Thiết kế, tổng hợp một số sensor huỳnh quang từ dẫn xuất của cyanine và coumarin để xác định biothiol và Hg(II)

The calculated results show that in the sensor AMC, the singlet electronic transition from S0 ground state to S1 excited state is the main transition with the greatest oscillator strength (f) of 0,5348 at 320,9 nm wavelength. The S0→S1 transition is the main contribution to transition from HOMO→LUMO, with a percentage contribution up to 96,21%. Besides, the overlap between HOMO and LUMO is very large, which shows that the transfer of electrons from HOMO to LUMO is favorable. The transition of other states have a small unnoticeable oscillator intensity (f). Meanwhile, with AMC-Cys, AMC-Hcy, and AMC-GSH, calculated data show that the singlet electronic transition from S0 to S2 is the main transition with oscillator intensity (f) of 0,3723; 0,3694 and 0,3801, respectively (much larger than other transfers) at the wavelength of 300,6; 300,4 and 300,7 nm, respectively. In the transition of the states, the transfer of electrons from HOMO-1→ LUMO is the main transition with a percentage contribution of 89,17; 89,05 and 89,24%, respectively. On the other hand, the overlap between HOMO-1 and LUMO is very large so the transfer of electrons from HOMO-1 to LUMO is very favorable. Other transitions of states have small, unnoticiable oscillator strength (f)

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nên không có quá trình PET nào can thiệp đến bước chuyển này. Kết quả này dẫn đến một kỳ vọng rằng L là hợp chất phát huỳnh quang. 3.1.2. Nghiên cứu thực nghiệm tổng hợp, đặc trưng và ứng dụng của sensor L 3.1.2.1. Thực nghiệm tổng hợp L Sau khi tổng hợp, cấu trúc của sản phẩm CBZT và L đã được khẳng định bởi phổ 1H-NMR, 13C-NMR và phổ FAB-MS. 3.1.2.2. Khảo sát thực nghiệm ứng dụng sensor L phát hiện ion Hg(II) a. Khảo sát phổ UV-Vis và phổ huỳnh quang của sensor L Hình 3.10. Phổ hấp thụ UV-Vis và phổ huỳnh quang của L: (a) Phổ UV-Vis, L (5,0 μM) trong C2H5OH/H2O (1/9, v/v), pH ~7,4; (b) Phổ huỳnh quang, L (5 μM) trong C2H5OH/H2O (1/9, v/v), pH ~7,4, bước sóng kích thích 540 nm 10 Như dự đoán từ tính toán, L phát huỳnh quang màu đỏ, với hiệu suất lượng tử huỳnh quang là 0,175; bước sóng huỳnh quang cực đại 585 nm, bước sóng hấp thụ cực đại 540 nm. b. Khảo sát phổ chuẩn độ UV-Vis và phổ huỳnh quang của sensor L phát hiện ion Hg(II) Hình 3.11 cho thấy, Hg(II) phản ứng và làm thay đổi phổ UV-Vis và phổ huỳnh quang của L. Cường độ huỳnh quang dung dịch L giảm dần khi tăng nồng độ Hg(II). c. Khảo sát phản ứng giữa sensor L với ion Hg(II) Hình 3.12 cho thấy, cường độ huỳnh quang dung dịch L giảm mạnh khi nồng độ ion Hg(II) tăng từ 0 đến 5,0 M; và sau đó giảm không đáng kể khi tiếp tục tăng nồng độ ion Hg(II). Điều này cho thấy L phản ứng với Hg(II) theo tỷ lệ mol 1:1. Hình 3.12. Đồ thị xác định quan hệ tỷ lượng phản ứng giữa ion Hg(II) với L (L (5,0 M) trong C2H5OH/H2O (1/9, v/v) ở pH ~7,4, bước sóng huỳnh quang 585 nm, bước sóng kích thích 540 nm Hình 3.11. Phổ chuẩn độ UV-Vis và phổ huỳnh quang của L bởi ion Hg(II): (a) Phổ UV-Vis, L (5,0 μM) trong C2H5OH/H2O (1/9, v/v), pH ~7,4, Hg(ClO4)2 (0 -5,0 μM); (b) Phổ huỳnh quang, L (5,0 μM) trong C2H5OH/H2O (1/9, v/v), pH ~7,4, Hg(ClO4)2 (0 -5,0 μM), bước sóng kích thích 540 nm 15 Hình 3.24 cho thấy, khi tăng dần Cys vào dung dịch phức Hg2L2: ở phổ UV-Vis, đỉnh hấp thụ cực đại ở bước sóng 460 nm dần dần biến mất, đồng thời xuất hiện một đỉnh hấp thụ cực đại mới với cường độ hấp thụ rất mạnh ở bước sóng 540 nm; ở phổ huỳnh quang, cường độ huỳnh quang tăng dần trở lại. b. Khảo sát ảnh hưởng của các amino acids cạnh tranh và phản ứng của Hg2L2 với các biothiol Kết quả trình bày ở Hình 3.25a cho thấy, chỉ các amino acids có chứa nhóm thiol mới làm thay đổi mạnh mẽ cường độ huỳnh quang của dung dịch. Các amino acids khác không chứa nhóm thiol hầu như không làm thay đổi tín hiệu huỳnh quang của dung dịch phức Hg2L2. Điều này cho thấy, phức Hg2L2 như một sensor huỳnh quang để phát hiện chọn lọc các biothiol trong sự hiện diện của các amino acids không chứa nhóm thiol. Kết quả thí nghiệm ở Hình 3.25b cho thấy, cường độ huỳnh quang tăng mạnh nhất là Cys, tiếp đến là GSH, Hcy. c. Khảo sát sử dụng Hg2L2 phát hiện định lượng Cys Trong khoảng nồng độ Cys từ 0 đến 5 μM, biến thiên cường độ huỳnh quang (F585) quan hệ tuyến tính với nồng độ Cys, thể hiện bởi phương trình F585 = (11,1 ± 5,9) + (133,3 ± 2,0) × [Cys], với R = Hình 3.25. (a) Phổ huỳnh quang của Hg2L2 (2,5 μM) trong C2H5OH/HEPES (pH =7,4, 1/9, v/v) tại 25 oC khi thêm các amino acids khác nhau (mỗi loại 10 μM), bao gồm Cys, Hcy, GSH, Ala, Asp, Arg, Gly, Glu, ILe, Leu, Lys, Met, Thr, Ser, Tyr, Trp, Val, và His (Others: hỗn hợp gồm tất cả các amino acids kể trên ngoại trừ Cys, Hcy và GSH). (b) Cường độ huỳnh quang (ở bước sóng phát quang 585 nm) của dung dịch Hg2L2 (2,5 μM) với các nồng độ khác nhau của Cys, GSH, Hcy, và các amino acids khác 14 3.1.4. Nghiên cứu sử dụng phức Hg2L2 phát hiện các biothiol 3.1.4.1. Nghiên cứu tính toán lý thuyết từ các phản ứng tạo phức Hằng số bền của phức đã được xác định bằng phương pháp chuẩn độ huỳnh quang. Kết quả tính toán đã xác định được hằng số bền của phức Hg2L2 bằng 1017,45 (M-3). Trong khi đó, hằng số cân bằng tạo phức Hg(RS)2 từ ion Hg(II) với các biothiol RSH, (2Hg(II) + 2RSH = Hg(SR)2 + 2H+, Ka) đối với Cys, GSH, Hcy tương ứng là 1020,1; 1020,2 và 1019,7. Vì vậy, phản ứng giữa Hg2L2 với các biothiol (Cys, GSH, Hcy) để tạo thành phức Hg(II) với các biothiol và giải phóng L tự do có thể xảy ra. Kết quả nghiên cứu về mặt nhiệt động của sự tương tác giữa ion Hg(II) với Cysteine (H2Cys) cho thấy, phản ứng (14) xảy ra vì có ΔG298 là âm nhất (ΔG298 = -821,6 kcal.mol -1) 2Hg2Cys + Hg(II) + 4OH- [Hg(Cys)2]2+ + 4H2O (14) Sự biến thiên của năng lượng tự do của phản ứng tạo Hg2L2 từ ion Hg(II) và sensor L là -410,2 kcal.mol‾1. Do đó, phản ứng sau xảy ra (vì có ΔG298 là -1232 kcal.mol-1): Hg2L2 + 4 H2Cys + 80H- 2- 2 + 8 H20 + 2LHg(Cys)2 3.1.4.2. Khảo sát thực nghiệm sử dụng phức Hg2L2 làm sensor huỳnh quang phát hiện các biothiol a. Khảo sát phổ chuẩn độ UV-Vis và phổ huỳnh quang của Hg2L2 Hình 3.24. Phổ chuẩn độ UV-Vis (a) và phổ huỳnh quang (b) của dung dịch Hg2L2 (2,5 μM) trong C2H5OH/HEPES (1/9, v/v), pH ~7,4, ở 25C khi thêm 0-10 μM Cys, bước sóng kích thích 540 nm, bước sóng phát huỳnh quang 585 nm 11 d. Khảo sát ảnh hưởng của các ion kim loại cạnh tranh Hình 3.13 cho thấy, không có bất kỳ sự thay đổi đáng kể nào trong phổ UV-Vis cũng như phổ huỳnh quang khi thêm các ion kim loại Cd(II), Fe(II), Co(III), Cu(II), Zn(II), Pb(II), Ca(II), Na(I), K(I) với nồng độ gấp 5 lần so với L. Như vậy, L có thể phát hiện chọn lọc ion Hg(II) trong sự hiện diện các ion này. e. Khảo sát sử dụng sensor L phát hiện định lượng ion Hg(II) Trong khoảng nồng độ ion Hg(II) từ 0 đến 400 μg/L: biến thiên mật độ quang (ΔA540) và biến thiên cường độ huỳnh quang (∆I585 ) quan hệ tuyến tính với nồng độ ion Hg(II) bởi các phương trình tương ứng: ΔA540= (0,01 ± 0,01) + (0,0011 ± 0,0000) × [Hg(II)], ∆I585= (-1,0 ± 0,4) + (0,3 ± 0,0) × [Hg(II)], với R=0,999. Giới hạn phát hiện và giới hạn định lượng bằng phương pháp trắc quang tương ứng là 15,3 μg/L và 51,2 μg/L hay 0,076 μM và 0,25 μM và phương pháp huỳnh quang tương ứng là 11,8 μg/L và 39,3 μg/L hay 0,059 μM và 0,19 μM. 3.1.3. Nghiên cứu lý thuyết ứng dụng sensor L phát hiện ion Hg(II) Hình 3.13. Phổ UV-Vis (a) và phổ huỳnh quang (b) của L (1,5 μM) với sự hiện diện của các ion kim loại Hg(II), Cd(II), Fe(II), Co(III), Cu(II), Zn(II), Pb(II), Ca(II), Na(I), K(I) (7,5 μM cho mỗi ion kim loại) trong C2H5OH/H2O (1/9, v/v), pH ~7,4, bước sóng kích thích 540 nm Hình 3.16. Hình học bền của phức Hg2L2 tại mức lý thuyết B3LYP/LanL2DZ 12 a. Nghiên cứu cấu trúc phân tử phức Hg2L2 Kết quả tính toán sự hình thành phức giữa ion Hg(II) và L theo tỷ lệ mol 1:1 ở mức lý thuyết B3LYP/LanL2DZ cho thấy, có một cấu trúc hình học bền được tìm thấy là Hg2L2 và được thể hiện ở Hình 3.16. Sự hình thành phức Hg2L2 là thuận lợi về mặt nhiệt động, với giá trị ∆G298 là -410,2 kcal mol -1. Các liên kết tạo phức gồm O25Hg93, O71Hg93, S55Hg93, và O26O71. Các liên kết này hình thành được cho là dựa trên kết quả tính toán khoảng cách giữa các nguyên tử nhỏ hơn đáng kể so với tổng bán kính Van der Waals của nguyên tử tham gia liên kết. Để khẳng định cấu trúc của phức Hg2L2, phân tích AIM đã được tiến hành. Kết quả phân tích cho thấy: có sự tồn tại các điểm tới hạn liên kết (BCPs) giữa các điểm tiếp xúc giữa các phối tử O, S với Hg(II), các liên kết này là liên kết cộng hóa trị và có sự tồn tại các điểm tới hạn vòng RCPs giữa các tiếp xúc O, S, N, Hg (phức có cấu trúc vòng) Nhằm giải thích tính chất huỳnh quang dựa vào bản chất electron của các liên kết, phân tích NBO cũng được tiến hành. Kết quả cho thấy, L tự do có cấu trúc kiểu: D-hệ liên hợp π-A (phát huỳnh quang); L trong phức: cặp electron của N7 không còn liên hợp vào hệ liên hợp π (cấu trúc D-hệ liên hợp π-A bị phá vỡ), nên có sự chuyển dịch electron dẫn đến dập tắt huỳnh quang của phức. b. Phân tích đặc tính huỳnh quang của phức Hg2L2 Kết quả tính toán ở Bảng 3.8 cho thấy, sự hình thành phức Hg2L2 đã dẫn đến sự chuyển dịch đáng kể mật độ electron từ các phối tử L đến các ion kim loại Hg(II) trung tâm và thu hẹp khoảng cách năng lượng giữa HOMO và LUMO. Kết quả, ở trạng thái kích thích chính (cường độ dao động lớn nhất và bằng 0,5913) từ S0→S2, với sự đóng góp chủ yếu từ bước chuyển HOMO→LUMO (53,12%), có năng lượng kích thích rất nhỏ là 1,37 eV. Điều này dẫn đến bước sóng phát xạ huỳnh quang của phức sẽ chuyển về vùng bước sóng dài, lớn hơn 900 nm. Vì vậy, trong thực tế không phát hiện được huỳnh quang từ phức Hg2L2. 13 Bảng 3.8. Năng lượng kích thích, cường độ dao động và các MO có liên quan đến quá trình kích thích chính của Hg2L2 ở mức lý thuyết B3LYP/LanL2DZ Bước chuyển MO Năng lượng (eV) Bước sóng (nm) f Tỷ lệ % đóng góp S0→S1 201→203 1,29 961,2 0,0838 4,63 202→203 30,06 202→204 59,58 S0→S2 201→203 1,37 903,4 0,5913 2,41 201→204 3,99 202→203 53,12 202→204 24,77 S0→S3 201→203 1,57 788,7 0,1063 38,83 201→204 39,46 S0→S4 201→203 1,59 778,5 0,0647 32,24 201→204 43,20 202→204 3,52 S0→S5 197→203 1,93 642,3 0,0183 2,66 199→203 50,52 199→204 10,31 200→203 25,64 S0→S5 201→205 2,29 S0→S6 198→204 1,95 636,4 0,0121 2,30 199→203 19,80 199→204 200→204 2,57 27,67 24 (c). Sensors Hg2L2 and AMC is for the detection of Cys in a small amount of organic solvent, the reaction time occurs fast can detect Cys with lower concentration than that in the intracellular and lower than that in the similar sensors of previous studies. 5. TD-DFT method is used to study the fluorescent properties of substances based on the optimized geometry at the ground state and the excited one in the combination with NBO analysis to consider the the change of the fluorescent properties of substances, based on the nature of bondings. The results of calcuations shows that ion Hg(II) creates complexion reactions with L, leading to the decrease in the energy distance between HOMO and LUMO in the meantime change the conjugated π-electron system, which is the cause for the fluorescence quenching in the complextion of Hg2L2. All the fluorescent emission of AMC, AMC-Cys, AMC-Hcy and AMC-GSH comes from the higher-lying electron excited states (S2, S4) to the ground state S0. This is an exceptional case of Kasha rule. 1 INTRODUCTION Cysteine (Cys), glutathione (GSH), and homocysteine (Hcy) are thiol compounds play vital roles in many biological processes. Mercury is one of popular dangerous pollutants which can cause serious effects to human’s health. Therefore, that the determination of biothiol in living cells and water sources helps diagnose related diseases and protect habitats and has caught much attention from local and oversea scientists. Many methods have been used for the detection of different biothiols and Hg(II) ions like high-performance liquid chromatography, mass spectrometry,,and fluorescent method. Among them, fluorescent method has outstanding benefits than other optical methods in term of investment of less expensive equipments and its simplicity. Furthermore, it can be applied to analyze many diffrent substances especially those in living cells. Professor Czarnik at Ohio University studied Fluorescent method and proposed a new approach to the field of sphere optical sensor in 1992. With advantages of fluorescent method, studies of fluorescent sensors to detect metal ions, anion, especially biomolecules is paid much attention by many local and oversea scientists, that have announced more and more Fluorescent sensors all over the world. In Vietnam, the study of the fluorescent sensors has been conducted by Duong Tuan Quang since 2007. To detect biothiols, the studies have designed fluorescent sensors based on the characterized reaction of biothiol, complex reactions (complex between fluorescence and ion Cu(II)..). The studies of fluorescent sensors detect Hg(II) based on the characterized reaction of ion Hg(II) and complex reactions between ion Hg(II) and legands - O,-N,-S in closed and opened circuit. However, most of these sensors have shortcomings like the use of a large amount of organic solvents, the limited detection just for high concentration, short excitation/emission wavelengths causing bad effects to living cells and slow reaction between sensors and analytes. Now, scientists are 2 continuing to study and design fluorescent sensors with high sensitivity and selectivity to detect biothiols and ions Hg(II). At present, quantum chemical calculations have been become an important tool in chemical studies in general and fluorescent sensors in particular. The combination between quantum chemical calculations and experimental studies is a modern trend in which, however, the numbers of studies announced in fluorescent sensors is still limited. From demand and situation of studies in the fluorescent sensors in the world and Vietnam, we have conducted the project “Design, synthesis of fluorescent sensors from cyanine and coumarin derivatives to detect biothiol and Hg(II)” New findings of the thesis: - A new fluorescent sensor L designed from derivatives of cyanine which has been reported, selective detection of Hg(II) ions, based on complexation reaction, ON-OFF mechanism; the complex of Hg(II) with L (Hg2L2) selective detection of Cys, based on decomplexation reaction and OFF-ON mechanism. The limit of detection and the limit of quantification for Hg(II) ions by L is 11,8 μg/L and 39,3 μg/L or 0,059 μM and 0,19 μM, respectively; limit of detection and limit of quantification Cys by Hg2L2 is 0,2 μM and 0,66 μM, respectively. - A new fluorescent sensor AMC designed from derivatives of coumarin which has been reported, selective detection of Cys based on Michael addition reaction, based on the change of ratiometry of fluorescent intensity at two different wavelengths. The limit of detection and limit of quantification Cys is 0,5 μM and 1,65 μM, respectively. - L and AMC studyed by flexible combination quantum chemical calculations and experimental studies. Chapter 1. OVERVIEW 1.1. Overview of fluorescent sensors 1.1.1. Current situation of fluorescent sensors 1.1.2. Operating principles of fluorescent sensors 1.1.3. Structure of fluorescent sensors 1.1.4. Design principles of fluorescent sensors 23 CONCLUSIONS 1. Flexible combination between quantum chemical calculations and experimental studies has been successfully applied for research and development of two new fluorescent sensors including L and AMC. This decreases the calculations of the theory and experiment, saving the time and expense for the chemicals, increase the possibility of success, clarify the nature of the proccesses and set ground for the further study. 2. Synthesis reactions sensor L and sensor AMC are studied, anticipated from calculations and afterwards verified from the synthesis results. 3. The structures, characteristics of sensor L and sensor AMC are determined at theoretical levels of B3LYP/LanL2DZ bringing about reliable results through contrastive tests and verification from experiment results. 4. (a). Sensor L is for selective detection of Hg(II) ions, in the presence of ther metal ions, based on fluorescent ON-OFF mechanism. The limit of detection and the limit of quantification for Hg(II) ions by colorimetric method is 0,076 μM and 0,25 μM; and by fluorescent method is 0,059 μM and 0,19 μM. Complextion of Hg2L2 is for selective detection of Cys in the presence amino acids without thiol groups based on decomplexation reaction and OFF-ON mechanism. The limit of detection and the limit of quantification Cys is 0,2 μM and 0,66 μM, respectively. Sensor L for the detection of ion Hg(II) and complextion Hg2L2 for the detection of Cys, based on complexation reaction between core ions Hg(II) with two ligands of L and Cys. (b). Sensor AMC is for the selective detection of biothiols (Cys, GSH, Hcy) in the presence of amino acids without thiol groups, based on the change of ratiometry of fluorescent intensity at two different wavelengths. The limit of detection and limit of quantification Cys is 0,5 μM and 1,65 μM, respectively. Sensor AMC reacts with biothiols (Cys, GSH, Hcy) based on Michael addition reaction. 22 For AMC-Cys, electron transitions from S1 to S0 at REES1 and REES2 are forbid. Meanwhile, electron transitions from S2 to S0 at REES1 and REES2 occur. In addition, because the oscillator intensity (f) of both processes are very large, while the oscillator intensity (f) at the wavelength of 340,3 nm is 0,5122, and obviously 0,3171 larger than that at the wavelength of 324,5 nm. This leads to the fact that fluorescence intensity of AMC-Cys observed in experiment is very strong, and that at the long wavelengths of 340,3 nm is stronger than short wavelengths of 324,5 nm. Besides, because the transitions processes of electron from S2 to S1 at REES1, with the S2 respectively do not have minimum energy, so processes (6) at Fig.3.48b is less dominant than processes (4) at Fig.3.48b. That may be another cause leading to fluorescence intensity at long wavelengths (340,3 nm) which is very stronger than the fluorescence intensity at short wavelengths (324,5 nm) as observed in experiment. This may be another cause to make fluorescence intensity at long wavelengths (469,5 nm) stronger than that at short wavelengths (417,4 nm), as observed in the experiment. For AMC-Hcy and AMC-GSH (similarly, AMC-Cys). As presented, the research results on optimum geometry with excited states of AMC, AMC-Cys, AMC-Hcy and AMC-GSH show that for AMC, there are twist angles between the coumarin moieties and acryloxy moieties at REES1 and REES2, causing the breakdown of the π- electron conjugate system between two moieties, which in turn leads to the fact that the electron density between the coumarin moieties and acryloxy moieties is strongly fragmented. As a result, there is very little overlap between the MOs in electron transfer at the excitation state of the sensor AMC. In contrast, at REES2 of AMC-Cys, AMC-Hcy and AMC-GSH, the coumarin moiety and acryloxy moiety are almost in the same plane. This is a favorable factor for the overlap between MOs in the state transitions. The above analysis shows that the fluorescence of the sensor AMC and its additive products with the biothiolis are not derived from the S1 state. This is an exceptional case of Kasha rule. 3 1.2. Roles of biothiols in cells and methods for detection 1.2.1. Biothiol và and roles of them 1.2.2. Method for detection of biothiols 1.3. The sources of pollution, toxicities and methods for detection of Hg(II) ions 1.3.1. The sources of pollution, toxicities of Hg(II) ions 1.3.2. Method for detection of Hg(II) ions 1.4. Fluorescent sensors for detection of biothiols 1.4.1. Based on the cyclization reactions with aldehydes 1.4.2. Based on the Michael addition reactions 1.4.3. Based on the native chemical ligation of peptide reactions 1.4.4. Based on the aromatic substitution-rearrangement reactions 1.4.5. Based on the cleavage of sulfonamide or sulfonate ester reactions by thiols. 1.4.6. Based on the cleavage of disulfides reactions by thiols 1.4.7. Based on the reactions complexation and decomplexation 1.4.8. Based on the mechanisms 1.5. Fluorescent sensors for detection of Hg(II) ions 1.5.1. Based on the reactions complexation with Hg(II) 1.5.2. Based on the characteristic reactions of Hg(II) ions. 1.6. Fluorescent sensors for detection of biothiol and Hg(II) ions based on the fluorophore are cyanine and coumarin 1.7. Overview of application of computational chemistry in the study on fluorescent sensors Chapter 2. RESEARCH CONTENTS AND METHODS 2.1. Research objectives 2.2. Research contents - Study on the design, synthesis, characteristics, and applications of cyanine derivatives based on sensor L for selective detection of biothiols and Hg(II) ions: + Theoretical study on design, synthesis and characteristics of sensor L. 4 + Experimental research on characteristics and application of sensor L. + Theoretical research on the application of the sensor L detects Hg (II). + Study the use of complex (form by Hg(II) ions with sensor L) detection of biothiol. In particular, theoretical research is conducted first to guide the study of the application of the next complex - Study on the design, synthesis, characteristics, and application of coumarin derivatives based on sensor AMC for selective detection of biothiols: + Study of design theory, synthesis of sensor AMC and reaction of sensor AMC with biothiols. + Experimental research on the synthesis, characteristics and applications of sensor AMC. + Theoretical study on characteristics and applications of sensor AMC. 2.3. Research methods 2.3.1. Theoretical calculation methods - The determination of the structure of geometry optimizations and single point energy was carried out by the density functional theory (DFT) method with the software of Gaussian 03. - The interaction energies adjusted for ZPE includesthe variation of enthalpy and variation of Gibbs free energy were derived as the differences between the total energy of the reaction products and the energy of the reactant substances. - The calculation of the excited state and the time-dependent factors was carried out using time-dependent density functional theory (TD-DFT) at the same theory level as the geometry optimisation procedure. - The analysis of AIM and NBO was executed at the B3LYP/LanL2DZ level of theory. 2.3.2. The experimental investigation methods - The structures of compounds were confirmed by 1H -NMR and 13C- NMR spectrum, mass spectrometry. 21 processes (3) and (4) above is not large enough (0,0137 and 0,0152), this results leads to the fact that fluorescence intensity of AMC is as small as observed in experiment. Moreover, because the excitation process from S0→S1 (process (1) in Figure 3.48a) has much greater oscillator intensity than that from from S0→S1 (2) in Fig 3.48a), so the transfer process of electrons from S1→S0 (process (3) in Fig. 3.48a) will be more dominant than that from S2→S0 (process (4) in Fig.3.48a). This may mainly cause the fluorescence intensity at long wavelengths (469,5 nm) stronger than that at short wavelengths (417,4 nm) as observed in the experiment. Fig.3.48. Energy diagrams of excitation processes and excitation energy release at geometry in ground state (RGS) and electron excitation states (REES1, REES2,...) at theoretical level B3LYP/LanL2DZ: (a) AMC; (b) AMC-Cys; (c) AMC-Hcy; (d) AMC-GSH (d) (d) (a) (b) (c) 20 state S1 (REES1), S2 (REES2), the coumarin moiety and acryloxy moiety are almost in two planes perpendicular to each other. For AMC-Cys, AMC-Hcy and AMC-GSH have twist angles between the coumarin moieties and acryloxy moieties in the RGS and REES1. In REES2 and the coumarin moiety and acryloxy moiety are almost in the same plane. 3.2.3.2. Research theories of spectral excitation and fluorescence spectrum a. Theoretical study on excitation and fluorescents pectra The calculated results show that in the sensor AMC, the singlet electronic transition from S0 ground state to S1 excited state is the main transition with the greatest oscillator strength (f) of 0,5348 at 320,9 nm wavelength. The S0→S1 transition is the main contribution to transition from HOMO→LUMO, with a percentage contribution up to 96,21%. Besides, the overlap between HOMO and LUMO is very large, which shows that the transfer of electrons from HOMO to LUMO is favorable. The transition of other states have a small unnoticeable oscillator intensity (f). Meanwhile, with AMC-Cys, AMC-Hcy, and AMC-GSH, calculated data show that the singlet electronic transition from S0 to S2 is the main transition with oscillator intensity (f) of 0,3723; 0,3694 and 0,3801, respectively (much larger than other transfers) at the wavelength of 300,6; 300,4 and 300,7 nm, respectively. In the transition of the states, the transfer of electrons from HOMO-1→ LUMO is the main transition with a percentage contribution of 89,17; 89,05 and 89,24%, respectively. On the other hand, the overlap between HOMO-1 and LUMO is very large so the transfer of electrons from HOMO-1 to LUMO is very favorable. Other transitions of states have small, unnoticiable oscillator strength (f). The analyzed results of the MO frontier also show that there is no overlap between HOMO and HOMO-1. Thus in AMC-Cys, AMC- Hcy and AMC-GSH do not occur in the PET process from HOMO to HOMO-1. As a result, AMC, AMC-Cys, AMC-Hcy and AMC-GSH are fluorescents as presented in the experiment. b. Theoretical study on fluorescent spectra For sensor AMC, at REES1, the electron transition from S1 and S2 to S0 are forbidden. At REES1, the lectron transition from S1 and S2 to S0 occur. In addition, because the oscillator intensity (f) of both 5 - The characteristics and applications of the sensors were performed by fluorescence spectroscopy and UV-Vis spectroscopy. - The conditions of sensors synthesis have been studied based on the predicted results from theoretical calculations and previous experimental results [2], [3], [29] on similar reactions. The synthesis process is summarized as follows: a. Sensor L synthesis: * The synthesis of CBTZ 2-methylbenzothiazole (3,0 g, 0,02 mol) and bromoacetic acid (4,18 g, 0,03 mol) were dissolved in 50 mL absolute ethanol. The mixture was boiled for 8 hours to, then cooled to room temperature until the precipitation was formed. This precipitation was washed with ethanol in alkali solution for several times, then dried to get the solid CBTZ (4,0 g, 75% yield). * The synthesis of L CBTZ (290 mg, 1 mmol) and 4-diethylamino-2- hydroxybenzaldehyde (190 mg, 1 mmol) were dissolved in 30 mL of absolute ethanol. With the addition of one drop of piperidine, the reaction solution turns red immediately. The reaction was kept boiling for 10 hours to cool to room temperature. The precipitation was formed and filtered, washed for several times with diethyl ether and then dried for the desired product L (3,0 g, 38% yield). b. Sensor AMC synthesis: 4-Methyl-7-hydroxylcoumarin (1,7 g, 9,4 mmol) and Et3N (7.9 mL, 56,4 mmol) were dissolved in CH2Cl2 (20 mL) with small addition of a catalyst amount of 4-dimethylaminopyridine to get a solution. The solution is made cool and keep at 0 oC. Each drop of acryloyl chloride (1,9 mL, 23,5 mmol) in CH2Cl2 (20 mL) is gradually added to the reaction solution in an hour. Then, the solution was stirred for 2 hours at room temperature and water was added to dissolve the amine salt. The organic phase was washed with aqueous solution and then dried over MgSO4. After the solvent was 6 evaporated, the product was purified by recrystallization from ethanol to form a white crystalline solid (1,0 g, 45%, yield). Chapter 3. RESULTS AND DISCUSSION 3.1. Design, synthesis, characteristics, and application of sensor L from cyanine derivatives for detection of biothiols and Hg(II) ions based on the reactions complexation 3.1.1. Theoretical study on design, synthesis, characteristics of sensor L 3.1.1.1. Theoretical study on design, synthesis of sensor L B3LYP/LanL2DZ levels of theory was applied for of research. Cyanine derivatives including R2N+=CH[CH=CH]n- NR2,Aryl=N+=CH[CH=CH]n-NR2, Aryl=N+=CH[CH=CH]n-N=Aryl, which all have the same structure, donor - π conjugated system - acceptor. Here, donor (the electron of push group) is an amino group; aceptor (the electron of withdrawal group) is amoni ions. They are known as color compounds with strong fluorescence [40]. Sensor L the design is planned as shown in the following synthesis diagram: N S N S -O2C BrCH2COOH N S -O2C N HO OHC N HO LCBZTBZT (I) (II) Fig. 3.1. Schematic design and synthesis sensor L Here, the fluorophore is cyanine, receptor is -COO- group, a strong affinity group with Hg(II) ion; The sensor L synthesis reaction occurs in two phases: phase (I) and phase (II). Reaction pairing the receptor to the fluorophore, the reaction (I) is based on the reaction between 4-methyl quinoline and carboxylic acid Fluorophore Receptor 19 For other amino acids without thiol groups do not change distinct fluorescence variations of sensor AMC solution (Fig.3.38a). The presence of this miscellaneous amino acids also do not have effect on the reation between AMC and biothiols (Cys, GSH and Hcy) with the clue that there is no significant difference between the fluorescence spectrum of solutions (AMC + biothiol + amino acids) with solutions (AMC + biothiol) (shown in Fig.3.38b). d. The survey on the use of sensor AMC to detect Cys In the concentration range of Cys from 0 to 10 μM, The ratiometric fluorescent intensity at two different wavelengths sóng 450 và 375 nm (F450/375) has a good linear relationship with Cys concentration in the equation: F450/375 = 1,5431 + 2,257 × [Cys], R = 0,982. The limit of detection and limit of quantitation for Cys are 0,5 μM and 1,65 μM, respectively. 3.2.3. Theoretical study on characteristics and application of sensor AMC 3.2.3.1. Optimized geometries (RGS, REES1, REES2) of AMC, AMC-Cys, AMC-Hcy, and AMC-GSH at electronically ground state and excited state In the ground state (S0) of sensor AMC, the coumarin moiety and acryloxy moiety are almost in the same plane, but at the excited Wavelength/nm Fig.3.38. (a) Fluorescence spectra of AMC (10 μM, ethanol/HEPES, pH 7,4, 1/4, v/v, at 25 oC) upon addition of Cys, Hcy, GSH, others amino acids (including Arg, Gly, Ala, Asp, Glu, Leu, Lys, Ile, Met, Thr, Ser, Trp, Tyr and Val). (b) Fluorescence spectra of AMC (10 μM, ethanol/HEPES, pH 7.4, 1/4, v/v, at 25 oC) in the presence of others amino acids mixture (including Arg, Gly, Ala, Asp, Glu, Leu, Lys, Ile, Met, Thr, Ser, Trp, Tyr and Val) when upon addition of Cys, Hcy, and GSH Wavelength/nm Wavelength/nm fl uo re sc en ce i n te n si ty ( a. u ) fl u or es ce n ce i n te n si ty ( a. u ) 18 increases more strongly than that of fluorescent emission at short wavelength. The fluorescence intensity change at both 375 and 450 nm wavelengths above leads to an ability to use AMC as a fluorescence sensor based on rate variation in fluorescence intensity at two wavelengths to detect Cys. b. Survey on reaction between sensor AMC and Cys When Cys were added from 0 to 10 μM to the sensor AMC solution (10 μM), the fluorescent ratio (F450/375) has a good linear relationship with the concentration of Cys. Then, this ratio has unnoticeable change if the concentration of Cys continues to increase. This shows that the reaction beween AMC and Cys occurs with 1:1 stoichiometry (similar for Hcy and GSH). This result is consistent with the result when determining the ratio of the reaction between AMC and Cys ratio with Job’s plot method and mass analysis of product reation betweent AMC and Cys. c. The survey on effects of the competitive amino acids These results of the survey reveal that when adding thiol- containing amino acids, the fluorescence intensity of AMC solution also increased markedly in both emission bands, in which the emission at 450 nm gave more fluorescent enhancement, whereas the emission at 375 nm produces a moderate increase. However, the the level of fluroescence enhancement is in this order: Cys>GSH> Hcy (Fig.3.38a). Fig.3.33. (a) Absorbance spectra of the sensor AMC (10 μM) in C2H5OH/HEPES (pH 7.4, 1/4, v/v) at 25 C upon addition of 20 μM of Cys. (b) Fluorescence spectra of AMC (10 μM) upon addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 equiv of Cys in C2H5OH/HEPES (pH 7.4, 1/4, v/v) at 25 C, excitation wavelength at 320 nm A bs o rb an ce fl u o re sc en ce i n te n si ty ( a. u ) Wavelength/nm Wavelength/nm 7 derivative [29] while reaction pairing to form fluorophore, the reaction (II) is based on the adol addition reaction ethanol and croton condensate [3]. a. Survey of the reactions of the phase (I) The reaction to form CBZT from BZT and bromoacetic acid is shown in Figures 3.2 and 3.3. The calculated results show that reaction between BZT and bromoacetic acid forms CBZT-3 and reaction between CBZT-3 and alkali solution forms CBZT, which is thermodynamically favorable. b. Survey of the reaction of phase (II) The reactions to form L from CBTZ and DHB can create four products (Fig.3.5). The calculated results have showed that the free energy (∆G298) of reaction (12) is negative. Accordingly, the reaction between CBZT and DHB in the trend to form L is clearly thermodynamically favorable. S N BrCH2COOH Br - S N+ COOHCBZT-1 (1) S N BrCH2COOH HBr S N+ COO- BZT CBZT-2 (2) S N BrCH2COOH S N+ COOH...Br-BZT CBZT-3 (3) S N BrCH2COOH S N+ COOHBZT CBZT-4 (4) Br- S N BrCH2COOH S N COOHBZT CBZT-5 Br (5) BZT Fig 3.2. The possible products formed from the reaction between BZT and bromoacetic acid CH3CH2OH Br- S N+ COO- CBZT S N+ COOH...Br- CH3CH2OH2 + H2O Br- S N+ COO- CBZT (7) S N+ COOH...Br- CBZT-3 H3O + OH- Br- S N+ COO- CBZT (8) S N+ COOH...Br- CBZT-3 H2O (6) CBZT-3 Fig 3.3. The reactions formed CBZT from CBZT-3 N+ S COO- N+ S COO- N HO OHC N HO CBZT (9) N+ S COO- L-1 (11) HO N L-3 H2O H2O N+ S COO- N (10) L-2 H2O HO N+ S COO- (12)N L H2O HO DHB Fig.3.5. The possible products formed from the reaction between CBTZ and DHB 8 3.1.1.2. Theoretical study on characteristics of L a. The molecular structure of L The bond lengths, bond angles, and dihedral angles of L were calculated. In particular, these values of BZT, bromacetic acid and DHB have little change compared with that from the beginning. In L, there is the formation of new bond between N7 and C11 and double one of bond C10 and C12. b. UV-Vis spectral analysis of sensor L The UV-Vis spectra of L at gains the maximum value at 452,6 nm. In a previous announcement, BZTVPA had the same structure as the sensor L with a maximum absorption wavelength of 405 nm, which is a strong fluorescent emission at 495 nm. This result led to an expectation that the fluorescent properties of L would be similar to BZTVPA fluorescence. c. The analysis of Fluorescent properties of sensor L Table 3.5. Calculated excitation energy (E), wavelength (λ), and oscillator strength (f) for low-laying singlet state of L at B3LYP/LanL2DZ State MO E (eV) λ (nm) f Percentage contribution(%) S0→S1 95→97 2,53 489,8 0,2566 56,44 96→97 35,80 S0→S2 93→97 2,74 452,6 0,5626 29,22 95→97 28,63 96→97 28,66 S0→S3 92→97 2,86 432,9 0,0097 5,90 93→97 8,83 94→97 77,56 Fig. 3.6. The optimized geometry of L at the B3LYP/LanL2DZ level of theory 17 The calculated results show that the free energy (∆298) of sensor AMC synthesis reaction is negative. Accordingly, its reaction is thermodynamically favorable. 3.2.1.2. Theoretical study on the reation between sensor AMC and biothiols According to the previously published works, the Michael addition reactions beween biothiols (Cys, Hcy and GSH) and esters created by acrylic acid and alcols (usually the fluorophore) initially generates thioethers, then followed by the formation of heterocycles compounds in the case of Cys and Hcy. Meanwhile, the thioether of GSH is usually stable with no subsequent ring formation. Different from the above study, the calculated results in term of the thermodynamics show that reactions between the sensor AMC and biothiols (including Cys, Hcy and GSH) to form thioether with the 1: 1 molar ratio is thermodynamically advantageous. 3.2.2. Experimental study on synthesis, characterization and application of sensor AMC 3.2.2.1. Experimental synthesis of sensor AMC After synthesis, the structure of AMC products was obtained with 1H- NMR and FAB-MS spectra. 3.2.2.2. Experimental study on the characteristics and application of sensor AMC a. Absorption and the fluorescence spectrum of the AMC sensor As shown in Fig.3.33a, the free sensor AMC shows a characteristic absorption band peaked at 275 and 320 nm. When Cys were added to the solution of sensor AMC, the absorption spectrum was negligibly changed. Meanwhile, the free sensor AMC displays fluorescence emission band peaked at 375 nm and 450 nm (Fig.3.33b). Fluorescence quantum yield (Φ) of sensor AMC was determined to be 0,05. When Cys were added to the solution of sensor AMC fluorescence tensity increases gradually at both emission bands. In particular, the intensity of fluorescent emission at long wavelength 16 not change the signals of the Hg2L2 solution’s fluorescence. This proves that the ensemble Hg2L2 complex is like a fluorescent sensor for the selective detection in the presence of amino acids without the thiol group. As shown in Fig.3.25b, fluorescence intensity gain strongest increase is Cys, then GSH and Hcy. c. Survey on the use of Hg2L2 for quantitative detection of Cys In the concentration range of Cys from 0 to 5 μM, there is a good linear relationship between the variation of fluorescence intensity (F585) of Hg2L2 and Cys concentration, shown in the following equation: F585 = (11,1 ± 5,9) + (133,3 ± 2,0) × [Cys], R = 0,998. The limit of detection and limit of quantitation for Cys are 0,2 μM and 0,66 μM, respectively. 3.2. Design, synthesis, characteristics, and application of sensor AMC from coumarin derivatives for the detection of biothiols based on the Michael addition reactions 3.2.1. Theoretical design, synthesis sensor AMC and reaction between AMC sensor with biothiols 3.2.1.1. Study on design, synthesis of sensor AMC 4-methyl-7-hydroxycoumarin compound gains maximum absorbance at 359 nm wavelength and maximum emission at 449 nm wavelength [159]. To design AMC fluorescence sensor (7- acryloyl -4- metylcouramin) from coumarin-based derivatives to detect biothiols based on the Michael addition reactions, 4-methyl-7-hydroxylcoumarin compounds are chosen as fluorophore and receptor as acryloyl chloride, because reaction of receptor to fluorophore is conducted easily with ester reation among phenol groups with the acid derivative [2] and this receptor can cause an addition reaction with the biothiols. The sensor AMC is designed in the following synthesis scheme: OO OH OO O O AMC O Cl + HCl+ (A) (B) Fig. 3.29. Schematic design and synthesis sensor AMC Receptor Fluorophore 9 State MO E (eV) λ (nm) f Percentage contribution(%) S0→S4 92→97 3,00 413,2 0,5815 5,42 93→97 49,94 94→97 10,62 95→97 9,35 96→97 11,07 S0→S5 92→97 3,05 406,0 0,0060 86,61 93→97 7,68 S0→S6 90→97 3,92 316,7 0,0051 44,35 91→97 41,32 96→97 8,40 Calculated results (Table 3.5) showed that, excited states have great oscillator strength namely S0→S1, S0→S2 at wavelengths 489,8 nm and 452,6 nm, respectively to make a significant contribution (35,80% and 28,66%, respectively) to transition from MO-96 to MO- 97. Because of continuous MOs, there is no PET to intervene this transition. This result led to an expectation that the L is fluorescence emission compound. 3.1.2. Experimental study on synthesis, characteristics and application of sensor L 3.1.2.1. Experimental study on synthesis of L After synthesis, the structure of CBZT and L products was obtained with 1H- NMR, 13C-NMR and FAB-MS spectra. 3.1.2.2. The experiamental survey of application of L for the detection of Hg(II) ions a. The survey of UV-Vis and fluorescence spectra of As expected from calculations, L performs red fluorescence with a quantum yield of 0,175. The maximum fluorescence wavelength is 585 nm and the maximum absorption wavelength is 540 nm (Fig.3.10). 10 b. They survey of UV-Vis and fluorescence titration spectra of L for the detection Hg(II) ions Fig.3.11 showed that Hg (II) ions reacted and changed the UV- Vis, and fluorescence spectra of L. The fluorescence intensity of L was decreased gradually while the concentration of Hg(II) ions is increased. c. The survey on the reaction between L and Hg(II) ions Fig 3.12 shows that, the fluorescence intensity of solution L decreases noticeably when the concentration of Hg (II) ions increases from 0 to 5.0 μM; and then decrease unsignificantly when we continue to decrease concentration of Hg (II) ions. This shows that L reacts with Hg (II) ions in a molar ratio of 1:1 Fig.3.11. The UV-Vis and fluorescence spectra of L with Hg(II) ions: (a) UV-Vis spectra, L (5 µM) in C2H5OH/H2O (1/9, v/v), pH ~7,4, Hg(ClO4)2 (0-5µM); (b) Fluorescence spectra, L (5 µM) in C2H5OH/H2O (1/9, v/v), pH ~7,4, Hg(ClO4)2 (0-5µM)), excitation wavelength at 540 nm Fig.3.10. UV-Vis and fluorescence spectra of L: (a) UV-Vis spectra, L (5 µM) in C2H5OH /H2O (1/9, v/v), pH ~ 7.4; (b) fluorescence spectra, L (5 µM) in C2H5OH/H2O (1/9, v/v), pH ~ 7.4, excitation wavelength at 540 nm. A b so rb an ce fl u o re sc en ce i n te ns it y ( a. u ) Wavelength/nm Wavelength/nm A bs o rb an ce fl u o re sc en ce i n te ns it y ( a. u ) Wavelength/nm Wavelength/nm 15 540 nm wavelength appears; in the fluorescence spectrum, the intensity of the fluorescence increases gradually again b. Survey on effects of competitive amino acids and reation between of Hg2L2 and biothiols The results presented in Fig.3.25a show that only the thiol- containing amino acids make strong change to fluorescence intensity of solution. In contrast, other amino acids without the thiol group do Fig.3.25. (a) Emission spectra of Hg2L2 (2,5 μM) C2H5OH/HEPES (pH =7,4, 1/9, v/v) at 25 oC upon addition of different amino acids (10 equiv each) including Cys, Hcy, GSH, Ala, Asp, Arg, Gly, Glu, ILe, Leu, Lys, Met, Thr, Ser, Tyr, Trp, and Val, respectively (Others: a mixture of miscellaneous amino acids without mercapto group). (b) Emission intensity at 585 nm of Hg2L2 (2,5 μM) vs different concentions of Cys, GSH, Hcy, and other amino acids. Fig.3.24. Absorbance spectra (a) and emission spectra (b) of Hg2L2 (2,5 μM) in C2H5OH/HEPES (1/9, v/v), pH ~7,4, at 25C upon addition of 0-10 equiv of Cys, excitation wavelength at 540 nm, emission wavelength at 585 nm A b so rb an ce fl u o re sc en ce i n te ns it y ( a. u ) fl u or es ce n ce i n te ns it y (a .u ) fl u or es ce n ce i n te ns it y (a .u ) Wavelength/nm Concentration (μM) Wavelength/nm Wavelength/nm 14 Calculated results in Table 3.8 show that the formation of Hg2L2 complex leads to the significant transfer of the electron density from L ligands to metal core Hg(II) ions and shorten the energy gap between HOMO and LUMO. As the results, at main excited states (maximum oscillator intensity is 0,5913) from S0→S2, with the main contribution of transition from HOMO to LUMO (53,12%), with a small amount of excitation energy of 1,37 eV. This leads to the fact that fluorescence emission wavelength of complex shifts to the long wavelength, greater than 900 nm. Therefore, in practice, there is no fluorescence from Hg2L2 complex to be detected. 3.1.4. Study on the use of Hg2L2 complex to detect biothiols 3.1.4.1. Study on theoretical calculations from complex reactions Constant association of complex was calculated by fluorescence titration method. The calculated results reveal that constant association of complex Hg2L2 is 10 17,45 (M-3). Meanwhile, the association constant of Hg(RS)2 from Hg(II) ions with biothiols RSH, (2Hg(II) + 2RSH = Hg(SR)2 + 2H+, Ka) for Cys, GSH, Hcy are 1020,1; 1020,2 and 1019,7, respectively. Hence, the reation between Hg2L2 with biothiols (Cys, GSH, Hcy) to format complex (Hg(II) with biothiols the freedom of L may be available. The research results in term of thermodynamics of reation between Hg(II) ions and Cysteine (H2Cys) show that the reation (14) occurs beacause ΔG298 is negative (value ΔG298 of -821,6 kcal.mol-1). 2Hg2Cys + Hg(II) + 4OH - [Hg(Cys)2] 2+ + 4H2O (14) The free energy (ΔG298) of reaction to form Hg2L2 from Hg(II) ions and L with a value ΔG298 of -410,2 kcal.mol‾1. Thus, the reation occurs (for the reason that the value ΔG298 is -1232 kcal.mol-1): Hg2L2 + 4 H2Cys + 80H- 2- 2 + 8 H20 + 2LHg(Cys)2 3.1.4.2. Experimental study on the use of Hg2L2 complex as fluorescence sensor to detect biothiol a. Survey on UV-Vis and fluorescence titration spectra of Hg2L2 Fig.3.24 indicates that when Cys is gradually added in Hg2L2 complex solution: in the UV-Vis spectra, the maximum absorption peak at 460 nm wavelength disappears constantly while a new maximum absorption peak with a very strong absorption intensity at 11 d. Survey on the effects of the competitive metal ions Fig.3.13 shows that there is no significant change in UV-Vis spectra as well as in fluorescence spectra when adding Cd (II), Fe (II), Co (III), Cu (II) , Zn (II), Pb (II), Ca (II), Na (I), K (I) with a 5 times higher concentration of L. Thus, L can detect selectively Hg (II) ions in the presence of these ions. e. The survey on the use of sensor L for quantitation detection of Hg(II) ions In the concentration range of Hg(II) ions from 0 to 400 μg/L: the variation of absorbance (ΔA540) and variation of fluorescence intensity (∆I585) maitain a good linear relationship with the ion concentration of Hg(II) with respective equations: ΔA540= (0,01 ± 0,01) + (0,0011 ± 0,0000) × [Hg(II)], R=0,999; ∆I585= (-1,0 ± 0,4) + (0,3 ± 0,0) × Fig.3.12. The graph for determination of the molar ratio of the reaction between Hg(II) ions and L: L 5 µM in C2H5OH/H2O (1/9, v/v) pH ~7,4, emission wavelength at 585 nm, excitation wavelength at 540 nm. Fig.3.13. Absorbance (a) and fluorescence (b) spectra of sensor L (1,5 μM) with different metal ions Hg(II), Cd(II), Fe(II), Co(III), Cu(II), Zn(II), Pb(II), Ca(II), Na(I), K(I) (5 equiv each) in C2H5OH/H2O (1/9, v/v), pH ~7,4, excitation wavelength at 540 nm fl u o re sc en ce i n te ns it y ( a. u ) Wavelength/nm fl u o re sc en ce i n te n si ty ( a. u ) fl u o re sc en ce i n te ns it y ( a. u ) A bs o rb an ce Wavelength/nm Wavelength/nm 12 [Hg(II)], R=0,999. The limit of detection and limit of quantitation for Hg(II) ions of colorimetric method are respectively 15,3 μg/L and 51,2 μg/L or 0,076 μM and 0,25 μM and the fluorescent method is 11,8 μg/L và 39,3 μg/L or 0,059 μM and 0,19 μM, respectively. 3.1.3. The theoretical study on the application of L for detection Hg(II) ions a. The study on the molecular structure of Hg2L2 The calculated results of the complex formation between Hg(II) ions and L with the molar ratio of 1: 1 at the theoretical level of B3LYP/LanL2DZ show that there is a unchanged geometrical structure found as Hg2L2, which is and shown in Fig.3.16. The formation of Hg2L2 complex is thermodynamically favorable, with a ∆G298 value of 410,2 kcal mol-1. The bonds of complexes include O25Hg93, O71Hg93, S55Hg93, and O26O71. These bonds are known to be formed based on the calculated results of the distances among atoms, significantly smaller than the sum of van der Waals radii of relevant atoms. To confirm that the structure of the Hg2L2 complex, AIM analysis was conducted. The results of the analysis show that there are bond critical points (BCPs) between the contact points between O, S ligands and Hg (II), which are covalent bonds with the existence of the ring critical points (RCPs) between contacts O, S, N, Hg points (complex have ring structure). To explain fluorescence characteristics based on nature of the electrons of the bonds, NBO analysis was conducted. The results show that free L has the structure: D-π conjugated - A (Fluorescent); L in complex: the electron pair of N7 is no longer conjugated to the conjugate π (the structure of D - π conjugated - A was broken), therefore there is an electron transfer leading to fluorescence quenching of the complex. Fig.3.16. The optimized geometry of Hg2L2 at the B3LYP/LanL2DZ level of theory 13 b. The analysis of fluorescent properties of Hg2L2 Table 3.8. Calculated excitation energy (E), wavelength (λ), and oscillator strength (f) for low-laying singlet state of Hg2L2 at B3LYP/LanL2DZ State MO E (eV) λ (nm) f Percentage contribution(%) S0→S1 201→203 1,29 961,2 0,0838 4,63 202→203 30,06 202→204 59,58 S0→S2 201→203 1,37 903,4 0,5913 2,41 201→204 3,99 202→203 53,12 202→204 24,77 S0→S3 201→203 1,57 788,7 0,1063 38,83 201→204 39,46 S0→S4 201→203 1,59 778,5 0,0647 32,24 201→204 43,20 202→204 3,52 S0→S5 197→203 1,93 642,3 0,0183 2,66 199→203 50,52 199→204 10,31 200→203 25,64 S0→S5 201→205 2,29 S0→S6 198→204 1,95 636,4 0,0121 2,30 199→203 19,80 199→204 200→204 2,57 27,67

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