Bioconjugate Chem. 2010, 21, 1305–1311
Bioconjugation of Hydroxylated Semiconductor Nanocrystals and Background-Free Biomolecule Detection Yongwook Kim, Wonjung Kim, Hye-Joo Yoon,* and Seung Koo Shin* Bio-Nanotechnology Center, Department of Chemistry, Pohang University of Science and Technology, San 31 Hyojadong Namgu, Pohang, Kyungbuk 790-784, Korea. Received March 2, 2010; Revised Manuscript Received June 1, 2010
Semiconductor nanocrystals emerge as fluorescent bioprobes for long-term imaging and multiplexed assays; however, there is a challenge of making nanocrystals biocompatible without nonspecific bindings to background molecules. Here, we report the bioconjugation of small-sized, hydroxylated nanocrystals, enabling highly sensitive detection of various biomolecules with little or no nonspecific binding. Zinc-blende CdSe/ZnS nanocrystals were passivated with 3-mercapto-1-propanol (MPO) and activated to amine-reactive succinimidyl carbonate derivatives and then covalently linked to amine-functionalized biomolecules, such as biotin, DNA, and hemagglutinin peptide, by forming a carbamate linkage. Tris(3-hydroxypropyl)phosphine was added to stabilize the zinc-thiolate linkage on nanocrystals. For comparison, CdSe/ZnS nanocrystals were passivated with 3-mercaptopropionic acid (MPA) and conjugated with aminated biomolecules. Photoluminescence properties of organic, water-soluble, and bioconjugated nanocrystals were characterized. Significantly, the bioconjugates of hydroxylated (CdSe/ZnS-MPO) nanocrystals exhibited brighter photoluminescence with longer lifetimes than those of carboxylated (CdSe/ ZnS-MPA) nanocrystals. Specific and nonspecific interactions between nanocrystals and biomolecules were examined by incubating nanocrystal-bioconjugates with avidin-agarose beads, anti-hemagglutinin affinity matrix, DNA glass slide, or avidin glass slide. CdSe/ZnS-MPO nanocrystals showed little or no nonspecific binding to both agarose beads and glass slides, whereas CdSe/ZnS-MPA nanocrystals exhibited significant nonspecific binding due to the carboxyl-amine interactions. Notably, CdSe/ZnS-MPO bioconjugates yield about 20 times brighter images than CdSe/ZnS-MPA bioconjugates in both DNA hybridization and biotin-streptavidin binding. Hydroxylated nanocrystals stabilized by hydroxyphosphine are small, bright, and photostable in physiological conditions, and their bioconjugates afford background-free detection of specific biomolecular interactions, positioning them for an ideal fluorescent probe to biological settings.
INTRODUCTION Core/shell CdSe/ZnS nanocrystals are a new class of fluorescent probes for biological studies (1, 2), from long-term imaging (3, 4) and tracking of biomolecules (5, 6) to biochip sensors (7, 8). Fluorescent semiconductor nanocrystals offer several advantages over organic dyes (7, 9), including sizetunable, narrow, and symmetric emission, broad excitation wavelengths, and strong resistance to photobleaching. However, organic-soluble CdSe/ZnS nanocrystals prepared by colloidal synthesis (10-13) are not directly compatible with biological settings (14-16). A nanocrystal surface must be made watersoluble and functionalized for bioconjugation. In recent years, organic-soluble nanocrystals have been made biocompatible in two ways: (1) direct ligand exchange with bifunctional hydrophilic thiols (17-19); (2) functional surface encapsulation using silica (20, 21), amphiphilic polymers (6, 22), or amphiphilic lipids (23). The surface encapsulation results in physiologically stable nanocrystals with multiple functionalities, but their hydrodynamic sizes are rather large and sometimes even larger than typical biomolecules (14-16, 24), thus hindering the transport and activity of conjugated biomolecules (14, 25). On the other hand, the ligand exchange with hydrophilic thiols yields nanocrystals with a small hydrodynamic size, but the resulting nanocrystals tend to aggregate into clusters over time and/or nonspecifically bind to biomolecules (26, 27), thereby obscuring the detection of specific biomolecular interactions (24). For instance, carboxylic acid-terminated nanocrystals with * To whom correspondence should be addressed. For H.-J. Y.: e-mail, [email protected]
For S. K. S.: e-mail, [email protected]
mercaptoalkanoic acid or dihydrolipoic acid nonspecifically bind to various biological surfaces presenting positively charged functional groups (28). To reduce the nonspecific binding to biological surfaces, nanocrystals have been functionalized with hydroxyl groups: Thompson and co-workers have shown little nonspecific binding of hydroxylated nanocrystals to DNA (19). Nie and co-workers (28) have minimized nonspecific binding to cellular membranes by functionalizing polymer-encapsulated nanocrystals with the hydroxyl group. Although the hydroxyl group is very promising in reducing nonspecific binding, the previously reported bioconjugation procedure for hydroxylated nanocrystals (19) is not as efficient as that for carboxylated nanocrystals (29-31). Carboxylic acid-terminated nanocrystals have been typically activated to the O-succinimide (OSu) esters in water and subsequently linked to amine-functionalized biomolecules (16). Meanwhile, hydroxyl-terminated nanocrystals have been activated to the imidazole carbamates in dioxane and conjugated with aminated biomolecules in dioxane/water (19). The OSu esters of carboxylated nanocrystals are highly soluble in water (2), but the imidazole carbamates of hydroxylated nanocrystals are sparingly soluble in dioxane/water (19). This solubility difference leads to a significant difference in reaction efficiency between the two bioconjugation procedures. Thus, the hydroxyl group is rarely used for direct conjugation with biomolecules. For instance, Klenerman and co-workers (29) have employed 11-meraptoundecyl-tri(ethylene glycol)acetic acid to covalently link probe oligonucleotides to nanocrystals and 11-meraptoundecyl-tri(ethylene glycol) alcohol to reduce nonspecific binding to target oligonucleotides. Obviously, there is a strong need for
10.1021/bc100114q 2010 American Chemical Society Published on Web 06/28/2010
1306 Bioconjugate Chem., Vol. 21, No. 7, 2010
efficient bioconjugation of hydroxylated nanocrystals. Herein, we present the bioconjugation of CdSe/ZnS core/shell nanocrystals capped with 3-mercapto-1-propanol (MPO), enabling background-free fluorescent detection of biomolecules. Recently, our group has reported synthesis and characterization of zinc blende (ZB) CdSe/ZnS core/shell nanocrystals and their high luminescence in water (13). Specifically, the ZB lattice structure has been used as a template for the uniform, epitaxial growth of the ZnS shell, and the ligand exchange with 3-mercaptopropionic acid (MPA) has resulted in ∼50% photoluminescence (PL) quantum efficiency (QE) in water (13). Using both organic- and water-soluble ZB CdSe/ZnS nanocrystals, we have further characterized the surface ligands by mass spectrometric imaging (32) and studied ligand- and shelldependent blinking of single nanocrystals (33, 34). In this paper, we established an efficient bioconjugation strategy for hydroxylated (CdSe/ZnS-MPO) nanocrystals by employing ZB core/ shell nanocrystals. To compare with hydroxylated nanocrystals, carboxylated (CdSe/ZnS-MPA) nanocrystals were also prepared. Both CdSe/ZnS-MPO and CdSe/ZnS-MPA nanocrystals were conjugated with amine-functionalized biomolecules, such as biotin, hemagglutinin (HA) peptide, and DNA. We characterized the lattice structure, size distribution, and optical properties of organic- and water-soluble nanocrystals and studied the effect of tris(3-hydroxypropyl)phosphine (THP) on the long-term stability of hydroxylated nanocrystals exposed to room light. Optical properties of nanocrystal-bioconjugates were also characterized. To examine specific and nonspecific interactions between nanocrystals and biomolecules, we incubated biotinylated, DNA-conjugated, and HA peptide-tagged nanocrystals with avidin-agarose beads/avidin glass slide, cDNA glass slide, and anti-HA affinity matrix, respectively. Our results demonstrate that CdSe/ZnS-MPO nanocrystals and their bioconjugates carry outstanding optical properties and thus offer highly sensitive and nonspecific interaction-free signals for biomolecule detection.
MATERIALS AND METHODS Materials. MPO (95%), MPA (99%), tetramethylammonium hydroxide (25% w/v in methanol), N,N-diisopropylethylamine (DIPEA, 99.5%), dimethyformamide (DMF, anhydrous, 99.8%), 4-morpholineethanesulfonic acid (MES, 99%), β-mercaptoethanol (98%), hydroxylamine (50% w/v in water), propylamine (98%), sodium bicarbonate (100%), magnesium chloride (99.5%), and dimethyl sulfoxide (DMSO, 99.8%) were purchased from Sigma-Aldrich. HPLC-grade methanol, acetone, chloroform, and ethyl acetate were from J. T. Baker. THP (80%) was obtained from Strem Chemicals. N,N′-Disuccinimidyl carbonate (DSC, g95%) was from Fluka. N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), sulfo-N-hydroxysuccinimide (sulfo-NHS), amine-functionalized biotin [(+)-biotinyl-3,6dioxaoctanediamine], streptavidin (affinity purified), D-Salt dextran desalting column, and avidin agarose beads were purchased from Thermo Scientific. Anti-HA agarose beads were from Roche Applied Science. Amine-functionalized DNA was obtained from Bionics. HA peptide (YPYDVPDYA) was from Peptron. NSB27-amine and NSB27-N-hydroxysuccinimide (NSB27-NHS) slides were purchased from Nano Surface Biosciences (NSB). Both NSB27 slides have 6-7 nm lateral spacing between surface functional groups (35). Preparation of Hydroxylated and Carboxylated Nanocrystals. Organic-soluble CdSe/ZnS core/shell nanocrystals were prepared, as presented in Supporting Information (SI), and characterized, as shown in Figures S1 and S2 (SI). Organicsoluble nanocrystals were converted to water-soluble ones by dissolving nanocrystal powder into a methanol solution containing MPA or MPO (0.1 M) for 3 h at 60 °C (32, 33). The solution
Kim et al.
pH was adjusted to >10 with tetramethylammonium hydroxide. Both CdSe/ZnS-MPO and CdSe/ZnS-MPA nanocrystals were precipitated and washed with ethyl acetate, harvested by centrifugation, and dried under vacuum. The hydrodynamic size distribution of water-soluble nanocrystals (∼1 µM) was measured in distilled water by dynamic light scattering (DLS) at 632.8 nm (Zetasizer Nano S, Malvern Instruments). The pHdependent solubility of water-soluble nanocrystals was tested at 10 different pH values between 3.5 and 8.0 using three buffer systems: propionate (pH 3.5-5.5), MES (pH 5.5-7.0), and phosphate buffered saline (PBS) (pH 7.0-8.0), as described in SI. Effects of THP on the Stabilization of Hydroxylated Nanocrystals. THP (260 nmol) was added to an aqueous solution (1 mL) containing CdSe/ZnS-MPO nanocrystals (0.1 µM). The amount of THP was 5 times the calculated amount of surface atoms on nanocrystals. The nanocrystal solution was sealed in a cuvette under argon in the presence or absence of THP and stored for several days at room temperature (rt) under room light. The hydrodynamic size distribution was measured over time by DLS, and fluorescent images were taken with a handheld UV (365 nm) lamp using a digital camera. Bioconjugation of Water-Soluble Nanocrystals. Biomolecules were conjugated to CdSe/ZnS-MPO nanocrystals through DSC coupling (36), as shown in Scheme 1a, or to CdSe/ ZnS-MPA nanocrystals through EDC/sulfo-NHS coupling (37), as illustrated in Scheme 1b. CdSe/ZnS-MPO powder (0.3 nmol) was dispersed into DMF, and the mixture was incubated with DSC (250 nmol) and DIPEA (500 nmol) for 2 h at rt to activate the hydroxyl groups to the succinimidyl carbonate derivatives. Then DSC-activated nanocrystals were precipitated and washed with diethyl ether, dried under vacuum, and dissolved in PBS (pH 7.4). CdSe/ZnS-MPA powder (0.3 nmol) was dissolved in water containing 0.1 M MES (pH 6.0), and the solution was incubated with EDC (2 µmol) and sulfo-NHS (5 µmol) for 15 min at rt to activate the carboxyl groups to the OSu esters. Excess EDC was quenched with β-mercaptoethanol (1.4 µL), and the reaction buffer was changed to PBS by using a dextran desalting column equilibrated with PBS to remove organic byproducts from the activated nanocrystals. Amine-functionalized biomolecules (3 nmol) such as biotin, HA peptide, and DNA (amine-C6-5′-GATCCAAATAAGTCACATGATGATA-3′) were reacted with the activated nanocrystals for 2 h at rt, and the reaction was quenched with hydroxylamine (10 µmol). The reaction mixture was passed through a dextran desalting size-exclusion column equilibrated with PBS. Nanocrystal-bioconjugates were stored at 4 °C in the dark. Specific and Nonspecific Bindings of Nanocrystal-Bioconjugates to Protein Beads and Functionalized Surfaces. Both avidin and anti-HA agarose beads were used as protein beads. They were washed three times with PBS, incubated with nanocrystals (10 nmol) in PBS for 1 h at rt, and washed twice with water. To examine specific binding, we incubated biotinylated nanocrystals with avidin beads and incubated HA-tagged nanocrystals with anti-HA beads. To examine nonspecific binding, both CdSe/ZnS-MPO and CdSe/ZnS-MPA nanocrystals were also separately incubated with agarose beads. Fluorescent images were taken with a microscope (Nikon, Eclipse 80i) using a mercury lamp as a light source. A bandpass filter (Omega 365WB50), a dichroic mirror (Omega 400DCLP), a long-pass filter (Omega 435ALP), and a color CCD camera (Nikon DXM1200F) were used for fluorescent detection. Both NSB27-amine and alkylated NSB27-NHS slides were employed as functionalized surfaces. The alkylated slide was prepared by conjugating propylamine (1 mM) onto a NSB27-
Bioconjugation of Hydroxylated Nanocrystals
Bioconjugate Chem., Vol. 21, No. 7, 2010 1307
Scheme 1. Activation and Bioconjugation of (a) CdSe/ZnS-MPO and (b) CdSe/ZnS-MPA Nanocrystals
NHS slide in sodium bicarbonate buffer (pH 8.5) for 7 h at rt. Both CdSe/ZnS-MPO and CdSe/ZnS-MPA nanocrystals were separately dissolved in PBS to 0.1 µM, and aliquots (1 µL each) were loaded on either an amine or alkyl surface. The slide was incubated in a humidity (100%) chamber for 1 h at rt, washed with PBST (0.1% Tween 20 in PBS, v/v) for 3 min, and rinsed twice with water. Fluorescent images of the glass slides were obtained using a gel imaging system (VersaDoc 5000MP, BioRad). DNA Hybridization and Biotin-Streptavidin Binding. Amine-functionalized target DNA (amine-C6-5′-TATCATCATGTGACTTATTTGGATC-3′) or biotin was dissolved to 1 mM in a buffer solution (pH 8.5) containing 25 mM sodium bicarbonate, 10 mM magnesium chloride, and 10% (v/v) DMSO (35). An aliquot (1 µL) of each solution was loaded on a NSB27NHS slide. The spot size was ∼2.5 mm in diameter. The slide was incubated in a humidity (100%) chamber for 12 h at rt and washed with PBST and water. NHS groups outside the spotted area were alkylated, as described above. For DNA hybridization, an aliquot (1 µL) of nanocrystal-DNA conjugates (0.1 µM) in PBS was placed on the target DNA-spot, and the slide was incubated in a humidity (100%) chamber for 1 h at 45 °C and then for 12 h at rt. For biotin-avidin binding, an aliquot (1 µL) of streptavidin (1 mg/mL) in PBS was loaded on the biotinspot, and the slide was incubated in a humidity (100%) chamber for 1 h at rt and washed sequentially with PBST and water. Then an aliquot (1 µL) of nanocrystal-biotin conjugates (0.1 µM) in PBS was loaded on the streptavidin-biotin-spot, and the slide was incubated again in a humidity (100%) chamber for 1 h at rt. Fluorescent images of the glass slides were recorded with a gel imaging system after washing with PBST and water and were processed with ImageJ software (NIH, Bethesda, MD).
Both the absorption/emission spectra and the PL decay of water-soluble nanocrystals are compared with organic-soluble ones in parts c and d of Figure 1, respectively. The band-edge absorption (λabs) occurs at 529 nm for CdSe/ZnS nanocrystals in chloroform and at 518 and 524 nm for CdSe/ZnS-MPO and CdSe/ZnS-MPA nanocrystals in water, respectively. The emission maxima (λem) appear at 549 nm for organic-soluble CdSe/ZnS nanocrystals and at 542 and 547 nm for water-soluble CdSe/ZnS-MPO and CdSe/ZnS-MPA nanocrystals, respectively, with full width at half-maximum (fwhm) of 30-32 nm. The QE decreases from 74% of CdSe/ZnS nanocrystals in chloroform to 56% and 51% after ligand exchange with MPO and MPA, respectively. The effective PL lifetime (τeff) is 16.9 ns for CdSe/ZnS nanocrystals in chloroform and 12.9 and 10.9 ns for CdSe/ZnS-MPO and CdSe/ZnS-MPA nanocrystals in water, respectively. These optical properties (λabs, λem, fwhm, QE, and τeff) are listed in Table S1 (SI). The thiolate capping
RESULTS AND DISCUSSION Size Distribution and Optical Properties of Water-Soluble Nanocrystals. The hydrodynamic size distributions of watersoluble CdSe/ZnS-MPO and CdSe/ZnS-MPA nanocrystals are presented in parts a and b of Figure 1, respectively. Their average size is 9.8 ( 1.6 and 11.7 ( 2.1 nm, respectively. CdSe/ ZnS-MPO nanocrystals appear to be smaller than CdSe/ ZnS-MPA nanocrystals. Nonetheless, both CdSe/ZnS-MPO and CdSe/ZnS-MPA nanocrystals display no indication of clusters larger than 20 nm.
Figure 1. Hydrodynamic size distributions (average of 15 measurements) of (a) CdSe/ZnS-MPO and (b) CdSe/ZnS-MPA nanocrystals (1 µM) in water. (c) Absorption (dashed line) and emission (solid line) spectra of CdSe/ZnS nanocrystals in chloroform and of CdSe/ ZnS-MPO and CdSe/ZnS-MPA nanocrystals in water. (d) PL decay and effective PL lifetime of CdSe/ZnS nanocrystals in chloroform and of CdSe/ZnS-MPO and CdSe/ZnS-MPA nanocrystals in water.
1308 Bioconjugate Chem., Vol. 21, No. 7, 2010
Kim et al.
Figure 2. Hydrodynamic size distributions of CdSe/ZnS-MPO nanocrystals (a) in the absence and (b) in the presence of THP over 96 h under exposure to room light. Insets show fluorescent images of nanocrystals in a cuvette after 96 h. Highly luminescent nanocrystals are precipitated at the bottom in the absence of THP (a) but uniformly dispersed in water in the presence of THP (b).
over the ZnS shell slightly changes optical properties of CdSe/ ZnS nanocrystals. Significantly, the MPO capping results in a longer PL lifetime than the MPA capping. CdSe/ZnS-MPO nanocrystals were stable (fluorescent and soluble) in water in the tested pH range from 3.5 to 8.0, whereas CdSe/ZnS-MPA nanocrystals were stable only at pH above 5.5, as shown in Figure S3 (SI). Below pH 5.0, CdSe/ ZnS-MPA nanocrystals were neutralized and precipitated because of intermolecular hydrogen bonding. Above pH 5.5, however, the carboxyl groups on CdSe/ZnS-MPA nanocrystals were ionized to become soluble in water. Nonetheless, both CdSe/ZnS-MPO and CdSe/ZnS-MPA nanocrystals were stable for 2 years in buffer as long as they were kept in the dark at 4 °C. Hydroxyphosphine (THP) Stabilization of Hydroxylated Nanocrystals. Although both CdSe/ZnS-MPO and CdSe/ ZnS-MPA nanocrystals are typically stable for the long-term in the dark at 4 °C, they tend to form aggregates over time when exposed to light. Between the two water-soluble nanocrystals, CdSe/ZnS-MPO nanocrystals are more sensitive to light than CdSe/ZnS-MPA nanocrystals. Hydrophilic thiols are reported to undergo photochemical oxidation to disulfides that dissolve into the solution (26). As the water-soluble ligands dissociate from the nanocrystal surface, nanocrystals become less and less soluble in water and assemble into clusters. By suppression of disulfide bond formation, nanocrystals could be kept from aggregation. Water-soluble hydroxyphosphine has been reported to reduce disulfides (38) more efficiently than tris(2-carboxyethyl)phosphine (39). Thus, we tested the effect of THP on the stability of CdSe/ZnS-MPO nanocrystals. The hydrodynamic size distribution and the PL intensity of hydroxylated nanocrystals were monitored in water in the absence or the presence of THP. Nanocrystals were exposed to room light under argon for 4 days. Fresh CdSe/ZnS-MPO nanocrystals exhibit a narrow hydrodynamic size distribution with no indication of clustering in water, as displayed in Figure 2 (time 0). They form aggregates that slowly precipitate over time in the absence of THP (Figure 2a). As the light induces the formation of disulfide bonds, nanocrystals keep losing thiolates from the surface, thereby precipitating out of the solution. In the presence of THP (Figure 2b), however, CdSe/ZnS-MPO nanocrystals undergo no aggregation or precipitation. Remarkably, THP stabilizes the zinc-thiolate linkage on CdSe/ ZnS-MPO nanocrystals. THP also increases the stability of CdSe/ZnS-MPA nanocrystals, as supported by the absence of aggregates over time (data not shown). Most recently, we have shown that the tertiary phosphine binds to and stabilizes the chalcogen atoms on CdSe semiconductor nanocrystals (40). THP seems to stabilize the surface chalcogen atoms on the CdSe/ ZnS nanocrystals and suppress the disulfide formation of thiolate ligands. Besides, addition of THP hardly modifies the optical properties (λabs, λem, fwhm, QE, and τeff) of water-soluble CdSe/ ZnS core/shell nanocrystals (data not shown). Also, THP has
Figure 3. (a) Emission spectra and (b) PL decay of CdSe/ZnS-MPO nanocrystals in water and their bioconjugates with biotin, DNA, and HA peptide in PBS. (c) Emission spectra and (d) PL decay of CdSe/ ZnS-MPA nanocrystals in water and their bioconjugates in PBS. λem, fwhm, and τeff are listed in Table S1 (SI).
been shown to be biocompatible under physiological conditions (39, 41). Therefore, we routinely added THP to nanocrystal preparations, particularly when nanocrystal probes were used for long-term bioimaging. Bioconjugation of Hydroxylated Nanocrystals. Both CdSe/ ZnS-MPO and CdSe/ZnS-MPA nanocrystals are activated to the succinimidyl derivatives and conjugated with biomolecules, as shown in Scheme 1. Although the previously reported imidazole carbamate derivatives of hydroxylated nanocrystals are sparingly soluble in dioxane/PBS, the succinimidyl carbonate derivatives are highly soluble in PBS. Consequently, the subsequent conjugation reaction with amine-functionalized biomolecules readily proceeds in PBS to near completion. The emission spectra and the PL decay of nanocrystalbioconjugates are presented in Figure 3. For all nanocrystals, the emission spectra look almost identical before and after bioconjugation. However, the PL lifetime decreases after bioconjugation from 12.9 to 8.4-9.0 ns for CdSe/ZnS-MPO nanocrystals and from 10.9 to 3.7-4.1 ns for CdSe/ZnS-MPA nanocrystals. To find out the origin of this lifetime shortening, we examined the optical properties of the succinimidyl derivatives of nanocrystals before and after DNA conjugation. The emission spectra and the PL decay obtained from another batch of nanocrystals are depicted in Figure S4 (SI). The activation of water-soluble nanocrystals to the succinimidyl derivatives reduces the emission intensity by ∼50% for CdSe/ZnS-MPO and by ∼90% for CdSe/ZnS-MPA. Meanwhile, the PL lifetime of CdSe/ZnS-MPO slightly increases, whereas that of CdSe/ZnS-MPA decreases. The subsequent DNA conjugation barely reduces the emission intensity of both nanocrystal-DNA conjugates with little change in lifetime. Evidently, the formation of the succinimidyl derivatives primarily reduces the emission intensity, whereas the subsequent bioconjugation induces little change in emission properties. Fluorescent Detection of Biomolecules Using Nanocrystal Probes. We applied nanocrystals to the fluorescent detection of biomolecules. In biological applications of fluorescent nanocrystals, nonspecific interactions between biomolecules and nanocrystals are one of the most serious problems (8, 14, 28). Thus, we first studied specific and nonspecific bindings between nanocrystal-bioconjugates and protein-agarose beads to detect
Bioconjugation of Hydroxylated Nanocrystals
Figure 4. Fluorescence microscope images of protein-immobilized agarose beads probed with (a) CdSe/ZnS-MPO-bioconjugates or (b) CdSe/ZnS-MPA-bioconjugates. Avidin-agarose beads were incubated with biotinylated nanocrystals (NC-biotin, top), and anti-HA-agarose beads were incubated with HA-tagged nanocrystals (NC-HA, top). CdSe/ZnS-MPO or CdSe/ZnS-MPA nanocrystals before bioconjugation were used as control (NC, bottom). Bright-field images (right) corresponding to the fluorescent images (left) are also shown. Nonspecific interaction between (c) CdSe/ZnS-MPO or (d) CdSe/ ZnS-MPA nanocrystals and the amine-functionalized glass slide (aminated glass, left) is shown. The amine surface was passivated with propylamine through DSC coupling to make the surface uncharged and hydrophobic (alkylated surface, right).
well-known biomolecular interactions, such as biotin-avidin and antigen-antibody recognition. Specific binding was examined by incubating biotinylated nanocrystals with avidinimmobilized agarose beads and incubating HA antigen-tagged nanocrystals with anti-HA antibody agarose beads. Nonspecific binding was tested by incubating the protein-agarose beads with water-soluble (both CdSe/ZnS-MPO and CdSe/ZnS-MPA) nanocrystals unconjugated with biomolecules. Fluorescence microscope images of agarose beads are presented in Figure 4. Both CdSe/ZnS-MPO-biotin and CdSe/ ZnS-MPA-biotin yield bright images by binding to avidin beads. HA-tagged nanocrystals also result in bright images by binding to anti-HA beads. In control experiments, CdSe/ ZnS-MPO nanocrystals show no fluorescent images with both avidin and anti-HA beads, indicating the absence of nonspecific interactions (Figure 4a, bottom). On the other hand, CdSe/ ZnS-MPA nanocrystals exhibit bright images with anti-HA beads but no fluorescence with avidin beads, suggesting the presence of nonspecific interactions between carboxylated nanocrystals and anti-HA beads (Figure 4b, bottom). Although the functional groups on the surface of protein-agarose beads are not well characterized, the positively charged amine groups on agarose beads could interact with the negatively charged carboxyl groups on nanocrystals in PBS (pH 7.4). To further examine nonspecific interactions between watersoluble nanocrystals and amine functional groups, we spotted nanocrystals on an amine-functionalized glass slide (NSB27amine) (35). As presented in Figure 4d, CdSe/ZnS-MPA nanocrystals indeed display a bright fluorescent image on the
Bioconjugate Chem., Vol. 21, No. 7, 2010 1309
Figure 5. Fluorescent images (top) and intensity profiles (bottom) of (a) DNA hybridization and (b) biotin-streptavidin interaction on glass slides using nanocrystal-bioconjugate probes.
amine surface at pH 7.4 (left), manifesting nonspecific interactions between the carboxyl groups on nanocrystals and the amine groups on the glass surface in PBS. In contrast, CdSe/ ZnS-MPO nanocrystals exhibit no fluorescent image (Figure 4c, left), confirming the absence of nonspecific binding to the amine surface. To solve the carboxyl-amine interaction problem, we modified the amine-functionalized glass surface with propylamine through DSC coupling. The propylamine passivation makes the glass surface hydrophobic with no charged functional groups. Consequently, CdSe/ZnS-MPA nanocrystals no longer bind to the alkylated surface (Figure 4d, right). CdSe/ ZnS-MPO nanocrystals again show no binding affinity to the hydrophobic surface (Figure 4c, right). This alkyl passivation of the amine surface is a simple and easy way to reduce the background noise when carboxylated nanocrystals are used as fluorescent probes. To broaden biological applications of our nanocrystal probes, we tested both CdSe/ZnS-MPO and CdSe/ZnS-MPA nanocrystals for the detection of DNA hybridization as well as biotin-streptavidin binding on NHS-functionalized glass chips (NSB27-NHS). Both DNA and biotin were immobilized on the NSB27-NHS slide, and then the unspotted area was passivated with propylamine to block nonspecific interaction between water-soluble nanocrystals and the glass surface. Fluorescent images and intensity profiles on the glass slide are displayed in Figure 5. The intensity profile across the horizontal line (green or red) is displayed below the image. In the case of DNA hybridization (Figure 5a), eight spots were tested for each type of nanocrystal. Of them, two spots, B2 and C2 for CdSe/ ZnS-MPA nanocrystals and F2 and G2 for CdSe/ZnS-MPO nanocrystals, have both target and probe DNA. The other six spots lack either one or both. Of the 16 spots for both nanocrystals, only those 4 spots having both target and probe DNA show fluorescent signals, demonstrating specific detection of DNA hybridization without nonspecific interactions. In the case of biotin-streptavidin binding (Figure 5b), of the eight spots tested for each type of nanocrystals, only one spot, C2 for CdSe/ZnS-MPA nanocrystals and G2 for CdSe/ZnS-MPO
1310 Bioconjugate Chem., Vol. 21, No. 7, 2010
nanocrystals, contains all three complementary components, biotin on the glass surface, streptavidin as a linker, and biotin on the nanocrystal. The other spots lack at least one component. Of the 16 spots for both nanocrystals, only C2 and G2 spots show fluorescent signals, and all other spots yield no fluorescence, manifesting background-free detection of biotinstreptavidin binding. Notably, CdSe/ZnS-MPO nanocrystalbioconjugates show the integrated signals about 20 times stronger than CdSe/ZnS-MPA nanocrystal-bioconjugates in both binding studies. In conclusion, the bioconjugates of CdSe/ZnS-MPO nanocrystals show higher photoluminescence intensities and longer lifetimes than those of CdSe/ZnS-MPA nanocrystals. Unlike CdSe/ZnS-MPA nanocrystals, CdSe/ZnS-MPO nanocrystals show no nonspecific binding to protein-immobilized agarose beads or to amine-functionalized glass slides. Nonspecific interactions between carboxylated nanocrystals and aminefunctionalized surface are minimized by alkylating the amine surface with propylamine. As a result, both CdSe/ZnS-MPO and CdSe/ZnS-MPA nanocrystals are applicable to fluorescent detection of biomolecules immobilized on glass slides with little or no nonspecific binding. CdSe/ZnS-MPO nanocrystals yield about 20 times stronger specific binding signals on the glass chip. Notably, tris(3-hydroxypropyl)phosphine greatly stabilizes the zinc-thiolate linkage on both CdSe/ZnS-MPO and CdSe/ ZnS-MPA nanocrystals. Addition of THP to CdSe/ZnS-MPO nanocrystals and their bioconjugates allows long-lasting, background-free fluorescent detection of biomolecules without aggregation of nanocrystals. CdSe/ZnS nanocrystals hydroxylated with 3-mercapto-1-propanol are a promising fluorescent probe in biological settings, which offers the small hydrodynamic size, high photoluminescence quantum yield, long-term photostability, and nonspecific interaction-free signals.
ACKNOWLEDGMENT We are thankful for the support from the Advanced Scientific Analysis Instruments Development Project administered by the Korea Research Institute of Standards and Science and the Nano Research and Development Program through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology (Grant No. 2008-04540). Supporting Information Available: Synthesis and characterization of CdSe core and CdSe/ZnS core/shell nanocrystals, powder X-ray diffraction patterns, high-resolution transmission electron microscope images, optical properties and pH-dependent solubility of nanocrystals, and fitting parameters of PL decay. This material is available free of charge via the Internet at http:// pubs.acs.org.
LITERATURE CITED (1) Bruchez, M., Jr., Moronne, M., Gin, P., Weiss, S., and Alivisatos, A. P. (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281, 2013–2016. (2) Chan, W. C. W., and Nie, S. (1998) Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281, 2016–2018. (3) Chan, W. C. W., Maxwell, D. J., Gao, X., Bailey, R. E., Han, M., and Nie, S. (2002) Luminescent quantum dots for multiplexed biological detection and imaging. Curr. Opin. Biotechnol. 13, 40–46. (4) Jaiswal, J. K., Mattoussi, H., Mauro, J. M., and Simon, S. M. (2003) Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat. Biotechnol. 21, 47–51. (5) Alivisatos, A. P. (2004) The use of nanocrystals in biological detection. Nat. Biotechnol. 22, 47–52.
Kim et al. (6) Gao, X., Cui, Y., Levenson, R. M., Chung, L. W. K., and Nie, S. (2004) In vivo cancer targeting and imaging with semiconductor quantum dots. Nat. Biotechnol. 22, 969–976. (7) Shin, S. K., Yoon, H.-J., Jung, Y. J., and Park, J. W. (2006) Nanoscale controlled self-assembled monolayers and quantum dots. Curr. Opin. Chem. Biol. 10, 423–429. (8) De, M., Ghosh, P. S., and Rotello, V. M. (2008) Applications of nanoparticles in biology. AdV. Mater. 20, 4225–4241. (9) Resch-Genger, U., Grabolle, M., Cavaliere-Jaricot, S., Nitschke, R., and Nann, T. (2008) Quantum dots versus organic dyes as fluorescent labels. Nat. Methods 5, 763–775. (10) Hines, M. A., and Guyot-Sionnest, P. (1996) Synthesis and characterization of strongly luminescing ZnS-capped CdSe nanocrystals. J. Phys. Chem. 100, 468–471. (11) Dabbousi, B. O., Rodriguez-Viejo, J., Mikulec, F. V., Heine, J. R., Mattoussi, H., Ober, R., Jensen, K. F., and Bawendi, M. G. (1997) (CdSe)ZnS core-shell quantum dots: synthesis and characterization of a size series of highly luminescent nanocrystallites. J. Phys. Chem. B 101, 9463–9475. (12) Yang, Y. A., Wu, H., Williams, K. R., and Cao, Y. C. (2005) Synthesis of CdSe and CdTe nanocrystals without precursor injection. Angew. Chem., Int. Ed. 44, 6712–6715. (13) Lim, S. J., Chon, B., Joo, T., and Shin, S. K. (2008) Synthesis and characterization of zinc-blende CdSe-based core/shell nanocrystals and their luminescence in water. J. Phys. Chem. C 112, 1744–1747. (14) Jaiswal, J. K., and Simon, S. M. (2004) Potentials and pitfalls of fluorescent quantum dots for biological imaging. Trends Cell Biol. 14, 497–504. (15) Michalet, X., Pinaud, F. F., Bentolila, L. A., Tsay, J. M., Doose, S., Li, J. J., Sundaresan, G., Wu, A. M., Gambhir, S. S., and Weiss, S. (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307, 538–544. (16) Medintz, I. L., Uyeda, H. T., Goldman, E. R., and Mattoussi, H. (2005) Quantum dot bioconjugates for imaging, labeling and sensing. Nat. Mater. 4, 435–446. (17) Mitchell, G. P., Mirkin, C. A., and Letsinger, R. L. (1999) Programmed assembly of DNA functionalized quantum dots. J. Am. Chem. Soc. 121, 8122–8123. (18) Mattoussi, H., Mauro, J. M., Goldman, E. R., Anderson, G. P., Sunder, V. C., Mikulec, F. V., and Bawendi, M. G. (2000) Selfassembly of CdSe-ZnS quantum dots bioconjugates using an engineered recombination protein. J. Am. Chem. Soc. 122, 12142–12150. (19) Pathak, S., Choi, S.-K., Arnheim, N., and Thompson, M. E. (2001) Hydroxylated quantum dots as luminescent probes for in situ hybridization. J. Am. Chem. Soc. 123, 4103–4104. (20) Gerion, D., Pinaud, F., Williams, S. C., Parak, W. J., Zanchet, D., Weiss, S., and Alivisatos, A. P. (2001) Synthesis and properties of biocompatible water-soluble silica-coated CdSe/ ZnS semiconductor quantum dots. J. Phys. Chem. B 105, 8861– 8871. (21) Parak, W. J., Gerion, D., Zanchet, D., Woerz, A. S., Pellegrino, T., Micheel, C., Williams, S. C., Seitz, M., Bruehl, R. E., Bryant, Z., Bustamante, C., Bertozzi, C. R., and Alivisatos, A. P. (2002) Conjugation of DNA to silanized colloidal semiconductor nanocrystalline quantum dots. Chem. Mater. 14, 2113–2019. (22) Wu, X., Liu, H., Liu, J., Haley, K. N., Treadway, J. A., Larson, J. P., Ge, N., Peale, F., and Bruchez, M. P. (2003) Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat. Biotechnol. 21, 41–46. (23) Dubertret, B., Skourides, P., Norris, D. J., Noireaux, V., Brivanlou, A. H., and Libchaber, A. (2002) In vivo imaging of quantum dots encapsulated in phospholipid micelles. Science 298, 1759–1762. (24) Smith, A. M., Duan, H., Rhyner, M. N., Ruan, G., and Nie, S. (2006) A systematic examination of surface coatings on the optical and chemical properties of semiconductor quantum dots. Phys. Chem. Chem. Phys. 8, 3895–3903.
Bioconjugation of Hydroxylated Nanocrystals (25) Choi, H. S., Ipe, B. I., Misra, P., Lee, J. H., Bawendi, M. G., and Frangioni, J. V. (2009) Tissue- and organic-selective biodistribution of NIR fluorescent quantum dots. Nano Lett. 9, 2354–2359. (26) Aldana, J., Wang, Y. A., and Peng, X. (2001) Photochemical instability of CdSe nanocrystals coated by hydrophilic thiols. J. Am. Chem. Soc. 123, 8844–8850. (27) Aldana, J., Lavelle, N., Wang, Y., and Peng, X. (2005) Sizedependent dissociation pH of thiolate ligands from cadmium chalcogenide nanocrystals. J. Am. Chem. Soc. 127, 2496–2504. (28) Kairdolf, B. A., Mancini, M. C., Smith, A. M., and Nie, S. (2008) Minimizing nonspecific cellular binding of quantum dots with hydroxyl-derivatized surface coatings. Anal. Chem. 80, 3029–3034. (29) Zhou, D., Ying, L., Hong, X., Hall, E. A., Abell, C., and Klenerman, D. (2008) A compact functional quantum dot-DNA conjugate: preparation, hybridization, and specific label-free DNA detection. Langmuir 24, 1659–1664. (30) Gerion, D., Parak, W. J., Williams, S. C., Zanchet, D., Micheel, C. M., and Alivisatos, A. P. (2002) Sorting fluorescent nanocrystals with DNA. J. Am. Chem. Soc. 124, 7070–7074. (31) Veronese, F. M. (2001) Peptide and protein PEGylation: a review of problems and solutions. Biomaterials 22, 405–417. (32) Min, H., Kim, Y., Yu, H., Moon, D. W., Lim, S. J., Yoon, H.-J., Lee, T. G., and Shin, S. K. (2008) Probing the surface of organic and bioconjugated nanocrystals by using mass spectrometric imaging. Chem.sEur. J. 14, 8461–8464. (33) Kim, Y., Song, N. W., Yu, H., Moon, D. W., Lim, S. J., Kim, W., Yoon, H.-J., and Shin, S. K. (2009) Ligand-dependent blinking of zinc-blende CdSe/ZnS core/shell nanocrystals. Phys. Chem. Chem. Phys. 11, 3497–3502.
Bioconjugate Chem., Vol. 21, No. 7, 2010 1311 (34) Chon, B., Lim, S. J., Kim, W., Seo, J., Kang, H., Joo, T., Hwang, J., and Shin, S. K. Shell- and ligand-dependent blinking of zinc-blende CdSe-based core/shell nanocrystals. Phys. Chem. Chem. Phys. [Online early access]. DOI: 10.1039/b924917F. (35) Hong, B. J., Sunkara, V., and Park, J. W. (2005) DNA microarrays on nanoscale-controlled surface. Nucleic Acids Res. 33, e106. (36) Miron, T., and Wilchek, M. (1993) A simplified method for the preparation of succinimidyl carbonate polyethylene glycol for coupling to protein. Bioconjugate Chem. 4, 568–569. (37) Staros, J. V., Wright, R. W., and Swingle, D. M. (1986) Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal. Biochem. 156, 220–222. (38) Levison, M. E., Josephson, A. S., and Kirschenbaum, D. M. (1969) Reduction of biological substance by water-soluble phosphine: gamma-globulin (IgG). Experientia 25, 126–127. (39) Cline, D. J., Redding, S. E., Brohawn, S. G., Psathas, J. N., Schneider, J. P., and Thorpe, C. (2004) New water-soluble phosphine as reductants of peptide and protein disulfide bonds: reactivity and membrane permeability. Biochemistry 43, 15195– 15203. (40) Kim, W., Lim, S. J., Jung, S., and Shin, S. K. (2010) Binary amine-phosphine passivation of surface traps on CdSe nanocrystals. J. Phys. Chem. C 114, 1539–1546. (41) Hansen, R. E., Roth, D., and Winther, J. R. (2009) Quantifying the global cellular thiol-disulfide status. Proc. Natl. Acad. Sci. U.S.A. 106, 422–427. BC100114Q