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The advent of immunotherapies with biological drugs has revolutionized the treatment of cancers and auto-immune diseases. However, in some patients, the production of anti-drug antibodies (ADAs) hampers the drug efficacy. The concentration of ADAs is typically in the range of 1-10 pm; hence their immunodetection is challenging. ADAs toward Infliximab (IFX), a drug used to treat rheumatoid arthritis and other auto-immune diseases, are focussed. An ambipolar electrolyte-gated transistor (EGT) immunosensor is reported based on a reduced graphene oxide (rGO) channel and IFX bound to the gate electrode as the specific probe. The rGO-EGTs are easy to fabricate and exhibit low voltage operations (≤ 0.3 V), a robust response within 15 min, and ultra-high sensitivity (10 am limit of detection). A multiparametric analysis of the whole rGO-EGT transfer curves based on the type-I generalized extreme value distribution is proposed. It is demonstrated that it allows to selectively quantify ADAs also in the co-presence of its antagonist tumor necrosis factor alpha (TNF-α), the natural circulating target of IFX., (© 2023 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

A new spectroelectrochemical two-enzyme sensor system has been developed for the detection of acetaldehyde in wine. A combination of spectroscopy and electrochemistry improves the analytical features of the electrochemical sensor because the optical information collected with this system is only associated with acetaldehyde and avoids the interferents also present in wines as polyphenols. Spectroelectrochemical detection is achieved by the analysis of the optical properties of the K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ] redox couple involved in the enzymatic process: aldehyde dehydrogenase catalyzes the aldehyde oxidation using β-nicotinamide adenine dinucleotide hydrate (NAD + ) as a cofactor and, simultaneously, diaphorase reoxidizes the NADH formed in the first enzymatic process due to the presence of K 3 [Fe(CN) 6 ]. An analysis of the characteristic UV-vis bands of K 3 [Fe(CN) 6 ] at 310 and 420 nm allows the detection of acetaldehyde, since absorption bands are only related to the oxidation of this substrate, and avoids the contribution of other interferents.

Health monitoring is experiencing a radical shift from clinic-based to point-of-care and wearable technologies, and a variety of nanomaterials and transducers have been employed for this purpose. 2D materials (2DMs) hold enormous potential for novel electronics, yet they struggle to meet the requirements of wearable technologies. Here, aiming to foster the development of 2DM-based wearable technologies, reduced graphene oxide (rGO)-based liquid-gated transistors (LGTs) for cation sensing in artificial sweat endowed with distinguished performance and great potential for scalable manufacturing is reported. Laser micromachining is employed to produce flexible transistor test patterns employing rGO as the electronic transducer. Analyte selectivity is achieved by functionalizing the transistor channel with ion-selective membranes (ISMs) via a simple casting method. Real-time monitoring of K + and Na + in artificial sweat is carried out employing a gate voltage pulsed stimulus to take advantage of the fast responsivity of rGO. The sensors show excellent selectivity toward the target analyte, low working voltages (<0.5 V), fast (5-15 s), linear response at a wide range of concentrations (10 µm to 100 mm), and sensitivities of 1 µA/decade. The reported strategy is an important step forward toward the development of wearable sensors based on 2DMs for future health monitoring technologies., (© 2022 The Authors. Small published by Wiley-VCH GmbH.)

Although antibody immobilization on solid surfaces is extensively used in several applications, including immunoassays, biosensors, and affinity chromatography, some issues are still challenging. Self-assembling protein layers can be used to coat easily different surfaces by direct deposition. A specific biofunctional layer can be formed using genetic engineering techniques to express fused proteins acting as self-immobilizing antibodies. In this study, fusion proteins combining the self-assembling adhesive properties of a fungal hydrophobin and the functionality of the single chain fragment variables (ScFvs) of two antibodies were produced. The chosen ScFvs are able to recognize marine toxins associated with algal blooms, saxitoxin, and domoic acid, which can bioaccumulate in shellfish and herbivorous fish causing food poisoning. ScFvs fused to hydrophobin Vmh2 from Pleurotus ostreatus were produced in Escherichia coli and recovered from the inclusion bodies. The two fusion proteins retained the functionality of both moieties, being able to adhere on magnetic beads and to recognize and bind the two neurotoxins, even with different performances. Our immobilization procedure is innovative and very easy to implement because it allows the direct functionalization of magnetic beads with ScFvs, without any surface modification. Two different detection principles, electrochemical and optical, were adopted, thus achieving a versatile platform suitable for different antigen detection methods. The sensitivity of the saxitoxin optical biosensor [limit of detection (LOD) 1.7 pg/ml] is comparable to the most sensitive saxitoxin immunosensors developed until now., Competing Interests: Authors DIB and MBGG are employed by the company Metrohm DropSens. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Stanzione, Izquierdo-Bote, González García, Giardina and Piscitelli.)

Enhancement of Raman intensity due to the electrochemical surface-enhanced Raman scattering (EC-SERS) effect is an interesting alternative to overcome the lack of sensitivity traditionally associated with Raman spectroscopy. Furthermore, activation of metallic screen-printed electrodes (SPEs) by electrochemical route leads to the reproducible generation of nanostructures with excellent SERS properties. EC-SERS procedure proposed in this work for the detection of several pesticides (thiram, imidacloprid and chlorpyrifos) with different nature, uses gold SPEs as SERS substrates, but also includes a preconcentration step as the initial and essential stage. Taking into account the small volume of solution employed, only 60 µL, the preconcentration cannot be performed for more than 15 min in order to ensure the proper contact of the solution with WE, RE and CE. Furthermore, selected temperature, 34 °C, is not very high to allow the exhaustive control of the drop volume. Optimization of preconcentration parameters (time and temperature) displays a crucial step, particularly in the detection of low concentrations of pesticides, because it will provide higher Raman intensity in EC-SERS experiments. After the initial step, gold SPEs were electrochemically activated by cyclic voltammetry, allowing the detection of very low concentration (µg·L -1 ) of pesticides due to the generation of fresh nanostructures with SERS effect., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A detailed analysis of the ECL interaction between luminol and tris(2,2'-bipyridyl)dichlororuthenium(ii) (Ru(bpy) 3 2+ ) is required before using them in ECL systems for multianalyte detection purposes. Spectro-electrochemiluminescence demonstrates that not only must the emission properties be considered, but also their additional optical characteristics are involved in the explanation of the interaction mechanism between these luminophores.

Metal phthalocyanines are well-known sensing phases with applications in different scientific fields due to their interesting properties. Detailed characterization by Raman spectroscopy was performed in order to study the shifting of the vibrational bands related to the coordination sphere of each metal phthalocyanine. In this work, a study involving the use of screen-printed electrodes (SPEs) with various metal phthalocyanines to electrochemically detect and quantify chlorine (Cl 2 ) gas is presented. The Cl 2 gas was generated in-situ via oxidation of the chloride present in form of aqueous salt solutions. The developed method offers not only the possibility to quantify chlorine, but also to discriminate among several chlorinated species due to the changes observed in the voltammetric profiles associated with the interaction between the specie assayed and the phthalocyanine metallic center. Optimization of detecting parameters was also performed to apply this procedure for the quantification of chlorine generated from commercial chlorine tablets. The development of this proof of concept shows interesting possibilities and easy-to-use applications with novel on metal phthalocyanines based SPE sensors., Competing Interests: The authors declare no conflict of interest.

Recent progress in the field of electroanalysis with metal nanoparticle (NP)-based screen-printed electrodes (SPEs) is discussed, focusing on the methods employed to perform the electrode surface functionalization, and the final application achieved with different types of metallic NPs. The ink mixing approach, electrochemical deposition, and drop casting are the usual methodologies used for SPEs' modification purposes to obtain nanoparticulated sensing phases with suitable tailor-made functionalities. Among these, applications on inorganic and organic molecule sensing with several NPs of transition metals, bimetallic alloys, and metal oxides should be highlighted.

Raman spectroelectrochemistry based on electrochemical surface-enhanced Raman scattering (EC-SERS) effect is an interesting alternative to overcome the lack of sensitivity of normal Raman spectroscopy. Electrochemical activation of metallic screen-printed electrodes (SPEs) leads to the reproducible generation of nanostructures with excellent SERS properties. In that way, gold SPEs circumvent the traditional reproducibility limitation and produce the enhancement of the Raman intensity to favor the detection of low concentrations. Furthermore, fingerprint features of Raman spectroscopy make possible the dynamic spectroelectrochemical analysis of B vitamins. The accuracy assignments of Raman bands associated with B1, B2, B3, B6 and B12 vitamins present in multivitamin complexes provides valuable information, allowing us not only the detection of B vitamin present in mixtures, but also to understand the interaction between vitamins and metallic SERS surfaces., (Copyright © 2019 Elsevier B.V. All rights reserved.)

NIR spectroelectrochemistry has scarcely been used for deconvolving aqueous mixtures due to the water restriction in this spectral range. However, this work offers an interesting approach for the study of mixtures of molecules with similar electrochemical and spectroscopic behaviour by overcoming the limitations of this hybrid technique. As a proof of concept, the resolution of mixtures of two dyes with similar chemical structures demonstrates the usefulness of NIR spectroelectrochemistry.

Spectroelectrochemical techniques are becoming increasingly versatile tools to solve a diverse range of analytical problems. Herein, the use of in situ real-time luminescence spectroelectrochemistry to quantify chloride ions is demonstrated. Utilizing the bleaching effect of chlorine-based electrogenerated products after chloride oxidation, it is shown that the fluorescence of the rhodamine 6G dye decreases proportionally to the initial chloride concentration in solution. A strong decrease of fluorescence is observed in acidic media compared to a lower decrease in alkaline media, which suggests that Cl 2 , favorably generated at low pH, could be the main species responsible for the fluorescence loss. This fact is corroborated with chronoamperometric measurements where the complete loss of fluorescence for the bulk solution is achieved. A fast mass transfer is needed to explain this behavior, in agreement with the generation of gaseous species such as Cl 2 . Chloride detection was performed in artificial sweat samples in less than 30 s with great accuracy. This electrochemical/optical combined approach allows us to quantify species that are difficult to measure by electrochemistry due to the inadequate resolution of their redox processes or being without significant optical properties.

Dynamic time-resolved Raman spectroelectrochemistry demonstrates the strong influence of nanostructuring and surface charge of in situ activated disposable substrates for SERS detection. Under specific conditions, a large enhancement factor and estimated calculations agree with the feasible detection of only a few molecules, approaching the limit of single-entity detection.

In this work, a turn-off photoluminescent sensing proof-of-concept based on blue luminescent graphene quantum dots (GQDs) as the fluorescent probe was developed. For that purpose, GQDs optical response was related with the catalytic enzymatic activity of alkaline phosphatase (ALP), in the presence of hydroquinone diphosphate (HQDP). The hydrolysis of HQDP by ALP generated hydroquinone (HQ). The oxidation of HQ, enzymatically produced, to p-benzoquinone (BQ) resulted in the quenching of GQDs fluorescence (FL). Therefore, the developed luminescent sensing mechanism allowed the FL quenching with ALP activity to be related and thus quantified the concentration of ALP down to 0.5 nM of enzyme. This innovative design principle appears as a promising tool for the development of enzymatic sensors based on ALP labeling with fluorescent detection or even for direct ALP luminescent quantification in an easy, fast and sensitive manner., (Copyright © 2018 John Wiley & Sons, Ltd.)

Naratriptan, active pharmaceutical ingredient with antimigraine activity was electrochemically detected in untreated screen-printed carbon electrodes (SPCEs). Cyclic voltammetry and differential pulse voltammetry were used to carry out quantitative analysis of this molecule (in a Britton-Robinson buffer solution at pH 3.0) through its irreversible oxidation (diffusion controlled) at a potential of +0.75V (vs. Ag pseudoreference electrode). Naratriptan oxidation product is an indole based dimer with a yellowish colour (maximum absorption at 320nm) so UV-VIS spectroelectrochemistry technique was used for the very first time as an in situ characterization and quantification technique for this molecule. A reflection configuration approach allowed its measurement over the untreated carbon based electrode. Finally, time resolved Raman Spectroelectrochemistry is used as a powerful technique to carry out qualitative and quantitative analysis of Naratriptan. Electrochemically treated silver screen-printed electrodes are shown as easy to use and cost-effective SERS substrates for the analysis of Naratriptan., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A new Screen-printed electrodes (SPE) integrated in one channel flow-cell was developed. The one channel flow-cell is attached and directly changeable with electrode. In the new flow-cell the injection is done through an "in-line luer injection port" which can be less aggressive than wall-jet flow cell for a biological recognition element immobilized on the surface of the electrode. The sample volume can be easily controlled by the operator through a syringe. In this novel thin layer flow-cell screen-printed electrodes, the working electrode was modified with graphene materials, and an enhancement of electroactive area to 388% over a standard electrode was found. This new configuration was applied to study the entrapped cellobiose dehydrogenase from the ascomycete Corynascus thermophilus (CtCDH) in a photocrosslinkable PVA-based polymer. The calibration curve of lactose using optimized parameters shows a wide linear measurement ranges between 0.25 and 5mM. A good operational stability of the CtCDH-PVA-modified graphene electrode is obtained, which keeps the same initial activity during 8h and exhibits a good storage stability with a decrease of only 9% in analytical response after 3 months storage at 4 ◦ C., (Copyright © 2016. Published by Elsevier B.V.)

Although with early treatment phenylketonuria patients may have average intelligence levels, it is important to optimize the nutritional management to maintain adequate phenylalanine levels, so that patients can develop their intellectal potential free of abnormalities in their daily activities due to deficits of cognitive executive functions. This study presents a series of 26 patients, diagnosed and treated early, who underwent a psychometric evaluation together with phenylalanine determinations along their lives, and at the time of doing the tests. A trend is observed towards a reverse relationship between IQ and concurrent phenylalanine concentration, phenylalanine median and phenylalanine/tyrosine ratio. Likewise, a trend towards a negative relationship is observed between executive functions and concurrent phenylalanine values along patients' lives., (Sociedad Argentina de Pediatría.)

Food allergens are a real threat to sensitized individuals. Although food labeling is crucial to provide information to consumers with food allergies, accidental exposure to allergenic proteins may result from undeclared allergenic substances by means of food adulteration, fraud or uncontrolled cross-contamination. Allergens detection in foodstuffs can be a very hard task, due to their presence usually in trace amounts, together with the natural interference of the matrix. Methods for allergens analysis can be mainly divided in two large groups: the immunological assays and the DNA-based ones. Mass spectrometry has also been used as a confirmatory tool. Recently, biosensors appeared as innovative, sensitive, selective, environmentally friendly, cheaper and fast techniques (especially when automated and/or miniaturized), able to effectively replace the classical methodologies. In this review, we present the advances in the field of food allergens detection toward the biosensing strategies and discuss the challenges and future perspectives of this technology.

Herein, a new miniaturized analytical instrumentation for electrochemiluminescence (ECL) assays is presented. A photodiode integrated in an ECL cell combined with a potentiostat/galvanostat, all integrated in a one-piece instrument (μSTAT ECL), was developed. In addition, a complementary micro-spectrometer integrated in a similar ECL cell for luminescence spectra recording is also proposed. Both cells are intended to be used with screen-printed electrodes and all the devices are portable and small sized. Their performance was corroborated with two innovative proofs-of-concept that centered on the luminol transduction chemistry: a first time reported ECL assay based on the enzymatic reaction between an indoxyl substrate and the enzyme alkaline phosphatase, and the electrochemiluminescence resonance energy transfer (ECL-RET) process triggered by the electro-oxidized luminol to the acceptor fluorescein. The photodiode system revealed to be more sensitive than the spectrometer device in collecting the light; however, with the latter, it is possible to discriminate different luminescent species according to their maximum wavelength emission, which is extremely useful for carrying out simple and simultaneous ECL multiplex analyzes. The spectrometer device works as an excellent accessory to couple with the μSTAT ECL instrument, complementing the experiments. Graphical abstract Schematic representation of the ECL-RET: from luminol-H2O2 system to fluorescein, the micro-spectrometer for the light collection and the 3D representation of the ECL-RET reaction.

Herein we present highly stable ferromagnetic nickel nanowires modified with streptavidin (NiNW-STR). This versatile functionalized nanomaterial works as an excellent biosensing platform for the immobilization of a wide range of biotinylated molecules. Moreover, these NWs can be employed in magnetic-based assays. Different proofs-of-concept such as streptavidin-biotin assays and capture of single and double stranded DNA were successfully carried out, corroborating NiNW-STR usefulness. Moreover, repeatability and stability studies were also effectively performed., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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

A gold nanoparticle-coated screen-printed carbon electrode was used as the transducer in the development of an electrochemical immunosensor for Ara h 1 (a major peanut allergen) detection in food samples. Gold nanoparticles (average diameter = 32 nm) were electrochemically generated on the surface of screen-printed carbon electrodes. Two monoclonal antibodies were used in a sandwich-type immunoassay and the antibody-antigen interaction was electrochemically detected through stripping analysis of enzymatically (using alkaline phosphatase) deposited silver. The total time of the optimized immunoassay was 3h 50 min. The developed immunosensor allowed the quantification of Ara h 1 between 12.6 and 2000 ng/ml, with a limit of detection of 3.8 ng/ml, and provided precise (RSD <8.7%) and accurate (recovery >96.6%) results. The immunosensor was successfully applied to the analysis of complex food matrices (cookies and chocolate), being able to detect Ara h 1 in samples containing 0.1% of peanut., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The use of the first flow-cell for magnetic assays with an integrated magnet is reported here. The flow injection analysis system (FIA) is used for biotin determination. The reaction scheme is based on a one step competitive assay between free biotin and biotin labeled with horseradish peroxidase (B-HRP). The mixture of magnetic beads modified with streptavidin (Strep-MB), biotin and B-HRP is left 15 min under stirring and then a washing step is performed. After that, 100 μL of the mixture is injected and after 30s 100 μL of 3,3',5,5'-Tetramethylbenzidine (TMB) is injected and the FIAgram is recorded applying a potential of -0.2V. The linear range obtained is from 0.01 to 1 nM of biotin and the sensitivity is 758 nA/nM. The modification and cleaning of the electrode are performed in an easy way due to the internal magnet of the flow cell., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A miniaturized electrochemical biosensor array with in situ detection of quantum dots (QDs) was developed for the detection of anti-transglutaminase IgG antibodies (a celiac disease biomarker) in human sera. For the fabrication of the sensor, a 8-channel screen-printed carbon electrochemical arrays were used as transducers and modified with tissue-transglutaminase by adsorption. The immunologic reaction was carried out in a few simple steps: reaction with human serum, which contains the analyte of interest, followed by the immunoreaction with anti-human IgG labeled with CdSe/ZnS QDs and electrochemical detection of Cd(2+) released from QDs. All steps were performed on the screen-printed arrays as the solid support, and the detection of Cd(2+) was performed in situ after acid attack of the QDs without a transfer step by voltammetric stripping. The electrochemical response was correlated with the anti-transglutaminase IgG concentration. The developed electrochemical immunosensor is a trustful screening tool for celiac disease diagnosis discriminating between positive and negative sera samples with high sensitivity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Human epidermal growth factor receptor 2 (HER2) is a breast cancer biomarker that plays a major role in promoting breast cancer cell proliferation and malignant growth. The extracellular domain (ECD) of HER2 can be shed into the blood stream and its concentration is measurable in the serum fraction of blood. In this work an electrochemical immunosensor for the analysis of HER2 ECD in human serum samples was developed. To achieve this goal a screen-printed carbon electrode, modified with gold nanoparticles, was used as transducer surface. A sandwich immunoassay, using two monoclonal antibodies, was employed and the detection of the antibody-antigen interaction was performed through the analysis of an enzymatic reaction product by linear sweep voltammetry. Using the optimized experimental conditions the calibration curve (ip vs. log[HER2 ECD]) was established between 15 and 100 ng/mL and a limit of detection (LOD) of 4.4 ng/mL was achieved. These results indicate that the developed immunosensor could be a promising tool in breast cancer diagnostics, patient follow-up and monitoring of metastatic breast cancer since it allows quantification in a useful concentration range and has an LOD below the established cut-off value (15 ng/mL)., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Mercury is a metal that has been extensively studied, in large part due to its high toxicity. Therefore, mercury levels must be monitored in different sample types using analytical methods. This review summarizes the electrochemical methods that have been used for mercury analysis in a variety of samples. A critical evaluation of the methods and electrode materials employed for mercury analysis is presented according to the following classifications: bare electrodes, chemically modified electrodes and nanostructured electrodes. The advantages and disadvantages of each type of electrode material regarding mercury analysis are also presented., (© 2013 Elsevier B.V. All rights reserved.)

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.

Different very simple single-use alcohol enzyme sensors were developed using alcohol oxidase (AOX) from three different yeast, Hansenula sp., Pichia pastoris and Candida boidinii, and employing three different commercial mediator-based Screen-Printed Carbon Electrodes as transducers. The mediators tested, Prussian Blue, Ferrocyanide and Co-phthalocyanine were included into the ink of the working electrode. The procedure to obtain these sensors consists of the immobilization of the enzyme on the electrode surface by adsorption. For the immobilization, an AOX solution is deposited on the working electrode and left until dried (1h) at room temperature. The best results were obtained with the biosensor using Screen-Printed Co-phthalocyanine/Carbon Electrode and AOX from Hansenula sp. The reduced cobalt-phthalocyanine form is amperometrically detected at +0.4V (vs. Ag pseudo reference electrode). This sensor shows good sensitivity (1211 nA mM(-1)), high precision (2.1% RSD value for the slope value of the calibration plot) and wide linear response (0.05-1.00 mM) for ethanol determination. The sensor provides also accurate results for ethanol quantification in alcoholic drinks., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The first fructose sensor using a commercial screen-printed ferrocyanide/carbon electrodes (SPFCE) is reported here. The ferrocyanide is included in the carbon ink of the commercial screen-printed carbon electrode. The immobilization of enzyme d-fructose dehydrogenase (FDH) was carried out in an easy way. An aliquot of 10μL FDH was deposited on the electrode surface and left there until dried (approximately 1h) at room temperature. The sensor, so constructed, shows a good sensitivity to fructose (1.25μA/mM) with a slope deviation of ±0.02μA/mM and a linear range comprised between 0.1 and 1mM of fructose, with a limit of detection of 0.05mM. These sensors show good intersensors reproducibility after a previous pretreatment and a high stability. Fructose was determined in real samples as honey, Cola, fruit juices (orange, tomato, apple and pineapple), red wine, red and white grapes, musts and liquor of peach with a good accuracy., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Celiac disease is a gluten-induced autoimmune enteropathy characterized by the presence of tissue tranglutaminase (tTG) autoantibodies. A disposable electrochemical immunosensor (EI) for the detection of IgA and IgG type anti-tTG autoantibodies in real patient's samples is presented. Screen-printed carbon electrodes (SPCE) nanostructurized with carbon nanotubes and gold nanoparticles were used as the transducer surface. This transducer exhibits the excellent characteristics of carbon-metal nanoparticle hybrid conjugation and led to the amplification of the immunological interaction. The immunosensing strategy consisted of the immobilization of tTG on the nanostructured electrode surface followed by the electrochemical detection of the autoantibodies present in the samples using an alkaline phosphatase (AP) labelled anti-human IgA or IgG antibody. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with a commercial ELISA kit indicating that the electrochemical immunosensor is a trustful analytical screening tool., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Voltammetric enzyme dual sensors for simultaneous determination of free and total prostate specific antigen (fPSA and tPSA) are described. Alkaline Phosphatase (AP) and a mixture solution of 3-indoxyl phosphate and silver ions were used as the enzymatic label and substrate, respectively. 8A6 or 5G6 antibodies specific for free and total PSA, respectively, were immobilized on different screen-printed electrodes (SPEs)--screen-printed carbon electrodes, screen-printed gold electrodes and screen-printed carbon electrodes modified with nanogold--in order to be able to select one of the surfaces as the most adequate one to develop the dual sensor. Screen-printed carbon electrodes modified with nanogold were the SPEs with the best analytical characteristics and lead to the most repeatable bioelectrodes, so they were selected for the development of the dual sensor. On Dualsensor-nAu electrodes, 8A6 antibody was immobilized on one working electrode and 5G6 antibody was immobilized on the other one by deposition of a drop of solution of each antibody and left overnight at 4 degrees C. Biotinylated anti-PSA antibody and streptavidin-AP conjugate were used as detection reagents, giving rise, to our knowledge, to the first simultaneous electrochemical biosensor for free and total PSA. The PSA dual sensor was used to monitor PSA production from three different cultures of human androgen-sensitive prostate tumor cells.

Two electrochemical DNA hybridization biosensors (genosensors) for the detection of a 30-mer sequence unique to severe acute respiratory syndrome (SARS) virus are described in this work. Both genosensors rely on the hybridization of the oligonucleotide target with its complementary probe, which is immobilized on positively charged polylysine modified screen-printed carbon electrodes (SPCEs), through electrostatic interactions. In one design, a biotinylated target is used and the detection of the hybridization reaction is monitored using alkaline phosphatase labeled streptavidin (S-AP). This enzyme catalyzes the hydrolysis of the substrate 3-indoxyl phosphate (3-IP) to indigo, which is then solubilized to indigo carmine and detected by means of cyclic voltammetry (CV). In the other design, the target is labeled using an Au(I) complex, sodium aurothiomalate, and the duplex formation is detected by measuring, for first time, the current generated by the hydrogen evolution catalyzed by the gold label. Using 30 min of hybridization time, a detection limit of 8 pM is calculated for the enzymatic genosensor. Although this good sensitivity cannot be reached with the metal label (0.5 nM), the use of this label allows a considerable decrease of the analysis time. Both genosensors do not require the modification of the oligonucleotide probe and using stringent experimental conditions (60 min of hybridization time and 50% formamide in the hybridization buffer) can discriminate between a complementary oligonucleotide and an oligonucleotide with a three-base mismatch.

Alkaline phosphatase (AP) is one of the most used enzymatic labels for the development of ELISAs, immunosensors, DNA hybridization assays, etc. This enzyme catalyzes the dephosphorylation of a substrate into a detectable product usually quantified by optical or electrochemical measurements. This work is based on a substrate (3-indoxyl phosphate) that produces a compound able to reduce silver ions in solution into a metallic deposit, which is localized where the enzymatic label AP is attached. The deposited silver is electrochemically stripped into solution and measured by anodic stripping voltammetry. Its application to an enzymatic genosensor on streptavidin-modified screen-printed carbon electrodes for the detection of virulence nucleic acid determinants of autolysin gene, exclusively present on the genome of the human pathogen Streptococcus pneumoniae, is described. Compared with the direct voltammetric detection of indigo carmine, the anodic stripping voltammetry of silver ions is 14-fold more sensitive.

In this work, a gold complex is used as electroactive label for monitoring hybridization assays on glassy carbon electrodes. Ionic gold is bound to a 30-mer sequence of the SARS (severe acute respiratory syndrome) virus, responsible for the atypical pneumonia, using sodium aurothiomalate. In order to label this single strand, a mixture of sodium aurothiomalate and the strand is prepared. Then, it is incubated for 24 h at 37 degrees C and, finally, free gold is separated from the labeled strand by a dialysis against a 0.15M NaCl solution (pH 7.5). The DNA hybridization sensor is designed immobilizing the complementary probe on the pre-treated electrode surface and, then, the hybridization reaction takes place with the gold labeled strand. The electrochemical determination is based on the catalytic effect of electrodeposited gold on the reduction of silver ions. In non-stringent experimental conditions, a limit of detection of 15 fmol (30 microL) is obtained, and discrimination between a complementary oligonucleotide and a three-based mismatch complementary oligonucleotide is achieved. For the discrimination of a single-base mismatch, is needed to use stringent conditions (50% of formamide in the hybridization buffer).

Hydrolysis of indoxyl-based substrates by hydrolytic enzymes is a commonly used semiquantitative detection system that generates a water-insoluble indigo dye which is difficult to quantify. This work describes the quantitative analysis and enzyme kinetics for alkaline phosphatase (AP) and 5-bromo-4-chloro-3-indoxyl phosphate (BCIP) in solution obtained by applying known solubilization methodology from the textiles industry to the enzymatic product. This proposal is based on the reduction of the tetrahalo-indigo blue dye in a basic medium with the aim of generating its aqueous-soluble parent compound termed indigo white, which gives a rich yellow color in solution and is fluorescent. A quantitative ELISA (where a soluble end product is required) is accomplished for first time using BCIP as substrate.

p-Nitrophenyl phosphate is one of the most widely used substrates for alkaline phosphatase in ELISAs because its yellow, water-soluble product, p-nitrophenol, absorbs strongly at 405 nm. p-Nitrophenol is also electroactive; an oxidative peak at 0.97 V (vs. an Ag pseudoreference electrode) is obtained when a bare screen-printed carbon electrode is used. When an amperometric detector was coupled to a flow-injection analysis system the detection limit achieved for p-nitrophenol was 2x10(-8) mol L(-1), almost two orders of magnitude lower than that obtained by measuring the absorbance of the compound. By use of this electrochemical detection method, measurement of 7x10(-14) mol L(-1) alkaline phosphatase was achieved after incubation for 20 min. The feasibility of coupling immunoassay to screen-printed carbon electrode amperometric detection has been demonstrated by performing an ELISA for detection of pneumolysin, a toxin produced by Streptococcus pneumoniae, which causes respiratory infections. The method is simple, reproducible, and much more sensitive than traditional spectrophotometry.

A new electroactive label has been used to monitor immunoassays in the determination of human serum albumin (HSA) using glassy-carbon electrodes as supports for the immunological reactions. The label was a gold(I) complex, sodium aurothiomalate, which was bound to rabbit IgG anti-human serum albumin (anti-HSA-Au). The HSA was adsorbed on the electrode surface and the immunological reaction with gold-labelled anti-HSA was then performed for one hour by non-competitive or competitive procedures. The gold(I) bound to the anti-HSA was electrodeposited in 0.1 mol L-1 HCl at -1.00 V for 5 min then oxidised in 0.1 mol L-1 H2SO4 solution at +1.40 V for 1 min. Silver electrodeposition at -0.14 V for 1 min followed by anodic stripping voltammetry were then performed in aqueous 1.0 mol L-1 NH(3)-2.0x10(-4) mol L-1 AgNO3. For both non-competitive and competitive formats, calibration plots in the ranges 5.0x10(-10) to 1.0x10(-8) mol L-1 and 1.0x10(-10) to 1.0x10(-9) mol L-1 HSA, respectively, with estimated detection limits of 1.5x10(-10) mol L-1 (10 ng mL-1) and 1.0x10(-10) mol L-1 (7 ng mL-1), respectively, were obtained. Levels of HSA in two healthy volunteer urine samples were also evaluated, using both immunoassay formats.

3,3',5,5'-Tetramethylbenzidine (TMB) is the most commonly used chromogen for horseradish peroxidase (HRP) and so its performance as an electrochemical substrate was evaluated. Measurements of HRP activity in solution were carried out by using an amperometric detector coupled to a flow injection analysis (FIA) system. The enzymatic product was easily detected at a potential of +0.1 V (vs. Ag-pseudoreference electrode) at a bare screen-printed electrode placed in a homemade electrochemical flow cell. A high flow rate (4.3 mL min(-1)) of 0.5 M H2SO4 was used to obtain repeatable signals and a short analysis time. The detection limit achieved after 15 min of incubation was 2x10(-14) M of HRP. The applicability of the amperometric detector to ELISAs was demonstrated by using a commercially available kit for the quantification of interleukin-6 (IL-6) without modifying the kit manufacturer's protocol or the reagents for this test.

Voltammetric genosensors on streptavidin-modified screen-printed carbon electrodes (SPCEs) for the detection of virulence nucleic acid determinants of pneumolysin (ply) and autolysin (lytA) genes, exclusively present on the genome of the human pathogen Streptococcus pneumoniae, were described. The oligonucleotide probes were immobilized on electrochemically pretreated SPCEs through the streptavidin/biotin reaction. After that, the hybridization reaction was carried out with labeled complementary targets on the electrode surface. The ply and lytA targets were labeled using the universal linkage system, which consists of the use of a platinum(II) complex that acts as coupling agent between targets and a, usually fluorescent, molecule label. In this case, the platinum(II) complex acts as a label itself because the analytical signal is achieved by measuring chronoamperometrically the current generated by the hydrogen evolution catalyzed by platinum. In nonstringent experimental conditions, these genosensors can detect 24.5 fmol of 20-mer oligonucleotide target and discriminate between a complementary oligo and an oligo with a three-base mismatch. In presence of 25% formamide in the hybridization buffer, a single-base mismatch on the oligonucleotide target can be detected.

In this work, two methods have been compared to produce enzymatic voltammetric immunosensors for the determination of Mycobacterium tuberculosis antigens (Ag360 and Ag231), using a pre-oxidised screen-printed carbon electrode (SPCE) as a signal transduction element. The enzyme alkaline phosphatase (AP) was used in combination with the substrate 3-indoxyl phosphate (3-IP). In one design, the immune complexes between M. tuberculosis antigens and monoclonal antibodies against M. tuberculosis were formed out of the electrode surface. Then, the immune complexes were captured by biotinylated rabbit anti-M. tuberculosis antibodies, immobilised on the streptavidin modified SPCEs through the streptavidin:biotin reaction. Finally, an alkaline phosphatase (AP) labelled rabbit IgG anti-mouse immunoglobulin G was used as a detector antibody. In the other design, the M. tuberculosis antigens were captured by monoclonal antibodies against M. tuberculosis, which were immobilised on the electrode surface through the reaction with rabbit IgG passively adsorbed on the SPCEs. The biotinylated rabbit anti-M. tuberculosis antibodies were used with an alkaline phosphatase labelled streptavidin as detector antibodies. The best results for M. tuberculosis antigen determination were obtained using the immunosensor on the streptavidin modified SPCEs and the immune complexes between antigen Ag231 and monoclonal antibodies MabF184-3, with a detection limit of 1.0 ng/ml. The immunosensor was also applied to Ag231 spiked proteic matrices.

Voltammetric enzyme immunosensors based on the employment of streptavidin modified screen-printed carbon electrodes (SPCEs) for the detection of rabbit IgG, as a model analyte, were described. Alkaline phosphatase (AP) and 3-indoxyl phosphate (3-IP) were used as the enzymatic label and substrate, respectively. The adsorption of streptavidin was performed by deposition of a drop of a streptavidin solution overnight at 4 degrees C on the pre-oxidized surface of the SPCEs. The analytical characteristics of these sensors were evaluated using biotin conjugated to AP. The immunosensor devices were based on a specific reaction of rabbit IgG with its biotinylated antibodies, which were immobilised on the modified screen-printed carbon electrodes through the streptavidin:biotin reaction. The immunosensors were used for a direct determination of AP labelled rabbit IgG, and for free rabbit IgG detection using a sequential competitive immunoassay. A calibration curve in the range of 5 x 10(-11) to 1 x 10(-9)M of rabbit IgG was obtained with a estimated detection limit of 5 x 10(-11)M (7.0ng/ml). These immunosensors were stable for 5 months if they were stored at 4 degrees C.

Voltammetric enzyme genosensors on streptavidin-modified screen-printed carbon electrodes (SPCEs) for the detection of virulence nucleic acid determinants of pneumolysin and autolysin genes, exclusively present on the genome of the human pathogen Streptococcus pneumoniae, were described. Alkaline phosphatase (AP) and 3-indoxyl phosphate were used as the enzymatic label and substrate, respectively. The oligonucleotide probes were immobilized on electrochemically pretreated SPCEs through the streptavidin/biotin reaction. The adsorption of streptavidin was performed by deposition of a drop of a streptavidin solution overnight at 4 degrees C on the surface of the SPCEs. After the hybridization reaction with FITC-labeled complementary targets, the enzyme is captured using an anti-FITC antibody conjugated to AP. In nonstringent experimental conditions, these genosensors can detect 0.49 fmol of 20-mer oligonucleotide target and discriminate between a complementary oligo and an oligo with a three-base mismatch. In the presence of 25% formamide in the hybridization buffer, a single-base mismatch on the oligonucleotide target can be detected.

The use of 3-indoxyl phosphate (3-IP) as an electrochemical substrate for ELISAs with voltammetric detection was investigated. Indirect measurements of alkaline phosphatase (AP) and horseradish peroxidase (HRP) activity in solution were carried out. Picomolar levels of both enzymes can be detected, which enables the design of electrochemical immunoassays using this substrate. The enzymatic turnover of the substrate gives indigo blue, insoluble in aqueous solutions. This product is easily converted into its soluble parent compound, indigo carmine (IC), by addition of fuming sulphuric acid to the reaction media. IC shows a reversible voltammetric peak at the formal potential of -0.15V (versus Ag pseudo-reference electrode) when a screen-printed carbon electrode (SPCE) is used. The peak current of this process constitutes the analytical signal. Using this approach an ELISA assay to quantify pneumolysin (PLY, a toxin related to respiratory infections) was carried out using AP or HRP as enzymatic label. Calibration plots obtained are reported. 3-IP is demonstrated to be the first suitable substrate for the two most common enzyme labels used in immunoassays.

A new electrochemical method to monitor biotin-streptavidin interaction, based on the use of colloidal gold as an electrochemical label, is investigated. Biotinylated albumin is adsorbed on the pretreated surface of a carbon paste electrode (CPE). This modified electrode is immersed in colloidal gold-streptavidin labelled solutions. Adsorptive voltammetry is used to monitor colloidal gold bound to streptavidin, obtaining a good reproducibility of the analytical signal (R.S.D. = 3.3%). A linear relationship between peak current and streptavidin concentration from 2.5 x 10(-9) to 2.5 x 10(-5) M is obtained when a sequential competitive assay between streptavidin and colloidal gold-labelled streptavidin is carried out. On the other hand, the adsorption of streptavidin on the electrode surface was performed, followed by the reaction with biotinylated albumin labelled with colloidal gold. In this way, a linear relationship between peak current and colloidal gold labelled biotinylated albumin concentration is achieved with a limit of detection of 7.3 x 10(9) gold particles per ml (5.29 x 10(-9) M in biotin).

Carbon paste electrodes, previously anodised in a basic media, are the basis for the development of a new voltammetric immunosensor device. Passive adsorption of the appropriate immunochemical reagent was performed onto the electrode surface. Alkaline Phosphatase labelled immunoglobulin was the tracer used in this work, 3-indoxyl phosphate being a very suitable enzymatic substrate for the electrochemical detection of the corresponding affinity reaction. The hydrolysis of this molecule generates indigo dimmer. This product was detected by alternating current voltammetry taking advantage of the adsorptive and inherent electrodic properties that it exhibits. The same electrochemical anodisation was used at the end of one assay to remove the entire protein layer attached to the carbon paste surface, allowing the formation of a new sensing phase and the use of the same support in several consecutive experiments. The methodology was applied to the design of two different immunoassays for the determination of human IgG. Good reproducibility of the electrodic signal and a limit of detection around 10(-10) M were achieved.

A new electrochemical method to monitor biotin-streptavidin interaction on carbon paste electrode, based on silver electrodeposition catalyzed by colloidal gold, was investigated. Silver reduction potential changed when colloidal gold was attached to an electrode surface through the biotin-streptavidin interaction. Thus, the direct reduction of silver ions on the electrode surface could be avoided and therefore, they were only reduced to metallic silver on the colloidal gold particle surface, forming a shell around these particles. When an anodic scan was performed, this shell of silver was oxidized and an oxidation process at + 0.08 V was recorded in NH3 1.0 M. Biotinylated albumin was adsorbed on the pretreated electrode surface. This modified electrode was immersed in colloidal gold-streptavidin labeled solutions. The carbon paste electrode was then activated in adequate medium (NaOH 0.1 M and H2SO4 0.1 M) to remove proteins from the electrode surface while colloidal gold particles remained adsorbed on it. Then, a silver electrodeposition at -0.18 V for 2 min and anodic stripping voltammetry were carried out in NH3 1.0 M containing 2.0 x 10(-5) M of silver lactate. An electrode surface preparation was carried out to obtain a good reproducibility of the analytical signal (5.3%), using a new electrode for each experiment. In addition, a sequential competitive assay was carried out to determine streptavidin. A linear relationship between peak current and logarithm of streptavidin concentration from 2.25 x 10(-15) to 2.24 x 10(-12) M and a limit of detection of 2.0 x 10(15) M were obtained.