Date of Award

2006

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Environmental Science

First Advisor

Dr. Sayed A. M. Marzouk

Second Advisor

Dr. Ahmed Almehdi

Third Advisor

Dr. Erno Lindner

Abstract

Sialic acid (SA) is a general term for a family composed of 43 derivatives of neuraminic acid. Whereas N-acetylneuraminic acid (NANA) is the most commonly occurring sialic acid in human. There has been a great interest in the determination of SA in humans because variations in SA level was linked to different medical conditions and diseases. In particular, serum SA are elevated in several types of cancers. SA also exists in Erythropoietin (EPO), a hormone, which induces the production of red blood cells and hence used in the treatment of anemia.

Although of the great physiological significance of SA and the attractive merits offered by the electrochemical techniques, there was a notable absence of literature describing electrochemical methods for the determination of SA. This surprising observation triggered this project to develop and to evaluate the first flow injection analysis (FIA) system as well as the first biosensor - based on amperometric transduction - for simple, fast, direct, and reliable determination of SA for clinical applications.

The principle of the present work is based on a sequence of two enzyme,s i.e., Nacetylneuraminic acid aldolase (NANA-aldolase) and pyruvate oxidase (PO) which catalyze a two-step conversion of SA into H2O2, which could be detected by anodic amperometry using platinum electrode polarized 0.6 V vs Ag/AgCI.

The first phase of the current project was to investigate the effect of different experimental variables on the generation of hydrogen peroxide by the sequence action of the two enzymes. This initial study is carried out using the two enzymes in the soluble form (i.e., homogenous enzyme catalysis). The obtained optimum experimental conditions for hydrogen peroxide generation and detection are 0.1 M phosphate buffer pH 6.3 at 37°C, using NANA-aldolase/PO activity ratio of 1.5 and a thiamine pyrophosphate (TPP) cofactor concentration of 0.5-2 mM.

The second phase aimed to construct an FIA system based on an immobilized enzyme reactor (IER) and an amperometric detector for the generated hydrogen peroxide. The IER is prepared by co-immobilization of the two enzymes on controlled pore glass beads activated with glutaraldehyde and packed in a glass tube (3-5 cm in length). A tubular platinum detector of large surface area is suggested in this work and proved efficient to enhance the sensitivity of SA determination by the proposed FIA system. The entire FIA system is evaluated under the optimum conditions obtained from the initial investigation. The obtained linear range, analysis time, and sensitivity could be easily tuned to meet the required performance characteristics by controlling the carrier buffer flow rate, and the injected sample volume. The determination of SA in real samples using the proposed FIA system is presented as well.

The third phase is devoted to the most challenging task in this project, i.e., to construct a prototype SA biosensor which necessitated co-immobilization of the two enzymes as well as their integration in a close proximity of platinum electrode surface. Although, three methods are tested for enzyme immobilization, the method based on glutaraldehyde crosslinking with BSA proved the most efficient. A novel microporous polyester membrane is used as a substrate for the enzyme layer, which provided high adhesion and reproducible fabrication of the enzyme layer. The optimum pH of the crosslinked enzyme system is ~ 1 pH higher (~ pH 7.3) than that obtained with homogenous catalysis. Careful optimization of enzyme layer composition and thickness allowed stable and fast response with detection limit of less than 10 µM SA .. Protection of the platinum surface with an inner electropolymeric layer enhanced the selectivity of the SA biosensor in the presence of interfering oxidizable species such as ascorbic and uric acids and acetaminophen. The favorable performance characteristics of the developed SA biosensor allowed its successful application in the determination of SA in simulated serum sample and real biological samples.

The obtained performance characteristics of the newly developed electrochemical methods suggest their wide use in the numerous clinical applications and in particular as a non-specific tumor marker and to monitor tumor therapy.

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