Functionalized quantum dots and its applications

FUNCTIONALIZED QUANTUM DOTS AND ITS APPLICATIONS XU JIA (B. Sc, Sichuan University) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE FOOD SCIENCE AND TECHNOLOGY PROGRAMME DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2008 Table of Contents Acknowledgement IV Summary V List of Tables Ⅶ List of Figures Ⅷ Chapter 1. General Introduction 1.1 Quantum Dots and Its Properties 1 1 1.1.1 Introduction to quantum dots 1 1.1.2 Quantum dots optical properties 2 1.2 Quantum Dots in Optical Sensing Applications 5 1.2.1 Anion and small molecules sensing 1.2.2 Hydrosulfide biology and its sensing 11 1.2.3 Aim and objectives 12 References Chapter 2. Thiolated Caffeic Acid Functionalized Qua ntum do ts 8 13 15 2.1 Introduction 15 2.2 Experimental Section 19 2.2.1 Materials and instruments 19 2.2.2 Synthesis of caffeic acid derivative 20 2.2.3 Quantum dots ligand exchange with thio-caffeic acid 25 -I- 2.3 Results and Discussion 26 2.4 Conclusion 33 References 35 C h a p t e r 3 . Tr o l o x F u n c t i o n a l i z e d Q u a n t u m d o t s 3.1 Introduction 35 35 3.1.1 Quantum dots and its synthesis 35 3.1.2 Quantum dots ligand exchange strategies 36 3.1.3 Electron transfer based sensor 40 3.2 Experimental Section 41 3.2.1 Materials and instruments 41 3.2.2 Experimental procedures 43 3.3 Results and Discussion 52 3.3.1 Synthesis and structural characterization of Trolox derivatives 52 3.3.2 Functionalization of quantum dots with Trolox derivative 56 3.3.3 Trolox-QDs sensing of peroxyl radicals 62 3.4 Conclusion References C h a p t e r 4 . Tr i s ( 2 - a m i n o e t h y l ) a m i n e ( Tre n ) F u n c t i o n a l i ze d Q D s 4.1 Introduction 68 70 71 71 - II - 4.2 Experimental Section 78 4.2.1 Materials and instruments 78 4.2.2 Sensing of metal ions 80 4.2.3 The effect of carbon disulfide on QDs-metal ions complex 81 4.2.4 Tren-CS2-metal complex synthesis and its reaction with sodium hydrosulfide 82 4.2.5 Tren-QDs-CS 2 -Fe(II) complex synthesis 82 4.2.6 Tren-QDs-CS2-Fe(II) complex sensing hydrosulfide anion and nitric oxide 83 4.3 Results and Discussion 84 4.3.1 Tren-QDs system 84 4.3.2 Optical characteristics of the Tren-QDs 86 4.3.3 Sensing of transition metals 88 4.3.4 Tren-QDs-CS 2 -Fe(II) complex system 95 4.3.5 Tren-QDs-CS2-Fe(II) complex synthesis and its application 4.4 Conclusion References 101 109 112 - III - Acknowledgement This research is financially supported by A*STAR’s Science and Engineering Research Council (SERC). The guidance and the fundamental works from Dr. Huang Dejian and Dr. Wang Suhua are acknowledged with gratitude. Miss Karen Hay and Miss Lim Pui Yee were also acknowledged for technical supports. - IV - Summary In this research the ability of several ligands bounding to the surface of ZnS capped CdSe quantum dots (QDs) was studied. Different ligands for quantum dots functionalization was synthesized and characterized by ESI-MS, NMR and FT-IR techniques. The ligand exchange was carried out between synthesized ligands and trioctylphosphine oxide (TOPO) functionalized CdSe/ZnS quantum dots under different conditions. The functionalized CdSe/ZnS quantum dots was purified and characterized to ensure the successful bounding. Preliminary sensing studies were carried out using the functionalized quantum dots sensing peroxyl radicals and hydrosulfide anion. The possibility for thiolated caffeic acid (3,4-dihydroxycinnamic acid) to serve as a quantum dot ligand was explored although the vulnerability of this ligand to air made ligand exchange difficult. The α-tocopherol (α-TOH) analogue Trolox (5,7,8-tetramethylchroman-2-carboxylic Acid) was modified to its diamine derivative. Using carbon disulfide as a bridging unit, Trolox diamine was capped onto CdSe quantum dots surface by ligand exchange process. The significant changes of functionalized QDs in solubility and optical properties, 31 P-NMR spectrum and FT-IR spectrum was consistent with literature reports which provide evidences for successful ligand exchange. The sensitivity of Trolox-QDs to peroxyl radicals and analysis to the reaction products showed us the promising future of this electron transfer based sensing Trolox-QDs system and the in depth studies are still -V- on going. The tris(2-aminoethyl)amine (Tren)-QDs system was developed in the same way as Trolox system but with a stronger bounding ligand. After ligand exchange, Tren-QDs can be well dissolved in water, PBS buffer and other polar solvents. It has been found the fluorescence of Tren-QDs has selective sensitivity to Co(III) and Cu(II). Different mechanisms for this effective quenching was proposed and the verification is still on the way. Moreover, Tren-QDs was used as the basis of development of new fluorescent probes. The Tren-QDs-CS2-Fe(II) complex material was synthesized and utilized for biologically important hydrosulfide anion (HS-) sensing. It was our speculation that a sulfide containing complex, which has strong absorbance at Tren-QDs excitation and emission wavelength was form from the reaction between the Tren-QDs-CS2-Fe(II) complex and HS- therefore the inner filter effect of this new complex quenched emission of Tren-QDs in the system. - VI - LIST OF TABLES Table 1.1 QD-based fluorescent probes for sensing of small molecules and ions Table 2.1 Scavenging properties of caffeic acid 9 17 Table 3.1 The rate constants of α-tocopheryl radical and the biological concentrations of the substrates 41 - VII - LIST OF FIGURES Figure 1.1 Orbital Energy levels in semiconductor QDs. Figure 1.2 Tuning the QD emission wavelength by changing the nanoparticle size or composition. 1 2 Figure 2.1 ESI-MS of thio-caffeic acid reaction mixture 22 Figure 2.2 Separation and purification of thio-caffeic acid 23 Figure 2.3 ESI-MS (negative) of thio-caffeic acid extraction 24 Figure 2.4 ESI-MS (negative) of purified thio-caffeic acid 24 Figure 2.5 ESI-MS result of DTT protective effect on thio-caffeic acid 31 Figure 3.1 ESI-MS (negative mode) spectrum of Trolox methyl ester 44 Figure 3.2 ESI-MS (positive mode) spectrum of Trolox methyl ester 44 Figure 3.3 1 45 Figure 3.4 13 45 Figure 3.5 ESI-MS spectrum of Trolox diamine 47 Figure 3.6 1 48 Figure 3.7 13 48 Figure 3.8 FT-IR spectra of Trolox methyl ester and Trolox diamine 53 Figure 3.9 Trolox methyl ester standard curve in methanol. 54 H-NMR spectrum of Trolox methyl ester C-NMR spectrum of Trolox methyl ester H-NMR spectrum of Trolox diamine C-NMR spectrum of Trolox diamine Figure 3.10 The reaction kinetic of AMVN(3.65 mM) and AAPH (3.35 mM) on Trolox methanol solution (4.52 mM) absorbance 55 Figure 3.11 ESI-MS (negative) spectrum of reaction mixture of Trolox methyl ester and AAPH Figure 3.12 Proposed Structure of Trolox functionalized quantum dots 56 57 Figure 3.13 Absorption spectrum and fluorescence emission spectra of TOPO Figure 3.14 QDs and Trolox functionalized QDs 58 31 60 P NMR spectra of TOPO-QDs and Trolox functionalized QDs Figure 3.15 FT-IR spectrum of Trolox functionalized QDs and TOPO QDs 61 Figure 3.16 Effect of AAPH and AMVN solution on the fluorescence of Trolox-QDs 63 Figure 3.17 Effect of AAPH solution on the fluorescence of Tren-QDs and Trolox-QDs 64 - VIII - Figure 3.18 FT-IR spectrum of Trolox-QDs and oxidized Trolox-QDs Figure 4.1 Ten distinguishable emission colors of ZnS-capped CdSe QDs excited with a near UV lamp with sizes of QDs increase from left to right Figure 4.2 66 73 Proposed surface structure of CdSe QDs capped by Tren ligands and their coordination complexes with transition metal ion. 85 Figure 4.3 Absorption spectrum and fluorescence emission spectra of Tren-QDs 86 Figure 4.4 The concentration-dependent of fluorescence property of QDs. 87 Figure 4.5 Effect of metal ions on the fluorescence of Tren-QDs. 88 Figure 4.6 Electronic spectra of Co(III) complexes of various concentrations in aqueous solution. Figure 4.7 90 Electronic spectra of Cu(II) complexes of various concentrations in aqueous solution. 91 Figure 4.8 Energy scheme of the Tren-QDs in the presence of Cu(II). 92 Figure 4.9 Effect of Cu(II) and Co(III) concentration on the fluorescence intensity of Tren-QDs. 93 Figure 4.10 Stern-Volmer plot of Cu(II) and Co(III) concentration dependence of the fluorescence intensity of Tren-QDs. Figure 4.11 Effect of metal ions and CS2 on the fluorescence of Tren-QDs. 95 97 Figure 4.12 UV-Vis spectrum of Tren-CS2-Fe(II) complex before and after NaSH addition 99 Figure 4.13 Effect of hydrosulfide anion on the UV-Vis absorbance of Tren-QDs-CS2-metal complex at 550nm. 100 Figure 4.14 Proposed surface structure of CdSe QDs capped by Tren ligands and their coordination complexes with Iron (II) ions. Figure 4.15 Spectra of Tren-QDs-CS2-Fe(II) complex quenched by NaSH solution 102 103 Figure 4.16 Effect of HS - concentration on the fluorescence intensity of Tren-QDs-CS2-Fe(II) complex 104 Figure 4.17 Effect of hydrosulfide anion concentration on the fluorescence intensity of Tren-QDs-CS2-Fe(II) complex 105 Figure 4.18 UV-VIS spectrum of Tren-QDs-CS2-Fe(II) complex before and after - IX - NaSH addition 106 Figure 4.19 Quenching Tren-QDs-CS2-Fe(II) complex by NaSH at different excitation wavelength 107 Figure 4.20 S p e c t r a o f Tr e n - Q D s - C S 2 - F e ( I I ) c o mp l e x q u e n c h e d b y nitric oxide solution 108 -X- Chapter 1 General Introduction 1.1 Quantum Dots and Its Properties 1.1.1 Introduction to quantum dots Quantum dots (QDs) are nanostructured semiconductor materials[1]. These colloidal nanocrystalline semiconductors comprising elements from the periodic groups II-VI, III-V or IV-VI, are featured with roughly spherical and with typical sizes (diameter) in the range 1-12 nanometer (nm). At such reduced sizes that close or smaller than dimensions of the exciton Bohr radius within the corresponding bulk material, these nanoparticles behave differently from bulk solids due to quantum confinement effects [2,3] . The result of quantum confinement are that the electron and hole energy states within the nanocrystals are discrete, but the electron and hole energy levels and therefore the band-gap is a function of the QDs diameter as well as composition[5]. The band-gap of semiconductor nanocrystals increase as their size decreases, resulting in shorter emission wavelength[6,7]. Hence, the quantum confinement effects are responsible for unique optoelectronic properties exhibited by QDs which includes high emission quantum yields, size-tunable emission profiles and narrow spectral bands[3,4]. Especially, their size-dependent properties result in a tunable emission that allows one to choose an emission wavelength that is well suited to a particular experiment and to synthesize the QD-based probe by using an appropriate semi-conductor material and nanocrystal size. -1- Semiconductor QDs are characterized by a band-gap between their valence and conduction electron bands (Figure 1). When a photon having an excitation energy exceeding the semiconductor band-gap is absorbed by a QD, electrons are promoted from the valence band to the high-energy conduction band. The excited electron may then relax to its ground state by the emission of another photon with energy equal to the band-gap[4]. Conduction Band e Band gap Hole Electron e + e e e e Valence Band Figure 1.1: Orbital Energy levels in semiconductor QDs. 1.1.2 Quantum dots optical properties In recent years there has been intense research in the fundamental study of the synthesis and photophysical properties of QDs[8-11]. Researches on II-VI semiconductor QDs, such as CdSe or CdS nanocrystals have been studied in order to characterize the relationship between size, shape and electronic properties[2-4]. And the initial applications of QDs were heavily focused on their use in microelectronics and opto-electrochemistry (e.g., light-emitting diodes, solar energy conversion et al.)[3,12,13]. -2- But soon the unique sized-dependent emission attracted enough attention from researchers and became probably the most intriguing and the most studied optical property of QDs. As the emission properties of semiconductor nanocrystals depend strongly upon the energy and the density of the electron state, they can be altered by engineering the size and the shape of their structure. As Figure 1.2 shows, different size CdSe nanoparticles can be tuned in the 500-700nm range. Moreover, by altering the chemical composition of QDs, fluorescence emission may be tuned from the near-infrared spectrum, spanning a broad wavelength range of 400-2000nm[14]. Figure 1.2 Tuning the QD emission wavelength by changing the nanoparticle size or composition. (A) The emission of a CdSe QD may be adjusted to anywhere within the visible spectrum (450-650 nm) by selecting a nanoparticle diameter between 2 and 7.5 nm (B) While keeping the nanoparticle size constant (5 nm diameter) and varying the composition of the ternary alloy CdSexTe1-x, the emission maximum may be tuned to any wavelength between 610 and 800 nm.(figure adapted from refs.16) Another advantage of quantum dots fluorescence emission is its typically narrow emission profile compared with traditional organic dyes with full width at half-maximum (FWHM) around 15-40 nm[1]. This property is due to QDs’ discrete, atom-like electronic structure. Since the emission lines are comparatively narrow, -3- detection of the QDs suffers much less from cross-talk that might result from the emission of a different fluorophore bleeding into the detection channel of analyte. On the other hand, QDs typically exhibit higher fluorescence quantum yields than conventional organic dyes which give it greater analytical sensitivity. The quantum yield of a fluorophore is a function of the relative influences of radiative recombination (producing light) and non-radiative recombination mechanisms. Non-radiative recombination, which largely occurs at the nanocrystal surface, is a faster mechanism than radiative recombination an is greatly influenced by the surface chemistry. By capping the nanocrystal with a shell of an inorganic wide-band semiconductor, such as ZnS, reduces such non-radiative deactivation and results in brighter emission[14]. The suitably surface protected QDs also have superior photoluminescent stability as compared to typical fluorescent organic dyes. Several studies have demonstrated that the photo luminescence properties of CdSe nanocrystals did not show any detectable change upon aging in air for several months [15] and were observed to be 100 times more stable than conventional organic fluorophores against photobleaching[12]. The long fluorescence lifetimes of QDs, on the order 10-50 ns, are advantageous for distinguishing QD signals from background fluorescence and for achieving high-sensitivity detection[17]. -4- 1.2 Quantum Dots In Optical Sensing Applications The application of luminescent QDs as biological labels was first reported in 1998 in two breakthrough papers published by A. P. Alivisatos at UC-Berkeley and S. M. Nie at Indiana University-Bloomington respectively[17,18]. Their research simultaneously demonstrated that semiconductors QDs could be made water soluble and could be conjugated with biological molecules by surface modification and bioconjugation methods. It was their pioneer work paved the way for QDs application as highly sensitive fluorescent bio-marker and biochemical probes. Developments in recent years have more focused on the importance of adequate surface modifications in developing luminescent QDs for labeling in bioanalysis and sensing. The nature of the ligands being coordinated to the QDs surface and the particular type of bonds which it forms with the nanocrystal surface atoms are of great importance to quantum dots researchers. By ligand designing and fabrication, several important properties of quantum dots can be tuned for specific purpose: processibility, reactivity and stability. All of these have direct consequences on quantum dots’ spectroscopic properties[19]. Three principal functions of the surface ligands can be described by Querner et al. as follows: (1) They prevent individual colloidal nanocrystals from aggregation. (2)They facilitate nanocrystals’ dispersion in a large variety of solvents. In the presence of surface ligands, the ability to disperse nanocrystals is governed by the difference between the ligand and the solvent solubility parameters, which can be precisely tuned. (3) Ligands containing appropriate functional groups may serve as bridging units for the coupling of -5- molecules or macromolecules to nanocrystals or their grafting on substrates[19]. As the luminescence of QDs is very sensitive to the surface states of the QDs, it is reasonable to expect that the chemical or physical interaction between a given chemical species and the surface of the nanoparticles would also result in changes in the efficiency of the core electron-hole recombination[20]. This has been the basis of the increase in research activity on the development of novel optical sensors based on QD probes. Following this approach, Cd-based QDs have been widely reported for optical sensing of small molecules and ions. In some pioneering works, the enhancement of fluorescence were reported by L. Spanhel et al. when Cd ions was added to a basic aqueous solution containing unpassivated CdS nanoparticles without detectable changes in particle sizes[10]. Similar phenomenon was observed when Zn and Mn ions was introduced to colloidal solutions of CdS or ZnS QDs[20,21]. These photoluminescence-activation effect could be attributed to passivation of surface trap sites that either being “filled” or energetically moved closer to the band edges. Besides the activation effect, QD-based optical sensing quenching strategies has also been proposed. The mechanism of the quenching by the analyte that affects the luminescence emission of the nanoparticle was summarized as: (1) inner filter effects; (2) non-radiative recombination pathways; (3) electron-transfer processes and (4) -6- ion-binding interactions. These above four quenching mechanisms have been proposed and intensely studied in recent years to elucidate QDs based sensing. Electron transfer between semiconductor nanoparticles and organic molecules bound to their surface is a fundamental process that has been studied extensively in the recent years, especially for the creation of solar cells and optoelectronic devices[22]. When electron transfer occurs, the nanoparticle and its attached molecule exist in highly reactive charged forms long enough to interact with the surrounding environment[22]. The redox potential of the organic ligand can be chosen or modified to maximized the efficiency of charge transfer or to yield a radical of the desired reactivity able to oxidize the target molecule. Currently most work in this area was performed with TiO2 nanocrystallites[23,24]. But research in recent years has also established the same system with CdSe and CdSe/ZnS quantum dots: D. S. Ginger et al reported photoinduced electron transfer from conjugated polymers to CdSe nanocrystals[25]; C. Landes et al used n-butylamine as an acceptor to occupies hole sites, thus blocking the recombination process, which results in decreasing the density of luminescent centers[26]. J. A. Kloepfer and coworkers observed CdSe solubilized with mercaptoacetic acid emission quenched by a hole acceptor adenine[27]; in a most recent publication S. J. Clarke et al reported electron transfer between neurotransmitter dopamine and CdSe/ZnS QDs[22]; Maurel et al. reported a non-linear quenching effects of fluorescent quantum dots by nitroxyl free radicals TEMPO (2,2,6,6-tetramethylpiperidine-N-oxide free radical) and suggested the -7- mechanism would involve electron transfer from the conduction band to the nitroxide (a mild acceptor), and back electron transfer from the nitroxide to the valence band, effectively leading to quenching using the nitroxide SOMO as a shuttle for electron and hole[28]. Later, in another publication them utilized 4-amino-TEMPO (4-AT) bounding on QDs surface to create a as highly selective prefluorescent sensors for the detection of carbon-centered free radicals[29]. The potential for the use of QD-electron-donor systems as biosensors is nonetheless great, as electron transfer eliminates (“on-and-off” system )or activate (“off-and-on” system ) fluorescence from the particle, thus providing a visible signal of its occurrence. In this work, we used derivatives of antioxidant caffeic acid and Trolox to study the electron transfer between small molecule ligands and CdSe/ZnS quantum dots. 1.2.1 Ions and small molecules sensing Methods based on chemical or physical interactions between target chemical species and the surface of the nanoparticles are very simple. But those methods appear to be restricted to sensing just a few reactive small molecules or ions (Table 1.1). -8- Table 1.1 QD-based fluorescent probes for sensing small molecules and ions (refs. 1) Chen et al. explained the quenching of L-cysteine capped QDs by Fe(III) is attributed to an inner filter effect as a result of the strong absorption by Fe(III) at the excitation wavelength used. This interference caused by Fe(III) can be eliminated by adding fluoride ions to form a colorless complex FeF63-, which will also dissociate from the surface of the QDs due to same charge repulsions. Also, the quenching of thioglycerol capped CdS QDs by Cu(II) is through an electron transfer from thioglycerol to Cu(II). The reduction of Cu(II) to Cu(I) by thioglycerol, formed CdS+-Cu+ on the surface, which has a lower energy level than pure CdS QDs, therefore causing a red-shift of fluorescence. Moreover, Cu(I) quenches by facilitating non-radiative recombination of excited electrons in the conduction band and holes in the valence band[30]. On the other hand, fluorescence enhancement of QDs has been reported by Moore et al. in 2001[20]. The reversible fluorescence activation process caused by Zn(II) and -9- Cd(II) adsorption on the surface of CdS was studied. These ions enhanced the fluorescence intensity of QDs, was attributed to some form of passivation of surface trap states. In 2002, Chen et al. worked on Zn(II) determination and found the formation of a Zn-cysteine complex on the surface of cysteine capping QDs which is believed responsible for the activation of the surface states and therefore exhibiting the fluorescence enhancement[30]. Apart from intensive studies for potential use of QDs in sensing cations, functionalized QDs were also used for detection of inorganic anions although they are still in embryonic stages. In 2002, Watanabe et al. reported that a gold nanoparticle capped with amide ligands showed enhanced optical sensing of anions. The presence of anions would cause a marked decrease in extinction as a result of anion-induced aggregation of amide-functionalized gold nanoparticle via the formation of hydrogen bonding between the anions and the interparticle amide ligands. However, there was no selectivity and the binding affinity of anions was low[31]. In 2005, Jin et al. developed water soluble fluorescent CdSe quantum dots which are capped with 2-mercaptoethane sulfonate (MES) for the selective detection of free cyanide. Consequently, a slight blue-shift fluorescence quenching with detection limit of 1.1 x 10-6 M was observed. The blue-shift implies the changes in size or surface properties of MES-CdSe QDs which brings about the decrease in fluorescence[32]. However, the mechanism of fluorescence quenching was not discussed. - 10 - In the present paper, transition metal ions sensing are performed by tris(2-aminoethyl)amine (Tren) capped QDs. However, only the quenching pathways of selective determination of Co(III) and Cu(II) by using CdSe QDs which is capped by Tren ligands are thoroughly investigated. Furthermore, the Tren-QDs and transition metal Fe(II)complex hybrid materials are also utilized for sensing physiologically important hydrosulfide anion. 1.2.2 Hydrosulfide biology and its sensing Hydrogen sulfide is the most recent small endogenously generated species touted as a biological signal species[33]. Like CO, H2S is not a radical but has the apparent ability to interact with and disrupt/modulate the actions of other radicals. In various papers, hydrogen sulfide was reported to cause vasorelaxation in the vascular system[34] and enhance the vasorlaxant effect of · NO[35]. In the brain, H2S can have numerous effects, one of them is the ability to act as a neuromodulator enhancing N-methyl-D-aspartate (NMDA) receptor responses[36], which was reported to be related to cAMP production[37]. H2S also have antioxidant properties, protecting neurons from oxidative stress[38] which was due to its ability to raise the intrcellular glutathione (GSH) concentration by as much as twofold without increasing oxidized GSH levels, as well as increasing the levels of the GSH biosynthetic enzyme γ-glutamylcystein synthase. It has also been demonstrated that H2S can increase the ability for the antioxidant enzyme superoxide dismutase (SOD) to scavenge superoxide[39]. Hydrogen sulfide has a pKa of 6.8, making the anionic species the predominant form - 11 - under most physiological conditions[33]. Hence the detection of HS- anion is of great importance to biological and medical sciences. The highly sensitive detection of HS(as low as 125 ± 9.8 nM ) can be achieved by anion chromatography with ultraviolet detection (IC/UV) method[40,41], but a easy, simple and inexpensive quantitative detection method has not yet been reported and is still required development. 1.2.3 Aim and Objectives In this paper, electron transfer meditated QDs quenching were studied with thio-caffeic acid functionalized QDs system and Trolox diamine functionalized QDs. Tris(2-aminoethyl)amine (Tren) functionalized QDs system was also established for transition metal ions sensing. Selective determination of Co(III) and Cu (II) by using CdSe QDs which is capped by Tren ligands are thoroughly investigated. Furthermore, the Tren-QDs and transition metal Fe (II) complex hybrid materials are also utilized for sensing hydrosulfide anion (HS-). - 12 - References [1] J. M. Costa-Fernandez, R. Pereiro, and A. Sanz-Medel, Trends in Analytical Chemistry, 2006, 25, 207-218 [2] A.P. Alivisatos, Science (Washington, DC), 1996, 271, 933. [3] C.J. Murphy, J.L. Coffer, Applied Spectroscopy, 2002, 56,16A. [4] H. Weller, Angew. Chem. Int. Ed. Engl., 1993, 32, 41. [5] L.E. Brus, the Journal of Chemical Physics, 1986, 90, 2555. [6] C.B. Murray, D.J. Norris, M.G. Bawendi, Journal of the American Chemical Society,1993, 115, 8706. [7] J.E. Bowen-Katari, V.L. Colvin, A.P. Alivisatos, Journal of Physical Chemistry, 1994, 98, 411. [8] L.E. Brus, the Journal of Chemical Physics, 1983, 5566. [9] L.E. Brus, the Journal of Chemical Physics, 1984, 80,4403. [10] L. Spanhel, M. Haase, H. Weller, A. Henglein, Journal of the American Chemical Society, 1987, 109, 5649. [11] C.B. Murray, D.J. Norris, M.G. Bawendi, Journal of the American Chemical Society, 1993, 115, 8706. [12] S. Chaudhary, M. Ozkan, W.C.W. Chan, Applied Physics Letters, 2004, 84, 2925. [13] V. Colin, M.C. Schlamp, A.P. Alivisatos, Nature (London), 1994, 370, 374. [14] B.O. Dabbousi, J. Rodriguez-Viejo, F.V. Mikulec, J.R. Heine, H. Mattoussi, R. Ober, K.F. Jensen, M.G. Bawendi, Journal of Physical Chemistry, 1997, 101, 9463. [15] L. Qu, X. Peng, Journal of the American Chemical Society, 2002, 124, 2049. [16] R. E. Bailey, S. M. Nie, Journal of the American Chemical Society, 2003, 125, 7100. [17] M. Bruchez, M. Moronne, P. Gin, S. Weiss, A.P. Alivisatos, Science (Washington, DC), 1998, 281, 2013. [18] W.C.W. Chan, S.M. Nie, Science (Washington, DC), 1998, 281, 2016. [19] C. Querner, P. Reiss, J. Bleuse, and A. Pron, Journal of the American Chemical Society, 2004, 126 , 11574-11582 [20] D.E. Moore, K. Patel, Langmuir, 2001, 17, 2541. [21] K. Sooklal, B.S. Cullum, S.M. Angel, C.J. Murphy, Journal of Physical Chemistry, 1996, 100, 4551. [22] S. J. Clarke, C. A. Hollmann, Z. Zhang, D. Suffern, S. E. Bradforth, N. M. Dimitrijevic, W. G. Minarik, J. L. Nadeau, Nature Materials, 2006, 5, 409 - 417 [23] N. A. Anderson, and T. Lian, Annual Review of Physical Chemistry, 2005, 56, 491–519. [24] N. M. Dimitrijevic, Z. V. Saponjic, B. M. Rabatic, and T. Rajh, Journal of the American Chemical Society, 2005, 127, 1344–1345. [25] D. S. Ginger, and N. C. Greenham, Physical Review B, 1999, 59, 10622–10629 . [26] C. Landes, C. Burda, M. Braun, and M. A. El-Sayed, Journal of Physical Chemistry B, 2001, 105, 2981–2986. [27] J. A. Kloepfer, S. Bradforth, and J. L. Nadeau, Journal of Physical Chemistry B, 2005, 109, 9996–10003. - 13 - [28] M. Laferrie`re, R. E. Galian, V. Maurel and J. C. Scaiano, Chemical Communications, 2006, 257–259 [29] V. Maurel, M. Laferrie`re, P. Billone, R. Godin, and J. C. Scaiano, Journal of Physical Chemistry B, 2006, 110, 16353-16358. [30] Y. F. Chen and Z. Rosenzweig, Analytical Chemistry, 2002, 74, 5132-5138. [31] S. Watanabe, M. Sonobe, M. Arai, Y. Tazume, T. Matsuo, T. Nakamura, and K. Yoshida, Chemical Communications, 2002, 2866-2867 . [32] W. J. Jin, M. T. Fernandez-Arguelles, J. M. Costa-Fernandez, R. Pereiro, and A. Sanz-Medel, Chemical Communications, 2005, 883-885. [33] W. A. Pryor, K. N. Houk, C. S. Foote, J. M. Fukuto, L. J . Ignarro, G. L. Squadrito and K. J. A. Davies, American Journal of Physiology, 2006, 291, R491-R511. [34] W. M.. Zaho, J. Zhang, Y. J. Lu, and R. Wang, EMBO J 20: 2001, 6008-6016 [35] R. Hosoki, N. Matsuki, and H. Kimura, Biochemical and Biophysical Research Communications, 1997, 237, 527-531. [36] R. Wang, FASEB J, 2002, 16, 1792-1798. [37] H. Kimura, Biochemical and Biophysical Research Communications, 2000, 267, 129-133. [38] Y. Kimura and H. Kimura, FASEB J, 2004, 18, 1165-1167. [39] D. G. Searcy, J. P. Whitehead, and M. J. Maroney, Archives of Biochemistry and Biophysics, 1995, 318, 251-263. [40] T. F. Rozan, and G. W. Luther, Marine Chemistry, 2002, 77, 1 – 6. [41] B. R. McCord, K. A. Hargadon, K. E. Hall and S. G. Burmeister, Anatytica Chimica Acta, 1994,288, 43-56. - 14 - Chapter 2 Thiolated Caffeic Acid Functionalized Quantum dots 2.1 Introduction Semiconductor nanocrystals (or quantum dots, QDs) have size-dependent fluorescent emissions wavelengths with high quantum yields and superior photostability comparing to organic dyes[1]. Thus QDs have great potential in various biological imaging applications, including labeling cells and other biological components. However, the prerequisite for the development of QD-based bioimaging systems is to gain access to photostable, and biocompatible nanocrystals. To achieve that, quantum dots synthesis and coating usually followed by surface modification as a preferred route for obtaining highly fluorescent water-soluble QDs[1]. Among the various techniques for surface modification, two general methods are most widely used: (1) Ligand exchange of hydrophobic surfactant molecules for bifunctional linker molecules (2) phase-transfer methods using amphiphilic molecules that act as detergents for solubilizing the QDs coated with hydrophobic groups[1]. Electron transfer between semiconductor nanocrystal and organic molecules bond to their surface has been studied extensively in recent years[2]. This kind of charge transfer has already been established with CdSe and ZnS coated CdSe quantum dots (CdSe/ZnS QDs) to create a sensor of changes in redox potential[2-5]. A suitable - 15 - ligand is the key to fabrication of such a sensor. Caffeic acid (3,4-dihydroxycinnamic acid) is a natural phenolic compound, which is reported to exist widely in vegetable and coffee products, and presents as a degradation product of chlorogenic acid [6-8] . Some pharmacological properties of this substance have been described previously. For example, it has a strong and specific inhibitory activity towards 5-1ipoxygenase, and can inhibit platelet aggregation and thromboxane biosynthesis [9-12]. Among the hydroxycinnamates, caffeic acid retains the structural features that maximize radical-scavenging activity in flavonoids, which is predictive of higher rate constants toward several types of oxidant species, as shown in Table 2.1[14]. It was argued that caffeic acid is the “active site ” of flavonoids for radical scavenging in particular flavones and flavonols. The o-dihydroxy group is typically the radical target site, which after one-electron oxidation produces a phenoxyl radical (o-semiquinone). Accordingly, the caffeic acid-derived o-semiquinone radical has been observed by electron paramagnetic resonance (EPR) after reaction of the phenolic acid with several oxidants, including peroxynitrite and ferrylmyogloben [15]. Even so, a recent study on the scavenging activity of caffeic acid derivatives against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical indicated that a saturated lateral group in the aromatic ring had a slightly higher inhibitory activity when compared with an unsaturated group [16]. - 16 - Oxidant Product/rate constant ROO. O2.OH. 1.5×107 M-1s-1 0.96×106 M-1s-1 3.24×109 M-1s-1 ONOO- Quinone a b b 5 -1 -1 (for chlorogenic1.6×10 M s ) . NO2 MbFeIV=O 8.6×10 M s b b MbFeIII Semiquinone radical c 8 9 -1 -1 -1 -1 N2O3 HOCl o-Quinone of chlorogenic 1 5.1×106 M-1s-1 O2 8.6×10 M s d e Table 2.1 Scavenging properties of caffeic acid (or Its Ester Derivative, Chlorogenic Acid) (refs.14) a Kinetic chemiluminescence in chlorobenzene at 50℃ and peroxyl radical derived from diphenyl methane, b Pulse radiolysis, c MbFeIV=O, ferrylmyoglobin; MbFeIII, metmyoglobin, d Competition spectrophotometric assay, e Time-resolved infrared phosphorescence in acetonitrile. It was claimed that the higher radical scavenging activities of caffeic acid can be ascribed to oxidative dimerization or even higher degrees of polymerization through which oxidizable -OH moieties are reproduced in the “oxidation” products after electrochemical studies[17 –19]. And it was also assumed that the oxidation of caffeic acid involves formation of dimer by a coupling reaction of the semiquinone radical as an intermediate of one-electron oxidation. Although various dimers of caffeic acid are reportedly formed by chemical autooxidations [8, 9], the electrochemical oxidation products could not be isolated despite considerable effort [17, 18], because the dimer(s) was susceptible to further polymerization. Caffeic acid is water soluble and vulnerable to free radicals, which indicate - 17 - possibility for this compound to serve as a quantum dot ligand. But the bounding between carboxyl group of caffeic acid and quantum dot surface was proved to be difficult by our preliminary ligand exchange experiments. Thiol compounds were reported to have the strongest affinity towards CdSe nanoparticles[19] although their has a major drawback: instability towards oxidation. The surface thiol ligands were found to undergo a photocatalytic oxidation using CdSe nanocrystals as photocatalysts and form the disulfides during the process. But severity of this oxidation varies with different thiol ligands, and some of them can sustain long enough in solution for practical applications[19]. Another disadvantage of Thiol ligands is that it can cause severe fluorescence quenching of quantum dots after ligand exchange process, but ZnS coating of quantum dots provides significant protection to QDs fluorescence against this quenching[20]. Except few reports on synthesis of thiol ester, so far has no literature report on thiolation of caffeic acid[21]. If we can synthesize thio-caffeic acid, it might be a good ligand with strong ability bounding to CdSe/ZnS surface, but whether CdSe/ZnS QDs can be stabilized well by thio-caffeic acid is yet to be found out. In addition, through ligand exchange process, we hope to establish a donor-receptor system between CdSe/ZnS QDs and thiolated caffeic acid. The quantum dots capped with thio-caffeic acid should be soluble in water or other polar solvent and still possess acceptable level of fluorescence. As electron transfer quenches or enhances fluorescence of quantum dot by interfering the efficiency of the CdSe core - 18 - electron-hole recombination, this “donor-receptor” caffeic acid-QDs system can provide a visible signal when it reacts with free radicals or other species and serves as a fluorescent probe. Our goal is to synthesize and purify thio-caffeic acid for quantum dot ligand exchange and try to replace the original hydrophobic trioctylphosphine oxide (TOPO) ligand of quantum dots with thio-caffeic acid. 2.2 Experimental Section 2.2.1 Materials and instruments All solvents used were of reagent grade unless otherwise specified. The tetrahydrofuran (THF) used in synthesis was dried over Na and distilled under N2. Caffeic acid, 4-dimercapto-2,3-butanediol (DTT) and dicyclohexylcarbodiimide (DCC) were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). 1 H and 13 C{1H} NMR spectra were recorded in deuterated methanol with a Bruker AC300 spectrometer (Karlsruhe, Germany) at 300 and 75 MHz, respectively. The electrospray ionization mass spectra were obtained from a Finnigan / MAT LCQ ion trap mass spectrometer (San Jose, CA, USA) equipped with an electrospray ionization (ESI) source. The heated capillary and voltage were maintained at 250 ℃ and 4.5 kV, respectively. The full-scan mass spectra from m/z 100 to 1000 were recorded. The samples were dissolved in methanol and the solution was introduced into the ion spray source with a syringe (100 μL). - 19 - 2.2.2 Synthesis of caffeic acid derivatives OH OH OH OH OH DCC OH 2 O O OH Caffeic Acid Fw: 180.16 O O Caffeic Acid Anhydride Fw: 342.30 Scheme 2.1 Synthetic route of caffeic acid anhydride Caffeic acid (1.000g, 5.5mmol) and dicyclohexylcarbodiimide (DCC) (0.453g, 2.2 mmol) were added into two schlenk flasks which were connected to a schlenk line, respectively. Freshly distilled tetrahydrofuran (THF) (20mL) was added into the two flasks to dissolve caffeic acid and DCC, respectively. The DCC solution was added dropwise into caffeic acid solution under vigorous stirring. The reaction mixture was kept stirring for 12 hours. The product mixture was later separated by filtration. The caffeic acid anhydride in filtrate was kept for further use (Scheme 2.1). The anhydride was directly used in next step of synthesis without characterization, but in unpurified reaction mixture it could be identified as a peak at 341 in ESI-MS negative model (Figure 2.2). The 4-dimethylaminopridine hydrosulfide salt precursor was prepared according literature reports. Freshly distilled THF (30 ml) was added into the a three-neck flask to dissolve 4-dimethylaminopridine (DMAP) 0.46 g (3.8 mmol). And the DMAP - 20 - solution was cooled in an ice bath. H2S gas was produced by dropping phosphoric acid on solid NaHS and dried over P2O5. The dried H2S gas was then bubbled through the cooled solution for 2 hours. The reaction was stopped when a slightly yellow color appeared in the reaction mixture (Scheme 2.2). N N N N H+ HS- H2S + Scheme 2.2 Preparation route of 4-dimethylaminopridine hydrosulfide salt The caffeic acid anhydride THF solution was added into the ice-cooled sulfur precursor solution and stirred for 3 hours. The temperature of cooled mixture was naturally increased to room temperature. Finally, a yellow precipitate appeared in reaction mixture, which can be separated by filtration. The precipitate is kept for characterization and further purification (Scheme 2.3). OH OH OH OH OH OH OH OH + M HS O O O C affeic Acid A nhydride O SH Thio-caffeic A cid O OH C affeic Acid Scheme 2.3 Synthetic route of thio-caffeic acid salt - 21 - 8 2.5x10 8 2.0x10 8 1.5x10 PPT_Positive 123 HN N Relative Abundance 8 1.0x10 7 5.0x10 0.0 8 1.0x10 100 150 200 8.0x10 7 6.0x10 135 161 150 350 400 195 341 359 200 250 300 Filtrate_Negative 7 1.2x10 6 9.0x10 350 400 385 O N OH C O 6 6.0x10 6 3.0x10 0.0 100 300 179 7 4.0x10 7 2.0x10 0.0 7 1.5x10 100 250 PPT_Negative 7 OH NH N-caffeic acid acyl DCC urea Fw: 386.48 150 200 250 300 350 400 m/z Figure 2.1 ESI-MS of thio-caffeic acid reaction mixture In ESI-MS spectra of this precipitate, thio-caffeic acid was corresponded by peak 195 in negative mode and coexist with caffeic acid peak 179 (Figure 2.1). - 22 - OH OH OH OH N S N H+ + Dissolved in H2O un-identified impurities + N 600 ml N H+ O pH = 6.1 measured O O Filtration Precipitated products OH OH N + + + HCl solution Transparent yellow solution pH = 3.2 pH = 6.5 OH OH N Cl H+ O SH OH Caffeic Acid Fw: 180.16 O Thio-caffeic Acid Fw: 196.22 Extraction with Diethyl ether OH OH OH O OH HO + + OH OH OH OH S SH O O S Fw: 390.43 O Figure 2.2 Separation and purification of thio-caffeic acid As illustrated in Figure 2.2, the precipitate obtained from last step was added into 600mL deionized water. Sodium hydroxide solution (0.1M) was added into the mixture to adjust its pH to 6.5. Then the mixture was filtrated to afford a transparent yellow solution. Hydrochloric acid (0.1 M) was added to adjust solution pH to 3.2. The solution was then extracted with 500mL diethyl ether. The organic phase turned yellow color after extraction and it was washed 5 times with deionized water. After that the organic phase was then collected and rotary evaporated, the yellow colored solid residue was kept under nitrogen. The extraction process was repeated 3 times. It was later confirmed that water phase mainly contained caffeic acid and organic phase contains thio-caffeic acid and impurities (Figure 2.3). And further purification was able to increase the thio-caffeic acid ratio in the mixture (Figure 2.4). - 23 - 6 2.0x10 179 6 Water phase 1.6x10 6 1.2x10 5 8.0x10 201 5 4.0x10 0.0 7100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 2.0x10 Organic phase 7 1.6x10 179 7 1.2x10 161 6 8.0x10 195 135 6 4.0x10 389 341 0.0 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 m/z Figure 2.3 ESI-MS (negative ) of thio-caffeic acid extraction 195 7 1.0x10 6 8.0x10 Relative Abundance 375 6 6.0x10 179 6 359 4.0x10 391 161 6 2.0x10 0.0 100 200 300 400 m/z Figure 2.4 ESI-MS (negative) of purified thio-caffeic acid - 24 - 2.2.3 Quantum dots ligand exchange with thio-caffeic acid The 100 nM quantum dots solution in 2mL chloroform and then added into 1mL 100 times equivalent amount of thio-caffeic acid diethyl ether solution dropwise. This mixture was vigorously stirred under nitrogen for 12 hours at room temperature in darkness. When the reaction was stopped, it was found the quantum dots precipitated out from solution. The quantum dots solid was centrifuged and collected. It was found this solid immiscible in any polar or non-polar solvent. 2.3 Result and Discussion The reaction between caffeic acid and dicyclohexylcarbodiimide (DCC) would form an intermediate after addition of reagents(Scheme 2.4). The caffeic acid-DCC intermediate is crucial to the following steps of synthesis. This intermediate would swiftly converted to desired product caffeic acid anhydride as well as another by-product N-acyl urea (Scheme 2.5). The two products were separated by filtration and the solid was conformed to be by-product. The anhydride formed from the intermediate stayed in solution (Scheme 2.6) and would be used in the next step of synthesis. - 25 - OH OH OH OH N + C N Fast O O OH O H N C N Intermediate Scheme 2.4 Formation of intermediate OH OH O N By-product reaction O THF O OH C O OH NH H N C N N-acyl urea Fw: 386.48 Intermediate Scheme 2.5 Formation route of by-product N-acyl urea OH OH OH OH OH OH + Desired reaction C O NH + Caffeic acid O NH O O H N C N O O Caffeic acid anhydride Intermediate DCC-urea; precipitate Fw: 342.30 Fw: 224.34 Scheme 2.6 Synthetic route of caffeic acid anhydride The instability of caffeic acid was a big problem we encountered during synthesis. - 26 - Caffeic acid has a pKa1 = 4.43±0.02 and pKa2 = 8.69±0.03[22]. It was found that caffeic acid tends to oxidize by oxygen in the atmosphere if environmental pH is above 7.0. As Figure 2.7 shows, the o-dihydroxy group would be easily oxidized to o-quinone. Not to mention the caffeic acid anhydride is extremely sensitive to water. For this reason, the synthetic process of caffeic acid anhydride was carried out under N2 and freshly distilled and dried THF was chosen as solvent. HO O O O pH > 7.0 HO O O2 O O Scheme 2.7 Oxidation of caffeic acid The synthesis was started with the formation of hydrosulfide anion donor. The commercially available sodium sulfide and sodium hydrosulfide were tried out, but their poor solubility in anhydrous THF was a serious problem in quantitative synthesis. This problem caused extremely slow reaction rate and low yield of thio-caffeic acid and also made experiment results not reproducible. Hence, 4-dimethylaminopridine (DMAP) hydrosulfide salt was prepared to serve as an alternative hydrosulfide anion donor (Scheme 2.2). The caffeic acid anhydride THF solution was added into the ice-cooled sulfur precursor solution and the temperature of cooled mixture was slowly increased to room temperature. A yellow precipitate appeared in reaction mixture and it was separated by filtration. The filtrate, which contained mainly by-product was discarded. From the ESI-MS result of the precipitates, 3 main components were - 27 - identified: caffeic acid (M.W.=180), thio-caffeic acid (M.W.=196) and unreacted caffeic acid anhydride (M.W.=342). The precipitate is kept for further purification (Figure 2.2). As Scheme 2.4 illustrated, the precipitates obtained from above steps were first dissolved in water and the pH was adjusted to 6.5 (below caffeic acid pKa2 and above pKa1). The mixture was filtered and the solid was discarded. Hydrochloric acid was added into the filtrate to adjust the pH to 3.2. Then the solution was extracted with diethyl ether. It was later confirmed that water phase mainly contained caffeic acid and unidentified impurities (Figure 2.3). The organic phase turned yellow color after extraction and it was washed 5 times with water. After that the organic phase was then collected and rotary evaporated, the yellow colored solid residue was kept under nitrogen. The extraction process was repeated 3 times to afford even purer thio-caffeic acid (before server oxidation took place), which is analyzed by ESI-MS and results are demonstrated in Figure 2.4. Caffeic acid and thio-caffeic acid are similar in chemical structure. Hence their solubility and polarity are also quite similar, which makes separation of pure thio-caffeic acid a tricky task. Furthermore, the amount of caffeic acid in the mixture was in largely excess comparing to the amount of thio-caffeic acid which makes purification even more difficult. The silica gel column chromatography separation method was taken into consideration, but later it was found that Rf value of the two - 28 - compound was too close for this kind of separation. And the vulnerability of thio-caffeic acid to oxygen also made this method impossible. Finally, we took advantage of difference in pKa of these two compounds. Caffeic acid has a pKa1 at 4.35 and its thiolated derivative has a pKa around 3.3. Although the two values are close, a progressive separation was proved possible. When we dissolved the mixture containing caffeic acid and thio-caffeic acid into water and adjusted its pH to 3.2, most of caffeic acids were not able to ionize and stay in solution in acid form. Thio-caffeic acid at this point was partially ionized and was safe from oxidation at this pH value. The mixture was first filtered and the filtrate was extracted with diethyl ether. The organic phase was separated and washed with water. The caffeic acid, which is more soluble in water (both ionized and unionized forms), was washed off by water repeatedly to afford a product with higher thio-caffeic acid ratio. Initial quantum dots ligand exchange was carried out with the thio-caffeic acid mixture. The quantum dots were dissolved in chloroform and then added into thio-caffeic acid diethyl ether solution (100 times equivalent amount) dropwise. This mixture was vigorously stirred under nitrogen for 12 hours at room temperature. When the reaction was stopped, it was found the quantum dots precipitated out from solution and was not soluble in any polar or non-polar solvent. - 29 - The ligand exchange step of thio-caffeic acid was not as successful as we expected. The possible reasons are: The ligand was oxidized or photooxidazied during the exchange process and formed disulfide dimmer. According to literatures, the disulfides would form micelle-like structure around the particles and kept them soluble in the solution at beginning. As the oxidation products diffused out of the ligand shell instead of surrounding the CdSe core, the quantum dots would gradually became smaller. At a critical point, the small CdSe inorganic core could not be supported by micelle-like ligand anymore and the whole system would become unstable and begin to precipitate out[19]. The thio-caffeic acid was more unstable than the caffeic acid because thio-caffeic acid has a lower pKa than caffeic acid which is around 3.3. If the environmental pH value is above 3.3 thio-caffeic acid tends to form disulfide dimmer even without photooxidation (Scheme 2.8). This dimmer formation could be protected by addition of 4-dimercapto-2,3-butanediol (DTT), which confirmed its nature of oxidation (Figure 2.5). With the addition of quantum dots solution, it was highly possible the oxidation process was photocatalized. And free thio-caffeic acid was converted to disulfide dimmer instead of bounding to CdSe/ZnS quantum dots surface. OH OH OH O2 2 OH OH HO pH = 5.5 O O SH O S S Fw: 390.43 - 30 - Scheme 2.8 Oxidation of thio-caffeic acid 6 3.5x10 6 3.0x10 6 2.5x10 OH OH OH HO 6 2.0x10 6 1.5x10 O O 161 389 S S Fw : 390.43 6 1.0x10 5 179 5.0x10 0.0 6100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 3.5x10 6 3.0x10 153 DTT 189 6 2.5x10 OH OH 6 195Thio-caffeic Acid 2.0x10 6 1.5x10 6 1.0x10 161 O S O O 193 O 5 5.0x10 S 0.0 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 m/z Figure 2.5 ESI-MS result of DTT protective effect on thio-caffeic acid; Above: without addition of DTT; Below: with addition of DTT The quantum dots ligand exchange with thio-caffeic acid needs further quantitative studies. It was reported by Peng et al.[19] that the amount of free thiol ligands is an important factor to the success of thiol ligand exchange. In this report, authors proposed that excessive thiol ligand could protect QDs by replacing the oxidized ligands so that they can keep quantum dot soluble in the solution for a longer period of time. And if there was insufficient free thiols in solution, quantum dot may not remain soluble through the exchange process. It is possible the amount of thio-caffeic acid we used in the preliminary studies was not sufficient for nanocrystals to survive from oxidation. Since precise control of amount of ligand is the key to make exchange process reproducible, the amount of thio-caffeic acid - 31 - requires further study and determination before anymore QDs exchange is conducted. Another factor that affects oxidation of quantum dot is reaction time. Many thiol ligand functionalized quantum dot has limited photochemical stability. From the literature report, the optical density of 3-mercaptopropionic acid functionalized quantum dots could start to change within 120 minutes[20]. For this reason, the experimental condition of thio-caffeic acid ligand exchange could be optimized and deduct the 12 hours reaction time to an acceptable level before serious oxidation of QDs or ligand occurs. It was also reported that the diffusion of oxygen was the rate-determining step for the photooxidation of quantum dots. Because the thiol ligand monolayer could serve as a barrier for the oxygen diffusion process[20]. Hence the quantum dots stability was closely related to packing of respective ligand. According to Peng et al., aromatic thiols can not stabilize quantum dots as good as aliphatic thiols with a similar length dimension because they can not pack as densely as the latter and the diffusion of oxygen is much easier through the loosely packed ligand shell. In this aspect, thio-caffeic acid may not be a good ligand to stabilize quantum dots in aqueous or polar solution[19]. However, other studies suggested carbodithiolic acid can be used to functionalized quantum dots regardless aromatic part in vicinity[20]. If we can modify caffeic acid with this carbodithiolic structure, maybe caffeic acid derivative could still be used as - 32 - a ligand to functionalize quantum dots or extend its use in other fields. 2.4 Conclusion By bounding antioxidant caffeic acid sulfide analogue to CdSe/ZnS quantum dots surface, we hope to create hydrophilic quantum dots which are also sensitive to oxidants like free radicals. The thiolization of caffeic acid, which has never been reported in literatures, was explored. The synthetic process, including formation of the caffeic acid-DCC intermediate and caffeic acid anhydrate was studied to provide information for further separation. The synthesis method was then optimized, which includes preparation of a new hydrosulfide anion donor. The pH-dependent thio-caffeic acid oxidation was also investigated by ESI-MS. The vulnerability of thio-caffeic acid to oxygen made it difficult for separation and further application. Hence the purification of thio-caffeic acid with water was stopped before serious oxidation occurred. Preliminary CdSe/ZnS quantum dots ligand exchange with thio-caffeic acid in chloroform was also carried out. The negative result of ligand exchanged step suggests the oxidation of thio-caffeic acid must be reduced before successful ligand exchange with CdSe/ZnS quantum dots. Possible reasons for this unsuccessful result was proposed and further studies on quantum dot ligand exchange using thio-caffeic acid is still underway. - 33 - References [1] R. E.Bailey, A. M.Smith, S. Nie, Physica E, 2004, 25, 1-12 [2] S. J. Clarke, C. A. Hollmann, Z. Zhang, D. Suffern, S. E. Bradforth, N. M. Dimitrijevic, W. G. Minarik, J. L. Nadeau, Nature materials, 2006, 5, 409-417 [3] D. S. Ginger; N. C. Greenham, Physical Review B, 1999, 59, 10622-10629 [4] A. J. Nozik, Physica E, 2002, 14, 115-120 [5] C. Landes, C. Burda, M. Braun, and M. A. El-Sayed, Journal of Physical Chemistry B, 2001, 105, 1981-1986 [6] T. P. Cameron, T. J. Hughes, P. E. Kirby, K. A. Palmer, V. A. Fung, V. C. Dunkel, Mutation Research/Genetic Toxicology, 1985, 155, 17-25. [7] H. Hsu, Y. Chen and M. Hong, The Chemical Constituents of Oriental Herbs, 1982, 1, 264 [8] A. Nahrstedt, M. Albrecht, V. Wray, H. G. Gumbinger, M. John, H. Winterhoff, F. H. Kemper, Planta Medica, 1990, 56,395. [9] M. Sugiura; et al, Chemical and Pharmaceutical Bulletin, 1989, 37, 1039. [10] H. Cho, M. Ueda, M. Tamaoka, M. Hamaguchi, K. Aisaka, Y. Kiso, T. Inoue, R. Ogino, T. Tatsuoka, and et al., Journal of Medicinal Chemistry, 1991, 34, 1503. [11]T. Okuyama, K. Fujita, S. Shibata, M. Hoson, T. Kawada, M. Masaki, N. Yamate, Planta Medica, 1986, 52, 171 [12] W. C. Chang and F. L. Hsu, Prostaglandins Leukotrienes and Essential Fatty Acids, 1992, 45, 307. [13] Y. Koshihara, et al, FEBS Letter, 1982, 143, 13. [14] L. Packer, E. Cadenas; Handbook of Antioxidants, second edition, Marcel Dekker, New York, 2002 [15]J. Laranjinha; E. Cadenas; IUBMB life, 1999, 48, 1-9 [16]CP. Chen,; et al, Experimental and Toxicologic Pathology, 1999,51,59-63 [17]Y. S. Uang; et al., J. Chromatogr. B ,1995, 673, 43-49 [18] R. Arakawa, M. Yamaguchi, H. Hotta, T. Osakai, T. Kimoto, Journal of the American Society for Mass Spectrometry, 2004, 15, 1228–1236 [19] J. Aldana, Y. A. Wang, and X. Peng, Journal of the American Society,2001,123,8844 [20] J. A. Kloepfer, S. E. Bradforth, and J. L. Nadeau, Journal of Physical Chemistry B, 2005,109, 9996-10003 [21] A. R. Kroon, W.D. Hoff, H. P.M. Fennema, J. Gijzen, G. Koomen, J. W. Verhoeven, W. Crielaard, and K. J. Hellingwerf, The Journal of Biological Chemistry, 1996, 271, 31949-31956 [22] IUPAC, Ionization Constants of Organic Acids in Aqueous Solutions, Pergamon Press, Oxford, 1979. - 34 - Chapter 3 Trolox Functionalized Quantum Dots 3.1 Introduction 3.1.1 Quantum dots and its synthesis Quantum dots (QDs) are spherical semiconductor nanoparticles that composed of atoms from groups II-VI or III-V elements in the periodic table, such as CdSe, ZnTe, GaAs and InSb. With quantum confinement effect, QDs present considerable advantages over traditional organic dyes. Their unique optical and electronic properties, including broad excitation spectra with high molar absorptivities, size-tunable emission properties, narrow and symmetric emission and comparatively bright and photostable properties[1]. For routine preparation, the CdSe core is usually capped with an organic layer such as trioctylphosphine/trioctylphosphine oxide (TOP/TOPO). This layer coordinates to Cd sites and stabilizes QD’s surface, preventing aggregation of the nanocrystals[2]. Since the ligands are hydrophobic, they make the QDs incompatible with aqueous solution and did not appear to have an immediate application for analytical system. Two breakthrough papers by Nie’s and Alivisatos’ groups in 1998 demonstrated the fluorescent QDs can be made water-soluble and biocompatible by surface modification and bioconjugation while maintaining their large fluorescent quantum yield[3,4]. Hence, by tailoring QDs with different capping ligands, they can serve for - 35 - different purposes. In the following years, there are two approaches to surface modification to obtain highly fluorescent and water-soluble QDs. Firstly, the formation of a layer of another semiconductor of higher band gap on the QDs surface such as ZnS, giving rise to a bi- or three-layered structure QD resulting in highly fluorescent QDs[5]. Another approach is to use competing capping agent such as mercaptosulphonic acid or dihydrolipoic acid to displace TOP/TOPO from the QDs surface and endow it solubility in polar solvents[6, 7]. 3.1.2 Quantum dots ligand exchange strategies The nature of the ligands being coordinated to the QDs surface and the particular type of bonds which it forms with the nanocrystal surface atoms are of great importance to quantum dots researchers. By ligand designing and fabrication, several important properties of quantum dots can be tuned for specific purpose: processibility, reactivity and stability. All of these have direct consequences on quantum dots’ spectroscopic properties[8]. Three principal functions of the surface ligands can be described by Querner et al. as follows: (1) They prevent individual colloidal nanocrystals from aggregation. (2)They facilitate nanocrystals’ dispersion in a large variety of solvents. In the presence of surface ligands, the ability to disperse nanocrystals is governed by the difference between the ligand and the solvent solubility parameters, which can be precisely tuned. (3) Ligands containing appropriate functional groups may serve as bridging units for the coupling of molecules or macromolecules to nanocrystals or their grafting on substrates[8]. - 36 - So far we have reported quantum dot ligand exchange with thio-caffeic acid. The ligand exchange step of thio-caffeic acid was not as successful as we expected because the thiol ligand was oxidized or photooxidazied during the exchange process and formed disulfide dimmer. According to Peng et al., the disulfides would form micelle-like structure around the particles and kept them soluble in the solution at beginning. As the oxidation products diffused out of the ligand shell instead of surrounding the CdSe core, the quantum dots would gradually became smaller. At a critical point, the small CdSe inorganic core could not be supported by micelle-like ligand anymore and the whole system would become unstable and begin to precipitate out[9]. Since thiols suffer from their major drawback, i.e. instability towards oxidation. Their alternatives was soon synthesized and applied to quantum dots ligand exchange. Alkyl or aryl derivatives of carbodithiolic acids (R-C(S)SH) were reported by Querner et al. in their publication. This family of ligands for CdSe nanoparticle surface functionalization has two main advantages comparing to thiols: they nearly quantitatively exchange the initial surface ligands (TOPO) in very mild conditions; they significantly improve the resistance of nanocrystals against photooxidation because of their ability of strong chelate-type binding to metal atoms[8]. However, one problem of this chelating-type ligands exchange method remains: a specific and adapted synthesis is required each time new ligands are needed. An improved method was proposed by Zhao et al.[10], by using dithiocarbamate - 37 - compounds they successfully assembled secondary amine containing ligands on Au surface. Recently this method was adapted by Dubois et al.[11], and it was used on CdSe/ZnS quantum dots for ligand exchange. Depending on the intrinsic nature of substituents borne by the amino group, different amine ligands were chelated to QDs surface and different properties were conferred to nanocrystals, such as solubility in different solvents. With these recent developments of quantum dots surface modification techniques, it would be possible for rational design and tailoring quantum dots’ ligand for specific purpose and application, such as biological sample labeling and radical sensing. The α-tocopherol (α-TOH), as a principal scavenger of peroxyl radicals in biological membranes, is probably the most important inhibitor of the free-radical chain reaction of lipid peroxidation in animal. Antioxidant properties of α-TOH appear to play a critical role in preventing oxidative injury by toxic and carcinogenic chemicals[12]. Research shows the lipid antioxidant activity of α-TOH comes from its preferential localization within the lipid, its high chemical reactivity toward radicals and the stability of the phenoxyl radical intermediate (α-TO.) generated[13]. Tocoherols inhibit lipid peroxidation largely because they scavenge lipid peroxyl (LO·2) radicals much faster than these radicals can react with adjacent fatty acid side-chains or with membrane proteins. Rate constants for the following reaction α-TOH + LO· α-TO· + LO2H are about 106M-1s-1, which is four orders of magnitude faster than those for reaction - 38 - of LO·2 radicals with lipids(~102M-1s-1) [13] . In addition, tocopherols both quench and react with singlet O2 and might protect membranes against this species. - α-tocopherol reacts slowly with O·2 and, like most other biological molecules, at an almost diffusion-controlled rate with OH·. The α-TO· radical might also react with a further peroxyl radical to give non-radical products [14]. α-TO· + LO2 · α-TOOOL That means theoretically one molecule of α-tocopherol is capable of terminating two peroxidation chains (Scheme 3.1). Products of the above reaction include eight α-substituted tocopherones and epoxy (hydroperoxy) tocopherones: the former would readily hydrolyze to tocopherylquinone and the latter to epoxyquinones[14]. Scheme 3.1 Peroxyl radical scavenging by α-TOH and α-TO· (refs 15) It has been shown that α-TO. radical reacts with peroxyl radical to give a peroxo adduct at the para 8a-position. It may react with another α-TO. radical to give a dimer. This α-TO. radical may be reduced by a reductant (e.g. ascorbic acid, uric acid, ubiquinol and polyphenolic compounds) to regenerate α-TOH. In some cases, this radical may react with the substrate and/or hydroperoxide to give active radicals, - 39 - (which may start a chain reaction) and acts as a chain-transfer agent[12]. The rate constants of α-TO. were obtained by E. Niki et al by a stopped-flow ESR method [12] (together with the biological concentrations of the substrates) are showed blow: Table 3.1 The rate constants of α-tocopheryl radical (obtained by a stopped-flow ESR method) and the biological concentrations of the substrates(refs 12) From Table 3.1, it is clear that α-TO. is sensitive to several biologically important · species but reacts most efficiently with LOO radical. 3.1.3 Electron transfer based sensor By bonding α-tocopherol analogue to quantum dots surface as ligand, we have reasons to expect a photoinduced electron transfer (PET) between this ligand and quantum dots excited state, which cause intramolecular emission quenching. This kind of quenching have already been observed by Oleynik et al. when they coupled a α-tocopherol analogue--Trolox (5,7,8-tetramethylchroman-2-carboxylic Acid) to a dipyrrometheneboron difluoride (BODIPY) fluorescent probe[15]. And trough radical-mediated oxidation of the - 40 - chromanol head (Trolox part), the Trolox-BODIPY complex significantly restored fluorescent emission by10-fold. The same kind of “off-and-on” phenomenon was also observed by Maurel et al. [16] . Their research showed the QDs capped by 4-amino-TEMPO (QD-4AT) which was initially quenched by the nitroxide in a QD-4AT complex and the fluorescent emission was readily restored when nitroxide moiety react with carbon-centered free radicals to form alkoxyamines. By synthesizing Trolox-QDs complex, which has never been reported before, we hope to establish a “off-and-on” system for sensitive peroxyl radical sensing. But a remaining problem is that Trolox’s carboxyl group can not directly bond to CdSe/ZnS quantum dots surface as it coupled to BODIPY. By modifying this carboxyl group to a amide or amino group, we can easily adopt the method developed by Dubois and coworkers, and take advantage of strong chelating ability of carbodithioate group after CS2 addition[11]. And finally make a strong bonding of Trolox on QDs’ surface. The ligand exchanged quantum dots would be applied for peroxyl radical sensing. 3. 2 Experimental Section 3.2.1 Materials All solvent used were of reagent grade unless otherwise specified. TOPO CdSe/ZnS quantum dots and Tren-CdSe/ZnS QDs were synthesized by Dr. Wang Shuhua in our research ((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic group. acid) Trolox and - 41 - 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) were purchased from Sigma-Aldrich Chemical 2,2'-azobis(2,4-dimethylvaleronitrile) Company (St. Louis, MO, USA). (AMVN) was purchased from cayman Cayman Chemical Company(Ann Arbor, MI, USA). Hydrochloric acid and sodium bicarbonate anhydrous were purchased from Fisher Scientific (Fair lawn, NJ, USA). Ethylenediamine were purchased from J.T. Baker (Phillipsburg, NJ, USA). Carbon Disulfide were purchase from BDH limited (Poole England). 1 H and 13 C{1H} NMR spectra were recorded in deuterated methanol with a Bruker AC300 spectrometer (Karlsruhe, Germany) at 300 and 75 MHz, respectively. The electrospray ionization mass spectra were obtained from a Finnigan / MAT LCQ ion trap mass spectrometer (San Jose, CA, USA) equipped with an electrospray ionization (ESI) source. The heated capillary and voltage were maintained at 250 ℃ and 4.5 kV, respectively. The full-scan mass spectra from m/z 50 to 1000 were recorded. The samples were dissolved in methanol and the solution was introduced into the ion spray source with a syringe (100 μL). UV-Vis spectra were recorded using a Shimadzu UK1601 spectrophotometer fitted with a quartz cell and Bio-TEK Synergy HT Plate Reader (Winnoski, Vermont, USA). Fluorescence measurements were performed using a Perkin–Elmer LS55 luminescence spectrometer with an excitation wavelength of 400 nm. FT-IR spectroscopy was taken from Perkin–Elmer Spectrum one-B spectrometer. - 42 - 3.2.2 Experimental Procedures Synthesis of 6-hydroxy-2,5,7,8-tetramathyl-chroman-2-carboxylic acid methyl ester (Trolox methyl ester) HO O MeOH OH O HO O Room Temp. OCH3 O Scheme 3.2 Synthetic route of trolox methyl ester Trolox (250mg) was weighed out and dissolved in MeOH (5mL). Five microliters of 3.3% (v/v) HCl/MeOH solution was added into this solution and stirred at room temperature for 12 hours (Scheme 3.2). When the reaction was stopped, solvent was removed by rotary evaporation to give white colored solids[17]. After separation by silica gel column with chloroform as mobile phase, 0.2351g white colored solid was obtained with a yield of 89.05%, rf value 0.35, and M.W.=264 (Figure3.1, Figure 3.2) The 1H-NMR results are: (δ)1.6 (s, 3H); 1.82–1.89 (m, 2H); 2.06, 2.15, 2.19 (3s, 9H); 2.39–2.61 (m, 2H); 3.67 (s, 3H). As Figure 3.7 shows, These result are consistent with literature report by Palozza et al[17]. The 13C-NMR was also carried out in CDCl3 and the results are: (δ)174.5, 145.5, 145.3, 122.5, 121.3, 118.4, 116.8, 52.3, 30.6, 25.3, 20.9, 12.1, 11.8, 11.2 (Figure 3.3, Figure 3.4). - 43 - troloxester #81 RT: 1.66 AV: 1 SB: 7 1.03-1.14 NL: 1.05E6 T: - c ESI Full ms [50.00-1000.00] 263.2 100 95 90 85 80 75 70 Relative Abundance 65 60 55 50 45 40 35 30 163.4 25 20 15 264.2 10 5 97.0 159.3 143.4 187.2 261.8 297.1 318.9 361.9 424.9 491.8 515.9 645.9 680.1 582.8 710.5 960.4 851.9 878.4 938.1 990.3 784.9 0 100 200 300 400 500 600 700 800 900 1000 m/z Figure 3.1 ESI-MS (negative mode) spectrum of Trolox methyl ester troloxester #91 RT: 1.88 AV: 1 SB: 16 1.35-1.66 NL: 4.53E7 T: + c ESI Full ms [50.00-1000.00] 282.0 100 95 90 85 80 75 265.0 70 Relative Abundance 65 60 55 50 45 40 35 30 205.2 550.9 25 20 287.1 15 10 5 92.9 196.1 206.1 165.2 545.7 551.9 315.1 364.6 416.0 429.4 481.7 526.1 588.1 630.9 687.2 742.3 809.1 852.8 906.0 934.2 992.6 0 100 200 300 400 500 600 700 800 900 1000 m/z Figure 3.2 ESI-MS (positive mode) spectrum of Trolox methyl ester - 44 - 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 (ppm) Figure 3.3 1H-NMR spectrum of Trolox methyl ester (CDCl3 ) 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 (ppm) Figure 3.4 13C-NMR spectrum of Trolox methyl ester (CDCl3) - 45 - Synthesis of N-(3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-carbonyl) ethylenediamine (Trolox diamine) HO O NH2CH2CH2NH2 OCH3 O HO O 110oC, 4Hours NHCH2CH2NH2 O Scheme 3.3 Synthesis of Trolox ethylenediamine Trolox methyl ester (500 mg, 1.9 mmol) and 5 mL of the ethylenediamine was heated at 110 °C and vigorously stirred for 4 h (Scheme 3.3). The reaction mixture was diluted with CHCl3 (5mL) and washed with 10 mL of saturated aqueous NaHCO3. The organic layer was dried with Na2SO4, and the solvent was evaporated in vacuo [18] . The remaining yellow colored solids was purified by silica gel chromatography using chloroform: MeOH (v/v=10:1) as mobile phase, and white colored solids of Trolox diamine was obtained with a yield of 76%, rf value 0.5, and M. W. 292 (Figure 3.5). - 46 - troloxamine #26 RT: 0.42 AV: 1 SB: 5 0.02-0.09 NL: 5.15E8 T: + c ESI Full ms [50.00-1000.00] 293.12 100 90 Relative Abundance 80 70 60 50 40 30 584.94 20 10 102.85 137.89 0 50 100 150 191.98 239.65 200 276.19 250 331.18 359.29 300 350 471.00 411.64 400 450 556.87 606.96 662.93 704.87 499.00 500 550 600 650 700 778.02 817.02 838.76 876.36 750 800 850 900 936.84 978.36 950 1000 m/z troloxamine #55 RT: 0.99 AV: 1 SB: 5 0.56-0.64 NL: 2.23E6 T: - c ESI Full ms [50.00-1000.00] 291.4 100 90 Relative Abundance 80 70 60 50 40 30 292.4 20 163.4 201.5 227.0 95.1 129.1 162.7 10 0 50 100 150 200 290.7 250 337.2 369.1 389.2 435.2 300 350 400 450 491.2 583.1 523.3 581.4 584.1 603.2 538.4 500 550 600 671.3 693.3 650 700 827.3 872.4 894.0 733.6 751.9 750 800 850 900 956.4 950 997.3 1000 m/z Figure 3.5 ESI-MS spectrum of Trolox diamine (up: positive mode; down: negative mode) The 1H-NMR results are: (δ)1.52 (s, 3H, CH3-C2), 1.91-1.82 (m, 1H, ArCH2CH2), 2.07, 2.16, 2.18(3s, 9H), 2.40-2.32 (m, 1H), 2.74-2.55 (m,4H), 3.37-3.13 (m, 2H), 6.77 (bs, 1H). Most apparently, the disappeared peak at 3.67 corresponding to methyl ester clearly indicates the success of conversion of methyl ester group to amine group(Figure 3.6). The 13 C-NMR results are: (δ)174.72, 145.72, 144.31, 122.04, 121.81, 119.57, 117.90, 41.82, 41.35, 29.60, 24.53, 20.56, 12.32, 11.92, 11.38( Figure 3.7). These results are consistent with literature report by Koufaki et al[18]. - 47 - 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 (ppm) Figure 3.6 H-NMR spectrum of Trolox diamine (CDCl3) 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 (ppm) Figure 3.7 13C-NMR spectrum of Trolox diamine (CDCl3) - 48 - Trolox methyl ester react with AMVN and AAPH Trolox methyl ester was dissolved in methanol to afford a sample solution (4.55×10-3M) for kinetic reaction. -3 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) methanol solution (40.25×10 M) and 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) methanol solution (36.87×10-3M) was prepared as free radical initiator. Synergy HT Plate Reader was to monitor absorbance change of sample solution. Each 300μL sample solution was transferred into 96 well plate followed by addition of 20μL free radical initiator solution. In each case, 300uL methanol was used as blank solution to deduct absorbance of the solvent. The machine internal temperature was adjusted to 37℃. A programmed shaking was used before each measurement. The data points were collected each 5 minutes and the entire kinetic test lasted 120 minutes. Quantum dot ligand exchange with Trolox diamine CdSe/ZnS quantum dots (10mL) was precipitated by adding 2mL of CHCl3 and 10mL of MeOH. The precipitates were reconstituted in 2 mL of toluene for the step of synthesis. The concentration of the solution was determined by Peng’s method [19]. The QDs in toluene solution (54.62×10-6 M, 2 mL , 109.23×10-9 moles) were precipitated out by adding MeOH (5 mL) and centrifugation at 4000rpm for 10 minutes. The supernatant was discarded and the QDs are redissolved in CHCl3 (1 - 49 - mL). Trolox diamine in largely excess (100 mg, 0.4 mmol) was added, followed by an equal molar quantity of carbon disulfide chloroform solution (1:4 v/v, 0.1mL). The solution was stirred for 12 hours at room temperature in darkness under nitrogen (Scheme 3.4). HO O HO NHCH2CH2NH2 O + O S CS2 R.T, 12 hours NHCH2CH2NH O S Scheme 3.4 Quantum dots ligand exchange with Trolox diamine Five milliliter of methanol was added into 1mL of reaction mixture and it was thoroughly mixed by vortexing, followed by centrifugation at 4000 rpm for ten minutes in order to precipitate out ligand exchanged quantum dots. The precipitates were reconstituted in 3mL of acetone. The reconstituted mixture was centrifuged at 3000 rpm for 5 minutes. The precipitates of this step were discarded and only the homogeneous supernatant was collected or diluted for further studies. The absorption and emission spectra of this ligand exchanged quantum dots were recorded. The size and extinction coefficient (ε) of QDs were determined by using equation [1] and [2] respectively[19]. D = (1.6122 x 10-9 )λ 4 -(2.6575 x 10-6 )λ 3 +(1.6242 x 10-3 )λ 2 -(0.4277)λ+41.57 [1] where D (nm) is the size of CdSe QDs sample and λ (nm) is the wavelength of the - 50 - first excitonic absorption peak. ε = 5857(D) 2.65 [2] The concentration of the QDs sample was then calculated using Lambert-Beer’s Law [3]: A=εCL [3] where A is the absorbance at the peak position of the first excitonic absorption peak of QDs sample, C is the molar concentration (M) of QDs sample and L is the path length (cm) of the radiation beam used for the recording of the absorption spectrum, and path length was set at 1 cm in this study. The sizes were determined to be 3.19 nm after the ligand exchange. Trolox functionalized quantum dots Sensing of Peroxyl Radicals The stock solution of AMVN and AAPH were prepared in Methanol. The concentration of these two free radical initiators were 40.15×10-6 M and 36.15×10-6 M respectively. The concentration of the ligand exchanged quantum dots was determined by equation [1], [2] and [3] and was diluted to be 235×10-9 M. Synergy HT plate reader was used in sensing peroxyl radicals with excitation wavelength at 360±40nm and emission wavelength at 540±25nm. Each type of - 51 - working solutions was injected into 3 plate wells repetitively for accuracy purposes. Besides, the total amount of working solutions in each plate well is kept the same to avoid any deviation came from the volume differences. The general procedure was: for each well, 200μL Trolox-QDs were added followed by 20μL of free radical initiator solutions. In each case 200μL methanol was sued as blank solution and 200 μL Trolox-QDs with 20μL methanol was used as control. 3.3 Results and Discussion 3.3.1 Synthesis and characterization of Trolox derivatives The white solid obtained from Trolox esterification has a 89% yield. The product obtained from purification was sent for ESI-MS, 1H-NMR and 13 C-NMR essay to confirm its structure. The molecular weight of the compound is determined by ESI-MS spectroscopy as 263 from the anionic mode MS (Figure 3.1) and 265 from the cationic mode (Figure 3.2). The major peak in Figure 3.6 is at m/z 265 corresponding to the unionized molecule (C15H20O4, requires 264.3). The yellow color Trolox diamine solid obtained from synthetic reaction has a 76% yield. The white solid obtained from purification was dried in vaccum and sent for ESI-MS, 1H-NMR and 13 C-NMR study to confirm its structure. As Figure 3.5 demonstrates, the molecular weight of the compound is determined from ESI-MS spectroscopy as 291 from the anionic mode MS, corresponding to the loss of one proton; and 263 from the cationic mode, corresponding to the obtaining of one proton (C16H24N2O3, requires 292.18). UV-Visible spectrum is a crucial character of Trolox diamine-QDs, therefore the UV-Vis and fluorescence spectra of the Trolox ligand were studied. The major - 52 - absorbance peaks of both Trolox methyl ester and diamine are at 290nm whereas the emission maximum is located at 400 nm (data now shown). Since the CdSe/ZnS quantum dots are normally exited at 400nm and emission signal collected at around 550nm, there would be no interference when we study the Trolox diamine-QDs in the same way. The Fourier Transform Infrared spectroscopy (FT-IR) was used to extract more information of the quantum dot ligand exchange reaction. The FT-IR spectrum of Trolox methyl ester and Trolox diamine was obtained before further functionalized Normalized Transmittance quantum dots characterization. Trolox methyl ester Trolox diamine 4000 3500 3000 2500 2000 1500 1000 500 -1 cm Figure 3.8 FT-IR spectra of Trolox methyl ester and Trolox diamine - 53 - In above Figure 3.8, the primary amino Trolox diamine has two absorption bands situated at 3418 cm-1 and 3341 cm-1 because of the symmetrical and asymmetrical stretching vibration of NH2, which clearly indicates the formation of amino group. Moreover, The C=O stretching band at 1737 cm-1 of Trolox methyl ester (Figure 3.8 redline above) was shifted to 1667cm-1 in the spectrum of diamine (Figure 3.8 black line below), and this could be the proof of formation of amide functional group. Figure 3.9 shows a plot of the absorbance versus concentration of Trolox methyl ester. By establishing quantitative relation between Trolox ester concentration in methanol and its UV-Vis absorbance, we hope to understand the kinetic effect of peroxyl radical on Trolox analogue concentration. 1 y = 0.1283x - 0.0448 2 R = 0.9944 A b s or b an c e 0.8 0.6 0.4 0.2 0 0 1 2 3 4 5 -0.2 Trolox methyl ester conc.(mM) Figure 3.9 Trolox methyl ester standard curve in methanol. Absorbance was recorded at 290nm 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AMVN) and - 54 - 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) was chosen to be the initiator of peroxyl radicals. The kinetic UV-Vis absorbance of mixture containing -3 -3 -3 4.52×10 M Trolox methyl ester and 3.65×10 M AMVN or 3.35×10 M AAPH was measured at 290nm for 120 minutes(reaction temperature 37℃). Figure 3.10 shows that the drop of absorbance occurred in both solutions containing AMVN and AAPH. This result demonstrates that AMVN reacts much faster with Trolox methyl ester than AAPH, and it made mixture absorbance decreased 13.9% whereas the one containing AAPH decreased only 6.4%. This result also indicates that both AMVN and AAPH could be used as effective and efficient free radical initiator to oxidize Trolox analogue within 120 minutes. And in future studies they would be used to challenge Trolox functionalized quantum dots. 1 0.98 Normalized Absorbance 0.96 0.94 0.92 0.9 0.88 0.86 0.84 0 20 40 60 80 100 120 140 Time (mins) Figure 3.10 The reaction kinetic of AMVN(3.65 mM) and AAPH (3.35 mM) on Trolox methanol solution (4.52 mM) absorbance in 120 minutes at room temperature. - 55 - To determine the nature of the reaction between Trolox methyl ester and peroxyl radical initiators, the mixture of Trolox methyl ester and AAPH was diluted 10 times and transferred into a round bottom flask. The mixture was stirred vigorously for 24 hours and sent for ESI-MS examination. According to MS spectrum result (Figure 3.11), the oxidation reaction completed in 24 hours and Trolox methyl ester was converted to more complicated oxidized products which we have not yet indentified. XJ1 #40-61 RT: 1.15-1.76 AV: 22 SB: 6 0.89-1.03 NL: 1.85E6 T: - c ESI Full ms [50.00-2000.00] 783.5 100 747.5 95 90 85 80 75 70 Relative Abundance 65 60 55 784.5 50 472.5 45 40 35 30 819.2 25 20 508.4 829.3 15 10 376.5 163.3 427.5 738.6 570.3 702.6 534.4 244.7 5 1085.3 831.3 847.2 955.3 152.2 1101.7 1330.6 0 200 400 600 800 1000 m/z 1200 1400 1512.8 1647.9 1718.2 1838.9 1935.0 1600 1800 2000 Figure 3.11 ESI-MS (negative) spectrum of reaction mixture of Trolox methyl ester and AAPH 3.3.2 Functionalization of quantum dots with Trolox derivatives As Dubois et al reported[11], dithiocarbamate moiety formed by amine and CS2 can provide strong coordination to CdSe/ZnS surface. The bidentate chelating of amine-CS2 ligand - 56 - displayed high affinity for metal atoms, hence they can readily increase stability of nanocrystals. And at the same time, this ligand exchange process can confer properties of the ligands to the nanoparticle, such as solubility in different solvents or affinity to other specific compounds. The hydrophilic Trolox diamine ligand was proved to be capable of significant changing QDs solubility. Depending on ligand/QDs ratio and the size of original TOPO quantum dots, functionalized QDs could be precipitated out from reaction mixture by diethyl ether and reconstituted in various solvent like acetone, chloroform, methanol and PBS buffer (Figure 3.12). HO OH O O O HN HN O NH - S S NH - S S CdSe S HN NH O S ZnS S - - S N H H N O O OH O HO Figure 3.12 Proposed structure of Trolox functionalized Quantum Dots - 57 - The fluorescence emission spectrum and absorption spectrum of Trolox functionalized QDs dissolved in acetone are shown in Figure 3.13. From this Figure, the absorption maximum is located at 557 nm whereas the emission maximum is located at 596.5nm. Both absorption and emission maximum has a about 3nm red shift comparing to TOPO QDs before ligand exchange process. Figure 3.13 Absorption spectrum and fluorescence emission spectra of TOPO QDs (black line) and Trolox functionalized QDs (red line) On the other hand, the concentration of Trolox-QDs was calculated by using equations [1], [2] and [3] following Peng’s method[19]. From the absorption spectrum (Figure 3.17), the absorbance value at first excitonic absorption peak (λ = 557nm) is 0.989. Therefore - 58 - D=(1.6122×10-9)λ4-(2.6575×10-9) λ3+(1.6242×10-3) λ2-(0.4277λ)+41.57 D=3.19 Extinction Coefficient, ε=5857(D)2.65 = 126683.64 By Lambert Beer’s law, A=εCL C=7.81×10-6 M The similar calculation steps were performed for obtaining the concentration of other solution of TOPO QDs or Trolox-QDs. To determine the fate of the original phosphine ligands upon exposure to dithiocarbamate formed by Trolox diamine and carbon disulfide, we carried out 31P NMR measurements and the results shown in Figure 3.14. - 59 - 1.0000 Integral 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 25 20 15 10 5 0 -5 (ppm) 85 80 75 70 65 60 55 50 0.5887 1.4876 1.0088 1.0000 Integral TOPO 45 40 35 30 (ppm) Figure 3.14 31P NMR spectra of TOPO-QDs (above) and Trolox functionalized QDs (below) The starting TOPO QDs exhibit only one peak at 55 ppm. After 12 hours of ligand exchange process, new peaks emerge in the reaction mixture although no new phosphine compound was introduced into this reaction. The appearance of the new peak situated at 48.7 ppm corresponds to free TOPO that released from the QDs surface. This phenomenon is in agreement with quantum dots ligand exchange study reported by Dubois [11]. Furthermore, other two new peaks at 54.2 ppm and 39.9ppm respectively suggest there might have chemical reaction between TOPO and Trolox - 60 - Normalized Transmittance diamine or CS2 and formed unidentified phosphine product in the system. Trolox-QDs 1514 cm-1 1651 cm-1 TOPO QDs 4000 3500 3000 2500 2000 1500 1000 500 -1 cm Figure 3.15 FT-IR spectrum of Trolox functionalized QDs (Red line above) and TOPO QDs (Black line below) The FT-IR spectrum of Trolox functionalized quantum dots is significantly different from the spectrum of ligand unchanged TOPO quantum dots(Figure 3.15). The peaks at 2928 cm-1 and 2860 cm-1, which originate from TOPO ligand’s C-H stretching vibration, are the most characteristic feature of TOPO quantum dots. But these two peak drastically diminished in Trolox-QDs’ spectrum. Instead, the two strong peaks featuring C=O stretching vibration of amide group of Trolox diamine ligand appear at 1651 cm-1 and 1514 cm-1 which proved the successful bonding of Trolox diamine - 61 - ligand and replacing of the original TOPO ligand. 3.3.3 Trolox-QDs sensing of peroxyl radicals The preliminary study of peroxyl radicals’ effect on Trolox functionalized quantum dots was performed using AMVN and AAPH as free radical initiators. Fluorescence response of 0.235×10-6 M Trolox-QDs acetone solution to peroxyl radical at concentration of 3.65×10-6 M (AMVN) and 3.35×10-6 M (AAPH) respectively are shown in Figure 3.16. It shows that the fluorescence intensity of Trolox-QDs was significantly quenched by peroxyl radical generated by AAPH at 37℃ in 120 minutes ( i.e. 66.7%) whereas the fluorescence was quenched moderately by AMVN ( i.e. 48.2%). Therefore, the sensing result demonstrates that Trolox-QDs system has highly sensitivity towards peroxyl radical. - 62 - 1.200 Normalized Fluorescence Intensity 1.000 0.800 0.600 0.400 0.200 0.000 Control AAPH AMVN Figure 3.16 Effect of AAPH and AMVN solution on the fluorescence of Trolox-QDs. Concentration of AAPH is 3.35×10-6 M and concentration of AMVN is 3.65×10-6 M. (The excitation and emission wavelength are 360 nm and 540 nm respectively.) Tris(2-aminoethyl)amine, which do not possess antioxidant ability, functionalized QD (Tren-QDs) was used for comparison. The Tren-QDs powder was dissolved in water to afford a 0.161×10-6 M Tren-QD water solution followed by addition of 3.35×10-6 M AAPH. After 120 minutes reaction at 37 ℃ , the fluorescence intensity of the mixture was measured by HT platereader. As Figure 3.17 shows, peroxyl radical generated by AAPH did not have profound effect on the fluorescence of Tren-QDs (i.e.11.2%) whereas it significantly quenched antioxidant functionalized Trolox-QDs (i.e. 66.7%) as we previously showed. Therefore, the sensing result demonstrates that Trolox-QDs system has highly sensitivity towards peroxyl radical. - 63 - 1.200 Normalized Fluorescence Intensity 1.000 0.800 0.600 0.400 0.200 0.000 Control Trolox QD Tren QD Figure 3.17 Effect of AAPH solution on the fluorescence of Tren-QDs and Trolox-QDs. Concentration of AAPH is 3.35×10-6 M. (The excitation and emission wavelength are 360 nm and 540 nm respectively.) We speculate this sensitivity to peroxyl radical related intramolecular emission quenching could be attributed to photoinduced electron transfer (PET) from quantum dots to the chromanol moiety of Trolox ligand. It is possible that peroxyl radicals oxidized Trolox ligand on QDs surface and produce Trolox phenoxyl radical intermediate (α-TO.), electron transfer from the conduction band of quantum dots to the α-TO., which is also a mild electron acceptor, and transfer back to QDs’ valence band to interfere the fluorescence excitation-relaxation process of QDs. In another words, the process of peroxyl radicals quenching Trolox-QDs fluorescence could be simply interpreted as the electron use the Trolox radical intermediate SOMO as a - 64 - shuttle and hole, therefore blocked the pathway of fluorescence. This result resemble the report of M. Laferriere et al. when they observed non-linear quenching of CdSe quantum dots by nitroxyl free radicals[20]. This preliminary study show Trolox-QDs system is promising for peroxyl radical sensing, but several problems remains. The photoluminescence profile of the ligand exchanged quantum dots depended greatly on the condition of TOPO coated quantum dots. The stability of Trolox-QDs vary from batch to batch which can make the ligand exchange results not repeatable. Generally ligand exchanged QDs can sustain their fluorescence for 4-8 hours in room temperature exposed to air and could be stable under nitrogen protection for nearly a week. But we also observed ligand exchanged QDs lost their fluorescence overnight after the functionalization and turned into a much darker color. These oxidized Trolox-QDs were precipitated out several times by diethyl ether and methanol and sent for FT-IR examination. - 65 - Normalized Transimittance 1633 cm-1 801 cm-1 Oxidized Trolox-QDs Trolox-QDs 1514 cm-1 -1 1651cm 4000 3500 3000 2500 2000 1500 1000 500 -1 cm Figure 3.18 FT-IR spectrum of Trolox-QDs (Red line below) and oxidized Trolox-QDs (Blue line above) As Figure 3.18 shows, the spectrum of Trolox-QDs and oxidized Trolox-QDs are compared to illustrate the nature of reaction. The peaks at 1651cm-1 and 1514 cm-1 which originate from C=O stretching vibration of Trolox diamine ligand significantly diminished. The former peak disappeared while the latter became a shoulder. This result is in accordance with that of the oxidation products of Trolox functionalized gold nano-particle reported by Z. Nie et al. [21]. Two new peaks of the oxidized Trolox-QDs appear at 1633cm-1 and 801cm-1. This two peaks’ wavenumber are similar to the oxidized Trolox product reported by Nie et al. [21] . From the spectrum of oxidized form of Trolox-QDs we speculate there might be two products of Trolox diamine ligand: one has a tetraalkylbenzoquinonoid group (IR 1633 cm-1), - 66 - one has a cyclohexanone structure conjugated with adiepoxide (IR 801 cm-1) Possible mechanism for this rapid oxidation remain unclear. Since severe oxidation was not observed when Trolox analogues were bounded to other compounds or chemicals [15], we speculate photooxidation catalyzed by quantum dots emission and traps on the quantum dot surface could be the culprits for oxidation and lose of fluorescence. Also according to Z. Nie et al., Trolox functionalized gold particle appeared to enhanced the activity of the Trolox derived ligand and increase the reaction rate to 8 times compared with Trolox monomer[21]. Their explanation to this phenomenon is that by self-assembling on gold nanoparticle surface, both reactants in close proximity create a highly effective concentration which would lead to enhanced reaction rate. If this speculation also applies to our case, Trolox functionalized CdSe QDs should be oxidized faster than the ligand--Trolox diamine alone and functionalized quantum dots would be more sensitive to oxidants or free radicals. Dubois et al. suggested [11] that the thickness and the type of the shell grown on the CdSe core of quantum dots are essential for the preservation of the QD optical properties. In the same paper, they also suggested multilayer CdSe/CdS/CdZnS/ZnS QDs should have greater resistance to the lose of fluorescence. Our future work is to carry out in depth study on oxidation of Trolox-QDs. The relationship between functionalized QDs’ solubility and “TOPO-QD: CS2: Ligand ratio” also requires investigation. Since Trolox-QDs tend to be oxidized in reaction mixture, reaction - 67 - time of ligand exchange step also needs further optimization to achieve balance between ligand exchange ratio and photoluminescence of product. Ideally, enough amount of Trolox diamine should be able to almost completely replace original TOPO ligand from QDs, and make functionalized product water soluble. Hydrophilic Trolox-QDs should have wide application in bio-compatible environment. Finally, multilayer CdSe/CdS/CdZnS/ZnS QDs could be developed to optimize Trolox-QDs system and provide us more stable quantum dots with a higher quantum yield. 3.4 Conclusion By quantum dots ligand exchange using Dubois’ method with this Trolox diamine, we successfully developed Trolox-QDs system. Characterization with FT-IR shows ligand exchange QDs obtained new peaks from Trolox ligand. And 31 P-NMR also illustrates release of TOPO ligand during ligand exchange process. Both of these, along with the change in solubility and optical properties all indicates modification of quantum dots surface and bounding between ligand and QDs surface. Preliminary peroxyl radicals sensing study was carried out. Trolox-QDs has significant response, which represented by quenching of fluorescence intensity, to radicals generated by AMVN and AAPH in 120 minutes. The oxidized Trolox-QDs was studied with FT-IR, and its spectroscopic changes were in accordance with literature report by Z. Nie et al. In future work, the sensitivity of Trolox system can still be optimized (by washing steps or dialysis ); Photostability can also be improved by developing more - 68 - robust multilayer quantum dots; the Trolox-QDs quenching mechanism requires more evidence to confirm the occurrences of electron transfer between ligand and quantum dots. - 69 - References [1] R. E. Bailey, A. M. Smith, S. Nie, Physica E, 2004, 25, 1-12. [2] R. Eran, The Journal of Chemical Physics, 2001, 115, 1493-1497. [3] W. C. Chan, nbsp, W, and S. Nie, Science, 1998, 281, 2016-2018. [4] M. Bruchez, M. Moronne, P. Gin, S. Weiss, and A. P. Alivisatos, Science, 1998, 281, 2013-2016. [5] S. J. Rosenthal, I. Tomlinson, E. M. Adkins, S. Schroeter, S. Adams, L. Swafford, J. McBride, Y. Wang, L. J. DeFelice, and R. D. Blakely, Journal of the American Chemical Society, 2002, 124, 4586-4594. [6] W. J. Jin, M. T. Fernandez-Arguelles, J. M. Costa-Fernandez, R. Pereiro, and A. Sanz-Medel, Chemical Communications, 2005, 883-885. [7] A. J. Sutherland, Current Opinion in Solid State and Materials Science, 2002, 6, 365-370. [8] C. Querner, P. Reiss, J. Bleuse, and A. Pron, Journal of the American Chemical Society, 2004, 126 , 11574-11582. [9] J. Aldana, Y. A. Wang, and Xiaogang Peng, Journal of the American Chemical Society, 2001, 123, 8844-8850. [10] Y. Zhao, W. P. Segarra, Q. Shi, and A. Wei, Journal of the American Chemical Society, 2005, 127, 7328-7329. [11] F. Dubois, B. Mahler, B. Dubertrer, E. Doris, and C. Mioskowski, Journal of the American Chemical Society, 2007, 129, 482-483. [12] E. Niki, and N. Nouguchi, Accounts of Chemical Research, 2004, 37, 45-51. [13] D. C. Liebler, J. A. Burr, S. Matsumoto, and M. Matsuo, Chemical Research in Toxicology, 1993, 6, 351-355. [14] B. Halliwell and J. M. C. Gutteridge, Free Radicals in Biology and Medicine, Third Edition, Oxford University Press, New York, 1999. [15] P. Oleynik, Y. Ishihara, and Gonzalo Cosa, Journal of American the Chemical Society, (Communication), 2007, 129, 1842-1843. [16] V. Maurel, M. Laferriere, P. Billone, R. Godin, and J. C. Scaiano, J. Phys. Cem. B, 2006, 110, 16353-16358. [17] P. Palozza, E. Piccioni, L. Avanzi, S. Vertuani, G. Calviello, and S. Manferdini, Free Radical Biology and Medicine, 2002, 33, 1724-1735. [18] M. Koufaki, T. Calogeropoulou, A. Detsi, A. Roditis, A. P. Kourounakis, P. Papazafiri, K. Tsiakitzis, C. Gaitanaki, I. Beis, and P. N. Kourounakis, Journal of Medicinal Chemistry, 2001, 44, 4301. [19] W. W. Yu, L. Qu, W. Guo, and Xiaogang Peng, Chemistry of Mateials, 2003, 15, 2855. [20] M. Laferriere, R. E. Galian, V. Maurel and J. C. Scaiano, Chemical Communications, 2006, 257-259. [21] Z. Nie, K. J. Liu, C. J. Zhong, L. F. Wang, Y. Yang, Free Radical Biology & Medicine, 2007, 43, 1243-1254. - 70 - Chapter 4 Tris (2-aminoethyl) amine (Tren) Functionalized QDs 4.1 Introduction The demand for chemosensors that are selective for specific target ionic species and radicals is continuously increasing. Most importantly in this aspect is the sensors that monitor transition metal ions, reactive oxygen species and other species which have played important roles on the human health and environment even at very low concentration level[1,2]. Currently, many fluorescent small molecules (organic dyes) have been used to detect transition metal ions[3,4]. However, most of them tend to have narrow excitation spectra, and often exhibit broad and asymmetric emission bands with red tailing, which makes simultaneous quantitative evaluation of relative amounts of different probes present in the same sample difficult due to spectral overlap[5]. Besides, organic dyes are also subject to photobleaching and instability. Quantum dots (QDs) are semiconductor nanoparticles that are composed of atoms from groups II-VI or III-V elements in the periodic table, with all three dimensions confined to the 1 to 10 nm length scale, such as those made of CdSe, ZnTe, GaAs and InSb. QDs present considerable advantages over organic dyes because they have unique optical and electronic properties, including broad excitation spectra with high molar absorptivities and tunable emission[6-8]. The former property is due to the relatively large densities of states and overlapping band structures. QDs are able to - 71 - absorb photons even when the excitation energy exceeds the band gap due to the presence of multiple electronic states in QDs[9]. Therefore it allows the ease of light sources usage from various wavelengths and also simultaneous excitation of QDs with a single light source. The latter property exhibits an attractive way of using QDs as fluorescent sensors for various ionic species and radicals since the wavelength of fluorescence emission of QDs can be tuned by altering the QDs size and its chemical composition. Besides, present methods of QDs synthesis are able to manufacture QDs with excellent control over the QDs size and its distribution, yielding QDs with narrow and symmetric emission peaks[6,10]. Those properties mentioned are as a result of quantum confinement effect[9] which occurs when the size of semiconductor crystal becomes small enough that it approaches the size of its Exciton Bohr radius, which is the physical separation in a crystal between an electron in the conduction band and the hole it leaves behind in the valence band. Under these conditions, the electron energy levels are treated as discrete and the semiconductor material ceases to resemble bulk and can be called a quantum dot. According to Planck’s law, E=hc/λ where E is the energy of light, h is Plank constant, c is speed of light and λ is the wavelength of light. The wider the QDs’ band gap (i.e. larger energy) which separates the conduction band and valence band, the bluer the fluorescence (i.e. shorter wavelength) and vice versa. Figure 4.1 shows the colors of - 72 - ZnS-capped CdSe QDs produced are size-dependent. On the contrary with organic dyes, QDs also has easily accessible synthetic routes, large fluorescence quantum yields, high extinction coefficients, low photobleaching and excellent photostability[6]. Figure 4.1: Ten distinguishable emission colors of ZnS-capped CdSe QDs excited with a near –UV lamp with sizes of QDs increase from left to right. (refs. 6) For routine preparation, the CdSe core is usually capped with an organic layer such as trioctylphosphine/trioctylphosphine oxide (TOP/TOPO). This layer coordinates to Cd sites and stabilizes QD’s surface, preventing an aggregation of the nanocrystals[11]. Since the ligands are hydrophobic, they make the QDs incompatible with aqueous solution and did not appear to have an immediate application for analytical system. Two breakthrough papers by Nie’s and Alivisatos’ groups in 1998 demonstrated the fluorescent QDs can be made water-soluble and biocompatible by surface modification and bioconjugation while maintaining their large fluorescent quantum yield[8,12]. Hence, by tailoring QDs with different capping ligands, they can serve for different purposes. In the following years, there are two approaches to - 73 - surface modification to obtain highly fluorescent and water-soluble QDs. Firstly, the formation of a layer of another semiconductor of higher band gap on the QDs surface such as ZnS, giving rise to a bi- or three-layered structure QD[13] resulting in highly fluorescent QDs[14]. An alternative approach is to use competing capping agent such as mercaptosulphonic acid[15] or dihydrolipoic acid[16] to displace TOP/TOPO from the QDs surface. Surface modification is important in designing QDs for different analytical applications. The conjugation of suitable recognition groups will target specific ionic species or radicals, creating a hybrid QDs-receptor sensing system. This is because the fluorescence of QDs arises from the recombination of the exciton, it is expected that the changes of surface components or charges of QDs would affect the efficiency of core exciton recombination and consequently the fluorescence of QDs. Therefore QDs in which activated by different ligands can act as novel fluorescence selective ion probes[10,15,17-23]. The key successful implementation of the uses relies on the ability to add functionality to QDs and to stabilize their emission. The earlier work performed on QDs focused mainly on the physical and photochemical properties. To date, QDs have been developed for their applications in sensing analytes. Until now, either fluorescence quenching or enhancement of functionalized QDs has been observed dependent on the nature and environment of the ionic species or radicals. There are three quenching mechanisms reported: inner - 74 - filter effects, electron transfer processes, and non-radiative recombination pathways. Chen et al. explained the quenching of cysteine capped QDs by Fe(III) is attributed to an inner filter effect as a result of the strong absorption by Fe(III) at the excitation wavelength used. This interference caused by Fe(III) can be eliminated by adding fluoride ions to form a colorless complex FeF63-, which will also dissociate from the surface of the QDs due to same charge repulsions Also, the quenching of thioglycerol capped CdS QDs by Cu(II) is through an electron transfer from thioglycerol to Cu(II). The reduction of Cu(II) to Cu(I) by thioglycerol, formed CdS+-Cu+ on the surface, which has a lower energy level than pure CdS QDs, therefore causing a red-shift of fluorescence. Moreover, Cu(I) quenches by facilitating non-radiative recombination of excited electrons in the conduction band and holes in the valence band[10]. On the other hand, fluorescence enhancement of QDs has been reported by Moore et al. in 2001. The reversible fluorescence activation process caused by Zn(II) and Cd(II) adsorption on the surface of CdS was studied. These ions enhanced the fluorescence intensity of QDs, was attributed to some form of passivation of surface trap states. In 2002, Chen et al. worked on Zn(II) determination and found the formation of a Zn-cysteine complex on the surface of cysteine capping QDs which is believed responsible for the activation of the surface states and therefore exhibiting the fluorescence enhancement[10]. - 75 - Apart from intensive studies for potential use of QDs in sensing cations, functionalized QDs were also used for detection of inorganic anions although they are still in embryonic stages. In 2002, Watanabe et al. reported that a gold nanoparticle capped with amide ligands showed enhanced optical sensing of anions. The presence of anions would cause a marked decrease in extinction as a result of anion-induced aggregation of amide-functionalized gold nanoparticle via the formation of hydrogen bonding between the anions and the interparticle amide ligands. However, there was no selectivity and the binding affinity of anions was low[24]. In 2005, Jin et al. developed water soluble fluorescent CdSe quantum dots which are capped with 2-mercaptoethane sulfonate (MES) for the selective detection of free cyanide. Consequently, a slight blue-shift fluorescence quenching with detection limit of 1.1 x 10-6 M was observed. The blue-shift implies the changes in size or surface properties of MES-CdSe QDs which brings about the decrease in fluorescence[15]. However, the mechanism of fluorescence quenching was not discussed. Currently, researchers also raise considerable attention on sensing the paramagnetic radicals such as reactive oxygen species and reactive nitrogen species by using QDs as fluorescence probes. This is due to the importance of radicals in maintenance of vascular homeostasis and injury in biological system. However, when these reactive species are overproduced (due to exogenous stimulation), they will become highly harmful, leading to cellular damage. Unfortunately, reactive species present some - 76 - characteristics that make functionalized QDs difficult to detect in biological system such as their very short lifetime and variety of antioxidants existing in vivo which capable of capturing these reactive species. Maurel et al. showed the QDs capped by 4-amino-TEMPO (QD-4AT) which was initially quenched by the nitroxide in a QD-4AT complex, was readily restored when carbon-centered free radicals were trapped by the nitroxide moiety to form alkoxyamines. The initial quenching was due to the electron exchange or assisted intersystem crossing[25] upon the binding of 4AT to QDs’ surface without displacing TOPO while the subsequent restoration was due to radical reaction with ligand and blocked this quenching effect[26]. In the other work, Neuman et al. described a fluorescence quenching pathway of CdSe/ZnS core/shell QDs, leading by NO production from trans-Cr(cyclam)(ONO)2+. These observations are interpreted in terms of Förster resonance energy transfer (FRET) mechanism and the presence of a number of Cr(III) cations within the QDs hydration sphere. FRET occurs via space in which the dipole oscillation in QDs is induced by the dipole oscillation in trans-Cr(cyclam)(ONO)2+. In the other words, FRET requires that the absorption spectrum of trans-Cr(cyclam)(ONO)2+ overlap the fluorescence emission spectrum of the QDs[27]. In the present paper, transition metal ions sensing are performed by tris(2-aminoethyl)amine (Tren)-QDs. However, only the quenching pathways of selective determination of Co(III) and Cu(II) by using CdSe QDs which is capped by Tren ligands are thoroughly investigated. Furthermore, the Tren-QDs and transition - 77 - metal complex hybrid materials are also utilized for preliminary sensing study of hydrosulfide anion and nitric oxide. 4.2 Experimental Section 4.2.1 Materials and Instruments Tren-CdSe/ZnS QDs were synthesized by Dr. Wang Suhua. Iron(II) sulfate heptahydrate, magnesium chloride hexahydrate, cadmium nitrate tetrahydrate, manganese(II)-chloride-2-hydrate, magnesium chloride hexahydrate, cadmium nitrate tetrahydrate and hydrogen peroxide were purchased from Merck (Germany). Chromium(II) fluoride, manganese(III) acetate dihydrate, vanadyl sulfate hydrate, ruthenium(II) nitrosyl chloride hydrate, Cobalt(II) tetrafluoroborate hexahydrate, manganese(III) acetate dehydrate, sodium hydrosulfide, L-Ascorbic Acid (Vitamin C) and dialysis tubing cellulose membrane were purchased from Sigma Aldrich (USA). Tris(2-aminoethyl)amine (Tren) and palladium(II) acetate were purchased from Fluka. Nickel(II) acetate tetrahydrate and pentaaminechloruthenium (III) dichloride were purchased from Alfa Aesar. Iron(III) chloride anhydrous was purchased from Comak. Copper(II) sulfate pentahydrate was from J.T. Baker (USA). Cuprous chloride was from British Drug House. Zinc chloride was from Riedel-de Haën. Carbon Disulfide were purchase from BDH limited (Poole England). All solvents used were analytical grade and used without any purification. Ultraviolet-visible (UV-Vis) absorption spectra were obtained using UV-Visible - 78 - Spectrophotometer UV-1601 (Shimadzu (Asia Pacific) Pte. Ltd, Singapore). The absorbance readings were obtained from 900 nm to 400 nm. The fluorescence was monitored by using Bio-tek Synergy HT (Winnoski, Vermont, USA) plate reader. Excitation and emission wavelengths were set at 360±40 nm and 540±25 nm respectively. The working solutions were injected into 384 Well HiBase polystyrene microplates and the fluorescence was measured with prior shaking for ten seconds with intensity of one for homogeneity. Temperature control was not necessary while reading fluorescence values. On the other hand, the fluorescence spectra were obtained on a Perkin Elmer LS55 Luminescence Spectrometer. The fluorescence spectra were obtained from 700 nm to 500 nm. The excitation slit width and the emission slit width were set at 10 nm and 20 nm respectively. The scan speed was set at 500 nm/min. PTFE vials (4mL, screw-top 15x45 vial with cap and bonded PTFE/silicone septa) from Waters® (USA) were utilized. All optical measurements were performed at room temperature under ambient conditions. After fluorescence measurement, the vials were further wrapped with aluminum foil to minimize the exposure to light. All pH measurements were made with Thermo Orion pH meter (Model 410). FT-IR spectroscopy was performed on Perkin–Elmer Spectrum one-B spectrometer. Choosing suitable pH of Phosphate Buffer Saline (PBS) for Tren-QDs system Different pH of PBS buffer were prepared by using commercial 10X Phosphate Buffer Saline which contains 137 mM NaCl, 2.7 mM KCl and 10 mM Phosphate Buffer. By adjusting pH of PBS buffer with NaOH and HCl and measuring the pH - 79 - value of PBS with pH meter, PBS 4.0, PBS 5.0, PBS 6.0, PBS 6.5, PBS 7.0, PBS 7.5, PBS 8.0, PBS 9.0 were prepared. The same amounts (1.2 mg) of Tren-QDs were then dissolved in 3.0 mL of different pH of PBS buffer respectively. After vortex each solution to make the solution homogenous, the fluorescence was measured for every five minutes (0-60 minutes). The stock solution of Tren-QDs was prepared by dissolving the powder form of Tren-QDs in PBS buffer, followed by centrifugation (1000 rpm) for three minutes in order to obtain saturated Tren-QDs in which they were well dispersed in PBS buffer. The precipitates were removed by centrifugation. 4.2.2 Sensing of metal ions The stock solutions with 1 mM concentration of Na(I), K(I), Mg(II), Ca(II), VO2+, Cr(II), Cr(III), Mn(II), Mn(III), Fe(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Ru(II), Ru(III), Pd(II), Ag(I) and Cd(II) were prepared by dissolving suitable amount of NaCl, KBr, MgCl2.6H2O, CaSO4.2H2O, VOSO4.xH2O, CrF2, CrCl3.6H2O, MnCl2.2H2O, (C2H3O2)3Mn.2H2O, FeSO4.7H2O, FeCl3, Co(BF4)2.6H2O, (CH3COO)Ni2.4H2O, CuSO4.5H2O, ZnCl2, Ru(NO)Cl3.xH2O, [RuCl(NH3)5]Cl2, Pd(OAc)2, AgNO3 and Cd(NO3)2.4H2O in 10 mL deionized water respectively and were further diluted when necessary. The stock solutions were prepared in 10 mL PTFE vials with further sealed with parafilm and kept under dark. However, freshly-prepared Fe(II) solution was needed due to the easy-oxidized property of Fe(II). The white precipitates adhere on the wall of vial indicated the oxidized Fe(II) solution. - 80 - The general detection procedures for metal ions were as follows: 3.0 mL of Tren-QDs in PBS buffer (stock solution) was introduced into a PTFE vials, followed by adding 20 μL of 1.0 mM standard solution of metal ions every time (0 -80 microliters). The fluorescence intensity was measured immediately after the working solution mixed thoroughly by vortex. 4.2.3 The effect of carbon disulfide on QDs-metal ions complex The stock solutions with 1 mM concentration of Na(I), Mn(III), Fe(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Ag(I) and Cd(II) were prepared by dissolving suitable amount of NaCl, (C2H3O2)3Mn.2H2O, FeSO4.7H2O, FeCl3, Co(BF4)2.6H2O, (CH3COO)Ni2.4H2O, CuSO4.5H2O, ZnCl2, AgNO3 and Cd(NO3)2.4H2O in 10 mL deionized water respectively and were further diluted when necessary. The stock solutions were prepared in 10 mL PTFE vials with further sealed with cap and kept in darkness. However, freshly-prepared Fe(II) solution was needed due to the easy-oxidized property of Fe(II). The white precipitates adhere on the wall of vial indicated the oxidized Fe(II) solution. The general detection procedures for metal ions were as follows: Trens-QDs methanol/H2O 1:1(v/v) solution (30nM) 10mL was mixed with 1mL CS2 methanol solution (30μM) and vigorously vortexed. This mixture was left in darkness for 30 minutes for stabilization and then transferred into NUNCTM 96 well micro plate followed by adding 20μL of various transition metal water solution (50μM) was - 81 - added for fluorescence reading. Each type of working solutions was injected into 3 plate wells repetitively for accuracy purposes. Besides, the total amount of working solutions in each plate well is kept the same to avoid deviation came from the volume differences. The fluorescence intensity was measured immediately after addition. 4.2.4 Tren-CS2-metal complex synthesis and reaction with sodium hydrosulfide Tren solution and CS2 solution was prepared by diluting these liquid into suitable amount of methanol. Tren solution (10mL, 2.67mM) and CS2 (10mL, 8.32mM) solution was mixed and vigorously stirred overnight. Each 5 mM of this reaction mixture was mixed with 1mL of copper(II) acetate methanol solution(6mM), Iron(II) acetate methanol solution (6mM), Iron(III) chloride methanol solution (6mM) and 1mL of methanol (as control) respectively. Above four mixtures were all fully vortexed following by adding of 20uL NaSH (200mM) water solution. Hence the finally concentration of each component was Tren: CS2: M+: HS-=0.56mM: 1.73mM: 0.5mM: 1.98mM. One milliliter of this mixture was withdrew and 2 times diluted to afford a 2 mL methanol solution for UV-Vis spectrum scan. 4.2.5 Tren-QDs-CS2-Fe(II) Complex synthesis Tren-QDs water solution(43nM, 10mL) was prepared by dissolving Tren-QDs powder into deionized water. The solution was fully vortexed and centrifuged at 1000rpm for 5minutes in order to obtain saturated Tren-QDs water solution. After that, the precipitates were removed - 82 - and the supernatant was transferred out for further applications. CS2 methanol solution (52μM, 2mL) was added into the saturated Tren-QDs water solution and fully vortexed for 30 seconds, followed by adding 1mL of Fe(OAc)2 water solution (135μM). The reaction mixture was vigorously stirred overnight in darkness at room temperature. When the reaction stopped, the mixture was withdrew from reaction vessel and purified by dialysis using Sigma-Aldrich dialysis tubing cellulose membrane. By one hour of dialysis in deionized water, the remaining solution of Tren-QDs-CS2- Fe(II) complex was withdrew from the membrane and stored in sealed PTEE vials in darkness and was ready for sensing or other applications. 4.2.6 Tren-QDs-CS2-Fe(II) complex sensing hydrosulfide anion and nitric oxide Hydrosulfide anion was provided by dissolving 265mg NaSH solid into 25mL deionized water(189× 10-3M). NO was generated by mixing NaNO2 and ascorbic acid water solution dropwise at room temperature. By passing NO gas trough deionized water, saturated NO water solution ( 2× 10-6M) was prepared. The general detection procedures for HS- and NO were as follow: 3.0 mL of Tren-QDs-CS2-Fe(II) complex in deionized water was introduced into a PTFE vials, followed by adding 20μL of NaSH (0-140μL)or 100μL NO water solution every times (0-500μL). The fluorescence intensity was measured on Perkin Elmer LS55 Luminescence Spectrometer five minutes after the working solution mixed thoroughly by vortex. - 83 - 4.3 Results and Discussion 4.3.1 Tren-QDs system The surface structure of QDs capped with Tren ligands and their interaction with transition metal ion are shown in Figure 4.2. Due to the presence of lone pairs on nitrogen atoms of amines groups in Tren ligands, they have high degree of basicity and are completely miscibility with water. Therefore, by capping QDs with Tren ligands, the functionalized QDs become water-soluble. Tren ligands are attached to the surface of QDs with the assistance of carbon disulfide which is bidentate chelating moieties with high affinity for metal atoms[28] and therefore leading to a secure coating of the QDs. The sulfur atoms of carbon disulfide are bound to the zinc atom of zinc disulfide via electrostatic interaction. One molecule of Tren consists of four amine groups. From Figure 4.2, N1 is tertiary amine and N2, N3 and N4 are primary amines. N2, N3 and N4 are chemically equivalent since they are in the same chemical environment. Therefore, either of them has the same reactivity and readily to react with carbon disulfide. This is because primary amines are more reactive than tertiary amines. Consequently, the remaining amines are believed corresponding to the coordination with transition metal ions. - 84 - NH2 4 M (Ln) N 1 NH2 3 NH2 (Ln) M HN 3 2 - S H2N S 4 N 1 S S CdSe - NH 2 - NH 2 S S N ZnS S H2N NH 4 N 2 1 S M NH2 3 (Ln) H2N 4 1 (Ln)M NH2 3 Figure 4.2: Proposed surface structure of CdSe QDs capped by Tren ligands and their coordination complexes with transition metal ion. M and Ln represent transition metal and other ligands. 4.3.2 Optical characteristics of the Tren-QDs The fluorescence emission spectrum and absorption spectrum of Tren-QDs are shown in Figure 4.3. The fluorescence spectrum shows a nonzero tail toward longer wavelength suggesting the presence of surface traps. This fluorescence is attributed to the recombination of the charge carriers immobilized in traps of different energies[29]. General fluorescence measurements were significantly simplified due to long wavelengths emission and a high separation between the excitation and emission wavelengths. - 85 - 600 559 0.10 Emission 0.09 Absorbance 0.08 500 400 361 0.07 300 0.06 200 0.05 100 0.04 300 350 400 450 500 550 600 650 0 700 F lu o rescen ce In ten sity A b so rb an ce 0.11 Wavelength (nm) Figure 4.3: Absorption spectrum and fluorescence emission spectra of Tren-QDs Figure 4.4 shows a plot of the fluorescence intensity versus concentration of QDs with a good linear relationship (inset in Figure 4.4). The result demonstrated that functionalized QDs were uniformly dispersed in PBS 7.4 buffer in a large concentration range. Furthermore, there was no fluorescence emission spectra band shift along with a serial representative emission spectrum at different concentration of QDs even at the lowest concentration of QDs. Tren-QDs can disperse uniformly and stably in aqueous solution for around one week without precipitation when preserved in the dark and under ambient conditions. - 86 - 1000 Fluorescence Intensity 900 800 700 [Tren-QDs] 600 500 400 300 200 100 0 400 450 500 550 600 650 Wavelength (nm) Figure 4.4: The concentration-dependent of fluorescence property of QDs. 4.3.3 Sensing of transition metals The effect of pH in a range between 4.0 and 9.0 was studied in order to find out the optimum conditions for the sensing of transition metals with Tren-QDs. The results obtained from this study showed Tren-QDs behave their highest stability between PBS 7.0 and PBS 7.5 (results not shown). A final PBS 7.4 buffer was selected for further experiments. Besides, pH 7.4 is also physiological pH and Tren-QDs system might be functioned in vivo in the future for sensing either transition metals or radicals. Responses to metal ions at concentration of 1.3 x 10-5 M except for Na(I), K(I), Mg(II) and Ca(II) of 13 x 10-5 M in 0.02 x 10-6 M Tren-QDs in PBS 7.4 buffer are shown in Figure 4.5. From Figure 4.5, it could be easily found that the fluorescence emission of Tren-QDs was quenched significantly by Co(III) and Cu(II) respectively. Although other transition metals (Ni(II), Fe(III), Fe(II), Mn(III), Mn(II), Cr(III), - 87 - Cr(II), VO2+, Ru(III), Ru(II) Ag(I), Pd(II)) also quenched the fluorescence of Tren-QDs to different extents but their quenching effects were not as strong as those of Co(III) and Cu(II). On the other hand, the fluorescence emission was slightly enhanced by Zn(II) and Cd(II) respectively. Furthermore, the effect of interferences of foreign substances such as Na(I), K(I), Mg(II) and Ca(II) was investigated as well. The result shows that they have hardly effect on Tren-QDs fluorescence even in high concentration. Therefore, the sensing result demonstrates that this method has high sensitivity and selectivity towards Co(III) and Cu(II). Hopefully, Tren-QDs system could be utilized to sense Co(III) and Cu(II) in biological system in the future since it would not be affected by the presence of interfering substances even in high 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 Ca(Ⅱ) Mg(Ⅱ) K(Ⅰ) Pd(II) Na(Ⅰ) Ag(I) Cd(Ⅱ) Ru(Ⅱ) Ru(Ⅲ) VO2+ Cr(Ⅱ) Cr(Ⅲ) Mn(Ⅱ) Mn(Ⅲ) Fe(Ⅱ) Fe(Ⅲ) Co(Ⅲ) Ni(Ⅱ) Cu(Ⅱ) Zn(Ⅱ) 0.0 Control Relative Fluorescence Intensity concentration. Metal Ions Figure 4.5: Effect of metal ions on the fluorescence of Tren-QDs. Concentrations of transition metal ions are all 1.3 x 10-5 M, and other alkali and alkali earth metal ions are 13 x 10-5M. Concentration of Tren-QDs is 2.0 x 10-8 M. The result of slight fluorescence enhancement of Tren-QDs by Zn(II) and Cd(II) is - 88 - consistent with Chen et al. reports[30] except they used L-cysteine functionalized CdS QDs instead of Tren-CdSe QDs . From the fluorescence spectra, these ions enhanced the band edge fluorescence intensity of the Tren-QDs upon introduction of salts of these ions, therefore it is attributed to some form of passivation of the surface trap states of QDs[31]. Although Co(II) source was used in this study, but Co(III) is likely the right candidate which coordinated with Tren-QDs. The high-spin Co(II) will readily become low-spin Co(II) when coordinated to amine ligands. The resulting Co(II) complex is known to react with oxygen and become Co(III). Indeed, we observed color changes of the solution upon addition of Co(II) solution to the Tren-QDs. Figure 4.6 displays the absorption behavior of Co(III) which coordinated with Tren ligands of various concentration in aqueous solution. There is an intense absorption occurred around 559 nm (i.e. the fluorescence emission maximum wavelength) when Co(III) was coordinated with Tren ligands. Therefore the absorption spectrum effective overlap with the band edge of fluorescence emission which ranging from around 500 – 600 nm (see Figure 4.3). This feature suggests that the energy transfer via Förster mechanism would be favorable. That is the energy transfer occurs from Tren-QDs donor to Co(III) acceptor via space in which the acceptor absorbs energy at the emission wavelength of the donor and quenching is observed when the donor does not re-emit the energy fluorescently itself. Apart from the spectral overlap between the donor emission and the acceptor absorption spectrum, energy transfer - 89 - also depends on the relative orientation and the distance between the donor and acceptor[32]. 0.20 Co(III) 0.15 1 eq Co(III):1 eq Tren Absorbance 1 eq Co(III):2 eq Tren 1 eq Co(III):3 eq Tren 1 eq Co(III):4 eq Tren 0.10 1 eq Co(III):5 eq Tren 1 eq Co(III):6 eqTren 1 eq Co(III):7 eq Tren 1 eq Co(III):8 eq Tren 0.05 0.00 400 500 600 700 800 900 Wavelength (nm) Figure 4.6: Electronic spectra of Co(III) complexes of various concentrations in aqueous solution. The absorption of Cu(II) complexes was also investigated and shown in Figure 4.7. There is only a slight absorption occurred around 559 nm (i.e. weak overlap with the band edge of fluorescence emission which ranging from around 500 – 600 nm) when Cu(II) was coordinated with Tren. Hence the quenching of Tren-QDs upon the addition of Cu(II) is only minor attributed by the energy transfer. Besides, from the absorbance spectrum of Cu(II) alone, there is a peak arises at around 780 nm which is attributed to the d-d transition of Cu(II) which energy gap is complementary to blue light and therefore contribute to the blue color of the copper(II) sulphate pentahydrate (source of Cu(II)). However, there is another peak arises at the shorter - 90 - wavelength upon the addition of Tren ligands. It is likely due to d-d transition as well. The blue shift is attributed to stronger ligand field of Tren than water. Since the absorbance coefficiency is still small judging from the UV-Vis spectrum and it can not be a charge transfer band. 0.20 d-d transition Absorbance 0.15 Cu(II) 1eq Cu(II):1 eq Tren 1eq Cu(II):2 eq Tren 1eq Cu(II):3 eq Tren 1eq Cu(II):4 eq Tren 1eq Cu(II):5 eq Tren 1eq Cu(II):6 eq Tren 1eq Cu(II):7 eq Tren 1eq Cu(II):8 eq Tren 0.10 d-d transition 0.05 0.00 400 500 600 700 800 900 Wavelength (nm) Figure 4.7: Electronic spectra of Cu(II) complexes of various concentrations in aqueous solution. Apart from the minor attribution of energy transfer, electron transfer pathway can also apply on the observed quenching effect of Tren-QDs by Cu(II). This is because Cu(II) is d9 ion and therefore Cu(II) tends to receive one additional electron in order to occupy d10 stable valence configuration. Hence, electron transfer is initiated from Tren-QDs to Cu(II) upon the coordination of Cu(II) with Tren-QDs. Future work shall be targeted at elucidating the fluorescence quenching mechanism. - 91 - However, the other quenching pathway proposed by Xie et al. group could also be plausible. Small transition metal ion Cu(II) could pass through the shell layer and interact with core if the QDs used were not perfectly capped with Tren ligands, resulting in the chemical displacement of Cd(II) by Cu(II). The displacement can be achieved because the solubility of CuSe is extremely low if compared to that of CdSe. So the quenching result could be explained in terms of strong binding of Cu(II) onto the surface of Tren-QDs. As shown in Figure 4.8, when the CuSe particles grew, its energy levels will become lower than that of pure QDs, facilitating the electron and hole transfer from the CdSe to CuSe energy levels rather then the process of fluorescence generation in the CdSe. Consequently, the fluorescence of Tren-QDs was quenched[18]. However, this assumption will be proved using high resolution transmission electron microscopy (TEM) in the future. By using TEM, one could image the surface structure of Tren-QDs. e- X h+ Tren-QDs CuSe Figure 4.8: Energy scheme of the Tren-QDs in the presence of Cu(II). - 92 - Figure 4.9 describes the effect of Co(III) and Cu(II) concentration dependence on the fluorescence intensity of Tren-QDs. For each of the transition metal ions, the fluorescence intensity all decreases greatly in exponential behavior along with the increase of the ion concentrations. The Tren-QDs in PBS 7.4 buffer are most likely to coordinate with Co(III) and Cu(II) respectively because the amount of those metal ions used was in excess with respect to the molar concentration of Tren-QDs. Furthermore, each molecule of QDs consists of many Tren ligands on its surface. Taking into account the reaction of Tren with each Tren ligand consists of more than one amine group, which therefore brings up the number of coordination atoms of Tren-QDs with metal ions. In the presence of Tren, the quenching mechanism of Tren-QDs is more of a surface alteration rather than formation of a completely new product. Consequently, the fluorescence intensity does not decrease linearly with increasing concentrations of the quencher (i.e. Co(III)/Cu(II)). Relative Fluorescence 1.20 1.00 0.80 Cu(II) 0.60 Co(III) 0.40 0.20 0.00 0 5 10 15 20 25 30 Concentration of ions (μM) Figure 4.9: Effect of Cu(II) and Co(III) concentration on the fluorescence intensity of Tren-QDs. - 93 - Furthermore, the Stern-Volmer quenching equations for Co(III) and Cu(II) were studied. Prior to discussing the results, it is important to introduce on the general characteristic of dynamic and static quenching which can be explained by Stern-Volmer plot. Basically, dynamic quenching occurs only when the excited fluorophore experiences contact with an atom or molecule that can facilitate non-radiative transitions to the ground state (i.e. diffusive encounters). In the simplest case of dynamic quenching, Stern-Volmer equation was held: I0/I = 1 + KSV[Q] = τo/τ where I0 and I are the fluorescence intensities observed in the absence and presence, respectively, of quencher, [Q] is the quencher concentration, KSV is the Stern-Volmer quenching constant, τo and τ are the excited state lifetime in the absence and presence of the quencher. In the situations where the fluorophore forms a ground-state complex with the quencher, and the complex causes quenching, then the static quenching occurs. In such a case, equation I0/I = 1 + Ka [Q] is held where Ka is the association constant of the complex. However, it is possible for both static and dynamic quenching to occur simultaneously. In Tren-QDs system, the Tren-QDs are fluorophores whereas the transition metals, Co(III) and Cu(II) are quenchers. Figure 4.10 shows Stern-Volmer quenching curves describing I0/I as a function of transition metal concentration. Both Co(III) and Cu(II) curves can fit into conventional linear Stern-Volmer equation with linear correlation coefficient of 0.978 and 0.910 respectively. Besides, the Stern-Volmer quenching constant (Ksv) are - 94 - found to be 0.2124 μM-1 and 0.1366 μM-1 for Co(III) and Cu(II) respectively. The higher the value of KSV, the more efficient the quenching effect by a quencher. Therefore in this case, Co(III) exhibits higher quenching ability than that of Cu(II). A linear Stern-Volmer plot is also indicative of a single class of fluorophores, all equally accessible to quencher. With the linearity, the concentration of transition metal ions can be monitored by measuring the fluorescence intensity of Tren-QDs. However, with the present data of Co(III) and Cu(II) study, it is not possible to definitely state which mechanism (dynamic or static) predominates since the lifetime study is not conducted in this experiment. The Stern-Volmer plots of other transition metals also can fit into linear Stern-Volmer equation (data not shown) but with lower KSV values than that of Co(III) and Cu(II), showing that they are poorer quenchers. 7.00 6.00 Io/I = 0.2124[Q] + 1 Io/I 5.00 2 Cu(II) Co(III) R = 0.978 4.00 3.00 Linear (Cu(II)) Linear (Co(III)) Io/I = 0.1366[Q] + 1 2 2.00 R = 0.910 1.00 0.00 0 5 10 15 20 25 30 Concentration of ions (μM) Figure 4.10: Stern-Volmer plot of Cu(II) and Co(III) concentration dependence of the fluorescence intensity of Tren-QDs. 4.3.4 Tren-QDs-CS2-Fe(II) complex system Tren-QDs was successfully coordinated with transition metal by our coworker. Tren-QDs-Cu(II) and Tren-QDs-Co(III) have been applied for sensing radicals and other oxygen species. - 95 - By screening with UV-Vis spectrum trough three metal ions Fe(II), Fe(III) and Cu(II), we try to design and synthesis another Tren-QDs metal complex which is sensitive to biologically important hydrosulfide anion and nitric oxide. The effect of carbon disulfide on QDs-metal ions complex The addition of carbon disulfide (CS2) was based on the idea that the free primary amino group of Tren ligand on Tren-QDs surface could react with CS2 to form a dithiocarbamate group, which can strongly chelate the transition metal ions. By using this chelating ability we can coordinate more metal ions on the surface of Tren-QDs and therefore create more sensitive fluorescent Tren-QDs-CS2-M+ metal complexes which can be applied to the sensing of hydrosulfide anion and nitric oxide. CS2 was know to have the ability to quench QDs fluorescence to a moderate degree, its synergetic effect with metal ions on Tren-QDs could severely quenching its fluorescence and compromise the sensitivity of the complex. Therefore, effect of metal ions and CS2 on the fluorescence of Tren-QDs is our first step of to study. - 96 - 1.2 Relative Fluorescence Intensity 1 0.8 0.6 0.4 0.2 0 Na(Ⅰ) Co(Ⅱ) Ag(Ⅰ) Mn(Ⅲ) Fe(Ⅱ) Fe(Ⅲ) Ni(Ⅱ) Metal Ions Cd(Ⅱ) Zn(Ⅱ) Cu(Ⅱ) Control Figure 4.11 Effect of metal ions and CS2 on the fluorescence of Tren-QDs. Concentration of metal ions are all 90.9×10-6M. Concentration of CS2 is 2.48×10-6M. Concentration of Tren-QDs is 24.8×10-9M. Fluorescent response to metal ions at concentration of 90.9×10-6M Tren-QDs ( 24.8×10-9M) and CS2 (2.48×10-6M) mixture are shown in Figure 4.11. CS2 was thoroughly mixed with Tren-QDs and left in darkness for 30 minutes, followed by adding of 10 kinds of metal ions water solution at the concentration of 90.9×10-6M. From Figure 4.11, it could be found that the fluorescence emission of Tren-QD-CS2 was quenched by Co(II) and Cu(II) most obviously. Although other transition metals (Mn(III), Fe(II), Fe(III), Ag(I), Ni(II) and Zn(II)) also quenched the fluorescence to different extents but their quenching effects were not as strong as those of Co(II) and Cu(II). Furthermore, the effect of interferences of foreign substances Na(I) was also investigated. The result shows it has hardly any effect on the fluorescence comparing to the control group. This result is in agreement with our previous observation in - 97 - PBS buffer without CS2 (Figure 4.5) except the fluorescence emission enhancement from addition of Zn(II) and Cd(II) was not reproduced. Instead a slight quenching was observed after addition of each ion. The addition of CS2 did not directly affect optical properties of Tren-QDs, which make it possible that CS2 serves as a bridging unit between Tren-QDs and transition metal ions. Synthesis of Tren-CS2-metal complex and its reaction with NaSH Copper(II), Iron(II) and Iron(III) was chosen for the first batch UV-Vis screening for selection of complex which has strong absorption peaks at Tren-QDs emission area (i.e. 500-600nm). The synthetic procedure was simply mixing Tren solution (10mL, 2.67mM) and CS2 (10mL, 8.32mM) solution was thoroughly and stirred vigorously overnight. When the reaction was stopped, each 5mL of the mixture was added into 1mL of copper(II), Iron(II) and Iron(III) water solution (6mM) respectively. The four mixtures were thoroughly vortexed, followed by the addition of 20uL NaSH (200mM) water solution respectively. The final concentration of component in each mixture was Tren: CS2: M+: HS-=0.51mM: 1.59mM: 0.91mM: 1.98mM. All mixture was 2 times diluted with methanol and studied by UV-Vis spectrum scan (200-900nm). Control group was the one without metal ion addition. The UV-Vis spectrum of Tren-CS2-Fe(II) complex with and without hydrosulfide are shown in Figure 4.12. - 98 - 3.0 Fe(II)+SH Fe(II) 2.5 Absorbance 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 200 300 400 500 600 700 800 900 Wavelength (nm) Figure 4.12 UV-Vis spectrum of Tren-CS2-Fe(II) complex before (red line) and after (black line) NaSH addition. (Tren: 0.255mM, CS2 :0.795mM, M+: 0.455mM, HS- :0.99mM.) Copper(II), Iron(II) and Iron(III) all caused drastic absorbance increase at 550nm, which is the maximum of Tren-QDs emission peak, when they were added into the mixture of Tren and CS2. In Figure 4.13, Fe(II) has the highest absorbance after HSaddition among all three metal ions tested, followed by Fe (III) and Cu (II). But 10 minutes after the UV-Vis essay, Fe(III) and Cu (II) complexes precipitated out from the solvent. Fe (II) complex, however, was able to sustain in solution several days after HS- addition. - 99 - 1 0.9 0.8 Normalized Absorbance 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Control Control+HS- Cu(Ⅱ) Cu(Ⅱ)+HS- Fe(Ⅲ) Fe(Ⅲ)+HS- Fe(Ⅱ) Fe(Ⅱ)+HS- Figure 4.13 Effect of hydrosulfide anion on the UV-Vis absorbance of Tren-QDs-CS2-metal complex at 550nm. Concentration of each component are Tren: 0.255mM, CS2 :0.795mM, metal ions: 0.455mM, HS- :0.99mM. The precipitate of the metal complex could be seemed as the proof of successful coordination (because Tren itself would not form insoluble precipitate with these metal ions in methanol), but the ultimate goal of this research is to coordinate metal ions on Tren-QDs surface, and precipitation of metal complex could result in precipitation of QDs itself from the solution. Therefore, only Iron(II) was suitable candidate for further investigation. In Figure 4.12, the UV-Vis spectrum of Tren-CS2-Fe(II) before and after NaSH addition was compared. It was clearly found that after HS- addition UV-Vis absorbance mainly increased at 200-600nm area. Hence the complex has the potential to block both the excitation light and the - 100 - emission of Tren-QDs. This observation is consistent with the Chen et al.’s report. In their publication the quenching effect of Fe3+ on the luminescence of L-cysteine and thioglycerol-capped was categorized as “inner filter effect”, which resulting from the strong absorption of excitation wavelength at 400 nm[10]. And the interference could be eliminated by adding fluoride ions to form a colorless complex FeF63-. 4.3.5 Tren-QDs-CS2-Fe(II) complex synthesis and its application With understanding of the optical properties of Tren-CS2-Fe(II), the surface of Tren-QDs was modified by the formation of Tren -CS2-Fe(II) complex. The synthesis step was simply started by mixing saturated Tren-QDs deionized water solution (43nM, 10mL) and CS2 methanol solution (52μM, 2mL) together and fully vortexed for 30 seconds. Following that, Fe(OAc)2 water solution (135μM, 1mL) was added to the mixture. The ratio of each component is Tren-QDs: CS2: Fe(II)=1:242:314.The reaction solution was vigorous stirred overnight in darkness. After reaction, the product mixture was purified by dialysis in Sigma-Aldrich dialysis membrane against deionized water for one hour to remove unreacted reagents in order to improve the sensitivity of the Tren-QDs-Fe(II) complex. The proposed complex structure was shown Figure 4.14. The purified complex was sealed in PTFE vials and kept in darkness at room temperature for further dilution or applications. - 101 - S Fe S HN H N S Fe S N Fe S NH S S - NH S ZnS H N N S - S S Fe S S - CdSe NH S S Fe S HN N N H HN - S S S S S Fe HN N H N Fe S NH S S Fe Figure 4.14 Proposed surface structure of CdSe QDs capped by Tren ligands and their coordination complexes with Iron (II) ions. Tren-QDs-CS2-Fe(II) complex sensing hydrosulfide anion The purified Tren-QDs-CS2-Fe(II) complex was diluted 2 times into PBS 7.4 buffer. Hydrosulfide anion was provided by NaSH deionized water (0.189M) solution. Each time 20μL of NaSH solution was added into 3mL of the complex, the working solution was fully vortexed and left in darkness for 5 minutes before fluorescence intensity measurement. Totally 140μL NaSH solution was added into the complex. Figure 4.15 describes the effect of HS- anion on the fluorescence emission spectrum of Tren-QDs-CS2-Fe(II) complex. - 102 - Tren-QDs-CS2-Fe(Ⅱ) 146.9 140 130 Tren-QDs-CS2-Fe(Ⅱ)+1.25 mM NaSH 120 Tren-QDs-CS2-Fe(Ⅱ)+2.49 mM NaSH 110 Tren-QDs-CS2-Fe(Ⅱ)+3.70 mM NaSH 100 Tren-QDs-CS2-Fe(Ⅱ)+4.91 mM NaSH 90 Tren-QDs-CS2-Fe(Ⅱ)+6.10 mM NaSH 80 Tren-QDs-CS2-Fe(Ⅱ)+7.27 mM NaSH 70 Tren-QDs-CS2-Fe(Ⅱ)+9.57mM NaSH 60 50 40 30 20 10 6.3 500.0 510 520 530 540 550 560 570 580 590 600 610 620 630 640 650 660 670 680 692.5 nm Figure 4.15 Spectra of Tren-QDs-CS2-Fe(II) complex quenched by NaSH solution Figure 4.16 shows the Effect of HS- concentration on the fluorescence intensity of Tren-QDs-CS2-Fe(II) complex. From this result, it is clear that the Tren-QDs-CS2-Fe(II) complex exhibits sensitivity to HS- anion. After addition of NaSH solution, the fluorescence intensity was quenched moderately (42.2%) by HSanion. - 103 - Normalized Fluorescence Intensity 1 Normalized Fluorescence Intensity 0.95 0.9 0.85 1 0.95 0.9 0.85 0.8 0.75 0.7 0.65 0.6 0.55 0.5 y = -0.0604x + 0.9868 2 R = 0.9832 0 1 2 3 4 5 6 7 NaSH concentration(mM) 0.8 0.75 0.7 0.65 0.6 0.55 0.5 0 1 2 3 4 5 6 NaSH Concentration(mM) 7 8 9 10 Figure 4.16 Effect of HS- concentration on the fluorescence intensity of Tren-QDs-CS2-Fe(II) complex To further understand the quenching mechanism and improve the sensitivity to HSanion, we modified component ratio of the complex Tren-QDs-CS2-Fe(II). The synthetic procedure was the same as the previous step, but the amount of CS2 (1mL, 430mM in methanol) and Fe(OAc)2 (460mM, 1mL in water) was significantly increased. Hence, the final ratio of the Tren-QDs-CS2-Fe(II) complex reaction mixture is QDs: CS2: Fe(II)=1:1000:1069. When the reaction was stopped, the mixture was withdrew and purified and the purified new complex was also 2 times diluted in PBS 7.4 buffer and sent for NaSH quenching test. - 104 - Normalized Fluorescence Intensity 1.00 Normalized Fluorescence Intensity 0.90 0.80 0.70 1.00 0.90 0.80 y = -0.1332x + 1.0094 R 2 = 0.9742 0.70 0.60 0.50 0.40 0.30 0.20 0 0.60 2 4 6 NaSH concentration (mM) 0.50 0.40 0.30 0.20 0 1 2 3 4 5 6 7 8 9 10 NaSH Concentration (mM) Figure 4.17 Effect of hydrosulfide anion concentration on the fluorescence intensity of Tren-QDs-CS2-Fe(II) complex (excited at 400nm) From above Figure 4.17, the fluorescence of new Tren-QDs-CS2-Fe(II) complex was more effectively quenched (73.6%) after addition of same amount NaSH solution (0.189M, 0-140μL). By comparing the slope of quenching plots with previous complex in the 0 mM to 6 mM range, the change of slope from 0.0604 (Figure 4.18 inserted graph) to 0.1332 (Figure 4.18 inserted graph) clearly indicates the improvement of sensitivity to HS- anion. On the other hand, this result proves our successful removal of extra Fe(II) ions from the reaction mixture. The addition of NaSH solution immediately turned the colorless new Tren-QDs-CS2-Fe(II) into yellow-brown colored solution. The UV-Vis absorption of this new Tren-QDs-CS2-Fe(II) complex was investigated and results are shown in Figure 4.18. Before addition of NaSH (blue solid line), the - 105 - complex itself has a strong absorption peak at around 340 nm, which is the same as Tren-Fe(II) complex (Figure 4.13 ), but it has no major absorption from 400 nm to 600 nm, which is the Tren-QDs excitation (400 nm) and fluorescence emission (550 nm-590 nm)area. After addition of NaSH, however, the absorption drastically increases in the area below 500 nm, and it has slight increased the absorbance from 500 nm to 600 nm. From this absorption increase we speculate a Tren-QDs-CS2-Fe(II)-SH complex with strong inner filter effect on Tren-QDs was formed. This sulfide containing complex has strong absorbance at 400 nm and greatly reduced the excitation light of Tren-QDs and consequentially resulted in low fluorescence emission. But the absorption at around 600 nm (Figure 4.18 insert) could also play some role in the quenching mechanism. 0.025 3 0.02 Absorbance Absorbance 2.5 2 0.015 0.01 0.005 1.5 0 500 510 520 530 540 550 560 Wavelength (nm) 570 580 590 600 1 0.5 0 300 350 400 450 500 550 600 650 Wavelength (nm) Figure 4.18 UV-VIS spectrum of Tren-QDs-CS2-Fe(II) complex before (solid line)and after(dotted line) NaSH addition - 106 - To confirm this hypothesis, the experiment was repeated at the excitation wavelength 480 nm, at which the Tren-QDs-CS2-Fe(II)-SH complex does not have absorption peaks. The results shown in Figure 4.19 demonstrates the fluorescence emission of Tren-QDs-CS2-Fe(II) was also quenched at excitation wavelength 480 nm (42.2%), but the quenching was not as effective as the complex exited at 400 nm (73.6%). By this comparison, we can conclude that the addition of NaSH solution caused formation of Tren-QDs-CS2-Fe(II)-SH and this complex quench fluorescence of Tren-QDs by strong inner filter effect which blocks both its excitation and emission wavelength of Tren-QDs. 1 Normalized Fluorescence Intensity 0.9 0.8 0.7 0.6 Before NaSH addition Affter NaSH addition 0.5 0.4 0.3 0.2 0.1 0 Excited at 400nm Excited at 480nm Different Excitation Wavelength Figure 4.19 Quenching Tren-QDs-CS2-Fe(II) complex by NaSH at different excitation wavelength - 107 - Because Tren-QDs fluorescence emission is pH sensitive, the pH value of the complex solution was measured to exclude other possibilities of the emission quenching. After addition, the original PBS 7.4 buffer solution was turned to a pH 10.2 solution. Since our previous study shows Tren-QDs are stable in pH ﹥7.4 environment, we can conclude the pH change is not responsible for the quenching. Tren-QDs-CS2-Fe(II) was also found to be sensitive to another biologically important specie nitro monoxide. The preliminary study showed addition of 0.5mL saturated NO water solution (2× 10-6M) can efficiently quench Tren-QDs-CS2-Fe(II) by 66.7% (Figure 4.20). The quenching was at first attributed to the formation of Tren-QDs-CS2-Fe(II)-NO complex. But the pH value of final solution was found to be acidified to pH 2.7, at which Tren-QDs’ hybrid structure is not stable, and therefore makes the quenching mechanism complicated. 108.2 Tren-QDs-CS2-Fe(Ⅱ) complex 105 100 95 90 85 80 75 70 Tren-QDs-CS2-Fe(Ⅱ) complex+0.06 μM NO 65 60 55 50 Tren-QDs-CS2-Fe(Ⅱ) complex+0.12 μM NO 45 40 Tren-QDs-CS2-Fe(Ⅱ) complex+0.18μM NO 35 30 Tren-QDs-CS2-Fe(Ⅱ) complex+0.24 μM NO 25 20 Tren-QDs-CS2-Fe(Ⅱ) complex+0.28μM NO 15 10 7.3 500.0 510 520 530 540 550 560 570 580 590 600 nm 610 620 630 640 650 660 670 680 690 700.0 Figure 4.20 Spectra of Tren-QDs-CS2-Fe(II) complex quenched by nitric oxide solution - 108 - Tren-QDs-CS2-Fe(II) still requires investigation on application of other metal ions; further characterization methods, like FT-IR may provide us more information of the complex bonding; its components ratio could also be optimized to be a more sensitive sensing probe; and the development of other primary amine containing ligands other than Tren would also be necessary for more effective coordination of metal ions on QDs surface. Further investigation in our lab is on going to screen for possible alternatives on Tren-QDs-CS2-Metal system. We believe more transition metal ions like Fe(II) could be used to coordinate with Tren-QDs. Rational design and fabrication of these QD-metal ion complex as fluorescent probes would be promising approach to radical, hydrosulfide and NO sensing. 4.4 Conclusion Fluorescent quantum dots (QDs) are an attractive alternative to fluorescent organic dyes due to their broad excitation spectra, narrow symmetric and tunable emission spectra, and excellent photostability. The functionalization of QDs confers this promising labeling material unique properties which can be applied in special areas. The present chapter described the application of tris(2-aminoethyl)amine (Tren) functionalized quantum dots and its metal complex in ions and hydrosulfide anion sensing. Water-soluble Tren-QDs were found to be a kind of satisfactory selective - 109 - sensing fluorescence probe in physiological buffer solution. Fluorescence response of Tren-QDs to sixteen physiologically important transition metal ions (Zn(II), Cu(II), Ni(II), Co(III), Fe(III), Fe(II), Mn(III), Mn(II), Cr(III), Cr(II), VO2+, Ru(III), Ru(II), Cd(II), Ag(I) and Pd(II)) was investigated. The fluorescence intensity of Tren-QDs was significantly quenched by Co(III) and Cu(II) respectively. The fluorescence quenching by Co(III) could be explained by Förster resonance energy transfer between QDs and the surface Tren-Co(III) complex upon the coordination of Co(III) with Tren-QDs. Besides, the probable fluorescence quenching by Cu(II) can be explained by a combination of energy transfer (minor) and electron (major) effects. However, the other quenching pathway could be plausible, that is the chemical displacement of surface Cd(II) by Cu(II) and the CuSe particles which can be formed at the surface of CdSe core will quench the recombination fluorescence of Tren-QDs by facilitating non-radiative electron and hole annihilation. Apart from fluorescence quenching, fluorescence enhancement of Tren-QDs was achieved by Zn(II) and Cd(II) respectively which is attributed to some form of passivation of the surface trap states of QDs. Based on the distinct fluorescence response of Tren-QDs to different transition metal ions, Tren-QDs can be developed to be a sensitive fluorescent probe for transition metal ions detection. Furthermore, the Tren-QDs-Fe(II)hybrid materials were used as hydrosulfide anion (HS-) sensing. The fluorescence intensities of Tren-QDs-Fe(II) were further quenched significantly by NaSH. And preliminary study has indicated this quenching was due to the formation of excitation and emission blocking metal complex. Therefore, hybrid materials - 110 - could be developed as potential HS- sensors. In future work, the factors causing the further significant quenching of fluorescence of transition metal and Tren-QDs hybrid material would be investigated. Also, lifetimes study for Stern-Volmer quenching and characterization of Tren-QDs by TEM would be carried out. References - 111 - [1] Y. K. Yang, K. J. Yook, and J. Tae, Journal of the American Chemical Society, 2005, 127, 16760-16761. [2] A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M. Huxley, C. P. McCoy, J. T. Rademacher, and T. E. Rice, Chemical Reviews, 1997, 97, 1515-1566. [3] M. R. Bryce, B. Johnston, R. Kataky, and K. Toth, The Analyst, 2000, 125, 861-866. [4] R. H. Yang, W. H. Chan, A. W. M. Lee, P. F. Xia, H. K. Zhang, and K. A. Li, Journal of the American Chemical Society, 2003, 125, 2884-2885. [5] H. Mattoussi, J. M. Mauro, E. R. Goldman, G. P. Anderson, V. C. Sundar, F. V. Mikulec, and M. G. Bawendi, Journal of the American Chemical Society, 2000, 122, 12142-12150. [6] W. C. W. Chan, D. J. Maxwell, X. H. Gao, R. E. Bailey, M. Y. Han, and S. M. Nie, Current Opinion in Biotechnology, 2002, 13, 40-46. [7] W. Horst, Angew. Chem. Inter. Ed. 1993, 32, 41-53. [8] W. C. Chan, nbsp, W, and S. Nie, Science, 1998, 281, 2016-2018 (1998). [9] W. Chen, J. Z. Zhang, and A. G. Joly, J.Nanosci. Nanotech. 4, 919-947 (2004). [10] Y. F. Chen and Z. Rosenzweig, Ana. Chem 74, 5132-5138 (2002). [11] R. Eran, The Journal of Chemical Physics, 2001, 115, 1493-1497. [12]M. Bruchez, M. Moronne, P. Gin, S. Weiss, and A. P. Alivisatos, Science, 1998, 281, 2013-2016. [13] S. R. Cordero, P. J. Carson, R. A. Estabrook, G. F. Strouse, and S. K. Buratto, the Journal of Physical Chemistry B, 2000, 104, 12137-12142 . [14] S. J. Rosenthal, I. Tomlinson, E. M. Adkins, S. Schroeter, S. Adams, L. Swafford, J. McBride, Y. Wang, L. J. DeFelice, and R. D. Blakely, Journal of the American Chemical Society, 2002, 124, 4586-4594. [15] W. J. Jin, M. T. Fernandez-Arguelles, J. M. Costa-Fernandez, R. Pereiro, and A. Sanz-Medel, Chemical Communications, 2005, 883-885. [16] A. J. Sutherland, Current Opinion in Solid State and Materials Science, 2002, 6, 365-370. [17] K. A. Gattas-Asfura and R. M. Leblanc, Chemical Communications, 2003, 2684-2685. [18] H. Y. Xie, H. G. Liang, Z. L. Zhang, Y. Liu, Z. K. He, and D. W. Pang, Spectrochimica Acta Part a-Molecular and Biomolecular Spectroscopy, 2004, 60, 2527-2530. [19] W. J. Jin, J. M. Costa-Fernandez, R. Pereiro, and A. Sanz-Medel, Analytica Chimica Acta, 2004, 522, 1-8 . [20] J. G. Liang, X. P. Ai, Z. K. He, and D. W. Pang, Analyst, 2004, 129, 619-622. [21] J. L. Chen and C. Q. Zhu, Analytica Chimica Acta, 2005, 546, 147-153 . [22] M. T. Fernandez-Arguelles, W. J. Jin, J. M. Costa-Fernandez, R. Pereiro, and A. Sanz-Medel, Analytica Chimica Acta, 2005, 549, 20-25. [23] J. L. Chen, Y. C. Gao, Z. B. Xu, G. H. Wu, Y. C. Chen, and C. Q. Zhu, Analytica Chimica Acta, 2006, 577, 77-84. [24] S. Watanabe, M. Sonobe, M. Arai, Y. Tazume, T. Matsuo, T. Nakamura, and K. Yoshida, Chemical Communications, 2002, 2866-2867. [25] M. Laferriere, R. E. Galian, V. Maurel, and J. C. Scaiano, Chemical Communications, 2006, 257-259. [26] V. Maurel, M. Laferriere, P. Billone, R. Godin, and J. C. Scaiano, the Journal of Physical Chemistry B, 2006, 110, 16353-16358. - 112 - [27] D. Neuman, A. D. Ostrowski, A. A. Mikhailovsky, R. O. Absalonson, G. F. Strouse, and P. C. Ford, Journal of the American Chemical Society, 2008, 130, 168-175. [28] F. Dubois, B. Mahler, B. Dubertret, E. Doris, and C. Mioskowski, Journal of the American Chemical Society, 2007, 129, 482-483. [29] S. F. Wuister, I. Swart, F. van Driel, S. G. Hickey, and C. de MelloDonega, Nano Letter, 2003, 3, 503-507. [30] J. Chen, A. Zheng, Y. Gao, C. He, G. Wu, Y. Chen, X. Kai, and C. Zhu, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2008, 69, 1044-1052. [31] X. J. Wang, M. J. Ruedas-Rama, and E. A. H. Hall, Anal. Letter, 2007, 40, 1497-1520. [32] D. M. Willard, T. Mutschler, M. Yu, J. Jung, and A. Van Orden, Anal. Bioanal. Chem., 2006, 384, 564-571. - 113 - [...]... labeling in bioanalysis and sensing The nature of the ligands being coordinated to the QDs surface and the particular type of bonds which it forms with the nanocrystal surface atoms are of great importance to quantum dots researchers By ligand designing and fabrication, several important properties of quantum dots can be tuned for specific purpose: processibility, reactivity and stability All of these... nitric oxide solution 108 -X- Chapter 1 General Introduction 1.1 Quantum Dots and Its Properties 1.1.1 Introduction to quantum dots Quantum dots (QDs) are nanostructured semiconductor materials[1] These colloidal nanocrystalline semiconductors comprising elements from the periodic groups II-VI, III-V or IV-VI, are featured with roughly spherical and with typical sizes (diameter) in the range 1-12 nanometer... - Chapter 2 Thiolated Caffeic Acid Functionalized Quantum dots 2.1 Introduction Semiconductor nanocrystals (or quantum dots, QDs) have size-dependent fluorescent emissions wavelengths with high quantum yields and superior photostability comparing to organic dyes[1] Thus QDs have great potential in various biological imaging applications, including labeling cells and other biological components However,... visible signal when it reacts with free radicals or other species and serves as a fluorescent probe Our goal is to synthesize and purify thio-caffeic acid for quantum dot ligand exchange and try to replace the original hydrophobic trioctylphosphine oxide (TOPO) ligand of quantum dots with thio-caffeic acid 2.2 Experimental Section 2.2.1 Materials and instruments All solvents used were of reagent grade unless... Quantum dots ligand exchange with thio-caffeic acid The 100 nM quantum dots solution in 2mL chloroform and then added into 1mL 100 times equivalent amount of thio-caffeic acid diethyl ether solution dropwise This mixture was vigorously stirred under nitrogen for 12 hours at room temperature in darkness When the reaction was stopped, it was found the quantum dots precipitated out from solution The quantum. .. thiol ligands were found to undergo a photocatalytic oxidation using CdSe nanocrystals as photocatalysts and form the disulfides during the process But severity of this oxidation varies with different thiol ligands, and some of them can sustain long enough in solution for practical applications[ 19] Another disadvantage of Thiol ligands is that it can cause severe fluorescence quenching of quantum dots. .. from the particle, thus providing a visible signal of its occurrence In this work, we used derivatives of antioxidant caffeic acid and Trolox to study the electron transfer between small molecule ligands and CdSe/ZnS quantum dots 1.2.1 Ions and small molecules sensing Methods based on chemical or physical interactions between target chemical species and the surface of the nanoparticles are very simple... semi-conductor material and nanocrystal size -1- Semiconductor QDs are characterized by a band-gap between their valence and conduction electron bands (Figure 1) When a photon having an excitation energy exceeding the semiconductor band-gap is absorbed by a QD, electrons are promoted from the valence band to the high-energy conduction band The excited electron may then relax to its ground state by the... these nanoparticles behave differently from bulk solids due to quantum confinement effects [2,3] The result of quantum confinement are that the electron and hole energy states within the nanocrystals are discrete, but the electron and hole energy levels and therefore the band-gap is a function of the QDs diameter as well as composition[5] The band-gap of semiconductor nanocrystals increase as their size... neurotransmitter dopamine and CdSe/ZnS QDs[22]; Maurel et al reported a non-linear quenching effects of fluorescent quantum dots by nitroxyl free radicals TEMPO (2,2,6,6-tetramethylpiperidine-N-oxide free radical) and suggested the -7- mechanism would involve electron transfer from the conduction band to the nitroxide (a mild acceptor), and back electron transfer from the nitroxide to the valence band, effectively ... Introduction 1.1 Quantum Dots and Its Properties 1 1.1.1 Introduction to quantum dots 1.1.2 Quantum dots optical properties 1.2 Quantum Dots in Optical Sensing Applications 1.2.1 Anion and small molecules... Pergamon Press, Oxford, 1979 - 34 - Chapter Trolox Functionalized Quantum Dots 3.1 Introduction 3.1.1 Quantum dots and its synthesis Quantum dots (QDs) are spherical semiconductor nanoparticles... several ligands bounding to the surface of ZnS capped CdSe quantum dots (QDs) was studied Different ligands for quantum dots functionalization was synthesized and characterized by ESI-MS, NMR and FT-IR