Ksantin molekülünün hassas elektrokimyasal tayini için elektrot modifikasyonu geliştirilmesi
Development of electrode modification for sensitive electrochemical determination of xanthine molecule
- Tez No: 955160
- Danışmanlar: DOÇ. DR. SEVGİ GÜNEY
- Tez Türü: Yüksek Lisans
- Konular: Kimya, Chemistry
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2025
- Dil: Türkçe
- Üniversite: İstanbul Teknik Üniversitesi
- Enstitü: Lisansüstü Eğitim Enstitüsü
- Ana Bilim Dalı: Kimya Ana Bilim Dalı
- Bilim Dalı: Kimya Bilim Dalı
- Sayfa Sayısı: 114
Özet
Günümüzde, farmasötik ilaçlar ve bu ilaçların metabolitleri, çevre kirleticiler, gıda katkı maddeleri gibi insan sağlığını etkileyen maddelerin belirlenmesine yönelik güvenilir, hızlı, hassas ve doğru analitik yöntemlerin geliştirilmesine yönelik talep artmaktadır. Biyolojik ve çevresel olarak önemli türlerin doğrudan veya dolaylı olarak belirlenmesine olanak sağlayan elektrokimyasal yöntemler, düşük maliyetli cihaz kullanımı, yüksek hassasiyet sergilemeleri, kararlılıkları, çevre dostu olmaları ve yerinde izleme imkanı sunmaları gibi özellikleriyle öne çıkmaktadır. Elektrokimyasal yöntemlerde karşılaşılan en büyük sorun, eş zamanlı bulunan bileşiklerin girişimidir. Bu sorunu aşmak için, çeşitli malzeme ve tekniklerin kullanılmasıyla Kimyasal Olarak Modifiye Edilmiş Elektrotlar (KME) hazırlanarak elektrotların performans özellikleri artırılabilir. KME'ler yüksek aşırı gerilime sahip türlerin modifiye edilmemiş elektrotlarda gerçekleşen indirgenme veya yükseltgenme tepkimelerini katalizleyerek pik potansiyellerini düşürürler. Ayrıca, analizin duyarlılığını artırır ve seçiciliğini geliştirirler. Pürinler, canlı organizmalarda fizyolojik işlevlerin gerçekleştirilmesinde önemli bir rol oynar. Nükleik asit sentezinin öncüleri olmalarının yanı sıra, metabolik sinyaller olarak görev yaparlar, enerji kaynağıdırlar ve birçok temel koenzimin parçasıdırlar. Bu nedenle, pürin metabolizmasındaki herhangi bir hata çeşitli bozukluklara neden olabilir ve bu yüzden fizyolojik sıvılardaki pürinlerin belirlenmesi klinik tanı açısından önemlidir. Ksantin (XA), insanlarda pürin metabolizmasının başlıca yan ürünlerinden biridir ve guanin ile hipoksantinin sırasıyla guanin deaminaz ve ksantin oksidaz enzimlerinin etkisiyle oluşur. Ardından ksantin, pürin metabolizmasının son ürünü olan ürik aside, ksantin oksidaz enzimi aracılığıyla dönüştürülür. Ksantin, birçok hastalığın zamanında teşhisi için güçlü bir biyobelirteç olarak hizmet eder. Fiziksel sıvılardaki anormal ksantin seviyeleri gut, ksantinüri, hiperürisemi, serebral iskemik durumlar, perinatal asfiksi ve böbrek yetmezliği gibi klinik durumların göstergesi olabilir. Bu nedenle, ksantinin belirlenmesi için basit, verimli ve düşük maliyetli, etkin analitik yöntemlerin geliştirilmesine her zaman büyük ihtiyaç vardır. Ksantinin belirlenmesinde diğer analitik yöntemlere kıyasla, elektrokimyasal yöntemler hızlı tepki süresi, basitlik, maliyet etkinliği ve minyatürleştirilebilme yetenekleri nedeniyle ön plana çıkmaktadır. Ksantin için enzimatik sensörler daha yüksek seçicilik sunsa da, bu sensörlerin kararsız ve pahalı olmaları nedeniyle enzimatik olmayan yöntemler daha fazla tercih edilmektedir. Bu çalışmada, insan metabolizmasında pürin degradasyonu sırasında oluşan ve biyolojik öneme sahip bir molekül olan ksantinin (XA) enzimsiz elektrokimyasal yöntem ile örnek ortamında belirlenmesi amacıyla bir KME geliştirilmiştir. Bu amaçla, amin fonksiyonlu ve elektrokimyasal olarak indirgenmiş grafen oksit (ErGO-NH2) sentezlendikten sonra camsı karbon elektrot yüzeyi bu malzeme ile modifiye edilmiş ve p-amino benzen sülfonik asit (ABSA)'in elektropolimerizasyonu ile elektrot yüzeyinde ince bir film tabakası oluşturulmuştur. XA'nın, hazırlanan modifiye elektrot üzerindeki elektrokimyasal yükseltgenmesine ait tarama hızı, pH, girişim etkisi gibi analitik parametreler, döngülü voltametri (CV) ve diferansiyel puls voltametrisi (DPV) yöntemleri kullanılarak incelenmiş ve XA'nın tespiti için en uygun deneysel koşullar belirlenmiştir. XA'nın geliştirilen modifiye elektrot sistemi üzerindeki elektrokimyasal yükseltgenmesine ait reaksiyon kinetiği ve reaksiyon mekanizması aydınlatılarak, belirlenen en uygun analiz koşullarında XA'nın biyolojik matrikse benzeyen bir ortamda analizi gerçekleştirilmiştir. Ayrıca, elde edilen sensörün XA analizindeki tekrarlanabilirliği, yeniden üretilebilirliği ve kararlılığı ortaya çıkarılmıştır. Geliştirilen modifiye elektrot, pürin degradasyonu sırasında XA ile birlikte biyolojik matrikste yer alan Hipoksantin (HXA) ve Ürik asit (UA) moleküllerinin varlığında da test edilmiştir. Elde edilen veriler, geliştirilen elektrokimyasal sensörün XA'nın HXA ve UA ile beraber yan yana analizlerinin gerçekleştirilebileceğini ortaya çıkarmıştır.
Özet (Çeviri)
Nowadays, there is an increasing demand for the development of reliable, rapid, sensitive, and accurate analytical methods for determining substances that affect human health, such as pharmaceutical drugs and their metabolites, environmental pollutants, and food additives. Electrochemical methods, which allow for the direct or indirect determination of biologically and environmentally important species, stand out due to low-cost instruments, high sensitivity, stability, environmental friendliness, and on-site monitoring capabilities. The biggest challenge encountered in electrochemical methods is the interference caused by compounds present simultaneously. To overcome this issue, the performance characteristics of electrodes can be improved by preparing Chemically Modified Electrodes (CMEs) using various materials and techniques. CMEs catalyze the reduction or oxidation reactions of species with high overpotentials on unmodified electrodes, thereby lowering peak potentials. Additionally, they increase the sensitivity and enhance the selectivity of the analysis. Xanthine (XA) has an important role in the metabolism of mammals and is present in the central nervous system. At high concentration levels, XA causes several serious diseases such as gout, Parkinson's disease, hyperuricemia, scurvy, and apraxia. Therefore, quantitative and qualitative determination of XA in human physiological fluids is significant. Various analytical methods have been developed for XA analysis, including high-performance liquid chromatography, enzymatic technology, and capillary electrophoresis. However, these methods have some restrictions, such as time-consuming processes, complex sample preparation, need for skilled personnel, and expensive equipment. As an alternative, electrochemical sensors are preferred due to their high sensitivity, rapid response, and practicality. In particular, non-enzymatic electrochemical sensors are more stable and cost-effective than enzymatic sensors that require specific enzymes like xanthine oxidase. In this study, a CME was developed for the determination of XA, a biologically important molecule formed during purine degradation in human metabolism, using an enzyme-free electrochemical method in sample media. For this purpose, amino-functionalized and electrochemically reduced graphene oxide (ErGO-NH₂) was synthesized, and the surface of a glassy carbon electrode was modified with this material. Subsequently, a thin film layer was formed on the electrode surface through the electropolymerization of para-aminobenzenesulfonic acid (ABSA). Analytical parameters such as scan rate, pH, and interference effects regarding the electrochemical oxidation of XA on the prepared modified electrode were investigated using cyclic voltammetry (CV) and differential pulse voltammetry (DPV), and the most suitable experimental conditions for XA detection were determined. The reaction kinetics and mechanism of the electrochemical oxidation of XA on the developed modified electrode system were elucidated, and analysis of XA was performed in an environment resembling a biological matrix under the determined optimal analysis conditions. Additionally, the repeatability, reproducibility, and stability of the obtained sensor in XA analysis were demonstrated. The developed modified electrode was also tested in the presence of Hypoxanthine (HXA) and Uric Acid (UA) molecules, which are present in biological matrices alongside XA during purine degradation. In the first part of the study, the electrochemical properties of XA on a glassy carbon electrode (GCE) surface were investigated using cyclic voltammetry. XA exhibited an oxidation peak at +0.86 V in a solution with a pH of 7.0, but no corresponding reduction peak was observed. This behavior indicates that XA undergoes an irreversible electrochemical reaction on the GCE surface. Due to the adsorption of the oxidation product of XA on the electrode surface, the peak current decreased with successive potential scans. The variation of peak currents with the square root of the scan rate was studied using cyclic voltammograms at different scan rates, and the specific surface area of the GCE was calculated as 0.06 cm². In the second part of the study, graphene oxide (GO) was synthesized and characterized using FTIR and UV spectroscopy. Additionally, glassy carbon electrodes modified with GO were prepared, and their electrochemical characterization in [Fe(CN)₆]³⁻/⁴⁻ solution was carried out using cyclic voltammetry. In the third part of the study, amino-functionalized graphene oxide (GO-NH₂) was synthesized and characterized with FTIR and UV spectroscopy. Glassy carbon electrodes modified with GO-NH₂ were also prepared and characterized electrochemically in [Fe(CN)₆]³⁻/⁴⁻ solution. Compared to GO/GCE, the GO-NH₂/GCE showed increased reduction and oxidation peak currents for the [Fe(CN)₆]³⁻/⁴⁻ couple. This increase in peak currents can be attributed to the enhancement of GO's conductivity by NH₂ groups. In the fourth part of the study, amino-functionalized graphene oxide was electrochemically reduced for the first time to form amino-functionalized reduced graphene oxide (ErGO-NH₂), which was then characterized. Reduction of GO was carried out using two different methods: constant potential and cyclic potential scanning. When the efficiency of electrodes prepared by both methods was compared in the oxidation of XA, the electrode modified by constant potential reduction (s-ErGO-NH₂/GCE) exhibited a higher peak current for XA oxidation. The optimal reduction potential and duration for GO under constant potential were determined as -1.2 V and 250 seconds, respectively. In the fourth and fifth parts of the study, amino-functionalized reduced graphene oxide/poly(p-aminobenzenesulfonic acid) material was synthesized electrochemically on a glassy carbon electrode surface for the first time. Using these PABSA/ErGO-NH₂/GCE modified electrodes, xanthine (XA) was determined. Electropolymerization of p-aminobenzenesulfonic acid on the ErGO-NH₂/GCE surface was performed by applying potential cycles in a neutral medium. An anodic peak at +0.98 V corresponding to the oxidation of the monomer was observed, and the peak current increased with the number of cycles. This increase indicates that the monomer polymerized on the electrode surface. When compared to PABSA/d-ErGO-NH₂/GCE prepared with GO reduced by potential cycling, the PABSA/s-ErGO-NH₂/GCE prepared with GO reduced by constant potential showed a higher peak current during XA oxidation. Therefore, it was concluded that reducing GO under constant potential before modification with PABSA is more suitable. When the peak currents observed during the electrochemical oxidation of XA were analyzed for PABSA/ErGO-NH₂/GCE electrodes prepared with varying numbers of potential cycles, it was found that the intensity of DPV peak currents increased with more cycles but decreased when more than 30 cycles were applied. Hence, the optimal number of cycles for polymerization was determined as 30. The electrode modified with ErGO-NH₂ showed an XA oxidation peak with a current of 18.5 µA, whereas the electrode modified with PABSA/ErGO-NH₂ exhibited a slightly shifted peak at a more negative potential with a current of 39.7 µA. As the concentration of GO-NH₂, which was dropped on the electrode surface and electrochemically reduced to ErGO before being modified with PABSA, increased, the reactive areas on the electrode surface also increased, resulting in a rise in peak current. However, beyond 7.0 µL, the film thickness increased, which blocked electron transfer between XA and the electrode surface. Thus, 7.0 µL was determined as the optimal GO-NH₂ concentration. The effect of solution pH on XA oxidation with the PABSA/ErGO-NH₂/GCE electrode was studied, and the optimal pH for XA analysis was found to be 7.0. Since the slope of the linear relationship between peak potential and pH was 0.058 V/pH, it was concluded that the electrochemical oxidation reaction of XA involves equal numbers of electrons and protons and is pH-dependent. Cyclic voltammograms obtained at scan rates ranging from 10 mV/s to 400 mV/s with the PABSA/ErGO-NH₂/GCE electrode showed that the peak potential of XA oxidation varied linearly with the square root of the scan rate, indicating an irreversible electrochemical reaction. Additionally, since the slope of the log(current) vs. log(scan rate) plot was 0.63, it was concluded that the oxidation reaction of XA on the PABSA/ErGO-NH₂/GCE surface is limited by both adsorption and diffusion. The linear variation of peak potential with the log of scan rate confirms the irreversible nature of XA's electrochemical oxidation. When the activities of different modified electrodes were compared, the peak current observed with PABSA-s-ErGO-NH₂/GCE was higher than those obtained with GCE and PABSA/GCE. Therefore, it was concluded that PABSA-s-ErGO-NH₂/GCE could be used as a sensor system for the electrochemical analysis of XA. Under the optimized analytical conditions, a calibration graph was created for XA oxidation on the PABSA/ErGO-NH₂/GCE modified electrode. Two linear concentration ranges were identified: 5.0×10-7 M – 1.0×10-5 M and 1.0×10-5 M – 1.5×10-4 M. The analytical application of the PABSA/ErGO-NH₂/GCE modified electrode was demonstrated by analyzing XA in a synthetic urine sample under the optimized experimental conditions. Recovery rates of 97.3%, 97.8%, 98.57% and 98.44% were obtained. In conclusion, in this study, the sensitivity and selectivity of the target xanthine molecule analysis were enhanced by modifying glassy carbon electrodes. Moreover, a new modification method combining amino-functionalized reduced graphene oxide and ABSA was proposed. The modified electrodes prepared by the proposed method demonstrated significantly higher catalytic activity in XA analysis compared to unmodified electrodes.
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