Kolesterolün ve fenolik bileşiklerin tayini için amperometrik enzim elektrotların hazırlanması

Abstract

Bu calismada, kolesterol ve fenolik bilesiklerin amperometrik tayini icin polimer film temelli amperometrik biyosensorler hazirlandi. Bu amacla, platin elektrotlar o-fenilendiamin (oPD) veya oPD ve 3-metiltiyofen (3MT) iceren asetonitril-su ortami kullanilarak donusumlu voltametri teknigi ile polimer filmlerle kaplandi. Kolesterol oksidaz (COx) veya polifenol oksidaz (POx) enzimleri Pt/PoDP ve Pt/PoPD-3MT elektrotlarin yuzeyine immobilize edildi. Pt/PoPD/COx ve Pt/PoPD-3MT/POx elektrotlarin sirasiyla kolesterol ve fenolik bilesiklerin tayini icin uygun olduklari gozlendi ve optimizasyon calismalari bu enzim elektrotlari kullanilarak yapildi. Kolesterol tayini enzimatik olarak olusan hidrojen peroksidin Ag/AgCl’e karsi +0,70 V’da yukseltgenmesine dayanilarak yapildi. Pt/PoPD/COx enzim elektrodu icin optimum tampon cinsi, tampon derisimi, pH’si, optimum calisma sicakligi sirasiyla fosfat tamponu; 0,05 M; 7,5 ve 40 °C olarak belirlendi. Enzim elektrodun, kolesterol icin dogrusal calisma araligi 9,8×10-6-1,1×10-2 mM, gozlenebilme siniri 9,8×10-6 mM, cevap suresi 150 s ve raf omru yaklasik bir hafta olarak bulundu. Kolesterol oksidaz enzim elektrot uzerine gercek numunelerde bulunan bazi muhtemel bozucu turlerin etkisi incelendi. Serum numunelerinde toplam kolesterol tayini hazirlanan Pt/PoPD/COx enzim elektrot kullanilarak yapildi ve sonuclarin spektroskopik olarak elde edilen sonuclarla uyumlu oldugu goruldu. Fenolik bilesikler, enzimatik olarak olusan o-kinonun Ag/AgCl’e karsi -0,20 V’da indirgenmesi yolu ile olculdu ve pirokatesol model substrat olarak kullanildi. Pt/PoPD-3MT/POx enzim elektrot icin optimum tampon cinsi, derisimi, pH’si, optimum calisma sicakligi ve optimum calisma potansiyeli sirasiyla fosfat tamponu; 0,10 M; 7,0; 25°C ve Ag/AgCl’e karsi -0,20 V olarak belirlendi. Enzim elektrodun, pirokatesol icin dogrusal calisma araligi 1,96×10-3-3,50×10-2 mM, alt tayin siniri 1,96×10-3 mM, cevap suresi 100 saniye ve raf omru yaklasik 2 hafta olarak bulundu. Ayrica, enzim elektrodun bazi diger fenolik bilesikler icin duyarliklari karsilastirildi ve standart referans maddede toplam fenolik bilesiklerin tayini yapildi.Abstract In this study, the polymer-based amperometric biosensors were prepared for amperometric detection of cholesterol and phenolic compounds. For this purpose, platinum electrodes were coated by polymer films from asetonitrile-water media containing o-phenylendiamine (oPD) or o-PD and 3-methylthiophene (3MT) with the cyclic voltammetry technique. Cholesterol oxidase (COx) or polyphenol oxidase (POx) enzymes were immobilized on to the surface of Pt/PoPD and Pt/PoPD-3MT electrodes. It was observed that Pt/PoPD/COx and Pt/PoPD-3MT/POx electrodes were appropriate for determination of cholesterol and phenolic compounds, respectively and these electrodes were used for optimization studies. The determination of cholesterol was performed via monitoring oxidation current of enzymatically produced H2O2 at +0.70 V vs. Ag/AgCl. Optimum buffer type, concentration, pH and optimum working temperature were determinated as phosphate, 0.05 M, 7.5 and 40° C, respectively for Pt/PoPD/COx named enzyme electrode. It was determined the linear working ranges of Pt/PoPD/COx enzyme electrode to cholesterol as 9.8×10-6-1.1×10-2 mM, detection limit as 9.8×10-6 mM, response time as 150 s and life time as about one week. The effects of possible interference species present in real samples on cholesterol oxidase enzyme electrode were examined. Analysis of total cholesterol in serum samples were performed by using proposed Pt/PoPD/COx enzyme electrode and good correlation was obtained between enzyme electrode results and those obtained by spectroscopic methods. Phenolic compounds were estimated by reduction of enzymatically liberated o-quinone at -0.20 V vs. Ag/AgCl and pyrocatechol was used as a substrate model. Optimum buffer type, concentration, pH, optimum working temperature, optimum working potential were determinated as phosphate, 0.10 M, 7.0, 25° C and -0.20 V vs. Ag/AgCl, respectively for Pt/PoPD-3MT/POx enzyme electrode. It was determined the linear working ranges of enzyme electrode for pirocatechol as 1.96×10-3-3.50×10-2 mM, detection limit as 1.96×10-3 mM, response time as 100 s and life time as about two weeks. Additionally, the sensitivity of enzyme electrode for many other phenolic compounds was compared and total phenolic compounds detection was performed in reference standard sample.