Langmuir 2000, 16, 3107-3118
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Preparation of Aminodextran-CdS Nanoparticle Complexes and Biologically Active Antibody-Aminodextran-CdS Nanoparticle Conjugates Ivan Sondi,† Olavi Siiman,*,‡ Steven Koester,‡ and Egon Matijevic´*,† Center for Advanced Materials Processing, Clarkson University, Potsdam, New York 13699-5814, and Advanced Technology, Beckman Coulter, 11800 SW 147th Avenue, P.O. Box 169015, Miami, Florida 33116-9015 Received August 17, 1999. In Final Form: December 29, 1999 Stable aqueous dispersions consisting of CdS nanoparticles having modal diameters, ranging between 2 and 8 nm, were prepared with amino-derivatized polysaccharides (aminodextrans, hence abbreviated as Amdex) as the stabilizing agents. The size, stability, and luminescence intensity of such dispersions were shown to be dependent on the types of the cadmium salts and aminodextrans used, as well as on the reactant concentrations. Specifically, it was demonstrated that the degree of substitution of amino groups in the aminodextran molecules greatly affected the properties of the dispersions; i.e., with higher degree of substitution, smaller CdS particles and higher luminescence intensity were achieved. It was also shown that the Amdex-CdS nanoparticle complexes could be activated and conjugated with antibody by conventional means. Molecular weight ranges of the Amdex and their complexes with CdS nanoparticles and the purity of antibody-Amdex-CdS nanoparticle conjugates were determined by polyacrylamide gel electrophoresis combined with Coomassie blue staining of resultant gel bands. The purified conjugate of the aminodextran-CdS nanoparticle complex with anti-CD4 monoclonal antibody was mixed with a whole blood control, followed by indirect sheep antimouse antibody-phycoerythrin (SAM-PE) labeling of washed cells incubated with T4-5X-Amdex-CdS. Red blood cells were then lysed and quenched, and the resulting mixture, which was run on a flow cytometer with 488.0 nm argon ion laser excitation, suggested that the T4 antibody from the conjugate was present specifically on lymphocytes.
Introduction Semiconductor nanoparticles, including those of CdS, showing the quantum size effect, also referred to as “quantum dots”, have attracted much interest, due to their unique photochemical and photophysical properties.1-10 The latter depend on the particle size and composition, which can be varied with the method of preparation,11-14 including the use of Langmuir-Blodgett films,15,16 reverse micelles,17,18 and vesicles,19 as well as different substrate supports, such as zeolites,20 clay minerals,21 glasses,22 † ‡
Clarkson University. Beckman Coulter.
(1) Weller, H. Angew. Chem., Int. Ed. Engl. 1993, 32, 41. (2) Henglein, A. Chem. Rev. 1989, 89, 1861. (3) Henglein, A. Ber. Bunsen-Ges. Phys. Chem. 1997, 101, 1562. (4) Kamat, P. V. Chem. Rev. 1993, 93, 267. (5) Brus, L. E. Appl. Phys. 1991, 53, 465. (6) Brus, L. E. J. Phys. Chem. 1986, 90, 2555. (7) Khairutdinov, R. F. Colloid J. 1997, 59, 535. (8) Fendler, J. H.; Tian, Y. In Nanoparticles and Nanostructured Films; Fendler, J. H., Ed.; Wiley: Weinheim, 1998. (9) Huang, H. H.; Yan, F. Q.; Kek, J. M.; Chew, C. H.; Xu, G. Q.; Ji, W.; Oh, P. S.; Tang, S. H. Langmuir 1997, 13, 172. (10) Spanhel, L.; Haase, M.; Weller, H.; Henglein, A. J. Am. Chem. Soc. 1987, 109, 5649. (11) Petit, C.; Pileni, M. P. J. Phys. Chem. 1988, 92, 2282. (12) Petit, C.; Lixon, P.; Pileni, M. P. J. Phys. Chem. 1990, 94, 1598. (13) Yao, H.; Takada, Y.; Kitamura, N. Langmuir 1998, 14, 595. (14) Murray, C. B.; Norris, D. J.; Bawendi, M. G. J. Am. Chem. Soc. 1993, 115, 8706. (15) Smotkin. E. S.; Chongmok, L.; Bard, A. J.; Campion, A.; Fox, M. A.; Mallouk, T. E.; Webber, S. E. Chem. Phys. Lett. 1988, 152, 265. (16) Xu, S.; Zhao, X. F.; Fendler, J. H. Adv. Mater. 1990, 2, 183. (17) Lianos, P.; Thomas, J. K. Chem. Phys. Lett. 1986, 125, 299. (18) Pileni, M. P.; Motte, L.; Petit, C. Chem. Mater. 1992, 4, 338. (19) Youn, H.-C.; Baral, S.; Fendler, J. H. J. Phys. Chem. 1988, 92, 6320. (20) Wang, Y.; Herron, N. J. Phys. Chem. 1987, 91, 257. (21) Stramel, R. D.; Nakamura, T.; Thomas, J. K. J. Chem. Soc., Faraday Trans. 1 1988, 84, 1287.
various polymer networks,23-27 and porous TiO2 layers.28,29 Polymers, including the recently described dendrimers,30-32 may be a good choice as stabilizers, because they can be designed to vary certain physical properties of semiconductor nanoparticles.33 This work describes the preparation of CdS quantum dots by the controlled double-jet precipitation process (CDJP) and by the rapid mixing of reactant solutions in the presence of different aminodextrans. The former method was originally employed for the production of larger particles, as exemplified by silver halide grains for photographic emulsions,34 but recently it was shown that it can be utilized for the synthesis of inorganic colloidal dispersions at relatively high concentrations of solids consisting of small particles.35-37 (22) Kuczynsky, J.; Thomas, J. K.J. Phys. Chem. 1985, 89, 2720. (23) Wang, Y.; Suna, M.; Mahler, M.; Kasowski, R. J. Phys. Chem. 1987, 87, 7315. (24) Olshavsky, M. A.; Allcock, H. R. Chem. Mater. 1997, 9, 1367. (25) Kuczynsky, J. P.; Milosavljevic, B. H.; Thomas, J. K. J. Phys Chem. 1984, 88, 980. (26) Wang, Y.; Suna, A.; McHugh, J.; Hilinski, E. F.; Lucas, P. A.; Johnson, R. D. J. J. Chem. Phys. 1990, 92, 6927. (27) Huang, J.; Yang, Y.; Yang, B.; Liu, S.; Shen, J. Polym. Bull. 1996, 36, 337. (28) Vogel, R.; Pohl, K.; Weller, H. Chem. Phys. Lett. 1990, 174, 241. (29) Weller, H. Ber. Bunsen-Ges. Phys. Chem. 1991, 95, 1361. (30) Dagani, R., Chem. Eng. News 1999, Feb 8, 33. (31) Zhao, M.; Sun, L.; Crooks, R. M. J. Am. Chem. Soc. 1998, 120, 4877. (32) Balogh, L.; Tomalia, D. A. J. Am. Chem. Soc. 1998, 120, 7355. (33) Stupp, S. I.; Braun, P. V. Science 1997, 277, 1242. (34) (a) Wey, J. S. In Preparation and Properties of Solid State Materials; Wilcox, W. R., Ed.; Dekker: New York, 1981. (b) Berry, C. R. In Precipitation and Growth of Silver Halide Emulsion Grains; James, T. H., Ed.; MacMillan: New York, 1977. (35) Wang, L.; Schultz, M.; Matijevic´, E. Colloid Polym. Sci. 1997, 275, 593. (36) Schultz, M.; Matijevic´, E. Colloids Surf. 1998, 131, 173. (37) Her, Y.-S.; Matijevic´, E. J. Mater. Res. 1995, 10, 3106.
10.1021/la991109r CCC: $19.00 © 2000 American Chemical Society Published on Web 02/23/2000
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Table 1. Amine Substitution Data for Aminodextrans
a
type of Amdex
MW (kDa)
glucose units per molecule
5X-Amdex, lot 1-5 5X-Amdex, lot 11-6 5X-Amdex, lot 2-2 1X-Amdex
34a 44.5a 170a 93a
210 275 1040 570
degree of substitution
mol of diaminopropane/ mol of Amdex
mol of amine/ mol of Amdex
2/5 1/3 1/3 1/20
84 91 350 29
170 180 690 57
Data reported in ref 43.
Furthermore, an extensive study has been carried out to evaluate the effects of the employed carriers (aminoderivatized polysaccharides), on the size, stability, and fluorescence of the resulting CdS nanoparticles. Amdex polymers were used as reducing and/or protective agents in the preparation or coating of monodispersed colloidal dispersions of magnetic ferrites,38 metals,39 polystyrene,40 and polystyrene-metal 41 particles. Amdex of sufficiently large molecular weight can accommodate multiple protein molecules. Previous experience 42 showed that the antiCD3 monoclonal antibody could be bound to Amdex under saturation conditions giving a T3 antibody conjugate, with a ratio of 37:1 of anti-CD3 monoclonal antibody molecules per molecule of 1X-Amdex, and 20:1 for T3 antibody/5XAmdex. Further, the preparation and structural characterization of various Amdex and their conjugates with antibody and phycoerythrin or its tandems with cyanin 5.1 and TEXAS RED are described elsewhere.43 It has been shown that a critical requirement in obtaining high luminescence emission intensities from CdS and other semiconductor nanoparticles is the capping of coordinatively unsaturated and electron-deficient surface metal atoms. Excitation of the band gap in the semiconductor nanoparticles can create excited electrons, which become trapped by uncapped surface cadmium atoms. Thus, the electron/hole recombination, which constitutes the luminescence emission, is inhibited causing very low luminescence intensities, due to the large ratio of surface atoms to total atoms in nanosize particles of 1-10 nm in diameter. To prevent the loss of emission intensity by nonradiative recombination, some specific electron-donating organic capping groups such as amines 44-46 and thiols47-50 have been used to eliminate these traps (38) Siiman, O.; Burshteyn, A. In situ use of gelatin or an aminodextran in the preparation of uniform ferrite particles. U.S. Patent No. 5,240,640, 1993. (39) Siiman, O.; Burshteyn, A. Formation of colloidal metal dispersions using aminodextrans as reductants and protective agents. U.S. Patent No. 5,248,772, 1993. (40) (a) Siiman, O.; Burshteyn, A.; Gupta, R. K. Biodegradable gelatinaminodextran particle coatings and processes for making same. U.S. Patent No. 5,466,609, 1995 and No. 5,707,877, 1998. (b) Siiman, O.; Burshteyn, A.; Gupta, R. K. Polymeric particles having a biodegradable gelatin or aminodextran coating and process for making same. U.S. Patent No. 5,639,620, 1997 and No. 5,776,706, 1998. (41) Siiman, O.; Burshteyn, A.; Cayer, M. Immobilized metal colloids on dispersed polymer microspheres. U.S. Patent No. 5,552,086, 1996. (42) Bolton, W. E.; Maples, J. A.; Siiman, O.; Kenyon, N. S.; Healy, C. G. Anti-CD3 antibody-aminodextran conjugates for induction of T-cell activation and proliferation. U.S. Patent No. 5,527,713, 1996 and No. 5,658,741, 1997. (43) Siiman, O.; Wilkinson, J.; Burshteyn, A.; Roth, P.; Ledis, S. Bioconjugate Chem. 1999, 10, 1090. (44) Dannhauser, T.; O’Neil, M.; Johansson, K.; Whitten, D.; McLendon, G. J. Phys. Chem. 1986, 90, 6074. (45) Cowdery-Corvan, J. R.; Whitten, D. G.; McLendon, G. L. Chem. Phys. 1993, 176, 377. (46) Peng, X.; Schlamp, M. C.; Kadavanich, A. V.; Alivisatos, A. P. J. Am. Chem. Soc. 1997, 119, 7019. (47) Henglein, A. Ber. Bunsen-Ges. Phys. Chem. 1995, 99, 903. (48) Lawless, D.; Kapoor, S.; Meisel, D. J. Phys. Chem. 1995, 99, 10329. (49) Bae, W.; Abdullah, R.; Mehra, R. K. Chemosphere 1998, 37, 363. (50) Bae, W.; Abdullah, R.; Henderson, D.; Mehra, R. K. Biochem. Biophys. Res. Commun. 1997, 237, 16.
for excited electrons. In addition, inorganic shells,46 such as Cd(OH)2 or ZnS on CdS cores10 and CdS or ZnS on CdSe cores,51-53 have been effective in capping. Herein, the combination of amino groups from Amdex polymer molecules and high pH have been employed to maximize luminescence emission intensity from CdS nanoparticles. The deployment of quantum dots as markers for labeling cells in luminescence-activated cell sorting is still in its infancy. Recently, two examples of CdSe core nanoparticles as labels for cells have been used for biological staining and observation with a fluorescence microscope.52,53 An important consideration in any flow cytometry experiment, which utilizes semiconductor nanoparticles as luminescent cell markers, is the luminescence lifetime of the nanoparticle emission, which for CdS nanoparticles can range widely from picoseconds to milliseconds.2,10,54 The mechanism of excitation-emission into semiconductor band gap states can explain this wide range. An emission corresponding to an electron-hole recombination will not occur at electron-deficient Cd sites on the surface of a CdS nanoparticle, unless these sites are capped by an electron pair donor, such as a nitrogen atom from Amdex or a hydroxide ion from the surface coating of Cd(OH)2. Generally, there will be some distribution of capped Cd sites and nonradiative Cd electron traps so that an excited electron will have to travel some variable distance and, thus, take some different times, before the recombination can take place to cause luminescence emission of radiation. For example, surface modification of CdS nanoparticles with mercaptoethylamine (e3 × 10-4 mol dm-3) gave average emission lifetimes from 82 to 611 ns.55 Consequently, sample flow rates and timing of the detection of emitted signals will have to be synchronized for maximum signal recovery. Further, the threshold luminescence intensity from nanoparticle probes, which allow their use as markers for analyzing cell populations in whole blood by flow cytometry, needs to be established and the compatibility of reagents, used in lysing and quenching of red blood cells with reactive inorganic nanoparticles, must be verified. Experimental Section Materials. All inorganic chemicals were reagent grade and were not further purified. Dextran-T-2M (Sigma) was used for the preparations of aminodextrans 1X-Amdex (lot-75) and 5XAmdex (lots 1-5, 2-2, and 11-6) by procedures described previously43 and in issued patents.38-42 The degree of substitution, unless otherwise noted, is defined as the total number of amino groups (primary and secondary) per monomeric glucose unit in the Amdex molecule (Table 1). Dextrans of different average molecular weights (20 and 500 kDa) were obtained from the Fluka (51) Hines, M. A.; Guyot-Sionnest, P. J. Phys. Chem. 1996, 100, 468. (52) Bruchez, M., Jr.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013. (53) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016. (54) Eychmuller, A.; Hasselbarth, A.; Katsikas, L.; Weller, H. Ber. Bunsen-Ges. Phys. Chem. 1991, 95, 79. (55) Kamat, P. V.; van Wijngaarden, M. L.; Hotchandani, S. Isr. J. Chem. 1993, 33, 47.
Dispersions of CdS Nanoparticles Company. Fura-2, pentapotassium salt, was obtained from Molecular Probes, Inc., Eugene, OR. All solutions used in the syntheses of CdS nanoparticles were freshly prepared and were filtered through 0.22 µm pore size membranes before each experiment. Sulfosuccinimidyl 4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) was obtained from Pierce, while 2-iminothiolane, digitonin, and saponin were obtained from Sigma. Coulter T4 monoclonal antibody, CD4 clone SFCI12T4D11 (IgG1), was derived from hybridization of mouse NS/1-AG4 cells with spleen cells of BALB/ cJ mice immunized with peripheral human T lymphocytes. AntiCD4-FITC and the whole blood control, IMMUNO-TROL, Beckman Coulter Part No. 6607077, are products of Coulter Corporation. Sheep antimouse antibody-phycoerythrin, SAMPE, was purchased from Silenus Laboratories, Hawthorne, Australia, and column packing materials, G-25 or G-50 Sephadex and Bio-Gel A-5m, were purchased from Pharmacia Biotech and Bio-Rad Laboratories, respectively. Phosphate-buffered saline, 1xPBS, of pH 7.1-7.3, and conductivity 13500-15500 µΩ-1 cm-1, was prepared by dilution with distilled water of a 20xPBS stock solution, which contained 26.9 g dm-3 K2HPO4, 6.4 g dm-3 KH2PO4, and 170.0 g dm-3 NaCl. Gel Electrophoresis. Gel electrophoresis of Amdex was carried out as described previously.43 The Pharmacia PHAST system, with a 4-15 gel gradient for the 30-300 kDa molecular weight range and sodium dodecyl sulfate (SDS) buffer strips, were used for the SDS-polyacrylamide gel electrophoresis (SDSPAGE) of Amdex at 400 V (65 V h-1). Amdex solutions containing 1 mg cm-3 polymer in 1xPBS buffer were mixed 1:1 with the SDS buffer and run together with high molecular weight (200, 116, 97, 66, and 45 kDa) standard samples. After the gel electrophoresis bands were stained with Coomassie blue, molecular weight ranges were estimated for the Amdex from the heaviest blue stain in each lane of the gel, in reference to the relatively narrow band positions of five standards in the same gel. 5XAmdex lots 1-5, 2-2, 11, and 11-6, and 1X-Amdex lot 75 gave molecular weight ranges of about 30-75, 55-175, 40-90, 30110, and 150-250 kDa, respectively. In most cases, the average molecular weight determined43 with the Viscotek triple detector (light scattering, viscosity, refractive index) system fell in the middle of the ranges shown by SDS-PAGE results. Preparation of the CdS Nanoparticles. Controlled DoubleJet Precipitation Process. The equipment for the controlled doublejet precipitation (CDJP) was described in detail previously.35-37 In a typical run, equal volumes (50 cm3) of solutions containing the Cd(II) salt and Na2S, respectively, were simultaneously introduced by peristaltic pumps at a constant flow rate of 10 cm3 min-1 into the jacketed reactor, containing 100 cm3 of an aqueous 5X-Amdex solution, stirred at 700 rpm. The outer jacket of the reactor was connected to a thermostated circulating water bath kept at 25.0 ( 0.1 °C. The mixing of the solutions immediately yielded CdS sols, which were further stirred for 15 min. The summary of experimental conditions used in these experiments is given in Table 1. To establish the dependence of the particle size and of the optical properties of the resulting CdS dispersions on the type of the anion present in Cd(II) salts, equal volumes of 1 × 10-3 mol dm-3 solutions of CdCl2, Cd(NO3)2, CdSO4, and Cd(ClO4)2, respectively, were reacted with a 1.0 × 10-3 mol dm-3 Na2S solution. The final concentrations of CdS and of 5X-Amdex were 2.5 × 10-4 mol dm-3 and 2.5 g dm-3, respectively. To establish the effect of the reactant concentrations, the experiments were also carried out using higher (up to 1.0 × 10-1 mol dm-3) concentrations of CdSO4 and Na2S. To enhance the luminescence intensity of the synthesized CdS dispersions, in some experiments a higher concentration of Cd(II) salts (1.3 ×10-3 mol dm-3) was used, while the concentration of Na2S was kept the same, still maintaining the same volumes of the reactant solutions. It was reported previously that the molar excess of Cd2+ ions over S2- ions during the particle preparation and a higher pH of the resultant CdS dispersions were two critical factors controlling the emission intensity.1,56 For this reason, a few drops of a 0.1 mol dm-3 NaOH solution were added to the prepared CdS dispersions to reach pH ∼ 11. (56) Henglein, A. Ber. Bunsen-Ges. Phys. Chem. 1982, 86, 301.
Langmuir, Vol. 16, No. 7, 2000 3109 Rapid Mixing of Reactant Solutions. This procedure consisted of rapidly adding 100 cm3 of a CdSO4 solution into 100 cm3 of a stirred Na2S solution containing 5 g dm-3 of 5X-Amdex, keeping the molar ratio [Cd2+]/[S2-] ) 1. The concentrations of the CdSO4 and Na2S solutions used in these experiments are given in Table 1. To evaluate the effects of different Amdex samples (5X-Amdex, lots 11-6, 1-5, and 2-2, and 1X-Amdex) on the size, stability, and luminescence, CdS nanoparticles were prepared by rapidly mixing 50 cm3 of a 1.5 × 10-3 mol dm-3 Cd(ClO4)2 solution with 50 cm3 of a solution containing 1.0 × 10-3 mol dm-3 Na2S and varying concentrations (0.05-16 g dm-3) of Amdex. The same experiments were carried out with dextran 500 000, in concentrations of 2, 5, and 20 g dm-3, and with 5 g dm-3 of dextran 20 000. The mixing of the solutions immediately yielded CdS sols, which were further stirred for 15 min. In all samples the pH was raised to ∼10.5 with NaOH. Particle Characterization. The UV-visible spectra of CdS dispersions were recorded with a Perkin-Elmer Lambda 6 UV/ vis or Beckman DU640 spectrophotometer. The size of CdS nanoparticles was determined from the onset of light absorption, based on the previously published calibration curve.57 The luminescence emission spectra of Amdex-CdS dispersions were recorded with the Spex Fluorolog instrument equipped with a Tracor Northern TN 6500 rapid scan spectrometer detection system, using 380 nm excitation. Emission and excitation spectra of T4-5X-Amdex-CdS conjugates were measured with a Shimadzu model RF5000U spectrofluorometer using a xenon lamp for excitation. Quasi-elastic light scattering, QELS (or photon correlation spectroscopy, PCS), data and their analysis were obtained with a Coulter model N4MD sub-micrometer particle analyzer with size distribution processor (SDP) analysis and multiple scattering angle detection with 632.8 nm He/Ne laser excitation. Preparation of T4 Antibody-5X-Amdex-CdS Conjugate. Standard primary amino functional group activation and conjugation procedures, described in detail in the Pierce Catalog58 and other monographs,59-61 were followed. Only 5X-AmdexCdS complexes were used in further antibody conjugations. In run 7, 10 cm3 of a 5X-Amdex-CdS dispersion, prepared by the rapid mixing protocol of 20 g dm-3 5X-Amdex (lot 1-5) solution, was purified and buffer exchanged on a 2.5 × 48 cm G-25 Sephadex column, then equilibrated and eluted with 1xPBS, and finally collected in 120-drop (∼3.6 cm3) fractions. A narrow first and a broader and brighter second luminescent band were observed with a 366 nm UV lamp (model UVL-21, Blak-Ray lamp, Ultra-Violet Products, Inc., San Gabriel, CA). Three of the brightest fractions (24-26) of the second band were combined into a 10.8 cm3 volume. A 0.20 cm3 portion of a solution containing 10 mg cm-3 of sulfo-SMCC solution in 1xPBS was used for activation. The system was roller mixed for 1 h and then separated on a G-25 Sephadex column, retaining the two brightest fractions (23 and 24) in a total volume of 7.2 cm3. A 25 mg (0.530 cm3) portion of a 47.2 mg cm-3 T4 antibody concentrate was activated with 0.16 cm3 of 2.0 mg cm-3 2-iminothiolane solution in 1xPBS, and 0.98 cm3 of 1xPBS buffer solution was added to the mixture, which was then roller mixed for 1 h. The latter was purified on G-50 Sephadex, retaining the middle fraction of the first band, containing 4.5 cm3 of 3.83 mg cm-3 IT-T4. The conjugation of 7.2 cm3 of the sulfo-SMCC-5X-Amdex-CdS suspension with 4.5 cm3 of IT-T4 solution was accomplished by roller mixing for 2 h. The conjugation mixture was separated on a Bio-Gel A-5m column, 2.5 cm × 48 cm, equilibrated with 1xPBS, and the eluant fractions were collected and monitored by A280 with a PharmaciaLKB Uvicord SII monitor. Two initial broad, poorly separated, (57) Weller, H.; Schmidt, H. M.; Koch, U.; Fojtik, A.; Baral, S.; Henglein, A.; Kunath, W.; Weiss, K.; Dieman, D. Chem. Phys. Lett. 1986, 124, 557. (58) Pierce Catalog and Handbook, Life Science & Analytical Research Products; Pierce Chemical Co.: Rockford, IL 1994. (59) Wong, S. S. Chemistry of Protein Conjugation and Cross-Linking; CRC Press: Boca Raton, FL, 1991. (60) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego, CA, 1996. (61) Aslam, M.; Dent, A. Bioconjugation-Protein Coupling Techniques for the Biomedical Sciences; Grove’s Dictionaries, Inc.: New York, 1998.
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Figure 1. Chromatograms of the A280 monitor versus fraction number on Bio-Gel A-5m for T4-5X-Amdex-CdS conjugation mixtures: A, run 7; B, run 9; C, run 10. On the right are SDSPAGE results as follows: (a) lane 1, pooled fractions 22-30; lane 2, pooled fractions 31-40; lane 3, purified T4 antibody; lane 4, standards; (b) lanes 1-3, successive fractions (23, 24, 25) of 5X-Amdex-CdS complex purified on Sephadex G-25; lane 4, IT-T4 antibody; lane 5, pooled fractions 31-40; lane 6, standards; (c) lane 4, standards; lanes 1, 2, 3, 5, and 6 are pooled fractions 31-35, 36-40, 41-45, 46-50, and 51-55, respectively. bands (32 cm3 of fractions 22-30 and 36 cm3 of 31-40) were retained, and each was pooled and concentrated in Amicon CentriPrep 30 tubes by centrifugation for 20 min at 2000 rpm in an IEC Centra-8 centrifuge to 3.7 and 2.8 cm3, respectively. Runs 9 and 10 were treated in a similar manner, except 15 cm3 of 5X-AmdexCdS sol was purified on G-25 Sephadex, using 0.3 cm3 of 10 mg cm-3 sulfo-SMCC solution for activation, and the two brightest fractions (22 and 23) of sulfo-SMCC-5X-Amdex-CdS were pooled and mixed with 17 mg (5.23 cm3) of IT-T4 in run 9. Run 10 was similar to run 9 except 0.4 cm3 of 10 mg cm-3 sulfo-SMCC solution was used and the four brightest fractions (21, 22, 23, and 24) of sulfo-SMCC-5X-Amdex-CdS were pooled and mixed with 17.9 mg (5 cm3) of IT-T4. Traces of the UV monitor-recorder for fractions collected from the Bio-Gel A-5m column for runs 7, 9, and 10 are shown in Figure 1, together with SDS-PAGE results for specified, pooled fractions from each run. Using the same level of activation of 5X-Amdex with sulfoSMCC as in previous conjugations of this polymer with CD3 antibody42 or with phycoerythrin and monoclonal antibody62,63 would require 0.45 cm3 of 10 mg cm-3 sulfo-SMCC solution or 1 × 10-5 mol sulfo-SMCC (MW 436.4 g mol-1) per 25 mg of 5XAmdex. For a two diaminopropane group repeating unit of 4 × (62) Siiman, O.; Smith, C.; Roth, P.; Burshteyn, A.; Raynor, R. Antibody-Aminodextran-Phycobiliprotein Conjugates. U.S. Patent No. 5,891,741, 1999; as presented at XIX Congress of International Society for Analytical Cytology, March 3-7, 1998, Colorado Springs, CO, Platform Presentation: Smith, C.; Wilkinson, J.; Roth, P.; Siiman, O. Detection of Low-Density Surface Markers Using Novel Amplified Fluorochrome-Conjugated Antibodies. In Cytometry; 1998; Supplement 9, p 56.
Sondi et al. 355 Da and 2 units of primary amine per repeating unit, there would be 0.035 mmol of amine groups per 25 mg of 5X-Amdex.The amine-to-sulfo-SMCC molar ratio was 3.5 (activation level ) 1/3.5), thus providing enough sulfo-SMCC to activate about 30% of the primary amine groups in 5X-Amdex. In the runs with 5X-Amdex-CdS conjugates the best results were obtained at about 10-fold lower activation levels where the amine-to-sulfoSMCC molar ratio was about 30-50, giving enough sulfo-SMCC to activate 2-3% of the primary amine groups in 5X-Amdex. Higher sulfo-SMCC amounts between 0.45 and 0.90 cm3 of 10 mg cm3 sulfo-SMCC solution in the above procedure reduced the luminescence intensity of CdS nanoparticles by about 5-10-fold to very low levels. Higher IT-T4 amounts (18-42 mg) during conjugation simply gave more excess antibody in fractions 3550 on the Bio-Gel A-5 column, causing a poorer separation of conjugates from the free antibody. Flow Cytometry. The whole blood control (IMMUNO-TROL, lot TL5) was delivered in 0.10 cm3 volumes into 12 × 75 mm tubes, and T4-5X-Amdex-CdS conjugate suspensions were then added. Samples were processed with either digitonin or saponin in 1xPBS to provide a non-acid-lyse of red blood cells. Titers of T4-5X-Amdex-CdS conjugate were carried out to establish saturation of lymphocyte CD4 receptor sites. A 0.5 cm3 portion of a 0.5 mg cm-3 digitonin or 0.1% saponin solution in 1xPBS was added, mixed, and incubated with sample mixtures in the tubes for 1 or 2 min. Cells were washed by adding 2 cm3 of PBSF (1xPBS with 0.01% sodium azide and 2.5% fetal bovine serum) to the tubes and centrifuged at 200g for 5 min, supernatant solutions discarded and 1 cm3 of PBSF was added. Each run included a nontreated (no T4-5X-Amdex-CdS) control to establish the fluorescence background for a negative gating region and a T4-5X-Amdex-CdS treated control to identify T4-5XAmdex-CdS conjugate binding. The treated control was obtained by further incubating the mixtures in tubes with SAM-PE for 15 min to identify the percent of cells with bound T4-5X-AmdexCdS conjugate. The processed IMMUNO-TROL cells were analyzed on a COULTER EPICS Elite ESP flow cytometer. The T4-5X-Amdex-CdS treated cell suspensions were excited with an argon ion laser tuned to 351-364 nm with 200 mW output laser power, whereas SAM-PE and CD4-FITC were excited at 488 nm with 15 mW of output laser power. The fluorescence emission from the 5X-Amdex-CdS treated cells was split using a 550 nm dichroic long pass filter and collected through a 525 nm band-pass filter. The CD4-FITC and SAM-PE signals were split by means of 550 and 600 nm dichroic long pass filters and collected through 525 and 575 nm long pass filters, respectively. The fluorescence was detected with photomultiplier tubes, by collecting 10 000 events for each sample after discriminating out electronic noise and debris. All inputs were stored in list mode files for further data reduction. For the CD4-Amdex-CdS conjugate titration, decreasing concentrations of CD4-Amdex-CdS conjugation mixtures from run 10 were added in 50 µL volumes, to 100 µL of whole blood, gently mixed, and incubated for 10 min at room temperature (RT). Following a wash in 2 cm3 of phosphate-buffered saline with 2.5% fetal bovine serum (PBSF), by centrifugation at 200g for 5 min, the supernatant solution was discarded and 100 µL of 1:30 sheep antimouse-PE (SAM-PE) in PBSF was added to the pellet of each sample, which were gently mixed and incubated for 10 min at RT, protected from light. Red blood cells were then lysed by a further addition of 500 µL of 0.1% saponin in PBS and incubated for 1 min at RT. Cells were washed once with 2 cm3 of PBSF and analyzed on the COULTER EPICS XL-MCL flow cytometer with 15 mW of 488.0 nm argon ion laser excitation. It is necessary to consider the consequences of the potential use of semiconductor nanoparticles of a wide luminescence lifetime range in flow cytometry. The scattered/emitted light from (63) Siiman, O.; Burshteyn, A.; Wilkinson, J.; Mylvaganam, R. Simultaneous Analyses of White Blood Cell Subsets Using Multi-Color, Multi-Intensity Fluorescent Markers in Flow Cytometry. U.S. Patent No. 5994089, 1999, as presented at XIX Congress of the International Society for Analytical Cytology, March 3-7, 1998, Colorado Springs, CO, Poster Presentation CT16: Mylvaganam, R.; Wilkinson, J.; Healy, C.; Bolton, W.; Siiman, O. Seven Markers, Four Colors, Single Laser Flow Cytometry Using Amplified Fluorochrome Conjugated Antibodies. In Cytometry; 1998; Supplement 9, p 117.
Dispersions of CdS Nanoparticles
Figure 2. Absorption spectra of CdS nanoparticles prepared by the controlled double-jet precipitation (CDJP) process using 1 × 10-3 mol dm-3 Na2S, 1 × 10-3 mol dm-3 (a) CdCl2, (b) Cd(ClO4)2, (c) Cd(NO3)2, and (d) CdSO4 solutions, and 5 g dm-3 of 5X-Amdex, lot 11-6.
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Figure 3. Absorption spectra of CdS samples collected after 6 s, 30 s, 3 min, and 5 min after the beginning of the CDJP process. The concentrations of CdSO4 and Na2S solutions were 1 × 10-1 mol dm-3 and the concentration of 5X-Amdex, lot 11-6, was 5 g dm-3.
an excited, 5 µm in diameter biological cell, labeled with nanoparticles in a flowing stream at a velocity of about 3 m s-1 (100 µm sort sense flow cell at 12 psi), which has a total transit time of about 6.7 µs through a cylindrical laser beam of about 15 µm in diameter, with a typical beam spot of 15 µm (H) × 60 µm (W), must be collected in the detector within 6.7 µs. The luminescence emission detector is triggered by light scattering (5 nm.2,57 None of these suspensions was luminescent. Increasing the concentrations of 1X-Amdex (lot-75) from 0.05 to 16 g dm-3, while keeping the same concentrations of other reactants, resulted in a shift of the onset of the absorption from 520 nm (spectrum h, particle size >5 nm) to 420 nm (spectrum a, particle size ) 2.3 nm) (Figure 9). The behavior of the 5X-Amdex (lot 2-2)-CdS dispersions is rather similar to that of 1X-Amdex-CdS dispersions as shown in Figure 10. Figure 11 compares the mean diameters of CdS particles as a function of the concentration of the two Amdex samples. The CdS dispersions prepared with these polymers in concentrations >1 g dm-3
Sondi et al.
Figure 7. Effect of the pH on the intensity of the luminescence emission of CdS dispersion prepared with CdSO4 and Na2S solutions under the same conditions as given in Figure 6.
Figure 8. Absorption spectra of CdS nanoparticles prepared with Cd(ClO4)2 and Na2S solutions using different concentrations of dextran 500 000 (2, 5, and 20 g dm-3) and 5 g dm-3 of dextran 20 000.
remained stable for about 1 month, while those with smaller amounts were stable only for a few days. Figure 12 shows that the relative luminescence intensity is stronger with increasing concentration of 1X-Amdex, with a maximum at 490 nm, shifting to 500 nm at the largest Amdex concentration, and a bandwidth of about 150 nm. Similar results were obtained for 5X-Amdex (Figure 13). Preparation of ZnS Nanoparticles by the CDJP Process. In the CDJP process, 50 cm3 of zinc sulfate (ZnSO4) and sodium sulfide (Na2S) solutions were separately and simultaneously introduced at the constant flow rate of 10 cm3 min-1 at 25 °C into 100 cm3 of an aqueous solution containing 5X-Amdex as the stabilizing agent. The dispersion was agitated with a stirrer at 700 rpm. The concentration of the reactants, i.e., zinc salt and sodium sulfide, was varied from 1 × 10-3 to 1 × 10-2 mol dm-3, while keeping constant the molar ratio [Zn2+]/[S2-] ) 1, and the concentration of 5X-Amdex at 5 g dm-3 (Table 3). The onset of the absorption determined with the 5 × 10-4 mol dm-3 ZnS dispersions shifted from 290 nm (sample a) to 306 nm for 5 × 10-3 mol dm-3 ZnS dispersion (sample c). These results correspond to quantum energies
Dispersions of CdS Nanoparticles
Figure 9. Absorption spectra of CdS nanoparticles prepared with Cd(ClO4)2 and Na2S solutions using different concentrations of 1X-Amdex: 16 (a), 8 (b), 4(c), 2(d), 1(e), 0.5 (f), 0.2 (g), and 0.05 (h) g dm-3.
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Figure 11. Plot of the mean diameter of CdS nanoparticles as a function of the concentration of 1X-Amdex, lot 75, and 5XAmdex, lot 2-2.
Figure 10. Absorption spectra of CdS nanoparticles prepared with Cd(ClO4)2 and Na2S solutions using different concentrations of 5X-Amdex (lot 2-2) as a stabilizer: 16 (a), 8 (b), 4(c), 2(d), 1(e), 0.5 (f), 0.2 (g), and 0.05 (h) g dm-3.
Figure 12. Luminescence spectra of CdS nanoparticles prepared with Cd(ClO4)2 and Na2S solutions using different concentrations of 1X-Amdex, lot 75: 16 (a), 8 (b), 4(c), 2(d), 1(e), 0.5 (f), and 0.2 (g) g dm-3.
larger than that of ZnS as a macroscopic solid (3.67 eV), and somewhat larger than those reported for ZnS quantum dots,64-67 indicating the formation of extremely small quantized zinc sulfide nanoparticles. Thus, 5X-Amdex also provides a good medium for the preparation of nanosized ZnS particles by the CDJP process. Preparation of Mixed CdS-ZnS Nanoparticles. To evaluate the possibility of synthesizing mixed ZnS and CdS nanoparticles, it was necessary to prepare dispersions of the individual components under the same conditions. Thus, single CdS or ZnS nanoparticles were generated by rapid mixing of 50 cm3 of 2 × 10-3 mol dm-3 CdSO4 or ZnSO4 solutions with 50 cm3 of 2 × 10-3 mol dm-3 Na2S solution containing 5 g dm-3 5X-Amdex. In the next step, 50 cm3 of solution containing CdSO4 and ZnSO4 in a total concentration of 2 × 10-3 mol dm-3 (but in different ratios)
was interacted with 50 cm3 of 2 × 10-3 mol dm-3 Na2S solution, which yielded mixed ZnxCd1-xS nanoparticles. The absorption spectra in Figure 14 show that single ZnS nanoparticles have an onset at 294 nm, which corresponds to a band gap of 4.22 eV, while single CdS particles show an absorption onset at 431 nm, corresponding to an average particle size of
