Surface Modification To Reduce Nonspecific Binding of Quantum Dots

Nov 1, 2005 - To advance studies of conjugated quantum dots for live cell applications, it is necessary to address the issue of nonspecific binding. I...
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Bioconjugate Chem. 2005, 16, 1488−1494

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Surface Modification To Reduce Nonspecific Binding of Quantum Dots in Live Cell Assays Elizabeth L. Bentzen,†,§ Ian D. Tomlinson,†,§ John Mason,‡ Paul Gresch,‡ Michael R. Warnement,† David Wright,† Elaine Sanders-Bush,† Randy Blakely,‡ and Sandra J. Rosenthal*,† Department of Chemistry, Vanderbilt University, Station B, 351822, Nashville, Tennessee 37235-1822, and Department of Pharmacology, Vanderbilt School of Medicine, Vanderbilt University, Nashville, Tennessee 37235-1822. Received July 8, 2005; Revised Manuscript Received September 7, 2005

Nonspecific binding is a frequently encountered problem with fluorescent labeling of tissue cultures when labeled with quantum dots. In these studies various cell lines were examined for nonspecific binding. Evidence suggests that nonspecific binding is related to cell type and may be significantly reduced by functionalizing quantum dots with poly(ethylene glycol) ligands (PEG). The length of PEG required to give a significant reduction in nonspecific binding may be as short as 12-14 ethylene glycol units.

INTRODUCTION

Fluorescence is a tool widely employed in biological assays. Current organic dyes suffer from many limitations including narrow excitation spectra, broad emission spectra, and photobleaching. Fluorescent semiconducting nanocrystals, also known as quantum dots, are beginning to find their way into the tool box of many biologists, chemists, and biochemists. Quantum dots are an attractive alternative to the traditional organic dyes due to their broad excitation spectra, narrow emission spectra, and photostability which allows for multiplexed experiments (1-10). During the synthesis of quantum dots, the size and composition can be varied in order to obtain emission wavelengths from blue to near-IR (1, 3, 4). Additionally, through the judicious choice of an appropriate excitation source, multiple color quantum dots may be excited simultaneously. Typically prepared CdSe/ZnS quantum dots are limited for biological uses because they are soluble only in nonpolar solvents making them incompatible with biological systems (5). This is overcome by an additional polymer shell consisting of amphiphilic poly (acrylic acid) (AMP) (6). The AMP coating can subsequently be conjugated, through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling (7) reaction, to biological molecules. Though the AMP-coated quantum dots are compatible with biological assays, studies are still needed to refine this functionalization strategy. Additional studies are also needed to investigate their stability in biological conditions, such as highly concentrated electrolyte solutions, and to minimize background fluorescence due to nonspecific binding. Currently, quantum dots are being explored as biological imaging agents in vitro (11, 12) and in vivo (13, 14). A number of groups have attached small molecules (1518), peptides (19), proteins (20), antibodies (21-24), and * To whom correspondence should be addressed. E-mail: [email protected]. Phone: 615-322-2633. Fax: 615-343-1234. † Department of Chemistry. ‡ Department of Pharmacology. § These authors contributed equally to this work.

DNA (25) to quantum dots and imaged their binding. It has been reported that the carboxyl groups on the surface of quantum dots lead to nonspecific binding to oligonucleotides (25), and we have observed nonspecific binding of mercaptoacetic acid-conjugated quantum dots to human epithelial kidney cells (HEK). While other studies have optimized labeling using quantum dots for their own particular applications, presented here are generalized strategies to reduce nonspecific binding of quantum dots through PEG functionalization of the quantum dot surface that can be utilized in a variety of applications. To advance studies of conjugated quantum dots for live cell applications, it is necessary to address the issue of nonspecific binding. It has been previously reported that quantum dots conjugated to methoxy-terminated PEG5000 (5000 g molecular wt) reduces nonspecific binding in mouse animal models (26). We assessed the degree of nonspecific binding of AMP-coated quantum dots (AMP dots) to cell surfaces of six different cell lines. We report that cell type is an important factor in the degree of nonspecific binding that is exhibited with quantum dots. The optimal conjugation conditions of PEG to AMP dots were investigated to ensure maximum fluorescence quantum yield after conjugation. The effect of ligand coverage of the quantum dot and PEG lengths required to sufficiently reduce background fluorescence caused by nonspecific binding of quantum dots was also evaluated in live cell assays. MATERIALS AND METHODS

Reagents. Poly(ethylene glycol) (PEG) 2000 monomethyl ether, 550 monomethyl ether, and 350 mono methyl ether were obtained from Fluka. Tosyl chloride, N-hydroxysuccinamide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), tetraethylene glycol, and methyl iodide were obtained from Aldrich. Potassium hydroxide was purchased from EM Science, Potassium phthalimide was obtained from Lancaster, and hydrazine hydrate was purchased from VWR. Borate buffer was obtained from Polysciences Inc (Warrington PA 18976); Sephadex G-50 was obtained from Amersham Biosciences (AB SE 751 84 Uppsala Sweden). All reagents were used as supplied without further purification.

10.1021/bc0502006 CCC: $30.25 © 2005 American Chemical Society Published on Web 11/01/2005

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Quantum Dots in Live Cell Assays Scheme 1. Synthetic Route for Creating Methoxy-Terminated Amino PEGs

Quantum Dots. Amphiphilic poly(acrylic acid) polymercoated quantum dots (AMP dots) were a gift from the Quantum Dot Corporation (Hayward, CA 94545). Core shell cadmium selenide/zinc sulfide quantum dots emitting at 605 nm were used in this study. Concentrations of quantum dots were measured using a Carey 50 UVvisible spectrophotometer. Their absorbance at 600 nm was measured, and the concentration was calculated based upon an extinction coefficient of 650 000.1 Spectroscopic Characterization. The PEG intermediates and the final product were characterized by NMR and MALDI mass spectroscopy. All NMR data was obtained using CDCl3 as a solvent unless otherwise stated and analyses were performed using a Brucker 300 MHz instrument. Chemical shifts were measured relative to TMS, and coupling constants are measured in Hz. Mass Spectroscopy. MALDI-TOF mass spectra were recorded on an Applied Biosystems Voyager mass spectrometer equipped with a 337 nm nitrogen laser. For each spectrum, 30 to 64 scans were averaged using an acceleration voltage of 25 kV. Samples were prepared, using a standard DBA matrix. A PEG standard prepared in the same manner as the other samples was used for mass calibration of the instrument. PEG Synthesis. Four methoxy terminated amino PEGs were used in this study. These were methoxyterminated amino PEG 2000, 550, 350 and the methoxyterminated amino PEG derived from tetra PEG (PEG 207). The synthetic route used to synthesize these compounds is shown in Scheme 1. The mono methoxy PEG derivative of tetra PEG was synthesized as described by Masci (27). Initially the PEG mono methyl ether was converted to a tosylate using the method described by Kegstra (28) resulting in tosylates 1a-d. The tosylate was displaced using potassium phthalimide by refluxing in acetonitrile for 18 h resulting in 2a-d. These were subsequently converted to the amino PEG derivative by stirring in ethanol with hydrazine hydrate giving 3a-d. Compounds 1a-d, 2a-d, 3a-d were characterized by NMR. Compounds 3a-d were further characterized by MALDI mass spectroscopy. The tosylates of the four PEG lengths were prepared as follows: 1

Brucez, M. Private communication.

Mono Tosyl Poly(ethylene glycol) 2000 Mono Methyl Ether (1a). PEG 2000 monomethyl ether (20 g) was dissolved in methylene chloride (200 mL) and cooled to 0 °C in an ice/acetone bath. p-Toluenesulfonyl chloride (2 g, 0.01 mol) was added, and the mixture was stirred at 0 °C for 30 min. Freshly powdered potassium hydroxide (4.5 g, 0.08 mol) was added, and the mixture was stirred at 0 °C for 6 h (28). After warming to room temperature, the solution was washed with water (2 × 20 mL) and dried over magnesium sulfate. The methylene chloride was removed under reduced pressure, and the product was purified by washing with hexanes (5 × 200 mL). This yielded 18 g of the product as a waxy solid. 1 H NMR (CDCl3) δ 2.42 (3H), 3.39 (3H), 3.60 (nH), 3.823.86 (2H), 4.10-4.14 (2H), 7.31 (2ArH), 7.75 (2 ArH). Mono Tosyl Poly(ethylene glycol) 550 Mono Methyl Ether (1b). An amount of 22 g of PEG 550 mono methyl ether gave 26 g of crude mono tosyl poly(ethylene glycol) 550 mono methyl ether, and this was purified by column chromatography on silica gel eluted with methylene chloride then methylene chloride/methanol (5%), giving 24 g of the product as a colorless oil. 1H NMR (CDCl3) δ 2.40 (3H), 3.33 (3H), 3.60 (nH), 4.10-4.12 (2H), 7.29 (2ArH), 7.76 (2 ArH). Mono Tosyl Poly(ethylene glycol) 350 Mono Methyl Ether (1c). An amount of 14 g of mono methyl PEG 350 gave 18 g of crude tosylate which was purified using column chromatography on silica gel eluted with methylene chloride and then a methylene chloride (98%)/ methanol (2%) mixture, resulting in 17.3 g of the product as colorless oil. 1H NMR (CDCl3) δ 2.37 (3H), 3.28 (3H), 3.46 (nH), 4.05 (2H), 7.27 (2ArH), 7.70 (2ArH). Toluene-4-sulfonic Acid 2-{2-[2-(2-Methoxyethoxy)ethoxy]ethoxy}ethyl Ester (1d). An amount of 10.4 g of 2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}ethanol gave 9.5 g of tosylate after column chromatography on silica gel eluted with methylene chloride 98%/methanol 2%. Mass spectroscopy indicated that this material was contaminated with 1-methoxy-2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}ethane (I). Attempts to remove this contaminant using column chromatography failed, and the product was used without further purification in the next synthetic step. 1H NMR (CDCl3) δ 2.27 (3H), 3.17 (nH), 3.47 (nH), 3.96 (2H), 7.19 (2ArH), 7.61 (2ArH). Mono Phthalimido Poly(ethylene glycol) 2000 Monomethyl Ether (2a). Mono tosyl PEG 2000 mono

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methyl ether (18 g) was dissolved in acetonitrile (200 mL) and heated at reflux with stirring for 18 h. The mixture was cooled and evaporated and dissolved in methylene chloride (200 mL). The organic solution was washed with water (2 × 50 mL) and dried over magnesium sulfate. After filtration and evaporation under reduced pressure, the product was purified by washing the crude product with hexanes (5 × 200 mL). This gave 7.7 g of the product as a colorless wax. 1H NMR (CDCl3) δ 3.36 (3H), 3.52 (nH), 3.68 (2H), 3.82 (2H), 7.69 (2ArH), 7.79 (2ArH). Mono Phthalimido Poly(ethylene glycol) 550 Monomethyl Ether (2b). The phthalimide was prepared as previously described for the phthalimide of mono methyl PEG 2000. Thus, 20 g of tosylate gave 15 g of crude phthalimide which was purified using column chromatography on silica gel eluted with a gradient system running from methylene chloride to 95% methylene chloride/5% methanol. This gave 14 g of the purified phthalimide as a yellow oil. 1H NMR (CDCl3) δ 3.20 (3H), 3.48 (nH), 3.56 (2H), 3.71 (2H), 7.57 (2ArH), 7.66 (2ArH). Mono Phthalimido Poly(ethylene glycol) 350 Monomethyl Ether (2c). An amount of 17.3 g of tosylate gave 8.7 g of phthalimide, after purification on silica gel eluted with a gradient system eluted with a solvent system from methylene chloride to methylene chloride (98%)/methanol (2%). 1H NMR (CDCl3) δ 3.30 (3H), 3.46 (nH), 3.66 (2H), 3.82 (2H), 7.65 (2ArH), 7.76 (2ArH). 2-(2-{2-[2-(2-Methoxyethoxy)ethoxy]ethoxy}ethyl)isoindole-1,3-dione (2d). An amount of 9.5 g of tosylate gave 3.9 g of phthalimide, after purification on silica gel eluted with a gradient system eluted with a solvent system from methylene chloride to methylene chloride (98%)/methanol (2%). Mass and NMR spectroscopy indicated that this material was contaminated with 1-methoxy-2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}ethane (I) and was used without further purification.1H NMR (CDCl3) 2.22 (nH), 3.45 (nH), 3.75 (2H), 7.57 (2ArH), 7.68 (2ArH). Mono Amino Poly(ethylene glycol) 2000 Monomethyl Ether (3a). Mono phthalimido PEG 2000 monomethyl ether (7.7 g) was dissolved in ethanol (200 mL), and hydrazine mono hydrate (10 mL) was added. The mixture was stirred at room temperature for 18 h and then evaporated under reduced pressure. The crude product was dissolved in methylene chloride and stirred for 18 h at room temperature. The solution was filtered and washed with water (2 × 50 mL) and dried over magnesium sulfate. After filtration and evaporation, the product was purified by washing with hexanes (5 × 200 mL). This gave the product as a colorless wax (5.9 g). 1H NMR (CDCl3) δ 3.32 (3H), 3.56 (nH), 3.69 (NH2). Mono Amino Poly(ethylene glycol) 550 Monomethyl Ether (3b). Mono amino poly(ethylene glycol) 550 monomethyl ether was prepared as previously described for mono amino poly(ethylene glycol) 2000 monomethyl ether. Thus 10 g of phthalimide resulted in 7.5 g of the desired product as colorless oil. 1H NMR (CDCl3) δ 2.73 (2H), 3.32 (3H), 3.44 (nH). Mono Amino Poly(ethylene glycol) 350 Monomethyl Ether (3c). Mono amino poly(ethylene glycol) 350 monomethyl ether was prepared as previously described for mono amino poly(ethylene glycol) 2000 monomethyl ether. Thus 8.7 g of phthalimide resulted in 7.5 g of the desired product as a colorless oil. 1H NMR (CDCl3) δ 2.81 (2H), 3.09 (3H), 3.36 (nH). 2-{2-[2-(2-Methoxyethoxy)ethoxy]ethoxy}ethylamine (3d). 2-{2-[2-(2-Methoxyethoxy)ethoxy]ethoxy}ethylamine (mono amino poly(ethylene glycol) 207 mono-

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methyl ether) was prepared as previously described for mono amino poly(ethylene glycol) 2000 monomethyl ether. Thus, 3.9 g of phthalimide resulted in 1.0 g of the desired product as colorless oil after distillation under reduced pressure at 1 mmHg (bp 115-125 °C). NMR and mass spectroscopy of this material indicated that it was a mixture of the desired product (70%) and 1-methoxy2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}ethane (I) (30%). As 1-methoxy-2-{2-[2-(2-methoxyethoxy)ethoxy]ethoxy}ethane is inert it was decided to couple this material to AMP dots without further purification. 1H NMR (CDCl3) δ 1.3 (NH2), 2.71 (2H), 3.22 (nH), 3.40 (nH). Coupling PEG Ligands to AMP Dots. During the course of our studies, we tried several different coupling methodologies using different ratios of EDC, NHS, and AMP dots. We observed that the best methodology used a ratio of 2000 PEGs, 1500 EDCs, 1500 NHS, and 1 dot. Thus, in a typical ligand exchange, 0.1 mL of a 8.9 µM solution of AMP dots was placed in a vial equipped with a stir bar. Mono methyl PEG 2000 amine (3.6 mg, 1.8 µM) was dissolved in borate buffer (0.3 mL) at pH 8.4, and this was added to the solution of the AMP dots. This mixture was stirred for 10 min at room temperature, and then N-hydroxysuccinamide (0.15 mg, 1.3µM) dissolved in borate buffer (0.1 mL) was added. The mixture was stirred for a further 10 min at room temperature, and then 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.25 mg, 1.3 µM) was added. The solution was stirred at room temperature for 2 h, and the pegylated dots were purified via size exclusion chromatography using sephadex (G50). Gel Electrophoresis. Conjugation was confirmed by gel electrophoresis. Gels were 1% agarose in Tris-EDTA acetate (TEA) buffer. Gels were run at 80 V for 80 min with TEA buffer as the running buffer. Gel images were taken on an AlphaImager 2000 imaging station (Alpha Innotech Corp.). Quantification of PEG on the Surface. The number of PEG molecules coupled to each quantum dot was determined by using the fluorescamine assay. An excess of fluorescamine (3 mg/mL) in acetone was added to each reaction mixture and vortexed for 15 min at room temperature. The fluorescence at 480 nm was measured and compared to the fluorescence produced by the same reaction mixture without EDC. The ratio of the fluorescence of these two reactions is the percentage of PEG still in solution, unconjugated to the quantum dots. This is subtracted by the amount of initial PEG and then divided by the number of quantum dots used in the reaction to give the number of PEG molecules conjugated to each quantum dot (equation given in Supporting Information). Cell Cultures. Several cell lines were chosen to compare the nonspecific binding of AMP dots to PEG conjugated AMP dots (PEG dots). These cell types were as follows: mouse fibroblasts (3T3); African green monkey kidney cells (COS7); Chinese hamster ovary (CHO), a gift from Dr. Tad Inagami (Vanderbilt University, Nashville, TN); human epithelial kidney (HEK); porcine renal epithelial (LLC); human epithelial larynx carcinoma (HEp-2). Approximately 5000 cells/well of 3T3 cells were plated in black/clear bottom 96-well plates and grown to confluency in DMEM (Gibco) containing 9% bovine serum, 5 units/mL penicillin, and 5 µg/mL streptomycin media. Approximately 10-15 000 cells/well of COS7, CHO, HEK, and LLC cells were plated in black/ clear bottom 96-well plates and grown to confluency in DMEM containing 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin media. Approximately 10 000 cells/well of

Quantum Dots in Live Cell Assays

Bioconjugate Chem., Vol. 16, No. 6, 2005 1491 RESULTS AND DISCUSSION

Figure 1. Fluorescent spectra used to determine relative quantum yield of PEGylated AMPdots with varied ratios of PEG:NHS:EDC. PEG2000-AMP-D (purple) and PEG2000-AMP-F (red) are 2000 equivalents of PEG to 1500 equivalents of NHS and EDC (separate reactions). PEG2000-AMP-E (yellow) is 2000 equivlaents of PEG, NHS and EDC. PEG2000-AMP-C (green) is 2000 equivalents of PEG to 1000 equivalents of NHS and EDC. PEG2000-AMP-B (blue) is 1000 equivalents of PEG to 3000 equivalents of NHS and EDC.

HEp-2 cells were plated in black/clear bottom 96-well plates and grown to confluency in OPTI-MEM containing 2% fetal bovine serum, 200 mM glutamine, 250 µg/mL of amphotericin-B, and 10 mg/mL gentamicin media. All cells were incubated at 37 °C with 5% CO2. Microscopy. Images of labeled cells were taken using a Nikon Diaphot epifluorescent microscope equipped with phase/fluorescent/camera and a Quixell automated micromanipulator on a Nikon D100 digital camera. Quantification of Nonspecific Binding. The nonspecific binding of the AMP dots and PEG dots to each cell type was quantified by fluorescence measurements of cell cultures grown in 96-well plates. Concentrations of PEG and AMP dots ranging from 1 nM to 50 nM were tested against each cell type by incubating three wells with each concentration for 30 min and then washing with excess KRH buffer. The fluorescent intensity of each well was measured using a BIO-TEK Synergy HT plate reader (excitation 250 nm/10, emission filter 590 nm/35, top optic position with a vertical offset of 1 mm and sensitivity ) 88.)

Conjugating PEG to the surface of the AMP dots was employed to reduce nonspecific binding in live cell assays. Previous studies indicated that the quantum efficiencies of the 605 nm AMP dots were affected by the conjugation chemistry (data not shown). Ratios of methoxy terminated amino PEG2000, NHS, and EDC were varied to determine the optimal conditions for coupling PEG to the surface of AMP dots without compromising the probe’s quantum yield. The relative quantum yields of 50 nM solutions of each of the PEG dots were determined by comparison of their emission intensity at 605 nm. The ratio 2000:1500:1500 of PEG/NHS/EDC equivalents per quantum dot (purple and red) (Figure 1) exhibited the least amount of quenching after conjugation. An excess of NHS and EDC (blue) displayed less than half of the original fluorescent emission, while lower ratios resulted in nonoptimal PEG coupling. The pH of the reactions was monitored to determine if the decrease in fluorescent intensity was due to etching of the quantum dot surface by hydrochloric acid liberated from EDC hydrolysis. Etching of the surface from HCl does not appear to be the cause of decreased fluorescence since the borate buffer maintained a pH of 8.5-8.2 throughout the 2 h coupling. All subsequent conjugations of PEG to AMP dots were carried at the 2000:1500:1500 ratio of PEG/ NHS/EDC equivalents per quantum dot. Six common cell lines, 3T3, LLC, COS7, CHO, HEp-2, and HEK, were chosen in order to determine the degree of nonspecific binding of AMP dots in live cell assays. Each cell line was incubated with 50 nM AMP dots for 30 min, washed, and imaged. While all cell lines showed some degree of nonspecific binding, the amount varied with each cell line (Figure 2: A1-F1). HEK cells (Figure 2: C1) displayed the most nonspecific binding while 3T3 cells exhibited the least (Figure 2:E1). The reduction of nonspecific binding was evaluated in parallel experiments by incubating the cell lines with 50 nM PEG functionalized AMP dots. PEG-conjugated quantum dots significantly reduced the nonspecific binding with all the selected cell types. Controls indicated that there was no binding of the AMP dots or the PEG conjugated quantum dots to the surface of the 96-well microtiter plates used for cells seeding, therefore, all binding was attributed to the cell surfaces. In cell lines that did not exhibit much nonspecific binding initially, like 3T3, there was a reduction of 18% when using 50 nM PEG dots. In cell lines like HEK, where there were very significant amounts of nonspecific binding PEG dots reduced nonspecific binding by as much as 90% at working concentrations of 50 nM (Supporting Information). While nonspecific bind-

Figure 2. Comparison of the nonspecific binding of 50 nM AMP dots (A1-F1) to PEGylated AMP dots (A2-F2). The comparison was done in six different cell lines: HEp-2 (A), LLC (B), HEK (C), COS7 (D), 3T3 (E), and CHO (F).

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Figure 3. Electrophoretic gels (1% agarose) of AMP dots and PEGylated AMP dots. The gel was run at 80 V for 80 min. The gel indicates that the conjugation was successful and that the AMP dots have been PEGylated with different equivalents of PEG2000. As expected, the more equivalents of PEG used the less electrophoretic movement.

ing is clearly related to the surface chemistry of the cells, the nature of the interaction that causes nonspecific binding likely arises from a variety of different interactions. It is possible to envision hydrophobic interactions occurring between tri-n-octylphosphine oxide (TOPO) on the surface of the quantum dot and lipids in cellular membranes. These hydrophobic interactions might occur from an incomplete coverage of the core/shell with the AMP coating. Different cell types express a variety of proteins or lipids on their surfaces that allow for interaction with exposed hydrophobic regions of the quantum dots. This variation of proteins and lipids expressed in a cell type dependent manner would give rise to some cell lines displaying larger amounts of nonspecific binding than others. Another possibility for nonspecific binding is the electrostatic interactions between the surfaces of cells and the carboxylic acids on the surfaces of the AMPcoated quantum dots. PEG reduces the ability of the quantum dots to form these interactions with the surface of the cell or proteins expressed in the extra cellular matrix, resulting in a reduction of nonspecific binding. Consequently, PEG functionalization yields an essentially passivated surface that minimizes cellular interaction. Further surface modifications of quantum dots with PEG will change the electrical double layer surrounding the quantum dots and this may also reduce the nonspe-

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cific binding. PEG is able to solvate cations in a manner similar to crown ethers (27). It is possible that cations surrounding the quantum dot are solvated by the PEGs on the surface of the quantum dot. Thus these cations may be less able to form interactions such as salt bridges between negatively charged groups on the cell surface and the quantum dot. Since HEK cells exhibited the most nonspecific binding and 3T3 cells the least, these cells lines were selected as the subjects for focused study of the effects of both the coverage and length of the PEG modulator on nonspecific cellular binding. The effect of ligand coverage was studied by coupling 2000, 500, and 100 equivalents of PEG2000 to AMP dots. Quantitation of the number of PEGs coupled to the surface was determined by a fluorescamine difference assay. The fluorescamine assay produces a fluorescent species when it reacts with primary amines thereby giving you the amount of amino PEGs that still have primary amines (26). Controls for Qdot interference and issues of solvent incompatibilities (i.e. presence of acetone) showed no interference. Assay results indicated approximately 380, 106, and 11 PEG modulators per quantum dot in the 2000, 500, and 100 equivalent reactions, respectively (Supporting Information). In the 2000 equivalent reaction it is expected that all surface accessible carboxyl groups of the AMP coating on the quantum dot are funtionalized. Subsequent characterization with gel electrophoresis (Figure 3) confirmed the coupling reaction and was consistent with the fluorescamine quantitation. Quantum dots with 380 PEG2000 displayed little to no nonspecific binding (Figure 4). Very little increase in nonspecific binding to HEK and 3T3 cells was observed with quantum dots which had about 106 PEG2000. However, when only 11 PEG2000 were coupled to the quantum dot, the nonspecific binding to HEK cells increased. Nevertheless, even though the surface has very few PEG molecules on it, it is still apparent that the nonspecific binding is reduced by about 50%. These results suggest that it may not be necessary to functionalize every site on the surface of the quantum dot with PEG to reduce nonspecific binding but the more complete functionalization of the AMP surface the better the reduction of nonspecific binding. There was little effect on the nonspecific binding of the quantum dots with only 11 PEG to the 3T3 cells, which had originally shown the least nonspecific binding to the AMPdots.

Figure 4. Graphs of the fluorescent intensity of AMP dots (0) and AMP dots PEGylated with 100 equivalent of PEG2000 (O), 500 equivalents of PEG2000 (9), and 2000 equivalents of PEG2000 (b) for 3T3 (A) and HEK (B).

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Figure 5. Graphs of the fluorescent intensity of AMP dots (0) and AMP dots PEGylated with 2000 equivalents of PEG350 (O), PEG550 (9), and PEG2000 (b) for 3T3 (A) and HEK (B).

The effect of PEG length was determined by comparing the amount of nonspecific binding AMPdots functionalized with PEG2000, PEG550, and PEG350 with uniform coverage (Figure 5). To ensure that nonspecific binding in these assays was a product of PEG length, all conjugations were done with 2000 equivalents of PEG as described above so that all surface available carboxyl groups were utilized. It has been observed by Ballou (26) et al. that when methoxy amino PEG5000 was attached to AMP dots only 100 PEGs were observed to bind, but when PEG750 was attached, 300 PEGs were observed to bind. Electrophoretic gels indicate that the mobility of PEG conjugated to quantum dots appear to behave as a function of the PEG length (Supporting Information). Consequently, with lengths shorter than PEG2000, steric hindrance does not appear to affect PEG conjugation but may be a factor in the reduced number of conjugations with longer PEGs. The length of the PEG chain does not seem to be important until it is shorter than about 14 units, and even PEG350 was able to eliminate most nonspecific binding. All lengths of PEG dots used reduced the nonspecific binding in both HEK cells and 3T3 cells. In the 3T3 cells, which had originally shown the least nonspecific binding to the AMP dots, length of the PEG had no effect on nonspecific binding. However, nonspecific binding of PEG dots to HEK cells did increase with decreasing PEG length (Figure 5). In HEK cells, the PEG2000 eliminated the nonspecific binding over the concentration range investigated by approximately 90%. While the PEG550 and PEG350 did show an increase in nonspecific binding, they still provide a significant reduction of 70% and 60%, respectively, over most concentrations examined. For most biological assays the working concentration of quantum dots will be around 10 nM, where the nonspecific binding for all lengths is very low. CONCLUSIONS

The nonspecific binding of AMP dots to cell surfaces is dependent upon the cell type. In our study, HEK cells exhibited the most nonspecific binding at concentrations of 50 nM. When AMP dots are conjugated to methoxy amino PEG2000, the nonspecific binding at 50 nM concentrations is greatly reduced. The PEG may be shortened to approximately 12-14 units without increasing nonspecific binding. While it may not be necessary to coat the entire quantum dot surface with PEG to reduce nonspecific binding, complete coverage of the AMP surface on the dots is optimal. While PEG coated dots do not provide a biologically interesting probe, this study shows that a small number

of PEGs are sufficient to reduce nonspecific binding. A coligand strategy could be implemented utilizing some of the available conjugation sites for PEG while other sites may be filled with ligands of biological relevance. Another strategy would be to fill available conjugation sites with customized ligands consisting of a PEG linker arm attached to a biologically relevant moiety. ACKNOWLEDGMENT

We would like to thank Quantum Dot Corporation for supplying the core/shell nanocrystals used in this study. We would like to thank David Hercules and Matthew Vergne for doing the MALDI mass spectrometry studies used in this work, and Marcus Voeler and Brian O. Bachman for providing the equipment to run and study agarose gels. This work was supported by the NIH under grant RO1 EB003728. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Alivisatos, A. P. (1996) Perspectives on the physical chemistry of semiconductor nanocrystals. J. Phys. Chem. 100, 13226-13239. (2) Schmelz, O., Mews, A., Basche´, T., Herrmann, A., and Mu¨llen, K. (2001) Supramolecular complexes from CdSe nanocrystals and organic fluorophors. Langmuir 17, 28612865. (3) Peng, Z. A., and Peng, X. (2001) Formation of high-quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor. J. Am. Chem. Soc. 123, 183-184. (4) Hines, M. A., and Guyot-Sinnest, P. (1996) Synthesis and characterization of strongly luminescing ZnS-capped CdSe nanocrystals. J. Phys. Chem. 100, 468-471. (5) Dabbousi, B. O., Rodriguez-Viejo, 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 Optical and Structural Characterization of a Size Series of Highly Luminescent Materials. J. Phys. Chem. B 101, 9463-9475. (6) Peng X., Schlamp, M. C., Kadavanich, A. V., and Alivisatos, A. P. (1997) Epitaxial growth of highly luminescent CdSe/ CdS core/shell nanocrystals with photostability and electronic accessibility J. Am. Chem. Soc. 119, 7019-7029. (7) Michalet, X., Pinaud, F., Lacoste, T. D., Dahan, M., Bruchez, M. P., Alivisatos, A. P., and Weiss, S. (2001) Properties of fluorescent semiconductor nanocrystals and their application to biological labeling. Single Mol. 2, 261-276. (8) Bruchez, M., Moronne, M., Gin, P., Weiss, S., and Alivisatos, A. P. (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281, 2013-2016.

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