Bazı nanoteknolojik immobilizasyon yöntemlerin katalaz enzimi aktivitesi üzerine etkisinin biyosensör yöntemi ile belirlenmesi
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Dosyalar
Tarih
2019
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu tez çalışmasının amacı son zamanlarda birçok araştırmacının dikkatini çeken nanoteknolojik immobilizasyon yöntemlerinin katalaz enzimi üzerine etkisinin biyosensör yöntemi ile tespit edilmesini sağlamaktır. Bu nanoteknolojik metaryallerin biyosensör üzerine etkisini incelemek için iki farklı immobilizasyon yöntemi ile tasarlanan enzim temelli biyosensörler şunlardır: I. Au/3-MPA/EDC-NHS/Cys/AuNP/CAT/GA enzim biyosensörü II. GCE/MWCNT/MWCNT-CAT enzim biyosensörü Au/3-MPA/EDC-NHS/Cys/AuNP/CAT/Glutaraldehit enzim biyosensörün geliştirilmesi için altın elektrot yüzeyinde kendiliğinden oluşan tek tabaka oluşumu 3-MPA ile sağlandı. 3-MPA'nın karboksil gruplarını aktive etmek için EDC-NHS kullanıldı. Daha sonra elektrot sisteamin çözeltisine daldırılarak inkübe edildi. Elektrot yüzeyine AuNP ve katalaz eklenerek biyoaktif tabakanın oluşması sonrası glutaraldehit ile katalaz enzim molekülleri arasında çapraz bağlanma gerçekleştirildi. GCE/MWCNT/MWCNT-CAT enzim biyosensörünün tasarlanmasında DMF içinde süspanse edilen çok duvarlı karbon nanotüp (MWCNT) kullanılmıştır. Biyosensör hazırlanmasında ilk aşama DMF içinde süspanse edilen MWCNT elektrot yüzeyine damlatılarak elektrot iletkenliğinin arttırılması sağlandı. İkinci aşamada ise MWCNT ile katalazın 1:3 (v/v) oranında karıştırılmasıyla oluşturulan çözeltinin sonikasyonu sonrası elektrot yüzeyine damlatılmasıyla biyosensör tasarımı tamamlandı. İki farklı immobilizasyon metodu kullanılarak geliştirilen biyosensörler ile hidrojen peroksit ölçümleri, -0,2 V & +0,5 V potansiyelleri arasında diferansiyel puls voltametrisi (DPV) yöntemi ile gerçekleştirilmiştir. Tasarlanan her iki biyosensör için ölçüm ortamında bulunan K3[Fe(CN)6]'ün -0,2 V & +0,5 V potansiyelleri arasında yükseltgenme piklerinde gözlenen değişimler ile hidrojen peroksit konsantrasyonu arasındaki doğrusal ilişkiden faydanılarak ölçümler alınmıştır. Tasarlanan enzim temelli biyosensörlerin çalışma koşullarının optimizasyonuna yönelik olarak sıcaklık, pH, iyon şiddeti ve farklı tamponlarda çalışmalar yapılmıştır. Tasarlanan enzim temelli biyosensörlerin karakterizasyonuna yönelik olarak yapılan çalışmalarda ise doğrusal tayin aralığının belirlenmesi, tekrarlanabilirlik ve sütte hidrojen peroksit tayini gibi parametreler belirlenmiştir. Deneysel çalışmalardan elde edilen sonuçlar incelendiğinde her iki immobilizasyon yöntemi ile geliştirilen enzim biyosensörleri için en iyi çalışma koşullarının 50 mM, pH: 7,0 potasyum fosfat tamponu ve 30ºC olduğu belirlenmiştir. Tasarlanan enzim temelli biyosensörler ile hidrojen peroksit için yapılan doğrusal tayin aralığı aynı olup 150 µM - 1000 µM arasında bulunmuştur. AuNP kullanılarak tasarlanan enzim biyosensörü için yapılan tekrarlanabilirlik çalışmalarında 600 µM hidrojen peroksit konsantrasyonunda (n=6) ortalama değer ( ) = 591.4 µM, standart sapma (S.S) = ± 6.54 µM ve % varyasyon katsayısı (% V.K) = 1.105 olarak bulunmuştur. MWCNT kullanılarak geliştirilen enzim temelli biyosensör için yapılan tekrarlanabilirlik denemelerinde 600 µM hidrojen peroksit konsantrasyonunda (n=6) ortalama değer ( ) = 596,3 µM, standart sapma (S.S) = ± 26.8 µM ve % varyasyon katsayısı (% V.K) = 4.5 olarak bulunmuştur. Bunların dışında enzim biyosensörleri ile süt örneklerinde hidrojen peroksit analizi de yapılmıştır.
The aim of this thesis is to determine the effect of nanotechnological immobilization methods on catalase enzyme which has attracted the attention of many researchers recently by biosensor method. The enzyme-based biosensors designed with two different immobilization methods to investigate the effect of these nanotechnological materials on the biosensor are: I. Au/3-MPA/EDC-NHS/Cys/AuNP/CAT/GA enzyme biosensor II. GCE/MWCNT/MWCNT-CAT enzyme biosensor For the development of the Au / 3-MPA / EDC-NHS / Cys / AuNP / CAT / Glutaraldehyde enzyme biosensor, self assambled monolayer formation on the gold electrode surface was provided with 3-MPA. EDC-NHS was used to activate the carboxyl groups of 3-MPA. The electrode was then immersed in cysteamine solution and incubated. After addition of AuNP and catalase onto the surface and forming the bioactive layer, cross-linking wal achieved between catalase enzyme molecules by glutaraldehyde. A multi-walled carbon nanotube (MWCNT) suspended in DMF was used to design the GCE / MWCNT / MWCNT-CAT enzyme biosensor. The first step in biosensor preparation was to drop the MWCNT susponded in DMF onto the electrode to increase the concluctivity of the electrode. In the second step the biosensor design was completed by the dropping the solution formed by mixing the MWCNT with 1/3 ratio (v/v) of the catalase after sonication, onto the electrode surface. Hydrogen peroxide measurements with the biosensors developed using two different immobilization methods were performed by differential pulse voltammetry (DPV) between -0.2 V & +0.5 V potentials. For both biosensors measurements were taken from the linear relationship between the observed changes in the oxidation peaks of K3[Fe (CN)6] and the hydrogen peroxide concentration between -0.2 V & +0.5 V potentials, In order to optimize the working conditions of designed enzyme-based biosensors, temperature, pH, ion intensity and different buffers were studied. For the characterization of the designed enzyme-based biosensors, same parameters such as determination of the linear range, repeatability and determination of hydrogen peroxide in milk were carried out. When the results obtanied from the experiments were examined, it was detected that the best working conditions for the enzyme biosensors developed by both immobilization methods were 50 mM, pH: 7.0 potassium phosphate buffer and 30ºC. The linear range for hydrogen peroxide was obtained the same in both biosensors designed as to be 150 µM -1000 µM. In the reproducibility studies performed for the enzyme biosensor designed using AuNP, the average value (n = 6) for 600 µM hydrogen peroxide concentration was found to be 591.4 µM, standard deviation (S.S) = ± 6.54 µM and coefficient of variation % (CV %) = 1.105 respectively. In the repeatability tests for the enzyme-based biosensor developed using MWCNT, the average value (n = 6) for 600 µM hydrogen peroxide concentration was found to be 597.3 µM, standard deviation (S.S) = ± 26.8 µM and coefficient of variation % (CV %) = 4.5 respectivity. In addition, hydrogen peroxide analysis was performed with enzyme biosensors in milk samples.
The aim of this thesis is to determine the effect of nanotechnological immobilization methods on catalase enzyme which has attracted the attention of many researchers recently by biosensor method. The enzyme-based biosensors designed with two different immobilization methods to investigate the effect of these nanotechnological materials on the biosensor are: I. Au/3-MPA/EDC-NHS/Cys/AuNP/CAT/GA enzyme biosensor II. GCE/MWCNT/MWCNT-CAT enzyme biosensor For the development of the Au / 3-MPA / EDC-NHS / Cys / AuNP / CAT / Glutaraldehyde enzyme biosensor, self assambled monolayer formation on the gold electrode surface was provided with 3-MPA. EDC-NHS was used to activate the carboxyl groups of 3-MPA. The electrode was then immersed in cysteamine solution and incubated. After addition of AuNP and catalase onto the surface and forming the bioactive layer, cross-linking wal achieved between catalase enzyme molecules by glutaraldehyde. A multi-walled carbon nanotube (MWCNT) suspended in DMF was used to design the GCE / MWCNT / MWCNT-CAT enzyme biosensor. The first step in biosensor preparation was to drop the MWCNT susponded in DMF onto the electrode to increase the concluctivity of the electrode. In the second step the biosensor design was completed by the dropping the solution formed by mixing the MWCNT with 1/3 ratio (v/v) of the catalase after sonication, onto the electrode surface. Hydrogen peroxide measurements with the biosensors developed using two different immobilization methods were performed by differential pulse voltammetry (DPV) between -0.2 V & +0.5 V potentials. For both biosensors measurements were taken from the linear relationship between the observed changes in the oxidation peaks of K3[Fe (CN)6] and the hydrogen peroxide concentration between -0.2 V & +0.5 V potentials, In order to optimize the working conditions of designed enzyme-based biosensors, temperature, pH, ion intensity and different buffers were studied. For the characterization of the designed enzyme-based biosensors, same parameters such as determination of the linear range, repeatability and determination of hydrogen peroxide in milk were carried out. When the results obtanied from the experiments were examined, it was detected that the best working conditions for the enzyme biosensors developed by both immobilization methods were 50 mM, pH: 7.0 potassium phosphate buffer and 30ºC. The linear range for hydrogen peroxide was obtained the same in both biosensors designed as to be 150 µM -1000 µM. In the reproducibility studies performed for the enzyme biosensor designed using AuNP, the average value (n = 6) for 600 µM hydrogen peroxide concentration was found to be 591.4 µM, standard deviation (S.S) = ± 6.54 µM and coefficient of variation % (CV %) = 1.105 respectively. In the repeatability tests for the enzyme-based biosensor developed using MWCNT, the average value (n = 6) for 600 µM hydrogen peroxide concentration was found to be 597.3 µM, standard deviation (S.S) = ± 26.8 µM and coefficient of variation % (CV %) = 4.5 respectivity. In addition, hydrogen peroxide analysis was performed with enzyme biosensors in milk samples.
Açıklama
Anahtar Kelimeler
Altın Nanopartikül, Çok Duvarlı Karbon Nanotüp, Hidrojen Peroksit, 3-Merkaptopropiyonik Asit, Sisteamin, Enzim Temelli Biyosensör, Gold Nanoparticle, Multi-Walled Carbon Nanotube, Hydrogen Peroxide, 3-Mercaptopropionic Acid, Cysteamine, Enzyme-Based Biosensor
