Fluorescent detection of inorganic mercury and methylmercury in aqueous solution with micelles and live cells using fluorescent probes based on Trp amino acid and cycloalkylamine-7-sulfonyl-2,1,3-benzoxadiazole = 마이셀 시스템 내 사이클릭 아민 고리가 포함된 아미노산 기반 형광 센서를 이용한 메틸수은(I) 및 무기수은(II)의 선택적 검출 연구

국문 초록 (Abstract)

수은과 메틸수은은 독성이 강한 물질로, 인간의 건강에 해로운 영향을 끼친다. 특히, 메틸수은은 수생 환경 및 어류에 의해 노출될 위험이 높고, 지질막을 쉽게 통과하여 신체 내에 축적되면 치명적인 영향을 끼칠 수 있다. 이러한 이유로 수용액에서 수은과 메틸수은을 검출하는 방법에 대한 개발은 필수적이다. 본 연구에서 합성한 형광 화학센서는 4- dimethyl-amine-7-sulfonyl-2,1,3-benzoxadiazole (ABD) 형광체와 트립토판 (Trp) 아미노산을 기반으로 구성되며, 형광체의 N,N-dimethylamine 작용기를 다양한 크기의 cyclic ring 으로 바꾼 유사체들을 합성하였다. 합성한 형광 화학센서는 인간 세포 지질막의 구성요소와 유사한 lyso- phosphatidyl choline 으로 형성된 마이셀 시스템에서 수은과 메틸수은을 성공적으로 검출하였다. 유사체들 중, 가장 hydrophobic 한 7-membered ring 을 포함한 화합물의 경우에는, 마이셀 시스템에서 메틸수은에 대한 형광변화가 매우 크게 나타났으며, 다른 유도체들에 비해 수은 종에 대한 결합 능력 (Kd = 15.8 nM for Hg2+, Kd = 24.1 μM for CH3Hg+) 이 가장 높았다. 이러한 결과를 기반으로, 우리는 다양한 유도체들의 검출 능력을 마이셀 시스템에서 비교함으로써 수생 환경에서 메틸수은을 예민하게 검출할 수 있는 프로브를 개발하는 데 성공하였다.

다국어 초록 (Multilingual Abstract)

Mercury species are well-known to be toxic to humans. Particularly, methylmercury is a highly toxic substance that can readily penetrate lipid membranes, accumulating in the body and causing lethal effects when exposed in substantial amounts. Thus, the selective detection of mercury and methylmercury in aquatic environments and live cells is crucial. In this study, we synthesized fluorescent chemosensors (1-5), primarily composed of a tryptophan amino acid (Trp) and cycloalkylamine-7-sulfonyl-2,1,3-benzoxadiazole fluorophores bearing a different size of cyclic amine moiety and developed new sensing systems for CH3Hg+ and Hg2+ by combining the fluorescent chemosensors with lyso-phosphatidylcholine (PC) micelles to resemble cytoplasmic lipid membranes of human cells. Fluorescent chemosensors (1-5) with visible light excitation exhibited significant enhanced emissions to Hg2+ among various metal ions in aqueous solutions. In PC micelles, fluorescent chemosensors (1-5) showed significant enhanced green and red emissions (500~670 nm) to CH3Hg+ and Hg2+ among various metal ions. Interestingly, the chemosensors have different binding affinities and fluorescent signal changes for CH3Hg+ and Hg2+ depending on the size of cyclic amine moiety. Among them, chemosensor 5 with 7-membered cyclic amino group, exhibited the largest fluorescence change for CH3Hg+ and demonstrated the highest binding affinity for Hg2+ (Kd = 15.8 nM for Hg2+ and Kd = 24.1 μM for CH3Hg+). Thus, we investigated the binding mode of chemosensor 5 with the mercury species in PC micelles using UV/visible absorbance titration, pH titration, and NMR titration experiments. Furthermore, chemosensor 5 that detected Hg2+ and CH3Hg+ was successfully applied to detect sensitively CH3Hg+ and Hg2+ inside of live cells through increase in both green and red emissions. Fluorescent chemosensors in PC micelle systems might serve as a promising new fluorescent method for detecting methylmercury in aqueous solutions and live cells.

국문 초록 (Abstract)

수은 종은 인간에게 독성이 높은 물질로 알려져 있으며 수생환경에서 무기수은 이온과 메틸수은을 정량하는 것은 중요하다. 지금까지는 수생 환경 및 생체 내에서 CH3Hg(I)을 정량하기 위한 비례 형광 프로브에 대한 보고가 없었다. 본 연구에서는 수은 종에 대한 자기조립 특성을 갖는 짧은 펩타이드와 이를 기반으로 한 벤조타이아졸-시아노비닐렌 형광체를 사용한 형광 프로브 (1)를 개발하였다. 이 형광체는 응집할 때 붉은색의 파장으로 이동하는 형광 특성을 나타낸다. 물에 높은 용해도를 가진 1은 Hg(II) 및 CH3Hg(I)를 순수한 수용액(1% DMSO)에서 감지하는 데 사용되었으며, 이때 수은 종의 첨가로 540 nm에서 580 nm으로 이동되고 형광 향상이 확인되었다. 1은 수용액 중 수은 종에 대한 강한 결합능력 및 낮은 검출한계 (Hg(II)의 경우 4.15 nM 및 CH3Hg(I)의 경우 105.4 nM)를 보여주었으며, 18종의 금속 이온 중 Hg(II)와 CH3Hg(I)에 대한 높은 선택성, CH3Hg(I)에 대한 즉각적인 반응 (<30초), 및 넓은 pH 범위(4.5~11)에서 작동했다. 1은 수생 환경 및 생체 내에서 Hg(II) 및 CH3Hg(I)를 정량화하는 데 성공적으로 적용되었다. DLS, IR, CD 및 NMR 분광법을 사용한 결합 모드 연구에서는 프로브의 펩타이드 수용체와 수은 종류의 결합으로 자기조립이 유도되어 나노 크기의 집합체가 형성되었음을 밝혀냈으며, 여기에 벤조타이아졸-시아노비닐렌 형광체가 현저한 비율형 형광 변화를 나타냈다. 프로브 1은 PheHis로 이루어진 펩타이드 부분이 CH3Hg(I)의 비례 형광 검출에 중요한 역할을 한다는 것을 나타냈다.

다국어 초록 (Multilingual Abstract)

As methylmercury is well known as a highly toxic substance to humans, it was highly required for quantification of inorganic mercury ions as well as methylmercury in aquatic environment. Until now, it was not reported that ratiometric fluorescent probes for quantification of CH3Hg(I) in aquatic environments as well as live cells. In this work, we developed a fluorescent probe (1) based on the short peptide with self-assembly property for mercury species and a benzothiazolyl-cyanovinylene fluorophore that exhibited red-shifted emissions when it aggregated. 1 with high solubility in water was proved to be a ratiometric fluorescent probe for detecting Hg(II) and CH3Hg(I) in purely aqueous solution (1% DMSO) because the addition of the mercury species induced enhanced and red-shifted emissions from 540 nm to 580 nm as a clear isoemissive point. 1 had the potent binding affinities and low detection limits (12.5 nM for Hg(II) and 248.6 nM for CH3Hg(I)) for the mercury species in aqueous solution, high selectivity for Hg(II) and CH3Hg(I) among 18 metal ions, instant responses (< 30 sec) to CH3Hg(I), and wide working pH range (4.5~11), 1 was successfully applied to quantify Hg(II) and CH3Hg(I) in aquatic environment as well as live cells. The binding mode study using DLS, CD, and NMR spectroscopy, revealed that the chelation of the mercury species with the peptide receptor of the probe induced the self-assembly, resulting in the formation of nano-sized aggregates, in which the benzothiazolyl-cyanovinylene fluorophore exhibited significant ratiometric fluorescent changes and the probe 1 revealed that the peptide part consisting of PheHis played a critical role for ratiometric sensing of CH3Hg(I).

목차 (Table of Contents)

• Part I. Fluorescent detection of inorganic mercury and methylmercury in aqueous solution with micelles and live cells using fluorescent chemosensors based on Trp amino acid and cycloalkylamine-7-sulfonyl-2,1,3-benzoxadiazole 1
• Abstract 2
• Introduction 4
• 1. Experimental 8
• 1.1. Materials and reagents 8
• 1.2. Solid phase synthesis 9
• 1.3. UV/Vis and fluorescence measurements 13
• 1.4. Dissociation constant determination 14
• 1.5. Determination of detection limit (LOD), and quantum yield 15
• 1.6. Cell viability and detection of CH3Hg+ and Hg2+ in cells by CLSM 16
• 2. Results and Discussion 17
• 2.1. Design of ABD-based amino acid probe for Hg2+ and CH3Hg+ 17
• 2.2. Fluorescent response of chemosensors to Hg2+ and CH3Hg+ in aqueous buffered solutions 18
• 2.3. Fluorescent response of chemosensors to Hg2+ and CH3Hg+ in micelle system 21
• 2.4. Job’s plot analysis of 5 for Hg2+ and CH3Hg+ 25
• 2.5. Binding affinity of chemosensors with Hg2+ and CH3Hg+ 26
• 2.6. Comparison of chemosensors 1-5 for quantum yield, binding affinity and fluorescence change in PC micelles 29
• 2.7. Metal selectivity study of chemosensors 1 and 5 31
• 2.8. Effect of pH on the fluorescence response of 5 for Hg2+ and CH3Hg+ 35
• 2.9. Fluorescence response of chemosensors to Hg2+ and CH3Hg+ at pH 10 37
• 2.10. Detection limit of 5 with Hg2+ and CH3Hg+ in PC micelles 39
• 2.11. Binding mode study of 5 for Hg2+ and CH3Hg+ 41
• 2.12. Reversible test for Hg2+ and CH3Hg+ using EDTA and Glutathione 45
• 2.13. Cell images of 5 with Hg2+ and CH3Hg+ 48
• 3. Conclusions 52
• 4. Characterization 53
• Reference 66
• Part II. Ratiometric sensing for Hg2+ and CH3Hg+ ions in aqueous solution and live cells using fluorescent probes based on PheHis 72
• Abstract 73
• Introduction 75
• 1. Experimental 79
• 1.1. Materials and reagents 79
• 1.2. Solid phase synthesis 80
• 1.3. UV/Vis and fluorescence measurements 82
• 1.4. Determination of dissociation constant and LOD 83
• 1.5. Dynamic light scattering (DLS) and circular dichroism (CD) 84
• 1.6. Fibril formation analysis using CLSM and TEM 85
• 1.7. Cell viability and detection of CH3Hg(I) and Hg(II) in cells 86
• 2. Results and Discussion 87
• 2.1. Design of excimer emission based self-assembly dipeptide probe for Hg(II) and CH3Hg(I) 87
• 2.2. Fluorescence response of probes to Hg(II) and CH3Hg(I) 88
• 2.3. Selectivity and interference test of metal ions 91
• 2.4. Study on reducing Cu(II) interference of 1 for Hg(II) and CH3Hg(I) 93
• 2.5. Time dependent fluorescence response of 1 95
• 2.6. Effect of pH on the fluorescence response of 1 for Hg(II) and CH3Hg(I) 97
• 2.7. Binding mode study of 1 with Hg(II) and CH3Hg(I) 99
• 2.8. Job’s plot analysis and dissociation constant of 1 104
• 2.9. Reversible test of 1 for Hg(II) and CH3Hg(I) 107
• 2.10. Fibril formation of 1 with Hg(II) 110
• 2.11. Application of 1 for detecting Hg(II) and CH3Hg(I) in cells 114
• 2.12. Detection limit of 1 for Hg(II) and CH3Hg(I) 116
• 3. Conclusions 118
• 4. Characterization 119
• Reference 124