Development of Galactose Biosensor Based on Ruthenium Mediator Complex

Öz

Measurement of galactose level is of clinical significance in terms of galactosemia and galactose intolerance. Especially in newborns, it can have fatal consequences if it is not detected immediately. After diagnosis, galactose should be removed from the diet. Therefore, the determination of galactose in both blood and diet, especially milk and dairy products, is of great importance. In the literature data, the methods developed for galactose determination are time consuming and relatively slow methods that require pre-processing and expensive equipment such as spectrophotometer, chromatography, mass spectrometer. The aim of this study is to develop a fast, specific, inexpensive electrochemical biosensor system for galactose determination.In this study, in which Galactose Oxidase (GaOX) enzyme was used as a biocomponent, a carbon paste electrode containing a ruthenium mediator complex was used as the working electrode. GaOX enzyme was immobilized in the paste structure. The originality of the study is the use of the Ruthenium mediator system used in the paste structure. Electrode construction stages were characterized by cyclic voltammetry (CV) method, while optimization and characterization studies were carried out by means of DP between 0 & 0.9 V and amperometric measurements at 0.5 V. The studies also used a 1 mM p-benzoquinone mediator system in working buffer. As a result of optimization studies, optimum pH was found to be 6.5 and optimum temperature was 30 oC. While the linear detection range of the developed biosensor was found between 0.05- mM, the repeatability was found to be 0.24 mM on average, the standard deviation ± 0.021 and the coefficient of variation (CV%) as 8.3% for 0.25 mM galactose. No interference was observed in the interference effect trial. Sample analysis was made on lyophilized serum and lactose-free milk samples and % recovery was calculated. The use of the Ruthenium mediator complex used in this project in the production of biosensors has been rarely encountered in the literature. However, with this study, it was used for the first time in a galactose biosensor and for the first time with a carbon paste electrode.

Anahtar Kelimeler

• rutenium mediator complex
• galactose
• galactosemia
• biosensor
• amperometry

Rutenyum Mediyatör Kompleksi Temelli Galaktoz Biyosensörü Geliştirilmesi

Öz

Galaktoz seviyesinin ölçümü, galaktozemi ve galaktoz intoleransı açısından klinik önem taşır. Özellikle yenidoğanlarda hemen tespit edilmemesi ölümcül sonuçlar doğurabilir. Teşhis konduktan sonra da galaktozun diyetten çıkarılması gerekir. Dolayısıyla hem kan hem de diyette özellikle süt ve süt ürünlerinde galaktozun tayini büyük önem taşır. Literatür verilerinde galaktoz tayini için geliştirilen yöntemler spektrofotometre, kromatografi, kütle spektrometresi gibi ön işlem ve pahalı ekipman gerektiren, zaman alıcı ve nispeten yavaş yöntemlerdir. Bu çalışmanın amacını da galaktoz tayini için hızlı, spesifik, ucuz elektrokimyasal bir biyosensör sistemi geliştirilmesi oluşturmaktadır. Biyokomponent olarak Galaktoz Oksidaz (GaOX) enzimi kullanılan bu çalışmada, çalışma elektrodu olarak Rutenyum mediyatör kompleksi içeren karbon pasta elektrot kullanılmıştır. GaOX enzimi pasta yapısı içerisinde immobilize edilmiştir. Çalışmanın özgünlüğünü pasta yapısı içerisinde kullanılan Rutenyum mediyatör sisteminin kullanılması oluşturmaktadır. Elektrot yapım aşamaları döngüsel voltammetri (CV) yöntemiyle karakterize edilirken, optimizasyon ve karakterizasyon çalışmaları 0&0.9 V arası DP, ve 0.5 V’ta amperometrik ölçümler yoluyla gerçekleştirilmiştir. Çalışmalarda ayrıca çalışma tamponu içerisinde 1 mM p-benzokinon mediyatör sistemi kullanılmıştır.Optimizasyon çalışmaları sonu optimum pH 6,5, optimum sıcaklık ise 30 oC olarak bulunmuştur. Geliştirilen biyosensörün doğrusal tayin aralığı 0.05 – 1 mM arası bulunurken, tekrarlanabilirliği 0.25 mM galaktoz için ortalama 0.24 mM, standart sapma ± 0.021 ve varyasyon katsayısı (%VK) %8,3 olarak bulunmuştur. Girişim etkisi denemesinde herhangi bir girişim gözlenmemiştir. Örnek denemesi liyofilize serum ve laktozsuz süt örneklerinde yapılmış ve % geri elde hesaplanmıştır. Bu projede kullanılan Rutenyum mediyatör kompleksinin biyosensör yapımında kullanılmasına literatürde çok az rastlanılmış olmakla birlikte bu çalışmayla ilk defa bir galaktoz biyosensöründe ve ilk defa bir karbon pasta elektrotla kullanımı gerçekleştirilmiştir.

Anahtar Kelimeler

• rutenyum mediyatör kompleksi
• galaktoz
• galaktozemi
• biyosensör
• amperometri

Kaynakça

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