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The study of the human microbiome is a multidisciplinary area that affects from the field of technology to that of personalized medicine. The possibility of using microbiota biomarkers for the improvement in the diagnosis and monitoring of diseases (e.g. cancer), health conditions (e.g. obesity) or clinical parameters (e.g. ageing) has aroused great expectations, also in the scope of bio-electroanalytical chemistry. The well-known advantages of electrochemical transduction: high sensitivity, rapid response and possibility of miniaturization, together with the potential of new nanomaterials to improve both the design of electrode platforms and the performance of the resulting electrochemical biosensors make presumably a huge development in the next few years providing a better understanding of human microbiome entities. In this review article, the recent applications of electrochemical (bio)sensors for monitoring microbial metabolites and biomarkers of diseases related to different types of the human microbiome, with a special focus on the gastrointestinal microbiome, are reviewed. Examples of electrochemical devices applied to real samples are critically discussed as well as the challenges and prospects to be faced.

The study of the human microbiome is a multidisciplinary area ranging from the field of technology to that of personalized medicine. The possibility of using microbiota biomarkers to improve the diagnosis and monitoring of diseases (e.g., cancer), health conditions (e.g., obesity) or relevant processes (e.g., aging) has raised great expectations, also in the field of bioelectroanalytical chemistry. The well-known advantages of electrochemical biosensors—high sensitivity, fast response, and the possibility of miniaturization, together with the potential for new nanomaterials to improve their design and performance—position them as unique tools to provide a better understanding of the entities of the human microbiome and raise the prospect of huge and important developments in the coming years. This review article compiles recent applications of electrochemical (bio)sensors for monitoring microbial metabolites and disease biomarkers related to different types of human microbiome, with a special focus on the gastrointestinal microbiome. Examples of electrochemical devices applied to real samples are critically discussed, as well as challenges to be faced and where future developments are expected to go.

The first electrochemical immunosensor for the determination of CXCL7 (chemokine (C-X-C motif) ligand 7) autoimmune biomarker is reported in this work. Click chemistry-assisted antibodies immobilization was performed by reaction of azide functionalized-multi-walled carbon nanotubes (MWCNTs) and ethynyl-IgG onto screen-printed carbon electrodes. The capture antibodies were further immobilized onto IgG-MWCNTs conjugates. After a blocking step with casein, a sandwich immunoassay was implemented involving biotinylated detector antibodies and alkaline phosphatase (AP)-streptavidin conjugate. Differential pulse voltammetry upon addition of 1-naphthylphosphate was used as the analytical readout. A linear calibration plot between 0.5 and 600 pg mL−1 CXCL7 and a LOD value of 0.1 pg mL−1 were obtained. The usefulness of the immunosensor was demonstrated by the successful analysis of serum samples from patients with rheumatoid arthritis.

A comparison of the performance of two electrochemical immunosensors for the determination of the anorexigen biomarker peptide YY (PYY) is reported by using as scaffolds screen printed gold electrodes modified either by oxidative grafting of p-aminobenzoic acid (p-ABA) or by assembling of a 4-mercaptobenzoic acid (4-MBA) SAM. Covalent immobilization of capture antibodies on the surface-confined carboxyl groups was carried out by EDC/NHSS chemistry, and competitive immunoassays between target PYY and Biotin-PYY were implemented. Upon labeling with alkaline phosphatase (AP)-streptavidin conjugate and 1-naphtyl phosphate addition, differential pulse voltammograms recorded between −0.2 and +0.7 V were used as analytical readout. All the steps involved in the functionalization of the electrodes and the preparation of the immunosensors were monitored by electrochemical impedance spectroscopy. The calibration plot for PYY using the AP-Strept-Biotin-PYY(PYY)-anti-PYY-Phe-N-SPAuE immunosensor provided a linear current vs. log [PYY] plot extending between 10−6 and 103 ng/mL PYY with a detection limit of 3 × 10−7 ng/mL. These analytical characteristics are remarkably better than those obtained with the immunosensor prepared with 4-MBA SAM-SPAuEs. The AP-Strept-Biotin-PYY(PYY)-anti-PYY-Phe-N-SPAuE immunosensor was used to analyze human serum and saliva samples spiked with PYY at concentrations fitting with normal levels in these biological fluids.

Rheumatoid arthritis is an autoimmune disorder characterized by persistent erosive synovitis, systemic inflammation and the presence of autoantibodies, which play an important role in inducing inflammation and joint damage, releasing pro-inflammatory cytokines from monocytes and macrophages [1,2]. Likewise, neutrophil activating protein-2 (CXCL7) is a platelet-derived growth factor belonging to the CXC chemokine subfamily, which is expressed in serum, synovial fluid and synovial tissue of patients developing rheumatoid arthritis during the first twelve weeks, being useful to reflect local pathological changes [3]. Besides, matrix metalloproteinase-3 (MMP-3), which is induced by inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α) in rheumatoid synovium, degrades several extracellular matrix components of cartilage and plays central roles in rheumatoid joint destruction [4]. Therefore, monitoring serum CXCL7 and MMP-3 levels is useful for predicting the disease activity in rheumatoid arthritis. In this work, the construction and analytical performance of a dual electrochemical platform for the simultaneous determination of CXCL7 and MMP-3 is described. After the optimization of experimental variables involved in the preparation and implementation of the biosensor, the analytical usefulness of the developed configuration was demonstrated by its application to the determination of these biomarkers in serum samples from healthy individuals and patients with rheumatoid arthritis. To carry out the simultaneous determination of CXCL7 and MMP3 in human serum, just a fifty-fold sample dilution in PBS of pH 7.4 was required. In addition, the results obtained using the dual immunosensor were compared with those provided by the respective ELISA immunoassays, yielding no significant differences between the two methods. It is important to highlight that reagents consumption, four times smaller using the dual immunosensor than that required in the ELISA protocol, and an assay time of 2 h 50 min versus almost 5 h, counted in both cases after incubation of the capture antibody, are advantageous features of the dual immunosensor [5].

This paper reports the preparation of the first dual electrochemical immunosensor for the simultaneous determination of the CXCL7 chemokine and the MMP3 metalloproteinase as relevant biomarkers for the better diagnosis and monitoring of rheumatoid arthritis derived from the multiple biomarkers measurement. The developed immunosensor involves the use of carboxylated magnetic beads (MBs) and dual screen-printed carbon electrodes (SPdCEs). Sandwich-type configurations implied the covalent immobilization of specific anti-CXCL7 (cAb1) or anti-MMP3 (cAb2) capture antibodies onto MBs and the use of biotinylated detection antibodies with further labelling with HRP-Strept conjugates. The resulting MBS bioconjugates were magnetically captured on the respective working electrode of the SPdCE and the determination of the antigens was accomplished by measuring the amperometric responses of H2O2 mediated by hydroquinone (HQ) at a potential value of −0.20 V. The dual immunosensor provided calibration plots with linear ranges between 1 and 75 ng mL−1 (CXCL7) (R2 = 0.997) and from 2.0 to 2000 pg mL−1 (MMP3) (R2 = 0.998) with detection limits of 0.8 ng mL−1 and 1.2 pg mL−1, respectively. The assay took 2 h 20 min for the simultaneous determination of both biomarkers. The dual immunosensor was successfully applied to the analysis of human serum from positive and negative RA patients.

This article critically discusses the latest advances in the use of voltammetric, amperometric, potentiometric, and impedimetric biosensors for forensic analysis. Highlighted examples that show the advantages of these tools to develop methods capable of detecting very small concentrations of analytes and provide selective determinations through analytical responses, without significant interferences from other components of the samples, are presented and discussed, thus stressing the great versatility and utility of electrochemical biosensors in this growing research field. To illustrate this, the determination of substances with forensic relevance by using electrochemical biosensors reported in the last five years (2015–2019) are reviewed. The different configurations of enzyme or affinity biosensors used to solve analytical problems related to forensic practice, with special attention to applications in complex samples, are considered. Main prospects, challenges to focus, such as the fabrication of devices for rapid analysis of target analytes directly on-site at the crime scene, or their widespread use and successful applications to complex samples of interest in forensic analysis, and future efforts, are also briefly discussed.

An electrochemical immunosensor was prepared for the simultaneous determination of two hormones, ghrelin (GHRL) and peptide YY (PYY), which play important roles in the regulation of hunger and saciety. Dual screen-printed carbon electrodes modified with reduced graphene oxide (rGO) were used as scaffolds for the immobilization of the corresponding capture antibodies. Grafting of the diazonium salt of 4-aminobenzoic acid (4-ABA) on the modified electrode surfaces allowed covalent immobilization of antibodies. Competitive immunoassays were employed and the affinity reactions were monitored by differential pulse voltammetry upon addition of 1-naphthyl phosphate. Under the optimized working conditions, linear current vs. log [hormone] plots extending between 10−3 and 100 ng/mL GHRL (r2 = 0.990), and 10−4 and 10 ng/mL PYY (r2 = 0.992) were obtained. These ranges are adequate for the determination of both hormones at clinical levels in serum and saliva. An excellent analytical performance in terms of detection limit, reproducibility of the measurements, storage stability and selectivity against other proteins was achieved. The usefulness of the approach was demonstrated by its application to spiked human serum and saliva.

Electrochemical devices making use of carbon nanomaterials have demonstrated great applicability. Single- or multi-walled nanotubes and graphene have been largely applied to the construction of electrochemical (bio)sensors improving two key aspects in this field: surface nanostructuration allowing the preparation of electrode platforms more conductive, selective and suitable for biomolecules immobilization, and the ability to design suitable strategies for signal amplification. Besides the above mentioned carbon nanomaterials, more recently new carbon nanoforms have appeared as suitable nanostructuration tools. Carbon nanohorns, double-walled carbon nanotubes, fullerene (C60), carbon nanoparticles, and graphene quantum dots are examples of novel nanomaterials whose particular characteristics make them well suited for the construction of bioelectrochemical devices. In this article, the use of these carbon nanomaterials for the development of electrochemical immunosensors is reviewed.

An electrochemical immunosensor for the simultaneous determination of ghrelin (GHRL) and peptide YY (PYY) using dual screen-printed carbon electrodes modified with reduced graphene oxide (rGO) is presented. Diazonium salt of 4-aminobenzoic acid (4-ABA) was electrochemically grafted on the modified electrodes allowing covalent immobilization of antibodies. After competitive immunoassays using alkaline phosphatase labelled antigens, the affinity reactions were monitored by DPV upon addition of 1-naphthyl phosphate. Calibration plots showed linear current vs. log [hormone] ranges from 10 -3 to 100 ng/mL GHRL, and 10 -4 to10 ng/mL PYY. The usefulness of dual immunosensor was demonstrated by analysis of spiked human serum and saliva.

Electro-click methodology was employed to prepare an electrochemical immunosensor for the cytokine interleukin 1β (IL-1β). The strategy involved binding of ethynylated IgG to azide-MWCNTs modified electrodes by Cu(I) catalyzed-cycloaddition reaction where the catalyst was electrochemically synthesized. This electro-click protocol is significantly faster and greener than the methods for catalyst generation through chemical reduction. The oriented immobilization of the capture antibody onto IgG-MWCNTs conjugates allowed the preparation of a sandwich-type immunosensor using biotinylated anti-IL-1β as detector antibody labeled with alkaline phosphatase-streptavidin (AP-strept). Differential pulse voltammetric transduction through the 1-naphthylphosphate/1-naphthol system was carried out. The analytical characteristics achieved with the electrochemical immunosensor showed a calibration curve exhibiting two linear ranges between 10 and 200 pg mL−1 (r2 = 0.998), and from 200 to 1200 pg mL−1 (r2 = 0.998), and a LOD value of 5.2 pg mL−1, an improvement compared with those claimed for commercial ELISA kits. In addition, the assay time was at least one hour shorter. Excellent performance was observed in the determination of IL-1β in saliva with no need for sample treatment, and by simple interpolation using a calibration plot constructed with standard solutions of the target cytokine.

An electrochemical immunosensor for ghrelin (GHRL) determination in saliva is reported. Anti-GHRL was immobilized onto Protein G-magnetic beads and a competitive immunoassay involving biotinylated GHRL and alkaline phosphatase-streptavidin was implemented. Once conjugate was magnetically captured on a screen-printed carbon electrode, GHRL quantization was accomplished by DPV of 1-naphtol formed upon addition of 1-naphtyl phosphate. A linear range between 10−3 and 103 ng/mL GHRL, and a LOD of 7 pg/mL, much smaller than those from commercial ELISA kits, were found. The usefulness of the immunosensor was demonstrated by analyzing human saliva spiked with GHRL at 0.01, 0.1, 1 and 10 ng/mL.

A biosensor for 3-hydroxybutyrate (3-HB) involving immobilization of the enzyme 3-hydroxybutyrate dehydrogenase onto a screen-printed carbon electrode modified with reduced graphene oxide (GO) and thionine (THI) is reported here. After addition of 3-hydroxybutyrate or the sample in the presence of NAD+ cofactor, the generated NADH could be detected amperometrically at 0.0 V vs. Ag pseudo reference electrode. Under the optimized experimental conditions, a calibration plot for 3-HB was constructed showing a wide linear range between 0.010 and 0.400 mM 3-HB which covers the clinically relevant levels for diluted serum samples. In addition, a limit of detection of 1.0 µM, much lower than that reported using other biosensors, was achieved. The analytical usefulness of the developed biosensor was demonstrated via application to spiked serum samples.

A novel electrochemical immunosensor for the determination of the hormone prolactin (PRL) is reported by using glassy carbon electrodes modified with a poly(pyrrolepropionic acid)/multiwalled carbon nanotubes hybrid nanomaterial as transducer. An indirect competitive assay was utilized where the antigen was covalently immobilized on the modified electrode and a fixed amount of anti-PRL labelled with alkaline phosphatase (AP) was reacted with PRL in the sample. After attachment of the remaining labelled antibody to the immobilized PRL, the affinity reaction was monitored by differential pulse voltammetry of the AP enzyme reaction product using 1-naphtyl phosphate as substrate. Upon optimization of the variables affecting the immunosensor behaviour, a calibration graph for PRL with a wide linear range between 10 −2 and 10 4 ng/mL was obtained. The calculated LOD value, 3 pg/mL resulted to be remarkably lower than those reported previously using other approaches. Moreover, excellent reproducibility of the voltammetric measurements and selectivity against other hormones was found. The immunosensor was successfully applied to the analysis of human serum and urine at the clinically relevant concentration levels.

Forensic analysis is an important branch of modern Analytical Chemistry with many legal and socially relevant implications. Biosensors can play an important role as efficient tools in this field considering their well known advantages of sensitivity, selectivity, easy functioning, affordability and capability of miniaturization and automation. This article reviews the latest advances in the use of biosensors for forensic analysis. The different methodologies for the transduction of the produced biological events are considered and the applications to forensic toxicological analysis, classified by the nature of the target analytes, as well as those related with chemical and biological weapons critically commented. The article provides several Tables where the more relevant analytical characteristics of the selected reported methods are gathered.

A label-free dual electrochemical immunosensor was constructed for the multiplexed determination of human growth (hGH) and prolactin (PRL) hormones. The immunosensor used an electrochemical platform composed of carbon nanotube-screen printed carbon electrodes (CNT/SPCEs) modified with poly(ethylene-dioxythiophene) (PEDOT) and gold nanoparticles, on which the corresponding hGH and PRL antibodies were immobilized. The affinity reactions were monitored by measuring the decrease in the differential pulse voltammetric oxidation response of the redox probe dopamine. The experimental variables involved in the preparation of both AuNP/PEDOT/CNT/SPC modified electrodes and the dual immunosensor were optimized. The immunosensor exhibited an improved analytical performance for hGH and PRL with respect to other electrochemical immunosensor designs, showing wide ranges of linearity and low detection limits of 4.4 and 0.22 pg mL(-1), respectively. An excellent selectivity against other hormones and in the presence of ascorbic and uric acids was found. The usefulness of the dual immunosensor for the simultaneous analysis of hGH and PRL was demonstrated by analyzing human serum and saliva samples spiked with the hormones at different concentration levels.

A composite electrode consisted of gold nanoparticles (AuNPs), multi-walled carbon nanotubes (MWCNTs) and Teflon, to which peroxidase (HRP) and ferrocene (Fc) were incorporated as auxiliary enzyme and redox mediator, respectively, was constructed. The enzymes creatininase, creatinase and sarcosine oxidase were then co-immobilized onto the surface of the resulting HRP/Fc/AuNPs/MWCNTs/Teflon electrode for the preparation of a creatinine biosensor. Amperometry in stirred solutions using a detection potential of 0.0 V vs Ag/AgCl allowed a linear calibration plot to be obtained in the 0.003–1.0 mM creatinine concentration range with a limit of detection of 0.1 μM (S/N = 3). The apparent Michaelis-Menten constant for creatininase was KMap = 4.1 ± 0.4 mM. The developed biosensor was validated with good results by determining creatinine in human serum and correlating with the spectrophotometric Jaffe's method.

A novel nanocomposite electrode material constituted of gold nanoparticles (AuNPs), multi-walled carbon nanotubes (MWCNTs) and n-octylpyridinium hexafluorophosphate (OPPF6) ionic liquid was prepared and checked for the development of electrochemical (bio)sensing devices. AuNPs/MWCNTs/OPPF6 paste electrodes with micrometer dimensions (500 μm, i.d.) were constructed and applied to the determination of cortisol and androsterone hormones. Regarding cortisol determination, the microsized paste electrode was used to detect 1-naphtol generated upon addition of 1-naphthyl phosphate as enzyme substrate in the competitive immunoassay between alkaline phosphatase-labelled cortisol and cortisol. Squarewave voltammetry allowed determining the hormone within the 0.1- to 10-ng/mL linear range (r = 0.990) with a detection limit of 15 pg/mL and a EC50 value of 0.46 ± 0.06 ng/mL cortisol. The method was applied to the determination of cortisol in urine and serum samples containing a certified cortisol content. Moreover, a microsized enzyme biosensor prepared by bulk modification of the AuNPs/MWCNTs/OPPF6 electrode with the enzyme 3α-hydroxysteroid dehydrogenase was used for the determination of androsterone through the amperometric detection of reduced nicotinamide adenine dinucleotide. A calibration plot with a linear range between 0.1 and 120 μg/mL (r = 0.993) and a limit of detection of 89 ng/mL were obtained. The biosensor was applied to the analysis of human serum spiked with androsterone at the 250 ng/mL concentration level.

Enzyme biosensors for cholesterol were prepared using a transduction platform constituted of a glassy carbon electrode (GCE) modified with gold nanoparticles (AuNPs)/poly-(diallyldimethyl-ammonium chloride) (PDDA)–multi-walled carbon nanotubes (MWCNTs) nanocomposites. This modified electrode exhibited an improved response to H 2 O 2 when compared with other configurations based on CNTs. The incorporation of cholesterol oxidase (ChOx) to this electrode matrix allows the preparation of a biosensor that responded linearly to cholesterol in the 0.02–1.2 mM range with a slope of 2.23 μA/mM and a limit of detection 4.4 μM. All the variables involved in the preparation and performance of the modified electrode and the enzyme biosensor were optimized. The interferences caused by the presence of ascorbic acid and/or uric acid were minimized by coating the electrode surface with a thin film of Nafion which also enhanced the stability of the biosensor. Moreover, the possibility of preparing bienzyme ChOx–HRP biosensors using the same electrode platform was demonstrated. This allowed a detection potential as low as −50 mV to be applied using hydroquinone as mediator. With this design, the sensitivity was almost one order of magnitude higher than that achieved with the single ChOx biosensor and no interference from ascorbic acid or uric acid was apparent. Both biosensors designs were used for the determination of cholesterol in human serum samples spiked at physiological levels with recoveries ranging between 98.2% and 104%.

A novel electrochemical platform for the preparation of disposable enzyme biosensors is reported in this work. This platform is constructed by electrodeposition of poly(3,4-ethylenedioxythiophene) (PEDOT) using the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF 6 ) as the electropolymerization solvent onto gold nanoparticles-modified screen-printed carbon electrodes (SPCE). The enzymes alcohol dehydrogenase (ADH) or Tyrosinase were entrapped onto the electrode surface during the electropolymerization step. The potentiostatic electropolymerization process of PEDOT on gold nanoparticles-modified SPCE was optimized and the resulting modified electrodes characterized voltammetrically and by electrochemical impedance spectroscopy (EIS). The NADH amperometric detection at PEDOT/nAu/SPCE was also optimized and compared with that produced at a PEDOT/SPCE. ADH/PEDOT/nAu/SPCEs were constructed. The measured current for ethanol was 30% larger than that obtained using ADH/PEDOT/SPCEs. At a detection potential of +300 mV, a calibration graph for ethanol with a linear range between 5 and 100 μM was obtained with a detection limit of 2 μM. The PEDOT/nAu/SPCEs electrodes were also tested for the preparation of Tyrosinase biosensors. Using a detection potential of −150 mV, a linear calibration graph for phenol was constructed over the 0.1–50 μM concentration range, with a limit of detection of 0.02 μM.

A novel electrochemical immunosensor for testosterone based on direct attachment of antitestosterone on hybrid gold nanoparticles/carbon nanotubes-Teflon composite electrodes is reported. This material combined the ability of gold nanoparticles for biomolecules immobilization retaining their biological activity with synergic electrocatalytic effect demonstrated by coupling gold nanoparticles and carbon nanotubes. Competitive assay between testosterone and HRP- testosterone for binding sites of antibodies was employed. Amperometry at −0.05 V vs. Ag/AgCl was used to monitor affinity reaction upon addition of H2O2 with catechol as redox mediator. Calibration plot was linear between 0.1 and 10 ng/mL testosterone. LOD value was 85±6 pg/mL. Good results were obtained in the application to spiked human serum.

A cytochrome c (Cyt c ) electrochemical biosensor constructed by immobilization of the biomolecule on a hybrid material consisting of the conducting polymer poly-(3-methylthiophene) (P3MT) and multi-walled carbon nanotubes (MWCNT) is reported. Cyt c was immobilized onto l-cysteine (l-Cys) self-assembled monolayer-modified P3MT/MWCNT/GCE at neutral pH, where positively charged Cyt c strongly interacts with l-Cys negatively charged sites. The electron transfer rate constant, k s , was 0.49 ± 0.01 s −1 , and the surface coverage of Cyt c , Γ , (1.6 ± 0.8) × 10 −11 mol cm −2 . Under the optimized conditions, the mediatorless biosensor exhibits good electrocatalytic activity towards H 2 O 2 reduction. The amperometric measurements of H 2 O 2 at an applied potential of 0.0 V in 0.05 M Tris–HCl buffer of pH 8.0 exhibited good repeatability (RSD = 3.1%, n = 10) and an acceptable reproducibility for the biosensor preparation (RSD = 8.1%, n = 5). The lifetime of the biosensor was evaluated. A linear calibration graph for H 2 O 2 ( r = 0.998) was obtained between 0.7 and 400 μM, with a detection limit of 0.23 μM. The analytical characteristics for H 2 O 2 determination were found to be better than those reported for other nanostructured cytochrome c biosensors. The apparent Michaelis–Menten constant for the reaction between Cyt c and H 2 O 2 was 451 μM. The developed Cyt c /l-Cys/P3MT/MWCNT/GCE biosensor was applied for the biocatalytic determination of nitrite through the oxidation of NO 2 - to NO 3 - by electrogenerated [Fe 4+ -Cyt c ] · species. Amperometry in stirred PBS solutions at a potential of +0.9 V allowed nitrite to be determined in a linear range between 10 and 100 μM, with a detection limit of 0.5 μM.

A novel electrochemical biosensor design for glucosinolate determination involving bulk-incorporation of the enzymes glucose oxidase and myrosinase into a colloidal gold - multiwalled carbon nanotubes composite electrode using Teflon as binder is reported. Myrosinase catalyzes the hydrolysis of glucosinolate forming glucose, which is enzymatically oxidized. The generated hydrogen peroxide was electrochemically detected without mediator at the nanostructured composite electrode at E=+0.5 V vs. Ag/AgCl. Under the optimized conditions, the bienzyme MYR/GOx-Aucoll-MWCNT-Teflon exhibited improved analytical characteristics for the glucosinolate sinigrin with respect to a biosensor constructed without gold nanoparticles, i.e. a MYR/GOx-MWCNT-Teflon electrode, as well as with respect to other glucosinolate biosensor designs reported in the literature. The biosensor exhibits good repeatability of the amperometric measurements and good interassay reproducibility. Furthermore, the biosensor exhibited a high selectivity with respect to various potential interferents. The usefulness of the biosensor was evaluated by the determination of glucosinolate in Brussel sprout seeds.

This review covers recent advances in the development of new designs of electrochemical sensors and biosensors that make use of electrode surfaces modification with carbon nanotubes. Applications based on carbon nanotubes-driven electrocatalytic effects, and the construction and analytical usefulness of new hybrid materials with polymers or other nanomaterials will be treated. Moreover, electrochemical detection using carbon nanotubes-modified electrodes as detecting systems in separation techniques such as high performance liquid chromatography (HPLC) or capillary electrophoresis (CE) will be also considered. Finally, the preparation of electrochemical biosensors, including enzyme electrodes, immunosensors and DNA biosensors, in which carbon nanotubes play a significant role in their sensing performance will be separately considered.

The construction of a colloidal gold-cysteamine-carbon paste electrode, Au coll -Cyst-CPE, for the electrochemical determination of homocysteine is reported. The improved voltammetric behaviour of homocysteine at Au coll -Cyst-CPE with respect to that observed at a gold disk electrode is attributed to an enhanced electron transfer kinetics as a consequence of the array distribution of gold nanoparticles immobilized onto the Cyst SAM. Cyclic voltammtery of homocysteine showed an adsorption-controlled current for scan rates between 500 and 5000 mV s −1 . The hydrodynamic voltammogram constructed for homocysteine allowed the selection of a potential value of +600 mV, where the background current is negligible, for the amperometric detection of the analyte at the Au coll -Cyst-CPE. Using a flow rate of 0.8 ml min −1 , the R.S.D. value for i p after 25 repetitive injections of homocysteine was of 4.3%, and one single electrode could be used for more than 15 days without any treatment or regeneration procedure of the modified electrode surface. An HPLC method for the separation and quantification of homocysteine and related thiols, using amperometric detection at the modified electrode has been developed. A mobile phase consisting of 2:98% (v/v) acetonitrile:0.05 mol l −1 buffer solution of pH 2.0, and a detection potential of +0.80 V were selected. Separation with baseline resolution and retention times of 3.00, 3.60, 4.52, 5.71 and 7.79 min were obtained for cysteine, homocysteine, glutathion, penicillamine and N -acetyl-cysteine, respectively. Calibration graphs were constructed for all the separated compounds. Detection limits ranged between 20 nM for cysteine and 120 nM for penilcillamine, with a value for homocysteine of 30 nM. These values compare advantageously with those achieved with previously reported HPLC methods using electrochemical, UV, fluorescence and MS detection modes. The developed method was applied to the determination of cysteine and homocysteine serum samples with good results.

The preparation of poly-(3-methylthiophene)—multi-walled carbon nanotubes hybrid composite electrodes is reported. The hybrid electrode shows a synergic effect of the electrocatalytic properties, and high active surface area of both the conducting polymer and carbon nanotubes, which gives rise to a remarkable improvement of oxidation of NADH with respect to polymer-modified electrodes, and CNTs-modified electrodes. SEM showed that carbon nanotubes served as nanosized backbone for P3MT electropolymerization. The amperometric NADH detection at +300 mV provided fast responses, a range of linearity between 5.0 × 10−7 and 2.0 × 10−5 mol l−1, and a detection limit of 1.7 × 10−7 mol l−1, which compares advantageously with those reported for other NADH CNT-based amperometric sensors. Furthermore, the direct electrochemistry of cytochrome c and FAD at the hybrid electrode is also checked.

The first electrochemical immunosensor for the determination of peptide YY is reported in this paper. A novel electrochemical platform, prepared by the electrochemical grafting of the diazonium salt of 4-aminobenzoic acid onto a reduced graphene oxide-modified glassy carbon electrode, was used, on which the covalent immobilization of specific anti-PYY antibodies was accomplished. The HOOC-Phe-rGO/GCEs were characterized using cyclic voltammetry and electrochemical impedance spectroscopy. The different variables affecting the preparation of the modified electrodes and the performance of the immunosensor were optimized. Under the optimized conditions, a calibration plot for PYY showing a linear range extending between 10(-4) and 10(2) ng mL(-1) was found. This range is adequate for the determination of this protein in real samples, since the expected concentration in human serum is around 100 pg mL(-1). The limit of detection was 0.01 pg mL(-1) of PYY. The immunosensor exhibited good reproducibility of the PYY measurements, excellent storage stability and selectivity, as well as a shorter assay time than those of ELISA kits. The usefulness of the immunosensor for the analysis of real samples was demonstrated by analyzing human serum samples spiked with PYY at three concentration levels.

The construction and characterization of a new carbon-felt electrode design of small dimensions is reported. The electrode was checked by testing the electrochemical behaviour of the Fe(CN) 6 3−/4− model system, as well as of several phenolic compounds with xenoestrogenic properties. The use of a carbon-felt cylinder electrode whose heigh was insulated with a poly(ethylene) cover and with two exposed bases of 2.0 mm diameter, resulted in an enhancement of the Fe(CN) 6 3−/4− voltammetric peak current with respect to a conventional glassy carbon electrode with a similar outer surface, which suggests a high contribution of the redox probe solution trapped in the three-dimensional structure of the electrode. The contribution of the electrolysis of the redox probe trapped in the electrode matrix to the voltammetric response, as well as that of the mass transfer from bulk solution, were investigated. The voltammetric behaviour of phenolic compounds with xenoestrogenic properties showed adsorption onto the carbon-felt electrode. Penetration of these compounds into the electrode at open circuit was demonstrated. Two possible applications of the new electrode design are outlined: flow analysis with electrochemical detection of phenolic endocrine disruptors and the possibility of using it for removal of these compounds.

The preparation and performance of a xanthine oxidase (XOD) biosensor, based on a carbon paste electrode (CPE) modified with electrodeposited gold nanoparticles (nAu), for the amperometric determination of hypoxanthine (Hx) is reported. Different XOD biosensor configurations were evaluated and compared with electrodes constructed by immobilizing XOD onto unmodified CPE and with biosensors prepared using glassy carbon electrodes and gold disk electrodes modified with electrodeposited gold. The XOD–nAu–CPE in which the enzyme was immobilized by cross-linking with glutaraldehyde (GA) and BSA exhibited the highest amperometric signal for Hx. Although Hx detection is usually carried out at potential values of around +600 mV versus Ag/AgCl, the GA–BSA–XOD–nAu–CPE allowed this detection to be carried out at 0.00 V, thus minimizing potential interferences from electrochemically oxidizable substances such as ascorbic acid. Experimental variables concerning the biosensor preparation were optimized. Calibration plots for Hx were constructed with the biosensor operating at +600 mV and at 0.00 V. The detection limit for Hx, 2.2 × 10 −7 mol l −1 , obtained using the latter potential value is similar to the best detection limits reported in the literature with other biosensor designs working at much more extreme potentials. The usefulness of the biosensor for the analysis of real samples was demonstrated by determining Hx in sardines and chicken meat.

The electrochemical behaviour of some nitroaromatic explosives (2,4,6-trinitrotoluene, TNT; 2,6-dinitrotoluene, 2,6-DNT; 2-nitrotoluene, 2-NT; 2-amino-4,6-dinitrotoluene, 2-A-4,6-DNT; 3,5-dinitroaniline, 3,5-DNA; and nitrobenzene, NB) at electrochemically activated carbon-fibre microelectrodes is reported. Electrochemical activation of such electrode material by repeated square-wave (SW) voltammetric scans between 0.0 and +2.6 V versus Ag/AgCl, produced a dramatic increase in the cathodic response from these compounds. This is attributed to the increase of the carbon-fibre surface area, because of its fracture, and the appearance of deep fissures along the main fibre axis into which the nitroaromatic compounds penetrate. Based on the important contribution of adsorption and/or thin layer electrolysis to the total voltammetric response, a SW voltammetric method for rapid detection of nitroaromatic explosives was developed. No interference was found from compounds such as hydrazine, phenolic compounds, carbamates, triazines or surfactants. The limits of detection obtained are approximately 0.03 microg mL(-1) for all the nitroaromatic compounds tested. The method was applied for the determination of TNT in water and soil spiked samples; recoveries were higher than 95% in all cases.

The electrochemical behavior of the nitrofuran antibiotics nitrofurantoin (NFA) and nitrofurazone (NFZ), as well as the related nitroimidazole drug, metronidazole (MNZ) at electrochemically activated carbon fibre microelectrodes (CFMEs) is reported. These nitrofurans have been widely used in the form of feed additives for the treatment of gastrointestinal infections in cattle, pigs and poultry. Electrochemical activation of CFMEs by repetitive square-wave (SW) voltammetric scans between 0.0 and +2.6 V, produced an unexpected high cathodic response from these compounds. This is attributed to the increase of the carbon fibre surface area due to its fracture and the appearance of deep fissures along the main fibre axis. Penetration of the nitroderivative compounds into the micro-channels of the activated fibres was profited to develop a sensitive and selective method for NFA based on stripping SW voltammetry. The metabolite of NFA, 1-aminohydantoin (AHD), did not exhibit cathodic response in the potential range between −0.9 and +0.1 V. However, AHD gave a SW oxidation signal at activated CFMEs, with a potential of +0.95 V, which did not appear at non-activated microelectrodes. Based on this finding, the simultaneous determination of NFA and AHD was performed by amperometry using two activated CFMEs in a parallel configuration connected to a multichannel detector. This methodology was also used to carry out degradation studies of NFA in acidic and basic media under photodegradation or thermal degradation conditions.

The optimal conditions for the preparation of colloidal gold‐carbon paste electrodes (CPEs) by using CPEs modified with cysteamine (Cyst) are discussed. Cysteamine modified‐CPEs (Cyst‐CPEs) were prepared by immersion in a Cyst solution, the experimental variables involved in this modification process being optimized. Different colloidal gold deposition methods on the Cyst‐CPEs were evaluated by recording cyclic voltammograms of methionine solutions. Better electrochemical responses were found for colloidal gold‐Cyst‐CPEs (nAu‐Cyst‐CPEs) prepared by immersion of Cyst‐CPEs in a colloidal gold suspension for 4 hr. Optimization of the different experimental variables involved (colloidal particle size and time of immersion) was also carried out. The surface of nAu‐Cyst‐CPEs was characterized by SEM, and the gold coating electrochemically tested. A surface coverage, Γ, of 3.07 × 10−10 mol cm−2 was obtained. The analytical suitability of nAu‐Cyst‐CPEs was verified by testing the voltammetric behavior of...

The flow-injection and HPLC determination of the diuretic drug furosemide using pulsed amperometric detection (PAD) at cylindrical carbon fibre microelectrodes (CFMEs) is reported. Experimental conditions such as pH (6.5) and buffer concentration (0.05 mol l(-1) HPO4(2-)/H2PO4(-)) were optimized using square-wave voltammetry (SWV). Repetitive flow-injection amperometric measurements at +1.25 V for furosemide showed a continuous decrease in the peak current, probably as a consequence of the microelectrode surface fouling. However, a suitable amperometric detection of furosemide was achieved using a PAD program consisting of a two-step potential waveform with alternating anodic and cathodic polarization. The anodic (detection) potential was +1.25 V (time of application 0.1 s), and the cathodic (cleaning) potential was -0.20 V (t=0.2 s). A linear calibration graph was obtained for furosemide in the 5.0 x 10(-7)-1.0 x 10(-4) mol l(-1) concentration range, with a limit of detection of 1.7 x 10(-7) mol l(-1). HPLC-PAD at carbon fibre microelectrodes was used for the determination of furosemide in the presence of several thiouracil drugs and oxytetracycline (OTC). The mobile phase selected was a 25:75 acetonitrile:5.0 x 10(-3) mol l(-1) NaH2PO4 (pH 5.0) mixture. A linear calibration graph was obtained for furosemide in the 1-100 microM range, with a limit of detection of 0.55 microM. The usefulness of this method for the determination of furosemide in real samples was evaluated by performing the analysis of commercial milk samples spiked with furosemide at a concentration level of 4.5 x 10(-7) mol l(-1) (150 ng ml(-1)), as well as with other thiouracil drugs and OTC. A mean recovery of 95+/-5% furosemide was obtained.

A voltammetric method for the determination of the antibiotic oxytetracycline (OTC) in food samples is reported. Carbon fiber microelectrodes (CFMEs), which allow voltammetric measurements to be performed in a small volume (1 mL) of the analyte extract from the samples, are employed. Repeatable electroanalytical responses were obtained with no need of applying cleaning treatments to the CFME. Under the optimized square-wave conditions, a linear calibration plot for OTC was obtained in the 1.0×10−6–1.0×10−4 mol L−1 range, with a detection limit of 2.9×10−7 mol L−1 (150 ng mL−1) OTC. The determination of OTC by a flow-injection method with amperometric detection using a homemade flow cell specially designed to work with CFMEs, was also evaluated using pure acetonitrile as the carrier. The SW voltammetric method was applied to the determination of OTC in spiked milk and eggs samples, at 100 ng mL−1 and 200 ng g−1 levels, respectively. The procedure involved the extraction of the analyte in ethyl acetate, evaporation of the solvent and reconstitution of the residue in acetonitrile −5.0×10−4 mol L−1 tetrabutylammonium perchlorate medium. Recoveries of 96±8 and 91±8% were obtained for milk and eggs, respectively, by applying the standard additions method.

Gold disk microelectrodes (AuMEs, 50 μm O) have been used for the determination of the fungicide disulfiram (DSF) at low concentration levels by differential pulse adsorptive stripping voltammetry (dp-AdSV). The AuMEs were fabricated in the laboratory and characterized by scanning electron microscopy and cyclic voltammetry. The AuME was pretreated daily by polishing with 3-μm diamond powder for 60 s, and by applying successively potentials of −0.4, −0.8, −1.0, −1.2 and −1.5 V for 30 s each in 0.1 mol L−1 H2PO4−/HPO42− (pH 6.0). In between measurements, the application of a potential of −1.5 V for 30 s was only necessary. The DSF adsorption on the AuME surface allows its determination at trace levels by dp-AdSV using low supporting electrolyte concentrations (3.0 mmol L−1) with an accumulation time of 120 s, a pulse amplitude of 50 mV, and a scan rate of 50 mV s−1. A detection limit of 6.3×10−8 mol L−1 and a RSD of 1.3% at a 5.0×10−7 mol L−1 DSF concentration level (n=10) were obtained with a two-electrode system and no stirring during the deposition step. The effect of the presence of several potential interferences on the DSF stripping signal has been tested. The developed method has been applied to the determination of DSF in spiked pea seeds with good results.

An amperometric flow-injection method for the determination of antithyroid drugs such as 6-methyl-2-thiouracil (MTU) using a carbon fiber cylindrical microelectrode-based detector is reported. A home-made flow-cell specially adapted for working with cylindrical microelectrodes ranging between 4 and 10 mm in length was used for this purpose. Methanol containing 0.05 mol l −1 tetrabutylammonium perchlorate as the supporting electrolyte was employed, and a potential of +1.6 V was selected for the amperometric detection of MTU. No cleaning or electrode surface regeneration of the fiber was necessary during the whole working day. A limit of detection of 2.6×10 −7 mol l −1 (37 μg MTU l −1 ) was achieved. The method was applied to the determination of MTU in spiked feed samples (peas and corn flour), at the 142 μg g −1 level. The procedure involved the extraction of the drug in methanol, a clean-up step using Florisil cartridges and the use of the standard additions method. Recoveries of 81±6 and 85±8% were obtained for peas and corn flour, respectively.

Cylindrical carbon fiber microelectrodes (CFMEs) (8 μm in diameter, 8 mm in length) are used to develop a voltammetric method for the determination of the thyreostatic drug methylthiouracil (MTU) in animal feed samples. Methanol, containing 0.05 mol L−1 tetrabutylammonium perchlorate as supporting electrolyte, was selected as working medium because of the high MTU solubility in this solvent, which is also used for the extraction of MTU from real samples. Reproducible electroanalytical responses were obtained with no need of applying cleaning treatments to the CFME. The net-current square-wave (SW) oxidation signal of MTU was selected to be exploited analytically. Under the optimized SW conditions, a linear calibration plot for MTU was obtained in the 1.0×10−6–1.0×10−4 mol L−1 range, with a detection limit of 3.8×10−7 mol L−1. The SW voltammetric behavior of other thyreostatic drugs was checked. A simple extraction procedure with methanol, followed by a clean up step, was used for the determination of MTU in a commercial dry fodder sample spiked with this drug at the 500 μg g−1 level. The detection limit in this type of sample was found to be 8.6×10−6 mol L−1.

Graphite-Teflon-peroxidase composite electrodes, constructed by simple physical inclusion of the enzyme into the bulk of the electrode matrix, have been used for the amperometric detection of 18 phenolic compounds. Monitoring of the enzyme reaction was carried out by the electrode reduction of the generated phenoxy radicals, in the presence of a constant concentration of 5.0×10−4 mol L−1 H2O2, at an applied potential of 0.00 V. Different aspects regarding the stability of the composite bioelectrode showed the robustness of the biosensor design. The kinetic constants of the enzyme reactions for the phenolic compounds tested were calculated. The limits of detection obtained were better than those found in the literature for peroxidase biosensors using other composite matrices, and the differences in sensitivity observed for the different phenolic substrates are discussed in terms of their pKa values and substituents. The analytical signals obtained with the graphite-Teflon-HRP electrodes have been compared with those yielded by a graphite-Teflon-tyrosinase composite electrode constructed in a similar way. With the aim of monitoring the largest possible number of phenolic compounds with the highest possible sensitivity, coimmobilization of peroxidase and tyrosinase into the composite electrode matrix was carried out.

The electrochemical behavior of the N , N -dialkyldithiocarbamate pesticide thiram at a Au microdisk electrode (10 μm φ) and at cylindrical carbon fiber microelectrodes (CFMEs) (8 μm φ, 8 mm length) in low-permitivity organic solvents such as toluene and ethyl acetate is reported. The obtained cyclic voltammograms were very different in shape depending on the microelectrode and the solvent used, as well as on thiram concentration. Analytically useless responses were obtained with the gold microelectrode. However, a good fitting of the voltammetric signals at cylindrical CFMEs with the theoretical model proposed by Aoki was found. The influence of variables such as the concentration of thiram and of the background electrolyte, the length of the CFMEs and the potential scan rate was tested by CV. The oxidation responses obtained by square-wave voltammetry at carbon fiber microelectrodes in ethyl acetate were found to be analytically suitable to develop a method for the determination of thiram at low concentration levels. A linear calibration graph in the 1.0×10 −6 –6.0×10 −4 mol l −1 concentration range was obtained, with a slope of (1.2±0.1)×10 4 μA l mol −1 . The limit of detection was 4.3×10 −7 mol l −1 thiram, and the relative standard deviation at a 5.0×10 −5 mol l −1 concentration level ( n =10) was of 1.6%. A good reproducibility of the electroanalytical measurements was found with no need of cleaning or pretreatment procedures of the CFMEs surface. The SWV method was applied with good results to the determination of thiram in spiked grapes, by measuring directly the net current in the analyte extract in ethyl acetate. Commercial fungicides and pharmaceutical preparations containing thiram were also analysed.

Cylindrical carbon fibre microelectrodes (CFMEs) (8 mm in diameter, 8 mm in length) were used to develop voltammetric methods for the determination of the antioxidant 4-tert-butylcatechol (TBC) in styrene, and of styrene in polystyrene samples. Acetone, containing 0.01 mol l−1 sodium perchlorate as supporting electrolyte, was selected as a suitable working medium. A good adjustment of the voltammetric responses to that predicted by the theory was found. Reproducible electroanalytical responses were obtained with no need of applying cleaning treatments to the CFME. Using square-wave voltammetry (SWV), linear calibration graphs were obtained for TBC and styrene in the 1.0 × 10−6–1.0 × 10−4 mol l−1 range, with detection limits of 4.3 × 10−7 mol l−1 and 4.0 × 10−7 mol l−1, respectively. The sensitivity of the method for styrene was around four orders of magnitude better than that reported for another microelectrode-based electroanalytical method. Voltammograms from mixtures of TBC and styrene showed two well-defined oxidation peaks with a difference between the peak potentials of 600 mV. Commercial styrene samples containing 0.005% of TBC were analyzed with good results. In addition, the method was applied to the determination of styrene in spiked polystyrene yoghurt packagings.

The use of cylindrical carbon fiber microelectrodes (CFMEs) of various mm in length modified with poly(3-methylthiophene) (P3MT) films as amperometric microsensors for the LC-EC detection of catecholamines is reported. Cyclic voltammetry at the modified microelectrodes of dopamine, epinephrine, catechol, serotonin and ascorbic acid showed electrocatalytic ability of the modified surface. This allowed the application of low potentials (a0.35 V) for the amperometric detection of the above-mentioned compounds. Flow injection measurements carried out using a 2:98 v=v methanol:0.025 mol L ˇ1 H2PO4 ˇ =HPO4 2ˇ solution of pH 7.0, showed no evidence of fouling of the modified microelectrode surface. No cleaning or regeneration treatment was needed when working with the same polymer modified microelectrode during the whole working day. Moreover, RSD values for ip lower than 5 % were obtained when amperometric measurements were carried out with three different P3MT-coated CFMEs. These modified microelectrodes have also shown to be suitable for amperometric detection in HPLC. A mobile phase composed of 2:98 % methanol:phosphate buffer solution of pH 7.0 allowed a good separation of mixtures of ascorbic acid, epinephrine, dopamine, serotonin and catechol with detection limits ranging between 14 and 48 pmol. Furthermore, as an application, the determination of those compounds in spiked serum samples, was accomplished with good results.

The use of cylindrical carbon fiber microelectrodes (CFMEs) modified with poly(3-methylthiophene) (P3MT) films as amperometric microsensors for continuous monitoring of some amino acids such as tyrosine (Tyr), tryptophan (Trp), L-dopa, and related compounds such as tyramine and ascorbic acid, as well as small peptides such as Trp–Ala and Tyr–Gly, is discussed. Important practical advantages with respect to conventional glassy carbon (GC) electrodes, and also with respect to P3MT-coated GC electrodes of conventional size are demonstrated. The electrocatalytic ability of the modified surface allows the possibility of applying moderate potentials for the amperometric detection of the above mentioned compounds. Flow-injection experiments carried out using a phosphate buffer solution of pH 7.0 as the carrier, and an applied potential of +0.8 V showed that no cleaning or regeneration pretreatment was needed when working with the same polymer modified electrode during the whole working day. The effect of the presence of acetonitrile or methanol in the flowing solution was evaluated. A flow-injection method with amperometric detection was developed for the determination of L-dopa in pharmaceutical preparations. Finally, the modified microelectrodes have also shown suitable for amperometric detection in liquid chromatography (LC). A mobile phase composed of 5 : 95 (v/v) methanol : phosphate buffer solution of pH 7.0 allowed a good separation of mixtures of ascorbic acid, L-dopa, Tyr, tyramine and Trp, with detection limits of around 160 pmol. Furthermore, the possibility of detection of Tyr- and Trp-containing oligopeptides was also demonstrated.