Seryum oksit esaslı hibrit materyalin peroksidaz aktivitesinin belirlenmesi
Determination of peroxidase activity of cerium oxide based hybrid material
Yazar: NAIBA JABIYEVA
Danışman: PROF. DR. ABDİL ÖZDEMİR
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı / Analitik Kimya Bilim Dalı
Derece: Yüksek Lisans, 2024
Konu: Kimya = Chemistry

Özet:
"Nanozimler" olarak adlandırılan yapay enzimler, enzim benzeri katalitik aktivitelere sahiptirler. Kolay, düşük maliyetli sentez, ayarlanabilir morfoloji ve katalitik aktivite, güçlü stabilite, zorlu koşullar altında yüksek katalitik aktivite ve zengin yüzey kimyası gibi üstün özellikleri nedeniyle biyosensörlerde destek malzemeleri, çevresel kirleticilerin parçalanması, hastalık teşhisi gibi çeşitli alanlarda araştırma yapan bilim insanlarının ilgisini çekmektedir.
Günümüzde gıda endüstrisinde, biyofarmasötikte, çevresel analizlerde ve klinik teşhiste hızlı, stabil ve hassas H2O2 tespiti büyük önem taşımaktadır. H2O2 analizi için kolorimetri, kemilüminesans/floresan bazlı sistemler, yüksek performanslı sıvı kromatografisi ve elektrokimyasal sistemler dahil olmak üzere birçok yöntem kullanılmıştır.
Peroksidazlar, hücresel ortamda sitotoksik H2O2'nin parçalanmasını katalize eder. Son zamanlarda, tek adımlı kolorimetrik ölçüm platformları üretmek için peroksidaz benzeri aktivite gösteren malzemeler önerilmektedir. H2O2'in miktar tayini için nanozimlerin kullanılması uygun görülmektedir.
Karbonlu malzemeler, MOF'ler, metal nanomalzemeler, kalkojenitler, metal oksitler dahil olmak üzere çeşitli nanomalzemelerin peroksidaz benzeri aktivite gösterdiği bulunmuştur.
Bunlar arasında metal oksitler, morfolojik olarak kontrol edilebilir üretimleri, yüksek stabiliteleri ve üstün peroksidaz benzeri aktivite göstermeleri nedeniyle daha fazla ilgi görmüştür.
Bu çalışmada, cam slayt üzerinde altın nanopartikülleri kaplı CeO2 ters opal fotonik kristallerinden (IOPC'ler) oluşan hibrit nanozim, avantajlı bir şekilde gözenekli morfolojiye sahip olarak üretildi. Yüzeydeki seryumun farklı değerlik durumları, yüksek yüzey alanı ve buna bağlı olarak bol miktarda aktif bölge içermesi nanozimin avantajları arasındadır. Nanozimin oksidaz ve peroksidaz enzim aktiviteleri, H2O2 varlığında gözle fark edilebilir bir şekilde kromojenik substrat 2,2'-azino-bis(3-etilbenzotiyazolin-6-sülfonik asit (ABTS)'nin katalitik oksidasyonuyla incelenmiştir. Ce3+ ve Ce4+'dan oluşan CeO2 IOPC'ler ve Au NP'ler peroksidaz benzeri aktivite göstererek H2O2 varlığında ABTS'yi oksitlediği gözlemlendi. Nanozimin peroksidaz benzeri aktivitesi, hibritin ışığa duyarlı doğası nedeniyle ışık varlığında büyük ölçüde iyileştirildi. Nanozimin peroksidaz benzeri aktivitesi, 2,7 μM LOD ile 0,009 ila 500 μM aralığında H2O2'nin doğrusal tespitini mümkün kıldı. Kabul edilebilir nanozim aktivitesi muhtemelen H2O2'nin nanozimin aktif bölgelerine kolay erişiminden kaynaklanmaktadır. Gözenekli yapı, reaktanların ve ürünlerin kolayca yönlendirilmesini sağladı. Nanozimin periyodik yapısı aynı zamanda iyi bir tekrarlanabilirliği de mümkün kıldı. İşlevselleştirme olmadan nanozim, hidrojen peroksit ölçümleri için peroksidaz enzimlerine iyi bir alternatif olabilir.

Abstract:
Enzymes are mostly sophisticated proteins that catalyze biological transformations and have substantial selective catalytic activity towards biologically, forensically, and environmentally important substances.
However, enzymes have some limitations including high-cost purification, labor-intensive preparations, high susceptibility to pH, temperature, ionic strength, organic solvents, surfactants, and low recyclability which hamper their widespread use. Additionally, proteases can digest the enzymes.
Artificial enzymes called "nanozymes" can resemble enzyme-like catalytic activities and have received increasing interest from researchers in various areas, such as support materials in biosensors, degradation of environmental pollutants, and disease diagnosis, owing to their superior properties such as facile, low-cost synthesis, adjustable morphology and catalytic activity, strong stability, high catalytic activity under harsh conditions, and rich surface chemistry.
Nowadays, fast, stable, and sensitive H2O2 detection in food industry, biopharmaceutical, environmental analysis, and clinical diagnosis is of great importance. H2O2 accumulation in biological fluids leads to biological harm that results in the development of progressive neurodegenerative diseases, Alzheimer's and Parkinson's diseases, cancer, etc.
Different methods have been utilized for H2O2 analysis, including colorimetry, chemiluminescence/fluorescence-based systems, high-performance liquid chromatography, and electrochemical systems.
Peroxidases catalyze the breakdown of cytotoxic H2O2 in the cellular environment. Recently, peroxidase-mimicking materials have been proposed to fabricate one-step colorimetric sensing applications. H2O2 monitoring is likely to be viable by using nanozymes.
Various nanomaterials, including carbonaceous materials (graphene oxide(Das ve ark, 2019), fullerenes, carbon nanotubes), MOFs, metal nanomaterials (Au NPs, Au NCs, Pd NPs, Pd nanosheets, Pt NPs), metal oxides (Fe3O4, Mn3O4, V2O5, CuO, SnO2, Co3O4, CeO2, CoFe2O4 MNPs), chalcogenides (CuS, MoSe2 nanosheets, PtS2 nanosheets), hemin etc. have been found to display peroxidase-like activity.
Among them, metal oxides gained more attention due to their controllable fabrication, high stability, and superior peroxidase-mimicking activity.
Nanozymes have integrated the outstanding merits of engineered nanomaterials and natural enzymes since the breakthrough work, which revealed the intrinsic peroxidase-like activity of magnetic Fe3O4 NPs.
The nanozymes are promising substitutes for high-cost enzymes due to their substantial catalytic stability and activity.
The catalytic activity of nanozyme can be adjusted by precise morphology engineering, surface functionalization, heterogeneous atom doping, NP size, and surface defects, viz. ledges, kinks, adatoms, and vacancies, which are coordinatively unsaturated and reactive.
The presence of dual oxidation states of the metal ions is favorable and the redox state of metal ions on the surface plays a pivotal role in redox enzyme-like activities.
However, nanozymes cannot catalyze one specific substrate as selectively as enzymes. The potential challenges guide the de novo nanozyme design and synthesis of nanozymes with uniform size and atomically precise structures.
Additionally, the catalytic activity of nanozymes has been boosted by enhancing the surface area, which can expose more active sites and preferential exposure of catalytically active atoms.
Among the oxidase-mimicking nanozymes, CeO2 with multiple oxidation states has garnered attention due to its low-cost production, substantial catalytic activity, rich chemical versatility, low toxicity, high stability, environmental friendliness, and extraordinary electronic and optical properties.
However, the improvement of enzyme mimicking activity of CeO2 is one of the potential challenges, and utilization of different morphologies can overcome this problem.
Noble metal nanoparticles such as Au NPs and their alloys have revealed oxidase-like activity owing to their substantial oxygen reduction catalytic activity and peroxidase-like activity. Au NPs have been widely used to catalyze glucose oxidation by molecular oxygen by generating H2O2.
Encouragingly, the catalytic performance and/or versatile activity of nanozymes have been significantly improved by functionally assembling several nanozymes, which show the same and/or different enzyme-mimicking activity for concurrent tandem catalysis and more sensitive-selective sensing of analytes.
To our knowledge, there is no report on IOPCs-based nanozyme material. Motivated by above consideration, we modified the morphology to increase the enzyme-mimicking activity, and the resulting hybrid nanomaterial showed promising peroxidase-like activity.
H2O2 measurement was successfully carried out with a good sensing performance by using the coupled CeO2 IOPCs/Au NPs nanozyme as a substitute for oxidase-peroxidase enzymes, and light energy enhanced the nanozyme activity.
Also, enzyme-mimicking behavior was surveyed by putting forward a mechanism of nanozyme activity. The study can give an impetus to develop IOPCs-based nanozymes.
In this study, the hybrid nanozyme gold nanoparticles deposited CeO2 inverse opal photonic crystals (IOPCs) on the glass slide were fabricated by bestowing the hybrid with the beneficial porous morphology with its unique advantages: its surface valence states, high surface area, and copious active sites.
The oxidase and peroxidase-mimicking activity was studied by observing the catalytic oxidation of chromogenic substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) in the presence of H2O2, which can be visualized by the eyes.
Au NPs and CeO2 IOPCs consisting of Ce3+ and Ce4+ can oxidize ABTS in the presence of H2O2 by mimicking peroxidase enzymes. The peroxidase-like activity of nanozyme was substantially improved by introducing light irradiation due to the photosensitive nature of the hybrid. The peroxidase-like activity of nanozyme enabled H2O2 linear detection over the range 9 to 500 μM, with a LOD of 2.7 μM.
Since CeO2 IOPCs and Au NPs exhibit peroxidase-like activity to some extent, the assembly of these nanozymes revealed remarkable H2O2 sensing performance.
The use of IOPCs grants access to high number of catalytically active sites and enhanced the catalytic activity. The high accuracy and precision results from the facilitated and rapid access of H2O2 to the proximity of the sensor surface. The hybrid nanozyme has been successfully utilized to detect H2O2, and the introduction of visible light substantially increased the detection sensitivity.
This work proves the simple and sensitive nanozyme based assays for detection of various analytes and the nanozyme research will find its unique niche in the sensor area.
An outstanding light-assisted nanozyme for hydrogen peroxide detection was fabricated with the distinct merits of simplicity and low cost. The solution-based nanozymes have an undesired interfering effect for measuring light absorption.
On the other hand, the free-standing nanozymes can be removed from the post-reaction medium and negate this interference. While individual CeO2 and Au NPs have been found to be excellent superoxide dismutase (SOD) and glucose oxidase substitutes, the hybrid of these materials exhibited remarkable peroxidase-like activity.
CeO2 IOPCs and Au NPs-based materials can also act as photocatalysts, implying that the hybrid can harvest solar energy for photocatalytic reactions. The color generation of chromogenic colorless substrate ABTS was visible to the naked eye, such that the oxidized green product was discernible for hydrogen peroxide concentrations as low as 30 μM.
CeO2-based nanomaterials have rich redox properties depending on their morphology and multiple enzyme-mimicking activity. CeO2 nanozymes can catalyze H2O2 to generate reactive oxygen species under acidic conditions by showing peroxidase-like activity.
The acceptable nanozyme activity is probably owing to the easy access of H2O2 to the active sites of nanozyme. The porous architecture led to the easy channeling of reactants and products.
The periodic architecture of the nanozyme also led to decent reproducibility. Without functionalization, the nanozyme can be a promising alternative to peroxidase enzymes for hydrogen peroxide measurements.