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Chemiluminescence resonance energy transfer in the luminol–CdTe quantum dots conjugates
ARTICLE IN PRESS Journal of Luminescence 130 (2010) 995–999
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Chemiluminescence resonance energy transfer in the luminol–CdTe quantum dots conjugates Zheng Li a,b, Yongxian Wang a,n, Guoxin Zhang a, Wanbang Xu c, Yanjiang Han a,b a
Radiopharmaceutical Centre, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China Graduate University of Chinese Academy of Sciences, Beijing 100039, China c Product and Quality Supervision and Testing Institute, Yueyang Bureau of Quality and Technical Supervision, Hunan 414000, China b
a r t i c l e in fo
abstract
Article history: Received 19 April 2009 Received in revised form 11 January 2010 Accepted 15 January 2010 Available online 25 January 2010
The luminol–CdTe quantum dots (QDs) conjugates were prepared through the reaction between –NH2 and –COOH. The resonance energy transfer between chemiluminescence donor (luminol–H2O2 system) and quantum dots (QDs, with different emission peaks) acceptors (CRET) was investigated. The luminescence of QDs in luminol–QDs conjugates in the process of CRET was influenced by the molar ratio of luminol/QDs. It could reach higher luminescence intensity while the luminol/QDs value was 1/1. Quantum yield of QDs and overlapping areas between the emission spectrum of luminol and adsorption spectrum of QDs played important roles in the CRET efficiency of luminol–QDs conjugates. The higher CRET efficiency (21.2%) was observed when the 540 nm QDs were used as acceptors. This work will offer helpful knowledge for the CRET studies based on QDs. & 2010 Elsevier B.V. All rights reserved.
Keywords: CRET CdTe quantum dots Luminol Luminescence
1. Introduction ¨ ‘‘Forster resonance energy transfer’’, termed in 1948 by ¨ German scientist Forster [1], describes an energy transfer mechanism between two chromophores. A donor chromophore in exciting state can transfer energy by a nonradiative, long-range dipole–dipole coupling mechanism to an acceptor chromophore in close proximity (typically o10 nm). When both molecules are fluorescent, the term ‘‘fluorescence resonance energy transfer’’ (FRET) is often used, though the energy is not actually transferred by fluorescence. Basically, FRET efficiency (E) depends on (1) the distance between the donor and the acceptor and (2) the degree by which the donor’s emission spectrum overlaps the acceptor’s absorption spectrum. For a definite donor–acceptor pair, due to the dipole–dipole coupling mechanism, E is decided by E ¼ ½1 þ ðr=R0 Þ6 1 ¨ where r is the donor–acceptor distance and R0 is the Forster distance at which E=50% for this pair of donor and acceptor. Therefore, FRET is an ideal technique to study molecular dynamics in biophysics and biochemistry, such as DNA conformational changes [2], protein conformational changes [3] and protein– protein interactions [4,5]. As a fluorescence material, II–VI and III–V semiconductor quantum dots (QDs) have excellent optical and electrical propern
Corresponding author. Tel.: + 86 21 59556884. E-mail address: [email protected] (Y. Wang).
0022-2313/$ - see front matter & 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2010.01.013
ties, with broader excitation spectrum, narrower emission bandwidth and less tendency to photobleach than organic dyes, allowing simultaneous excitation of particles of different sizes at a single wavelength because of the size-dependence of the fluorescence wavelength [6–8]. Due to these advantages over organic dyes in FRET-based studies, QDs have been widely applied to FRET as donor [9–14] and acceptor [15], such as gene-based fluorescent probes [16], optical biosensor [17], FRET microscopy [18] and proteolytic activity monitored [19]. Recently, the QDs conjugates used in bioluminescence resonance energy transfer (BRET) [20,21] in the absence of external excitation, which is suitable for multiplexed in vivo imaging, have developed fast. It could overcome the disadvantage of QDs used in in vivo imaging because of strong background autofluorescence [22] and absorption or scattering of optical photons [23]. Huang et al. [24] and Wang et al. [25] investigated the chemiluminescence resonance energy transfer (CRET), which is similar to BRET, by using luminol–H2O2 system as energy donor and quantum dot as acceptor. CRET, which occurs by oxidation of a luminescent substrate without an excitation source, could reduce dramatically the background autofluorescence and fluorescence bleaching. Hence, it is a promising technique in biochemical application. In this paper, we report a CRET process with the CdTe QDs as acceptor and the chemiluminescence of luminol as donor. Unlike Refs. [24,25], we do not adopt the QDs–HRP (horseradish peroxidase) conjugates but link the CdTe QDs with luminol directly. Luminol is used to excite CdTe QDs through the luminol/ hydrogen peroxide CL reaction in the presence of NaClO [26]. The chemiluminescence specta of luminol–QDs conjugates were
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investigated. The results showed that the molar ratio of luminol/ QDs and different QDs acceptors would influence the process of CRET dramatically.
was carried out on a ThermoNicolet Avatar 370 Fourier Transform Infrared Spectrometer in the 500–4000 cm 1 range. TEM images were taken by a Hitachi H-800 transmission electron microscope.
2. Experimental
2.2. Preparation of TGA-capped CdTe QDs and luminol–QDs conjugates
2.1. Materials and apparatus Luminol was purchased from Yacoo Chemical Reagent Co. Ltd. Te powder; CdCl2 2.5 H2O and 1-[3-(dimethylamino)propyl]-3ethylcarbodiimide methiodide (EDC) were from Aldrich. Thioglycolic acid (TGA), NaBH4, H2O2 and NaOH were from Sinopharm Chemical Reagent Co., Ltd. All other chemicals and materials were of analytical grade. UV–vis absorption spectrum was obtained by using a Hitachi U-3010 spectrophotometer. Fluorescence spectrum was achieved with a Hitachi F-4500 FL spectrophotometer at room temperature. Chemiluminescence of luminol and the CRET spectrum of the luminol–QDs system were measured by using the Hitachi F-4500 FL spectrophotometer while the excitation light was blocked. FTIR
TGA-capped CdTe QDs were synthesized according to the procedure described in Ref. [27] with some changes. Briefly, NaHTe solution, prepared from Te powder and NaBH4, was added to N2-saturated CdCl2 solution in the presence of thioglycolic acid (TGA). The molar ratio of Cd2 + :Te2 :TGA was fixed at 1.0:0.5:2.0. Then, the solution was adjusted to pH9.0–9.5 with 1 M NaOH solution. After mixing, the reaction solution was heated to 100 1C and refluxed for different times to prepare TGA-capped CdTe QDs with different sizes. Luminol was conjugated to TGA-capped CdTe QDs using EDC as a coupling reagent. Firstly, 1 mL of as-prepared TGA-capped CdTe QDs was precipitated with NaOH and methanol, and then they were isolated by centrifugation and dried in a vacuum drying
Fig. 1. Preparation of luminol–CdTe QDs conjugates.
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Wavenumber(cm-1) Fig. 2. FTIR spectrum of TGA-capped CdTe QDs, luminol and luminol–CdTe QDs conjugate: (a) luminol–CdTe QDs conjugate; (b) TGA-capped CdTe QDs and (c) luminol.
ARTICLE IN PRESS Z. Li et al. / Journal of Luminescence 130 (2010) 995–999
Nomalized intensity (a.u.)
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stretching vibrations of C–N and benzene ring can be found in Fig. 2(c) also. The band of N–H changes to sharp and there is only a C= O stretching vibration at 1680 cm 1 in Fig. 2(a) because there is only secondary amide in the luminol–CdTe QDs conjugate due to the reaction. Furthermore, the band of C–N and benzene ring can still be found in Fig. 2(a). However, the C–S (1018 cm 1) and CH stretching vibrations appear and there is no stretching vibration of OH compared to Fig. 2(b). The analysis of the FTIR spectrum of TGA-capped CdTe QDs, luminol and luminol–CdTe QDs conjugate confirms that the luminol–CdTe QDs conjugate is prepared successfully, which could insure the accomplishment of CRET in luminol–CdTe QDs conjugate.
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Fig. 3. The CL spectrum of luminol–QDs conjugates with different luminol/QDs molar ratios: (a) luminol/QDs= 2/1; (b) luminol/QDs= 1.5/1; (c) luminol/QDs= 0.5/1 and (d) luminol/QDs= 1/1.
3.2. Absorption and emission spectrum of QDs and luminol–QDs conjugates
As shown in Fig. 1, the –COOH on the surface of CdTe QDs could react with –NH2 of the luminol in the presence of EDC and form a steady amido link. The FTIR spectrum of TGA-capped CdTe QDs is shown in Fig. 2(b), in which two intense bands are seen at 3240 and 1710 cm 1, respectively. Since OH and C= O stretching vibrations are known to be generally intense in FTIR spectrum, the intense bands at 3294 and 1710 cm 1 can readily be assigned to the OH and C= O oscillator of TGA. Two weak bands also appear at 3006 and 2860 cm 1, where CH stretching vibrations are
Luminol is linked to CdTe QDs through –COOH on the surface of QDs. So, the amount of luminol bonded with QDs may have some influences on the luminescence of QDs. The luminol H3BO3– KOH buffer solution with different concentration was added into QDs powders to prepare luminol–QDs conjugates with different luminol/QDs molar ratio. The CL spectra of the luminol–QDs conjugates are shown in Fig. 3. Similar to the QDs–HRP–luminol system [24,25], an efficient CRET was observed in luminol–QDs conjugates. The chemiluminescence luminescence of luminol did not shift obviously. However, the luminescence of QDs in luminol–QDs conjugates was intensively dependent on the luminol/QDs molar ratio, which has not been reported before. The luminescence intensity of QDs is weaker while the luminol/ QDs value is lower because the amount of luminol bonded with QDs is smaller. In this case, the energy of chemiluminescence luminescence is lower and the corresponding energy transferred to QDs nonradiatively is not enough to engender brighter fluorescence. However, it would also lead to a weaker luminescence of QDs while the luminol/QDs value is higher. The reasons may be that the particle sizes of luminol–QDs conjugates are larger and they would aggregate together, which could be seen in Fig. 4, when the QDs capped with too much of luminol. The superfluous luminol would be adsorbed on the surface of QDs simply, blocking the absorption and emission (luminescence) of energy of QDs. The luminescence intensity of QDs is highest while the luminol/QDs value is 1/1. Moreover, the luminescence emission’s peak position of QDs was slightly blue shifted with the increase of luminol/QDs value. The possible reactions, which lead to the CL, are described by the following reactions:
expected. On the other hand, the weak band at 1030 cm 1 can be assigned to a C–S stretching vibration. In the spectrum of luminol (Fig. 2(c)), the band from 3500 to 3100 cm 1 is almost certainly resulted from the N–H stretching vibration of NH2 and NH. There are also C =O stretching vibrations at 1680 and 1690 cm 1 of primary and secondary amides. Besides, the
Excited state luminol–QDs would advance the irreversible photooxidation of QDs. The amount of excited state luminol–QDs is larger when there are more luminol on the surface of QDs. So, the irreversible photooxidation of QDs would occur more frequently. As a result of photooxidation, macroscopic oxide layers were created and the effective QDs size was reduced. The
oven. 1 millilitre of luminol H3BO3–KOH buffer solution with different concentrations and 0.01 g of EDC were added into asprepared QDs powders to prepare luminol–QDs conjugates with different luminol/QDs molar ratio. Then, the mixture was stirred under room temperature for 2 h. Finally, the prepared solutions were purified by precipitating with NaOH and methanol, centrifuging to remove superfluous luminol and EDC. Then, a few drops of 5.0 10 3 mol/L H2O2 and NaClO solution were added into the luminol–CdTe QDs conjugates solution to engender chemiluminescence.
3. Result and discussion 3.1. Infrared spectrum of the luminol–QDs conjugates
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Fig. 4. TEM images of luminol–QDs conjugates: (a) luminol/QDs= 1/1 and (b) luminol/QDs = 2/1.
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Fig. 5. Absorption spectrum of CdTe QDs at different emission peaks and CL emission spectrum of luminol.
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Fig. 7. PL emission spectrum of CdTe QDs (emission peaks at 530, 540, 550, 565, 575, 590 and 605 nm) of different sizes; lex = 440 nm.
0.8 Table 1 Quantum yield and CRET efficiency of different CRET acceptors.
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Fig. 6. The CL emission spectrum of luminol–CdTe QDs conjugates. Seven different sized CdTe QDs (with emissions at a: 530, b: 540, c: 550, d: 565, e: 575, f: 590 and g: 605 nm) were used as acceptors.
new surface defects could also be generated by oxidation of the QDs surface. Hence, the luminescence intensity decreased and blue shift could be observed [26,28]. As Fig. 5 shows, there are overlapping areas between the absorption spectrum of CdTe QDs with different sizes and emission spectrum of luminol, which would make the process of CRET possible. So, we investigated the CRET between luminol
a The QY was measured according to the literature [29]. Solutions of both standard (quinine sulfate) and samples (CdTe QDs) were adjusted to exhibit the low absorption intensity at the excitation wavelength of 350 nm. b CRET efficiency was evaluated by equation Ee =FQDs/Fcon; here Ee is the estimate efficiency of CRET, FQDs is the integral area of QDs emission spectrum of luminol–QDs conjugate, take for example, 530 nm QDs then FQDs is the integral area from 520 to 700 nm in the spectrum of luminol–QDs conjugate, and Fcon is the integral area of the whole spectrum of luminol–QDs conjugate.
and QDs conjugates in the H2O2–NaClO system and the results obtained are shown in Fig. 6, in which seven different sized QDs were used as acceptors. All of them emerge as two peaks, the first of which almost keeps at the same position (440 nm) and the second possessess different peak positions and intensities.
ARTICLE IN PRESS Z. Li et al. / Journal of Luminescence 130 (2010) 995–999
Compared with Figs. 5 and 7, we could find that they are the emission peaks of luminol and QDs excited by CL of luminol. It is confirmed that an efficient CRET occurred in luminol–QDs conjugates in this process. The luminescence emission peak position of QDs in luminol–QDs conjugates was determined by the property of single QDs. However, the luminescence intensity would be influenced by luminol and QDs jointly. The CRET efficiency of luminol–QDs conjugates was taken to assess the difference of different luminol–QDs conjugates. The CRET efficiency of different luminol–QDs conjugates is shown in Table 1. We found that the differences of CRET efficiency are contributed to the quantum yield of QDs, chiefly. It would reduce with the decrease of the quantum yield of QDs, which has been reported in Ref. [25]. Furthermore, the CRET efficiency has something to do with the overlapping areas between emission spectrum of luminol and adsorption spectrum of QDs (Fig. 5). So, although the quantum yield of QDs with its emission peak at 550 nm (550 nm QDs) is higher, the CRET efficiency is higher while the CRET acceptor is 540 nm QDs because the overlapping area between emission spectrum of luminol and adsorption spectrum of 540 nm QDs is larger than that of 550 nm QDs.
4. Conclusion CRET is a potential technique for in vivo imaging because it does not need an exciting light source and could dramatically reduce the fluorescence bleaching and lessen the autofluorescence of the system. The synthesis and CRET of luminol–QDs conjugates have been, for the first time, reported in this paper. The experiment results showed that CRET could occur in the luminol–QDs conjugates in the system of H2O2–NaClO similar to the QD–HRP–luminol system. The CRET efficiency of luminol–QDs conjugates was dependent on the quantum yield of QDs chiefly. Besides, it had something to do with the overlapping areas
999
between the emission spectrum of luminol and adsorption spectrum of QDs. The cytotoxicity of luminol–QDs conjugates must be studied in the future to make sure that the CRET technique could be used for in vivo imaging successfully.
References ¨ [1] T. Forster, Ann. Phys. 437 (1948) 55. [2] G. Stengel, J.P. Gill, P. Sandin, L.M. Wilhelmsson, Biochemistry 46 (2007) 12289. [3] Q. Ni, D.V. Titov, J. Zhang, Methods 40 (2006) 279. [4] P. Gottfried, M. Kolot, N. Silberstein, E. Yagil, FEBS Lett. 577 (2004) 17. [5] M. Merzlyakov, E. Li, R. Casas, K. Hristova, Langmuir 22 (2006) 6986. [6] A.P. Alivisatos, Science 271 (1996) 933. [7] W. Cai, D.W. Shin, K. Chen, O. Gheysens, Nano Lett. 6 (2006) 669. [8] H. Peng, L. Zhang, C. Soeller, J. Travas-Sejdic, J. Lumin. 127 (2007) 721. [9] L. Shi, V.D. Paoli, N. Rosenzweig, Z. Rosenzweig, J. Am. Chem. Soc. 128 (2006) 10378. [10] A.R. Clapp, I.L. Medintz, J.M. Mauro, B.R. Fisher, J. Am. Chem. Soc. 126 (2004) 301. [11] E. Alphande´ry, L.M. Walsh, Y. Rakovich, A.L. Bradley, Chem. Phys. Lett. 388 (2004) 100. [12] D. Zhou, J.D. Piper, C. Abell, D. Klenerman, Chem. Commun. 38 (2005) 4807. [13] S. Sadhu, A. Patra, Chem. Phys. Chem. 9 (2008) 2052. [14] S. Sadhu, Amitava Patra, Appl. Phys. Lett. 93 (2008) 183104. [15] M. Achermann, M.A. Petruska, S. Kos, D.L. Smith, Nature 429 (2004) 642. [16] V.R. Hering, G. Gibson, R.I. Schumacher, Bioconjugate Chem. 18 (2007) 1705. [17] R. Gill, M. Zayats, I.R. Willner, Angew. Chem. Int. Ed. 47 (2008) 7602. [18] Y. Ebenstein, T. Mokari, U. Banin, J. Phys. Chem. B 108 (2004) 93. [19] I.L. Medintz, A.R. Clapp, F.M. Brunel, T. Tiefenbrunn, Nat. Mater. 5 (2006) 581. [20] Y. Xing, M. So, A.L. Koh, R. Sinclair, Biochem. Biophys. Res. Commun. 372 (2008) 388. [21] M. So, C. Xu, A.M. Loening, Nat. Biotechnol. 24 (2006) 339. [22] T. Troy, D. Jekic-McMullen, L. Sambucetti, B. Rice, Mol. Imaging 3 (2004) 9. [23] V. Tuchin, Tissue Optics, SPIE Press, Washington, 2000. [24] X. Huang, L. Li, H. Qian, Angew. Chem. Int. Ed. 45 (2006) 5140. [25] H.Q. Wang, Y.Q. Li, J.H. Wang, Anal. Chim. Acta 610 (2008) 68. [26] M. Voicescua, M. Vasilescua, T. Constantinescua, A. Meghea, J. Lumin. 97 (2002) 60. [27] W.W. Yu, Y.A. Wang, X. Peng, Chem. Mater. 15 (2003) 4300. [28] Y. Zhang, J. He, P. Wang, J. Am. Chem. Soc. 128 (2006) 13396. [29] J.N. Demasa, G.A. Crosby, J. Phys. Chem. 75 (1971) 991.
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Chemiluminescence resonance energy transfer in the luminol–CdTe quantum dots conjugates Zheng Li a,b, Yongxian Wang a,n, Guoxin Zhang a, Wanbang Xu c, Yanjiang Han a,b a
Radiopharmaceutical Centre, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China Graduate University of Chinese Academy of Sciences, Beijing 100039, China c Product and Quality Supervision and Testing Institute, Yueyang Bureau of Quality and Technical Supervision, Hunan 414000, China b
a r t i c l e in fo
abstract
Article history: Received 19 April 2009 Received in revised form 11 January 2010 Accepted 15 January 2010 Available online 25 January 2010
The luminol–CdTe quantum dots (QDs) conjugates were prepared through the reaction between –NH2 and –COOH. The resonance energy transfer between chemiluminescence donor (luminol–H2O2 system) and quantum dots (QDs, with different emission peaks) acceptors (CRET) was investigated. The luminescence of QDs in luminol–QDs conjugates in the process of CRET was influenced by the molar ratio of luminol/QDs. It could reach higher luminescence intensity while the luminol/QDs value was 1/1. Quantum yield of QDs and overlapping areas between the emission spectrum of luminol and adsorption spectrum of QDs played important roles in the CRET efficiency of luminol–QDs conjugates. The higher CRET efficiency (21.2%) was observed when the 540 nm QDs were used as acceptors. This work will offer helpful knowledge for the CRET studies based on QDs. & 2010 Elsevier B.V. All rights reserved.
Keywords: CRET CdTe quantum dots Luminol Luminescence
1. Introduction ¨ ‘‘Forster resonance energy transfer’’, termed in 1948 by ¨ German scientist Forster [1], describes an energy transfer mechanism between two chromophores. A donor chromophore in exciting state can transfer energy by a nonradiative, long-range dipole–dipole coupling mechanism to an acceptor chromophore in close proximity (typically o10 nm). When both molecules are fluorescent, the term ‘‘fluorescence resonance energy transfer’’ (FRET) is often used, though the energy is not actually transferred by fluorescence. Basically, FRET efficiency (E) depends on (1) the distance between the donor and the acceptor and (2) the degree by which the donor’s emission spectrum overlaps the acceptor’s absorption spectrum. For a definite donor–acceptor pair, due to the dipole–dipole coupling mechanism, E is decided by E ¼ ½1 þ ðr=R0 Þ6 1 ¨ where r is the donor–acceptor distance and R0 is the Forster distance at which E=50% for this pair of donor and acceptor. Therefore, FRET is an ideal technique to study molecular dynamics in biophysics and biochemistry, such as DNA conformational changes [2], protein conformational changes [3] and protein– protein interactions [4,5]. As a fluorescence material, II–VI and III–V semiconductor quantum dots (QDs) have excellent optical and electrical propern
Corresponding author. Tel.: + 86 21 59556884. E-mail address: [email protected] (Y. Wang).
0022-2313/$ - see front matter & 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2010.01.013
ties, with broader excitation spectrum, narrower emission bandwidth and less tendency to photobleach than organic dyes, allowing simultaneous excitation of particles of different sizes at a single wavelength because of the size-dependence of the fluorescence wavelength [6–8]. Due to these advantages over organic dyes in FRET-based studies, QDs have been widely applied to FRET as donor [9–14] and acceptor [15], such as gene-based fluorescent probes [16], optical biosensor [17], FRET microscopy [18] and proteolytic activity monitored [19]. Recently, the QDs conjugates used in bioluminescence resonance energy transfer (BRET) [20,21] in the absence of external excitation, which is suitable for multiplexed in vivo imaging, have developed fast. It could overcome the disadvantage of QDs used in in vivo imaging because of strong background autofluorescence [22] and absorption or scattering of optical photons [23]. Huang et al. [24] and Wang et al. [25] investigated the chemiluminescence resonance energy transfer (CRET), which is similar to BRET, by using luminol–H2O2 system as energy donor and quantum dot as acceptor. CRET, which occurs by oxidation of a luminescent substrate without an excitation source, could reduce dramatically the background autofluorescence and fluorescence bleaching. Hence, it is a promising technique in biochemical application. In this paper, we report a CRET process with the CdTe QDs as acceptor and the chemiluminescence of luminol as donor. Unlike Refs. [24,25], we do not adopt the QDs–HRP (horseradish peroxidase) conjugates but link the CdTe QDs with luminol directly. Luminol is used to excite CdTe QDs through the luminol/ hydrogen peroxide CL reaction in the presence of NaClO [26]. The chemiluminescence specta of luminol–QDs conjugates were
ARTICLE IN PRESS 996
Z. Li et al. / Journal of Luminescence 130 (2010) 995–999
investigated. The results showed that the molar ratio of luminol/ QDs and different QDs acceptors would influence the process of CRET dramatically.
was carried out on a ThermoNicolet Avatar 370 Fourier Transform Infrared Spectrometer in the 500–4000 cm 1 range. TEM images were taken by a Hitachi H-800 transmission electron microscope.
2. Experimental
2.2. Preparation of TGA-capped CdTe QDs and luminol–QDs conjugates
2.1. Materials and apparatus Luminol was purchased from Yacoo Chemical Reagent Co. Ltd. Te powder; CdCl2 2.5 H2O and 1-[3-(dimethylamino)propyl]-3ethylcarbodiimide methiodide (EDC) were from Aldrich. Thioglycolic acid (TGA), NaBH4, H2O2 and NaOH were from Sinopharm Chemical Reagent Co., Ltd. All other chemicals and materials were of analytical grade. UV–vis absorption spectrum was obtained by using a Hitachi U-3010 spectrophotometer. Fluorescence spectrum was achieved with a Hitachi F-4500 FL spectrophotometer at room temperature. Chemiluminescence of luminol and the CRET spectrum of the luminol–QDs system were measured by using the Hitachi F-4500 FL spectrophotometer while the excitation light was blocked. FTIR
TGA-capped CdTe QDs were synthesized according to the procedure described in Ref. [27] with some changes. Briefly, NaHTe solution, prepared from Te powder and NaBH4, was added to N2-saturated CdCl2 solution in the presence of thioglycolic acid (TGA). The molar ratio of Cd2 + :Te2 :TGA was fixed at 1.0:0.5:2.0. Then, the solution was adjusted to pH9.0–9.5 with 1 M NaOH solution. After mixing, the reaction solution was heated to 100 1C and refluxed for different times to prepare TGA-capped CdTe QDs with different sizes. Luminol was conjugated to TGA-capped CdTe QDs using EDC as a coupling reagent. Firstly, 1 mL of as-prepared TGA-capped CdTe QDs was precipitated with NaOH and methanol, and then they were isolated by centrifugation and dried in a vacuum drying
Fig. 1. Preparation of luminol–CdTe QDs conjugates.
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Wavenumber(cm-1) Fig. 2. FTIR spectrum of TGA-capped CdTe QDs, luminol and luminol–CdTe QDs conjugate: (a) luminol–CdTe QDs conjugate; (b) TGA-capped CdTe QDs and (c) luminol.
ARTICLE IN PRESS Z. Li et al. / Journal of Luminescence 130 (2010) 995–999
Nomalized intensity (a.u.)
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stretching vibrations of C–N and benzene ring can be found in Fig. 2(c) also. The band of N–H changes to sharp and there is only a C= O stretching vibration at 1680 cm 1 in Fig. 2(a) because there is only secondary amide in the luminol–CdTe QDs conjugate due to the reaction. Furthermore, the band of C–N and benzene ring can still be found in Fig. 2(a). However, the C–S (1018 cm 1) and CH stretching vibrations appear and there is no stretching vibration of OH compared to Fig. 2(b). The analysis of the FTIR spectrum of TGA-capped CdTe QDs, luminol and luminol–CdTe QDs conjugate confirms that the luminol–CdTe QDs conjugate is prepared successfully, which could insure the accomplishment of CRET in luminol–CdTe QDs conjugate.
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Fig. 3. The CL spectrum of luminol–QDs conjugates with different luminol/QDs molar ratios: (a) luminol/QDs= 2/1; (b) luminol/QDs= 1.5/1; (c) luminol/QDs= 0.5/1 and (d) luminol/QDs= 1/1.
3.2. Absorption and emission spectrum of QDs and luminol–QDs conjugates
As shown in Fig. 1, the –COOH on the surface of CdTe QDs could react with –NH2 of the luminol in the presence of EDC and form a steady amido link. The FTIR spectrum of TGA-capped CdTe QDs is shown in Fig. 2(b), in which two intense bands are seen at 3240 and 1710 cm 1, respectively. Since OH and C= O stretching vibrations are known to be generally intense in FTIR spectrum, the intense bands at 3294 and 1710 cm 1 can readily be assigned to the OH and C= O oscillator of TGA. Two weak bands also appear at 3006 and 2860 cm 1, where CH stretching vibrations are
Luminol is linked to CdTe QDs through –COOH on the surface of QDs. So, the amount of luminol bonded with QDs may have some influences on the luminescence of QDs. The luminol H3BO3– KOH buffer solution with different concentration was added into QDs powders to prepare luminol–QDs conjugates with different luminol/QDs molar ratio. The CL spectra of the luminol–QDs conjugates are shown in Fig. 3. Similar to the QDs–HRP–luminol system [24,25], an efficient CRET was observed in luminol–QDs conjugates. The chemiluminescence luminescence of luminol did not shift obviously. However, the luminescence of QDs in luminol–QDs conjugates was intensively dependent on the luminol/QDs molar ratio, which has not been reported before. The luminescence intensity of QDs is weaker while the luminol/ QDs value is lower because the amount of luminol bonded with QDs is smaller. In this case, the energy of chemiluminescence luminescence is lower and the corresponding energy transferred to QDs nonradiatively is not enough to engender brighter fluorescence. However, it would also lead to a weaker luminescence of QDs while the luminol/QDs value is higher. The reasons may be that the particle sizes of luminol–QDs conjugates are larger and they would aggregate together, which could be seen in Fig. 4, when the QDs capped with too much of luminol. The superfluous luminol would be adsorbed on the surface of QDs simply, blocking the absorption and emission (luminescence) of energy of QDs. The luminescence intensity of QDs is highest while the luminol/QDs value is 1/1. Moreover, the luminescence emission’s peak position of QDs was slightly blue shifted with the increase of luminol/QDs value. The possible reactions, which lead to the CL, are described by the following reactions:
expected. On the other hand, the weak band at 1030 cm 1 can be assigned to a C–S stretching vibration. In the spectrum of luminol (Fig. 2(c)), the band from 3500 to 3100 cm 1 is almost certainly resulted from the N–H stretching vibration of NH2 and NH. There are also C =O stretching vibrations at 1680 and 1690 cm 1 of primary and secondary amides. Besides, the
Excited state luminol–QDs would advance the irreversible photooxidation of QDs. The amount of excited state luminol–QDs is larger when there are more luminol on the surface of QDs. So, the irreversible photooxidation of QDs would occur more frequently. As a result of photooxidation, macroscopic oxide layers were created and the effective QDs size was reduced. The
oven. 1 millilitre of luminol H3BO3–KOH buffer solution with different concentrations and 0.01 g of EDC were added into asprepared QDs powders to prepare luminol–QDs conjugates with different luminol/QDs molar ratio. Then, the mixture was stirred under room temperature for 2 h. Finally, the prepared solutions were purified by precipitating with NaOH and methanol, centrifuging to remove superfluous luminol and EDC. Then, a few drops of 5.0 10 3 mol/L H2O2 and NaClO solution were added into the luminol–CdTe QDs conjugates solution to engender chemiluminescence.
3. Result and discussion 3.1. Infrared spectrum of the luminol–QDs conjugates
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Fig. 4. TEM images of luminol–QDs conjugates: (a) luminol/QDs= 1/1 and (b) luminol/QDs = 2/1.
1.0
0.8 0.6
0.5
0.4 0.2 0.0
0.0 400
450
500 550 Wavelength (nm)
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3500 3000 2500 2000 1500 1000
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Fig. 5. Absorption spectrum of CdTe QDs at different emission peaks and CL emission spectrum of luminol.
1.0 Nomalized PL intensity (a.u.)
530nmCdTe 540nmCdTe 550nmCdTe 565nmCdTe 575nmCdTe 590nmCdTe 605nmCdTe
4000
PL intensity(a.u.)
530 nm Cd Te 540 nm Cd Te 550 nm Cd Te 565 nm Cd Te 575 nm Cd Te 590 nm Cd Te 605 nm Cd Te LMO
Nomalized PL intensity (a.u.)
Nomalized absorbance (a.u.)
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0 350
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500 550 600 Wavelength (nm)
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Fig. 7. PL emission spectrum of CdTe QDs (emission peaks at 530, 540, 550, 565, 575, 590 and 605 nm) of different sizes; lex = 440 nm.
0.8 Table 1 Quantum yield and CRET efficiency of different CRET acceptors.
0.6 a
b
c Emission peak positions of CdTe QDs(nm)
d
Quantum yield (%)a
CRET efficiency (%)b
17.5 32.1 36.4 30.5 27.9 20.0 12.9
15.8 21.2 19.3 18.2 15.3 10.0 2.9
0.4 530 540 550 565 575 590 605
e f 0.2
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g
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Fig. 6. The CL emission spectrum of luminol–CdTe QDs conjugates. Seven different sized CdTe QDs (with emissions at a: 530, b: 540, c: 550, d: 565, e: 575, f: 590 and g: 605 nm) were used as acceptors.
new surface defects could also be generated by oxidation of the QDs surface. Hence, the luminescence intensity decreased and blue shift could be observed [26,28]. As Fig. 5 shows, there are overlapping areas between the absorption spectrum of CdTe QDs with different sizes and emission spectrum of luminol, which would make the process of CRET possible. So, we investigated the CRET between luminol
a The QY was measured according to the literature [29]. Solutions of both standard (quinine sulfate) and samples (CdTe QDs) were adjusted to exhibit the low absorption intensity at the excitation wavelength of 350 nm. b CRET efficiency was evaluated by equation Ee =FQDs/Fcon; here Ee is the estimate efficiency of CRET, FQDs is the integral area of QDs emission spectrum of luminol–QDs conjugate, take for example, 530 nm QDs then FQDs is the integral area from 520 to 700 nm in the spectrum of luminol–QDs conjugate, and Fcon is the integral area of the whole spectrum of luminol–QDs conjugate.
and QDs conjugates in the H2O2–NaClO system and the results obtained are shown in Fig. 6, in which seven different sized QDs were used as acceptors. All of them emerge as two peaks, the first of which almost keeps at the same position (440 nm) and the second possessess different peak positions and intensities.
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Compared with Figs. 5 and 7, we could find that they are the emission peaks of luminol and QDs excited by CL of luminol. It is confirmed that an efficient CRET occurred in luminol–QDs conjugates in this process. The luminescence emission peak position of QDs in luminol–QDs conjugates was determined by the property of single QDs. However, the luminescence intensity would be influenced by luminol and QDs jointly. The CRET efficiency of luminol–QDs conjugates was taken to assess the difference of different luminol–QDs conjugates. The CRET efficiency of different luminol–QDs conjugates is shown in Table 1. We found that the differences of CRET efficiency are contributed to the quantum yield of QDs, chiefly. It would reduce with the decrease of the quantum yield of QDs, which has been reported in Ref. [25]. Furthermore, the CRET efficiency has something to do with the overlapping areas between emission spectrum of luminol and adsorption spectrum of QDs (Fig. 5). So, although the quantum yield of QDs with its emission peak at 550 nm (550 nm QDs) is higher, the CRET efficiency is higher while the CRET acceptor is 540 nm QDs because the overlapping area between emission spectrum of luminol and adsorption spectrum of 540 nm QDs is larger than that of 550 nm QDs.
4. Conclusion CRET is a potential technique for in vivo imaging because it does not need an exciting light source and could dramatically reduce the fluorescence bleaching and lessen the autofluorescence of the system. The synthesis and CRET of luminol–QDs conjugates have been, for the first time, reported in this paper. The experiment results showed that CRET could occur in the luminol–QDs conjugates in the system of H2O2–NaClO similar to the QD–HRP–luminol system. The CRET efficiency of luminol–QDs conjugates was dependent on the quantum yield of QDs chiefly. Besides, it had something to do with the overlapping areas
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between the emission spectrum of luminol and adsorption spectrum of QDs. The cytotoxicity of luminol–QDs conjugates must be studied in the future to make sure that the CRET technique could be used for in vivo imaging successfully.
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