Eloy Salinas

Enzymatic rotating biosensor for cysteine and glutathione determination in a FIA system

The high sensitivity that can be attained using an enzymatic system and mediated by catechols has been verified by on-line interfacing of a rotating biosensor and continuous flow/stopped-flow/continuous-flow processing. Horseradish peroxidase, HRP, [EC 1.11.1.7], immobilized on a rotating disk, in presence of hydrogen peroxide catalyzed the oxidation of catechols, whose back electrochemical reduction was detected on glassy carbon electrode surface at -150mV. Thus, when l-cysteine (Cys) or glutathione (GSH) was added to the solution, these thiol-containing compounds participate in Michael addition reactions with catechols to form the corresponding thioquinone derivatives, decreasing the peak current obtained proportionally to the increase of its concentration. Cys was used as the model thiol-containing compound for the study. The highest response for Cys was obtained around pH 7. This method could be used to determine Cys concentration in the range 0.05-90muM (r=0.998) and GSH concentration in the range 0.04-90muM (r=0.999). The determination of Cys and GSH were possible with a limit of detection of 0.7 and 0.3nM, respectively, in the processing of as many as 25 samples per hour. Current response of the HRP-rotating biosensor is not affected by the oxidized form of GSH and Cys (glutathione disulfide, GSSG, and l-cystine, respectively), by sulfur-containing and alkyl-amino compounds such as methionine and lysine, respectively. The interferences from easily oxidizable species such as ascorbic acid and uric acid are lowest.

Enzymatic rotating biosensor for ciprofloxacin determination

The high sensitivity that can be attained using an enzymatic system and mediated by catechol has been verified by on-line interfacing of a rotating biosensor and continuous flow/stopped-flow/continuous-flow processing. Horseradish peroxidase, HRP [EC 1.11.1.7], immobilized on a rotating disk, in the presence of hydrogen peroxide, catalyzed the oxidation of catechol, whose back electrochemical reduction was detected on a glassy carbon electrode surface at -200mV. Thus, when ciprofloxacin (CF) was added to the solution, this piperazine-containing compound participate in Michael addition reactions with catechol to form the corresponding piperazine-quinone derivatives, decreasing the peak current obtained, in proportion with the increase of its concentration. The highest response for CF was obtained around pH 7. This method could be used to determine CF concentration in the range of 0.02-65muM (r=0.999). The determination of CF concentration was possible with a detection limit of 0.4nM, in the processing of as many as 25 samples per hour. Application of this analysis to different pharmaceutical samples containing CF supports the utility of the HRP-rotating biosensor.

Continuous-flow system for horseradish peroxidase enzyme assay comprising a packed-column, an amperometric detector and a rotating bioreactor

The roles of chemical kinetics and mass transfer in three types of bioreactors (packed-column reactors, rotating disk bioreactors and amperometric detector), used with continuous-flow sample/reagent(s) processing, are discussed in detail. A normalized quantitative comparison between these types of reactors clearly shows that rotating disk reactors afford a significantly more efficient utilization of active sites and permit the effective utilization of very small amounts of biocatalysts. Horseradish peroxidase (EC 1.11.1.7), in presence of hydrogen peroxide catalyses the oxidation of [Os(bpy)(2)Cl(pyCOOH)]Cl. The electrochemical reduction back of this cosubstrate is detected on glassy carbon electrode surface at 0.00V. Furthermore, the critical effect of substrate and cosubstrate concentration on amperometric immunosensors construction in which HRP is used as an enzymatic label was studied.

Milk lactate determination with a rotating bioreactor based on an electron transfer mediated by osmium complexes incorporating a continuous-flow/stopped-flow system

The high sensitivity that can be attained using a bienzymatic system and mediated by the redox polymer [Os(bpy)2ClPyCH2NHpoly(allylamine)] (Os-PAA), has been verified by on-line interfacing of a rotating bioreactor and continuous-flow/stopped-flow/continuous-flow processing. When the hydrogen peroxide formed by LOx layer reaches the inner layer, the electronic flow between the immobilized peroxidase and the electrode surface produces a current, proportional to lactate concentration. The determination of lactate was possible with a limit of detection of 5 nmol l−1 in the processing of as many as 30 samples per hour. This arrangement allows working in undiluted milk samples with a good stability and reproducibility. Horseradish peroxidase [EC 1.11.1.7] and Os-PAA were covalently immobilized on the glassy carbon electrode surface (upper cell body), lactate oxidase [EC 1.1.3.x] was immobilized on a disk that can be rotated.

Immuno-column for on-line quantification of human serum IgG antibodies to Helicobacter pylori in human serum samples.

This study report an human serum IgG antibodies to Helicobacter pylori quantitation procedure based on the multiple use of an immobilized H. pylori antigen on an immuno-column incorporated into an a flow-injection (FI) analytical system. The immuno-adsorbent column was prepared by packing 3-aminopropyl-modified controlled-pore glass (APCPG) covalently linking H. pylori antigens in a 3-cm of Teflon tubing (0.5 i.d.). Antibodies in the serum sample are allowed to react immunologically with the immobilized H. pylori antigen, and the bound antibodies are quantified by alkaline phosphatase (AP) enzyme-labeled second antibodies specific to human IgG. p-Aminophenyl phosphate (pAPP) was converted to p-aminophenol (pAP) by AP and an electroactive product was quantified on glassy carbon electrode (GCE) modified with multiwall carbon nanotubes (MWCNT) (GCE-CNTs) at 0.30 V. The total assay time was 25 min. The calculated detection limits for amperometric detection and the ELISA procedure are 0.62 and 1.8 UmL(-1), respectively. Reproducibility assays were made using repetitive standards of H. pylori-specific antibody and the intra- and inter-assay coefficients of variation were below 5%. The immuno-affinity method showed higher sensitivity and lower time-consumed, demonstrate its potential usefulness for early assessment of human serum immunoglobulin G (IgG) antibodies to H. pylori.

Use of multiple sequential injections of equal volumes to determine the apparent binding constant for antibody-antigen complexes by capillary electrophoresis

A new modified version of the well-known flow-through partial-filling technique [viz. multiple sequential injection of equal volumes (MSI-EV) of neutral marker, antigen (Ag) and antibody (Ab)] was used to calculate the apparent binding constant (Ka) of monoclonal Ab (mAb) and polyclonal Ab (pAb) to their specific antigens (Ags). Such a constant is very important in immunoassays. The procedure involves the sequential injection of small, identical volumes of a neutral marker (dimethyl sulfoxide, DMSO), an Ag and an Ab into a capillary column for electrophoresing. The apparent Ka values thus obtained from a Scatchard plot were 0.76+/-0.15 mg(-1) mL for the complex of anti-canine Immunoglobulin G (IgG) as mAb and canine IgG as Ag, and 0.79+/-0.14 mg(-1) mL for that between anti-human IgG as pAb and human IgG as Ag. These values are of the same order to those provided by indirect competition enzyme-linked immunosorbent assay (ELISA) (viz. 0.42+/-0.28 mg(-1) mL for the mAb-Ag complex and 0.81+/-0.09 mg(-1) mL for the pAb-Ag complex). The high sensitivity of the MSI-EV-CE technique affords the detection of very low concentrations of Ab.

CONTINUOUS-FLOW/STOPPED-FLOW SYSTEM INCORPORATING A ROTATING BIOREACTOR BASED ON A DOUBLE REDOX CATALYTIC CYCLE AND ELECTRON TRANSFER MEDIATED BY OSMIUM COMPLEXES. APPLICATION IN THE DETERMINATION OF EXTREMELY LOW LEVELS OF GLUCOSE

ABSTRACT The high sensitivity that can be attained by enzymatic amplification via substrate cycling and mediated by the redox polymer [Os(bpy)2ClPyCH2NHpoly(allylamine) (Os-PAA), has been verified by on-line interfacing of a rotating bioreactor and continuous-flow/stopped-flow/continuous-flow processing system. The determination of glucose was possible with a limit of detection of 20 fmol L−1 in the processing of as many as 30 samples per hour. Determination at such low levels is of interest in several situations encountered in fermentation biotechnology and clinical chemistry, and for the determination in culture broths; it illustrates the capabilities of the proposed approach. Glucose oxidase and Os-PAA were covalently immobilized on a glassy carbon electrode surface (upper cell body), Glucose dehydrogenase [EC 1.1.1.47] was immobilized on a disk that can be rotated. Substrate cycling was realized via NADH/NAD+ that, in conjunction with glucose dehydrogenase, regenerates glucose, the substrate in the glucose oxidase-catalyzed reaction.

Electrochemical Study of the Antioxidant Activity and the Synergic Effect of Selenium with Natural and Synthetic Antioxidants

In this paper we propose two different electrochemical methods such as Cyclic Voltammetry (CV) and Osteryoung Square Wave Voltammetry (OSWV) to study the free radical scavenging ability of Selenium (Se) and natural and synthetic antioxidants. The originality of this paper is based on the study of the synergic effect of Se, not only with α-Tocopherol, but with a variety of antioxidants. As a result, we find an important synergism, in vitro, between Se and some other natural and synthetic antioxidants in the aqueous medium.

Detection transposable elements in Botrytis cinerea in latent infection stage from symptomless apples

Fil: Fernandez, Jorge Gaston. Universidad Nacional de San Luis. Facultad de Quimica, Bioquimica y Farmacia. Departamento de Quimica. Area de Quimica Analitica; Argentina