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New highly fluorescent biolabels based on II–VI semiconductor hybrid organic–inorganic nanostructures for bioimaging
Applied Surface Science 255 (2008) 790–792
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
New highly fluorescent biolabels based on II–VI semiconductor hybrid organic–inorganic nanostructures for bioimaging B.S. Santos a,e,*, P.M.A. Farias b,e, F.D. Menezes a,c,e, A.G. Brasil Jr.a,e, A. Fontes b,e, L. Roma˜o d, J.O. Amaral d, V. Moura-Neto d, D.P.L.A. Teno´rio e, C.L. Cesar f, L.C. Barbosa f, R. Ferreira c,e a
Depto. de Cieˆncias Farmaceˆuticas, UFPE, CDU, 50740 120 Recife, PE, Brazil Depto. Biofı´sica e Radiobiologia, UFPE, CDU, 50670 901 Recife - PE, Brazil Depto. Quı´mica Fundamental, UFPE, CDU, 50670 901 Recife, PE, Brazil d Depto. Anatomia, UFRJ, 21941-590 Rio de Janeiro, RJ, Brazil e Grupo de Pesquisas em Nanoestruturas e Interfaces Biolo´gicas, CDU, 50670 901 Recife, PE, Brazil f Depto. Eletroˆnica Quaˆntica, IFGW, UNICAMP, Campinas, SP, 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 quantum dots based on II–VI materials may be prepared to develop good biolabeling properties. In this study we present some well-succeeded results related to the preparation, functionalization and bioconjugation of CdY (Y = S, Se and Te) to biological systems (live cells and fixed tissues). These nanostructured materials were prepared using colloidal synthesis in aqueous media resulting nanoparticles with very good optical properties and an excellent resistance to photodegradation. ß 2008 Elsevier B.V. All rights reserved.
Available online 4 July 2008 Keywords: Quantum dots Nanoparticles CdS CdTe Biolabel Bioimaging
1. Introduction Nanostructured materials based on semiconductor quantum dots (QDs) revealed to be a powerful tool for labeling biological systems in the last decade. Their nanoscale size range is compatible to most of the metabolic and internalization processes observed in cells [1,2]. Compared to organic fluorophores QDs show an exceptional resistance to photodegradation and photobleaching, narrower photoluminescence with high quantum yield, broader absorption bands, large effective Stokes shifts and higher absorption coefficients. In order to target them to a specific molecule (e.g. proteins, peptides, organic and inorganic polymers, DNA, carbohydrates) present in the biological system under study, their surface must be chemically modified with organic species. This procedure is called the functionalization of the nanoparticles and the resulting system may be considered a hybrid organic– inorganic nanostructure [3]. The resulting systems possess the
* Corresponding author at: Depto. de Cieˆncias Farmaceˆuticas, UFPE, CDU, R. Prof. Artur de Sa, S/N, Cidade Universita´ria, 50740 120 Recife, PE, Brazil. Tel.: +55 81 99688268; fax: +55 81 2126 8511. 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.2008.07.024
combined properties of both forming elements: the optical properties of the QDs and the biological functionalities leading to specific association to biosystems. These nanobiolabels may be used in monitoring biological processes at molecular level. In this work we show several well-succeeded examples of the functionalization of CdS/Cd(OH)2, CdSe/CdS and CdTe/CdS nanoparticles (d = 2–6 nm) and their biolabeling to different biological systems (cancer cells and tissues, live parasites and yeast cells). 2. Experimental details CdY (Y = S, Se, Te) nanocrystals were prepared in colloidal aqueous medium by a controlled precipitation procedure using polyphosphate (PolyP) or mercapto-acetic acid (MAA) as stabilizing agents [4,5]. In a typical CdSe (or CdTe) procedure, Se2 (or Te2 ) ions were produced reducing the Se (or Te) metal with NaBH4 in aqueous solution and adding it to a solution containing the Cd2+–MAA complexes. A further thermal treatment was performed to produce a CdS shell by the hydrolysis of the thiol groups of the MAA molecules [6]. The CdS/Cd(OH)2 crystals were produced by adding H2S(g) to a Cd2+ solution containing PolyP. The passivation procedure of the CdS particles consists of the controlled precipitation of a Cd(OH)2 layer using OH followed by
B.S. Santos et al. / Applied Surface Science 255 (2008) 790–792
791
Fig. 1. Schematic representation of a functionalized core–shell semiconductor quantum dot.
Fig. 3. Glioblastomas labeled with CdS/Cd(OH)2 6 nm QDs and visualized with fluorescence microscopy. Exc: 488 nm.
Fig. 2. (a) Emission spectrum of 2,4 nm CdTe/CdS nanocrystals in aqueous medium and (b) temporal evolution of the emission intensity shown in (a) under excitation 337 nm at room temperature (lmax = 530 nm).
an excess of Cd2+ ions. The chemical and structural analysis of these systems was performed by absorption electronic spectroscopy, X-ray diffraction and transmission electron microscopy. In the functionalization step the particles in physiological medium were chemically associated to either glutaraldehyde (Glut) via a one-pot cross linking glutaraldehyde procedure for non-specific labeling. Fig. 1 shows a scheme of a functionalized QD. The specific labeling was accomplished by binding the nanoparticles either bare or functionalized with glutaraldehyde to target biomolecules, such as poly(ethylene glycol)-1000, glucose-1-phosphate, concanavalin-A and antibodies. In a typical bioconjugation procedure the functionalized nanocrystals’ colloidal suspensions were incubated with the biological systems under study (e.g. live cells in culture medium
Fig. 4. A representative scheme of a specific conjugation of the QDs to cell membrane proteins through an antibody–antigen association.
of hydrated tissue sections) at temperatures from 25 to 37 8C for different time periods (1–30 min). 3. Results and discussion The size of the particles was estimated to be 1.9–3.0 nm for the CdSe (wurzite type crystal structure) and CdTe crystals (cubic
Fig. 5. Confocal micrograph of A+ type red blood cells labeled with CdS/Cd(OH)2/Glut/Anti-A: (a) phase contrast image and (b) under 543 nm excitation [7].
792
B.S. Santos et al. / Applied Surface Science 255 (2008) 790–792
phase) and 5–7 nm for the CdS crystals (zinc blend crystal structure). These colloidal systems show high intensity fluorescent maxima tunable from 480 to 600 nm (FWMH = 50 nm) and excellent resistance to photobleaching, as exemplified in Fig. 2, for CdTe/CdS nanocrystals in aqueous medium. The fluorescence is used as a primary tool in order to explore and differentiate the labeling of the samples. Tissues and cells conjugated with QDs were analyzed by the laser scanning confocal microscopy. The glutaraldehyde-functionalized nanocrystals when bioconjugated show an inspecific labeling pattern. This is explained in terms of the reaction of the aldehyde groups with any amine groups present in the biosystems. Fig. 3 shows the labeling of glioblastomas (cancer brain cells) with CdS/Cd(OH)2 6 nm QDs. On the other hand, specific labeling is accomplished when a targeting molecule is tagged to the QDs. A successful example of specific labeling is the antibody–antigen driven interaction. Fig. 4 shows a representative conjugation scheme of specific labeling while Fig. 5 shows the comparative marking of red blood cells with CdS/Cd(OH)2/Glut/Anti-A antibody assembly. All the live biological systems conjugated to these nanocrystals prepared in aqueous medium showed no cell damage and could be kept alive for more than 5 days in vitro in culture medium. The microscopy images obtained either by confocal or conventional fluorescence microscopy showed a remarkable resistance to photobleaching (more than 30 min of continuous excitation in a fluorescence or confocal microscope) and conserve their fluorescence pattern for over 2 years.
4. Summary The characteristic properties of the hybrid organic–inorganic fluorescent nanostructures shown in this study (e.g. high intensity fluorescent pattern, very low photodegradation rates, simpler synthetic and bioconjugation procedures) demonstrate their great potential as tools to be used in fluorescence and confocal microscopic analysis, common tools used to investigate biological systems at a molecular level. Acknowledgements The authors are thankful for the financial support received from CNPq, RENAMI, Philips, FACEPE. References [1] D. Gerion, F. Pinaud, S.C. Williams, W.J. Parak, D. Zanchet, S. Weiss, A.P. Alivisatos, J. Phys. Chem. B 105 (2001) 8861. [2] A.N. Rogach, D. Nagesha, J.W. Ostrander, M. Giersig, N.A. Kotov, Chem. Mater. 12 (2000) 2676. [3] I.L. Medintz, H.T. Uyeda, E.R. Goldman, H. Mattoussi, Nat. Mater. 4 (2005) 435. [4] D. Petrov, B.S. Santos, G.A.L. Pereira, C. de, M. Donega´, J. Phys. Chem. B 106 (2002) 5325. [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] 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. [7] P.M.A. Farias, B.S. Santos, F.D. Menezes, C.L. Cesar, A. Fontes, P.R.M. Lima, M.L. Barjas-Castro, V. Castro, R. Ferreira, J. Microsc. 219 (2005) 103.
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
New highly fluorescent biolabels based on II–VI semiconductor hybrid organic–inorganic nanostructures for bioimaging B.S. Santos a,e,*, P.M.A. Farias b,e, F.D. Menezes a,c,e, A.G. Brasil Jr.a,e, A. Fontes b,e, L. Roma˜o d, J.O. Amaral d, V. Moura-Neto d, D.P.L.A. Teno´rio e, C.L. Cesar f, L.C. Barbosa f, R. Ferreira c,e a
Depto. de Cieˆncias Farmaceˆuticas, UFPE, CDU, 50740 120 Recife, PE, Brazil Depto. Biofı´sica e Radiobiologia, UFPE, CDU, 50670 901 Recife - PE, Brazil Depto. Quı´mica Fundamental, UFPE, CDU, 50670 901 Recife, PE, Brazil d Depto. Anatomia, UFRJ, 21941-590 Rio de Janeiro, RJ, Brazil e Grupo de Pesquisas em Nanoestruturas e Interfaces Biolo´gicas, CDU, 50670 901 Recife, PE, Brazil f Depto. Eletroˆnica Quaˆntica, IFGW, UNICAMP, Campinas, SP, 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 quantum dots based on II–VI materials may be prepared to develop good biolabeling properties. In this study we present some well-succeeded results related to the preparation, functionalization and bioconjugation of CdY (Y = S, Se and Te) to biological systems (live cells and fixed tissues). These nanostructured materials were prepared using colloidal synthesis in aqueous media resulting nanoparticles with very good optical properties and an excellent resistance to photodegradation. ß 2008 Elsevier B.V. All rights reserved.
Available online 4 July 2008 Keywords: Quantum dots Nanoparticles CdS CdTe Biolabel Bioimaging
1. Introduction Nanostructured materials based on semiconductor quantum dots (QDs) revealed to be a powerful tool for labeling biological systems in the last decade. Their nanoscale size range is compatible to most of the metabolic and internalization processes observed in cells [1,2]. Compared to organic fluorophores QDs show an exceptional resistance to photodegradation and photobleaching, narrower photoluminescence with high quantum yield, broader absorption bands, large effective Stokes shifts and higher absorption coefficients. In order to target them to a specific molecule (e.g. proteins, peptides, organic and inorganic polymers, DNA, carbohydrates) present in the biological system under study, their surface must be chemically modified with organic species. This procedure is called the functionalization of the nanoparticles and the resulting system may be considered a hybrid organic– inorganic nanostructure [3]. The resulting systems possess the
* Corresponding author at: Depto. de Cieˆncias Farmaceˆuticas, UFPE, CDU, R. Prof. Artur de Sa, S/N, Cidade Universita´ria, 50740 120 Recife, PE, Brazil. Tel.: +55 81 99688268; fax: +55 81 2126 8511. 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.2008.07.024
combined properties of both forming elements: the optical properties of the QDs and the biological functionalities leading to specific association to biosystems. These nanobiolabels may be used in monitoring biological processes at molecular level. In this work we show several well-succeeded examples of the functionalization of CdS/Cd(OH)2, CdSe/CdS and CdTe/CdS nanoparticles (d = 2–6 nm) and their biolabeling to different biological systems (cancer cells and tissues, live parasites and yeast cells). 2. Experimental details CdY (Y = S, Se, Te) nanocrystals were prepared in colloidal aqueous medium by a controlled precipitation procedure using polyphosphate (PolyP) or mercapto-acetic acid (MAA) as stabilizing agents [4,5]. In a typical CdSe (or CdTe) procedure, Se2 (or Te2 ) ions were produced reducing the Se (or Te) metal with NaBH4 in aqueous solution and adding it to a solution containing the Cd2+–MAA complexes. A further thermal treatment was performed to produce a CdS shell by the hydrolysis of the thiol groups of the MAA molecules [6]. The CdS/Cd(OH)2 crystals were produced by adding H2S(g) to a Cd2+ solution containing PolyP. The passivation procedure of the CdS particles consists of the controlled precipitation of a Cd(OH)2 layer using OH followed by
B.S. Santos et al. / Applied Surface Science 255 (2008) 790–792
791
Fig. 1. Schematic representation of a functionalized core–shell semiconductor quantum dot.
Fig. 3. Glioblastomas labeled with CdS/Cd(OH)2 6 nm QDs and visualized with fluorescence microscopy. Exc: 488 nm.
Fig. 2. (a) Emission spectrum of 2,4 nm CdTe/CdS nanocrystals in aqueous medium and (b) temporal evolution of the emission intensity shown in (a) under excitation 337 nm at room temperature (lmax = 530 nm).
an excess of Cd2+ ions. The chemical and structural analysis of these systems was performed by absorption electronic spectroscopy, X-ray diffraction and transmission electron microscopy. In the functionalization step the particles in physiological medium were chemically associated to either glutaraldehyde (Glut) via a one-pot cross linking glutaraldehyde procedure for non-specific labeling. Fig. 1 shows a scheme of a functionalized QD. The specific labeling was accomplished by binding the nanoparticles either bare or functionalized with glutaraldehyde to target biomolecules, such as poly(ethylene glycol)-1000, glucose-1-phosphate, concanavalin-A and antibodies. In a typical bioconjugation procedure the functionalized nanocrystals’ colloidal suspensions were incubated with the biological systems under study (e.g. live cells in culture medium
Fig. 4. A representative scheme of a specific conjugation of the QDs to cell membrane proteins through an antibody–antigen association.
of hydrated tissue sections) at temperatures from 25 to 37 8C for different time periods (1–30 min). 3. Results and discussion The size of the particles was estimated to be 1.9–3.0 nm for the CdSe (wurzite type crystal structure) and CdTe crystals (cubic
Fig. 5. Confocal micrograph of A+ type red blood cells labeled with CdS/Cd(OH)2/Glut/Anti-A: (a) phase contrast image and (b) under 543 nm excitation [7].
792
B.S. Santos et al. / Applied Surface Science 255 (2008) 790–792
phase) and 5–7 nm for the CdS crystals (zinc blend crystal structure). These colloidal systems show high intensity fluorescent maxima tunable from 480 to 600 nm (FWMH = 50 nm) and excellent resistance to photobleaching, as exemplified in Fig. 2, for CdTe/CdS nanocrystals in aqueous medium. The fluorescence is used as a primary tool in order to explore and differentiate the labeling of the samples. Tissues and cells conjugated with QDs were analyzed by the laser scanning confocal microscopy. The glutaraldehyde-functionalized nanocrystals when bioconjugated show an inspecific labeling pattern. This is explained in terms of the reaction of the aldehyde groups with any amine groups present in the biosystems. Fig. 3 shows the labeling of glioblastomas (cancer brain cells) with CdS/Cd(OH)2 6 nm QDs. On the other hand, specific labeling is accomplished when a targeting molecule is tagged to the QDs. A successful example of specific labeling is the antibody–antigen driven interaction. Fig. 4 shows a representative conjugation scheme of specific labeling while Fig. 5 shows the comparative marking of red blood cells with CdS/Cd(OH)2/Glut/Anti-A antibody assembly. All the live biological systems conjugated to these nanocrystals prepared in aqueous medium showed no cell damage and could be kept alive for more than 5 days in vitro in culture medium. The microscopy images obtained either by confocal or conventional fluorescence microscopy showed a remarkable resistance to photobleaching (more than 30 min of continuous excitation in a fluorescence or confocal microscope) and conserve their fluorescence pattern for over 2 years.
4. Summary The characteristic properties of the hybrid organic–inorganic fluorescent nanostructures shown in this study (e.g. high intensity fluorescent pattern, very low photodegradation rates, simpler synthetic and bioconjugation procedures) demonstrate their great potential as tools to be used in fluorescence and confocal microscopic analysis, common tools used to investigate biological systems at a molecular level. Acknowledgements The authors are thankful for the financial support received from CNPq, RENAMI, Philips, FACEPE. References [1] D. Gerion, F. Pinaud, S.C. Williams, W.J. Parak, D. Zanchet, S. Weiss, A.P. Alivisatos, J. Phys. Chem. B 105 (2001) 8861. [2] A.N. Rogach, D. Nagesha, J.W. Ostrander, M. Giersig, N.A. Kotov, Chem. Mater. 12 (2000) 2676. [3] I.L. Medintz, H.T. Uyeda, E.R. Goldman, H. Mattoussi, Nat. Mater. 4 (2005) 435. [4] D. Petrov, B.S. Santos, G.A.L. Pereira, C. de, M. Donega´, J. Phys. Chem. B 106 (2002) 5325. [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] 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. [7] P.M.A. Farias, B.S. Santos, F.D. Menezes, C.L. Cesar, A. Fontes, P.R.M. Lima, M.L. Barjas-Castro, V. Castro, R. Ferreira, J. Microsc. 219 (2005) 103.