Bifunctional Au@Pt/Au core@shell Nanoparticles As Novel Electrocatalytic Tags in Immunosensing: Application for Alzheimer's Disease Biomarker Detection.

In this work, bifunctional core@shell Au@Pt/Au NPs are presented as novel tags for electrochemical immunosensing. Au@Pt/Au NPs were synthesized following a chemical route based on successive metal depositions and galvanic replacement reactions from the starting AuNPs. Au protuberances growth on the surface of Au@Pt NPs allowed their easy bioconjugation with antibodies, while the high catalytic Pt surface area was approached for their sensitive detection through the electrocatalyzed water oxidation reaction (WOR) at neutral pH. Moreover, the synergy between Au and Pt metals on the NP surface also lead to an increased catalytic activity, improving the sensitivity of the NP detection. Cyclic voltammetry and chronoamperometry were used for the evaluation of the Au@Pt/Au NPs electrocatalytic activity toward WOR. The chronoamperometric current recorded at a fixed potential of +1.35 V was selected as the analytical signal, allowing the quantification of Au@Pt/Au NPs at 1013 NPs/mL levels. The optimized electrocatalytic method was applied to the quantification of conformationally altered p53 peptide Alzheimer's disease (AD) biomarker in a competitive immunoassay using magnetic bead (MB) platforms at levels as low as 66 nM. The performance of the system in a real scenario was demonstrated analyzing plasma samples from a cognitively healthy subject. This novel Au@Pt/Au NPs-based electrocatalytic immunoassay has the advantage, over common methods for NP tags electrochemical detection, of the signal generation in the same neutral medium where the immunoassay takes place (0.1 M PBS pH 7.2), avoiding the use of additional and more hazardous reagents and paving the way to future integrated biosensing systems.

Electrochemical quantification of Ag2S quantum dots: evaluation of different surface coating ligands for bacteria determination.

In this work, novel silver sulphide quantum dots (Ag2S QD) are electrochemically quantified for the first time. The method is based on the electrochemical reduction of Ag+ to Ag0 at -0.3 V on screen-printed carbon electrodes (SPCEs), followed by anodic stripping voltammetric oxidation that gives a peak of currents at +0.06 V which represents the analytical signal. The optimized methodology allows the quantification of water-stabilized Ag2S QD in the range of approximately 2 × 109-2 × 1012 QD·mL-1 with a good reproducibility (RSD: 5%). Moreover, as proof-of-concept of relevant biosensing application, Ag2S QD are evaluated as tags for Escherichia coli (E. coli) bacteria determination. Bacteria tagged with QD are separated by centrifugation from the sample solution and placed on the SPCE surface for quantitative analysis. The effect of two different Ag2S QD surface coating/stabilizing agents on both the voltammetric response and the bacteria sensing is also evaluated. 3-mercaptopropionic acid (3-MPA) is studied as model of short length coating ligand with no affinity for the bacteria, while boronic acid (BA) is evaluated as longer length ligand with chemical affinity for the polysaccharides present in the peptidoglycan layer on the bacteria cells surface. The biosensing system allows to detect bacteria in the range 10-1-103 bacteria·mL-1 with a limit of detection as low as 1 bacteria·mL-1. This methodology is a promising proof-of-concept alternative to traditional laboratory-based tests, with good sensitivity and short time and low cost of analysis. Graphical abstractNovel silver sulphide quantum dots (Ag2S QD) are electrochemically quantified for the first time. Moreover, Ag2S QD are evaluated as tags for Escherichia coli bacteria determination. The effect of two different QD surface coating ligands is also evaluated.

A monoclonal antibody-based immunosensor for the electrochemical detection of imidacloprid pesticide.

Imidacloprid (IMD) is one of the most used pesticides worldwide as a systemic insecticide as well as for pest control and seed treatment. The toxic and potential carcinogenic character of IMD makes its monitoring of great relevance in the field of agriculture and environment, so sensitive methodologies for in field analysis are strongly required. In this context, we have developed a competitive immunoassay for the determination of IMD using specific monoclonal antibodies followed by electrochemical detection on screen-printed carbon electrodes (SPCE). The optimized immunosensor exhibited a good reproducibility (RSD of 9%) and a logarithmic response in the range 50-10 000 pM of IMD, with an estimated detection limit (LOD) of 24 pM, which was below the maximum levels allowed by the legislation. High-Performance Liquid Chromatography-Mass Spectrometry-Mass Spectrometry (HPLC-MSMS) and Enzyme-Linked Immunosorbent Assay (ELISA) analysis were also performed for comparison purposes, where the electrochemical immunosensor exhibited a wider range of response and a lower detection limit. Matrix effects below 6.5% were obtained using tap water samples. All these characteristics make our electrochemical immunosensor a valid and advantageous tool for the in field determination of IMD.

Nanoparticles as Emerging Labels in Electrochemical Immunosensors.

This review shows recent trends in the use of nanoparticles as labels for electrochemical immunosensing applications. Some general considerations on the principles of both the direct detection based on redox properties and indirect detection through electrocatalytic properties, before focusing on the applications for mainly proteins detection, are given. Emerging use as blocking tags in nanochannels-based immunosensing systems is also covered in this review. Finally, aspects related to the analytical performance of the developed devices together with prospects for future improvements and applications are discussed.

Electrogeneration of Gold Nanoparticles on Porous-Carbon Paper-Based Electrodes and Application to Inorganic Arsenic Analysis in White Wines by Chronoamperometric Stripping.

This paper describes the development of simple, sustainable, and low-cost strategies for signal enhancement on paper-based carbon platforms through gold nanoparticles electrogenerated from small volumes of tetrachloroauric (III) acid solutions. Carbon ink is deposited on a hydrophilic working area of the paper delimited with hydrophobic wax. This maskless procedure is fast and cuts down ink waste. The connection of this working electrode to the potentiostat is ensured with the use of screen-printed electrodes (SPEs). Close contact of the whole area of both carbon electrodes improves the precision of the nanostructuration. Resulting gold-modified paper-based carbon working electrodes (AuNPs-PCWEs) were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), and electron dispersion X-ray spectrometry (SEM/EDX). This methodology was applied for the first time to the inorganic arsenic determination in commercial white wines by chronoamperometric stripping of the electrodeposited As(0). In an optimized system, As(III) was reduced and deposited as As(0) on the nanostructured surface by applying a potential of -0.3 V during 180 s. Then, anodic stripping chronoamperometry was performed at +0.4 V. The analytical signal was the current recorded at 30 s. On the other hand, As(V) was chemically reduced to As(III) with 0.2 M KI, and total determination of arsenic could be carried out. As(V) was determined as the difference between total As and As(III). Then, this fast, simple and low-cost method can be employed for speciation purposes. Limits of detection for As(III) and total arsenic (in the presence of KI) are 2.2 µg L-1 and 2.4 µg L-1, respectively, and indicate that this method is suitable for regulated quality control.

Enhanced detection of quantum dots by the magnetohydrodynamic effect for electrochemical biosensing.

In this work, we describe the use of a magnetoelectrochemical support for screen-printed electrodes to improve the anodic stripping voltammetry of cadmium due to the generated magnetohydrodynamic (MHD) effect. To create a significant MHD effect, Fe(iii) was added at mM concentrations to the solution. The reduction of Fe(iii) simultaneously with the cadmium deposition on the electrode surface allowed the production of a high cathodic current, which generated a large Lorentz force capable of exerting a convective effect on the solution in the presence of the magnetic field. This convective effect allowed the increase in the mass transfer in the quiescent solution, enhancing the deposition of cadmium as observed by an increased stripping peak current. The optimized method was applied to the detection of CdSe/ZnS quantum dots (QDs) in solution. Using the magnetoelectrochemical support, we were able to detect extremely low concentrations of QDs, with a detection limit of 100 amol of QDs (in particle number). The great performance shown by this system was evaluated in biosensing applications. Firstly, detection of biotin was carried out using a competitive bioassay between biotin and QD-labelled biotin, obtaining good analytical results (0.6 × 10-10 M as the limit of detection). Then, the magnetoelectrochemical support was tested in a more complex biosensor for the determination of anti-transglutaminase IgA antibodies, a celiac disease biomarker. This work shows that the improvement in the metal electrodeposition caused by the MHD effect can be used successfully for the development of disposable electrochemical biosensors with great performance using screen-printed electrodes.

Stabilization of electrogenerated copper species on electrodes modified with quantum dots.

Quantum dots (QDs) have special optical, surface, and electronic properties that make them useful for electrochemical applications. In this work, the electrochemical behavior of copper in ammonia medium is described using bare screen-printed carbon electrodes and the same modified with CdSe/ZnS QDs. At the bare electrodes, the electrogenerated Cu(i) and Cu(0) species are oxidized by dissolved oxygen in a fast coupled chemical reaction, while at the QDs-modified electrode, the re-oxidation of Cu(i) and Cu(0) species can be observed, which indicates that they are stabilized by the nanocrystals present on the electrode surface. A weak adsorption is proposed as the main cause for this stabilization. The electrodeposition on electrodes modified with QDs allows the generation of random nanostructures with copper nanoparticles, avoiding the preferential nucleation onto the most active electrode areas.

Electrochemical Study and Applications of Selective Electrodeposition of Silver on Quantum Dots.

In this work, selective electrodeposition of silver on quantum dots is described. The particular characteristics of the nanostructured silver thus obtained are studied by electrochemical and microscopic techniques. On one hand, quantum dots were found to catalyze the silver electrodeposition, and on the other hand, a strong adsorption between electrodeposited silver and quantum dots was observed, indicated by two silver stripping processes. Nucleation of silver nanoparticles followed different mechanisms depending on the surface (carbon or quantum dots). Voltammetric and confocal microscopy studies showed the great influence of electrodeposition time on surface coating, and high-resolution transmission electron microscopy (HRTEM) imaging confirmed the initial formation of Janus-like Ag@QD nanoparticles in this process. By use of moderate electrodeposition conditions such as 50 µM silver, -0.1 V, and 60 s, the silver was deposited only on quantum dots, allowing the generation of localized nanostructured electrode surfaces. This methodology can also be employed for sensing applications, showing a promising ultrasensitive electrochemical method for quantum dot detection.

Pin-Based Flow Injection Electroanalysis.

This work describes the use of mass-fabricated stainless-steel pins as new low-cost electrodes for a flow injection analysis (FIA) system with electrochemical detection. The pins serving as electrodes are directly punched in the tubing where solutions flow, being one of the simplest flow cells for FIA. This cell consists of a carbon ink coated pin as working electrode and two bare pins as counter and reference electrodes. The pins are able to perform at least 300 measurements. Moreover, they can be easily replaced showing good repeatability and reproducibility (RSD lower than 6% in all the cases). As a proof-of-concept, the feasibility of the system to determine glucose was evaluated by an enzymatic assay using glucose oxidase, horseradish peroxidase, and ferrocyanide as electron-transfer mediator. The application of this system to real food samples has shown accurate results.

Methylene blue covalently attached to single stranded DNA as electroactive label for potential bioassays.

Methylene blue is an electroactive molecule that has been employed for the detection of the DNA hybridization event in electrochemical sensors. However, its use as a covalent label is very scarce and in most of the cases, non-covalent interactions (hydrophobic, electrostatic) are employed. Although it has advantages as simplicity and fewer number of procedure steps, the covalent attachment is less exploited in the development of these sensors. In this article, the electrochemical behavior of methylene blue attached to different DNA-strands is studied. Several lengths (15- and 30-mer) and different degree of DNA modification (MB-DNA, MB-DNA-MB and MB-DNA-SH) have been studied. The highest signals were obtained for longer strands with two MB molecules. In all the cases the signal is enhanced by CNT-nanostructuration of the electrode. Adsorption on these modified screen-printed electrodes allowed the amplification by employing an accumulation time. In this way, a sensitivity of -0.2864 µA µM-1 and a limit of detection of 800 nM for a 120 s accumulation time were obtained.

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.

Development and evaluation of an electrochemical biosensor for creatinine quantification in a drop of whole human blood.

An electrochemical biosensor for creatinine determination in a drop of whole human blood was developed and applied to the determination of creatinine in real clinical samples. It is based on the modification of a dual carbon working electrode with a combination of three enzymes: creatinine amidohydrolase (CNN), creatine amidinohydrolase (CRN) and sarcosine oxidase (SOX). Electrochemical transduction is performed using horseradish peroxidase (HRP) and potassium hexacyanoferrate(II) as mediator. A drop of human blood is enough to carry out the measurements by differential chronoamperometry where one carbon electrode detects creatine and the other both creatine and creatinine. The integrated differential signal obtained in the biosensor is linear with the concentration of creatinine in blood in the range 0.5-15 mg/dL and the enzyme-modified electrodes are stable for at least 3 months at 4 °C. The biosensor was lined to a reference method based on Isotope Dilution Mass Spectrometry (IDMS) with 50 real human blood samples and the results compared with those obtained by alternative routine techniques based on Jaffé method and an enzymatic method (Cobas 8000 Roche®, Crep2 Roche®). There were no significant differences between the creatinine concentrations found by the routine techniques and the developed biosensor.

Microchip electrophoresis with amperometric detection for a novel determination of phenolic compounds in olive oil.

The relevance of the development of microchip electrophoresis applications in the field of food analysis is considered in this work. A novel method to determine important phenolic compounds in extra virgin olive oil samples using a miniaturized chemical analysis system is presented in this paper. Three interesting phenolic compounds in olive oil and fruit (tyrosol, hydroxytyrosol and oleuropein glucoside) were studied by end-channel amperometric detection using a 100 µm gold wire as working electrode in glass microchip electrophoresis. The electrochemical behavior of these compounds was studied and the medium to carry out their detection was selected (0.1 M aqueous sulfuric acid). The best conditions for the separation were achieved in sodium tetraborate (10% methanol, pH 9.50) with different concentrations for the sample and the running buffer in order to allow the sample stacking phenomenon. The injection was carried out using 600 V for 3 s and the separation voltage was set at 1000 V. The quality of the method was evaluated through its analytical figures of merit and by its performance on real extra virgin olive oil samples. Determination of these compounds was carried out using the standard addition calibration method with good recoveries.

Unfolded p53 as a Marker of Oxidative Stress in Mild Cognitive Impairment, Alzheimer's and Parkinson's Disease.

AIMS: There are several candidate biomarkers for AD and PD which differ in sensitivity, specificity, cost-effectiveness, invasiveness, logistical and technical demands. This study is aimed to test whether plasma concentration of unfolded p53 may help to discriminate among the neurodegenerative processes occurring in Mild Cognitive Impairment, Alzheimer's disease and Parkinson's disease. METHODS: An electrochemical immunosensor was used to measure unfolded p53 in plasma samples of 20 Mild Cognitive Impairment (13 males/7 females; mean age 74.95±5.31), 20 Alzheimer's (11 males/9 females; mean age: 77.25±7.79), 15 Parkinson's disease patients (12 males/3 females; mean age: 68.60 ± 7.36) and its respective age/sex/studies-matched controls. RESULTS: We observed a significantly higher concentration of unfolded p53 in the plasma of patients of each of the three pathologies with respect to their control groups (p=0.000). Furthermore, the plasma concentration of unfolded p53 was significantly higher in Alzheimer's disease patients in comparison with Mild Cognitive Impairment patients (p=0.000) and Parkinson's disease patients (p=0.006). No significant difference between Mild Cognitive Impairment and Parkinson's disease patients was observed (p=0.524). CONCLUSION: Our results suggest that unfolded p53 concentration in the plasma may be a useful biomarker for an undergoing neuropathological process that may be common, albeit with different intensity, to different diseases.

Celiac disease diagnosis and gluten-free food analytical control.

Celiac disease (CD) is an autoimmune enteropathy, characterized by an inappropriate T-cell-mediated immune response to the ingestion of certain dietary cereal proteins in genetically susceptible individuals. This disorder presents environmental, genetic, and immunological components. CD presents a prevalence of up to 1% in populations of European ancestry, yet a high percentage of cases remain underdiagnosed. The diagnosis and treatment should be made early since untreated disease causes growth retardation and atypical symptoms, like infertility or neurological disorders. The diagnostic criteria for CD, which requires endoscopy with small bowel biopsy, have been changing over the last few decades, especially due to the advent of serological tests with higher sensitivity and specificity. The use of serological markers can be very useful to rule out clinical suspicious cases and also to help monitor the patients, after adherence to a gluten-free diet. Since the current treatment consists of a life-long gluten-free diet, which leads to significant clinical and histological improvement, the standardization of an assay to assess in an unequivocal way gluten in gluten-free foodstuff is of major importance.

Paper-Based Working Electrodes Coated with Mercury or Bismuth Films for Heavy Metals Determination.

Paper-based carbon working electrodes were modified with mercury or bismuth films for the determination of trace metals in aqueous solutions. Both modification procedures were optimized in terms of selectivity and sensitivity for the determination of different heavy metals, aiming their simultaneous determination. Cd (II), Pb (II) and In (III) could be quantified with both films. However, Cu (II) could not be determined with bismuth films. The modification with mercury films led to the most sensitive method, with linear ranges between 0.1 and 10 µg/mL and limits of detection of 0.4, 0.1, 0.04 and 0.2 µg/mL for Cd (II), Pb (II), In (III) and Cu (II), respectively. Nevertheless, the bismuth film was a more sustainable alternative to mercury. Tap-water samples were analyzed for the determination of metals by standard addition methodology with good accuracy, by using a low-cost and easily disposable paper-based electrochemical platform. This system demonstrated its usefulness for monitoring heavy metals in water.

Electrochemical (Bio)Sensors for Pesticides Detection Using Screen-Printed Electrodes.

Pesticides are among the most important contaminants in food, leading to important global health problems. While conventional techniques such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS) have traditionally been utilized for the detection of such food contaminants, they are relatively expensive, time-consuming and labor intensive, limiting their use for point-of-care (POC) applications. Electrochemical (bio)sensors are emerging devices meeting such expectations, since they represent reliable, simple, cheap, portable, selective and easy to use analytical tools that can be used outside the laboratories by non-specialized personnel. Screen-printed electrodes (SPEs) stand out from the variety of transducers used in electrochemical (bio)sensing because of their small size, high integration, low cost and ability to measure in few microliters of sample. In this context, in this review article, we summarize and discuss about the use of SPEs as analytical tools in the development of (bio)sensors for pesticides of interest for food control. Finally, aspects related to the analytical performance of the developed (bio)sensors together with prospects for future improvements are discussed.

Competitive electrochemical immunosensor for the detection of unfolded p53 protein in blood as biomarker for Alzheimer's disease.

Alzheimer's disease is one of the most common causes of dementia nowadays, and its prevalence increases over time. Because of this and the difficulty of its diagnosis, accurate methods for the analysis of specific biomarkers for an early diagnosis of this disease are much needed. Recently, the levels of unfolded isoform of the multifunctional protein p53 in plasma have been proved to increase selectively in Alzheimer's Disease patients in comparison with healthy subjects, thus entering the list of biomarkers that can be used for the diagnosis of this illness. We present here the development of an electrochemical immunosensor based on nanostructured screen-printed carbon electrodes for the quantification of unfolded p53 in plasma samples. The sensor shows a suitable linear range (from 2 to 50 nM) for its application in real blood samples and a very low limit of detection (0.05 nM). The concentration of unfolded p53 has been accurately detected in plasma of elderly people in healthy conditions, subjects with mild cognitive impairment (MCI) and Alzheimer's Disease (AD) subjects, obtaining results with no significant differences to those provided by an ELISA assay. These results support the possibility of measuring unfolded p53 levels with a cheap, simple and miniaturized device with a promising future for point-of-care applications in the early diagnosis of Alzheimer's dementia.

Direct competitive immunosensor for Imidacloprid pesticide detection on gold nanoparticle-modified electrodes.

A direct competitive immunosensor for the electrochemical determination of Imidacloprid (IMD) pesticide on gold nanoparticle-modified screen-printed carbon electrodes (AuNP-SPCE) is here reported for the first time. Self-obtained specific monoclonal antibodies are immobilized on the AuNP-SPCE taking advantage of the AuNPs biofunctionalization abilities. In our biosensor design, free IMD in the sample competes with IMD conjugated with horseradish peroxidase (IMD-HRP) for the recognition by the antibodies. After that, 3,3',5,5'-Tetramethylbenzidine (TMB) is enzymatically oxidized by HRP, followed by the oxidized TMB reduction back at the surface of the SPCE. This process gives an associated catalytic current (analytical signal) that is inversely proportional to the IMD amount. The main parameters affecting the analytical signal have been optimized, reaching a good precision (repeatability with a RSD of 6%), accuracy (relative error of 6%), stability (up to one month), selectivity and an excellent limit of detection (LOD of 22 pmol L-1), below the maximum levels allowed by the legislation, with a wide response range (50-10000 pmol L-1). The detection through antibodies also allows to have an excellent selectivity against other pesticides potentially present in real samples. Low matrix effects were found when analysing IMD in tap water and watermelon samples. The electrochemical immunosensor was also validated with HPLC-MS/MS, the reference method used in official laboratories for IMD analysis, through statistical tests. Our findings make the electrochemical immunosensor as an outstanding method for the rapid and sensitive determination of IMD at the point-of-use.

MCE-electrochemical detection for following interactions of ssDNA and dsDNA with methylene blue.

The interaction between the organic dye, methylene blue and DNA has been studied by MCE with electrochemical detection. Interaction produces two different signals, one corresponding to free methylene blue and other, for the complex methylene blue-DNA. The hybridization between a ssDNA and a complementary sequence, specific to the severe acute respiratory syndrome virus, has been performed and studied in a thermoplastic olefin polymer of amorphous structure CE-microchip with an end-channel gold wire detector. Moreover, studies with a longer dsDNA, an expression vector involved in the transitory or stable expression in mammals cells, pFLAG-CMV4, has also been performed.