Sisteamin-tek duvarlı karbon nanotüp modifiye altın elektrot temelli tirozinaz biyosensörü geliştirilmesi ve bazı tirozinaz inhibitörlerinin tayininde kullanılma potansiyelinin araştırılması
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Dosyalar
Tarih
2017
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Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Tez kapsamında Sisteamin/SWCNT/Glutaraldehit modifiye bir tirozinaz biyosensörü geliştirilerek bir tirozinaz inhibitörü olan kojik asitin tayin edilmesi amaçlanmıştır. Kojik asit tayini enzimin substratı olan katekol varlığında, yarışmalı inhibisyona dayalı olarak gerçekleştirilmiştir.
Biyosensör tasarımında altın elektrot yüzeyine kendiliğinden oluşan tek tabaka (SAM) oluşumu sisteamin ile gerçekleştirilmiştir. Bu modifikasyon sonrasında elektrot yüzeyine tek duvarlı karbon nanotüp bağlanması gerçekleştirilerek tirozinaz enzimi bağlanarak glutaraldehitle çapraz bağlama yapılmıştır.
Tasarlanan tirozinaz biyosensörü ile kojik asit tayini (-0.2) - (0.6) V arasında diferansiyel puls voltammetrisi yöntemiyle gerçekleştirilmiştir. Ölçümler K3[Fe(CN)6] içeren ortamda tirozinaz enziminin katekolün o-benzokinona dönüşümününü kojik asitin inhibe etmesi prensibine dayanmaktadır.
Tasarlanan biyosensörde kullanılan modifiye elektrodun karakterizasyonu çalışmaları gerçekleştirilmiştir. Bu amaçla enzim miktarı, sisteaminde bekletme süresi optimizasyonu, SWCNT miktarı ve SWCNT' te bekletme süresi optimizasyonu ve son olarak glutaraldehit konsantrasyonu optimizasyonu çalışmaları yapılmıştır. Çalışma koşullarına yönelik optimizasyon çalışmaları bağlamında sıcaklık, pH, tampon konsantrasyonu etkisine bakılmıştır. Tirozinaz biyosensörünün karakterizayonuna yönelik çalışmalarda ise doğrusal tayin aralığı, tekrar üretilebilirlik, biyosensöre farklı türlerin etkisi ve diğer türlerin tek tek cevabına bakılmıştır.
Tüm bu çalışmalar nihayetinde en uygun enzim miktarı 0,58 U/μL , sisteaminde bekletme süresi 9 saat, SWCNT miktarı olarak 1mg/ml, SWCNT' e bekletme süresi olarak 4 saat ve hacimsel glutaraldehit konsantrasyonu olarak %2,5 belirlenmiştir. Tirozinaz biyosensöründe tampon çözeltisi olarak 50 mM pH 7.0 potasyum fostat tamponu kullanılmıştır. Çalışma sıcaklığı 25°C olarak belirlenmiştir. Tirozinaz biyosensörünün doğrusal çalışma aralığı 10 - 150 μM olarak belirlenmiştir.
In the thesis, it was aimed to develop a modified tyrosinase biosensor of cysteamine / SWCNT / glutaraldehyde to determine kojic acid, a tyrosinase inhibitor. The kojic acid assay was performed in the presence of catechol, the substrate of the enzyme, based on competitive inhibition. In biosensor design, self assembled monolayer (SAM) formation on gold electrode surface was performed with cysteamine. After this modification, single walled carbon nanotubes were attached to the electrode surface and crosslinked with glutaraldehyde by tyrosinase binding. Determination of kojic acid in the presence of catechol with tyrosinase biosensor was performed between (-0.2) & (+0.6) V potential with differential pulse voltammetry. Measurements were performed in K3[Fe(CN)6] and the principle of inhibition of tyrosinase conversion of catechol o-benzoquinone with kojic acid. The characterization studies of the modified electrode used in the designed biosensor have been carried out. For this purpose, optimization of the amount of enzyme, cysteamine incubation time, optimization of SWCNT amount and SWCNT incubation time and finally optimization of glutaraldehyde concentration were performed. Temperature, pH, buffer concentration effect was examined for optimization studies for working conditions. In studies to characterize the tyrosinase biosensor, linear range, reproducibility, the effect of different species on the biosensor response and the individually responses of the other species are examined. At the end of these studies, the optimum amount of enzyme was determined as 0.58 U / μL, the incubation time of cysteamine was 9 hours, the SWCNT amount was 1 mg / mL, the incubation time of SWCNT was 4 hours and the volumetric glutaraldehyde concentration was 2.5%. 50 mM pH 7.0 potassium phosphate buffer was used as the buffer solution in the tyrosinase biosensor. The working temperature is 25 ° C. The linear working range of the tyrosinase biosensor was determined to be 10 - 150 μM.
In the thesis, it was aimed to develop a modified tyrosinase biosensor of cysteamine / SWCNT / glutaraldehyde to determine kojic acid, a tyrosinase inhibitor. The kojic acid assay was performed in the presence of catechol, the substrate of the enzyme, based on competitive inhibition. In biosensor design, self assembled monolayer (SAM) formation on gold electrode surface was performed with cysteamine. After this modification, single walled carbon nanotubes were attached to the electrode surface and crosslinked with glutaraldehyde by tyrosinase binding. Determination of kojic acid in the presence of catechol with tyrosinase biosensor was performed between (-0.2) & (+0.6) V potential with differential pulse voltammetry. Measurements were performed in K3[Fe(CN)6] and the principle of inhibition of tyrosinase conversion of catechol o-benzoquinone with kojic acid. The characterization studies of the modified electrode used in the designed biosensor have been carried out. For this purpose, optimization of the amount of enzyme, cysteamine incubation time, optimization of SWCNT amount and SWCNT incubation time and finally optimization of glutaraldehyde concentration were performed. Temperature, pH, buffer concentration effect was examined for optimization studies for working conditions. In studies to characterize the tyrosinase biosensor, linear range, reproducibility, the effect of different species on the biosensor response and the individually responses of the other species are examined. At the end of these studies, the optimum amount of enzyme was determined as 0.58 U / μL, the incubation time of cysteamine was 9 hours, the SWCNT amount was 1 mg / mL, the incubation time of SWCNT was 4 hours and the volumetric glutaraldehyde concentration was 2.5%. 50 mM pH 7.0 potassium phosphate buffer was used as the buffer solution in the tyrosinase biosensor. The working temperature is 25 ° C. The linear working range of the tyrosinase biosensor was determined to be 10 - 150 μM.
Açıklama
Anahtar Kelimeler
Tirozinaz, İnhibitör, Kojik Asit, Enzim Biyosensörü, Tyrosinase, Inhibitor, Kojic Acid, Enzyme Biyosensor
