Hedef kimyasala duyarlı floresan malzemelerin sentezi ve kimyasal sensörlerde kullanımı
Synthesis of fluorescent materials sensitive to target chemicals and usage in chemical sensors
Yazar: FEHMİ KARAGÖZ
Danışman: DOÇ. DR. ORHAN GÜNEY
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı
Derece: Doktora, 2015
Konu: Kimya = Chemistry

Özet:
Spektrofotometrik analiz, mikromolar seviyelerin altındaki konsantrasyonların algılanması ve kullanılmasındaki kolaylık nedeniyle etkin bir sensör yaratmak için önemli bir yöntemdir. Floresans sensör en önemli kimyasal sensörlerden biridir ve hedef molekülleri ve iyonları canlı organizma içinde görüntülemek için de güçlü bir alettir. Çünkü yüksek hassasiyet ve aynı anda görüntüleme özelliğine sahiptir. Floresans sensör bir analit ile etkileşimi sonucu algılanabilir spektral değişiklik üreten bir molekül veya polimer olabilmektedir. Floresan bir malzemenin tasarlanması, floresans cevabın algılama elemanının içine yerleştirilmesini gerektirmektedir. Bu yüzden, belirli bir analite sensör yaratmak için hem tanıma hem de floresans sinyalin optimize edilmesine ihtiyaç vardır. Polimer matriks içinde floroforun tuzaklanması, malzeme içinde homojensizlik oluşturacağından ve floresans klavuzun dışa salınması da kararsızlık problemi oluşturacağından, sensörün kullanım ömrü ve tekrarlanabilirlik özelliği azalacaktır. Bu malzemelerin kararlılığını iyileştirmek için polimerik matrikse klavuz molekülünün kimyasal olarak bağlanması alternatif olarak ortaya çıkmaktadır.  
Toksik ağır metal iyonlarının belirlenmesi için seçici kemosensörlerin geliştirilmesi, bu metal iyonlarının yaygın kullanımı ve çevresel olarak önemli etkileri sebebiyle özel ilgi çekmektedir. Atomik absorpsiyon spektrometresi, indüktif eşleşmiş plazma-kütle spektrometresi, indüktif eşleşmiş plazma-atomik emisyon spektrofotometri gibi analitik teknikler ve iyon-seçici potansiyometri gibi oldukça hassas elektrokimyasal teknikler metal iyonlarının tayininde kullanılmaktadır. Bununla birlikte, bu tekniklerde numune hazırlamanın karmaşık bir prosedür olması, yüksek enstrüman maliyeti ve bir elementin farklı yük durumu için farklı modlar olması gibi birçok dezavantaj meydana getirmektedir. Bu yüzden, basit spektrometrik teknikler ile metal iyonlarının düşük konsantrasyonlarının belirlenmesi önem arz etmektedir. Çeşitli spektroskopik yöntemler arasında, metal iyonlarının tespiti için floresans tekniklerin kullanımı yüksek hassasiyet, seçicilik, hızlı tepki süresi, düşük maliyet ve basit işlem prosedürü açısından sayısız avantajlar sağlamaktadır. 
Çalışmanın birinci kısmında, 9-aminoakridin (Ac) ve akriloyil klorür reaksiyonundan 9-acrylamidoacridine (VAc) sentezlendi ve FTIR ve NMR spektroskopisi ile karakterize edildi. VAc nin fotofiziksel özellikleri UV-vis ve floresans ölçümleriyle açığa çıkarıldı. Kuantum verimi etanol içerisinde hesaplandı (φf=0,15). Etanol-su tampon çözeltisi içinde (1:1v/v) pH=6,0 da Hg2+ iyonları ile titrasyonuna bağlı olarak VAc nin floresans artışı sergilediği bulundu. Oysa, diğer divalent geçiş-metal iyonlarının ilave edilmesine bağlı olarak VAc floresans emisyonu sönümlendi. Ayrıca, VAc nin pH a bağlı floresans özelliğinde değişim gözlendi ve pH a karşı integrasyon yapılan emisyon şiddeti dataları kullanılarak pKa değeri belirlendi. Bu kayda değer özellik hem çözeltinin pH sını hem de diğer metal iyonları arasında Hg2+ iyonlarının seçici tanınmasının gerçekleşmesine olanak sağlar. Buna ilaveten, VAc ve Hg2+ iyonu arasındaki kompleks stokiyometrisi spektroflorometrik titrasyon uygulanmasıyla   (1:2) olarak açığa çıkartıldı ve assosiasyon sabiti K=1,42x108 M-2 olarak hesaplandı.
İkinci kısımda, floresans algılayıcı olarak, izosiyanatopropil trimetoksisilan ile aşılandırarak (graft) elde edilmiş 9-amino akridin (AcI) sentezlendi ve FTIR ve NMR spektroskopileriyle karakterize edildi. AcI nın pH a bağlı floresans davranışı ve fotofiziksel özellikleri araştırıldı ve AcI nin uranil iyonu ile kompleks stokiyometrisi açığa çıkartıldı. Çözeltinin pH sı ile AcI nın floresans emisyonunda değişim gözlendi ve pH a karşı integrasyon ile edilen emisyon şiddeti dataları kullanılarak pKa değeri belirlendi. AcI nın floresans emisyonlarının etkilenmediği, etanol-tampon çözeltisi karışımı içerisinde uranil iyonlarıyla titrasyonuna bağlı olarak molekül içi yük transferine (Intra-molecular Charge Transfer) atfedilen AcI nın floresans artış sergilediği bulunmuştur. Oysa ki, AcI nın floresans emisyonu Civa (II) iyonları hariç diğer divalent geçiş metal iyonlarının ilavesiyle etkilenmemiştir. Diğer taraftan hem floresans hem de UV-vis titrasyon ölçümleri civa (II) iyonlarının girişim etkisi üzerinde uranil iyonları için seçicilik açığa çıkarmıştır. Ayrıca, AcI ile uranilin etkileşmesine bağlı denge sabiti K=7,41×106 M−2/3 ve kompleks stokiyometrisinin   (2:3) yapısında olduğu spektroflorometrik titrasyon ile belirlenmiştir. Kompleks tarafından sergilenen floresans artış üzerine bazı katyonların girişim etkisi test edilmiştir.
Üçüncü kısımda, sol-jel prosesinin spin kaplamayla birleştirilmesi sonucunda iyon-imprint floresans sensörü elde edildi. Floresans fonksiyonel silan önce sentezlendi ve sol-jel türevli malzemelerin elde edilmesi için tetrametoksisilan ile reaksiyona sokuldu ve daha sonra sulu çözeltide Hg2+ nin seçici olarak uzaklaştırılması ve tayini için reseptör olarak kullanıldı. Hg2+ varlığında sentezlenen iyon-imprint malzeme, FTIR, SEM, N2 adsorpsiyon–desorpsiyon analizi ve termogravimetik analiz ile karakterize edilerek elde edilen sonuçlar Hg2+ nin bulunmadığı durumda elde edilen non-imprint malzemeyle karşılaştırıldı. Azot adsorpsiyon profilinden, elde edilen malzemelerin yüzey alanı, gözenek hacmi ve çapı analiz edildi. Floresan klavuz molekülü içeren sol-jel filmin Hg2+ ile etkileşime bağlı olarak floresans emisyonunda ayırt edici değişikler sergilediği gözlendi. Filmlerin spektral karakterizasyonu için kararlı hal floresans deneyleri yapıldı ve sol-jel reaksiyonun ilerleyişi takip edildi. Farklı matrikslerin emisyon maksimumundaki kayma ve diğer spektral değişikler incelenerek sol-jel ağının jelleşme zamanı, pre-jel çözeltisindeki floresans fonksiyonel silanın emisyonunun izlenmesiyle belirlendi. Imprint floresans filminin Hg2+ nin diğer analoglarına karşılık gelen seçicilik faktörleri açığa çıkartılmıştır.

Abstract:
One of the fundamental goals for chemists is to design and synthesis of a sensitive and selective fluorescent sensor since fluorescent devices for the sensing and reporting of chemical species are currently of significant importance for chemistry, biology, and environmental science. Fluorescence detection with metal ion-responsive chemosensors offers a promising approach for simple and rapid tracking of metal ions for biological, toxicological, and environmental monitoring. Increasing attention has been given to the exploitation of a simple synthesis reagent and the development of a rapid, sensitive, and practical fluorescence analysis method to determine heavy metal ions. An important practical challenge to achieving this goal is devising water-soluble fluorescent dyes that detect metal ions selectively over other competing contaminants of metal ions.  Although heavy metal ions are relatively easy to chelate and detect in organic solvents, they are rather difficult to recognize directly in aqueous environments due to their strong hydrations. Concerns over toxic exposure to metal ions provide motivation to explore new methods for monitoring aqueous metal ions from biological and environmental samples. Analytical techniques such as atomic absorption spectrometry, inductively coupled plasma-mass spectrometry, inductively coupled plasma-atomic emission spectro-photometry and highly sensitive electrochemical techniques such as ion-selective potentiometry offer unparalleled sensitivity. However, they bear several disadvantage such as high instrument cost, a complicated procedure for sample preparation, and diﬀerent modes for diﬀerent charged states of an element. Therefore, detection of low concentrations of metal ions with simple spectrometric techniques is crucial. Among the various spectroscopic methods, use of fluorescent techniques for the detection of metal ions oﬀers numerous advantages in terms of high sensitivity, selectivity, rapid response time, low cost and simple operation procedure. Most small-molecule based fluorescent detection relies on changes in the signal resulting from metal–ligand binding events. 
Mercury and its derivatives threaten the environment and human health because of their durability, easy transference and high biological accumulation. Mercury pollution is a global problem and a major source of human exposure stems from contaminated natural waters. Exposure of mercury ions even at low concentration can damage the heart, stomach, intestine, and kidney. Additionally, Hg2+ in aquatic eco systems can be converted into lipophilic, readily absorbed, and poorly excreted organomercury species, such as methyl mercury species, which affect many different areas of the brain and their associated functions. Thus, the development of methods for selective and sensitive determination of Hg2+ is very important to understand its distribution and implement more detailed toxicological studies. Fluorescence method is highly sensitive, non sample-destructing or less cell-damaging, and can offer fast analysis with spatial resolution. These unique features make it favorable for both detection and imaging of Hg2+ in biological samples. Hg2+ is a heavy metal ion that is recognized as a fluorescence quencher due to the enhancement of spin–orbit coupling commonly associated with the heavy atom effect. Chemosensors that display fluorescence enhancement (fluorescence "turn-on") upon Hg2+ complexation are favored in terms of sensitivity concerns, over those showing fluorescence quenching (fluorescence "turn-off") depending on binding of Hg2+. In addition, fluorescence "turn-off" probes may report inaccurate results caused by other quenchers in practical samples and are undesirable for practical analytical applications. Therefore, it would be advantageous to design fluorescent sensors that can turn on fluorescence and provide a specific response following Hg2+ recognition. Different methods of fluorescence sensing of mercury ion are based on photo induced electron transfer (PET), intra-molecular charge transfer (ICT), chelation enhanced fluorescence (CHEF), excimer/exciplex formation, chemodosimeter, and fluorescence resonance energy transfer (FRET). Most of the Hg2+ selective fluorescent probes have suffered at least from one of the following parameters, which hinder them to be a novel fluorescent probe viz. cost of synthesis, number of synthesis steps, selectivity, LOD, detection medium, low binding constant. Spectrofluorimetric methods for the determination of mercury are mainly based on the coordination of Hg2+ ion with fluorescent reagents have been examined for fluorescence Hg2+ detection. To date, a number of small-molecule Hg2+ ion detection methods have been examined. Most of these systems have limitations, which include interference from other metal ions, delayed response to Hg2+ , and/or a lack of water solubility, requiring the use of organic solvent. Although there are lots of examples of chemosensors for Hg2+ based on fluorescent quenching in organic or aqueous solution, there are less chemosensors with fluorescence enhancement for Hg2+. Therefore, development of selective chemosensors based on the fluorescence enhancement for Hg2+ in aqueous solution is the target of the many researchers. 
Uranium is a soil and water contaminant at during the processing of uranium mining and nuclear fuel production. Under environmental conditions, uranium typically occurs in the hexavalent form as the mobile aqueous uranyl ion ( ), which can be found in soils around nuclear waste sites and processing facilities. Uranyl ion is transported through the most soil matrices and the rate of uranyl migration depends on a variety of parameters including soil porosity and composition, water content and temperature. Uranium and its compounds are highly toxic and may lead to kidney failure and death. The inhalation of uranium compounds results in deposition of uranium in lungs, which reach kidneys through the blood stream. There has been a growing interest in low-cost rapid techniques for measuring heavy metal ions and polluting wastes in environmental water. The determination of uranium is crucial in a number of nuclear related applications, such as environmental monitoring, fuel preparation and reprocessing. The sensitive and selective photometric reagents and improving existing procedures have been dedicated to develop simple and accessible procedures for the analysis of uranyl ion. Spectrophotometric methods are still indispensable because of their simplicity, rapidity and wide applications. Spectrophotometric analysis will diminish the demand to apply techniques that require expensive equipment with higher operation costs such as inductively coupled plasma–mass spectrometry, alpha spectrometry, neutron activation analysis, X-ray fluorescence, gamma spectrometry, laser fluorimetry. Although the above methods have good sensitivity, they all have some drawbacks, some of which require extensive chemical manipulation and wellcontrolled experimental conditions, and some may suffer from many types of interferences.
In first part of study, 9-acrylamidoacridine (VAc) was synthesized by the reaction of 9-aminoacrydine (Ac) and acryloyl chloride and characterized by FTIR and NMR spectroscopy. The photophysical properties of VAc were elucidated by UV–vis and ﬂuorescence measurements and quantum yield in ethanol was calculated (φF=0.15). We have found that VAc exhibited selective ﬂuorescence enhancement upon titration with Hg2+ ion in buffered aqueous-ethanol solution (1:1, v/v) at pH 6.0, whereas the ﬂuorescence emission of VAc was quenched upon addition of other divalent transition metal ions. Further, pH-dependent change in ﬂuorescent property of VAc was observed and pKa value was determined using data of integrated emission intensity versus pH. This unique property allows the evaluation of both pH of the solution and the selective recognition of Hg2+ ion among the other metal ions. In addition, the stoichiometry of complex between VAc and Hg2+ ion was elucidated as K=1.42x108 M-2. We have described the synthesis and photophysical evaluation of an acridine-derivated ﬂuorescent receptor, 9-acrylamidoacridine for sensitive pH and selective screening of mercury metal ion. The pKa value of VAc was found to be 5.76 from ﬂuorescence response curve versus pH and the ﬂuorescence intensity is almost a constant minimal value when pH>8.0 in a solution. The PET chemosensor, VAc for recognition of mercury metal ion displays high selectivity by ﬂuorescence enhancement, and a red shift of about 19 nm were observed in the presence of Hg2+ ion by comparison with that of VAc alone. The stoichiometry of coordination complex between VAc and Hg2+ ion was found to be  (1:2). VAc exhibits excellent selectivity for Hg2+ over competing environmentally relevant the other transition metal ions, Pb2+, Ag+, Fe2+, Cd2+, Cu2+, Ni2+ known as ﬂuorescent quenchers. The method developed in this study provides a useful starting point for developing new mercury contamination screens for a wide range of biological, toxicological, and environmental samples.
In second part of study, a fluorescent receptor, isocyanatopropyl trimethoxysilane grafted 9-amino acridine (AcI), was synthesized and characterized by FTIR and NMR spectroscopy. Photophysical properties and pH-dependent fluorescence behavior of AcI were investigated and its complex stoichiometry with uranyl ion was elucidated. Change in fluorescence emission of AcI with pH of the solution was observed and pKa value was determined by using integrated emission intensity versus pH. It was found that AcI exhibited fluorescence enhancement, which can be attributed to an internal charge transfer (ICT) mechanism, upon titration with uranyl ions in mixture of ethanol-buffer solution while the fluorescence emission of AcI was not affected by addition of other divalent transition metal ions except mercury (II) ions. On the other hand, the both fluorescence and UV-vis titration measurements revealed unique selectivity for uranyl ions over the interfering mercury (II) ions. The spectrofluorometric titration clarified that uranyl interacted with AcI to form   (2:3) complex structure with an apparent association constant of K=7.41×106 M−2/3. The interference effect of some cations on fluorescence enhancement exhibited by complex was also tested. The study presents the appropriate selectivity for the determination of  ion based on fluorescence enhancement of AcI upon complex formation with uranyl ion. AcI exhibits turn-on type fluorescence phenomena towards uranyl ion in pH-acidic solution. Fluorescence emission intensity of AcI increased upon titration of uranyl ions and red shift of about 7 nm was observed. In order to eliminate the interference effect of mercury (II) ion on assay of uranyl ion, UV-vis titration was carried out. It was observed that peak maximum of AcI in UV-vis spectrum shifted to shorter wavelength region and absorbance value increased upon titration with uranyl ion. These behaviors of AcI, i.e. functioning in a turn-on mode, displaying high selectivity over mercury ion and other cations, and having a unique reversible function, make AcI a promising candidate as a fluorescent sensor for uranyl ion.
In third part of study, an ion-imprinted fluorescent sensor by combining sol–gel processing and spin coating was developed. Fluorescent functional silane was first synthesized and reacted with tetramethoxysilane to obtained sol-gel-derived materials and then used as a receptor for selective removal and sensing of Hg2+ in aqueous solution. Ion-imprinted material synthesized in the presence of Hg2+ was characterized by Fourier transform-infrared spectroscopy, scanning electron microscopy, N2 adsorption–desorption analysis and thermogravimetric analysis by comparing with non-imprinted material obtained in the absence of Hg2+. Surface area, pore volume and diameter were analyzed from the profile of nitrogen adsorption. It was observed that fluorescent sol-gel film exhibits distinctive change in fluorescence emission upon interaction with Hg2+. Steady-state fluorescence experiments were performed for spectral characterization of the films and the advancement of the reaction was defined. The shift in emission maxima and other spectral changes of the different matrices have been identified and gelation time of sol-gel network was revealed by monitoring the emission of fluorescent silane in pre-gel solution. The corresponding selectivity factors of the imprinted fluorescent film toward Hg2+ against the other analogues were evaluated. In summary, we reported the synthesis and characterization of sol–gel derived ion-imprinted materials for Hg2+ ion recognition using optical sensing method. Based on the in situ measurement of FFS fluorescence during the gelation of the system, duration for sol-gel transition was found to be higher for Hg-imp than that of N-imp since interaction of FFS with Hg2+ diminish the effect of basic catalyst in the initiation step of sol-gel process. The sol-gel film exhibited a pH sensitive fluorescent behavior with a dual-emission in the range 2.0 to 4.0. Shift in fluorescence response of FFS immobilized in sol-gel film indicates the removal of the imprinted Hg2+ ion template creating geometrically oriented cavities within the material pore wall with selective rebinding characteristics. By exhibiting higher fluorescence enhancement behavior, Hg-imp sol-gel film showed better selectivity for the targeted Hg2+ ions than their non-imprinted analogues. The present study emphasizes problems associated with molecular imprinting as well as the significance of material structural properties. The system not only can detect the presence of Hg2+, but also could be used as an adsorbent for the removal of mercuric ions from contaminated aqueous solutions. The high surface area, thermal stability, easy preparation and high selectivity of the ion-imprinted sol-gel material will lead to the development of sorbents with an ordered microporous structures for a range of applications including selective recognition, separation and sensor devices.