Lipid tayini için enzim esaslı biyosensör geliştirilmesi
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Dosyalar
Tarih
2012
Yazarlar
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Yayıncı
Trakya Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu tez çalışmasında, lipidlerin tayin edilmesine yönelik, lipaz enzimi esaslı bir pH biyosensörü geliştirilmesi amaçlandı. Cam pH elektrodu üzerine, jelatin matriksi, çapraz bağlayıcı glutaraldehit ve seçilen enzim lipaz kullanılarak pH biyosensörü hazırlandı. Önce hazırlanan pH biyosensörü ile, substrat spesifitesi çalışması yapıldı. Seçilen substrat kullanılarak, biyosensörün biyoaktif tabaka bileşenlerinin ve çalışma koşullarının optimizasyonları yapıldı. pH biyosensörünün belirlenen optimum koşullarda karakterizasyon çalışmaları gerçekleştirildi. Son olarak geliştirilen pH biyosensörü ile optimum koşullarda, çeşitli kan örneklerinde lipid tayini için uygulanabilirlik çalışmaları yapıldı. Tez çalışmasında; pH biyosensörü geliştirmede kullanılacak lipaz enzimi olarak; domuz pankreatik lipazı ve Candida rugosa lipazı kullanıldı. pH biyosensöründe daha yüksek sensör cevapları Candida rugosa lipazı ile elde edildi. Biyosensörün substrat seçimi için, triasetin, triolein, tristearin, tribütirin, trikaprilin denendi. Seçilen lipazla hazırlanan biyosensör, içlerinden en yüksek spesifisiteyi tribütirine karşı gösterdi. Belirlenen enzim ve substrat kullanılarak, biyoaktif tabaka bileşenlerini oluşturan enzimin ve jelatinin miktarları, gluteraldehidin % oranının biyosensör cevapları üzerine etkisi incelendiğinde; optimum değerler sırasıyla 9 mg, 20 mg ve % 2.5 olarak belirlendi. Çalışma koşullarının optimizasyon çalışmalarında; hazırlanan pH biyosensörünün biyosensör cevapları üzerine, reaksiyon ortamı tampon pH'ı, tampon konsantrasyonu ve tampon sıcaklığının etkileri incelendi. Çalışmalar sonunda; Tris-HCl tampon sistemi pH'ı 7.5, derişimi 200 mM, ve optimum sıcaklığı ise 35 ºC olarak bulundu. pH biyosensörünün karakterizasyon çalışmalarında; optimum koşullarda hazırlanan biyosensörün lipid tayini için, tribütirin substratına göre doğrusal tayin aralığı 16.5 ? 82.5 mM olarak belirlendi. pH biyosensörünün analiz sonuçlarının tekrarlanabilirliği çalışması için; 82.5 mM substrat ile arka arkaya ölçümler alındı. Xort= 91.25 mM, standart sapma (S.D.) ± 4.732, varyasyon katsayısı (C.V.) % 10.97 olarak belirlendi. İşlem kararlılığı belirlenmesi için; hazırlanan biyosensör ile optimum koşullarda tribütirin subsratının sabit derişimiyle ölçümler alındı. Biyosensörün depo kararlılığı çalışmalarında; optimum koşullarda hazırlanan pH biyosensörünün 25 saat boyunca belirli aralıklarla substrata karşı pH değişim ölçümleri yapıldı. İlk 8 saat aktivitenin %50'si korundu. Biyosensörün canlı sıvı örneklerinden kan serumunda lipid tayinlerinde uygulanabilirlik çalışmasında; yapılan ölçümlere ait standart sapma ve varyasyon katsayıları hesaplandı. Anahtar kelimeler: Lipaz, Lipid, pH biyosensörü, Trigliserid, Candida rugosa
Abstract
In this thesis study, for the determination of lipids to develop an lipase enzyme-based pH biosensor was aimed. The pH biosensör was prepared on glass pH electrode using gelatine as a matrix, glutaraldehyde as a crosslinking and selected lipase enzyme. Primarily, substrate specificity studied by prepared pH biosensor. Using the prepared biyosensor and the selected substrate, the optimizations of the components of the bioactive layer of biosensor and working conditions were made. pH biosensor?s characterization studies were carried out at determined optimum conditions. Finally, the applicability of lipid determinations of various blood examples with developed pH biosensor was carried out under the optimum conditions. In thesis study; as the lipase enzyme; porcine pancreatic lipase and Candida rugosa lipase were used in development of pH biosensor. In the pH biosensor higher sensor responses were obtained with Candida rugosa lipase. For biyosensor?s substrate selection as substrates were tested by using triacetine, trioleine, tristearin, tricaprylne and tributyrine. Biyosensor prepared with selected lipase showed highest specificity against tributyrine. By using determined enzyme and substrate, layer of bioactive components that make up the amounts of the enzyme and gelatin, glutaraldehyde rate %, response on the impact of biosensors is examined, the optimum values was determined 9 mg, 20 mg and 2.5 %, respectively. In the optimization working conditions; effects of reaction medium of buffer pH, buffer concentration and buffer temperature on biosensor responses were examined by prepared pH biosensor. At the end of studies, optimum values were found to be Tris-HCl buffer system pH:7.5, buffer concenration 200 mM and optimum temperature 35 ºC, respectivelly. In the pH biosensor characterization studies; The linear measurement range of the developed pH biosensor with tributyrine substrate was determined between 16,5-82,5 mM. in the optimum working conditions. For determination of repeatability of analysis results; measurements were done at 82,5 mM concentration of tributyrine. Xort= 91.25 mM, S.D. ± 4.732, C.V. % 10,97 were determined. For determination of operational stability of prepared biosensor; the experiments were carried out with constant concentration of tributyrine substrate at optimum conditions. For determination of storage stability of biosensor; changes in pH against to substrate were measured interval at certain during 25 hours with prepared biosensor in optimum conditions. The first 8 hours, the activity was protected constantly %50. Applicability of lipid determinations in human blood samples of human; the standard deviation and the coefficient of variation of the measurements were calculated. Keywords: Lipase, Lipid, pH biosensor, Triglyceride, Candida rugosa
Abstract
In this thesis study, for the determination of lipids to develop an lipase enzyme-based pH biosensor was aimed. The pH biosensör was prepared on glass pH electrode using gelatine as a matrix, glutaraldehyde as a crosslinking and selected lipase enzyme. Primarily, substrate specificity studied by prepared pH biosensor. Using the prepared biyosensor and the selected substrate, the optimizations of the components of the bioactive layer of biosensor and working conditions were made. pH biosensor?s characterization studies were carried out at determined optimum conditions. Finally, the applicability of lipid determinations of various blood examples with developed pH biosensor was carried out under the optimum conditions. In thesis study; as the lipase enzyme; porcine pancreatic lipase and Candida rugosa lipase were used in development of pH biosensor. In the pH biosensor higher sensor responses were obtained with Candida rugosa lipase. For biyosensor?s substrate selection as substrates were tested by using triacetine, trioleine, tristearin, tricaprylne and tributyrine. Biyosensor prepared with selected lipase showed highest specificity against tributyrine. By using determined enzyme and substrate, layer of bioactive components that make up the amounts of the enzyme and gelatin, glutaraldehyde rate %, response on the impact of biosensors is examined, the optimum values was determined 9 mg, 20 mg and 2.5 %, respectively. In the optimization working conditions; effects of reaction medium of buffer pH, buffer concentration and buffer temperature on biosensor responses were examined by prepared pH biosensor. At the end of studies, optimum values were found to be Tris-HCl buffer system pH:7.5, buffer concenration 200 mM and optimum temperature 35 ºC, respectivelly. In the pH biosensor characterization studies; The linear measurement range of the developed pH biosensor with tributyrine substrate was determined between 16,5-82,5 mM. in the optimum working conditions. For determination of repeatability of analysis results; measurements were done at 82,5 mM concentration of tributyrine. Xort= 91.25 mM, S.D. ± 4.732, C.V. % 10,97 were determined. For determination of operational stability of prepared biosensor; the experiments were carried out with constant concentration of tributyrine substrate at optimum conditions. For determination of storage stability of biosensor; changes in pH against to substrate were measured interval at certain during 25 hours with prepared biosensor in optimum conditions. The first 8 hours, the activity was protected constantly %50. Applicability of lipid determinations in human blood samples of human; the standard deviation and the coefficient of variation of the measurements were calculated. Keywords: Lipase, Lipid, pH biosensor, Triglyceride, Candida rugosa
Açıklama
Yüksek Lisans Tezi
Anahtar Kelimeler
Lipaz, Lipid, pH Biyosensörü, Trigliserid, Candida Rugosa, pH Biosenso, Lipase, Triglyceride, Candida Rugosa
