Baixe Functionalizing Nanoparticles for Biomedical Use: Silica Coating Approaches e outras Notas de estudo em PDF para Engenharia Elétrica, somente na Docsity!
Langmuir XXXX, XXX(XX), XXX–XXX DOI: 10.1021/la903512m A
pubs.acs.org/Langmuir © XXXX American Chemical Society
Functional and Multifunctional Nanoparticles for Bioimaging and Biosensing
Subramanian Tamil Selvan,* ,†^ Timothy Thatt Yang Tan, ‡^ Dong Kee Yi, §^ and Nikhil R. Jana )
†Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602, ‡Division of Chemical and Biomolecular Engineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, §Division of Bionanotechnology, Kyungwon University, Seong Nam City, Republic of Korea, and ) Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata-700032, India
Received September 17, 2009. Revised Manuscript Received November 10, 2009
Herein, we describe the synthesis of functional and multifunctional nanoparticles (NPs), derived from our recent work, for bioimaging and biosensing applications. The functionalized NPs involve quantum dots (QDs), magnetic particles (MPs) and noble metal NPs for the aforementioned applications. A diverse silica coating approaches (reverse microemulsion and thin silanization) are delineated for the design of water-soluble NPs. We also review the synthesis of silica-coated bifunctional NPs consisting of MPs and QDs for live cell imaging of human liver cancer cells (HepG2) and mouse fibroblast cells (NIH-3T3). Using silica coated NPs, various NPs that are functionalized with antibody, oligonucleotide, biotin and dextran are efficiently used for protein detection.
1. Introduction
This feature article is organized as follows: (1) In the Introduc- tion, we describe recent advances in inorganic nanoparticles (NPs) with more emphasis on quantum dots (QDs), magnetic NPs (MPs), and multifunctional NPs. (2) The significance of the coating is described for the creation of water-soluble functional NPs. (3) Silica coating approaches are delineated for the direct encapsulation of hydrophobic QDs and MPs or bifunctional QDs and MPs within a silica shell using a reverse microemulsion method, derived from our recent work. We also describe a thin silica coating approach for the preparation of functional metallic NPs (e.g., Au and Ag), MPs, and QDs. (4) The functionalization of NPs with various chemicals and biomolecules are discussed. (5) The biomedical applications of functional and multifunctional NPs with respect to live cell imaging and biosensing are described. (6) Finally, our conclusions and outlook are given. Advances in Inorganic NPs. Inorganic NPs for biomedical applications have advanced rapidly in recent years because of their excellent optical and magnetic properties. An extensive amount of work has been done in the area of synthesis and surface modification of NPs.^1 Semiconductor, noble metal, and metal oxide NPs of 1-100 nm have unique size-dependent properties, which prompted the researchers to investigate them in various imaging and sensing applications. The emerging applications of NPs in bioimaging and biosensing would lead to a variety of essential tools in medical diagnostics. Highly fluor- escent semiconductor nanocrystals (NCs) or QDs (e.g., CdSe-ZnS) have emerged as a potential fluorescent label, owing to their remarkable optical properties.^2 Compared to fluorescent dyes, QDs do not suffer the setback of photobleaching and their emission colors are tunable from the visible to the NIR region by varying the size or composition of QDs.
Recent advances in organometallic synthesis have enabled the size- and shape- dependent preparation of magnetic NPs (MPs) (e.g., Fe 2 O 3 , Fe 3 O 4 , Co, and FePt) and QDs (e.g., CdSe- ZnS, CdTe, and CdS) for applications in biology. A wide variety of contrast agents and optical labels are required for different types of imaging and detection modalities such as magnetic resonance imaging (MRI), positron emission tomo- graphy (PET), optical coherence tomography (OCT), and fluorescence-based imaging. 3 Although each method has inhe- rent advantages and drawbacks, MRI and PET are ideal for in vivo imaging, whereas fluorescence-based imaging is most widely employed for in vitro imaging. Noble metal NPs such as gold (Au) and silver (Ag) are considered to be promising materials and are emerging as alter- natives to semiconductor NCs.^4 Noble metal NCs have surface plasmon resonance that induces a drastic enhancement in their absorption and scattering cross sections. This surface plasmon band is tunable from the visible to the NIR region by changing the particle size and shape from 1-100 nm length scale. They are also used as optical-imaging-based contrast agents for the detection of proteins and DNA. Although individual particles of nanometer size can be imaged using scattered light or by the photothermal method, their application in bioimaging is largely limited because of background scattering from cellular organelles. Recent advan- ces in synthetic methods have made it possible to explore a variety of metallic nanostructures as optical contrast agents.5,6^ Other types of magnetic oxide NPs have also been investigated for various biomedical applications.3,7^ Superparamagnetic NPs are emerging as versatile probes in biomedical applications, especially in the area of MRI. Among these, iron oxide (γ-Fe 2 O 3 or Fe 3 O 4 ) MPs have been widely used as T 2 (negative) contrast agents. Additionally, manganese oxide (MnO) MPs are emerging as
*Corresponding author. Tel: þ65 6874-5249. Fax: þ65 6774-4657. E-mail: [email protected]. (1) Jun, Y.-W.; Lee, J.-H.; Cheon, J. Angew. Chem., Int. Ed. 2008 , 47 , 5122–
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(3) Weissleder, R. Science 2006 , 312 , 1168–1171. (4) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005 , 105 , 1547–1562. (5) Jana, N. R. Small 2005 , 1 , 875–882. (6) Murphy, C. J.; Gole, A. M.; Stone, J. W.; Sisco, P. N.; Alkilani, A. M.; Goldsmith, E. C.; Baxter, S. C. Acc. Chem. Res. 2008 , 41 , 1721–1730. (7) Huh, Y. M.; Jun, Y. W.; Song, H. T.; Kim, S.; Choi, J. S.; Lee, J. H.; Yoon, S.; Kim, K. S.; Shin, J. S.; Suh, J. S.; Cheon, J. J. Am. Chem. Soc. 2005 , 127 , 12387–
B DOI: 10.1021/la903512m Langmuir XXXX, XXX(XX), XXX–XXX
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promising contrast agents because of their size-dependent mag- netic properties.^7 These oxide MPs are also used in the magnetic harvesting of proteins and cells. In clinical diagnosis, MRI is increasingly being used as an adjunct. To increase the tissue contrast in MRI applications, a number of contrast agents have been developed. Generally, the contrast agents are classified into two types: (a) positive and (b) negative. Positive and negative contrast agents are characterized by their ability to shorten either the longitudinal relaxation time, T 1 , resulting in the brightening (hyperintense) of MR images, or the transversal relaxation time, T 2 , resulting in the darkening (hypointense) of MR images, respectively. The shortening of the relaxation time of water protons in the tissues dictates the utility and effectiveness of the contrast agents. The intrinsic T 1 and T 2 values of water are best described by their molar relaxivities, r 1 and r 2 , which are expressed in mM-^1 s-^1. In clinical imaging, gadolinium-based paramagnetic chelates (e.g., Gd-DTPA (DTPA is diethylene triamine pentaacetic acid)) are mainly used as T 1 contrast agents.^8 Introducing multifunc- tionalities such as fluorescence and a drug-targeting moiety onto Gd-DTPA platforms has been found to be problematic. Con- versely, iron oxide-based MPs could easily be modified to introduce a wide range of biological and pharmacological target- ing/delivery moieties. Recently, MPs (Fe 2 O 3 , Fe 3 O 4 , and MFe 2 O 4 where M = Ni, Co, Mn, or Fe) have been used as negative T 2 contrast agents for MRI.^9 Luminescent gadolinium oxide (Gd 2 O 3 ) hybrid MPs have been used as T 1 contrast agents for both in vivo fluorescence and MR imaging.^10 Nanoshells com- posed of Au 3 Cu 1 have also been used as MR contrast agents.^11 Advances in Multifunctional NPs. The fabrication of bi- functional or multifunctional NPs has received a great deal of attention in recent years because of their various biomedical applications. Hybrid inorganic NPs are emerging as useful probes for magnetic-based targeting, delivery, cell separation, MRI, and fluorescence-based biolabeling applications. Gold (Au) NPs have been extensively used to prepare multifunctional composites with QDs and MPs. Typical examples include Fe 3 O 4 - Au,^12 CdSe-Au,^13 and PbSe-Au-Fe 3 O 4.^14 Multifunctional NPs have been actively explored for the en- hancement of imaging, targeting, and delivery. In the field of biological and biomedical imaging, QDs and MPs have been enjoying greater roles in biolabeling^2 and MRI,^15 respectively. A combination of optical and magnetic properties in a single material would enable simultaneous biolabeling/imaging and cell sorting/ separation.16,17^ Nanocomposites consisting of semiconductor
and magnetic NPs, known as magnetic quantum dots (MQ- Ds),^18 are of great interest as a new class of materials. Hybrid nanocomposites such as Fe 3 O 4 - Au and CoPt-Au have shown the potential applications of such bifunctional NPs.^19 More recently, silica-coated MPs with either QDs or dyes have been used in the aforementioned applications. We have developed silica coating methods for CdSe-ZnS^20 and PbSe QDs^21 and bifunctional NPs22,23^ consisting of CdSe-ZnS QDs and γ-Fe 2 O 3 MPs. Despite all of these advances, the application of multi- functional NPs in in vivo imaging is still in its infancy. In this feature article, we intend to discuss the synthesis and application of functional and multifunctional NPs derived from our recent work.
2. Functional and Multifunctional NPs: Significance of
Coating
Water-soluble functional NPs are indispensable for various biomedical applications. However, the synthesis of robust func- tional NPs is very challenging because most of the good synthetic methods available for noble metal, QD, and magnetic oxides produce hydrophobic NPs as a result of the hydrophobic surfac- tant coating. Thus, water solubilization and functionalization are the key issues prior to their application, and herein lies the significance of the coating.24,25^ The coating helps to convert hydrophobic NPs into hydrophilic water-soluble particles and introduce chemical functionality onto the particle surface so that different chemicals and biomolecules can be covalently attached. There are two common coating strategies that exist in order to convert hydrophobic NPs into hydrophilic and functional NPs. The first approach involves the ligand exchange of the original surfactant by hydrophilic ligands such as thiols or other func- tional groups.^26 Thiol-based ligand exchange is most common for noble metal NPs compared to other systems. This is because thiol brings about strong chemisorption on noble metal surfaces. A variety of thiol-based functional NPs of Au and Ag were synthesized.^4 In addition, various approaches of thiol-based methods were developed to make a stable coating, which involves the use of ligands with either multiple thiols, thiolated dendrimers, dendrons, or the cross-linking of surface ligands.2, The second approach involves the interdigited bilayer forma- tion between amphiphilic molecules/polymers and the passivating surfactant layer surrounding NPs.^27 These approaches have been successfully applied to noble metal NPs, in comparison with iron oxide MPs and QDs. Several methods exist in the literature on the design of water-soluble QDs.^27 -^30 One method involves an
(8) Aime, S.; Frullano, L.; Geninatti, C. S. Angew. Chem., Int. Ed. 2002 , 41 , 1017 – 1019. (9) Lee, J.-H.; Huh, Y.-M.; Jun, Y.-w.; Seo, J.-w.; Jang, J.-t.; Song, H.-T.; Kim, S.; Cho, E.-J.; Yoon, H.-G.; Suh, J.-S.; Cheon, J. Nat. Med. 2007 , 13 , 95–99. (10) Bridot, J.-L.; Faure, A.-C.; Laurent, S.; Riviere, C.; Billotey, C.; Hiba, B.; Janier, M.; Josserand, V.; Coll, J.-L.; Elst, L. V.; Muller, R.; Roux, S.; Tillement, O. J. Am. Chem. Soc. 2007 , 129 , 5076–5084. (11) Su, C.-H.; Sheu, H.-S.; Lin, C.-Y.; Huang, C.-C.; Lo, Y. -W.; Pu, Y. -C.; Weng, J. -C.; Shieh, D. -B.; Chen, J. -H.; Yeh, C. -S. J. Am. Chem. Soc. 2007 , 129 , 2139 – 2146. (12) Yu, H.; Chen, S.; Rice, P. M.; Wang, S. X.; White, R. L.; Sun, S. Nano Lett. 2005 , 5 , 379–382. (13) Mokari, T.; Rothenberg, E.; Popov, I.; Costi, R.; Banin, U. Science 2004 , 304 , 1787–1790. (14) Shi, W.; Sahoo, Y.; Zeng, H.; Ding, Y.; Swihart, M. T.; Prasad, P. N. Adv. Mater. 2006 , 18 , 1889–1894. (15) de Vries, I. J.; Lesterhuis, W. J.; Barentsz, J. O.; Verdijk, P.; van Krieken, J. H.; Boerman, O. C.; Oyen, W. J.; Bonenkamp, J. J.; Boezeman, J. B.; Adema, G. J.; Bulte, J. W.; Scheenen, T. W.; Punt, C. J.; Heerschap, A.; Figdor, C. G. Nat. Biotechnol. 2005 , 23 , 1407–1413. (16) Salgueirino-Maceira, V.; Correa-Duarte, M. A.; Spasova, M.; Liz-Marzan, L. M.; Farle, M. Adv. Func. Mater. 2006 , 16 , 509–514. (17) Quarta, A.; Di Corato, R.; Manna, L.; Ragusa, A.; Pellegrino, T. IEEE Transactions on Nanobioscience 2007 , 6 , 298–308.
(18) Ang, C. Y.; Giam, L.; Chan, Z. M.; Lin, A. W. H.; Gu, H.; Devlin, E.; Papaefthymiou, G. C.; Selvan, S. T.; Ying, J. Y. Adv. Mater. 2009 , 21 , 869–873. (19) Cozzoli, P. D.; Pellegrino, T.; Manna, L. Chem. Soc. Rev. 2006 , 35 , 1195–
(20) Selvan, S. T.; Tan, T. T.; Ying, J. Y. Adv. Mater. 2005 , 17 , 1620–1625. (21) Tan, T. T.; Selvan, S. T.; Zhao, L.; Gao, S.; Ying, J. Y. Chem. Mater. 2007 , 19 , 3112–3117. (22) Yi, D. K.; Selvan, S. T.; Lee, S. S.; Papaefthymiou, G. C.; Kundaliya, D.; Ying, J. Y. J. Am. Chem. Soc. 2005 , 127 , 4990–4991. (23) Selvan, S. T.; Patra, P. K.; Ang, C. Y.; Ying, J. Y. Angew. Chem., Int. Ed. 2007 , 46 , 2448–2452. (24) Gerion, D.; Pinaud, F.; Williams, S. C.; Parak, W. J.; Zanchet, D.; Weiss, S.; Alivisatos, A. P. J. Phys. Chem. B 2001 , 105 , 8861–8871. (25) Pellegrino, T.; Manna, L.; Kudera, S.; Liedl, T.; Koktysh, D.; Rogach, A. L.; Keller, S.; Radler, J.; Natile, G.; Parak, W. J. Nano Lett. 2004 , 4 , 703–707. (26) Aldana, J.; Lavelle, N.; Wang, Y.; Peng, X. J. Am. Chem. Soc. 2005 , 127 , 2496 – 2504. (27) Duan, H.; Nie, S. J. Am. Chem. Soc. 2007 , 129 , 3333–3338. (28) Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat. Biotechnol. 2003 , 21 , 41–46. (29) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002 , 298 , 1759–1762. (30) Susumu, K.; Uyeda, H. T.; Medintz, I. L.; Pons, T.; Delehanty, J. B.; Mattoussi, H. J. Am. Chem. Soc. 2007 , 129 , 13987–13996.
D DOI: 10.1021/la903512m Langmuir XXXX, XXX(XX), XXX–XXX
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introduction into the reverse microemulsion medium for silica coating. This direct silica-coating approach has enabled a wide variety of hydrophobic QDs (e.g., CdSe, CdSe-ZnS, and PbSe), MPs (e.g., Fe 2 O 3 ), and bifunctional NPs or heterodimers (e.g., CdSe-Fe 2 O 3 ) to be encapsulated within spherical silica particles.^20 -^23 To ex- amine the reliability of the reverse microemulsion approach in preparing silica-coated QDs (SiO 2 /CdSe), CdSe QDs with differ- ent surface-passivating ligands such as TOPO and octadecylpho- sphonic acid (ODPA) or TOPO and hexadecyl amine (HDA)^42 were examined. At first, hydrophobic NPs have to be transferred to the hydrophilic interior of the micelles where silica growth takes place. The particles bearing a hydrophobic surface have to be exchanged with a hydrophilic ligand. The TOPO-capped QDs are introduced to the reverse micelles, where Igepal could be ex- changed partially or completely with TOPO. The resulting QDs become amenable for incorporation into the aqueous domains of the reverse microemulsion, and TEOS is added as a sol- gel precursor for silica polymerization within the aqueous domains (Scheme 2).
The incorporation mechanism of hydrophobic QDs into silica spheres is clearly elucidated by Koole et al. in a recent paper.^43 The transfer of hydrophobic QDs into aqueous domains of a reverse microemulsion has been probed by fluorescence spectro- scopy. It has been found that the hydrolyzed TEOS has a high affinity for the QD surface and replaces the hydrophobic amine ligands (octadecylamine, ODA). This replacement facilitates the transfer of the QDs into the hydrophilic interior of the micelles, where silica growth takes place. If the QDs contain stronger- binding thiol ligands, then the replacement by TEOS is hindered partially or fully, resulting in off-centered or surface-incorporated QDs. This work has probed the mechanism thoroughly and allowed for the highly controlled incorporation of hydrophobic single QDs (CdSe-CdS-ZnS core-shell-shell and PbSe QDs) exactly in the middle of monodisperse silica spheres by the reverse microemulsion method.^43 A similar reverse microemulsion met- hod was used earlier to incorporate hydrophilic CdTe QDs^44 and
Scheme 2. Encapsulation of Hydrophobic TOPO-Capped CdSe QDs or Oleic Acid-Capped PbSe QDs (a) within a Reverse Microemulsion and (b) within a Silica Shell and (c) Synthesis of Silica-Coated Nanocomposites of MPs and QDs
(42) Qu, L.; Peng, X. J. Am. Chem. Soc. 2002 , 124 , 2049–2055.
(43) Koole, R.; Van Schooneveld, M. M.; Hilhorst, J.; Donega, C. D.; 0 t Hart, D. C.; van Blaaderen, A.; Vanmaekelbergh, D.; Meijerink, A. Chem. Mater. 2008 , 20 , 2503 – 2512. (44) Yang, Y. H.; Jing, L. H.; Yu, X. L.; Yan, D. D.; Gao, M. Y. Chem. Mater. 2007 , 19 , 4123–4128. (45) Darbandi, M.; Thomann, R.; Nann, T. Chem. Mater. 2005 , 17 , 5720–5725.
Langmuir XXXX, XXX(XX), XXX–XXX DOI: 10.1021/la903512m E
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hydrophobic CdSe-ZnS^45 or PbSe^46 QDs in silica particles. As demonstrated in our work and also by others, the reverse microemulsion method is more advantageous because it is less complicated. In comparison with the modified-St€ober approach, the reverse microemulsion method provides better control over particle size and the size dispersion is higher;^45 furthermore, no prior ligand exchange is required. Figure 1 shows an array of TEM images depicting the efficacy of the reverse microemulsion method in encapsulating different hydrophobic QDs, MPs, and hybrid NPs within silica. Single or multiple QDs (Figures 1a-d) can be encapsulated within each silica NP.20,21^ The silica shell thickness can be altered by varying the TEOS concentration. Varying the amount of water and ammonia in the microemulsion also affected the silica shell thickness because this altered the aqueous domain size. The encapsulated MPs are monodisperse (11.8 ( 1.3 nm in diameter), and multiple (single, double, and triple) MPs are clearly seen in the image (Figure 1e).^22 X-ray diffraction (XRD) pattern as well as X-ray photoelectron Fe 2p3/2 and Fe 2p1/2 peaks at 711.4 and 724.7 eV, respectively (not shown here), confirmed that the MPs are γ-Fe 2 O 3 crystallites. Uniform particles containing a single MP and a tailored silica shell thickness could be derived under controlled experimental conditions. In our microemulsion synthesis, MPs (∼11.8 nm) and QDs (∼3.5 nm) were encapsulated within spherical silica shells after 48 h of reaction, yielding SiO 2 /MP-QD NPs.^22 After the encapsula- tion of QDs and MPs, silica NPs appear to be elongated (Figures 1f,g). Energy dispersive X-ray (EDX) analysis confirmed the presence of CdSe, Fe 2 O 3 , and silica. Even at a lower QD concentration of 0.5 mg/mL cyclohexane, both QDs and MPs (marked by an arrow) are successfully encapsulated in the silica shell (Figure 1g). Figure 2a shows the photostability of silica-coated QDs (SiO 2 / CdSe) with and without aminopropyl trimethoxysilane (APS) functionalization in a mixture of water and phosphate-buffered saline (PBS). The APS functionalization improves the emission intensity. The initial increase in the quantum yield (QY) relative to the uncoated CdSe QDs in toluene might be due to the
photobrightening of QDs caused by photoionization. Figure 2a clearly illustrates that the SiO 2 /CdSe samples exhibit superior photostability in water, compared to the uncoated CdSe QDs in toluene.^20 In another embodiment, polyethylene glycol (PEG)-modi- fied silica-coated core-shell (SiO 2 /CdSe-ZnS) NPs have been synthesized by a similar reverse microemulsion method. The QDs are encapsulated within 25 nm silica NPs. The addition of PEG reduces the uniformity of the silica shell thickness. 20 Figure 2b compares the QY of PEG-modified SiO 2 /CdSe-ZnS NPs in water as a function of silica coating time, illustrating an optimal QY of 17% with a silica coating time of 90 h. Recently, Kooley et al. studied the mechanism by which the QDs transfer into aqueous domains of the reverse microemulsion and obtained a higher QY of 30%. 43 In our case, the maximum QY (17%) attained by PEG-modified SiO 2 /CdSe-ZnS NPs in water is higher than that of the uncoated CdSe-ZnS in organic solvent (10%). Remarkably, it did not deteriorate even after 4 weeks of storage in oxygen. This is because the silica coat- ing provides a robust barrier against oxidation of the QD core. The PEG-modified SiO 2 /QDs also exhibited high stability in water (pH 7) over 4 weeks. At high salt concentrations, the particles precipitated but remained fluorescent. The pre- sence of PEG enhances the water solubility of NPs by pre- venting their coagulation and by improving their hydro- philicity. Other types of functionalized PEGs could also be incorporated onto the silica by a similar approach for biocon- jugation applications. The PEG groups not only enhance water solubility but also reduce the nonspecific adsorption of the particles. 23 Cytotoxicity studies of CdSe and CdSe-ZnS QDs with differ- ent surface coatings are well documented in the literature.^47 The in vitro cytotoxicity studies^20 conducted on different cell lines (e.g., HepG2 human liver cancer cells and NIH-3T3 mouse fibroblast cells) showed that silica-coated QDs are much less toxic compared to other water-soluble QDs coated with mercaptoacetic acid (MAA) and poly(maleic anhydride-alt-1-tetradecene) (PMA). It has been found that SiO 2 /CdSe and PEG-modified SiO 2 / CdSe-ZnS NPs maintained over 50% cell viability (IC 50 ) even
Figure 1. TEM images of (a-c) CdSe QDs, (d) PbSe, (e) γ-Fe 2 O 3 MPs, and (f, g) hybrid NPs consisting of CdSe QDs and γ-Fe 2 O 3 MPs encapsulated within silica NPs. (a-c) Multiple 5 nm CdSe QDs are encapsulated within 25-100 nm silica NPs. (b) Free, uncoated QDs are observed because of the high concentration of QD. Single, double, and triple MPs are seen in image e. Silica NPs appear to be elongated in images f and g after the encapsulation of QDs and MPs. Adapted from (b, c) ref 20 and (e-g) ref 22 with permission (copyright 2005, Wiley-VCH and ACS, respectively).
(46) Darbandi, M.; Lu, W. G.; Fang, J. Y.; Nann, T. Langmuir 2006 , 22 , 4371–
- (47) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Nano Lett. 2004 , 4 , 11–18.
Langmuir XXXX, XXX(XX), XXX–XXX DOI: 10.1021/la903512m G
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colloidal stability. Recently, PEG-silane-coated MPs have been used as contrast agents to visualize tumors by MRI.^51
Scheme 3 summarizes the silica-coating strategy for different NPs. The silanes are chosen in such a way that silica shell
Figure 3. Photographs of MQDs under white light (a) before and (b) after magnetic harvesting. (c) Harvested red MQDs showing the magnetic and luminescence properties under UV excitation at 365 nm. (d) Harvested and chloroform-dispersed green and orange MQDs under UV excitation at 365 nm. (e, f ) TEM micrographs of heterodimers of MQDs after magnetic harvesting. The larger particles are Fe 2 O 3 MPs (8-10 nm), and the smaller particles are CdSe QDs (4-5 nm). (g) Absorption and (h) emission spectra of various SiO 2 /MQDs in PBS. The absorption and emission wavelengths are characteristic of the CdSe dot size. Adapted from ref 23 with permission (copyright 2007, Wiley-VCH).
Scheme 3. Thin Silica Coating Chemisty for Different Types of NPsa
a (^) Adapted from ref 50 with permission (copyright 2007, ACS).
H DOI: 10.1021/la903512m Langmuir XXXX, XXX(XX), XXX–XXX
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formation is initiated at the NP surface. Trimethoxy silanes can be directly polymerized on the surface of oxide NPs (such as Fe 3 O 4 and ZnO) under basic conditions because oxide NPs have a dominant hydroxide-terminated surface and thus silane hydro- lysis and polymerization initiate from the NP surface. In the case of Au, Ag NPs, and CdSe-ZnS QDs, a linker silane (merca- propropyltrimethoxy silane, MPS) is used to adsorb on the particle surface via a thiol group and the silane group initiates the silica shell formation. Depending on the nature of the silane used, it is possible to introduce different chemical functional groups such as amine, phosphate, and polyethylene glycol. Figure 4 shows the TEM images of various silanized NPs obtained through Scheme 3. Because the silica shells are very thin, they are invisible under the electron microscope but their presence is determined by other methods such as NMR and FTIR. The average silica shell thickness of 1.5-3 nm is estimated by comparing the TEM and dynamic light scattering (DLS) measurements, which suggest that the overall small coated NPs can be prepared by this approach.^50
4. Functionalization of NPs with Various Chemicals
and Biomolecules
NPs with an appropriate biofunctionality on their surface are required for enhanced labeling and specific detection. Antibodies, peptides, aptamers (small oligonuclotides that bind strongly with proteins, small molecules, or other ions), vitamins, and carbo- hydrates are examples of such molecules. However, the linking of these molecules to NPs is very challenging and requires an appropriate bioconjugation strategy.^52 Silica-coated NPs can be successfully used for such functionalization using commercially
available conjugation reagents (e.g., N-hydroxysuccinimide NHS-based reagents and 1-ethyl-3-(3-dimethylaminopropyl) cabodiimide, EDC). Scheme 4 presents diverse strategies used to make various nanobioconjugates by linking the NPs with a variety of biomo- lecules. Primary-amine- or carboxylate-terminated NPs are deri- ved from the silica coating, and then they are linked with different molecules via bioconjugation chemistry. For example, biotin is linked to primary-amine-terminated NPs by reacting with the commercially available NHS-biotin; amine-terminated oligonu- cleotides and antibodies are linked to primary-amine-terminated NPs using a bifunctional NHS-PEG-NHS reagent; carboxy- late-terminated NPs are linked to primary amine groups of the oligonucleotide/antibody via EDC coupling. To prepare robust functional NPs, primarily covalent linkage between NPs and biomolecules has been investigated. Because our silica coating is robust, we are able to prepare robust functional NPs. Using silica- coated NPs, we have prepared various NPs that are func- tionalized with antibodies, oligonucleotides, biotin, and dex- tran.49,50,53-^55 The successful functionalization indicates that the silica coating is a unique method of deriving functional and multifunctional NPs.
5. Biomedical Applications
5.1. Live Cell Imaging. One of the emerging applications of QDs is cell labeling/imaging. We have used silica-coated QDs and MQDs for live cell imaging. Silanization in a reverse microemul- sion produces a thin silica coating on bare CdSe QDs or MQDs with surface NH 2 groups (scheme in Figure 5). The addition of a
Figure 4. Representative TEM micrographs of various silanized NPs. Compared to Figure 1, the silica shells are invisible because they are too thin to observe under TEM. (Insets) Respective optically clear aqueous solutions containing 10-20 mg/mL NPs. Adapted from ref 50 with permission (copyright 2007, ACS).
(52) Faure, A. -C.; Hoffmann, C.; Bazzi, R.; Goubard, F.; Pauthe, E.; Marquette, C. A.; Blum, L. J.; Perriat, P.; Roux, S.; Tillement, O. ACS Nano 2008 , 2 , 2273–2282.
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J DOI: 10.1021/la903512m Langmuir XXXX, XXX(XX), XXX–XXX
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nanomolar concentration range in the membrane-based particle enlargement method. This is possible due to the unique silaniza- tion process, which provides a thin silica coating that protects the Au cores from the environment without blocking NP enlargement in the signal-enhancement process. In other interesting work, we have demonstrated the use of avidin-coated QD multilayer thin films (fabricated by the Langmuir-Blodgett (LB) technique) for fluorescence resonance energy transfer (FRET) sensing.^56 The avidin-coated QD films are excellent substrates for the conjugation of biotinylated Au particles.
6. Conclusions and Outlook
In this feature article, we have shown the importance of inorganic NPs, particularly, QDs, MPs, and other metal NPs, in bioimaging and biosensing applications. In the area of bioima- ging, silica-coated QDs and MPs derived from our earlier work clearly demonstrate the efficacy of the silica coating method in live cell imaging. For protein detection, functionalized Au and Ag NPs with dextran, aptamers, and antibodies have been efficiently used. Multifunctional NPs consisting of MPs and QDs or organic dyes with drug molecules are emerging candidates for fluores- cence-based biolabeling and magnetic-based targeting, delivery, cell separation, and MRI applications. Although there have been considerable advances in recent years, the application of
Figure 5. (a) Scheme depicting the silica-coated CdSe QDs or Fe 2 O 3 - CdSe MQDs with APS in a reverse microemulsion and dispersed NPs in PBS buffer. The surface NH 2 groups are then reacted with BAM to form a covalent amide bond. The oleyl groups are used to anchor to the cell membrane in biolabeling. CLSM images showing the labeling of (b) HepG2 and (c) NIH-3T3 cell membranes using BAM/SiO 2 /CdSe QDs. Adapted from ref 23 with permission (copyright 2007, Wiley-VCH).
Figure 6. Glycoprotein (Con A) detection using dextran-functionalized NPs. (a) Ag, (b) Fe 3 O 4 , and (c) CdSe-ZnS. In each sample, Con A was added to a final concentration of 10 μM, and the absorption spectrum was obtained after (black line) 0 min, (blue line) 1 h, and (red line) 2 h. A magnet is shown to remove the aggregated Fe 3 O 4 MPs from the solution in panel b. Adapted from ref 54 with permission (copyright 2008, ACS).
(56) Gole, A.; Jana, N. R.; Selvan, S. T.; Ying, J. Y. Langmuir 2008 , 24 , 8181–
Langmuir XXXX, XXX(XX), XXX–XXX DOI: 10.1021/la903512m K
Selvan et al. Invited Feature Article
multifunctional NPs in in vivo imaging is still in its infancy. This invites the development of more multifunctional systems with less-toxic fluorescent probes, excluding carcinogenic elements such as Cd and Pb. Very recently, up-conversion NPs (UCNPs) have emerged as a new type of multimodal imaging probe for both optical imaging and MRI.57,58^ The deep penetration depth of NIR excitation, excellent photostability, nonblinking, and ab- sence of autofluorescence of UCNPs make them attractive imaging probes for applications such as the targeting of tumor tissues in vivo and long-term cellular and animal imaging. The challenge that we face in the future involves the develop- ment of “smart” contrast agents that are capable of monitoring specific cellular and molecular events in vivo. Our current work is
focused on the development of versatile multifunctional para- magnetic NPs for multimodal imaging in a number of clini- cal pathologies such as early cancer diagnosis and cellular trafficking in stem cell therapy and immunological interventions. The targeted delivery of drugs is an important area in health care. By conjugating multiple components such as fluorescent QDs or dyes or UCNPs, tumor-targeting groups, anticancer drugs, or siRNA to the MPs, future work would seek to provide solutions to early cancer diagnosis and the targeted delivery of therapeutics.
Acknowledgment. Most of this work was carried out at the Institute of Bioengineering and Nanotechnology (IBN), Singapore. We thank Prof. Jackie Y. Ying for giving us the opportunity to work at IBN and all of the students and researchers who have contributed to the work described in this feature article.
Figure 7. Protein detection based on aptamer- and antibody-functionalized Au NPs. Adapted from ref 55 with permission (copyright 2008, Wiley-VCH).
(57) Li, Z. Q.; Zhang, Y.; Jiang, S. Adv. Mater. 2008 , 20 , 4765–4767. (58) Zhang, Y.; Das, G. K.; Xu, R.; Tan, T. T. Y. J. Mater. Chem. 2009 , 19 , 3696 – 3703.
