Highly-sensitive, label-free detection of microorganisms and viruses via interferometric reflectance imaging sensor

Pathogenic microorganisms and viruses can easily transfer from one host to another and cause disease in humans. The determination of these pathogens in a time- and cost-effective way is an extreme challenge for researchers. Rapid and label-free detection of pathogenic microorganisms and viruses is critical in ensuring rapid and appropriate treatment. Sensor technologies have shown considerable advancements in viral diagnostics, demonstrating their great potential for being fast and sensitive detection platforms. In this review, we present a summary of the use of an interferometric reflectance imaging sensor (IRIS) for the detection of microorganisms. We highlight low magnification modality of IRIS as an ensemble biomolecular mass measurement technique and high magnification modality for the digital detection of individual nanoparticles and viruses. We discuss the two different modalities of IRIS and their applications in the sensitive detection of microorganisms and viruses.Published versio

Atıksu Ön Arıtımı için Koimmobilize Enzim Tasarımı

ABSTRACT CO-IMMOBILISED ENZYME DESIGNING FOR WASTEWATER PRETREATMENT SİNEM DİKEN GÜR Doctor of Philosophy, Department of Biology Supervisor: Prof. Dr. Nilüfer AKSÖZ December 2016, 90 Pages Immobilized enzymes are defined as enzymes physically confined or localized in a particular region with retention of their catalytic activities, and which can be used continuously and stored for a long period; take the place of harsh chemical agents and long-lasting biological processes in the wastewater treatment process. In this study Na alginate, Na alginate-κ carrageenan hydrogels, Na alginate activated with glutaraldehyde, grafted Na alginate and chitosan activated with glutaraldehyde were used with intent to co-immobilization of protease, pectinase and α amylase enzymes. The study was continued by using Na alginate beads and glutaraldehyde-activated chitosan beads in which three enzymes could be successfully co-immobilized. The enzymes were covalently bond to the surface of chitosan beads activated by glutaraldehyde while entrapped into the Na alginate beads. Characterization studies were performed with scanning electron microscopy and Fourier transform infrared spectroscopy. Enzymes, co-immobilized using two different support materials and methods, were compared with each other in terms of temperature and pH stability, reusability and storage stability characteristics. Wastewater pretreatment process was performed by using glutaraldehyde-activated chitosan beads which have a higher immobilization yield and relative activity and are more appropriate in respect iv to reproducibility and storage stability. For this purpose, synthetic manucipial wastewater, real wastewater sample and sea water were used in which waste materials were added. In sea water, while pectinase enzyme was inhibited, the maximum removal quantity was determined by α amylase and protease. In terms of synthetic and municipal wastewater, while the removal rates of co-immobilized α amylase and protease enzymes showed very close results, it was determined that the product formation of pectinase in manucipal wastewater was higher. In this study, pectinase, protease and α amylase enzymes were co-immobilized using glutaraldehyde activated chitosan beads for pre-treatment of wastewater. We believe that this developed material will be a model for the design of multi enzyme co-immobilization studies and will shed light on future researches on this topic.İÇİNDEKİLER Sayfa ÖZET ................................................................................................................................................ i ABSTRACT ................................................................................................................................... iii TEŞEKKÜR .................................................................................................................................... v İÇİNDEKİLER…………………………………………………………………………………..vi SİMGELER VE KISALTMALAR ................................................................................................. x ŞEKİLLER DİZİNİ ...................................................................................................................... xiii ÇİZELGELER DİZİNİ ................................................................................................................ xvi 1. GİRİŞ .......................................................................................................................................... 1 2. GENEL BİLGİ ............................................................................................................................ 4 2.1. Enzimler ................................................................................................................................... 4 2.1.1. Atıksu Arıtımında Enzimler .................................................................................................. 4 2.1.2. α amilaz Enziminin Özellikleri ve Atıksu Arıtımında Kullanımları ..................................... 5 2.1.3. Pektinaz Enziminin Özellikleri ve Atıksu Arıtımında Kullanımları ..................................... 7 2.1.4. Proteaz Enziminin Özellikleri ve Atıksu Arıtımında Kullanımları ....................................... 8 2.2. Enzim İmmobilizasyonu .......................................................................................................... 9 2.2.1. Enzim İmmobilizasyonunun Avantajları ............................................................................ 10 2.2.2. Enzim İmmobilizasyon Metotları ........................................................................................ 10 2.2.2.1. Taşıyıcıya Bağlanma ........................................................................................................ 11 2.2.2.1.1. Fiziksel Adsorbsiyon ..................................................................................................... 11 2.2.2.1.2. Kovalent Bağlanma ....................................................................................................... 11 2.2.2.1.3. İyonik Bağlanma ........................................................................................................... 12 2.2.2.2. Çapraz Bağlanma ............................................................................................................. 13 2.2.2.3. Tutuklama ve Kapsülleme ................................................................................................ 14 2.2.3. Enzim İmmobilizasyonunda Destek Materyal Olarak Kullanılan Bazı Doğal Polimerler . 14 2.2.3.1. Aljinat ............................................................................................................................... 15 2.2.3.2. Karragenan ....................................................................................................................... 16 2.2.3.3. Kitosan ............................................................................................................................. 17 2.2.4. Destek Materyallerin Aktive Edilmesinde Kullanılan Bazı Ajanlar ................................... 18 2.2.4.1. Glutaraldehit ..................................................................................................................... 18 2.2.4.2. Polietilenimin ................................................................................................................... 20 2.2.5. Enzim Ko-immobilizasyonu ............................................................................................... 20 vii 3. MATERYAL VE YÖNTEM .................................................................................................... 22 3.1. Kullanılan Enzimler ............................................................................................................... 22 3.2. Enzimatik Aktivite Tayini ...................................................................................................... 22 3.2.1. α amilaz Aktivite Tayini ...................................................................................................... 22 3.2.2. Pektinaz Aktivite Tayini ...................................................................................................... 22 3.2.3. Proteaz Aktivite Tayini ....................................................................................................... 22 3.3. İmmobilizasyon Verimi ve Yükleme Etkinliği Değerlerinin Hesaplanması ......................... 23 3.4. α amilaz, Pektinaz ve Proteaz Enzimlerinin Ko-immobilizasyonu için Uygun Destek Materyal ve İmmobilizasyon Metodunun Seçimi ................................................................. 23 3.4.1. Enzimlerin Aljinat-κ Karragenan Hidrojel ile Tutuklama Yöntemiyle Ko-immobilizasyonu ............................................................................................................................................... 23 3.4.2. Enzimlerin Glutaraldehit ile Aktive Edilen Na Aljinat Boncuklarda Kovalent Bağlanma ile Ko-immobilizasyonu ............................................................................................................. 24 3.4.3. Enzimlerin Ekleme Yapılmış Na Aljinat Boncuklar ile Ko-immobilizasyonu ................... 24 3.4.4. Enzimlerin Na aljinat Jelde Tutuklama Yöntemiyle Ko-immobilizasyonu ........................ 25 3.4.5. Na Aljinat Jelde Tutuklama Yöntemiyle Ko-İmmobilizasyon Koşullarının Optimizasyonu ............................................................................................................................................. 26 3.4.5.1. Na aljinat Konsantrasyonu ............................................................................................... 26 3.4.5.2. CaCl2 Konsantrasyonu ..................................................................................................... 26 3.4.5.3. Enzim Konsantrasyonları ................................................................................................. 27 3.4.6. Enzimlerin Glutaraldehit Aracılığıyla Kitosan Boncuk Yüzeyine Kovalent Bağlanma Yöntemiyle Ko-immobilizasyonu ........................................................................................ 27 3.4.6.1. Kitosan Boncukların Hazırlanması .................................................................................. 27 3.4.6.2. Kitosan Boncukların Glutaraldehit ile Aktivasyonu ........................................................ 27 3.4.6.3. α amilaz, Pektinaz ve Proteaz Enzimlerinin Aktive Kitosan Boncuklara Koİmmobilizasyonu ................................................................................................................. 28 3.4.7. Ko-İmmobilizasyon Koşullarının Optimizasyonu .............................................................. 29 3.4.7.1. Kitosan Konsantrasyonu .................................................................................................. 29 3.4.7.2. Glutaraldehit Konsantrasyonu .......................................................................................... 29 3.4.7.3. Aktivasyon süresi ............................................................................................................. 30 3.4.7.4. İmmobilizasyon Süresi ..................................................................................................... 30 3.4.8. Kitosan Boncukların Karakterizasyonu .............................................................................. 30 3.4.8.1. Kitosan Boncukların Taramalı Elektron Mikroskobu (SEM) Analizi ............................. 30 viii 3.4.8.2. Fourier Dönüşümlü Kızılötesi Spektroskopisi (FTIR) ..................................................... 30 3.5. Serbest ve İki Farklı Destek Materyal Kullanılarak Ko-immobilize Edilen Enzimlerin Bazı Özelliklerinin Saptanması ..................................................................................................... 30 3.5.1. Optimum Çalışma Sıcaklıklarının Belirlenmesi ................................................................. 30 3.5.2. Optimum Çalışma pH’ larının Belirlenmesi ....................................................................... 31 3.5.3. Termal Kararlılıklarının Saptanması ................................................................................... 31 3.5.4. pH Kararlılıklarının Saptanması .......................................................................................... 31 3.5.5. Depolama Karaklılıklarının Saptanması ............................................................................. 31 3.5.6. Operasyon Kararlılıklarının Saptanması ............................................................................. 32 3.6. Aktive Edilmiş Kitosan Boncuklara Ko-immobilize Enzimlerle Atıksu Ön Arıtımı ............ 32 4. SONUÇ ve TARTIŞMA ........................................................................................................... 34 4.1. α amilaz, Pektinaz ve Proteaz Enzimlerinin Ko-immobilizasyonu İçin Uygun Destek Materyal ve İmmobilizasyon Metodunun Seçimi ................................................................. 34 4.2. α amilaz, Pektinaz ve Proteaz Enzimlerinin Na aljinat Jelde Tutuklama Yöntemiyle Koimmobilizasyonu .................................................................................................................... 35 4.2.1. İmmobilizasyon Koşullarının Optimizasyonu .................................................................... 36 4.2.1.1. Na Aljinat Konsantrasyonu .............................................................................................. 36 4.2.1.2. CaCl2 Konsantrasyonu ..................................................................................................... 37 4.2.1.3. Yüklenen Enzim Konsantrasyonu .................................................................................... 39 4.3. Enzimlerin Glutaraldehit Aracılığıyla Kitosan Boncuk Yüzeyine Kovalent Bağlanma Yöntemiyle Ko-immobilizasyonu ........................................................................................ 40 4.3.1. Kitosan Boncukların Hazırlanması ..................................................................................... 40 4.3.2. Kitosan Boncukların Glutaraldehit ile Aktivasyonu ........................................................... 41 4.3.3.1 Kitosan Konsantrasyonu ................................................................................................... 42 4.3.3.2. Glutaraldehit Konsantrasyonu .......................................................................................... 44 4.3.3.3. Aktivasyon Süresi ............................................................................................................ 46 4.3.3.4. İmmobilizasyon Süresi ..................................................................................................... 47 4.3.4. Glutaraldehit ile Aktive Edilmiş Kitosan boncukların karakterizasyonu ............................ 49 4.3.4.1. Taramalı Elektron Mikroskobu (SEM) Analizi ............................................................... 49 4.4. Serbest ve İki Farklı Destek Materyal Kullanılarak Ko-immobilize Edilen Enzimlerin Bazı Özelliklerinin Saptanması ..................................................................................................... 53 4.4.1. Optimum Çalışma Sıcaklıklarının Belirlenmesi ................................................................. 53 4.4.2. Optimum Çalışma pH’ larının Belirlenmesi ....................................................................... 57 4.4.4. pH Kararlılıklarının Saptanması .......................................................................................... 62 4.4.5. Depolama Kararlılıklarının Saptanması .............................................................................. 63 4.4.6. Tekrar Kullanım Kararlılıklarının Saptanması .................................................................... 68 4.4.7. Atıksuda Ko-immobilize Enzimlerle Ön Arıtım ................................................................. 72 5.YORUM ..................................................................................................................................... 75 KAYNAKLAR .............................................................................................................................. 77 ÖZGEÇMİŞ .................................................................................................................................. 89ÖZET ATIKSU ÖN ARITIMI İÇİN KO-İMMOBİLİZE ENZİM TASARIMI SİNEM DİKEN GÜR Doktora, Biyoloji Bölümü Tez Danışmanı: Prof. Dr. Nilüfer AKSÖZ Aralık 2016, 90 Sayfa Belirli bir bölgeye katalitik aktivitesini koruyarak yerleştirilmiş, sürekli kullanılabilir ve uzun süre saklanabilir enzimler olarak tanımlanan immobilize enzimler, atıksu arıtım işlemlerinde sert kimyasal ajanların ve uzun süren biyolojik işlemlerin yerini almaktadır. Bu çalışmada proteaz, pektinaz ve α amilaz enzimlerinin ko-immobilizasyonu amacıyla Na aljinat, Na aljinat-κ karragenan hidrojel, glutaraldehit ile aktive edilen Na aljinat, ekleme yapılmış Na aljinat ve glutaraldehit ile aktive edilen kitosan boncuklar kullanıldı. Üç enzimin de başarılı bir şekilde ko-immobilize edilebildiği Na aljinat boncuklar ve glutaraldehit ile aktive edilen kitosan boncuklar kullanılarak çalışmaya devam edildi. Enzimler, Na aljinat boncuklara hapsedilirken glutaraldehit kullanılarak aktive edilen kitosan boncukların yüzeyine kovalent olarak bağlandılar. Karakterizasyon çalışmaları taramalı elektron mikroskobu (SEM) ve Fourier dönüşümlü kızılötesi spektroskopisi (FTIR) ile gerçekleştirildi. İki farklı destek materyal ve metot kullanılarak ko-immobilize edilen enzimler sıcaklık ve pH stabilitesi, tekrarlanabilirlik ve saklama stabilitesi gibi özellikleri bakımından birbirleriyle karşılaştırıldılar. İmmobilizasyon verimi ve bağıl aktivitesi daha yüksek olan, tekrarlanabilirlik ve saklama stabilitesi bakımından daha uygun olan enzim ko-immobilize kitosan boncuklar kullanılarak atıksu ii ön arıtım işlemi gerçekleştirildi. Bu amaçla sentetik evsel atıksu, gerçek bir atıksu örneği ve deniz suyu içerisine atık maddeler eklenerek kullanıldı. Deniz suyunda pektinaz enzimi inhibe olurken, α amilaz ve proteazda maksimum giderim miktarları saptandı. Sentetik ve gerçek evsel atıksu örneklerinde ise, ko-immobilize α amilaz ve proteaz enzimlerinin giderim oranları birbirlerine yakın sonuçlar gösterirken, pektinaz için gerçek evsel atıksudaki ürün oluşumu daha yüksek olarak saptandı. Bu çalışmada atıksu ön arıtımı için glutaraldehit ile aktive kitosan boncuklar kullanılarak α amilaz, proteaz ve pektinaz enzimleri ko-immobilize edildi. Geliştirilen bu materyalin çoklu enzim koimmobilizasyon çalışmalarının tasarlanması için bir model olacağına ve gelecekte bu konuda yapılacak çalışmalara ışık tutacağına inanmaktayız

Sterilization studies of hydrogel nanocomposites designed for possible biomedical applications before in vivo research

This study emphasized the importance of hydrogel-based therapy in repairing cartilage tissue and discussed the nanoscopic requirements for the physical sterilization of hydrogels, which are repairable, biochemically compatible with cartilage structure, and shape memory under mechanical effects.The nanostructured and the shape memory hydrogel composites, previously designed, synthesized, and nano -structurally characterized by our group, were used as material in the present study. Samples are including poly (N,N-dimethylacrylamide) (poly (DMAA) chains, n-octadecyl acrylate (C18A) segments and with/without lauryl methacrylate (LM).The study consists of four main sections in which physical sterilization processes (with electromagnetic waves from low energy (UV) to high energy (X-ray and Gamma-ray) are applied, structural changes are determined at microscopic and nanoscopic scale, and biofilm formations in the mentioned hydrogel materials are evaluated.The present study investigated these hydrogels' potential as artificial cartilage or cartilage tissue scaffolds. To initiate in vivo studies, it was aimed to determine the most appropriate physical sterilization method.In the result of the study, the most convenient hydrogel sample for surgical (in vivo) research, the useful physical sterilization methods, and the ability to resist biofilm formation was determined for the sample of N:3, [DMMA/C18A/LM, (70/30/0.0) l (Pre stretching ratio) = 1.8]. UV applications were also determined as the most generally suitable sterilization method for these hydrogels. As the pre-stretching ratio increases, the emergence of more compact and globular nano formations in hydrogel structures also affects the bioactive properties. It was also shown that, with the help of the usage of energetic electromagnetic waves for sterilizations, the new 3D nano aggregation morphologies might be created in the hydrogel structures

Highly-Sensitive, Label-Free Detection of Microorganisms and Viruses via Interferometric Reflectance Imaging Sensor

Pathogenic microorganisms and viruses can easily transfer from one host to another and cause disease in humans. The determination of these pathogens in a time- and cost-effective way is an extreme challenge for researchers. Rapid and label-free detection of pathogenic microorganisms and viruses is critical in ensuring rapid and appropriate treatment. Sensor technologies have shown considerable advancements in viral diagnostics, demonstrating their great potential for being fast and sensitive detection platforms. In this review, we present a summary of the use of an interferometric reflectance imaging sensor (IRIS) for the detection of microorganisms. We highlight low magnification modality of IRIS as an ensemble biomolecular mass measurement technique and high magnification modality for the digital detection of individual nanoparticles and viruses. We discuss the two different modalities of IRIS and their applications in the sensitive detection of microorganisms and viruses

Solid-Phase Optical Sensing Techniques for Sensitive Virus Detection

Viral infections can pose a major threat to public health by causing serious illness, leading to pandemics, and burdening healthcare systems. The global spread of such infections causes disruptions to every aspect of life including business, education, and social life. Fast and accurate diagnosis of viral infections has significant implications for saving lives, preventing the spread of the diseases, and minimizing social and economic damages. Polymerase chain reaction (PCR)-based techniques are commonly used to detect viruses in the clinic. However, PCR has several drawbacks, as highlighted during the recent COVID-19 pandemic, such as long processing times and the requirement for sophisticated laboratory instruments. Therefore, there is an urgent need for fast and accurate techniques for virus detection. For this purpose, a variety of biosensor systems are being developed to provide rapid, sensitive, and high-throughput viral diagnostic platforms, enabling quick diagnosis and efficient control of the virus's spread. Optical devices, in particular, are of great interest due to their advantages such as high sensitivity and direct readout. The current review discusses solid-phase optical sensing techniques for virus detection, including fluorescence-based sensors, surface plasmon resonance (SPR), surface-enhanced Raman scattering (SERS), optical resonators, and interferometry-based platforms. Then, we focus on an interferometric biosensor developed by our group, the single-particle interferometric reflectance imaging sensor (SP-IRIS), which has the capability to visualize single nanoparticles, to demonstrate its application for digital virus detection