Abstract— In order to detect and quantify the phenolic contents of a wastewater with biosensors, two working electrodes based on modified Poly(Pyrrole) films were fabricated. Enzyme horseradish peroxidase was used as biomolecule of the prepared electrodes. Various phenolics were tested at the biosensor. Phenol detection was realized by electrochemical reduction of quinones produced by enzymatic activity. Analytical parameters were calculated and the results were compared with each other. Keywords—Carbon nanotube, Phenol biosensor, Polypyrrole, Poly(glutaraldehyde). I. INTRODUCTION HENOLIC compounds are among the major contaminants in medical, food and environmental matrices [1]. Some techniques such as spectrophotometry and chromatography have been employed for the determination of phenol. However, these methods are usually expensive, time- consuming, and sometimes require sample pretreatment that increases the risk of sample loss and their sensitivity and procedures limit the in-situ applications [2]. Instead of these conventional methods, biosensors could be a cheap and easy alternative measurement method, getting increasing attention in the literature [3], [4]. A biosensor is a self-contained integrated device, consisting of a biological recognition element in direct contact with a transduction element, which converts the biological recognition event into a useable output signal [5]. Owing to their specificity and sensitivity, amperometric enzyme biosensors have been developed for many applications such as electrochemical immunoassays, water pollutants detection and monitoring of biological metabolites [6]. Most amperometric biosensors for the detection of phenolic compounds have been introduced as a mono-enzyme system using tyrosinase, laccase or horseradish peroxidase (HRP) [7]. HRP based biosensors are most sensitive for a great number of phenolic compounds since phenols can be act as electron donors for peroxidase [8]. Recent research activities have focused on the design and construction of modified working electrodes which are most effective to achieve faster enzymatic reaction and electron flow in biosensors. S. Korkut is with the Department of Environmental Engineering, Bulent Ecevit University, Zonguldak, 67100 Turkey (corresponding author phone: +90 372 2574010; fax: +90 372 2574023; e-mail: s.korkut@beun.edu.tr). M. S. Kilic is with the Department of Chemistry, Bulent Ecevit University, Zonguldak, 67100 Turkey (e-mail: sametkilic_4274@hotmail.com). E. Erhan is with the Department of Environmental Engineering, Gebze Institute of Technology, Gebze-Kocaeli, 41400 Turkey (e-mail: e.erhan@gyte.edu.tr). In this paper, we discussed measurement performance of two different modified Poly(pyrrole) (PPy) film based phenol biosensors prepared by immobilizing the HRP on gold electrodes via entrapment and chemical bond. Various phenolic compounds were tested at each electrode. Analytical measurement parameters of the biosensors were calculated and compared with each other. II. MATERIALS AND METHODS A. Reagents HRP (E.C.1.11.1.7) with an activity of 10.000U/vial (according to pyrogallol method performed by the supplier), aqueous solution of hydrogen peroxide (35%), phenol (purity of 99%), glutaraldehyde (25% w/v), lithium chloride, di- potassium hydrogen phosphate and potassium di-hydrogen phosphate were purchased from Merck. Pyrrole and sodium dodecyl sulfate (SDS) and phenol derivatives were obtained from Sigma. Multiwalled carbon nanotubes (CNTs) were obtained from Nanocs. Inc., Newyork, USA. B. Experimental Setup All electrochemical experiments were performed by using a CHI 800B Model electrochemical analyzer. Three-electrode system included the gold (Au) working electrode, a Pt wire counter electrode, Ag/AgCl (3 M NaCl) reference electrode and a conventional three-electrode electrochemical cell was obtained from CH Instruments. Amperometric measurements were conducted at a fixed potential of -50 mV. The reaction medium consisted of 100 mM, pH 7 potassium phosphate buffer including, 0.7 mg/mL of lithium chloride as the supporting electrolyte. Three-electrode system was immersed into the reaction medium and, analyzer was started. After reaching a steady-state current, increasing concentrations of phenolic compounds were added to the medium. The amperometric currents were recorded as current-time recordings. The experimental setup was presented in Fig. 1. C. Fabrication of Working Electrodes PGA/PPy/HRP/Au working electrode was prepared as follows: polyglutaraldehyde (PGA) solution was prepared by adding 2 mL of 0.1 M NaOH and 2 mL of 25% glutaraldehyde into 10 mL of distilled water. The final solution was stirred at 600 rpm for 30 minutes up to the reaching a final pH of 9-10. The pyrrole polymerization medium was comprised of 100 mM pyrrole and 0.6 mg/mL SDS in 10 mL of PGA solution. The potential was scanned between 0 and +1.2V for electropolymerization of pyrrole. Au electrode was immersed in 25% glutaraldehyde solution and stored at +4°C overnight Modified Poly(pyrrole) Film Based Biosensors for Phenol Detection S. Korkut, M. S. Kilic, E. Erhan P World Academy of Science, Engineering and Technology International Journal of Environmental and Ecological Engineering Vol:9, No:3, 2015 439 International Scholarly and Scientific Research & Innovation 9(3) 2015 scholar.waset.org/1307-6892/10000759 International Science Index, Environmental and Ecological Engineering Vol:9, No:3, 2015 waset.org/Publication/10000759