and highly sensitive detection (14.6% reduction mL pg −1 ) of Aβ with low-cost and opens the possibilities for diagnostic ex vivo applications and research-based in vivo studies. [ABSTRACT FROM AUTHOR]

Abstract: Celiac disease (CD) is a gluten-induced autoimmune enteropathy characterized by the presence of antibodies against gliadin (AGA) and anti-tissue transglutaminase (anti-tTG) antibodies. A disposable electrochemical dual immunosensor for the simultaneous detection of IgA and IgG type AGA and anti-tTG antibodies in real patient's samples is presented. The proposed immunosensor is based on a dual screen-printed carbon electrode, with two working electrodes, nanostructured with a carbon–metal hybrid system that worked as the transducer surface. The immunosensing strategy consisted of the immobilization of gliadin and tTG (i.e. CD specific antigens) on the nanostructured electrode surface. The electrochemical detection of the human antibodies present in the assayed serum samples was carried out through the antigen–antibody interaction and recorded using alkaline phosphatase labelled anti-human antibodies and a mixture of 3-indoxyl phosphate with silver ions was used as the substrate. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with commercial ELISA kits indicating that the developed sensor can be a good alternative to the traditional methods allowing a decentralization of the analyses towards a point-of-care strategy. [Copyright &y& Elsevier]

• Electroanalysis of ecstasy (MDMA) using screen-printed carbon electrodes (SPCEs). • Molecularly imprinted polymers (MIPs) were developed for MDMA. • The polymerization of o -phenylenediamine (o -PD) and its interaction with MDMA was simulated by DFT methods. • The SPCE-MIP selectively analysed MDMA by square-wave voltammetry (SWV). • First time a SPCE-MIP sensor was developed for MDMA. This study demonstrates the ability of an electrochemical sensor based on molecularly imprinted polymers (MIPs) to selectively quantify 3,4-methylenedioxymethamphetamine (MDMA), also known as ecstasy, in biological samples. The device was constructed using ortho -phenylenediamine (o -PD) as the MIP's building monomer at the surface of a screen-printed carbon electrode (SPCE). The step-by-step construction of the SPCE-MIP sensor was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Density functional theory (DFT) calculations and modelling were performed not only to understand template-monomer interaction but also to comprehend which possible polymer structure - linear or ramified poly(o -PD) – indeed interacts with the analyte. The prepared sensor worked by directly measuring the MDMA oxidation signal through square-wave voltammetry (SWV) after an incubation period of 10 min. Several parameters were optimized, such as the monomer/template ratio, the number of electropolymerization scanning cycles, and the incubation period, to obtain the best sensing efficiency. Optimized sensors exhibited suitable selectivity, repeatability (2.6%), reproducibility (7.7%) and up to one month of stable response. A linear range up to 0.2 mmol L−1 was found with an r2 of 0.9990 and a limit of detection (LOD) and quantification (LOQ) of 0.79 and 2.6 μmol L−1 (0.15 and 0.51 μg mL−1), respectively. The proposed sensor was successfully applied to human blood serum and urine samples, showing its potential for application in medicine and in forensic sciences. [ABSTRACT FROM AUTHOR]

Highlights • Simultaneous electrochemical detection of two Roundup Ready™ soybean events is feasible. • Core@shell Fe 3 O 4 @Au MNPs proved to be a suitable nanosupport. • Detection limits at the subnM level were achieved. • Methodology specific for these events with no cross-reactivity. Abstract Development of expeditious analytical methods for the detection of genetically modified organisms (GMOs) is increasingly necessary, not only to verify compliance with labelling, but also to help industry to efficiently control the reception of raw materials. On the basis of this, a disposable electrochemical magnetogenoassay is proposed for simultaneous detection of two Roundup Ready (RR) soybean lines GTS 40-3-2 and MON89788, using gold-coated magnetic nanoparticles (Fe 3 O 4 @Au) as nanosupport. To perform this magnetogenoassay, a sandwich-type hybridization assay was used with different enzymatic labelling systems (fluorescein isothiocyanate and digoxigenin) and dual screen-printed carbon electrodes (SPdCEs), which allowed the simultaneous readout of each target. A linear relationship ranging from 0.1 to 2.5 nM and from 0.1 to 1.0 nM was achieved for GTS 40-3-2 and MON89788 events, respectively, and both assays showed a similar detection limit of about 0.1 nM. Furthermore, a good performance in terms of precision and selectivity was achieved. The proposed approach is a step forward for event-specific multiplex detection. [ABSTRACT FROM AUTHOR]

The extracellular domain of the human epidermal growth factor receptor 2 (HER2-ECD) is a protein breast cancer biomarker. Its quantification in peripheral blood could provide an important contribution to diagnostics and patient follow-up. In this work an electrochemical molecularly imprinted polymer (MIP) sensor for the quantification of HER2-ECD was developed. The MIP was electropolymerized by cyclic voltammetry using a solution containing phenol and HER2-ECD on a screen-printed gold electrode (AuSPE). The sensor was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The analysis of HER2-ECD was performed by differential pulse voltammetry using ([Fe(CN) 6 ] 3−/4− as redox probe. The linear range was established in the concentration interval from 10 to 70 ng/mL HER2-ECD, with a limit of detection of 1.6 ng/L and a limit of quantification of 5.2 ng/mL. Through the analysis of other protein biomarkers, the MIP sensor was found to be selective. Furthermore, these proteins did not interfere in the analysis of the selected biomarker. The developed sensor was used for the analysis of spiked human serum samples, providing adequate recovery values and precise results. The outcomes of this study indicate that the developed MIP sensor could be useful in the non-invasive analysis of HER2-ECD in breast cancer patients. [ABSTRACT FROM AUTHOR]

The incidence of breast cancer has been increasing over the years. To control and monitor this disease several tumor biomarkers have been proposed for early diagnosis, patient follow-up and/or treatment guidance. The only serum breast cancer biomarker in current use is the cancer antigen 15-3 (CA 15-3). In this work a molecularly imprinted polymer (MIP)-based electrochemical (voltammetric) sensor to monitor breast cancer was developed, based on direct surface imprinting of CA 15-3 on a screen-printed gold electrode (Au-SPE). The imprinting was performed in two steps: (1) adsorption of CA 15-3 on the surface of the Au-SPE and (2) electropolymerization of 2-aminophenol around the adsorbed protein. After extraction of the imprinted protein voltammetric analysis was conducted using hexacyanoferrate(II/III) as redox probe, measuring the signals before and after protein binding. The sensor was characterized by voltammetric techniques and electrochemical impedance spectroscopy, and the analytical responses of imprinted and non-imprinted polymer sensors were studied. A linear relationship between the peak current intensity of the redox probe and the logarithm of CA 15-3 concentration was established between 5 and 50 U mL −1 , achieving a limit of detection of 1.5 U mL −1 . The prepared MIP-sensor provides fast (15 min) analysis and is cheap, easy to prepare, disposable and could easily be integrated in small portable point-of care devices. [ABSTRACT FROM AUTHOR]

Cancer Antigen 15-3 (CA 15-3) and the extracellular domain of the human epidermal growth factor receptor 2 (HER2-ECD) are independent breast cancer biomarkers. The combination of their profiles (presence and concentration) could provide an important contribution to diagnostics and patient follow-up. Therefore, a disposable electrochemical immunosensor for the simultaneous detection of CA 15-3 and HER2-ECD was developed in this work. The immunosensor was constructed on a customized dual screen-printed carbon electrode. The carbon working electrodes' surfaces were first modified with in situ electrodeposited gold nanoparticles and then individually coated with either a monoclonal anti-human CA 15-3 or a monoclonal anti-human HER2-ECD antibody. After incubation with the biomarkers and monoclonal biotin-labelled detection antibodies, the antigen-antibody interactions were detected by linear sweep voltammetric analysis of enzymatically (alkaline phosphatase) generated metallic silver. The immunosensor’s limits of detection for the selected biomarkers were 5.0 U mL −1 for CA 15-3 and 2.9 ng mL −1 for HER2-ECD. These values could allow the use of the sensor in the non-invasive control of these biomarkers in breast cancer patients. [ABSTRACT FROM AUTHOR]

Naloxone (NLX) is a pharmaceutical used as opioid antagonist. A molecular imprinted polymer electrochemical sensor for simple and rapid detection of NLX was prepared through the modification of commercial available screen printed carbon electrode (SPCE). The SPCE was modified with multi-walled carbon nanotubes (MWCNT) by drop coating to increase the signal response and improve the sensitivity. The MIP preparation was carried out via in situ electropolymerization using 4-aminobenzoic acid (4-ABA) as functional monomer. The morphology of the obtained sensor was characterized by scanning electron microscopy (SEM). Several parameters controlling the preparation and performance of the MIP sensor were studied and optimized. The electrochemical behavior of NLX at MIP and control non-imprinted (NIP) sensor was evaluated by differential pulse voltammetry (DPV), demonstrating a better MIP response and the success of the imprinting. The proposed MIP/MWCNT/SPCE sensor showed a linear relationship between peak current intensity and NLX concentration in the range between 0.25 and 10.0 μM, with limits of detection (LOD) and quantification (LOQ) of 0.20 μM and 0.67 μM respectively. The repeatability and reproducibility were also tested with relative standard deviations (RSD) of 4.6 and 9.6% respectively. Moreover, the applicability of the method was successfully confirmed with detection of NLX in biological samples (urine and human serum). The sensor is promising to be used for screening NLX in point-of-care people with opioid overdose. [ABSTRACT FROM AUTHOR]

Regardless the controversies surrounding genetically modified organisms (GMO), their cultivation is constantly increasing and in according to the EU legislation, labeling is mandatory for products containing EU-authorized-GMO higher than 0.9%. Thereby, new analytical strategies for rapid and effective detection of GMO on foodstuffs are required. In this work, an electrochemical immunosensor for effective determination of Cry1Ab protein from MON810 transgenic maize (EU-authorized-GMO) is described. The immunosensor was developed onto indium tin oxide (ITO) electrodes modified by 3-aminopropyltrimethoxysilane (APTES) monolayer to covalently immobilize Anti-Cry1Ab polyclonal antibodies. The protein interaction with the polyclonal antibody (PAb) recognition platform was directly monitored and measured by cyclic voltammetry and electrochemical impedance spectroscopy using commercially Cry1Ab protein. After the analytical features optimization a linear response from 1 to 10 ng mL −1 , a limit of detection (LOD) of 0.37 ng mL −1 and a limit of quantification (LOQ) of 1.23 ng mL −1 – which provided accurate results (RSD < 7.5%) – were achieved. The immunosensor allowed a simple and fast detection of Cry1Ab protein extracted from maize seeds with different GM maize mass percentages (0%, 0.5%, 1%, 2.5% and 5%). To crosscheck the detection of Cry1Ab protein, an enzyme-linked immunosorbent assay (ELISA) was used. The results indicate that the immunosensor is suitable for the transgenic protein Cry1Ab detection in GM maize representing a successfully tool to verify the compliance of the EU regulations. [ABSTRACT FROM AUTHOR]

Simple, cost-effective and reliable tools for the quantification of genetically modified organisms (GMO) in food and feed are highly demanded to enforce labelling legislation in the EU. Herein, we report a novel method for quantitative analysis of genetically modified soybean with the event GTS-40-3-2, also known as Roundup Ready (RR) soybean, using magnetoassays with electrochemical detection, coupled to DNA amplification by end-point polymerase chain reaction (PCR). For the proposed work, two DNA sequences were targeted via hybridisation onto magnetic beads, one specific for the transgenic event and the other for the taxon or species-specific lectin gene. Enzymatic labelling was performed to obtain an electrochemically active product measured by chronoamperometry. By optimising the number of PCR cycles, among other parameters, two magnetoassays coupled to PCR were successfully accomplished and linearity was obtained in the ranges of 53–4425 and 1093–88,496 DNA copies for the event-specific and lectin sequences, respectively. The proposed method provides accurate and precise RR soybean quantitative results, being effectively compared to those obtained by real-time PCR, as the reference method. These findings confirm the suitability of the method as an alternative tool for GMO quantification. [ABSTRACT FROM AUTHOR]

In this work, a norfloxacin selective modified glassy carbon electrode (GCE) based on a molecularly imprinted polymer (MIP) as electrochemical sensor was developed. A suspension of multi-walled carbon nanotubes (MWCNTs) was deposited on the electrode surface. Subsequently, a molecularly imprinted film was prepared by electropolymerization, via cyclic voltammetry of pyrrole (PPy) in the presence of norfloxacin (NFX) as the template molecule. A control electrode (NIP) was also prepared. Scanning electron microscopy (SEM) and cyclic voltammetry in a ferrocyanide solution were performed for morphological and electrochemical characterisation, respectively. Several experimental parameters were studied and optimised. For quantification purposes the MIP/MWCNT/GCE was immersed in NFX solutions for 10 min, and the detection was performed in voltammetric cell by square wave voltammetry. The proposed sensor presented a linear behaviour, between peak current intensity and logarithmic concentration of NFX between 1 × 10 −7 and 8 × 10 −6 M. The obtained results presented good precision, with a repeatability of 4.3% and reproducibility of 9% and the detection limit was 4.6 × 10 −8 M ( S/N = 3). The developed sensor displayed good selectivity and operational lifetime, is simple to fabricate and easy to operate and was successfully applied to the analysis of NFX in urine samples. [ABSTRACT FROM AUTHOR]

For the first time, a glassy carbon electrode (GCE) modified with novel N-doped carbon nanotubes (CNT-N) functionalized with MnFe 2 O 4 nanoparticles (MnFe 2 O 4 @CNT-N) has been prepared and applied for the electrochemical determination of caffeine (CF), acetaminophen (AC) and ascorbic acid (AA). The electrochemical behaviour of CF, AC and AA on the bare GCE, CNT-N/GCE and MnFe 2 O 4 @CNT-N/GCE were carefully investigated using cyclic voltammetry (CV) and square-wave voltammetry (SWV). Compared to bare GCE and CNT-N modified electrode, the MnFe 2 O 4 @CNT-N modified electrode can remarkably improve the electrocatalytic activity towards the oxidation of CF, AC and AA with an increase in the anodic peak currents of 52%, 50% and 55%, respectively. Also, the SWV anodic peaks of these molecules could be distinguished from each other at the MnFe 2 O 4 @CNT-N modified electrode with enhanced oxidation currents. The linear response ranges for the square wave voltammetric determination of CF, AC and AA were 1.0 × 10 −6 to 1.1 × 10 −3 mol dm −3 , 1.0 × 10 −6 to 1.0 × 10 −3 mol dm −3 and 2.0 × 10 −6 to 1.0 × 10 −4 mol dm −3 with detection limit ( S / N = 3) of 0.83 × 10 −6 , 0.83 × 10 −6 and 1.8 × 10 −6 mol dm −3 , respectively. The sensitivity values at the MnFe 2 O 4 @CNT-N/GCE for the individual determination of AC, AA and CF and in the presence of the other molecules showed that the quantification of AA and CF show no interferences from the other molecules; however, AA and CF interfered in the determination of AC, with the latter molecule showing the strongest interference. Nevertheless, the obtained results show that MnFe 2 O 4 @CNT-N composite material acted as an efficient electrochemical sensor towards the selected biomolecules. [ABSTRACT FROM AUTHOR]

A novel electrochemical sensor for ochratoxin A (OTA) detection was fabricated through the modification of a glassy carbon electrode (GCE) with multiwalled carbon nanotubes (MWCNTs) and a molecularly imprinted polymer (MIP). The MWCNTs dramatically promoted the sensitivity of the developed sensor, while polypyrrole (PPy) imprinted with OTA served as the selective recognition element. The imprinted PPy film was prepared by electropolymerization of pyrrole in the presence of OTA as a template molecule via cyclic voltammetry (CV). The electrochemical oxidation of OTA at the developed sensor was investigated by CV and differential pulse voltammetry (DPV). The developed MIP/MWCNT/GCE sensor showed a linear relationship, when using DPV, between peak current intensity and OTA concentration in the range between 0.050 and 1.0 μM, with limits of detection (LOD) and quantification of 0.0041 μM (1.7 μg/L) and 0.014 μM (5.7 μg/L) respectively. With the developed sensor precise results were obtained; relative standard deviations of 4.2% and 7.5% in the evaluation of the repeatability and reproducibility, respectively. The MIP/MWCNT/GCE sensor is simple to fabricate and easy to use and was successfully applied to the determination of OTA in spiked beer and wine samples, with recoveries between 84 and 104%, without the need of a sample pre-treatment step. [ABSTRACT FROM AUTHOR]

• First time a selective MIP electrochemical sensor was developed for Furazolidone (FZD). • QM calculations and MD simulations were used to select the best functional monomer and the optimum polymerisation conditions. • The sensor constructed based on MIP-CPE-MWCNTs showed high selectivity and sensitivity. • The proposed sensor was successfully applied for FZD detection in water samples without any pre-treatment step. Determination of antibiotics in environmental waters is an important global issue. Although furazolidone (FZD) was banned from use in food-producing animals, owing to its mutagenic and carcinogenic effects, this antibiotic has been illegally used across the world and its presence in environment have being noted. In this work, the first selective molecularly imprinted polymer (MIP) was developed for electrochemical detection of FZD. It was constructed based on the modification of the traditional carbon paste electrode (CPE) with MIP microparticles, followed by introduction of multi-walled carbon nanotubes (MWCNTs). Quantum mechanical (QM) calculations and molecular dynamics (MD) simulations were performed to allow rational selection of an appropriate functional monomer and to simulate the best pre-polymerisation conditions, respectively. The MIP were synthetized by polymerization using 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) as monomer and FZD as template molecule. The MIP microparticles were then incorporated on CPE-MWCNTs and the electrochemical analysis of FZD were evaluated by differential pulse voltammetry (DPV). After optimisation of experimental conditions, the MIP-CPE-MWCNTs sensor exhibited a good linear response over the concentration range of 0.01 μM to 1 μM with a correlation coefficient of 0.9995. The limit of detection (LOD) was found to be 0.03 μM (S/N = 3). Due to high imprinting efficiency the sensor displayed selectivity to recognise FZD molecules and it was successfully applied in water samples where excellent recovery values (over 90 %) were obtained. The proposed sensor provides an efficient and promising sustainable strategy for monitorisation of FZD in environmental waters. [ABSTRACT FROM AUTHOR]

• Detection of cancer biomarkers in biological fluids is useful for early diagnosis. • An electrochemical immunomagnetic bioassay was developed for HER2-ECD detection. • Magnetic beads constitute a versatile tool for the construction of the assay. • The assay was selective towards the target protein and live breast cancer cells. • The assay was successfully tested in the analysis of human serum. Despite the evolution of targeted therapies in oncology, some challenges such as screening and early diagnosis of cancer-related biomarkers still remain. The analysis of the Human Epidermal growth factor Receptor 2 (HER2) in biological fluids provides essential information for effective treatments. In this work we report the development of an electrochemical immunomagnetic bioassay for the analysis of the extracellular domain of HER2 (HER2-ECD) in human serum and cancer cells. Biomodified carboxylic acid functionalized magnetic beads (COOH-MBs) were used as the capture probe and an antibody labelled with alkaline phosphatase (AP) as the signalling probe. In the presence of HER2-ECD a sandwich complex was formed on the MBs, which were magnetically attracted to the surface of a screen-printed carbon electrode (SPCE). After the addition of 3-indoxyl phosphate and silver ions, used as the enzymatic substrate, the immunological interaction was detected by linear sweep voltammetry. Two linear concentration ranges were established: one between 5.0 and 50 ng/mL and another between 50 and 100 ng/mL. The developed assay provided a clinically useful detection limit (2.8 ng/mL) and has an adequate precision (V x0 < 5%). The assay provided accurate results and was selective towards the target biomarker. Additionally, CTCs were analysed in human serum and a detection limit of 3 cells/mL was achieved for the HER+ breast cancer cell line SK-BR-3. [ABSTRACT FROM AUTHOR]