Biomedical subjects

Fernando Battaglini

Publications and source records attributed to Fernando Battaglini.

3 recordsLinked to original sources

PubMed2006

Endotoxin detection in a competitive electrochemical assay: synthesis of a suitable endotoxin conjugate.

A biotin-lipopolysaccharide (biotin-LPS) conjugate was synthesized from LPS smooth from Salmonella minnesota, yielding a conjugate with a biotin/LPS ratio equal to 1:1 and endotoxic activity of 0.08 EU ng(-1). The conjugate was used in an amperometric competitive assay to determine endotoxins with endotoxin-neutralizing protein (ENP) as the recognition element. The assay is performed on a modified electrode, involving the covalent binding of carboxymethyl dextran (CMDex) to a cystamine-modified gold electrode and then the covalent binding of the recognition protein, ENP, to CMDex. The assay is carried out by incubating the modified electrode in an LPS sample to which biotin-LPS was added. Both species compete for the recognition sites on the modified surface. After the incubation stage and a careful rinsing, the electrode is immersed in a solution containing neutravidin-horseradish peroxidase conjugate (N-HRP), which binds to the sites containing biotin-LPS on the electrode. The system is rinsed and a current signal is generated by the addition of hydrogen peroxide and a redox mediator. The assay is able to detect LPS from Salmonella minnesota at concentrations as low as 0.1 ng ml(-1), equivalent to 0.07 EU ml(-1).

Biotin↗

PubMed2005

Use of an antimicrobial protein for endotoxin detection in a competitive electrochemical assay.

Endotoxins, also referred to as pyrogens, are part of the cellular walls of Gram-negative bacteria and are capable of inducing fever when entering into the blood stream. Current methods of detection involve the use of either amoebocytes from horseshoe crab that clots upon exposure to endotoxin or a live assay on rabbits. In this work, the detection of endotoxins by an electrochemical competitive assay is presented. Two configurations of modified electrodes were constructed using a recombinant endotoxin neutralizing protein (ENP) as recognition element. A modified lipopolysaccharide with horseradish peroxidase was used for the competitive assay. Modified electrodes constructed by electrostatic interaction of ENP and an electroactive polymer can detect the presence of endotoxins in concentrations as low as 0.2EU mL(-1), below the limit imposed for water in injectable drugs in the American Pharmacopoeia. Modified electrodes constructed by covalent linking of ENP to a carboxymethyl dextran matrix bound to the electrode show a better dynamic range but a higher detection limit.

Adsorption↗

PubMed2004

Continuous-flow/stopped-flow system for enzyme immunoassay using a rotating bioreactor: determination of Chagas disease.

The high sensitivity that can be attained using an immunoassays coupled to a rotating bioreactor with electrochemical detection mediated by [Os(bpy)2Cl(pyCOOH)]Cl, has been verified for the detection of Trypanozoma cruzi (T. cruzi), This protozoan parasite causes Chagas disease, affecting more than 18 million people in central and south America. Antibodies in the serum sample are allowed to react immunologically with whole homogenates of the parasite as antigen that are immobilized on a rotating disk. The bound antibodies are quantified by a horseradish peroxidase (HRP) enzyme labeled second antibodies specific to human IgG in presence of hydrogen peroxide using an osmium complex [Os(bpy)2Cl(pyCOOH)]Cl as enzymatic mediators. The amperometric measurement performed at 0.00 V versus Ag/AgCl can be done within 2 min and the analysis time does not exceed 23 min. The calculated detection limits was 0.01 mIU ml(-1). Reproducibility assays were made using repetitive serum of 0.182 mIU ml(-1) T. cruzi specific antibody (measured as the activity of the correspondent anti-serum's enzyme conjugated); the percentage standard error was less than 5%. The amperometric immunoreactors showed significantly higher sensitivity and lower time consumed than the standard spectrophotometric detection ELISA method.

Animals↗