Serological diagnosis of Toxoplasmosis disease using a fluorescent immunosensor with chitosan-ZnO-nanoparticles.

This article describes a microfluidic LIF immunosensor for the quantitative determination of anti-Toxoplasma gondii IgG (anti-T. gondii) specific antibodies. The serological detection of these antibodies plays a crucial role in the clinical diagnosis of toxoplasmosis. Zinc oxide nanoparticles (ZnO-NPs) obtained by wet chemical procedure were covered with chitosan and then used to conjugate T-gondii antigens into the central microfluidic channel. Serum samples containing anti-T-gondii IgG antibodies were injected into the immunosensor where they interact immunologically with T. gondii antigens. Bound antibodies were quantified by the addition of anti-IgG antibodies labeled whit alkaline phosphatase (ALP). ALP enzymatically converts the non-fluorescent 4-methylumbelliferyl phosphate (4-MUP) to soluble fluorescent methylumbelliferone that was measured using excitation at 355 nm and emission at 440 nm. The relative fluorescent response of methylumbelliferone is proportional to the concentration of anti-T. gondii IgG antibodies. The coefficients of variation are less than 4.73% for within-day assays and less than 6.34% for between-day assays. Results acquired by LIF immunosensor agree with those obtained by enzyme-linked immunosorbent assay method, suggesting that the designed sensor represents a promising tool for the quantitative determination of anti-T. gondii IgG antibodies of clinical samples.

Novel Electrochemical Paper-Based Immunocapture Assay for the Quantitative Determination of Ethinylestradiol in Water Samples.

We report a novel and innovative electrochemical paper-based immunocapture assay (EPIA) to address the need for ultrasensitive detection of emerging pollutants without regulatory status and whose effects on environment and human health are not completely yet understood. In particular, we present the application of this system toward highly sensitive detection of the emerging pollutant ethinyl estradiol (EE2). The EPIA approach is based on the use of paper microzones modified with silica nanoparticles (SNs) and anti-EE2 specific antibodies for capture and preconcentration of EE2 from river water samples. After the preconcentration procedure, the paper microzones are placed onto a screen-printed carbon electrode modified with electrochemically reduced graphene (RG). The bound EE2 is subsequently desorbed adding a diluted solution of sulfuric acid on the paper microzones. Finally, recovered EE2 is electrochemically detected by OSWV. The proposed novel methodology showed an appropriate LOD and linear range for the quantification of EE2 for water samples with different origins. The nonsophisticated equipment required, the adequate recovery values obtained (from 97% to 104%, with a RSD less than 4.9%), and the appropriate LOD and linear range value (0.1 ng L-1 and 0.5-120 ng L-1, respectively) achieved by our immunocapture sensor present significant analytical figures of merit, particularly when the routine quantification of EE2 is considered. In addition, our system was based on electrochemical paper-based technology, which allows obtainment of portable, easy-to-use, inexpensive, and disposable devices. The EPIA can also serve as a general-purpose immunoassay platform applicable to quantitation of other drugs and emerging pollutants in environmental samples.

Silica nanoparticle-based microfluidic immunosensor with laser-induced fluorescence detection for the quantification of immunoreactive trypsin.

The purpose of this study was to develop a silica nanoparticle-based immunosensor with laser-induced fluorescence (LIF) as a detection system. The proposed device was applied to quantify the immunoreactive trypsin (IRT) in cystic fibrosis (CF) newborn screening. A new ultrasonic procedure was used to extract the IRT from blood spot samples collected on filter papers. After extraction, the IRT reacted immunologically with anti-IRT monoclonal antibodies immobilized on a microfluidic glass chip modified with 3-aminopropyl functionalized silica nanoparticles (APSN-APTES-modified glass chips). The bounded IRT was quantified by horseradish peroxidase (HRP)-conjugated anti-IRT antibody (anti-IRT-Ab) using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as enzymatic mediator. The HRP catalyzed the oxidation of nonfluorescent ADHP to highly fluorescent resorufin, which was measured by LIF detector, using excitation lambda at 561nm and emission at 585nm. The detection limits (LODs) calculated for LIF detection and for a commercial enzyme-linked immunosorbent assay (ELISA) test kit were 0.87 and 4.2ngml(-1), respectively. The within- and between-assay variation coefficients for the LIF detection procedure were below 6.5%. The blood spot samples collected on filter papers were analyzed with the proposed method, and the results were compared with those of the reference ELISA method, demonstrating a potential usefulness for the clinical assessment of IRT during the early neonatal period.

Zinc oxide nanoparticles based microfluidic immunosensor applied in congenital hypothyroidism screening.

In this article, we present an innovative approach for congenital hypothyroidism (CHT) screening. This pathology is the most common preventable cause of mental retardation, affecting newborns around the world. Its consequences could be avoided with an early diagnosis through the thyrotropin (TSH) level measurement. To accomplish the determination of TSH, synthesized zinc oxide (ZnO) nanobeads (NBs) covered by chitosan (CH), ZnO-CH NBs, were covalently attached to the central channel of the designed microfluidic device. These beads were employed as platform for anti-TSH monoclonal antibody immobilization to specifically recognize and capture TSH in neonatal samples without any special pretreatment. Afterwards, the amount of this trapped hormone was quantified by horseradish peroxidase (HRP)-conjugated anti-TSH antibody. HRP reacted with its enzymatic substrate in a redox process, which resulted in the appearance of a current whose magnitude was directly proportional to the level of TSH in the neonatal sample. The structure and morphology of synthesized ZnO-CH NBs were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The calculated detection limits for electrochemical detection and the enzyme-linked immunosorbent assay procedure were 0.00087 µUI mL(-1) and 0.015 µUI mL(-1), respectively, and the within- and between-assay coefficients of variation were below 6.31% for the proposed method. According to the cut-off value for TSH neonatal screening, a reasonably good limit of detection was achieved. These above-mentioned features make the system advantageous for routine clinical analysis adaptation.

Chemometrically assisted differential pulse voltammetry for simultaneous and interference-free quantification of gallic and caffeic acids.

This article explores the application of chemometric tools including multivariate curve resolution with alternating least squares for the simultaneous determination of gallic and caffeic acids on the surface of a glassy carbon electrode without additional modification. Gallic and caffeic acids are primary polyphenols, the most abundant in red wines produced in Argentina, and are often used as quality markers for them. These polyphenols significantly contribute to the organoleptic properties of wines from this origin, but their electrochemical signals overlap significantly, making simultaneous quantification challenging without additional experiments such as electrode modification or alternative analytical techniques beyond differential pulse voltammetry. This study successfully quantified these compounds in complex mixtures by generating second-order data from differential pulse voltammetry experiments conducted at various potential steps and subsequently applying multivariate curve resolution with alternating least squares. The use of constraints during optimization prevented rotational ambiguities common in this modeling, leading to unique results in validation samples. The limits of detection (LOD) found for gallic and caffeic acids were 1.6 and 7.6 mg L-1, which are in excellent agreement with the expected concentrations of these compounds in red wines. The concentration ranges analyzed showed a linear dependency (between the LOD and 300 mg L-1) with the signals estimated by the model for both analytes. Advantages such as simplicity, low cost, and high speed, as well as not requiring electrode modification, combined with excellent results obtained for real samples, make it a promising alternative for polyphenol analysis in the wine industry.

Modification of electrodes with polymer nanocomposites: Application to the simultaneous determination of Zn(II), Cd(II), and Cu(II) in water samples.

Currently, there is a need for fast and sensitive analytical methods for monitoring metals in water due to the progressive increase in the presence of metal ions in the environment. These metals reach the environment mainly from industrial activity and heavy metals are non-biodegradable. The present work evaluates different polymeric nanocomposites to carry out the simultaneous electrochemical determination of Cu, Cd, and Zn in water samples. Screen-printed carbon electrodes (SPCE) were modified with the nanocomposites, which were obtained by a mixture of graphene, graphite oxide, and polymers, such as polyethyleneimide, gelatin, and chitosan. These polymers have amino groups in their matrix, giving the nanocomposite the ability to retain divalent cations. However, the availability of these groups plays a fundamental role in the retention of these metals. The modified SPCEs were characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. The electrode that presented the best performance was selected to determine the concentration of metal ions in water samples by square-wave anodic stripping voltammetry. The obtained detection limits were 0.23 µg L-1, 0.53 µg L-1, and 1.52 µg L-1 for Zn(II), Cd(II), and Cu(II), respectively, with a lineal range of 0.1-50 µg L-1. The obtained results made it possible to conclude that the method developed using the SPCE modified with the polymeric nanocomposite presented adequate LODs, reasonable sensitivity, selectivity, and reproducibility. Besides, this platform is an excellent tool for developing devices to simultaneously determine heavy metals in environmental samples.

Novel solid inks based on beeswax, graphite and graphene applied to the fabrication of paper-based sensor for galactose determination.

In this study, we present for the first time a simple and novel method for the fabrication of paper-based electrochemical sensors. The device development was carried out in a single stage with a standard wax printer. Hydrophobic zones were delimited with commercial solid ink, while electrodes were generated using new composite solid inks of graphene oxide/graphite/beeswax (GO/GRA/beeswax) and graphite/beeswax (GRA/beeswax). Subsequently, the electrodes were electrochemically activated by applying an overpotential. Various experimental variables for the GO/GRA/beeswax composite synthesis and electrochemical system obtention were evaluated. The activation process was examined by SEM, FTIR, cyclic voltammetry, electrochemical impedance spectroscopy and contact angle measurement. These studies showed morphological and chemical changes in the electrode active surface. As a result, the activation stage considerably improved the electron transfer on the electrode. The manufactured device was successfully applied for galactose (Gal) determination. This method presented a linear relation in the Gal concentration range from 84 to 1736 µmol L-1, with a LOD of 0.1 µmol L-1. The variation within and between-assay coefficients were 5.3% and 6.8%, respectively. The strategy here exposed for paper-based electrochemical sensors design is an unprecedented alternative system and represents a promising tool for mass production of economic analytical devices.

Microfluidic Platform Integrated with Carbon Nanofibers-Decorated Gold Nanoporous Sensing Device for Serum PSA Quantification.

Prostate cancer is a disease with a high incidence and mortality rate in men worldwide. Serum prostate-specific antigens (PSA) are the main circulating biomarker for this disease in clinical practices. In this work, we present a portable and reusable microfluidic device for PSA quantification. This device comprises a polymethyl methacrylate microfluidic platform coupled with electrochemical detection. The platinum working microelectrode was positioned in the outflow region of the microchannel and was modified with carbon nanofibers (CNF)-decorated gold nanoporous (GNP) structures by the dynamic hydrogen bubble template method, through the simultaneous electrodeposition of metal precursors in the presence of CNF. CNF/GNP structures exhibit attractive properties, such as a large surface to volume ratio, which increases the antibody's immobilization capacity and the electroactive area. CNFs/GNP structures were characterized by scanning electron microscopy, energy dispersive spectrometry, and cyclic voltammetry. Anti-PSA antibodies and HRP were employed for the immune-electrochemical reaction. The detection limit for the device was 5 pg mL-1, with a linear range from 0.01 to 50 ng mL-1. The coefficients of variation within and between assays were lower than 4.40%, and 6.15%, respectively. Additionally, its clinical performance was tested in serum from 30 prostate cancer patients. This novel device was a sensitive, selective, portable, and reusable tool for the serological diagnosis and monitoring of prostate cancer.

Microfluidic amperometric immunosensor based on porous nanomaterial towards claudin7 determination for colorectal cancer diagnosis.

In this work, we present a microfluidic amperometric immunosensor for cancer biomarker claudin7 (CLD7) determination in circulating extracellular vesicles (EVs) as well as its validation in colorectal cancer (CC) patients. The device is based on synthetized nanosized MIL-125-NH2 particles, covalently anchored to the central channel of the microfluidic immunosensor. This nanomaterial was employed as efficient platform for anti-CLD7 monoclonal antibodies immobilization for specifically recognize and capture CLD7 in EVs samples. Afterwards, the amount of this trapped CLD7 was quantified by HRP-conjugated anti-CLD7-antibody. HRP reacted with its enzymatic substrate in a redox process which resulted in the appearance of a current whose magnitude was directly proportional to the level of CLD7 in the sample. This immunosensor, under optimum conditions, gave the limit of detection for CLD7 of 0.1 pg mL-1, with a wide linear range from 2 to 1000 pg mL-1. The results reported herein open up the use of porous open framework platforms for sensing applications for biomedicine and diagnosis.

Highly sensitive and selective nanostructured microbiosensors for glucose and lactate simultaneous measurements in blood serum and in vivo in brain tissue.

Highly sensitive and selective nanostructured lactate and glucose microbiosensors for their in vivo simultaneous determination in rat brain were developed based on carbon fiber microelectrodes (CFM) modified with nanoporous gold (NPG) using the Dynamic Hydrogen Bubble Template (DHBT) method. Electrodeposition of platinum nanoparticles (PtNP) onto the NPG film enhances the sensitivity and the electrocatalytic properties towards H2O2 detection. The nanostructured microelectrode platform was modified by glucose oxidase (GOx) and lactate oxidase (LOx) enzyme immobilization. High selective measurements were achieved by covering with a perm-selective layer of electropolymerized m-phenylenediamine, deposition of a Nafion® film and by using a null sensor. The morphological characteristics and electroanalytical performance of the microbiosensors were assessed, by scanning electron microscopy and electrochemical techniques, respectively. The PtNP/NPG/CFM shows a high sensitivity to H2O2 (5.96 A M-1 cm-2) at 0.36 V vs. Ag/AgCl, with a linear range from 0.2 to 200 µM, and an LOD of 10 nM. The microbiosensors were applied to the simultaneous determination of lactate and glucose in blood serum samples. Moreover, the basal extracellular concentrations of lactate and glucose were measured in vivo in four different rat brain structures. These results support the potential of the microbiosensor to be used as a valuable tool to investigate brain neurochemicals in vivo.

Development of an indirect competitive enzyme-linked immunosorbent assay applied to the Botrytis cinerea quantification in tissues of postharvest fruits.

BACKGROUND: Botrytis cinerea is a phytopathogenic fungus responsible for the disease known as gray mold, which causes substantial losses of fruits at postharvest. This fungus is present often as latent infection and an apparently healthy fruit can deteriorate suddenly due to the development of this infection. For this reason, rapid and sensitive methods are necessary for its detection and quantification. This article describes the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for quantification of B. cinerea in apple (Red Delicious), table grape (pink Moscatel), and pear (William's) tissues. RESULTS: The method was based in the competition for the binding site of monoclonal antibodies between B. cinerea antigens present in fruit tissues and B. cinerea purified antigens immobilized by a crosslinking agent onto the surface of the microtiter plates. The method was validated considering parameters such as selectivity, linearity, precision, accuracy and sensibility. The calculated detection limit was 0.97 µg mL-1 B. cinerea antigens. The immobilized antigen was perfectly stable for at least 4 months assuring the reproducibility of the assay. The fungus was detected and quantified in any of the fruits tested when the rot was not visible yet. Results were compared with a DNA quantification method and these studies showed good correlation. CONCLUSIONS: The developed method allowed detects the presence of B. cinerea in asymptomatic fruits and provides the advantages of low cost, easy operation, and short analysis time determination for its possible application in the phytosanitary programs of the fruit industry worldwide.

Microfluidic immunosensor with gold nanoparticle platform for the determination of immunoglobulin G anti-Echinococcus granulosus antibodies.

This article describes a microfluidic immunosensor, developed for the detection of IgG antibodies specific to Echinococcus granulosus in human serum samples, which represents an alternative tool that can be used for the immunodiagnosis of hydatidosis in an automated way. Our device consists of a Plexiglas system with a central channel and a gold electrode. For immobilization of the E. granulosus antigen, a gold electrode was modified with the incorporation of gold nanoparticles. Immobilized antigen was allowed to react with IgG-anti-E. granulosus antibodies in samples, and these were quantified by horseradish peroxidase (HRP) enzyme-labeled secondary antibodies specific to human IgG using catechol (Q) as enzymatic mediator. HRP in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the oxidation of Q to o-benzoquinone (P). The electrochemical reduction back to Q was detected on the gold electrode (AuE) at -0.15 V. The current obtained was proportional to the activity of the enzyme and to the concentration of antibodies of interest. The detection limit for electrochemical detection was 0.091 ng ml(-1), and the within- and between-assay coefficients of variation were below 6.7%. The proposed system presents many benefits, the more relevant are: reduced complexity and costs that are considered as the most wanted features for the clinical-immunodiagnostic field.

Determination of Ochratoxin A in apples contaminated with Aspergillus ochraceus by using a microfluidic competitive immunosensor with magnetic nanoparticles.

Ochratoxin A (OTA) is a mycotoxin produced by filamentous fungi of the genus Aspergillus and Penicillium that presents carcinogenic, teratogenic and nephrotoxic properties. In this work, we have developed, characterized and applied an immunoassay methodology comprised of magnetic nanoparticles (MNPs) as platform for immobilizing bioactive materials incorporated into a microfluidic system for rapid and sensitive quantification of Ochratoxin A (OTA) in apples (Red Delicious) contaminated with Aspergillus ochraceus. The sensor has the potential for automation and the detection of OTA was carried out using a competitive indirect immunoassay method based on the use of anti-OTA monoclonal antibodies immobilized on 3-aminopropyl-modified MNPs. The total assay time into the microfluidic competitive immunosensor was 16 min, and the calculated detection limit was 0.05 µg kg(-1). Moreover, the intra- and inter-assay coefficients of variation were below 6.5%. The proposed method can be a very promising analytical tool for the determination of OTA in apparently healthy fruits post-harvest and for its application in the agricultural industry.

A microfluidic device based on a screen-printed carbon electrode with electrodeposited gold nanoparticles for the detection of IgG anti-Trypanosoma cruzi antibodies.

In this article we report the development of an integrated microfluidic system coupled to a screen-printed carbon electrode (SPCE) applied to the quantitative determination of IgG specific antibodies present in serum samples of patients that suffer from Chagas disease. This relevant parasitic infection caused by the hemoflagellate protozoan Trypanosoma cruzi represents a major public health concern in Latin America. In order to perform the detection of mentioned antibodies, SPCE coupled to a microfluidic device was modified by electrodeposition of gold nanoparticles (AuNPs) and functionalized with Trypanosoma cruzi proteins from epimastigote membranes. The developed microfluidic immunosensor with immobilized T. cruzi proteins on the SPCE surface was successfully applied in the detection of specific IgG anti-T. cruzi antibodies, which were allowed to react immunologically with immobilized T. cruzi antigen. After that, labelled antibodies were quantified through the addition of horseradish peroxidase (HRP) enzyme-labeled secondary antibodies specific to human IgG, using 4-tert-butylcatechol (4-TBC) as enzymatic mediator. HRP in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the oxidation of 4-TBC whose back electrochemical reduction was detected on a modified electrode at -100 mV. The calculated detection limit for electrochemical detection was 3.065 ng mL(-1) and the intra- and inter-assay coefficients of variation were below 6.95%.

Online immunoaffinity assay-CE using magnetic nanobeads for the determination of anti-Helicobacter pylori IgG in human serum.

About two-thirds of the world's population is infected with Helicobacter pylori (H. pylori). This Gram-negative bacterium is the most important etiological agent of chronic active type B gastritis and peptic ulcer diseases. Conventional methods such as gastric biopsy, ELISA and culture, require a long time for the determination of H. pylori infections. Moreover, the antibodies in human serum sample are capable to react immunologically with the purified H. pylori antigens immobilized on different kinds of support like magnetic nanobeads. In this study, we have developed an online immunoaffinity assay-CE to determine the concentration of anti-H. pylori IgG using magnetic nanobeads as a support of the immunological affinity ligands and an LIF as a detector. The separation was performed in 0.1 M glycine-HCl, pH 2, as the background electrolyte. The linear calibration curve to predict the concentration of H. pylori-specific immunoglobulin G antibodies in serum was produced within the range of 0.12-100 U/mL. The linear regression equation was i = 492.86+96.03 × C(anti-H. pylori), with the linear regression coefficient r(2) = 0.999. The LOD calculated by fluorescence detection procedure was of 0.06 U/mL. The whole assay was done in no more than 35 min and it was entirely automatized. The development of immunoaffinity assay-CE in this study demonstrates that there is a large possibility to introduce nanotechnology in several fields with significant advantages over the classic methodologies. Our proposition comprises the diagnosis and screening field.

Determination of melatonin in wine and plant extracts by capillary electrochromatography with immobilized carboxylic multi-walled carbon nanotubes as stationary phase.

The finding of melatonin, the often called "hormone of darkness" in plants opens an interesting perspective associated to the plethora of health benefits related to the moderate consumption of red wine. In this study, the implementation of a new method for the determination of melatonin in complex food matrices by CEC with immobilized carboxylic multi-walled carbon nanotubes as stationary phase is demonstrated. The results indicated high electrochromatographic resolution, good capillary efficiencies and improved sensitivity respect to those obtained with conventional capillaries. In addition, it was demonstrated highly reproducible results between runs, days and columns. The LOD for melatonin was 0.01 ng/mL. The method was successfully applied to the determination of melatonin in red and white wine, grape skin and plant extracts of Salvia officinalis L.

Determination of the beta-glucosidase activity in different soils by pre capillary enzyme assay using capillary electrophoresis with laser-induced fluorescence detection.

Enzyme activities can provide indication for quantitative changes in soil organic matter (SOM). It is known that the activities of most enzymes increase as native SOM content reflecting larger microbial communities and stabilization of enzymes on humic materials. Beta-glucosidase (beta-Glu) activities have been frequently used as indicators of changes in quantity and quality of SOM. In this study we propose a simple and very sensitive method, which has lower limit of detection compared with classic spectrophotometric method with the aim of determinate the beta-Glu activity in soil samples using Fluorescein mono-beta-D-glucopyranoside (FMGlc) as a substrate. The fluorescein released by the enzymatic reaction was quantified by capillary electrophoresis-laser induced fluorescence (CE-LIF) method. The background electrolyte (BGE) consisted in 40 mM phosphate buffer, pH 6. The LOD and LOQ for fluorescein were 1.3 10(-7) mg mL(-1) and 6.4 10(-6) mg mL(-1), respectively. This work deals with the minimization of the mixture for the enzymatic reaction and with the optimization conditions of CE separation. To the best of our knowledge, this is the first time that an enzymatic activity was detected in soil using CE-LIF system.

IgG anti-gliadin determination with an immunological microfluidic system applied to the automated diagnostic of the celiac disease.

In the present article, a novel microfluidic immunosensor coupled with electrochemical detection for anti-gliadin IgG antibody quantification is proposed. This device represents an important tool for a fast, simple, sensitive, and automated diagnostic for celiac disease, which is carried out through detection of anti-gliadin IgG antibodies present in human serum samples. Celiac disease (CD) is an autoimmune disease generated by gluten protein fractions called prolamins. This pathology affects about one in 250 people around the world, produces intestinal inflammation, villous atrophy, and crypt hyperplasia, which causes a range of symptoms including altered bowel habits, malnutrition and weight loss. Our immunosensor consists of a Plexiglas device coupled to a gold electrode, with a central channel containing 3-aminopropyl-modified controlled pore glass (AP-CPG). The quantification of anti-gliadin IgG antibodies was carried out using a heterogeneous, non-competitive enzyme-linked immunosorbent assay (ELISA) in which IgG antibodies bound to gliadin protein, immobilized on AP-CPG, were determined by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. The p-aminophenyl phosphate (p-APP) was converted to p-aminophenol (p-AP) by AP, and the electroactive product was quantified on a gold electrode at 0.250 V. The calculated detection limits for electrochemical detection and the ELISA procedure were 0.52 and 2.72 UR mL(-1), respectively, and the within- and between-assay coefficients of variation were below 5.8%. The optimized procedure was applied to the determination of anti-gliadin IgG antibodies in human serum samples.

Determination of beta-glucosidase activity in soils with a bioanalytical sensor modified with multiwalled carbon nanotubes.

Soil microorganisms and enzymes are the primary mediators of soil biological processes, including organic matter degradation, mineralization, and nutrient recycling. They play an important role in maintaining soil ecosystem quality and functional diversity. Moreover, enzyme activities can provide an indication of quantitative changes in soil organic matter. Beta-glucosidase (beta-Glu) activity has been found to be sensitive to soil management and has been proposed as a soil quality indicator because it provides an early indication of changes in organic matter status and its turnover. The aims of the present study were to test and use a simple and convenient procedure for the assay of beta-Glu activity in agricultural soil. The method described here is based on the enzymatic degradation of cellobiose by beta-Glu present in the soil sample and the subsequent determination of glucose produced by the enzymatic reaction using screen-printed carbon electrodes modified with multiwalled carbon nanotubes (SPCE-CNT) equipped with coimmobilized glucose oxidase and horseradish peroxidase enzymes. The potential applied to the SPCE-CNT detection was -0.15 V versus a Ag/AgCl pseudo-reference electrode. A linear calibration curve was obtained in the range 2.7-11.3 mM with a correlation coefficient. In the present study, an easy and effective SPCE-CNT-modified electrode allowed an improved amperometric response to be achieved and this is attributed to the increased surface area upon electrode modification.

Electrochemical microfluidic immunosensor based on TES-AuNPs@Fe3O4 and CMK-8 for IgG anti-Toxocara canis determination.

We report a microfluidic immunosensor for the electrochemical determination of IgG antibodies anti-Toxocara canis (IgG anti-T. canis). In order to improve the selectivity and sensitivity of the sensor, core-shell gold-ferric oxide nanoparticles (AuNPs@Fe3O4), and ordered mesoporous carbon (CMK-8) in chitosan (CH) were used. IgG anti-T. canis antibodies detection was carried out using a non-competitive immunoassay, in which excretory secretory antigens from T. canis second-stage larvae (TES) were covalently immobilized on AuNPs@Fe3O4. CMK-8-CH and AuNPs@Fe3O4 were characterized by transmission electron microscopy, scanning electron microscopy, energy dispersive spectrometry, cyclic voltammetry, electrochemical impedance spectroscopy, and N2 adsorption-desorption isotherms. Antibodies present in serum samples immunologically reacted with TES, and then were quantified by using a second antibody labeled with horseradish peroxidase (HRP-anti-IgG). HRP catalyzes the reduction from H2O2 to H2O with the subsequent oxidation of catechol (H2Q) to p-benzoquinone (Q). The enzymatic product was detected electrochemically at _100 mV on a modified sputtered gold electrode. The detection limit was 0.10 ng mL-1, and the coefficients of intra- and inter-assay variation were less than 6%, with a total assay time of 20 min. As can be seen, the electrochemical immunosensor is a useful tool for in situ IgG antibodies anti-T. canis determination.