María I. Sanz

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.

Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast

In the present article, we describe a study of antitumor activity in breast cell lines using silver nanoparticles (Ag NPs) synthesized by a microbiological method. These Ag NPs were tested for their antitumor activity against MCF7 and T47D cancer cells and MCF10-A normal breast cell line. We analyzed cell viability, apoptosis induction, and endocytosis activity of those cell lines and we observed that the effects of the biosynthesized Ag NPs were directly related with the endocytosis activity. Moreover, Ag NPs had higher inhibition efficacy in tumor lines than in normal lines of breast cells, which is due to the higher endocytic activity of tumor cells compared to normal cells. In this way, we demonstrate that biosynthesized Ag NPs can be an alternative for the treatment of tumors.

Microfluidic Immunosensor with Micromagnetic Beads Coupled to Carbon-Based Screen-Printed Electrodes (SPCEs) for Determination of Botrytis cinerea in Tissue of Fruits

A wide range of plant species, including economically important crops such as vegetables, ornamentals, bulbs, and fundamentally fruits, can be affected by gray mold caused by the fungal pathogen Botrytis cinerea . This paper describes the development of a microfluidic immunosensor with micromagnetic beads (MMBs) coupled to carbon-based screen-printed electrodes (SPCEs) for the rapid and sensitive quantification of B. cinerea in apple (Red Delicious), table grape (pink Moscatel), and pear (Williams) tissues. The detection of B. cinerea was carried out using a competitive immunoassay method based on the use of purified B. cinerea antigens immobilized on 3-aminopropyl-modified MMBs. The total assay time was 40 min, and the calculated detection limit was 0.008 μg mL(-1). Moreover, the intra- and interassay coefficients of variation were below 7%. The developed method allowed detects B. cinerea even in asymptomatic fruits and promises to be particularly useful for application in the agricultural industry.

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.

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.

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