Journal of Medical and Biological Engineering, 26(3): 131-135
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Fluorescence Properties of Colloidal CdSe/ZnS Quantum Dots with Various Surface Modifications Wu-Ching Chou*
Der-San Chuu
Hong-Sheng Lin2
Ruoh-Chyu Ruaan2
Chi-Tsu Yuan
Walter H. Chang1
Department of Electrophysics, National Chiao Tung University, HsinChu, 300, Taiwan, ROC Department of Biomedical Engineering, Chung Yuan Christian University, ChungLi, 320, Taiwan, ROC 2 Department of Chemical and Materials Engineering, National Central University, ChungLi, 320, Taiwan, ROC 1
Received 31 Mar 2006; Accepted 26 Jun 2006
Abstract Colloidal CdSe/ZnS quantum dots (QDs) were modified by various functional ligands to study the effect of ligand modification on the fluorescence emission wavelength, efficiency, and mechanism for their application in nano-bio-photonics. The fluorescence spectra were investigated by the time-integrated and time-resolved as well as spatial-resolved micro-fluorescence measurements. Fluorescence spectra and decay profiles were significantly changed by the introduction of various ligands. The spatial dependent fluorescence of colloidal QDs in the ambient environment was observed. Fluorescence intensity imaging of colloidal QDs attached to Mercaptopropionic acid (MPA) ligands show a doughnut-like shape. Keywords: Quantum dots, Surface modifications, Time-resolved fluorescence spectra, Time-integrated fluorescence spectra, Fluorescence image
Introduction Tunability in emission color, high emission efficiency and high photo-stability make the colloidal semiconductor QDs very attractive to scientists for their potential application in bio-photonics.[1-2] Colloidal QDs exhibit high emission efficiency due to the enhanced quantum confinement of a much smaller size of a few nanometers compare to the bulk exciton Bohr radius. However, due to a high surface to volume ratio, e.g. there are more than 30% surface atoms for a 4 nm QD, the surface states could quench the radiative emission and result in a fatal problem for the application in bio-photonics. In addition, for the in vivo bio-photonics application, the solubility of QDs in aqueous solution could be an important issue. The development of various techniques of surface modification to increase the solubility and maintain the excellent fluorescence properties is urgently needed. Recently, the original hydrophobic TOPO (trioctylphosphine oxide) surfactant were successfully replaced by the mercaptoacetic acid (MPA) ligands or intercalated comb-like polymers (maleic anhydride alt-1-tetradecene) to achieve nice solubility in aqueous solution and provide specific functionality.[3-4] * Corresponding author: Wu Ching Chou Tel: +886-3-5712121-56129; Fax: +886-3- 5725230 E-mail:
[email protected]
Colloidal QDs with different organic or inorganic surface modifications were also tested to improve emission properties.[5] Moreover, it was demonstrated that the confinement states of excitons are sensitive to the surface layer of colloidal QDs and the ambient environment [6]. Especially, lifetime constant was shown to be a sensitive parameter to its size [7], surface [8], and environments. Fluorescence decay profiles could be very different at various surroundings even though the emission peak positions are very close. Furthermore, in order to incorporate the nano-crystals into semiconductors devices [9], scientists would like to disperse QDs onto the substrate instead of the solvent. As nano-crystals form a thin film after solvent evaporation, the optical properties might be changed due to the variation in the surrounding environment. In this study, time-integrated, time-resolved, and spatial-resolved micro-fluorescence properties of colloidal CdSe/ZnS QDs with various surface modification onto a glass coverslip following solvent evaporation were investigated at room temperatures. Not only fluorescence spectra but also the decay profiles changed due to the introduction of various ligands. The fluorescence intensity images of colloidal QDs with MPA ligands show doughnut-like shapes. We found that smaller nano-crystals with a shorter decay lifetime prefer to arrange themselves at the surface of the doughnut-like spot.
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Wavelength ( nm ) Figure 1. Fluorescence spectra of colloidal CdSe/ZnS QDs with various surface modifications.
Experiments Colloidal CdSe/ZnS QDs with hydrophobic TOPO surface ligands were wet chemically synthesized by conventional methods.5 In order to obtain water-soluble colloidal QDs, two kinds of molecules, MPA and polymer, were used to modify the TOPO surface. The first one replaced the TOPO surfactant by MPA, which is a bi-functional molecule. One end of MPA molecule is hydrophilic carboxyl and the other is thiol. Thiol is used to bind to the surface of the ZnS shell, and carboxyl plays the role of hydrophilic. After original TOPO precipitation and purification in chloroform by adding methanol, colloidal CdSe/ZnS QDs with hydrophobic TOPO surface ligands were mixed with 1.0 M or 10 mM MPA for one day. Followed by the addition of 1.0 M or 10 mM 4-(dimethylamino)pyridine, which was dissolved in chloroform. After discarding supernatant, the precipitation of water soluble QDs dispersed in pH 7.4, 10 mM phosphate buffer with sonication for 30 mins. The second method is coating the colloidal CdSe/ZnS QDs, which has hydrophobic TOPO surface ligands, by polymer. Polymer and mono-dispersed QDs in chloroform were mixed and stirred. After solvent evaporation, bis(6-aminohexyl)amine in chloroform was added to cross-link the polymer shell. The solution was sonicated and the solvent was then evaporated. The solid was dissolved in a diluted TBE (Tris-Borate EDTA) buffer solution. After sonicating, the nanocrystals dissolved completely and the solution was filtered to remove the excess unbound polymer. To enhance the uptake efficiency of biological cells, we also coated additional HSA (Human serum album) protein around the surface of carboxyl QDs. A reaction mixture containing 0.36μM MPA or polymer coated CdSe/ZnS QDs, 0.72 μ M HSA, 0.68 mM EDC (1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride) and sulfo-NHS (N-hydroxysulfo-succinimide) in 10 mM phosphate buffer was prepared and kept at room temperature for 4 hrs. The precipitate is removed by centrifugation, and the HSA-QDs complexes were examined by SDS-PAGE (Sodium
dodecyl sulphate-Polyacrylamide Gel Electrophoresis) and purified by SEC (size exclusion chromatography). The addition of NaCl to MPA or polymer coated QD was used to test salt stability. The experimental setup of fluorescence measurements consists of three segments. The first part is a scanning confocal microscope equipped with a nanometers-accuracy piezo-scanner for spatial-resolved micro-fluorescence measurements. The second part is a ultra-fast laser pulse at a wavelength of 400 nm with 150 fs pulse duration and 76 MHz repetition rate provided by a Ti:sapphire laser opterating at 800 nm (pumped by a 4 W solid state laser at 532 nm) followed through a nonlinear BBO crystal for doubling frequency. The third part is a detection system including a low temperature charge couple devices attached to a spectrometer for fluorescence spectral analysis and a single photon avalanche photodiodes (SPAD) for fluorescence intensity and lifetime detection by use of time-correlated single-photon counting (TCSPC) techniques. Excitation laser pulse was focused by a microscope objective (100X, NA=0.7) to form a 1µm2 spot onto the samples. Fluorescence signals were collected through the same objective and guided into a low temperature CCD or SPAD after passing through a confocal pinhole.
Results and discussion In Figure 1, fluorescence spectra of colloidal CdSe/ZnS QDs with/without surface modification were shown. Colloidal CdSe/ZnS QD without surface modification has TOPO hydrophobic surfactant. In the current study, the QDs were on glass cover-slips following solvent evaporation at room temperature. In order to more accurately determine the energy position of the PL peak, the spectra were fitted by a Guassian formula. The PL peak positions for the TOPO, MPA, MPA/HSA, polymer, and polymer/HSA are 531, 534, 536, 525, and 530 nm, respectively. In addition, the PL peak wavelengths of the MPA and MPA/HSA modified QDs exhibit red-shift. On
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the other hand, the emission wavelengths of the polymer and polymer/HSA QDs present blue-shift. The red-shift is attributed to the extension of exciton wave-function to the outer MPA or MPA/HSA ligands. It results in a decrease in the confinement energy of exciton and the wavelength red-shift. In the case of polymer and polymer/HSA modified QDs, polymers were intercalated instead of covalently bound to QDs. Hence, the extension of exciton wavefunction to the outer intercalated polymers is not possible. However, the reason for blue-shift is not clear. Compared with the hydrophobic TOPO QDs, which have no surface modification, the PL intensities of the MPA, MPA/HSA, polymer, and polymer/HSA modified QDs are suppressed. However, when comparing MPA with MPA/HSA and polymer with polymer/HSA, the further modification by HSA improves the emission efficiency. After carefully examining the spectrum of the MPA ligand attached QDs, another feature at a longer wavelength of 650 nm is revealed, which comes from the surface trap states. By the further attachment of HSA, the emission from trap states vanished. In addition, the fluorescence intensity was enhanced by five times and the full width at half maximum narrowed. This is attributed to the passivation of the surface trap states by the introduced HSA protein. In Figure 2, the fluorescence decay profiles of colloidal QDs with various functional groups are shown. All spectra exhibit bi-exponential decay behavior. In the previous study, a fast decay lifetime of ~1 ns is caused by the recombination of delocalized exciton in the internal core states and a slow time constant of 5~12 ns varies with different surface modifications attributed to the localized exciton in the surface state [6]. In the current investigation, a shorter lifetime is insensitive to the surface modification, but the longer one alters dramatically with the different surface modification. The experimental results agree with the previous assignment which attributed the longer lifetime to the exciton localized at the surface. The respective lifetime for the MPA, MPA/HAS, polymer, and polymer/HSA modified CdSe/ZnS QDs are 13, 11, 10, and 5 ns. With the comparison of static and dynamic fluorescence
Figure 3. 10 µm by 10 µm fluorescence intensity image of the colloidal CdSe/ZnS QDs with MPA ligands modification.
spectra of Figure 1 and Figure 2, we discovered that the additional HSA protein modification not only enhance the fluorescence intensity but also shorten the emission lifetime. Figure 3 is a 10 µm by 10 µm fluorescence intensity image of the colloidal CdSe/ZnS QDs with MPA ligands modification. Aggregation of QDs is observed. For an aggregation size of over 0.5 µm, a doughnut-like image was observed. It implies QDs prefer to aggregate at the edge of a disk instead of at the center during the solvent evaporation. However, the doughnut-like image turns to a disk image after a few days of exposure in air. This implies that the residual surface charges of MPA modified QDs repel each other during the aggregation process. Finally, after being further exposed to air for a few days, residual surface charges were compensated. The image of the MPA modified QDs stays the shape of a disk. In the case of TOPO QDs without surface modification, no residual surface charge exists. Only a disk image instead of a doughnut-like image is observed. In order to study the size distribution in these aggregated QDs, spatial-resolved micro-fluorescence measurement was performed. Figure 4
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shows the fluorescence spectra of colloidal QDs obtained at different locations, (1) center, (2) surface and (3) a high concentration area (bright), marked by a circle in Figure 3. The respective decay lifetime profiles were shown in Figure 5. The short wavelength peaks of the fluorescence spectra are attributed to the band edge emission and the emission peaks at a longer wavelength arising from the surface state emissions. The PL peak wavelength of the band edge emission for QDs at the circle edge is situated at 520 nm, which is shorter than 530 nm of the band edge emission for QDs at the center. It implies that QDs of smaller size tends to diffuse away from the center during the solvent evaporation. As a result, the fluorescence decay lifetime obtained from the emission at the center is longer because a larger QD has a longer radiative lifetime.
Conclusion The static and dynamic fluorescence properties of colloidal CdSe/ZnS QDs with surface modification by MPA or intercalated comb-like polymer were studied at room temperature. The fluorescence intensities of colloidal QDs with MPA or intercalated comb-like polymer modification and further HSA protein over-coating were enhanced. In addition, the decay profiles are sensitive to the surface modification. The spatial dependent fluorescence of colloidal QDs in the ambient environment was observed. Fluorescence intensity imaging of colloidal QDs attached to MPA ligands show a doughnut-like shape. We conclude that carboxyl colloidal QDs with HSA surface ligands are suitable candidates for biological application.
Fluorescence Properties of Quantum Dots
Acknowledgement
[5]
This work was supported by MOE-ATU and the National Science Council under the grant numbers of NSC95-2112-M-009-047 and 94-2120-M-033-001.
[6]
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