An Electrochemical Competitive Assay for the Detection of Endotoxins Using a Single Addressable Electrode Array System

PALLAROLA DIEGO; PRIANO GRACIELA; GONZÁLEZ GRACIELA; BATTAGLINI FERNANDO

Sevilla, España

Congreso; 59th Annual Meeting of The International Society of Electrochemistry; 2008

International Society of Electrochemistry

A complete system for the detection of lipopolysaccharides (LPS) was developed. LPS, also known as endotoxins, have potent immunostimulating activity that sometimes causes lethal sepsis when entering into the blood stream. Localization of LPS-binding sites and identification of membrane receptors are important for developing antisepsis drugs. In this work several issues have been taking into account for the development of an electrochemical competitive assay for LPS determination, this involves a single addressable 8 gold electrode array, the synthesis of an electroactive conjugate, HRP-LPS, and the electrode modification with a recognition element. The electrode array was constructed by a combination of screen printing techniques and gold electrodeposition, yielding a disposable cost-effective system, that can be easily implemented in any amperometric immunoassay. The conjugation of LPS from S. minnesota with horseradish peroxidase (HRP) through activation with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) in an aqueous medium is presented. In order to promote the reaction a series of amphiphilic compounds were tested, being sodium deoxycholate the most suitable.  The method presents several advantages: it is carried out in a mild environment, good conjugation ratios are obtained, the endotoxic activity of LPS is preserved and a good enzymatic activity is observed for HRP. Finally, it was carried out the development of a recognition surface, taking advantage of the chemical interactions between lipopolysaccharides and antibacterial agents produced by many organisms. These findings allow the development of assays equivalent to those based upon antigen-antibody interaction as an alternative for the commercially available LAL test method, among the available recognition element a short peptide named CAP-18 and an endotoxin neutralizing protein (ENP) were tested. The final design consists in an arrangement of eight modified gold electrodes with one  of the LPS recognition element. The modified electrodes were constructed by covalent linking of the recognition element to a carboxymethyl dextran matrix. The modified lipopolysaccharide with horseradish peroxidase is used as the signaling element. This arrangement avoid nonspecific adsorption of LPS leading to a good signal/background ratio capable of detecting the presence of endotoxins in the order of ng mL-1.