Compact and Versatile Nickel-Nitrilotriacetate-Modified Quantum Dots

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Compact and Versatile Nickel-Nitrilotriacetate-Modified Quantum Dots for Protein Imaging and F€orster Resonance Energy Transfer Based Assay Hye-Young Park,† Keumhyun Kim,† Sukmin Hong,† Heeyeon Kim,† Youngseon Choi,† Jiyoung Ryu,† Doyoon Kwon,‡ Regis Grailhe,*,‡ and Rita Song*,† †

Nano/Bio Chemistry Laboratory and ‡Neurodegenerative Disorder Laboratory, Institut Pasteur Korea (IP-K), 696 Sampyeong-dong, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea Received November 5, 2009. Revised Manuscript Received December 6, 2009

The generation of compact quantum dots (QDs) probes is of critical importance for visualizing molecular interaction occurring in biological context, particularly when using the F€orster resonance energy transfer (FRET) approach. This Article reports novel water-soluble compact CdSe/ZnS QDs prepared by ligand exchange reaction using thiolated nitrilotriacetate (NTA). The resulting NTA-QDs revealed higher stability and remarkable conjugation efficiency compared to the other QDs prepared with different ligands by using the ligand exchange method. The Ni-NTA group is a well-known binding moiety for the detection and purification of oligohistidine-tagged recombinant proteins. We demonstrated that NiNTA-QDs prepared by Ni2þ complexation exhibited highly specific binding ability toward 6-histidine (His)-tagged peptides present in various experimental conditions (buffer solution, agarose beads, and HEK cells). Importantly, the compact NiNTA-QDs serve as an efficient FRET donor. These results suggest that the stable and highly selective multifunctional NTA-QDs can be useful for labeling and tracking molecular interactions within biological context.

Introduction Semiconductor nanocrystals or quantum dots (QDs) have been studied extensively for their potential applications in laser optics,1,2 light emitting devices,3,4 electronics, and biological imaging5,6 due to their attractive optical properties, such as narrow emission and broad excitation bands enabling the use of a single excitation source for multiplexing detection. In particular, over the past few years, studies on the development of QDs as biological probes for diagnostics and bioimaging have been of considerable interest. The synthesis of QDs in a nonpolar hot organic solvent has provided the most efficient way of obtaining QDs with excellent monocrystallinity, photoluminescence properties, and colloidal stability. Moreover, the ability to manipulate the surface properties of QDs is important for their successful adaptation for biological application. The major concern of water solubilization of hydrophobic ligand-capped QDs is to maintain their original stability, quantum yield (QY), and controllable hydrodynamic diameter. In this context, considerable efforts have been made to improve the surface properties of hydrophilic QDs. The most well-known methods are based on a ligand exchange *To whom correspondence should be addressed. (R.S.) Telephone: þ82-318018-8230. Fax þ82-31-8018-8014. E-mail: [email protected]. (R.G.) Telephone: þ82-31-8018-8004. Fax þ82-31-8018-8010. E-mail: regis.grailhe@ ip-korea.org. (1) Wuister, S. F.; Swart, I.; van Driel, F.; Hickey, S. G.; Donega, C. D. Nano Lett. 2003, 3, 503–507. (2) Klimov, V. I.; Mikhailovsky, A. A.; Xu, Su; Malko, A.; Hollingsworth, J. A.; Leatherdale, C. A.; Eisler, H. J.; Bawendi, M. G. Science 2000, 290, 314. (3) Colvin, V. L.; Schlamp, M. C.; Alivisatos, A. P. Nature 1994, 370, 354–357. (4) Seth, C.; Wing-Keung, W.; Moungi, B.; Vladimir, B. Nature 2002, 420, 800. (5) Lidke, D. S.; Nagy, P.; Heintzmann, R.; Arndt-Jovin, D. J.; Post, J. N.; Grecco, H. E.; Jares-Erijman, E. A.; Jovin, T. M. Nat. Biotechnol. 2004, 22, 198–203. (6) Mihaljevic, T.; Kim, S.; Lim, Y. T.; Lee, J.; Nakayama, A.; Parker, J. A.; Laurence, R. G.; Soltesz, E. G.; De Grand, A. M.; Dor, D. M.; Cohn, L. H.; Bawendi, M. G.; Frangioni, J. V. Nat. Biotechnol. 2004, 22, 93–97. (7) 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.

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reaction using hydrophilic small molecules,1 surface silanization,7-9 and polymer coating.10-13 The polymer coating method has gained popularity for the water solubilization of QDs due to the physicochemical stability, high QY, and resistance to particle aggregation. While polymercoated QDs are stable in a wide range of biological media and have potential for in vitro and in vivo imaging, their size usually exceeds 15 nm. The applications requiring an analysis of the close interaction between QDs and other biomolecules, as in the case of F€orster resonance energy transfer (FRET)-based applications, still need a compact capping of QDs. The ligand exchange method is based on the reaction where hydrophobic capping ligands on QDs are displaced by heterobifunctional ligands possessing both a hydrophilic moiety and thiol group. Conventional ligands of choice are usually mercaptopropionic acid (MPA), mercaptoundecanoic acid (MUA), and dihydrolipoic acid (DHLA). Among them, bidentate DHLA ligand has been widely used because of the improved stability due to the chelation effect.1,14,15 However, DHLA-QDs still have limitations in terms of stability in various media.16-18 (8) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013–2016. (9) Warner, J. H.; Hoshino, A.; Yamamoto, K.; Tilley, R. D. Angew. Chem., Int. Ed. 2005, 44, 4550–4554. (10) Jeong, S.; Achermann, M.; Nanda, J.; Lvanov, S.; Klimov, V. I.; Hollingsworth, J. A. J. Am. Chem. Soc. 2005, 127, 10126–10127. (11) 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. (12) Nann, T. Chem. Commun. 2005, 1735–1736. (13) Janczewski, D.; Tomczak, N.; Khin, Y. W.; Han, M. Y.; Vancso, G. J. Eur. Polym. J. 2009, 45, 3–9. (14) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 12142–12150. (15) Medintz, I. L.; Uyeda, H. T.; Goldman, E. R.; Mattoussi, H. Nat. Mater. 2005, 4, 435–446. (16) Susumu, K.; Uyeda, H. T.; Medintz, I. L.; Pons, T.; Delehanty, J. B.; Mattoussi, H. J. Am. Chem. Soc. 2007, 129, 13987–13996. (17) Wang, X. Q.; Wu, J. F.; Li, F. Y.; Li, H. B. Nanotechnology 2008, 19, 1–8. (18) Wang, X. S.; Dykstra, T. E.; Salvador, M. R.; Manners, I.; Scholes, G. D.; Winnik, M. A. J. Am. Chem. Soc. 2004, 126, 7784–7785.

Published on Web 12/23/2009

DOI: 10.1021/la9041887

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The conjugation of the poly(ethylene glycol) (PEG) moiety to improve the stability of QDs has been reported to address the stability issue.16,19 However, the FRET efficiency can be lowered by the presence of bulky PEG with a large molecular weight. In this context, the development of compact QDs with high stability and small hydrodynamic size is important for FRET-based biological applications. The nitrilotriacetate (NTA) ligand contains tricarboxylates that improve the stability of QDs in aqueous media. Furthermore, NiNTA-QDs prepared by a simple Ni2þ complexation reaction can be used as a labeling/detection probe for oligohistidine-tagged biomolecules, since the NiNTA group has been widely used for the separation, surface immobilization, and in vitro detection of recombinant histidine-tagged proteins.20-27 This Article reports the preparation of novel water-soluble CdSe/ZnS QDs via ligand exchange reaction using carefully designed thiolated NTA along with their potential biological applications using a variety of model systems. The stability, optical properties, and reactivity on chemical modifications were also investigated. In addition, the targeting/tagging ability of the NiNTA-QDs was examined in 6His-modified agarose beads and human cells expressing cytoplasmic membrane 6His-protein. Finally, NiNTA-QDs were tested for their potential as a specific fluorescence donor in FRET-based studies.

Experimental Section Reagents. 16-Mercaptohexadecanoic acid (MHDA), mercaptopropionic acid (MPA), acetyl chloride, acetic acid, dicyclohexyl carbodiimide (DCC), dicyclohexylurea (DCU), 1,2-dimethoxyethane (DME), hydrazine acetate, sodium myristate (99%), cadmium nitrate tetrahydrate (99.99%), selenium powder (99.99%), 1-octadecene (ODE, 90%), oleic acid (90%), 1-hexadecylamine (HAD, 90%), trioctylphosphine (TOP, 90%), diethylzinc (1.0 M solution in hexane), trioctylphosphine oxide (TOPO, 99%), trihexamethyldisilthiane (TMS), imidazole, paraformaldehyde (PFA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and tetramethylammonium hydroxide (TMAOH) were purchased from Aldrich. Thioctic acid (Aldrich) was reduced by borohydride to give DHLA for ligand exchange reaction. N-Hydroxysuccinimide (NHS) and N,N-bis(carboxymethyl)-L-lysine from Fluka, Zn powder from Daejung (South Korea), CHCl3 from Baker, NaHCO3 from Junsei, dimethylformamide (DMF) from Acros, Dulbecco’s modified Eagle’s medium (DMEM) from Gibco, NH2PEG-OMe (MW 2000) from Sunbio, and BCA protein assay kit from Thermo Scientific were used without further purification. Peptides, Gly-Gly-His-His-His-His-His-His (2G6H) and Gly-GlyGly-Gly-Gly-His-Gly-Gly (5GH2G), 5-carboxy tetramethylrhodamine (TAMRA)-2G6H, and TAMRA-5GH2G peptides were obtained from Peptron Inc. (Daejon, Korea). Glyoxal-agarose beads were purchased from ABT (Tampa, FL). Fetal bovine serum (FBS), penicillin-streptomycin, bovine serum albumin (BSA), phosphate buffered saline (PBS), and tris/borate/EDTA (19) Mei, B. C.; Susumu, K.; Medintz, I. L.; Delehanty, J. B.; Mountziaris, T. J.; Mattoussi, H. J. Mater. Chem. 2008, 18, 4949–4958. (20) Lata, S.; Reichel, A.; Brock, R.; Tampe, R.; Piehler, J. J. Am. Chem. Soc. 2005, 127, 10205–10215. (21) Buchel, C.; Morris, E.; Orlova, E.; Barber, J. J. Mol. Biol. 2001, 312, 371–379. (22) Guignet, E. G.; Hovius, R.; Vogel, H. Nat. Biotechnol. 2004, 22, 440–444. (23) Kapanidis, A. N.; Ebright, Y. W.; Ebright, R. H. J. Am. Ceram. Soc. 2001, 123, 12123. (24) Xu, C. J.; Xu, K. M.; Gu, H. W.; Zhong, X. F.; Guo, Z. H.; Zheng, R. K.; Zhang, X. X.; Xu, B. J. Am. Chem. Soc. 2004, 126, 3392–3393. (25) Roullier, V.; Clarke, S.; You, C.; Pinaud, F.; Gouzer, G.; Schaible, D.; Marchi-Artzner, V.; Piehler, J.; Dahan, M. Nano Lett. 2009, 9, 1228–1234. (26) Gupta, M.; Caniard, A.; Touceda-Varela, A.; Campopiano, D. J.; Mareque-Rivas, J. C. Bioconjugate Chem. 2008, 19, 1964–1967. (27) Bae, P. K.; Kim, K. N.; Lee, S. J.; Chang, H. J.; Lee, C. K.; Park, J. K. Biomaterials 2009, 30, 836–842.

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(TBE) buffers were purchased from Invitrogen. Fibronectin and Fugene 6 were obtained from BD Bioscience and Roche, respectively. Characterization. Morphology and size distribution of TOPO-QDs and NTA-QDs were investigated by scanning transmission electron microscopy (STEM, Philips CM-30) at an accelerating voltage of 50-300 kV. Absorption and emission spectra of QDs were obtained by using a UV-vis spectrometer (CARY 5000, Varian) and fluoro-spectrophotometer (Fluorolog, Horiba Jovin Yvon), respectively. Size and zeta potential of QDs were measured using a dynamic light scattering (DLS) analyzer (Zetasizer-Nano-ZS, Malvern). The electrophoretic mobility of the QDs was investigated in agarose gel using a gel station (MupidEXu, Advance) and image analyzer (LAS 3000, Fujifilm). An Olympus IX71 inverted fluorescence microscope (Olympus IX71) and a confocal microscope (Zeiss LSM5) were utilized for fluorescence imaging of agarose beads and cells, respectively, using a band-pass (BP) excitation filter (420-480 nm) and BP emission filter (495-555 nm). For the image anaylsis, Image J software (NIH, Bethesda, MD) was utilized to obtain pixel intensities for each image. Ni2þ elemental analysis was performed using an ELAN DRC (Perkin-Elmer) inductively coupled plasma mass spectrometer (ICP-MS) equipped with a quadrupole mass analyzer. Synthesis of TOPO-CdSe/ZnS QDs. TOPO-capped CdSe/ ZnS (core-shell) QDs were synthesized using the procedure reported in the literature (see the Supporting Information).28 Synthesis of NTA Ligand. The thiolated NTA molecule was synthesized according to modification of the procedure reported in the literature (see the Supporting Information).24 NTA Ligand Exchange Reaction of TOPO-QDs. The thiolated NTA ligand (100 μmol) dissolved in dimethyl sulfoxide (DMSO) was added to as-synthesized TOPO-QDs in chloroform. The mixture was stirred for 24 h at room temperature, and then an aqueous solution of TMAOH was added. After the aqueous phase had been separated, the solution was washed several times with borate buffer using a membrane filter. EDC Coupling Reaction of NTA-QDs with PEG. For the stability test of NTA-QDs in the EDC coupling reaction, different amounts of EDC (0.1k-250k equiv) in borate buffer (pH 8) or PBS (pH 7.4) were added to the NTA-QD solution (10 μL, 10 μM). After the QDs were reacted for 1 h with EDC at room temperature, the EDC-activated NTA-QDs were washed several times with deionized (DI) water. The same set of experiments was carried out using DHLA-QDs for comparison. For conjugation with PEG, 1000 or 10 000 equiv of EDC was added to the NTAQDs and reacted for 1 h at room temperature. The EDC-activated NTA-QDs were then reacted with various amounts of NH2-PEGOMe (MW 2000). After the coupling reaction, the QDs were washed three times with DI water and characterized by agarose gel electrophoresis. Gel electrophoresis was run with a 1% agarose gel in 0.5 TBE buffer at 100 V for 30 min. The gel image was obtained using a LAS-3000 image reader. Quantitation of 6His Binding on NiNTA-QDs. The NTAQDs were complexed with various amounts of NiCl2 (0-500 equiv) and then reacted with 200 equiv of the 2G6H peptide. 5GH2G peptide was used as a control. After the reaction, the QDs were purified using a membrane filter (MWCO, 30 kD). Agarose gel electrophoresis was performed with a 1% agarose gel in 0.5 TBE buffer (pH 8.5) at 100 V for 15 min. The 6His on NiNTAQDs was quantified by collecting the unreacted peptide followed by a BCA peptide assay. The absorbance of the solution was measured using a UV-vis spectrometer, and the absorbance at 562 nm of each sample was plotted as a function of the amount of peptide that reacted with the NiNTA-QDs.

Binding Study of NiNTA-QDs with 6His-Functionalized Agarose Beads. Two different agarose beads (ABs) functionalized (28) Jun, S.; Jang, E. Chem. Commun. 2005, 4616–4618.

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Article Scheme 1. Water Solubilization of TOPO-QDs via Ligand Exchange Reaction with NTA

with different amino acid sequences were prepared in order to examine the potential of NiNTA-QDs as site-specific and selective fluorescent probes for the 6His-tagged proteins. ABs have previously been modified by reductive amination of the glyoxal group with the N-terminal amino group of 6His. The functionalized bead with two glycine (G) and six histidine (H) groups, AB-2G6H, was used as a model for cells expressing 6His on their membranes. AB-5GH2G, which represents a random amino acid sequence, was used as a negative control. Both agarose beads were incubated with NiNTA-QDs at room temperature for 15 min, and the unbound QDs were removed by washing with 10 mM PBS. The fluorescence images of the beads were obtained using a fluorescence microscope. Cell Imaging. HEK-293 cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 in DMEM supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 IU/mL streptomycin. Media were changed 2-3 times per week. For plasmid DNA transfection, cells were trypsinized and then seeded in a Lab-Tek 8 well chamber slide coated with 50 μg/mL fibronectin at a density of 8  103 cells per well. A day after, the cells were then transfected with a plasmid expressing N-terminal 6His-tagged 5HT2C serotonin receptor using a Fugene 6 transfection reagent, following the manufacturer’s instructions: DNA/lipid complexes were prepared by mixing 0.3 μg of 6His-5HT2C plasmid with 0.9 μL of Fugene 6 diluted in serum-free medium and added to cells. Then 48 h later HEK293 cells expressing 6His-5HT2C and naı¨ ve HEK-293 cells were blocked with 5% BSA, incubated for 30 min with 0, 10, 25, 50, 75, 100, 150, and 200 nM NiNTA-QDs at 37 °C, washed with PBS twice, and fixed with 4% PFA. Confocal microscopy was performed on the prepared cells with a Zeiss LSM5 live system using excitation at 405 nm and a BP 495-555 emission filter. FRET Experiment. The NiNTA-QDs in deionized water were added to various equivalents of TAMRA-2G6H (or TAMRA-5GH2G as a negative control) in PBS. The final QDs solution was diluted to 1 μM. After 30 min incubation at room temperature, the NiNTA-QDs were washed several times with PBS using membrane filtration (MWCO, 30 kD) and the photoluminescence (PL) spectra were collected. The PL recovery test on the prequenched NiNTA QD-6H2G-TAMRA complex was performed by adding a 0.5 M imidazole solution.

Results and Discussion Ligand Exchange of QDs by NTA. The synthesis of thiolated NTA ligand was performed following the previously reported method with a slight modification (see the Supporting Information for detailed synthetic process.).24 The TOPO-capped QDs dissolved in organic solution were transferred to water via ligand exchange with NTA (Scheme 1). In the ligand exchange process, the thiol functionality of NTA ligand readily forms the coordination bond with ZnS through a strong metal-sulfur interaction. The exchange reaction was evidenced by the complete transfer of QDs into the aqueous phase and by the characterization of their optical and binding properties (vide infra) (see Supporting Information Figure S3). The resulting watersoluble NTA-QDs were found to retain their spectral properties and monodispersity of original TOPO-QDs (see Supporting Information Figures S4 and S5). Langmuir 2010, 26(10), 7327–7333

Figure 1. Stability test of (a) NTA-QDs, (b) DHLA-QDs, and (C) MPA-QDs in various media (after 1 month). Samples 1 to 7 represent aqueous solutions of pH 3, 5, 7, 10, PBS (pH 7.4), borate (pH 8.5), and 1 M NaOH, respectively. (d) Photostability test of MPA-QDs (dark gray), DHLA-QDs (gray), and NTA-QDs (white) in various solution conditions. The inset picture is the QDs in citrate buffer (pH 3) observed after 6 h.

Stability of NTA-QDs in Aqueous Solutions. NTA-QDs must retain their stability in aqueous media to be utilized in wide variety of biological applications. The colloidal and optical stability of NTA-QDs were tested in various aqueous media and compared with MPA- and DHLA-QDs. Figure 1 shows the NTA-QDs well dispersed in all the media tested without any visible precipitation even in acidic conditions (pH 3 and 5) and under the harsh conditions, such as 1 M NaOH after 1 month of storage at 4 °C. On the other hand, the MPA-QDs and DHLAQDs began to precipitate at pH 3 and 5 in the short term observations (6 h). PL intensity measured also revealed higher photostability of NTA-QDs than MPA- and DHLA-QDs in the various media (data points of the samples forming precipitates were eliminated in the plot). The overall improved colloidal and photostability of NTA-QDs over MPA- or DHLA-QDs at prolonged exposure to various media can be attributed to the combined stabilizing effect from the long hydrophobic alkyl chain and tricarboxyl groups. Especially, the remarkable solubility of DOI: 10.1021/la9041887

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NTA-QDs in weak acidic media can be explained by the low pKa value (pKa 1.84) of the NTA ligand, which means that most of NTA-QDs exist in anionic form even in pH 3-5. The NTA-QDs were found to have a higher negative charge on the surface than DHLA- and MPA-QDs as verified by zeta potential measurement. The zeta potential values of NTA-QDs, DHLA-QD, and MPA-QD in PBS (pH 7.4) were -26.8, -5.93, and -22.6 mV, respectively. The ability to create QDs with a small hydrodynamic diameter is the most important advantage of the ligand exchange method. The hydrodynamic diameter of the resulting NTA-QDs was determined to be 9 ( 1.4 nm which is much smaller than that of the polymer-coated QDs which are typically much larger than 15 nm. The quantum yield (25%) of the as-prepared NTA-QDs was comparable to that of DHLA-QDs. These results confirm the compactness, strong negative charge, and high quantum yield of the NTA-QDs. Conjugation of PEG on NTA-QDs. Chemical stability and conjugation efficiency are of critical importance for the successful preparation of biologically applicable QDs. We have tested the chemical conjugation ability of the NTA-QDs using PEG molecules. PEG is a biologically and chemically important molecule due to its ability to decrease the nonspecific adsorption and increase the solubility and biocompatibility of nanoparticles.29,30 A wide range of PEG-derivatives have been used successfully in biomedical research as well as in the medicinal and cosmetic industries. The conjugation of NTA-QDs with amino-PEG (MW 2000) was carried out using EDC coupling agent to stabilize the QDs and observe the capability of forming covalent bonding with other molecules. For the conjugation of PEG onto NTA-QDs, first the NTA-QDs were tested for their stability in EDC-containing buffers. The stability of the DHLA-QDs was also tested under the same conditions for comparison. As shown in Figure 2, the NTA-QDs showed higher stability than DHLA-QDs in a solution containing a large excess of EDC. Figure 2b shows gel electrophoresis analysis of NTA-QDs reacted

with different amounts of amino-PEG in the presence of 2500 equiv of EDC. The gel mobility of the QDs decreased with increasing amino-PEG due to the increase in molecular weight and decrease in overall negative surface charge. The zeta potential measurement showed the decrease in the values. Reaction of NTA-QDs with 0, 50, 100, 500, and 1000 equiv of PEG resulted in zeta potentials of -37.38, -35.2, -32.6, -25.13, and -21.02 mV, respectively, in borate buffer, pH 8.5. On the other hand, the NTA-QDs reacted with amino-PEG without EDC did not show any significant change in mobility from the original QDs, confirming the successful conjugation of amino-PEG on NTA-QDs. The results indicate that other biologically important molecules such as small molecules, peptides, and DNA can be readily conjugated on the NTA-QDs. Ni2þ Complexation and 6His Binding Test. The NiNTA moiety is a well-known chelating agent for 6His and is used in the separation, surface modification, and in vitro detection of recombinant His-tagged proteins.20-26 Recently, imaging of 6His-tagged membrane protein in live cells using NiNTAconjugated polymer-coated QDs and their application in Western blot analysis were reported.31,32 In this section, we have tested the ability of the compact NTA-QDs for the detection of 6His-tagged proteins. NTA-QDs were reacted with various equivalents of NiCl2 and analyzed by agarose gel electrophoresis and ICP-MS. The electrophoretic mobility of the NiNTA-QDs decreased with increasing amounts of Ni2þ (Figure 3). ICP-MS elemental analysis confirmed almost stoichiometric complexation of Ni2þ on NTA-QDs (see Supporting Information Table S1). The stability of the NiNTA-QDs was affected by the amount of Ni2þ complexation due to the reduced negative charge on the QD surface. NiNTA-QDs began to slightly precipitate with 500 equiv of Ni2þ complexation. To optimize the maximum NiNTA coverage and stability at the same time, NiNTA-QDs complexed with 250 Ni2þ were utilized in the binding test otherwise specified. The specific binding of NiNTA-QDs toward the 6His moiety was tested by a reaction of NiNTA-QDs with short length peptides, 2G6H and 5GH2G. The latter was chosen as a control peptide. First, the effect of Ni2þ on the 6His binding property was tested and characterized by gel electrophoresis. NTA-QD (no Ni2þ) and Ni-NTA-QDs prepared with 20, 200, and 500 equiv of Ni2þ were reacted with 200 equiv of 2G6H or 5GH2G peptide. In Figure 4a, lanes 1, 3, 5, and 7 are NTA-QDs (no Ni2þ) and NTA-QDs complexed with 20, 200, and 500 equiv of Ni2þ, respectively, after the reaction with 2G6H. Therefore, comparing lanes 1, 3, 5, and 7 provides information on the effect of the amount of Ni2þ complexed on NTA-QDs. As more Ni2þ was complexed, QDs displayed more retardation in the mobility and the effect reached maximum when NTA-QDs complexed with 200 (lane 5) and 500 (lane 7) equiv of Ni2þ. The result clearly demonstrates that the presence of Ni2þ plays an important role on the binding to 2G6H. Lanes 2, 4, 6, and 8 serve as a control for each QD as these lanes correspond to NTA-QD (no Ni2þ) and NTA-QDs complexed with 20, 200, and 500 equiv of Ni2þ, respectively, after the reaction with 5GH2G. For all the tested QDs, minimal binding to 5GH2G was observed, which is the indication of specific binding of NiNTA-QDs to the 2G6H peptide. In the second set of experiments, the binding ability of the NiNTA-QDs with the 6His peptide was further tested by varying the amount of 2G6H. As shown in Figure 4b, the QDs migrated slowly as more 2G6H was bound to the NiNTA-QDs, indicating the quantitative and

(29) Otsuka, H.; Akiyama, Y.; Nagasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2001, 123, 8226–8230. (30) Araki, J.; Wada, M.; Kuga, S. Langmuir 2001, 17, 21–27.

(31) Kim, M. J.; Park, H. Y.; Kim, J.; Ryu, J.; Hong, S.; Han, S. J.; Song, R. Anal. Biochem. 2008, 379, 124–126. (32) Kim, J.; Park, H. Y.; Kim, J.; Ryu, J.; Kwon, D. Y.; Grailhe, R.; Song, R. Chem. Commun. 2008, 1910–1912.

Figure 2. (a) Stability test of NTA-QDs in EDC containing solution and (b) gel electrophoresis of NTA-QDs and PEG-conjugated NTA-QDs.

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Figure 4. Analysis of the reaction between NiNTA-QDs and 2G6H and 5GH2G peptides. (a) Gel electrophoresis image of the reaction of NTA-QDs (lanes 1 and 2) and NiNTA-QDs (20, 200, and 500 equiv of NiCl2 for lanes 3-4, lanes 5-6, and lanes 7-8, respectively.) with 200 equiv of 2G6H (lanes 1, 3, 5, and 7) and 5GH2G (lanes 2, 4, 6, and 8). (b) Gel electrophoresis image of NiNTA-QDs reacted with various amounts of 2G6H (0, 20, 40, 100, 200, 400, 800, and 1600 equiv of 2G6H). Figure 3. Ni2þ complexation reaction on NTA-QDs. (a) Scheme

of Ni2þ complexation on NTA-QDs. (b) Agarose gel electrophoresis of NiNTA-QDs. Each lane represents QDs reacted with various NiCl2 equivalents (0, 20, 100, 200, 500, 1000, 2000, and 5000, respectively). (c) Stability test of NiNTA-QDs reacted with NiCl2 equivalents of 0, 20, 100, 200, 500, 1000, 2000, and 5000 in PBS, respectively.

specific binding of 2G6H on NiNTA-QD. BCA assay also confirmed the quantitative specific binding of NiNTA-QDs to the 2G6H peptide (Figure S6, Supporting Information). The 6His peptide binding affinity of NiNTA-QDs was further tested using peptide-conjugated agarose beads. The bead-based assay has been developed in our lab as previously reported31,32 to confirm the specific binding ability of the NiNTQ-QD probe to the 6His moiety. Glyoxal-agarose beads (AB, 45-180 μM in diameter) were used to prepare AB-2G6H and AB-5GH2G. The glyoxal group reacts with the amine group of peptides of interest via reductive amination. This reaction is very efficient compared to the typical conjugation method, which utilizes carboxylate or a thiolate activation linker, for example, EDC or sulfo-SMCC. Figure 5a shows a typical fluorescence image of the AB-2G6H that reacted with NiNTA-QDs. The strong green fluorescence of AB-2G6H comes from the binding of NiNTA-QDs on AB2G6H. On the other hand, virtually no fluorescence was observed from the reaction of AB-5GH2G with NiNTA-QDs (Figure 5b). The sigmoidal increase in the fluorescence intensity of AB-2G6H incubated with NiNTA-shows an EC50 value of NiNTA-QDs of 80.2 nM. The high affinity of NiNTA-QDs can be ascribed to the multivalent chelator head effect resulting from the multiple NiNTA binding on the QD surface.20 When the bead test was performed using NTA-QDs, a moderate fluorescence signal was observed (data not shown) probably due to the nonspecific electrostatic binding. The elucidation and quantification of the nature of the nonspecific binding between NTA-QDs and 6His moiety is under investigation. Langmuir 2010, 26(10), 7327–7333

The NiNTA-QDs were further tested for biological labeling properties for the 6His-tagged recombinant protein expressed in human cells. HEK-293 cells were transfected to express on the extracellular compartment 6His such that the histidine tag was attached on the NH2 terminal extremity of the 5HT2C serotonin receptor. Both transfected and untransfected cells were incubated with various concentrations of NiNTA-QDs. Since highly negatively charged NiNTA cannot penetrate through the cellular membrane in given experimental time, only 5HT2C receptors expressed on the plasma membrane are able to interact with the NiNTA-QDs present in solution. After 30 min of incubation, the cells were fixed with PFA and washed several times with PBS and DMEM media to remove the unbound NiNTA-QDs. Figure 5d and e shows the fluorescence confocal microscopy images of the cells after incubation with the NiNTA-QDs. Binding of the NiNTA-QDs was only observed on the surface of the cells expressing 6His-5-HT2C (Figure 5d), whereas almost no significant labeling was observed on the untransfected cells (Figure 5e). The PL intensity as a function of the concentration of NiNTA-QDs showed a sigmoidal increase with an EC50 value of 74.8 nM. Test NTA-QD as FRET Donor. In addition to the specific binding properties of the NiNTA-QDs, the compactness of the QDs allowed them to be useful for FRET application. Although polymer-coated QDs are used widely owing to their stability, their size usually exceeds 15 nm. This excessive size limits the use of polymer-coated QDs for FRET application that can only track biomolecular interactions below the 10 nm range. In this regard, the generation of compact NiNTA-QDs can be extremely valuable for FRET-based applications. To test the ability of NiNTAQDs to be used in FRET-based assays, 2G6H-conjugated TAMRA dye (abs. 560 nm, em. 582 nm) was used as an acceptor for QD512 (Scheme 2). The NiNTA-QDs were incubated with various equivalents of TAMRA-2G6H. The same experiment was performed for TAMRA-5GH2G as a control test. DOI: 10.1021/la9041887

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Figure 5. Reaction of NiNTA-QDs with peptide-functionalized agarose bead (a-c) and 6His tagged 5-HT2C receptors expressed in HEK 293 cells (d-f). Fluorescence microscope images of (a) 2G6H-Agarose bead reacted with NiNTA-QD and (b) 5GH2G-Agarose bead reacted with NiNTA-QD. (c) PL intensity of 2G6H-AB (9) and that of 5GH2G-AB (2) after reaction with NiNTA-QDs. Confocal microscope images of (d) 6His-5-HT2C receptor expressing HEK-293 cells incubated with NiNTA-QDs, (e) untransfected HEK-293 cells incubated with NiNTA-QDs. (f) Normalized PL intensity of NiNTA-QDs after cell binding.

Figure 6. FRET study using NiNTA-QDa and TAMRA-2G6H. (a) Upper 1 to 8, NiNTA-QD reacted with 0, 1, 2, 5, 10, 15, 20, and 30 equiv, respectively, of TAMRA-2G6H; lower 1 to 8, NiNTA-QD reacted with 0, 1, 2, 5, 10, 15, 20, and 30 equiv, respectively, of TAMRA-5GH2G. (b) Plot of the PL intensity of NiNTA-QD reacted with various molar ratios of TAMRA-2G6H to QDs. (c) PL recovery test of NiNTA-QDs after the addition of imidazole. The red circle indicates the PL spectrum of the NiNTA-QDs after reaction with 10 equiv of TAMRA-2G6H. The blue down triangle represents the PL recovery of the NiNTA-QDs after adding an excess of imidazole (0.5 M).

After 30 min of incubation, the PL spectra of the NiNTA-QDs were collected. As shown in Figure 6, the fluorescence intensity of the NiNTA-QDs decreased with increasing amount of TAMRA2G6H with a concomitant color change from green to red. Interestingly, a visually detectable color change of NiNTA-QDs was observed even with 2 equiv of TAMRA-2G6H. The fluorescence 7332 DOI: 10.1021/la9041887

of the QDs was quenched almost completely (>95%) by binding with 20 equiv of TAMRA-2G6H. On the other hand, the NiNTA-QDs reacting with TAMRA-5GH2G did not show a color change, even at the highest equivalent of TAMRA-5GH2G. In this experiment, the calculated F€orster radius (R0) and the donor-acceptor distance (r) values were 5.46 and 3.3 nm, Langmuir 2010, 26(10), 7327–7333

Park et al. Scheme 2. Schematic of FRET Mechanism of NiNTA-QDs Reacted with 6His-TAMRA

respectively (for the equation, see the Supporting Information).33,34 The specific binding between 6His and NiNTA was further confirmed by the strong PL recovery of the prequenched [TAMRA-2G6H]-[NiNTA-QD] complex upon the addition of excess of imidazole (Figure 6c). The use of NTA-QDs can be extended to the systems using different acceptors, including other organic dyes and gold nanoparticles. Although careful selection of the acceptors to maximize FRET efficiency is necessary, the system has more flexibility in the choice of the acceptor dyes owing to the fluorescence tunability of the QDs. These results undoubtedly demonstrate both the highly specific binding ability and the excellent FRET efficiency of NiNTA-QDs, suggesting their great potential for detecting proteins of interest and tracking the molecular interactions in solution and cells. (33) Clapp, A. R.; Medintz, I. L.; Uyeda, H. T.; Fisher, B. R.; Goldman, E. R.; Bawendi, M. G.; Mattoussi, H. J. Am. Chem. Soc. 2005, 127, 18212–18221. (34) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic/Plenum Publishers: New York, 1999; pp 368-374.

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Article

Conclusions The specifically designed NTA ligand was used to generate highly stable water-soluble QDs. The resulting NTA-QDs showed remarkable particle stability and photostability over an extended range of biological media. NTA-QDs were successfully evaluated for their stability and accessibility to EDC conjugation reaction. NiNTA-QDs prepared by Ni2þ complexation of NTAQDs showed the selective and quantitative binding for the 6His peptide in various conditions. In addition, the selective labeling of 6His-tagged recombinant protein in HEK cells and the use of NiNTA-QDs as a valuable FRET donor has been demonstrated. Further studies on the specific labeling and FRET approaches in live cells are under investigation. The compactness and unique 6His binding ability of NTA-QDs provides access to a highly stable, selective, and sensitive multifunctional nanoprobe for specific cell labeling and tracking molecular interactions within biological context. Acknowledgment. This work was supported by Ministry of Education and Science and Technology (MEST) and Korea Science and Engineering Foundation (KOSEF) through its National Nuclear Technology Program (Nos. 2009-0062234 and 2009-2006216) and National R&D Program (No. 2009-0082751). Supporting Information Available: Detailed synthesis of TOPO-QDs and NTA ligand, TEM images, FTIR spectra, UV-vis spectra, and fluorescence spectra of TOPO-QDs and NTA-QDs, ICP-MS data. This material is available free of charge via the Internet at http://pubs.acs.org.

DOI: 10.1021/la9041887

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