İçeceklerde kafein tayini için yeni bir biyosensör hazırlanması
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Dosyalar
Tarih
2010
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Trakya Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Günümüzde kafein tayininde çeşitli kromatografik, spektrofotometrik yöntemler kullanılmaktadır. Ancak bunlar özel ekipmanlar gerektiren zaman alıcı sistemlerdir. Biyosensörler; enzim, hücre ve doku gibi biyolojik unsurların uygun bir iletim sistemiyle birleştirilmesi ile oluşan biyoanalitik cihazlardır. Biyosensörlerin diğer yöntemlere karşı en önemli avantajı, tayin edilecek maddeler için ekonomik, pratik ve spesifik ölçümlere imkan vermesidir. Saf enzimleri içeren dokuların kullanılmasıyla hazırlanan doku temelli biyosensörler, saf enzimlerin immobilize edilmesiyle hazırlanan biyosensörlerle kıyaslandıklarında, oldukça ucuz, yüksek kararlılıklı ve yüksek aktiviteli tayin sistemlerini oluşturmaktadır. Bu çalışmada, kahve çekirdeği, çay yaprağı, taze fasulye ve taze bakla olmak üzere 4 farklı bitkisel doku, kafein oksidaz aktivitesi bakımından tarandı. Dokulardan, nispeten daha yüksek kafein oksidaz aktivitesi gösteren taze bakla dokusu kullanılarak kafein tayini için doku temelli bir biyosensör geliştirildi. Bu amaç doğrultusunda taze bakla bitki dokusu, immobilizasyon materyali olarak jelatin ve çapraz bağlayıcı olarak glutaraldehitin kullanılmasıyla camsı karbon elektrot yüzeyinde immobilize edildi. Ölçümler, kafein konsantrasyonu ile orantılı olarak azalan akım şiddetinin belirlenmesiyle elde edilen standart grafikler yardımıyla gerçekleştirildi. İmmobilizasyon ve çalışma koşulların optimizasyonu için, optimum glutaraldehit yüzdesi, doku miktarı, jelatin miktarı, pH, tampon konsantrasyonu ve sıcaklık değerleri taze bakla biyosensörü için sırasıyla % 1, 20.59 mg/cm2, 2.37 mg/cm2, 6.5, 100 mM, 37.5 °C olarak bulundu. Bunun yanı sıra purin halkası içeren 6 farklı bileşik substrat olarak kullanılarak taze bakla biyosensörünün verdiği cevaplar incelendi. Geliştirilen biyosensörün depolama kararlılığı, analiz sonuçlarının tekrarlanabilirliği ve bazı kimyasal maddelerin biyosensör cevabı üzerine etkisi araştırıldı. Taze bakla biyosensörü için kafein lineer tayin aralığı, 1.5-4.0 mM olarak bulundu. Biyosensörün standart sapma ve varyasyon katsayısı değerleri, sırasıyla ±0.040866 ve % 1.36 olarak bulundu. Çalışmanın son kısmında çeşitli içecek örneklerinde kafein miktarı standart ekleme metoduyla belirlendi.
Asbtract
Nowadays, several methods can be used for caffeine determination such as chromatographic and spectrophotometric methods. However, these methods are time consuming and require expensive equipments. Biosensors are defined as a analytical device incorporating a biological sensing element such as enzyme, cell and tissue with a transducer. In contrast the other methods, the most important advantage of the biosensor is to offer specific, economic and portable diagnostic for the target biological substances. As compared with prepared biosensor with immobilized pure enzymes, tissue based biosensors, which have including these enzymes, show potential advantages of low cost, high stability and activity. In this study, firstly, four different tissues as tea leaf, fresh bean, fresh broad, unroasted coffee bean were scanned for caffeine oxidase activity. The tissue based biosensor was prepared for determination of caffeine by using fresh broad, which have the highest caffeine oxidase activity. For this purpose, by using gelatin as the immobilization material and glutaraldehyde as cross-linking agent, the tissues were immobilized on the surface of glassy carbon electrode. Measurements were taken by standart curves which were obtained by the determination of decreasing current level related to caffeine compound concentration. For the optimization of the immobilization and experimental parameters such as optimum glutaraldehyde percentages, amounts of tissue homogenate, amounts of gelatin, pH, buffer concentrations and temperatures were founded to be 1 %, 20.59 mg/cm2, 2.37 mg/cm2, 6.5, 100 mM, 37.5 °C for fresh broad based biosensor, respectively. Substrate specifities of biosensor were studied by using six different substrates which contain purine system. The usability and storage of the biosensor were investigated in optimum conditions. The effects of some chemical compounds on biosensor response were studied by using some salts and sugars. The typical calibration curve for fresh broad biosensor revealed a linear range of 1.5- 4.0 mM caffeine. Standart deviation and variation coefficient of the biosensor were calculated ±0.040866 and % 1.36 for fresh broad based biosensor, respectively. Finally, concentration of caffeine were determined with developed biosensor by using standart addition method in common drinks used in daily life.
Asbtract
Nowadays, several methods can be used for caffeine determination such as chromatographic and spectrophotometric methods. However, these methods are time consuming and require expensive equipments. Biosensors are defined as a analytical device incorporating a biological sensing element such as enzyme, cell and tissue with a transducer. In contrast the other methods, the most important advantage of the biosensor is to offer specific, economic and portable diagnostic for the target biological substances. As compared with prepared biosensor with immobilized pure enzymes, tissue based biosensors, which have including these enzymes, show potential advantages of low cost, high stability and activity. In this study, firstly, four different tissues as tea leaf, fresh bean, fresh broad, unroasted coffee bean were scanned for caffeine oxidase activity. The tissue based biosensor was prepared for determination of caffeine by using fresh broad, which have the highest caffeine oxidase activity. For this purpose, by using gelatin as the immobilization material and glutaraldehyde as cross-linking agent, the tissues were immobilized on the surface of glassy carbon electrode. Measurements were taken by standart curves which were obtained by the determination of decreasing current level related to caffeine compound concentration. For the optimization of the immobilization and experimental parameters such as optimum glutaraldehyde percentages, amounts of tissue homogenate, amounts of gelatin, pH, buffer concentrations and temperatures were founded to be 1 %, 20.59 mg/cm2, 2.37 mg/cm2, 6.5, 100 mM, 37.5 °C for fresh broad based biosensor, respectively. Substrate specifities of biosensor were studied by using six different substrates which contain purine system. The usability and storage of the biosensor were investigated in optimum conditions. The effects of some chemical compounds on biosensor response were studied by using some salts and sugars. The typical calibration curve for fresh broad biosensor revealed a linear range of 1.5- 4.0 mM caffeine. Standart deviation and variation coefficient of the biosensor were calculated ±0.040866 and % 1.36 for fresh broad based biosensor, respectively. Finally, concentration of caffeine were determined with developed biosensor by using standart addition method in common drinks used in daily life.
Açıklama
Yüksek Lisans Tezi
Anahtar Kelimeler
Amperometrik Biyosensör, Kafein Oksidaz, Doku Biyosensörü, Kafein, Amperometric Biosensor, Caffeine Oxidase, Caffeine, Tissue Biosensor
