Palladium nanoclusters as a label to determine GFAP in human serum from donors with stroke by bimodal detection: inductively coupled plasma-mass spectrometry and linear sweep voltammetry.

Water-soluble, stable, and monodisperse palladium nanoclusters (PdNCs) were synthesized using NaBH4 as a reductant and lipoic acid as a ligand. PdNCs, measured by high-resolution transmission electron microscopy, showed a round shape and a diameter of 2.49 ± 0.02 nm. It was found that each PdNC contains 550 Pd atoms on average. These PdNCs offer high amplification as a label of biochemical reactions when inductively coupled plasma-mass spectrometry (ICP-MS) is used as a detector. In addition, PdNCs have catalytic activity on electrochemical reactions, allowing detection by linear sweep voltammetry (LSV). As a proof of applicability, a competitive immunoassay based on PdNC labels was developed for the determination of glial fibrillary acidic protein (GFAP) in human serum, comparing ICP-MS and LSV detection. GFAP is a biomarker for differentiating between patients with ischemic stroke (IS) and hemorrhagic stroke (HS). The limit of detection (LoD), corresponding to IC10 (4-parameter logistic curve), was 0.03 pM of GFAP, both by ICP-MS and LSV, being lower than the 0.31 pM LoD provided by the ELISA commercial kit. Using the error profile method, 0.03 pM and 0.11 pM LoDs were obtained respectively by ICP-MS and LSV: LoD is lower by ICP-MS due to the better precision of the measurements. The analyses of human serum samples from IS, HS, and control (CT) donors using PdNC labels and detection by ICP-MS and LSV were validated with a commercial ELISA kit (for CT donors only ICP-MS provided enough sensitivity). Results point out toward the future use of PdNCs as a label in other immunoprobes for the determination of specific proteins requiring very low LoDs as well as the development of electrochemical decentralized methodologies.

Electrochemical Detection for Isothermal Loop-Mediated Amplification of Pneumolysin Gene of Streptococcus pneumoniae Based on the Oxidation of Phenol Red Indicator.

A highly sensitive electrochemical methodology for end-point detection of loop-mediated isothermal nucleic acid amplification reactions was developed. It is based on the oxidation process of phenol red (PR), commonly used as a visual indicator. The dependence of its redox process on pH, which changes during amplification, allows performing quantitative measurements. Thus, the change in the oxidation potential of PR during the amplification is used, for the first time, as the analytical signal that correlates with the number of initial DNA copies. As a proof-of-concept, the amplification of the pneumolysin gene from Streptococcus pneumoniae, one of the main pathogens causing community-acquired pneumonia, is performed. Combination of isothermal amplification with electrochemical detection, performed on small-size flexible electrodes, allows easy decentralization. Adaptation to the detection of other pathogens causing infectious diseases would be very useful in the prevention of future epidemics.

Paper-Based Enzymatic Electrochemical Sensors for Glucose Determination.

The general objective of Analytical Chemistry, nowadays, is to obtain best-quality information in the shortest time to contribute to the resolution of real problems. In this regard, electrochemical biosensors are interesting alternatives to conventional methods thanks to their great characteristics, both those intrinsically analytical (precision, sensitivity, selectivity, etc.) and those more related to productivity (simplicity, low costs, and fast response, among others). For many years, the scientific community has made continuous progress in improving glucose biosensors, being this analyte the most important in the biosensor market, due to the large amount of people who suffer from diabetes mellitus. The sensitivity of the electrochemical techniques combined with the selectivity of the enzymatic methodologies have positioned electrochemical enzymatic sensors as the first option. This review, focusing on the electrochemical determination of glucose using paper-based analytical devices, shows recent approaches in the use of paper as a substrate for low-cost biosensing. General considerations on the principles of enzymatic detection and the design of paper-based analytical devices are given. Finally, the use of paper in enzymatic electrochemical biosensors for glucose detection, including analytical characteristics of the methodologies reported in relevant articles over the last years, is also covered.

Paper-based platforms with coulometric readout for ascorbic acid determination in fruit juices.

There is growing interest in the development of simple, fast, sustainable and low-cost analytical methodologies on paper-based platforms. However, sensitive detection strategies that fit properly with these devices are still required. In this work, a calibration-free method is proposed for analytical determinations performed on paper-based electrochemical devices, in this case, for ascorbic acid. Carbon ink is deposited on a hydrophilic working area of the paper delimited with a hydrophobic wax. This maskless procedure is fast and cuts down ink waste. The connection of this working electrode to the potentiostat is provided by reusable gold-plated connector headers that provide also the reference and counter electrodes. The thickness of the paper substrate defines the electrochemical cell and confines a sample volume, ideal for thin-layer coulometry. Controlled-potential coulometry is performed applying a potential of +0.6 V for 50 s. The charge is calculated by measuring the area under the fast chronoamperogram and the concentration is determined following Faraday's law (known number of transferred electrons). This methodology was applied to the determination of ascorbic acid, with a limit of detection of 40 µM. Its concentration in commercial fruit juices can be directly determined in diluted samples. The absence of matrix effects is observed by comparing the results obtained before and after enzymatic reaction of the sample with cucumber ascorbate oxidase. Good accuracy and precision makes this method suitable for quality control of ascorbic acid in commercial juices. Underexploited coulometric readout can be applied as a fast (calibration-free) and low-cost (standards not required) transduction principle for the newly developed paper devices.

Obtaining information from the brain in a non-invasive way: determination of iron in nasal exudate to differentiate hemorrhagic and ischemic strokes.

Background Differentiation between hemorrhagic and ischemic stroke is currently made by brain imaging or analyzing blood and cerebrospinal fluid (CSF) samples. After describing a new drainage route from brain to nasal mucosa, nasal exudate samples can be considered a new and promising source of biomarkers. Saliva can also be evaluated. Methods We determined iron in nasal exudate and saliva samples from patients of acute stroke during the first 48 h from onset. A simple, non-invasive sampling procedure was employed to obtain information from the brain. Samples were taken with a pre-weighed swab, solved in a 2% nitric acid solution and iron was measured by inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS). Results A significant difference in the dispersion of results of iron concentration for both stroke subtypes was observed in nasal exudate samples. The interquartile range was 0.608 nmol mg-1 of iron for hemorrhagic strokes and only 0.044 nmol mg-1 for ischemic strokes. In saliva samples, however, the values were 0.236 vs. 0.157 nmol mg-1. A cut-off limit of 0.102 nmol of iron per mg of nasal exudate provides a methodology with a 90% of sensitivity and a 90% of specificity. The value of the area under (AUC) the receiver operating characteristic curve (ROC) for nasal exudate samples is 0.960, considered as very good in which regards to its predictive value. Conclusions Non-invasive samples of nasal secretion have allowed obtaining, for the first time, information from the brain. Determination of iron in nasal exudate by ICP-MS allowed differentiation between ischemic and hemorrhagic strokes.

Folding-Based Electrochemical Aptasensor for the Determination of ß-Lactoglobulin on Poly-L-Lysine Modified Graphite Electrodes.

Nowadays, food allergy is a very important health issue, causing adverse reactions of the immune system when exposed to different allergens present in food. Because of this, the development of point-of-use devices using miniaturized, user-friendly, and low-cost instrumentation has become of outstanding importance. According to this, electrochemical aptasensors have been demonstrated as useful tools to quantify a broad variety of targets. In this work, we develop a simple methodology for the determination of ß-lactoglobulin (ß-LG) in food samples using a folding-based electrochemical aptasensor built on poly-L-lysine modified graphite screen-printed electrodes (GSPEs) and an anti-ß-lactoglobulin aptamer tagged with methylene blue (MB). This aptamer changes its conformation when the sample contains ß-LG, and due to this, the spacing between MB and the electrode surface (and therefore the electron transfer efficiency) also changes. The response of this biosensor was linear for concentrations of ß-LG within the range 0.1-10 ng·mL-1, with a limit of detection of 0.09 ng·mL-1. The biosensor was satisfactorily employed for the determination of spiked ß-LG in real food samples.

Open-Source Potentiostat for Wireless Electrochemical Detection with Smartphones.

This paper describes the design and characterization of an open-source "universal wireless electrochemical detector" (UWED). This detector interfaces with a smartphone (or a tablet) using "Bluetooth Low Energy" protocol; the smartphone provides (i) a user interface for receiving the experimental parameters from the user and visualizing the result in real time, and (ii) a proxy for storing, processing, and transmitting the data and experimental protocols. This approach simplifies the design, and decreases both the size and the cost of the hardware; it also makes the UWED adaptable to different types of analyses by simple modification of the software. The UWED can perform the most common electroanalytical techniques of potentiometry, chronoamperometry, cyclic voltammetry, and square wave voltammetry, with results closely comparable to benchtop commercial potentiostats. Although the operating ranges of electrical current and voltage of the UWED (±1.5 V, ±180 µA) are more limited than most benchtop commercial potentiostats, its functional range is sufficient for most electrochemical analyses in aqueous solutions. Because the UWED is simple, small in size, assembled from inexpensive components, and completely wireless, it offers new opportunities for the development of affordable diagnostics, sensors, and wearable devices.

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.

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.

Folding analytical devices for electrochemical ELISA in hydrophobic R(H) paper.

This work describes a device for electrochemical enzyme-linked immunosorbent assay (ELISA) designed for low-resource settings and diagnostics at the point of care. The device is fabricated entirely in hydrophobic paper, produced by silanization of paper with decyl trichlorosilane, and comprises two zones separated by a central crease: an embossed microwell, on the surface of which the antigen or antibody immobilization and recognition events occur, and a detection zone where the electrodes are printed. The two zones are brought in contact by folding the device along this central crease; the analytical signal is recorded from the folded configuration. Two proof-of-concept applications, an electrochemical direct ELISA for the detection of rabbit IgG as a model antigen in buffer and an electrochemical sandwich ELISA for the detection of malarial histidine-rich protein from Plasmodium falciparum (Pf HRP2) in spiked human serum, show the versatility of this device. The limit of detection of the electrochemical sandwich ELISA for the quantification of Pf HRP2 in spiked human serum was 4 ng mL(-1) (10(2) pmol L(-1)), a value within the range of clinically relevant concentrations.

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.

Electrocatalytic Palladium Nanoclusters as Versatile Indicators of Bioassays: Rapid Electroanalytical Detection of SARS-CoV-2 by Reverse Transcription Loop-Mediated Isothermal Amplification.

Quantitative polymerase chain reaction (qPCR) is considered the gold standard for pathogen detection. However, improvement is still required, especially regarding the possibilities of decentralization. Apart from other reasons, infectious diseases demand on-site analysis to avoid pathogen spreading and increase treatment efficacy. In this paper, the detection of SARS-CoV-2 is carried out by reverse transcription loop-mediated isothermal amplification, which has the advantage of requiring simple equipment, easily adaptable to decentralized analysis. It is proposed, for the first time, the use of palladium nanoclusters (PdNCs) as indicators of the amplification reaction at end point. The pH of the medium decreases during the reaction and, in turn, a variation in the catalytic activity of PdNCs on the oxygen reduction reaction (ORR) can be electrochemically observed. For the detection, flexible and small-size screen-printed electrodes can be premodified with PdNCs, which together with the use of a simple and small electrochemical equipment would greatly facilitates their integration in field-deployable devices. This would allow a faster detection of SARS-CoV-2 as well as of other future microbial threats after an easy adaptation.

Electrochemical micropipette-tip for low-cost environmental applications: Determination of anionic surfactants through their interaction with methylene blue.

Miniaturization is one of the main requirements in the design of portable devices that allow in-field analysis. This is especially interesting in environmental monitoring, where the time of the sample-to-result process could be decreased considerably by approaching the analytical platforms to the sampling point. We employed traditional mass-produced and low-cost elements (micropipette tips and pins) in an out-of-box application to generate an innovative and cost-effective platform for analytical purposes. We have designed simple and easy-to-use electrochemical cells inside polypropylene micropipette tips with three stainless-steel pins acting as the working, reference and counter electrodes of a potentiostatic system. The pin acting as working electrode was previously coated with carbon ink, meanwhile the rest were used unmodified. In this way, electrochemical in-the-tip measurements were done directly using low volumes (µL) of sample. The devices showed good reproducibility, with a relative standard deviation of 7% (n = 5) for five different tip-based complete electrochemical cells. As a proof-of-concept, its utility has been probed by the determination of an anionic surfactant (sodium dodecyl sulphate, SDS) in water through its interaction with methylene blue (MB). Two different alternatives were presented based on the: 1) increase in the current intensity of the cathodic peak of MB due to the presence of SDS; 2) electrochemical determination of the MB remaining in the aqueous phase after extraction of the pair SDS-MB to an organic medium.

Micropipette Tip-Based Immunoassay with Electrochemical Detection of Antitissue Transglutaminase to Diagnose Celiac Disease Using Staples and a Paper-Based Platform.

In this work, 1-200 µL polypropylene micropipette tips were used as platforms for performing immunoassays after converting their inner surfaces on a capture zone for the analyte of interest. We have used a micropipette-tip immunoelectroanalytical platform for the detection of antitissue transglutaminase (IgA), the main biomarker for celiac disease. Modification of the tip wall with poly-l-lysine allowed adsorption of tissue transglutaminase (tTG), which will capture later anti-tTG (IgA) antibodies developed in celiac-affected people. A sandwich-type format was followed, incubating simultaneously the analyte and the detection antibody, labeled with horseradish peroxidase. With this new application for an extremely common lab material, we can perform quantitative analysis by dispensing the liquid into a low-cost and miniaturized staple-based paper electrochemical platform. The analytical signal was the reduction of the enzymatically oxidized substrate, recorded chronoamperometrically (i-t curve). The intensity of the current obtained at a fixed time after the application of the cathodic potential followed a linear relationship with anti-tTG (IgA) concentration. The relative standard deviation obtained for immunoassays performed in different tips indicates the adequate precision of this new methodology, which is very promising for decentralized analysis. Negative and positive controls produced results that were in accordance with those obtained with spectrophotometric enzyme linked-immunosorbent assays.

Fabrication and evaluation of single- and dual-channel (Pi-design) microchip electrophoresis with electrochemical detection.

A new dual-channel microchip capillary electrophoresis (MCE) has been developed on glass substrates for the first time with electrochemical detection. Dual-channel (called Pi-design) as well as single-channel microchips have been fabricated on soda-lime glass using photolithography, wet etching and thermal bonding. Moreover, a laser writing system has been applied for the fabrication of photomasks with the different microchip designs (single- and dual-channel configurations). The microfabricated channels have been characterized by optical, confocal and scanning electron microscopy. The resulting single- and dual-channel microchips have been evaluated using an end-channel amperometric detector based on one (single-channel) or two (dual-channel) 100-mum gold wires aligned at the outlet of the separation channel. Parameters affecting the separation of several phenolic compounds (dopamine, p-aminophenol and hydroquinone) have been studied in the glass microchips. Thus, the influence of separation voltage, detection potential and background electrolyte has been examined in the single-channel microchip. Different total length microchannel has been compared. Furthermore, the possibility of carrying out two simultaneous measurements has been demonstrated in the new dual-channel microchip electrophoresis. The injection format has been checked and resulted to be critical, in such a way that a special and new form is employed for obtaining simultaneous signals at both channels. Analytical characteristics, such as sensitivity and reproducibility have been evaluated and resulted very adequate.

In situ gold-nanoparticle electrogeneration on gold films deposited on paper for non-enzymatic electrochemical determination of glucose.

This work describes the development and evaluation of a new electrochemical platform based on the sustainable generation of gold-nanoparticles on paper-based gold-sputtered electrodes. The disposable porous paper electrode is combined with screen-printed electrodes for ensuring a precise electrogeneration of nanoparticles and also for the evaluation of these simple, versatile and low-cost microfluidic devices. Two types of chromatographic paper with different thicknesses have been evaluated. Paper gold working electrodes modified with gold nanoparticles were characterized by scanning electron microscopy and cyclic voltammetry using potassium ferrocyanide as a common redox probe, showing an improved electrochemical performance when compared to bare gold electrodes. The platform has been applied to the non-enzymatic determination of glucose, molecule of enormous interest. The porous gold structure made by sputtering on paper, modified with electrogenerated nanoparticles allowed precise and accurate determination of the analyte in beverages at low potential.

DNA single-base mismatch study with an electrochemical enzymatic genosensor.

A thorough selectivity study of DNA hybridization employing an electrochemical enzymatic genosensor is discussed here. After immobilizing on a gold film a 30-mer 3'-thiolated DNA strand, hybridization with a biotinylated complementary one takes place. Then, alkaline phosphatase is incorporated to the duplex through the interaction streptavidin-biotin. Enzymatic generation of indigo blue from 3-indoxyl phosphate and subsequent electrochemical detection was made. The influence of hybridization conditions was studied in order to better discern between fully complementary and mismatched strands. Detection of 3, 2 and 1 mismatch was possible. The type and location of the single-base mismatch, as well as the influence of the length of the strands was studied too. Mutations that suppose displacement of the reading frame were also considered. The effect of the concentration on the selectivity was tested, resulting a highly selective genosensor with an adequate sensitivity and stability.

Oriented immobilisation of anti-pneumolysin Fab through a histidine tag for electrochemical immunosensors.

Orientation of reagents is a key step in the construction of immunosensors. When the immunoreagent is a recombinant protein, this can be achieved by employing hexahistidine tags. The orientation of recombinant histidine-tagged Fab fragments of monoclonal anti-pneumolysin antibodies on gold films is evaluated. Using histidine as a chelator of Ni or employing an anti-polyhistidine antibody for capturing the His6 residue is considered. Measurements are based in the signal of indigo, which comes from the hydrolysis of 3-indoxylphosphate by alkaline phosphatase (AP). The attachment of the enzyme occurs through the interaction of biotin with AP-labelled streptavidin or employing AP-conjugated immunoreagents. In the case of the interaction Ni-histidine, for the study of the self-assembling process a His-tagged and biotinylated protein (His6-GST-B) was employed. General conditions were studied and non-specific adsorption was avoided with the use of 1-hexanethiol. Improvements of the signal compared with the direct adsorption were only achieved by the use of histidine capturing antibodies. With an optimised ratio anti-polyhis:His6-Fab the signal increases approximately a 100%. Precision is adequate and the response is linear with the concentration of pneumolysin between 0.1 and 10 ng/mL.

Pin-based electrochemical glucose sensor with multiplexing possibilities.

This work describes the use of mass-produced stainless-steel pins as low-cost electrodes to develop simple and portable amperometric glucose biosensors. A potentiostatic three-electrode configuration device is designed using two bare pins as reference and counter electrodes, and a carbon-ink coated pin as working electrode. Conventional transparency film without any pretreatment is used to punch the pins and contain the measurement solution. The interface to the potentiostat is very simple since it is based on a commercial female connection. This electrochemical system is applied to glucose determination using a bienzymatic sensor phase (glucose oxidase/horseradish peroxidase) with ferrocyanide as electron-transfer mediator, achieving a linear range from 0.05 to 1mM. It shows analytical characteristics comparable to glucose sensors previously reported using conventional electrodes, and its application for real food samples provides good results. The easy modification of the position of the pins allows designing different configurations with possibility of performing simultaneous measurements. This is demonstrated through a specific design that includes four pin working-electrodes. Different concentrations of antibody labeled with alkaline phosphatase are immobilized on the pin-heads and after enzymatic conversion of 3-indoxylphosphate and silver nitrate, metallic silver is determined by anodic stripping voltammetry.

Point-of-need simultaneous electrochemical detection of lead and cadmium using low-cost stencil-printed transparency electrodes.

In this work, we report a simple and yet efficient stencil-printed electrochemical platform that can be integrated into the caps of sample containers and thus, allows in-field quantification of Cd(II) and Pb(II) in river water samples. The device exploits the low-cost features of carbon (as electrode material) and paper/polyester transparency sheets (as substrate). Electrochemical analysis of the working electrodes prepared on different substrates (polyester transparency sheets, chromatographic, tracing and office papers) with hexaammineruthenium(III) showed that their electroactive area and electron transfer kinetics are highly affected by the porosity of the material. Electrodes prepared on transparency substrates showed the best electroanalytical performance for the simultaneous determination of Cd(II) and Pb(II) by square-wave anodic stripping voltammetry. Interestingly, the temperature and time at which the carbon ink was cured had significant effect on the electrochemical response, especially the capacitive current. The amount of Cd and Pb on the electrode surface can be increased about 20% by in situ electrodeposition of bismuth. The electrochemical platform showed a linear range comprised between 1 and 200 µg/L for both metals, sensitivity of analysis of 0.22 and 0.087 µA/ppb and limits of detection of 0.2 and 0.3 µg/L for Cd(II) and Pb(II), respectively. The analysis of river water samples was done directly in the container where the sample was collected, which simplifies the procedure and approaches field analysis. The developed point-of-need detection system allowed simultaneous determination of Cd(II) and Pb(II) in those samples using the standard addition method with precise and accurate results.