Bis-neokuproin bakır(II) kompleksine dayalı optik ve elektrokimyasal sensör/biyosensörlerin geliştirilmesi ve biyoteknolojik uygulamaları
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Tarih
2024
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Çanakkale Onsekiz Mart Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu tez çalışmasında, bakır(II) bis neokuproin kompleksinin (CUPric Reducing Antioxidant Capacity, CUPRAC reaktifi) kullanımına dayalı elektrokimyasal ve optik olmak üzere iki farklı sensör/biyosensör çalışması geliştirilmiştir. Tez çalışmanın ilk kısmında, elektrokimyasal olarak tersinir davranış sergileyen CUPRAC reaktifi çok duvarlı karbon nanotüp (Multi walled carbon nanotube, MWCNT) modifiye camımsı karbon elektrot (Glassy carbon electrode, GCE) yüzeyine modifiye edilerek, antioksidan kapasite tayini akışa enjeksiyon analizi (Flow injection analysis, FIA) sistemiyle amperometrik olarak ilk defa bu tez kapsamında gerçekleştirilmiştir. Model olarak seçilen trolox, kuersetin ve kateşin antioksidan örnekleri için optimize edilen koşullarda 21 farklı antioksidan için doğrusal çalışma aralığı, LOD ve LOQ değerleri hesaplanmış, girişim ve gerçek örnek çalışmaları elektrokimyasal olarak FIA sisteminde amperometri yöntemi kullanılarak gerçekleştirilmiştir. Çalışmanın ikinci kısımında, CUPRAC reaktifi kullanılarak üç farklı enzim grubu için i) O2 ve substrat varlığında H2O2 üreten oksidaz (Glukoz oksidaz-GOx, Kolesterol oksidaz-KOx, Laktat oksidaz-LOx ve Pirüvat oksidaz-POx), enzimlerine dayalı glukoz, kolesterol ve laktat tayini için; ii) kofaktör NAD+ ve substrat varlığında NADH üreten dehidrogenaz Glukoz dehidrogenaz-GDH) enzimine dayalı glukoz tayini için (iii) asetiltiyokolin varlığında tiyokolin üreten Asetilkolinesteraz-AChE enziminin inhibisyonuna dayalı pestisit tayini için optik biyosensörler geliştirilmiştir. Her bir enzim silanlanmış manyetik nanopartiküllere (SiO2@Fe3O4) immobilize edilerek kullanılmıştır. Tez çalışması kapsamında GDH, GOx, KOx, ve LOx-POx enzimleriyle hidrofilik-politetrafloroetilen (H-PTFE) membrana dayalı fiber optik reflektometrik, GOx, KOx ve LOx-POx enzimleriyle şeffaf Nafyon (Nf) membrana dayalı fiber optik absorptimetrik biyosensör tasarımı gerçekleştirilmiştir. Son olarak AChE'nin inhibisyonuna bağlı pestisit biyosensörü geliştirilmiştir. Bu amaçla klorpirfos (CPF) ve paraoxon etil (POX-E) kullanılarak her iki pestisit için inhibisyon süresi optimize edilerek analitik performans çalışmaları gerçekleştirilmiştir. Sonuç olarak bu tez çalışmasıyla optik sensörlerde yaygın olarak kullanılan yararlı kromojenik yükseltgen CUPRAC reaktifi ile i) hem antioksidanların hem de diğer indirgenme ajanlarının doğrudan, ii) indirgen olmayan substratların, enzimatik tepkime sonucu oluşan CUPRAC aktif ürünlerle dolaylı ve iii) enzimleri inhibe eden pestisitlerin dolaylı olmak üzere üç farklı grubun analiz edebileceği belirlenmiştir.
This thesis presents the development of two distinct research projects using sensors and biosensors, one utilizing electrochemical methods and the other utilizing optical methods. Both studies are centered on the application of the copper(II) bis neocuproin complex (CUPric Reducing Antioxidant Capacity, CUPRAC reagent). The first part of the thesis involved adding the CUPRAC reagent, which has electrochemically reversible properties, to the surface of a glassy carbon electrode (GCE) that had multi-walled carbon nanotubes (MWCNT) added to it. The antioxidant capacity was then found using an amperometric method with a flow injection analysis (FIA) system. This was the first time that this method was used in this thesis. Analyzed in this study were trolox, quercetin, and catechin, chosen as representative antioxidant samples. The linear operating range, limit of detection (LOD), and limit of quantification (LOQ) values for 21 different antioxidants were found through optimization studies. The amperometry method of electrochemical analysis in a flow injection analysis (FIA) system was used to look into interference and real sample studies. The trolox-equivalent antioxidant capacity (TEAC coefficient) was computed for each antioxidant. In the second part of the study, CUPRAC reagent was used for three different enzyme groups i) Oxidase enzymes that produce H2O2 in the presence of O2 and substrate (Glucose Oxidase-GOx, Cholesterol Oxidase-KOx, Lactate Oxidase-LOx and Pruvate Oxidase-POx), ii) dehydrogenase enzyme (Glucose Dehydrogenase-GDH), which produces NADH in the presence of cofactor NAD+ and substrate, iii) Acetylcholinesterase-AChE, which produces thiocholine in the presence of acetylthiocholine. The silanized (SiO2@Fe3O4) magnetic nanoparticles were used to immobilize each enzyme. The thesis study involved designing fiber optic reflectometric biosensors using hydrophilic polytetrafluoroethylene (H-PTFE) membranes with GDH, GOx, KOx, and LOx-POx enzymes. Additionally, fiber optic absorptimetric biosensors were designed using a transparent Nafion (Nf) membrane with GDH, GOx, and KOx enzymes. Optimization studies were carried out to determine the optimal conditions for the substrates involved, and interference and real sample studies were performed. We have effectively developed a pesticide biosensor that employs the inhibition of AChE. To achieve this objective, we initially calibrated the inhibition time for chlorpyrifos (CPF) and paraoxon ethyl (POX-E), and subsequently conducted analytical performance tests. As a result, it has been determined in this thesis that three different groups can be analyzed with the useful chromogenic oxidizing CUPRAC reagent, which is widely used in optical sensors: i) both antioxidants and other reducing agents directly, ii) non-reducing subtrates, indirectly by CUPRAC active products formed as a result of enzymatic reaction, and iii) pesticides that inhibit enzymes indirectly.
This thesis presents the development of two distinct research projects using sensors and biosensors, one utilizing electrochemical methods and the other utilizing optical methods. Both studies are centered on the application of the copper(II) bis neocuproin complex (CUPric Reducing Antioxidant Capacity, CUPRAC reagent). The first part of the thesis involved adding the CUPRAC reagent, which has electrochemically reversible properties, to the surface of a glassy carbon electrode (GCE) that had multi-walled carbon nanotubes (MWCNT) added to it. The antioxidant capacity was then found using an amperometric method with a flow injection analysis (FIA) system. This was the first time that this method was used in this thesis. Analyzed in this study were trolox, quercetin, and catechin, chosen as representative antioxidant samples. The linear operating range, limit of detection (LOD), and limit of quantification (LOQ) values for 21 different antioxidants were found through optimization studies. The amperometry method of electrochemical analysis in a flow injection analysis (FIA) system was used to look into interference and real sample studies. The trolox-equivalent antioxidant capacity (TEAC coefficient) was computed for each antioxidant. In the second part of the study, CUPRAC reagent was used for three different enzyme groups i) Oxidase enzymes that produce H2O2 in the presence of O2 and substrate (Glucose Oxidase-GOx, Cholesterol Oxidase-KOx, Lactate Oxidase-LOx and Pruvate Oxidase-POx), ii) dehydrogenase enzyme (Glucose Dehydrogenase-GDH), which produces NADH in the presence of cofactor NAD+ and substrate, iii) Acetylcholinesterase-AChE, which produces thiocholine in the presence of acetylthiocholine. The silanized (SiO2@Fe3O4) magnetic nanoparticles were used to immobilize each enzyme. The thesis study involved designing fiber optic reflectometric biosensors using hydrophilic polytetrafluoroethylene (H-PTFE) membranes with GDH, GOx, KOx, and LOx-POx enzymes. Additionally, fiber optic absorptimetric biosensors were designed using a transparent Nafion (Nf) membrane with GDH, GOx, and KOx enzymes. Optimization studies were carried out to determine the optimal conditions for the substrates involved, and interference and real sample studies were performed. We have effectively developed a pesticide biosensor that employs the inhibition of AChE. To achieve this objective, we initially calibrated the inhibition time for chlorpyrifos (CPF) and paraoxon ethyl (POX-E), and subsequently conducted analytical performance tests. As a result, it has been determined in this thesis that three different groups can be analyzed with the useful chromogenic oxidizing CUPRAC reagent, which is widely used in optical sensors: i) both antioxidants and other reducing agents directly, ii) non-reducing subtrates, indirectly by CUPRAC active products formed as a result of enzymatic reaction, and iii) pesticides that inhibit enzymes indirectly.
Açıklama
Lisansüstü Eğitim Enstitüsü, Kimya Ana Bilim Dalı
Anahtar Kelimeler
Biyoteknoloji, Biotechnology, Kimya
