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Semiconductor nanocrystals obtained by colloidal chemistry for biological applications
Applied Surface Science 255 (2008) 796–798
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Semiconductor nanocrystals obtained by colloidal chemistry for biological applications B.S. Santos a,d,*, P.M.A. Farias b,d, A. Fontes b,d, A.G. Brasil Jr.a,d, C.N. Jovino a,d, A.G.C. Neto d, D.C.N. Silva d, F.D. de Menezes a,c,d, R. Ferreira c,d a
Depto. Cieˆncias Farmaceˆuticas, UFPE – CDU, 50740-120 Recife, PE, Brazil Depto. Biofı´sica e Radiobiologia, UFPE – CDU, 50670-901 Recife, PE, Brazil Department Quı´mica Fundamental, UFPE – CDU, 50740-120 Recife, PE, Brazil d Grupo de Pesquisas em Nanoestruturas e Interfaces Biolo´gicas, CDU, 50670 901 Recife, PE, Brazil b c
A R T I C L E I N F O
A B S T R A C T
Article history:
Semiconductor nanoparticles in the quantum confinement regime used as biolabels present many advantages over the other chemical species used as fluorophores. They are composed of 2000–6000 atoms rendering a far greater photostability and allowing for long time bioimaging experiments. In this work we present a synthetic route for the obtention of large quantities of highly fluorescent CdSe and CdTe/CdS core–shell nanocrystals based on aqueous colloidal chemistry. The methodologies were optimized and the systems were characterized by optical spectroscopy, transmission electronic microscopy and X-Ray diffractometry. The fluorescent biolabels were tested in live macrophages. ß 2008 Elsevier B.V. All rights reserved.
Available online 4 July 2008 Keywords: Quantum dots CdSe CdTe Bioimaging Colloidal Nanoparticles
1. Introduction Semiconductor nanocrystals are being used by several research groups worldwide for different applications. This high applicability results mainly from the quantum confinement effect in which the optical properties of the nanocrystals (quantum dots) can be tuned by varying their size and/or nature [1–3]. There is an increasing interest of semiconductor nanostructured materials as efficient tools in fluorescent biolabeling specially II–VI semiconductor materials. Compared to organic biolabels these quantum dots show unique spectroscopic properties such as (i) greater resistance to photodegradation, (ii) narrower photoluminescence with high quantum yield, (iii) broader absorption bands, (iv) large effective Stokes shifts and (v) higher absorption coefficients [4]. The fluorescent properties of these materials are highly dependent on their surface defect concentration. It has been established that the reduction of these defects may be accomplished by coating the particle with a higher band-gap semiconductor layer. This process, known as surface passivation, usually makes use of highly toxic organic species which are not
* Corresponding author at: Depto. Cieˆncias Farmaceˆuticas, UFPE – CDU, 50740120 Recife, PE, Brazil. Tel.: +55 81 2126 8511; fax: +55 81 2126 8510. E-mail address: [email protected] (B.S. Santos). 0169-4332/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2008.07.026
suitable to transpose for a large scale production. In this work, we report a synthetic route of highly fluorescent II–VI semiconductor nanocrystals (CdTe/CdS and CdSe) based on aqueous colloidal chemistry and discuss on their optical properties. 2. Experimental details The nanoparticles were prepared at room temperature, under inert atmosphere and in aqueous medium using Cd(ClO)4, NaOH, Te or Se as the chemical precursors and mercaptoacetic acid (MAA) as the stabilizing agent (typically Cd:metal:AMA = 1:1:6) adapted from [5]. Se2 and Te2 are highly unstable in aqueous medium and were obtained in situ by the controlled reduction of the respective metals using NaHB4 at a pH 12. Freshly prepared CdSe nanoparticles were submitted to a controlled growth process using two routes: (1) promoting Cd surface rich particles and slowly injection of Se2 at T = 80 8C and (2) adding consecutive amounts of Cd2+ and Se2 at T = 80 8C. The colloidal particles were characterized by transmission electronic microscopy (TEM), X-ray diffractometry (XRD) and absorption, excitation and emission spectroscopies. Several experimental conditions (e.g. pH, Cd/ chalcogenide and the concentration of the stabilizing agent) were varied in order to optimize the chemical stability and the fluorescent properties of the colloidal particles. The application of CdTe systems as biolabels was tested using in situ experiments
B.S. Santos et al. / Applied Surface Science 255 (2008) 796–798
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Fig. 1. Powder X-ray diffraction pattern of freshly prepared CdSe and CdTe particles.
Fig. 3. Emission and absorption pattern of a CdTe nanocrystal’s suspension with average size d = 2.5 nm.
Fig. 2. Absorption spectra of CdSe suspensions. (a) Freshly prepared; (b) after 2 h growth following method (1); (c) after 2 h growth following method (2).
about 3–4 monolayers and is in the strong quantum confinement regime for this semiconductor material. These particles were grown up to 2.3 nm and by analyzing the evolution of their absorption spectra we suggest that the formation of Cd2+ rich particles followed by Se2 injection (method 1) promoted the growth of the particles (Fig. 2(c)) while method 2 also resulted in the growth of different CdSe populations, increasing their final size dispersion (Fig. 2(b)). Chemically stable and luminescent CdTe nanoparticles were obtained with an initial size of d = 2.5 nm, also in the strong quantum confinement regime. The photoluminescence (PL) of the colloidal suspensions of CdSe and CdTe nanoparticles show different characteristics. The emission of the CdSe suspensions is observed right after their preparation, but the spectra show broad emission bands associated with trap emission. The growth of the particles up to 2.3 nm did improve its luminescence intensity increasing it up to 100 but maintained its broadband profile. The band-edge CdTe emission, on the other hand, developed slowly after its synthesis taking up to several days to become readily observable even at daylight. A representative spectrum of the CdTe emission pattern is shown in Fig. 3. This suggests a slow kinetic mechanism of surface defect suppression. Some authors have reported on the nature of the surface of these particles using different spectroscopic techniques [6–8]. They suggested that the increase in the PL results from the decomposition of the thiol groups of the stabilizing agent and incorporation of the sulfide ion in the structure. This corresponds to coating the particle with CdS, which has a greater band-gap compared to CdTe, rendering CdTe/CdS core–shell type structures.
by incubating live macrophages (a type of white blood cell) with the nanocrystals suspension at pH 7.2. 3. Results and discussion The crystalline phase of both sets of nanoparticles was determined to be cubic (zinc blend structure). Fig. 1 shows the powder diffraction pattern of freshly prepared systems. The CdSe arrested precipitation resulted in very small crystals with sizes around 1.9 nm determined by analyzing the XRD pattern, TEM images and the first maximum of the absorption spectra of the suspensions (Fig. 2(a)). This means that each nanoparticle has
Fig. 4. Confocal microscopy image of a macrophage labeled with CdTe/CdS nanocrystals (a) under 543 nm laser excitation; (b) phase contrast image.
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B.S. Santos et al. / Applied Surface Science 255 (2008) 796–798
In a typical biolabeling experiment live macrophages were incubated with CdTe/CdS-AMA 2.5 nm nanocrystals for about 20 min at room temperature and the resulting image obtained by confocal microscopy is shown in Fig. 4. The marked cells showed no signs of cell damage and maintained their brightness even after a long time laser exposition (up to 30 min) showing a very high resistance to photodegradation. 4. Summary Here we described the synthesis of CdSe and CdTe/CdS semiconductor quantum dots. These colloidal systems show fluorescence with maxima ranging from 480 to 640 nm depending on the semiconductor particle’s size and nature. The preparation of these nanocrystals in aqueous medium is the greatest advantage of this synthetic route enabling a large scale production. Luminescent CdTe/CdS nanocrystals were tested in primary living cells showing a great potential as new probes in the investigation of biological systems.
Acknowledgments The authors are thankful for the financial support received during the development of this work from CNPq, FACEPE, RENAMI and PHILIPS. References [1] M. Bruchez, M. Moronne, P. Gin, S. Weiss, A.P. Alivisatos, Science 281 (1998) 2013. [2] W.C.W. Chan, D.J. Maxwell, X. Gao, R.E. Bailey, M. Han, S. Nie, Curr. Opin. Biotechnol. 13 (2002) 40. [3] H. Wu, K.N. Liu, J.A. Haley, J.P. Treadway, N. Larson, F. Ge, M.P. Peale, Bruchez, Nat. Biotechnol. 21 (2003) 41. [4] B.M. Lingerfelt, H. Mattoussi, E.R. Goldman, J.M. Mauro, G.P. Anderson, Anal. Chem. 75 (2003) 4043. [5] F.D. Menezes, A.G. Brasil Junior, W.L. Moreira, L.C. Barbosa, C.L. Cesar, R. Ferreira, P.M.A. Farias, B.S. Santos, Microelectr. J. 36 (2005) 989. [6] J. Rockenberger, L. Tro¨ger, A. Kornowski, T. Vossmeyer, A. Eychmu¨ller, J. Feldhaus, H. Weller, B. Bunsenges, Phys. Chem. 101 (1997) 1613. [7] H. Borchert, D.V. Talapin, N. Gaponik, C. McGinley, S. Adam, A. Lobo, T. Mo¨ller, H. Weller, J. Phys. Chem. B 107 (2003) 9662. [8] H. Zhang, Z. Zhou, B. Yang, M. Gao, J. Phys. Chem. B 107 (2003) 8.
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Semiconductor nanocrystals obtained by colloidal chemistry for biological applications B.S. Santos a,d,*, P.M.A. Farias b,d, A. Fontes b,d, A.G. Brasil Jr.a,d, C.N. Jovino a,d, A.G.C. Neto d, D.C.N. Silva d, F.D. de Menezes a,c,d, R. Ferreira c,d a
Depto. Cieˆncias Farmaceˆuticas, UFPE – CDU, 50740-120 Recife, PE, Brazil Depto. Biofı´sica e Radiobiologia, UFPE – CDU, 50670-901 Recife, PE, Brazil Department Quı´mica Fundamental, UFPE – CDU, 50740-120 Recife, PE, Brazil d Grupo de Pesquisas em Nanoestruturas e Interfaces Biolo´gicas, CDU, 50670 901 Recife, PE, Brazil b c
A R T I C L E I N F O
A B S T R A C T
Article history:
Semiconductor nanoparticles in the quantum confinement regime used as biolabels present many advantages over the other chemical species used as fluorophores. They are composed of 2000–6000 atoms rendering a far greater photostability and allowing for long time bioimaging experiments. In this work we present a synthetic route for the obtention of large quantities of highly fluorescent CdSe and CdTe/CdS core–shell nanocrystals based on aqueous colloidal chemistry. The methodologies were optimized and the systems were characterized by optical spectroscopy, transmission electronic microscopy and X-Ray diffractometry. The fluorescent biolabels were tested in live macrophages. ß 2008 Elsevier B.V. All rights reserved.
Available online 4 July 2008 Keywords: Quantum dots CdSe CdTe Bioimaging Colloidal Nanoparticles
1. Introduction Semiconductor nanocrystals are being used by several research groups worldwide for different applications. This high applicability results mainly from the quantum confinement effect in which the optical properties of the nanocrystals (quantum dots) can be tuned by varying their size and/or nature [1–3]. There is an increasing interest of semiconductor nanostructured materials as efficient tools in fluorescent biolabeling specially II–VI semiconductor materials. Compared to organic biolabels these quantum dots show unique spectroscopic properties such as (i) greater resistance to photodegradation, (ii) narrower photoluminescence with high quantum yield, (iii) broader absorption bands, (iv) large effective Stokes shifts and (v) higher absorption coefficients [4]. The fluorescent properties of these materials are highly dependent on their surface defect concentration. It has been established that the reduction of these defects may be accomplished by coating the particle with a higher band-gap semiconductor layer. This process, known as surface passivation, usually makes use of highly toxic organic species which are not
* Corresponding author at: Depto. Cieˆncias Farmaceˆuticas, UFPE – CDU, 50740120 Recife, PE, Brazil. Tel.: +55 81 2126 8511; fax: +55 81 2126 8510. E-mail address: [email protected] (B.S. Santos). 0169-4332/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2008.07.026
suitable to transpose for a large scale production. In this work, we report a synthetic route of highly fluorescent II–VI semiconductor nanocrystals (CdTe/CdS and CdSe) based on aqueous colloidal chemistry and discuss on their optical properties. 2. Experimental details The nanoparticles were prepared at room temperature, under inert atmosphere and in aqueous medium using Cd(ClO)4, NaOH, Te or Se as the chemical precursors and mercaptoacetic acid (MAA) as the stabilizing agent (typically Cd:metal:AMA = 1:1:6) adapted from [5]. Se2 and Te2 are highly unstable in aqueous medium and were obtained in situ by the controlled reduction of the respective metals using NaHB4 at a pH 12. Freshly prepared CdSe nanoparticles were submitted to a controlled growth process using two routes: (1) promoting Cd surface rich particles and slowly injection of Se2 at T = 80 8C and (2) adding consecutive amounts of Cd2+ and Se2 at T = 80 8C. The colloidal particles were characterized by transmission electronic microscopy (TEM), X-ray diffractometry (XRD) and absorption, excitation and emission spectroscopies. Several experimental conditions (e.g. pH, Cd/ chalcogenide and the concentration of the stabilizing agent) were varied in order to optimize the chemical stability and the fluorescent properties of the colloidal particles. The application of CdTe systems as biolabels was tested using in situ experiments
B.S. Santos et al. / Applied Surface Science 255 (2008) 796–798
797
Fig. 1. Powder X-ray diffraction pattern of freshly prepared CdSe and CdTe particles.
Fig. 3. Emission and absorption pattern of a CdTe nanocrystal’s suspension with average size d = 2.5 nm.
Fig. 2. Absorption spectra of CdSe suspensions. (a) Freshly prepared; (b) after 2 h growth following method (1); (c) after 2 h growth following method (2).
about 3–4 monolayers and is in the strong quantum confinement regime for this semiconductor material. These particles were grown up to 2.3 nm and by analyzing the evolution of their absorption spectra we suggest that the formation of Cd2+ rich particles followed by Se2 injection (method 1) promoted the growth of the particles (Fig. 2(c)) while method 2 also resulted in the growth of different CdSe populations, increasing their final size dispersion (Fig. 2(b)). Chemically stable and luminescent CdTe nanoparticles were obtained with an initial size of d = 2.5 nm, also in the strong quantum confinement regime. The photoluminescence (PL) of the colloidal suspensions of CdSe and CdTe nanoparticles show different characteristics. The emission of the CdSe suspensions is observed right after their preparation, but the spectra show broad emission bands associated with trap emission. The growth of the particles up to 2.3 nm did improve its luminescence intensity increasing it up to 100 but maintained its broadband profile. The band-edge CdTe emission, on the other hand, developed slowly after its synthesis taking up to several days to become readily observable even at daylight. A representative spectrum of the CdTe emission pattern is shown in Fig. 3. This suggests a slow kinetic mechanism of surface defect suppression. Some authors have reported on the nature of the surface of these particles using different spectroscopic techniques [6–8]. They suggested that the increase in the PL results from the decomposition of the thiol groups of the stabilizing agent and incorporation of the sulfide ion in the structure. This corresponds to coating the particle with CdS, which has a greater band-gap compared to CdTe, rendering CdTe/CdS core–shell type structures.
by incubating live macrophages (a type of white blood cell) with the nanocrystals suspension at pH 7.2. 3. Results and discussion The crystalline phase of both sets of nanoparticles was determined to be cubic (zinc blend structure). Fig. 1 shows the powder diffraction pattern of freshly prepared systems. The CdSe arrested precipitation resulted in very small crystals with sizes around 1.9 nm determined by analyzing the XRD pattern, TEM images and the first maximum of the absorption spectra of the suspensions (Fig. 2(a)). This means that each nanoparticle has
Fig. 4. Confocal microscopy image of a macrophage labeled with CdTe/CdS nanocrystals (a) under 543 nm laser excitation; (b) phase contrast image.
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B.S. Santos et al. / Applied Surface Science 255 (2008) 796–798
In a typical biolabeling experiment live macrophages were incubated with CdTe/CdS-AMA 2.5 nm nanocrystals for about 20 min at room temperature and the resulting image obtained by confocal microscopy is shown in Fig. 4. The marked cells showed no signs of cell damage and maintained their brightness even after a long time laser exposition (up to 30 min) showing a very high resistance to photodegradation. 4. Summary Here we described the synthesis of CdSe and CdTe/CdS semiconductor quantum dots. These colloidal systems show fluorescence with maxima ranging from 480 to 640 nm depending on the semiconductor particle’s size and nature. The preparation of these nanocrystals in aqueous medium is the greatest advantage of this synthetic route enabling a large scale production. Luminescent CdTe/CdS nanocrystals were tested in primary living cells showing a great potential as new probes in the investigation of biological systems.
Acknowledgments The authors are thankful for the financial support received during the development of this work from CNPq, FACEPE, RENAMI and PHILIPS. References [1] M. Bruchez, M. Moronne, P. Gin, S. Weiss, A.P. Alivisatos, Science 281 (1998) 2013. [2] W.C.W. Chan, D.J. Maxwell, X. Gao, R.E. Bailey, M. Han, S. Nie, Curr. Opin. Biotechnol. 13 (2002) 40. [3] H. Wu, K.N. Liu, J.A. Haley, J.P. Treadway, N. Larson, F. Ge, M.P. Peale, Bruchez, Nat. Biotechnol. 21 (2003) 41. [4] B.M. Lingerfelt, H. Mattoussi, E.R. Goldman, J.M. Mauro, G.P. Anderson, Anal. Chem. 75 (2003) 4043. [5] F.D. Menezes, A.G. Brasil Junior, W.L. Moreira, L.C. Barbosa, C.L. Cesar, R. Ferreira, P.M.A. Farias, B.S. Santos, Microelectr. J. 36 (2005) 989. [6] J. Rockenberger, L. Tro¨ger, A. Kornowski, T. Vossmeyer, A. Eychmu¨ller, J. Feldhaus, H. Weller, B. Bunsenges, Phys. Chem. 101 (1997) 1613. [7] H. Borchert, D.V. Talapin, N. Gaponik, C. McGinley, S. Adam, A. Lobo, T. Mo¨ller, H. Weller, J. Phys. Chem. B 107 (2003) 9662. [8] H. Zhang, Z. Zhou, B. Yang, M. Gao, J. Phys. Chem. B 107 (2003) 8.