Enzyme Biosensors for Phenols Detection

เอนไซม์ไบโอเซนเซอร์สำหรับการตรวจวัดฟีนอล

Name: Patsamon Rijiravanich

Organization : King Mongkut's University of Technology Thonburi. School of Bioresources and Technology. Biotechnology

keyword: Phenols

; Biosensors

Abstract: This dissertation concerns the construction of enzyme biosensors and the measurementof phenols under batch conditions. Three aspects are considered the electrochemicalreaction of catechol at polyphenol oxidase (PPO) immobilized on latex spheres (PPOlatex),the adsorption of PPO-Iatex on micro-cyclinder carbon fibre electrodes forphenol and catechol determination, and a dual-enzyme biosensor for total phenoldetermination.The first approach used PPO immobilized on the polystyrene latex particles in order tocontrol the amount of enzyme. The PPO-latex particles composed of the corepolystyrene and four successive coating layers: polystyrene sulfonate, polyallylamine,PPO and polyallylamine, built up by the layer-by-layer technique. The particles showedcatalytic currents for the enzymatic oxidation of catechol to a-quinone. The enzymeactivity per particle was evaluated as 2.3 x 10-7 Units from UV absorption of thea-quinone. The relation between the catalytic current and concentration of catechol leadto a Michelis-Menten type kinetic equation. The layer-by-layer method was found tohave a deactivating effect on enzyme activity. In spite of this, the catechol oxidationcurrent was larger than the current from free PPO at a common value of enzyme activity'per volume. This was ascribed to strong adsorption of the latex particles on theelectrode, leading to the enhancement in the local concentration of PPO.PPO-Iatex particles adsorbed onto electrochemically pretreated carbon fibres were usedto construct microelectrode sensors for phenol and catechol. The steady-state responsesof the fibres were analyzed in terms of a cylindrical diffusion-kinetic model. It wasdeduced that the adsorbed latex particles provided a relatively open film structure,resulting in a diffusion coefficient only one order of magnitude lower than the solutionvalue, and that at least 9% of the immobilized enzyme was catalytically active. Theoptimized sensors exhibited linear ranges to phenol and catechol of 7-56.5 u.M and2-19.7 ~LM respectively, with sensitivities of 0.15 A M-l cm-2 and 1.72 A Ivr 1 cm-2respectively. The limiting factor to sensor stability was desorption of latex from thefibres.In the third approach, a biosensor for total phenol determination was investigated, basedon a dual enzyme electrode. Three types of enzyme, PPO, laccase (Lac) and horseradishperoxidase (HRP) , were studied. PPO and Lac were immobilized byelectropolymerization of poly(pyrrole) on a platinum electrode. HRP was bound by thesequential immobilization of sulfonic thiol, HRP and polystyrene sulfonate (PSS) onto agold electrode. The steady-state response of each enzyme electrode was noted whilevarying film thickness, loading enzyme, rate of convection and pH. Eight phenolderivatives were screened by each biosensor. The different enzymes showed differentamperometric responses to the different derivatives. Hence, a dual-biosensor, based onPPO- and HRP-modified electrodes, was used for total phenols measurement. Thebiosensor could determine total phenol concentration in a mixture of two to fourderivatives with standard error of 0.18 to 1.95 using, the proposed mathematics model.However, passivation by quinone derivatives limited the sensor stability.

King Mongkut's University Technology Thonburi. Library

Address: กรุงเทพมหานคร (Bangkok)

Email: info.lib@mail.kmutt.ac.th

Name: Werasak Surareungchai

Role: Advisor

Name: Mithran Somasundrum

Role: Advisor

Created: 2004

Issued: 2007-06-15

Modified: 2007-07-20

วิทยานิพนธ์/Thesis

ISBN: 9741847963

CallNumber: BIT526

eng

DegreeName: Doctor of Philosophy

Level: Doctoral Degree

Descipline: Biotechnology

Grantor: King Mongkut's University of Technology Thonburi

©copyrights King Mongkut's University of Technology Thonburi

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