Anal. Chem. 2007, 79, 8520-8530
Spectral Bar Coding of Polystyrene Microbeads Using Multicolored Quantum Dots Shyam V. Vaidya,† M. Lane Gilchrist,† Charles Maldarelli,†,‡ and Alexander Couzis*,†
Chemical Engineering Department, The City College and The Graduate Center of the City University of New York, and Levich Institute, The City College of the City University of New York, New York, New York 10031
This paper focuses on encoding polystyrene microbeads, 10-100 µm in diameter, with a luminescent spectral bar code composed of mixtures of quantum dots (QDs) emitting at different wavelengths (colors). The QDs are encapsulated in the bead interior during the bead synthesis using a suspension polymerization, and the bar code is constructed by varying both the number of colors included in the bead and, for each color, the number of QDs of that color. Confocal laser scanning microscopy images of the beads demonstrate that the multicolored QDs are pushed together into inclusions within the bead interior. The encoded bead emission spectrum indicates that the peak position of the included colors does not shift relative to the corresponding peaks of the spectra recorded for the nonaggregated QDs at identical loading concentrations. Due to the spatial proximity of the QDs in the inclusions, electronic energy transfer from the lower wavelength emitting QDs to the higher emitting QDs changes the relative intensities of the colors compared to the values in the nonaggregated spectra. We show that this energy transfer does not obscure the spectral uniqueness of the different codes. Ratiometric encoding, in which the bar code is read as relative color intensity, is shown to remove the dependence of the code on the bead size. This study focuses on optically encoding polystyrene (PS) microbeads using a procedure in which beads are encapsulated with luminescent semiconductor nanocrystals (quantum dots or QDs) during a suspension polymerization to form an intensity/ color spectral bar code. Photoluminescent spectral encoding of microbeads has emerged as a very effective strategy for particle labeling (for reviews see Finkel et al.1 and Wang et al.2) and has generated interest primarily due to their use in bead-based platforms to meet the demand for high-throughput multiplexed screening assays.3-15 Luminescent encoding has initially been * To whom correspondence should be addressed. E-mail: couzis@ ccny.cuny.edu. † Chemical Engineering Department. ‡ Levich Institute. (1) Finkel, N.; Lou, X.; Wang, C.; He, L. Anal. Chem. 2004, 76, 353A-359A. (2) Wang, F.; Tan, W. B.; Zhang, Y.; Fan, X. P.; Wang, M. Q. Nanotechnology 2006, 17, R1-R13. (3) Fulton, R. J.; McDade, R. L.; Smith, P. L.; Kienker, L. J.; Kettman, J. R., Jr. Clin. Chem. 1997, 43, 1749-1756. (4) Vignali, D. A. A. J. Immunol. Methods 2000, 243, 243-255. (5) Battersby, B. J.; Lawrie, G.; Johnston, A.; Trau, M. Chem. Commun. 2002, 14, 1435-1441.
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developed using polymer latex beads encoded by encapsulating organic chromophores (or luminescent lanthanide chelate complexes), but these have several disadvantages. These luminescent molecules readily photobleach, can be chemically unstable, have different excitation wavelengths, and are characterized by broad and asymmetric emission spectra. Luminescent semiconductor nanocrystals (or QDs) are potentially more suitable as fluorescent labels to encode beads. Owing to quantum confinement effects, the peak emission wavelength is tunable by the size of the crystal as well as the composition, and as a result, different sized QDs can provide a source for a large number of tags with distinct spectral signatures. The emission spectrum for each QD is Gaussian, symmetric, and relatively narrow (20-30 nm full width at half-maximum), and their absorption spectra all exhibit peak absorption in a common band of UV wavelengths, which simplifies the excitation process in reading the bar code. The incorporation of QDs in microbeads for encoding has been undertaken using three principal routes: (i) the QDs are trapped or affinity partitioned directly into a preformed bead, (ii) a shell of QDs is encrusted on the surface of a bead, or (iii) the QDs are incorporated in the beads during the bead synthesis, either by encapsulation or by chemical grafting. Nie et al.16-19encoded polystyrene microbeads with QDs by directly impregnating the QDs into preformed beads. Polystyrene beads were first dissolved in a chloroform/alcohol solution, which penetrates the network of mesopores of the beads causing the particles to swell and the pores to enlarge. The swelling solution also contained hydrophobically capped CdSe/ZnS core shell QDs, which are soluble in (6) Rosenthal, S. J. Nat. Biotechnol. 2001, 19, 621-622. (7) Nolan, J. P.; Sklar, L. A. Trends Biotechnol. 2002, 20, 9-12. (8) Trau, M.; Battersby, B. J. Adv. Mater. 2001, 13, 975-979. (9) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792. (10) Ferguson, J. A.; Boles, T. C.; Adams, C. P.; Walt, D. R. Nat. Biotechnol. 1996, 14, 1681-1684. (11) Ferguson, J. A.; Steemers, F. J.; Walt, D. R. Anal. Chem. 2000, 72, 56185624. (12) Epstein, J. R.; Leung, A. P. K.; Lee, K.-H.; Walt, D. R. Biosens. Bioelectron. 2003, 18, 541-546. (13) Wilkins, Stevens, P.; Kelso, D. M. Anal. Chem. 2003, 75, 1147-1154. (14) Battersby, B. J.; Bryant, D.; Meutermans, W.; Matthews, D.; Smythe, M. L.; Trau, M. J. Am. Chem. Soc. 2000, 122, 2138-2139. (15) Grondahl, L.; Battersby, B. J.; Bryant, D.; Trau, M. Langmuir 2000, 16, 9709-9715. (16) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631-635. (17) Chan, W. C. W.; Maxwell, D. J.; Gao, X.; Bailey, R. E.; Han, M.; Nie, S. Curr. Opin. Biotechnol. 2002, 13, 40-46. (18) Gao, X.; Nie, S. Anal. Chem. 2004, 76, 2406-2410. (19) Gao, X.; Nie, S. J. Phys. Chem. B 2003, 107, 11575-11578. 10.1021/ac0710533 CCC: $37.00
© 2007 American Chemical Society Published on Web 10/11/2007
the infiltrating/swelling solution. The QDs diffuse through the pores of the swelled beads and adsorb onto the hydrophobic pore walls; removal of the swelling solvent contracts the pores and effectively traps the QDs. Nie et al. loaded QDs with a single color (monochromatic) and demonstrated that the photoluminescence of a single bead was linearly proportional to the number of QDs embedded. Nie et al. also loaded beads with three colors and obtained a spectrum in which the peak emission wavelengths of the colors were identical to the values recorded dispersed in an nonaggregated form in a nonpolar solvent. Using the same impregnation procedure, Vincent et al.20 used confocal microscopy to obtain serial sections of the photoluminescence of beads with monochromatic QDs and showed that the depth of impregnation into the bead increases with the chloroform content of the swelling solution. Infiltration incorporation of monochromatic hydrophobically capped QDs in polystyrene microbeads were also undertaken by Nabiev et al.21 and Riegler et al.,22 and for hydrophilically capped, water-soluble QDs into hydrogels by Mohwald et al.23,24 Zhao et al.25 used the infiltration method to incorporate in polystyrene beads hydrophobically capped QDs with two different colors; they demonstrated that by varying the molar ratio of the two QDs in the impregnating solution, the intensities of the two colors in the bead emission spectra can be varied. The process of arranging QDs into a shell around the periphery of a preformed microbead was undertaken by Bawendi et al.26 on silica beads by reacting QDs with a siloxane surface functionalization with silica seed particles in an ethanol dispersion by Sto¨ber growth (see also Hirai et al.27 for thiol binding of QD shells). Ruhl et al.28 obtained a QD shell by first adsorbing poly(vinylpyrrolidone) (PVP) to hydrophobically capped CdSe/ZnS QDs to make them hydrophilic, then adsorbing the PVP-modified QDs directly onto amino-functionalized silica particles, and finally using a Sto¨ber growth to coat the particles with a shell of silica and seal the QDs in the bead. The incorporation of monochromatic QDs in concentric shells around a seed particle was obtained by Rogach et al.29-31 using a layer-by-layer (LBL) assembly technique in which polyelectrolyte layers of alternate charge and polar-capped QDs are sequentially deposited onto a surface and held together by electrostatic interactions. The LBL technique was also used by Su et al.32 to load QDs with two different colors, at different loading ratios. (20) Bradley, M.; Bruno, N.; Vincent, B. Langmuir 2005, 21, 2750-2753. (21) Stsiapura, V.; Sukhanova, A.; Artemyev, M.; Pluot, M.; Cohen, J. H. M.; Baranov, A. V.; Oleinikov, V.; Nabiev, I. Anal. Biochem. 2004, 334, 257265. (22) Riegler, J.; Ehlert, O.; Nann, T. Anal. Bioanal. Chem. 2006, 384, 645-650. (23) Kuang, M.; Wng, D.; Bao, H.; Gao, M.; Mohwald, H.; Jiang, M. Ad. Mater. 2005, 17, 267-270. (24) Gong, Y.; Gao, M.; Wang, D.; Mohwald, H. Chem. Mater. 2005, 17, 26482653. (25) Wang, H.-Q.; Huang, Z.-L.; Liu, T.-C.; Wang, J.-H.; Cao, Y.-C.; Hua, X.-F.; Li, X.-Q.; Zhao, Y.-D. J. Fluoresc. 2007, 17, 133-138. (26) Chan, Y.; Zimmer, J.; Sroh, M.; Steckel, J.; Jain, R.; Bawendi, M. Adv. Mater. 2004, 16, 2092-2096. (27) Hirai, T.; Saito, T.; Komasawa, I. J. Phys. Chem. B 2001, 105, 9711-9714. (28) Graf, C.; Dembski, S.; Hofmann, A.; Ruhl, E. Langmuir 2006, 22, 56045610. (29) Rogach, A.; Susha, A.; Caruso, F.; Sukhorukov, G.; Kornowski, A.; Kershaw, S.; Mohwald, H.; Eychmuller, A.; Weller, H. Adv. Mater. 2000, 12, 333337. (30) Wang, D.; Rogach, A. L.; Caruso, F. Nano Lett.2002, 2, 857-861. (31) Gaponik, N.; Radtchenko, I. L.; Sukhorukov, G. B.; Weller, H.; Rogach, A. L. Adv. Mater. 2002, 14, 879-882. (32) Ma, Q.; Wang, X.; Li, Y.; Shi, Y.; Su, X. Talanta 2007, 72, 1446-1452.
The direct in situ encapsulation of QDs in microbeads during the bead polymerization has utilized two routes. In the first, beads form from nuclei that are dispersed in a continuous phase. Monomer, initiator, and QDs, cap exchanged to make them dispersible in the continuous phase and functionalized to make them reactive to the polymer, are required to transport to the nuclei and become incorporated in the growing bead. Bawendi et al.33 encapsulated monochromatic QDs in polystyrene beads in an ethanol dispersion polymerization. Scanning transmission electron microscopy sections of the beads showed that the QDs were dispersed uniformly, but the bead size had decreased, and the distribution had become broadened relative to the beads formed without the QDs. In addition, nonintegrated QDs were found in the dispersion. Bawendi et al. concluded that the inability to quantitatively load the beads, and the disruption of the size distribution, make this route problematic for bar coding. Monochromatic QDs have also been encapsulated in polystyrene beads during the polymerization step using the micelles of an emulsion as the nucleating centers (emulsion polymerization34,35) and in silica beads36 in a silicate polymerization. A second route is to use suspension polymerization,37 in which monomer, polymerizing initiator, and QDs are first mixed together and dispersed as microdroplets in a continuous phase in which the QDs and monomer are not soluble. Polymerization is then initiated in the droplet phase by activation of the initiator, and the QDs, owing to their insolubility in the continuous phase, remain sequestered in the droplets as the droplets evolve to a polymer bead. This route has been used for the encapsulation of QDs in polystyrene beads by O’Brien et al.38,39 by dispersing nonpolar styrene and initiator in an aqueous phase and loading the QDs in the styrene phase. O’Brien et al. used monochromatic QDs, which were functionalized with vinyl groups to enable polymerization with styrene, and later showed that QDs without a polymerizable ligand can also be incorporated as long as they are dispersible in styrene. The suspension technique was also utilized to encode polystyrene nanobeads40 with QDs by using a miniemulsion procedure41 in which styrene monomer with hydrophobic QDs is dispersed into nanodroplets in an aqueous phase by the addition of surfactant to lower the surface energy of the droplets and by sonication to disperse the monomer to the nanodroplet size (see also Fleischhaker and Zentel42). Transmission electron microscopy sections of these beads showed that the QDs were dispersed throughout the bead interior but also indicated that there was aggregation. The aggregation was attributed to a phase separation between the QDs and the polystyrene as the polymerization proceeded. O’Brien et al.,39 using the suspension technique to fabricate micrometer sized (33) Sheng, W. K. S.; Lee, J.; Kim, S. W.; Jensen, K.; Bawendi, M. G. Langmuir 2006, 22, 3782-3790. (34) Sherman, R. L.; Ford, W. T. Langmuir 2005, 21, 5218-5222. (35) Yang, X.; Zhong, Y. Langmuir 2004, 20, 6071-6073. (36) Rogach, A. L.; Nagesha, D.; Ostrander, J. W.; Giersig, M.; Kotov, N. A. Chem. Mater. 2000, 12, 2676-2685. (37) Dowding, P. J.; Vincent, B. Colloids Surf., A 2000, 161, 259-269. (38) O’Brien, P.; Cummins, S.; Darcy, D.; Ryley, S.; Sutherland, A. Chem, Commun. 2003, 18, 2532-2533. (39) Li, Y.; Liu, E. C. Y.; Pickett, N.; Skabara, P. J.; Cummins, S. S.; Ryley, S.; Sutherland, A. J.; O’Brien, P. J. Mater. Chem. 2005, 15, 1238-1243. (40) Joumaa, N.; Lansalot, M.; Theretz, A.; Elaissari, A.; Sukhanova, A.; Artemyev, M.; Nabiev, I.; Cohen, J. H. M. Langmuir 2006, 22, 1810-1816. (41) Landfester, K. Macromol. Rapid Commun. 2001, 22, 896-936. (42) Fleisschhaker, F.; Zentel, R. Chem. Mater. 2005, 17, 1346-1351.
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particles, have also noted regions within the beads where QDs concentrate, noting that they can occur at defects in the polymer matrix. Gao et al.43 used the suspension polymerization method to form polystyrene beads encapsulating vinyl-capped QDs with two different colors. Particle solvent evaporation/casting techniques are routes to incorporating QDs into polymer microbeads, which are conceptually similar to suspension polymerization methods in the sense that the beads are formed from liquid droplet precursors.44,45 In these techniques, an organic solvent is used to form a solution containing dispersed, hydrophobically capped QDs and dissolved polymer, and droplets of this solution are dispersed in water. The solvent partitions from the droplets into the water during the curing process, forming polymeric particles, which entrap the QDs. Both monochromatic and multicolor encoded beads have been fabricated with this process. In this paper, we use the suspension polymerization method to embed QDs into microbeads. This technique is chosen because it allows the facile, quantitative loading of QDs in the beads, which is a prerequisite for accurate encoding. Our investigation has the following two objectives. The first is to examine the effect, on the spectral code, of electronic interactions among the QDs sequestered in the beads when the QDs are electronically excited by radiation. As we noted above, when a suspension polymerization is used to embed QDs in polymer beads, evidence suggests that the QDs aggregate or phase separate into microdomains as they are rejected from the polymerizing matrix of the beads. In these microdomains, the QDs can conceivably be pushed to within nanometers of one another. Several studies have been undertaken of the effect on the luminescence of electronic interactions of QDs, which are separated by nanometer scales. Particularly relevant are studies of electronic interactions in QD assemblies consisting of evaporated films,46-54 monolayers and multilayers of QDs formed by Langmuir-Blodgett or layer-by-layer deposition,52,55 and QD “molecules” consisting of covalently conjugated or electrostatically cojoined dots.56-58 These studies have shown that electronic (43) Yang, Y.; Wen, Z.; Dong, Y.; Gao, M. Small 2006, 2, 898-901. (44) Xu, C.; Bakker, E. Anal. Chem. 2007, 79, 3716-3723. (45) Yin, W.; Liu, H.; Yates, M. Z.; Du, H.; Jiang, F.; Guo, L.; Krauss, T. D. Chem. Mater. 2007, 19, 2930-2936. (46) Kagan, C. R.; Murray, C. B.; Bawendi, M. G. Phys. Rev. B 1996, 54, 8633. (47) Kagan, C. R.; Murray, C. B.; Nirmal, M.; Bawendi, M. G. Phys. Rev. Lett. 1996, 76, 1517. (48) Micic, O. I.; Jones, K. M.; Cahill, A.; Nozik, A. J. J. Phys. Chem. B 1998, 102, 9791-9796. (49) Artemyev, M. V.; Bibik, A. I.; Gurinovich, L. I.; Gaponenko, S. V.; Woggon, U. Phys. Rev. B 1999, 60, 1504. (50) Micic, O. I.; Ahrenkiel, S. P.; Nozik, A. J. Appl. Phys. Lett. 2001, 78, 40224024. (51) Dollefeld, H.; Weller, H.; Eychmuller, A. J. Phys. Chem. B 2002, 106, 56045608. (52) Crooker, S. A.; Hollingsworth, J. A.; Tretiak, S.; Klimov, V. I. Phys. Rev. Lett. 2002, 89, 186802. (53) Wuister, S. F.; Koole, R.; deMelloDonega, C.; Meijerink, A. J. Phys. Chem. B 2005, 109, 5504-5508. (54) Koole, R.; Liljeroth, P.; deMelloDonega, C.; Vanmaekelbergh, D.; Meijerink, A. J. Am. Chem. Soc. 2006, 128, 10436-10441. (55) Achermann, M.; Petruska, M. A.; Crooker, S. A.; Klimov, V. I. J. Phys. Chem. B 2003, 107, 13782-13787. (56) Franzl, T.; Koktysh, D. S.; Klar, T. A.; Rogach, A. L.; Feldmann, J.; Gaponik, N. Appl. Phys. Lett. 2004, 84, 2904-2906. (57) Osovsky, R.; Shavel, A.; Gaponik, N.; Amirav, L.; Eychmuller, A.; Weller, H.; Lifshitz, E. J. Phys. Chem. B 2005, 109, 20244-20250. (58) Ramadurai, D.; Geerpuram, D.; Alexson, D.; Dutta, M.; Kotov, N.; Tang, Z.; Stroscio, M. Superlattices Microstruct. 2006, 40, 38-44.
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interactions in the form of electronic coupling and exciton energy transfer can affect the QD absorption and emission spectra. In electronic coupling,49,50 electronic excitations delocalize across multiple dots, leading to states described by the superpositions of electron and hole wave functions. These coherent interactions require a high degree of structural order and have been observed as a red shift in the adsorption and emission spectra for very small interdot separations (
