Synthesis and Fluorescent Properties of CdSe Quantum Dots for the

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Abstract— As novel quantum fluorescent materials, QDs have longer term photo-stability, narrower emission range, stronger intensity, and broader UV excitation ...
Proceedings of the 13th IEEE International Conference on Nanotechnology Beijing, China, August 5-8, 2013

Synthesis and Fluorescent Properties of CdSe Quantum Dots for the Detection of Single Cells array* D. H. Ren, Y. Q. Xia, and Z. You 

Abstract— As novel quantum fluorescent materials, QDs have longer term photo-stability, narrower emission range, stronger intensity, and broader UV excitation spectra than that of normal fluorescent materials. Due to quantum dots’ (QD) quantum scale effect, QDs of different sizes can emit fluorescence of different colors with the same excitation light, making them suitable bio-probes in multiplexed detection, by which we can achieve multiplexed analysis of cells’ behaviors in real time. In this paper, we first synthesized CdSe QDs in ODE and TOPO system. To enlarge the fluorescent range and get more fluorescent colors, we utilized paraffin liquid as the solvent instead of QDE and oleic acid (OA) as the stabilizer instead of TOPO. Finally, we obtained CdSe QDs emitting colors varying from purple to red in a simple and green experimental condition, reaching the requirement of multiplexed detection. By this method, we synthesized high-quality QD probes for multi-analysis which is low cost, less contaminating and less dependent on equipments.

I. INTRODUCTION Cells isolation and patterning as well as the further intracellular analysis on single-cell scale are of great significance to biomedical research. The single cells chip can be an effective tool for the investigation of cells behavior. Also it can be employed for drug screening and tissue engineering. We have presented an approach for cells patterning based on bioMEMS (biomedical microelectromechanical systems) by generating a two-dimensional hexamethyldisilazane monolayer on the glass substrate as a protein adsorption template as well as the following poly(ethylene glycol) treatment to prevent the non-target adhesion, by which we got a template for the specific antibodies immobilization. Based on the binding between these antibodies and the cell surface immobilized ligands, we finally achieved a two-dimensional patterning of HL-60 cells, which can well match the designed * This work is supported by the National Natural Science Foundation of China (Project No: 61071002), National Program for Significant Scientific Instruments Development of China (Project No: 2011YQ030134), the Funds for State Key Laboratory of China and the Scientific Research Foundation for Returned Overseas Chinese Scholars. D. H. Ren is with the State Key Laboratory of Precision Measurement Technology & Instruments, Department of Precision Instrument, Tsinghua University, Beijing, 100084, China (corresponding author, phone: 86-10-62776000, e-mail: [email protected]). Y. Q. Xia is with the State Key Laboratory of Precision Measurement Technology & Instruments, Department of Precision Instrument, Tsinghua University, Beijing, 100084, China (e-mail: [email protected]). Z. You is with the State Key Laboratory of Precision Measurement Technology & Instruments, Department of Precision Instrument, Tsinghua University, Beijing, 100084, China (e-mail: [email protected]).

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template. Figure 1 demonstrates a cell patterning result for HL-60. Typical result for single cells isolation can be found in references [1] and [2].

Figure 1. A demonstration result of HL-60 cells patterning

Fluorochrome is very important tool in cellular and biochemical analysis. However, the peak emission spectrum and the excitation spectrum peak are close in common organic dyes. Also the asymmetry of the peak shapes and the "trailing" phenomenon are serious in such materials. These lead to the overlap of the different fluorescence emission peaks. It is hard to generate a variety of fluorescence under one excitation light. Moreover, the brightness is relatively low and there is serious light fade in common organic dye. Therefore, they are difficult to meet the requirement of high throughout analysis for thousands of proteins, genes and other biochemical substances. As novel quantum fluorescent materials, QDs have longer term photo-stability, narrower emission range, stronger intensity, and broader UV excitation spectra than that of normal fluorescent materials. Especially, QDs of different sizes can emit fluorescence of different colors with the same excitation light, making multiplexed labeling easily [3]. Therefore, QDs are useful qualitative and quantitative probes, which can be used as suitable probes for DNA and protein analysis in real time [4][5]. QDs are often synthesized in water or oil-soluble system, where which synthesized in oil-soluble system have better spectral properties. However, as they are oil-soluble, they should be modified to be water-soluble when used in DNA or protein detection. Usually, there are four methods for hydrophilic modification, which are compounds [6], silanization [7], microspheres [8] and electrostatic interaction [9]. In 1989 and 1993, using Cd(CH3)2, Stigerwald [10] and Bawendi [11] synthesized CdS, CdSe and CdTe QDs in oil-soluble systems respectively. In the following years, Alivisators [12][13], Hines [19] and Peng [14][15] improved the synthesizing methods. However, during the synthesizing, it is easy to explode, leading to a though experiment condition in QDs preparation. To eliminate the danger of explosion, in

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2001, Peng [16][17] used CdO, Cd(Ac)2 and CdCO3 instead of Cd(CH3)2 to gain high-quality QDs in QDE and TOPO system. In 2005, Deng [18] used paraffin liquid and QA instead of expensive and poisonous QDE and TOPO, achieving a low-cost and green method to synthesize CdSe QDs.

excitation light, we can excite all of these different QDs. Moreover, According to the emission spectrum (Figure 3), we can find that the emission wavelength increases from 628.5nm to 638.5nm as the reaction time increases.

In this paper, we first synthesized CdSe QDs in ODE and TOPO system. To enlarge fluorescent range and get more fluorescent colors, we utilized paraffin liquid as the solvent instead of QDE and oleic acid (OA) as the stabilizer instead of TOPO. By this improved method, we obtained CdSe QDs emitting colors varying from purple to red in a simple and green experimental condition, reaching the requirement of multiplexed detection.

II. SYNTHESIS OF CDSE QDS IN ODE AND TOPO SYSTEM The materials and chemicals employed in this paper (Part Ⅱand part Ⅲ) include: Selenium (Se) powder, Cadmium oxide (CdO), octadecene (ODE) (analytical grade), trioctylphosphine oxide (TOPO) (analytical grade), tributyl phosphate (TBP) (analytical grade), octadecylamine (ODA) (analytical grade) oleic acid (OA) (analytical grade), cyclohexane (analytical grade), paraffin liquid (analytical grade).

Figure 2. Excitation spectrum of sample A, B, and C

A. Procedures for the Synthesis of CdSe QDs First, we mixed 0.6mmol CdO, 0.8ml OA and 10ml ODE and heated the mixture at 200℃ under nitrogen. Until all CdO dissolved, we got Cd stock solution. Second, we mixed 0.036mmol Se powder and 10ml ODE, and heated them slowly to 250℃ with rapid stirring under nitrogen until all Se powder dissolved, by which we got Se stock solution. Third, we added 1.5g TOPO and 4.5g ODA to the Cd stock solution, and heated the mixture with rapid stirring under 250℃. After all TOPO and ODA dissolved, we injected the Se stock solution into the heating mixture, leading to a red homogeneous solution. To let the CdSe QD grow, we kept heating at 250℃. After synthetizing, we washed the QDs in cyclohexane and airproofed them in cyclohexane at 4℃. B. Influence of Reaction Time to QDs’ Fluorescent Properties QDs’ size is an important parameter to QDs’ fluorescent properties, especially their fluorescent colors. Since the reaction time determines the growth time of QDs and the ligand TBP affects the growth speed of QDs, these two reaction parameters are important to QDs’ fluorescent properties. First, we investigated the influence of the reaction time to QDs’ fluorescent properties. We synthesized CdSe QDs in 50s, 200s and 350s (Sample A, B, C, respectively) without TBP, and then measured these QDs’ excitation spectrum and emission spectrum. According to the broad range of excitation spectrum (Figure 2), we can conclude that just under one

Figure 3. Emission spectrum of sample A, B, and C

C. Influence of Ligand TBP to QDs’ Fluorescent Properties Second, we consider the influence of TBP to QDs’ fluorescent properties. We synthesized CdSe QDs still in 50s and 200s with 1ml TBP (Sample D and E, respectively), and then measured these QDs’ excitation spectrum and emission spectrum. According to the broad range of excitation spectrum (Figure 4), we can conclude that we can excite all of these QDs with one excitation light. According to the emission spectrum (Figure 5), we can find that both in 50s and 200s, QDs emission wavelength increased after adding TBP, that is, from 628.5nm to 629.5nm and from 630.5nm to 634.5 nm, respectively.

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synthetizing, we washed the QDs in cyclohexane and airproofed them in cyclohexane at 4℃. B. Influence of the Reaction Time to QDs’ Fluorescent Properties With the improved method, reaction time still determines the growth time of CdSe QDs, affecting the fluorescent properties of QDs. To study the influence of reaction time, we synthetized CdSe QDs in different time (from 10s to 30 minutes) under 200 ℃ respectively, and observed their fluorescent colors under UV light (emission wavelength: 350nm). From the fluorescent colors of these six QDs samples, we found that the colors range from purple to yellow, reaching a boarder spectrum range. Meanwhile, the longer the reaction time is, the longer the QDs’ emission wavelength is, which can be easily explained according to the reaction process. Since the growth time of QDs is equal to the reaction time, when the reaction time gets longer, the diameters of QDs get bigger, leading to a red-shift of QDs’ emission wavelength. However, by comparing QDs’ fluorescent color to the lights’ wavelength, we also found that as the time goes by, the growth speed of QDs is decreasing, so is the speed of emission wavelength’s red-shift. Specially, when we increased the reaction time to 1.5h, the fluorescent color of QDs still remained yellow. To gain a longer fluorescent range, only increasing reaction time is not enough. Therefore, we should try to increase the growth speed of QDs.

Figure 4. Excitation spectrum of sample D and E

C. Influence of the Reaction Fluorescent Properties Figure 5. Emission spectrum of sample A and D, B and E

However, synthetizing QDs in ODE and TOPO system by above methods, the emission wavelength ranges only from 628.5 to 638.5nm, all in red spectrum, which are difficult to differentiate with the naked eye. To gain QDs of other fluorescent colors, we should largely shorten the reaction time. But it also brings difficulties to control QDs’ size in experiment. Therefore, with this method, it is hard to gain a broad fluorescent color range of QDs, which cannot meet the requirement of multiplexed detection. Moreover, the cost of chemicals such as ODE and TOPO are high and TOPO is also poisonous. III. SYNTHESIS OF CDSE QDS IN PARAFFIN LIQUID AND OA A. Procedures for the Synthesis of CdSe QDs First, we mixed 10mmol CdO, 10ml OA and 20ml paraffin liquid and heated the mixture at 150 ℃ until all CdO dissolved, by which we got Cd stock solution. Second, we mixed 0.05mmol Se powder and 30ml paraffin liquid, and heated them slowly to 200 ℃ with rapid stirring under nitrogen. Until all Se powder dissolved, we got Se stock solution. Then, we injected 3ml Cd stock solution into the heating mixture, leading to a red homogeneous solution. To let the CdSe QD grow, we kept heating at 200℃. After

Temperature

to

QDs’

As mentioned above, to gain CdSe QDs of a larger fluorescent color range in a shorter reaction time, we should increase the growth speed of QDs. During QDs synthesizing, there are two parameters, which can be easily controlled and can influence the growth speed of QDs, that is, the reaction temperature and the rate of Cd to Se. First, we considered the influence of reaction temperature to QDs’ fluorescent properties. We synthetized CdSe QDs under 250℃ instead of 200℃ in different time (from 10s to 30 minutes) respectively, and observed their fluorescent colors under UV light (emission wavelength: 350nm). With the same reaction time, the emission wavelength of QDs synthetized under 250℃ is longer than that of 200℃. Especially, when the reaction time is 1h, we can gain QDs of red fluorescence under 250℃. Therefore, we can conclude that as the reaction temperature increases, the growth speed of QDs also increases, reaching a red-shift of their emission wavelength. Although we can gain QDs of red fluorescence under 250 ℃, the reaction time is still long, sometimes even more than 1h. However, if only increasing the reaction temperature, a very high temperature is needed to synthetize QDs of a long emission wavelength in a short time, which also increases the

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danger of the experiment. Therefore, we should also figure out other approaches to solve this problem. D. Influence of the Rate of Cd to Se to QDs’ Fluorescent Properties Here, we considered the influence of the rate of Cd/Se to QDs’ fluorescent properties. With the rate of Cd/Se 2 and 4, we synthetized CdSe QDs in different time (from 10s to 30 minutes) under 200℃, and observed their fluorescent colors under UV light (emission wavelength: 350nm). With the same reaction time, the emission wavelength of QDs synthetized when the rate is 4 is longer than that of 2. Especially, when the reaction time is 10min, we can gain orange fluorescence QDs with the rate of 4, while with the rate of 2, the fluorescence is green. Therefore, by comparison, we can greatly increase the growth speed of QDs by raising the rate of Cd/Se. According to above studies of reaction parameters’ influence to QDs’ fluorescent properties, we can increase the growth time by raising the reaction time, and increase the growth speed by raising the reaction temperature and the rate of Cd/Se. Therefore, by changing these three parameters, we can easily control the fluorescent colors of QDs, achieving multicolor QDs whose emission fluorescence ranges from purple to red under a simple experimental condition and in a short reaction time. Figure 6 shows the multicolor QDs with wide emission fluorescence range synthesized in paraffin liquid and OA by adjusting above parameters.

Figure 6. Multicolor QDs with wide emission fluorescence range synthesized in paraffin liquid and OA

IV. CONCLUSION In this paper, we first gained CdSe QD in ODE and TOPO system. However, the emission wavelength ranges only from 628.5 to 638.5nm, all in red spectrum, which cannot meet the requirement of multiplexed detection. Moreover, the cost of chemicals such as ODE and TOPO are high and TOPO is poisonous. Therefore, we use paraffin liquid as the solvent instead of QDE and oleic acid (OA) as the stabilizer instead of TOPO. By this improved method, we obtained CdSe QDs emitting colors varying from purple to red in a simple and green experimental condition, reaching the requirement of multiplexed detection. In the following studies, to make these oil-soluble CdSe QDs hydrophilic, we will modify CdSe QDs, making them applicable in multiplexed DNA and protein detection.

ACKNOWLEDGMENT We would like to thank Professor Yinye Wang and Dr. Xiaoyan Liu of Beijing University Health Science Center for the cell supports of HL-60. The authors also thank Professor Jinying Yuan of the Department of Chemistry of Tsinghua University for her help of the centrifuge. REFERENCES [1] D. H. Ren, M. Y. Cui, J. Wang, Y. Q. Xia, Z. You, “A single cells patterning approach for human promyelocytic leukemia cells”, 3rd International Conference of CSMNT, 2012. 11. [2] D. H. Ren, Z. You, N. Li, C. M. Ho, “A bioMEMS based cells array chip for drug screening”, 4th TU-SNU-UT Joint Symposium, Tokyo, Mar. 2010. [3] M. Y. Han, X. H. Gao, J. Z. Su and S. M. Nie, “Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules,” Nature Biotechnology, vol. 19, pp. 631-635, Jul. 2001. [4] Dubertret, P. Skourides, D. J. Norris, et al., “In vivo imaging of quantum dots encapsulated in phospholipid micelles,” Science, vol. 298, pp. 1759-1762, Nov. 2002. [5] U. Resch-Genger, M. Grabolle, S. Cavaliere-Jaricot, R. Nitschke, T. Nann, “Quantum dots versus organic dyes as fluorescent labels,” Nature Method, vol. 5, pp. 763–775, Aug. 2008. [6] W. C. W. Chan and S. M. Nie, “Quantum dot bioconjugates for ultrasensitive nonisotopic detection,” Science, vol. 281, pp. 2016-2018, Sep. 1998. [7] P. Alivisatos, “Semiconductor clusters, nanocrystals, and quantum dots,” Science, vol. 271, no. 5251, pp. 933-937, Feb. 1996. [8] X. H. Gao, T. Nie, “Doping mesoporous materials with multicolor quantum dots,” J. Phys. Chem. B, vol. 107, no. 42, pp. 11575-11578, Sep. 2003. [9] H. Mattoussi, J. M. Mauro, E. R. Goldman, et al., “Self-assembly of CdSe−ZnS quantum dot bioconjugates using an engineered recombinant protein,” J. Am. Chem. Soc., vol. 122, no. 49, pp. 12142-12150,Nov. 2000. [10] S. M. Stuczynski, J. G. Brennan and M. L. Steigerwald, “Rmation of metal-chalcogen bonds by the reaction of metal-alkyls with silyl chalcogenides,” Inorganic Chemistry. vol. 28, no. 25, pp. 4431-4432, Dec. 2009. [11] B. Murray, D. J. Norris and M. G. Bawendi, “Synthesis and characterization of nearly monodisperse CdE (E = sulfur, selenium, tellurium) semiconductor nanocrystallites,” J. Am. Chem. Soc., vol. 115, no. 19, pp. 8706-8715, Sep. 1993. [12] J. E. B. Katari, V. L. Colvin and A. P. Alivisatos, “X-ray photoelectron spectroscopy of CdSe nanocrystals with applications to studies of the nanocrystal surface,” J. Phys. Chem., vol. 98, no. 15, pp. 4109-4117, Apr. 1994. [13] X. G. Peng, J. Wickham and A. P. Alivisatos, “Kinetics of II-VI and III-V colloidal semiconductor nanocrystal growth: “Focusing” of size distributions,” J. Am. Chem. Soc., vol. 120, no. 21, pp. 5343-5344, May. 1998. [14] M. A. Hines, S. P. Guyot, “Synthesis and characterization of strongly luminescing ZnS-capped CdSe nanocrystals,” J. Phys. Chem, vol. 100, no. 2, pp. 468-471, Jan. 1996. [15] Z. A. Peng and X. G. Peng, “Formation of high-quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor,” J. Am. Chem. Soc., vol. 123, no. 1, pp. 183-184, Dec. 2000. [16] Z. A. Peng and X. G. Peng, “Mechanisms of the shape evolution of CdSe nanocrystals,” J. Am. Chem. Soc., vol. 123, no. 7, pp. 1389-1395, Jan. 2001. [17] L. Qu, Z. A. Peng and X. G. Peng, “Alternative routes towards high quality CdSe nanocrystals,” Nano Letters, vol. 1, no. 6, pp. 333-337, Jun. 2001. [18] Z. T. Deng, G. Li, F. Q. Tang, B. S. Zou, “A new route to zinc-blende CdSe nanocrystals: Mechanism and synthesis,” J. Phys. Chem. B. vol. 109, no. 35, pp. 16671-16675, Aug. 2005.

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