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Evanescent Wave Excited Luminescence from Levitated Quantum Dot Modified Colloids W. Neil Everett,† Richard E. Beckham,‡ Kenith Meissner,§ and Michael A. Bevan*,‡ Department of Mechanical Engineering, Department of Chemical Engineering, and Department of Biomedical Engineering, Texas A&M UniVersity, College Station, Texas 77843 ReceiVed April 6, 2007. In Final Form: June 6, 2007 Evanescent wave excited luminescence of quantum dot modified polystyrene (QDPS) colloids is investigated to measure potential energy profiles of QDPS colloids electrostatically levitated above a planar glass surface. Luminescence is characterized for three different-sized PS colloids modified with three different-sized QDs using confocal microscopy, emission spectra, flow cytometry, and temporal measurements of levitated and deposited colloids. Colloid-surface potential energy profiles constructed from scattering and luminescence intensity data display excellent agreement with each other, theoretical predictions, and independently measured parameters. QDPS luminescence intensity is indirectly confirmed to have an exponential dependence on height similar to conventional colloidal evanescent wave scattering. Our findings indicate that evanescent wave excited QDPS luminescence could enable total internal reflection microscopy measurements of index-matched hard spheres, multiple specific biomolecular interactions via spectral multiplexing, enhanced morphology-dependent resonance modes, and integrated evanescent wave-video-confocal microscopy experiments not possible with scattering.
Introduction Measurement of absorbance, fluorescence, and scattering of macromolecules and colloids in evanescent waves (EW) provides the basis for a variety of techniques for characterizing interfacial chemical and physical phenomena on nanometer dimensions. Examples of such techniques include attenuated total reflection,1 total internal reflection fluorescence (TIRF),2 surface plasmon resonance,3 and total internal reflection microscopy (TIRM).4 When EW methods are combined with optical microscopy and digital imaging methods, it is possible to simultaneously measure spatial and temporal behavior of single particles and particle ensembles near surfaces.5-7 With TIRM,8,9 EW scattering is typically used to monitor Brownian motion of single colloids levitated above planar surfaces, which can be used to quantify potential energy profiles10-16 and hydrodynamic interactions.17-19 * To whom correspondence should be addressed. E-mail: mabevan@ tamu.edu. † Department of Mechanical Engineering. ‡ Department of Chemical Engineering. § Department of Biomedical Engineering. (1) Harrick, N. J. Internal Reflection Spectroscopy; Wiley: New York, 1967. (2) Axelrod, D.; Burghardt, T. P.; Thompson, N. L. Ann. ReV. Biophys. Bioeng. 1984, 13, 247-268. (3) Stenberg, E.; Persson, B.; Roos, H.; Urbaniczky, C. J. Colloid Interface Sci. 1991, 143, 513-526. (4) Prieve, D. C.; Luo, F.; Lanni, F. Faraday Discuss. 1987, 83, 297-307. (5) Brockman, J. M.; Nelson, B. P.; Corn, R. M. Annu. ReV. Phys. Chem. 2000, 51, 41-63. (6) Qu, X.; Wu, D.; Mets, L.; Scherer, N. F. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11298-11303. (7) Wu, H.-J.; Everett, W. N.; Anekal, S. G.; Bevan, M. A. Langmuir 2006, 22, 6826-6836. (8) Walz, J. Y. Curr. Opin. Colloid Interface Sci. 1997, 2, 600-606. (9) Prieve, D. C. AdV. Colloid Interface Sci. 1999, 82, 93-125. (10) Bike, S. G.; Prieve, D. C. Int. J. Multiphase Flow 1990, 16, 727-740. (11) Walz, J. Y.; Prieve, D. C. Langmuir 1992, 8, 3043. (12) Sober, D. L.; Walz, J. Y. Langmuir 1995, 11, 2352-2356. (13) Liebert, R. B.; Prieve, D. C. Biophys. J. 1995, 69, 66-73. (14) Bevan, M. A.; Prieve, D. C. Langmuir 1999, 15, 7925-7936. (15) Bevan, M. A.; Prieve, D. C. Langmuir 2000, 16, 9274-9281. (16) Rudhardt, D.; Bechinger, C.; Leiderer, P. Phys. ReV. Lett. 1998, 81, 13301333. (17) Frej, N. A.; Prieve, D. C. J. Chem. Phys. 1993, 98, 7552-7564. (18) Pagac, E. S.; Tilton, R. D.; Prieve, D. C. Chem. Eng. Commun. 1996, 148, 105.
More recently, TIRM has been combined with video microscopy to track three-dimensional excursions of many colloids near surfaces to measure single and ensemble colloid-surface potentials,20 particle-particle potentials,21 particle interactions with patterns,7 and local protein-macromolecule interactions.22 The utility of TIRM lies in its ability to sensitively resolve colloid-surface interactions on the kT energy scale and nanometer length scales. While kT-scale energy resolution is obtained via a clever experimental design and a statistical mechanical inversion, access to nanometer dimensions with TIRM is due to the properties of colloidal EW scattering. Figure 1a schematically depicts a single colloid in an EW generated via total internal reflection of a laser at a glass-water interface.9 In this configuration, the scattering intensity, I, is exponentially sensitive to the instantaneous particle-surface separation height, h, as23,24
I(h) ) I0 exp(-βh)
(1)
β ) (4π/λ)[(ng sin θ)2 - nw2]1/2
(2)
where I0 is the intensity at colloid-surface contact (h ) 0), and β is the inverse of the EW decay length determined by glass and water refractive indices, ng and nw, and the laser’s incident angle, θ. Although the validity of eqs 1 and 2 has recently been contested for variations of the arrangement in Figure 1a (e.g., adsorbed films, AFM tips, small βa),25-27 the overwhelming majority of TIRM studies have directly or indirectly confirmed the exponential (19) Bevan, M. A.; Prieve, D. C. J. Chem. Phys. 2000, 113, 1228-1236. (20) Wu, H. J.; Bevan, M. A. Langmuir 2005, 21, 1244-1254. (21) Wu, H.-J.; Pangburn, T. O.; Beckham, R. E.; Bevan, M. A. Langmuir 2005, 21, 9879-9888. (22) Everett, W. N.; Wu, H.-J.; Anekal, S. G.; Sue, H.-J.; Bevan, M. A. Biophys. J. 2007, 92, 1005-1013. (23) Chew, H.; Wang, D. S.; Kerker, M. Appl. Opt. 1979, 18, 2679. (24) Prieve, D. C.; Walz, J. Y. Appl. Opt. 1993, 32, 1629-1641. (25) McKee, C. T.; Clark, S. C.; Walz, J. Y.; Ducker, W. A. Langmuir 2005, 21, 5783-5789. (26) Eremina, E.; Grishina, N.; Eremin, Y.; Helden, L.; Wriedt, T. J. Opt. A: Pure Appl. Opt. 2006, 8, 999-1006. (27) Riefler, N.; Eremina, E.; Hertlein, C.; Helden, L.; Eremin, Y.; Wriedt, T.; Bechinger, C. J. Quant. Spectrosc. Radiat. Transfer 2007, 106, 464-474.
10.1021/la701012j CCC: $37.00 © 2007 American Chemical Society Published on Web 07/17/2007
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Figure 1. (left-to-right, top-to-bottom) (a) Schematic of height-dependent scattering (IS) and luminescence (IL) intensities from a levitated QDPS colloid in an EW (not to scale; for accurate scaling, colloid diameter, 2a, relative to the EW thickness (3β-1) should be about (2/3)βa ≈ 15-23). (b) CSLM image of nominal 4, 5, and 6 µm QDPS colloids excited with a 488 nm laser. Artificially colored CCD images of 6 µm QDPS (c) scattering at 488 nm and (d) luminescing at 640 nm with insets showing contour plots of the spatial intensity distribution.
relationship between scattering intensity and height via colloidsurface interaction measurements.4-24 Although TIRF is commonly used to investigate fluorescent macromolecules near interfaces, only a few studies have investigated EW excitation of fluorescent colloids, which have tended to focus on measurements of interfacial particle image velocimetry28-30 rather than colloid-surface potential energy profiles. Reasons for the lack of such studies probably include lower absolute fluorescence intensities compared to scattering and the role of photobleaching in producing undesirable temporal intensity changes in addition to the height dependence in eq 1. Despite these drawbacks, monitoring fluorescence has many benefits including capabilities for (1) spectral multiplexing of multiple specific biomolecular interactions,31,32 (2) measurements in index-matched media that minimize van der Waals interactions,33 (3) observation of enhanced morphology dependent resonance/whispering gallery modes,34,35 (4) lower signal-tonoise ratios by limiting background scattering,36 and (5) avoiding (28) Zettner, C. M.; Yoda, M. Exp. Fluids 2003, 34, 115-121. (29) Pouya, S.; Koochesfahani, M.; Snee, P.; Bawendi, M.; Nocera, D. Exp. Fluids 2005, 39, 784-786. (30) Huang, P.; Guasto, J. S.; Breuer, K. S. J. Fluid Mech. 2006, 566, 447464. (31) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631-635. (32) Chan, W. C.; Maxwell, D. J.; Gao, X.; Bailey, R. E.; Han, M.; Nie, S. Curr. Opin. Biotechnol. 2002, 13, 40-46. (33) Helden, L.; Roth, R.; Koenderink, G. H.; Leiderer, P.; Bechinger, C. Phys. ReV. Lett. 2003, 90, 048301. (34) Liu, C.; Kaiser, T.; Lange, S.; Schweiger, G. Opt. Commun. 1995, 117, 521-531. (35) Snee, P. T.; Chan, Y.; Nocera, D. G.; Bawendi, M. G. AdV. Mater. 2005, 17, 1131-1136. (36) Temple, P. A. Appl. Opt. 1981, 20, 2656-2664.
interparticle optical interference37-39 in integrated evanescent wave-video-confocal microscopy measurements.7,20-22 Here, we monitor EW excited luminescence of quantum dot (QD) modified polystyrene (PS) colloids (QDPS) to measure potential energy profiles of ensembles of QDPS colloids levitated above glass. The rationale for using QDs in the present work is to exploit their superior quantum yield in terms of absolute intensity, their resistance to photobleaching, and the large range of emission wavelengths accessible with a single excitation wavelength. The goal is to determine whether the heightdependent EW excited QDPS luminescence is accurately described by eq 1 to allow quantitative, nanometer-scale measurements of colloid-surface interactions. Potential issues are whether the luminescent shell-nonabsorbing core geometry influences the validity of eq 1 and whether temporal luminescence variations due to the ensemble QD response for each QDPS colloid are significant.40 To investigate the use of QDPS with EW excitation, we first characterize QDPS luminescent properties using confocal scanning laser microscopy (CSLM), emission spectra, and flow cytometry (FC) for three different-sized PS colloids modified with three different-sized QDs. We then compare scattering and luminescence temporal intensity variations for electrostatically levitated and irreversibly deposited QDPS colloids. Finally, we (37) Baumgartl, J.; Bechinger, C. Europhys. Lett. 2005, 71, 487-493. (38) Baumgartl, J.; Arauz-Lara, J. L.; Bechinger, C. Soft Matter 2006, 2, 631635. (39) Ramirez-Saito, A.; Bechinger, C.; Arauz-Lara, J. L. Phys. ReV. E 2006, 74. (40) Chung, I.; Witkoskie, J. B.; Zimmer, J. P.; Cao, J.; Bawendi, M. G. Phys. ReV. B 2007, 75, 045311.
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measure colloid-surface potential energy profiles using both scattering and luminescence intensity measurements. Our results demonstrate that EW excited luminescence of QDPS colloids can be used to obtain quantitative colloid-surface potential energy profiles. This finding should enable the use of similar QDPS colloids in future studies that exploit their unique spectral and optical properties for TIRM measurements of index-matched colloids without particle-surface van der Waals attraction and of multiple specific biological interactions via spectral multiplexing.
(QM-4, Photon Technology Int., NJ). FC (FACSCalibur, BectonDickinson, CA) results were acquired using two band-pass filters (530 ( 15 nm, 585 ( 20 nm) and a high-pass filter (>650 nm). Potential Energy Profiles. Luminescent and scattering intensities were analyzed in an identical fashion to construct potential energy profiles, u(h), which is the standard single and ensemble TIRM analysis described in detail elsewhere.20 Briefly, single colloid height histograms, n(h), are constructed from many independent height observations using eqs 1 and 2, which are related to u(h) via a Boltzmann inversion as
Experimental Section
u(h) - u(hm) n(hm) ) ln kT n(h)
Materials. Coverslips and microscope slides (Corning Inc., Corning, NY) were cleaned with piranha solution (1:3 of H2O2/ H2SO4) and rinsed with deionized water prior to use. Surfactantfree, sulfate-stabilized PS colloids (Interfacial Dynamics Corp., Eugene, OR) had manufacturer-reported diameters of d ) 2a ) 4.00 ( 0.17, 5.2 ( 0.29, and 5.9 ( 0.58 µm and densities of FPS ) 1.055 g/cm3. Trioctylphosphine oxide (TOPO), trioctylphosphine (TOP), cadmium oxide, selenium, hexamethyldisilathiane, dimethyl zinc, sodium chloride, chloroform, sulfuric acid, hydrogen peroxide, methanol, and toluene were purchased from Sigma-Aldrich. Additional purchased chemicals included 1-butanol (Fisher Scientific), 200-proof ethanol (Aaper Alcohol, Shelbyville, KY), and tetradecylphosphonic acid (Alfa Aesar). All chemicals were used as received without further purification. Colloid Preparation. Three sizes of CdSe nanocrystals were synthesized from CdO and Se in TOPO41 and then capped with ZnS.42 Different sizes and emission characteristics were achieved by quenching the reaction at predefined times. QDs were transferred to chloroform following removal of excess TOPO via repeated washing with methanol and hexane (i.e., centrifugation, solvent replacement, redispersion). Three batches of QDs with nominal emission maxima at 540, 590, and 640 nm were used to modify three different sets of PS colloids with nominal sizes of 4, 5, and 6 µm using a variation of a literature protocol.31 After transferring PS colloids from water to 1-butanol via multiple washing steps, QDs in chloroform at 5% by volume were added and shaken for 12 h. Following this step, the mixture was repeatedly washed in 1-butanol to recover QDPS and to remove excess QDs and chloroform media. QDPS colloids were then washed twice in ethanol, transferred to deionized water, and stored at 4 °C. Microscopy and Spectroscopy. Three-dimensional colloid trajectories near glass surfaces were monitored using an integrated EW and video microscopy apparatus described elsewhere.20,21 The present work employs a 150 mW, p-polarized, 488 nm Ar ion laser (Melles Griot, Carlsbad, CA) and 68° dovetail prism for EW generation. Sedimentation cells fabricated from microscope slides, coverslips, and polydimethylsiloxane O-rings were optically coupled to the prism using index matching oil (n ) 1.515). All EW experiments used an Axioplan 2 microscope (Zeiss, Germany) and a 12-bit monochrome CCD camera (ORCA-ER, Hamamatsu, Japan). Highresolution images and movies were obtained using 96 nm/pixel, 1× CCD binning, 4 frames/s, and a 63× (NA ) 1.4) oil-immersion objective, and images for potential energy profile analyses were obtained using 1215 nm/pixel, 8× CCD binning, 43 frames/s, and a 40× (NA ) 0.6) air objective. Luminescent signals excited with a 488 nm laser were filtered with a high-pass filter (>500 nm). Image analysis algorithms written in Fortran were used to integrate colloid scattering and luminescence intensities and track the colloid centers with half-pixel resolution. Potential energy profiles were constructed from 90 000 images. CSLM of QDPS modified colloids was performed using either a Leica TCS SP5 or Zeiss Axiovert 200M through a 100× (NA ) 1.4) oil-immersion objective (Zeiss, Germany) with a 488 nm laser for excitation. Emission spectra for QDs dispersed in chloroform were measured using 400 nm excitation in a spectrofluorometer (41) Peng, Z. A.; Peng, X. G. J. Am. Chem. Soc. 2001, 123, 183-184. (42) Talapin, D. V.; Rogach, A. L.; Kornowski, A.; Haase, M.; Weller, H. Nano Lett. 2001, 1, 207-211.
[ ]
(3)
where k is Boltzmann’s constant, T is absolute temperature, and hm is the most probable height. Ensemble analyses combine height excursions of many colloids in an average height histogram, which is also analyzed using eq 3.20 Because colloids are free to diffuse laterally over the surface, each single colloid-surface u(h) is an average over many surface locations, and the ensemble average u(h) is an average over many surface locations and all colloids. Potential energy profiles are analyzed as the superposition of gravitational and DLVO electrostatic potentials. In the case where such expolinear potentials are referenced to the potential energy minimum (location of hm, where ΣF ) 0), potential energy profiles obtained via eq 3 can be fit using9 u(h) - u(hm) G ) kT kT
{
}
exp[-κ(h - hm)] - 1 + (h - hm) κ
(4)
where G is the buoyant colloid weight given as G ) (4/3)πa3(Fp - Ff)g
(5)
where Fp and Ff are the colloid and fluid densities, g is acceleration due to gravity, and the inverse of the Debye length, κ, is given for a single component, 1:1, univalent electrolytes as κ ) [(2e2CNA)/(kT)]1/2
(6)
where is the dielectric permittivity of water, e is the elemental charge, C is the bulk electrolyte concentration, and NA is Avogadro’s number.
Results and Discussion Quantum Dot Modified Colloids in Evanescent Waves. Figure 1b-d shows images of scattering and luminescence from PS colloids (4.0 µm, 5.2 µm, 5.9 µm) modified with QDs having different emission wavelengths (540 nm, 590 nm, 640 nm) (movies in Supporting Information). Each image includes an inset contour plot showing the spatial distribution of the scattering or luminescence intensity. Figure 1b is a true-color, multichannel CSLM image of all three QDPS that demonstrates the ability to perform spectral multiplexing by identifying different QDPS colloidal probes via their emission wavelengths.7,20-22 Although the present work only explores QD luminescence as a “tag” for colloid size, which can be verified independently via direct visualization, the following results demonstrate how such an approach could be exploited to probe specific biomolecular interactions in a combinatorial fashion. The angular independence of the luminescence intensity from each QDPS colloid in Figure 1b with uniform illumination indicates the homogeneous distribution of the QD coatings and absence of QD aggregates of dimensions discernible by CSLM. The CSLM image in Figure 1b also shows the general uniformity in intensity between different QDPS colloids which could offer a number of potential advantages in ensemble TIRM measurements (e.g., single particle intensities do not have to be
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Figure 2. (left-to-right, top-to-bottom) (a) Emission spectra of three different-sized CdSe-ZnS QDs with luminescence maxima at 542, 590, and 640 nm. (b) FC results for three different QDPS colloids shown in Figure 1b. Color variations within each scatter plot correspond to a histogram based on absolute counts. (c) Scattering (blue) and luminescence (red) intensities for 488 nm excitation of single 6 µm levitated colloids in 1 mM NaCl as a function of time. (d) Scattering (red) and luminescence (blue) intensities from stuck colloids as a function of time. Intensities are normalized by the maximum values for each data set.
renormalized for ensemble analyses).7,20,21 Because the apparent thickness of the bright shell region in the CSLM image is approaching the visible diffraction limit (∼200 nm), it is not possible to accurately determine the radial shell thickness that contains the majority of the QDs, except that it is probably less than ∼100 nm. Figure 1c,d shows images of scattering and luminescence of levitated QDPS colloids in a 488 nm wavelength EW, which are artificially colored 12-bit monochromatic images. In the scattering case, QD luminescence is not filtered, since the scattering dominates luminescence by as much as a factor of 103, whereas in the luminescence case, the scattered light is filtered (>500 nm). Although both scattering and luminescence arise from EWs with the same penetration depth (β-1 ) 88 nm), the resulting intensity distribution is distinctly different in the two images. The measured spatial intensity distributions depend on the microscope objective numerical aperture in terms of the solid angle over which light is collected. When comparing the images in Figure 1, it should be noted that a 100× (NA ) 1.4) objective was used for the CSLM image in Figure 1b and a 63× (NA ) 1.4) objective was used for the EW images in Figure 1c,d. (As a side note, potential energy profiles in Figure 3 were measured using a 40× (NA ) 0.6) objective.) In the case of scattering in Figure 1c, two spots appear at forward and backward scattering positions with significantly
brighter forward scattering. The luminescence in Figure 1d appears to be more distributed over the colloid, although local maxima still occur at the forward and backward scattering positions. Of course, these two signals are not unconnected; the intensity distribution that results from scattering is essentially the excitation source for luminescence, and attenuation in the scattering pattern due to local absorbance will be captured in the luminescence intensity distribution. One advantage of luminescence over scattering is that background scattering of the EW from other particles and surface roughness36 is removed by highpass filtering to produce a lower signal-to-noise ratio and higherresolution measurements of colloid-surface interactions.20,21 Multiple-particle EW luminescence may also be less of a problem than multiple-particle EW scattering after including the net effect of excitation light attenuation, reabsorbance of emitted light, and the isotropic nature of luminescence, although a thorough examination of this topic is well beyond the scope of the present work. Quantum Dot Modified Colloid Luminescence. Before analyzing QDPS luminescence for the purpose of measuring colloid-surface interactions, results in Figure 2 more fully characterize spectral properties and dynamic aspects of QDPS luminescence. Figure 2a shows emission spectra for three different-sized QDs using a 400 nm excitation source (before their addition to PS colloids). Despite the considerable overlap
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among emission spectra, presumably as the result of QD polydispersity, the ensemble luminescence from QDPS colloids (with as many as 104 QDs/colloid40) is clearly distinguishable, as already shown in Figure 1b. Figure 2b shows FC results for the three sets of QDPS colloids, each modified with one of the three batches of QDs characterized in Figure 2a. The two band-pass filters in Figure 2b capture luminescence signatures in three distinct groups. These results show retention of spectral separation from ensemble QD luminescence signals for each of the three batches of QDPS colloids. In addition to the CSLM image in Figure 1b that demonstrates detection of each QDPS population on a separate channel, the FC data in Figure 2b provide statistical information on the distribution and separation of spectral properties for each set of QDPS colloids including all variations introduced during their synthesis and preparation. The results in Figure 2a,b along with the CSLM image in Figure 1b clearly demonstrate that such QDPS colloids could be used in spectral multiplexing applications. Because single QDs and QD ensembles are known to display time-variant luminescence,40,43 Figure 2c reports temporal intensity fluctuations from EW scattering and luminescence of single, levitated QDPS colloids. Regression lines indicate that scattering gradually decreases with time, whereas luminescence gradually increases with time. In both cases, the short-term intensity fluctuations are primarily the result of Brownian excursions of the levitated colloids within the EW.19 In the scattering case, the long-term decrease in the average intensity with time is probably due to drift from several sources including the laser, microscope, and detection system. The gradually increasing slope in the luminescence data is comparable in magnitude to the decreasing slope in the scattering data and could also be attributed to long-term drift. However, luminescent QDPS colloids can display time-dependent intensity fluctuations over a broad range of time scales based on the collective statistical behavior of QD ensembles,40 which cannot be dismissed as a contributing factor to the long-term increase in Figure 2c. Because the intensity variations in Figure 2c are dominated by Brownian excursions of the levitated QDPS, Figure 2d reports time-dependent scattering and luminescence intensity data for QDPS colloids irreversibly deposited on the underlying glass surface. The intensity fluctuations in the scattering case now characterize the cumulative noise inherent to our apparatus, since Brownian excursions no longer contribute to the intensity fluctuations. While scattering intensity fluctuations due to residual thermal motion of deposited colloids cannot be absolutely dismissed (possibly allowed by surface roughness, adsorbed molecules, etc.), such contributions have previously been shown to be minimal compared to system noise.44 The luminescence intensities from deposited QDPS in Figure 2d display small-amplitude fluctuations due to noise that are superimposed on a more obvious non-monotonic, long-term dependence. As noted earlier, the signal-to-noise ratio in the luminescent case is greater than that in the scattering case, since background scattering of the EW is removed via filtering. Luminescence results in Figure 2d show an initial ∼5% intensity increase occurring over ∼100 s, which is followed by a ∼10% decrease over a ∼2000 s period. These variations are not unlike other literature results40 in that an initial short-term increase is observed, followed by a longer decay. However, the time constants associated with these intensity changes in Figure 2d appear to be different from other ensemble QD measurements, which might
result from the glancing illumination of the QDPS colloid bottom surface by the EW (see Figure 1a). Because intensity data are often acquired over ∼2000 s periods in TIRM experiments, the temporal luminescence variations of ∼10% in Figure 2d could adversely affect potential energy profile measurements. However, an inconsistency to be resolved between Figure 2 parts c and d is the gradually increasing luminescence intensity for the levitated colloid in Figure 2c in contrast to the deposited colloid in Figure 2d. One explanation is that free rotation of levitated QDPS colloids affects the residence time of QDs that experience full EW illumination, which could influence their net temporal intensity response compared to irreversibly deposited QDPS colloids that remain fixed. In addition, deposited colloids have ∼10% of their surfaces illuminated by the EW (spherical cap intersecting EW within 4 β-1) compared to ∼2% for levitated colloids in ∼1 mM aqueous media. It is possible that the increasing intensity in Figure 2c is the result of QDs with shorter residence times in the EW effectively all being on the initial part of the time-dependent luminescence curves in Figure 2d. Ultimately, it is not trivial to know exactly the time-dependent ensemble QD luminescence characteristics for levitated QDPS colloids without extensive control experiments and modeling work beyond the scope of this initial investigation. The problem is much more complex than simply including the spherical cap of colloids directly illuminated by the EW, since scattering excites QDs at varying intensities within the entire colloid (see Figure 1c,d).23,24 In addition, the incident EW intensity experienced by QDPS colloids fluctuate in time as a result of Brownian excursions. The average rotational diffusion coefficient of levitated QDPS colloids also depends on the relative frequency at which different heights are sampled above the surface, which in turn depends on the colloid-surface interaction potential.19 With consideration of these complexities and the fact that the long-term luminescence change in Figure 3c could be attributed to drift in our apparatus, we proceed with measurements of potential energy profiles to assess the validity of eq 1 for describing height-dependent EW excited QDPS luminescence. Colloid-Surface Potential Energy Profiles. We analyze EW excited QDPS luminescence in the same manner that heightdependent EW scattering is typically analyzed in TIRM9 to construct potential energy profiles, u(h), and to indirectly test the validity of eq 1. Although it would be desirable to directly measure the height-dependent EW luminescence intensity profile for QDPS colloids, established methods for performing such measurements using deposition on MgF2 films24,45 or AFM cantilever tips25,46 would interfere with QDPS Brownian rotation and dynamic aspects of their luminescence as discussed above. Instead, we simply check whether u(h) are obtained in agreement with theory as evidence that the exponential intensity dependence is retained for the luminescent, levitated QDPS colloids investigated in this work. Figure 3 shows six plots of u(h) obtained from both scattering and luminescence measurements of ensembles of QDPS colloids that were characterized in Figures 1 and 2. Each of the three QDPS batches is measured at two ionic strengths that are sufficiently low so that van der Waals attraction does not contribute to the net u(h). The absence of van der Waals attraction simplifies the analysis of measured potentials, since the effects of roughness, retardation, QD dielectric properties, and so forth do not have to be considered.14 Measurements of u(h) via luminescence were performed immediately prior to scattering
(43) Shimizu, K. T.; Neuhauser, R. G.; Leatherdale, C. A.; Empedocles, S. A.; Woo, W. K.; Bawendi, M. G. Phys. ReV. B 2001, 63, 205316. (44) Bevan, M. A. Ph.D. Dissertation, Carnegie Mellon University, 1999.
(45) Radler, J.; Sackmann, E. J. Phys. II 1993, 3, 727-748. (46) Sarkar, A.; Robertson, R. B.; Fernandez, J. M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 12882-12886.
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Figure 3. (left-to-right, top-to-bottom) Ensemble average colloid-surface potential energy profiles for (0) scattering and (∆) luminescence from QDPS colloids with sizes/emission maxima of (a,b) 4 µm/540 nm, (c,d) 5 µm/590 nm, (e,f) 6 µm/640 nm. Ionic strengths are (a,c,e) 0.1 mM, (b,d) 1.0 mM, and (f) 0.5 mM. Insets in each plot show single colloid and ensemble average potential energy profiles. Ensemble average potential energy profiles in each main plot are fit with eq 4 to give the parameters reported in Table 1.
measurements to minimize temporal luminescent intensity variations that could occur as the result of scattering 488 nm light for 40 min prior to measuring luminescence. Simultaneous measurement of several colloids in each case was used to compare single and ensemble colloid-surface u(h). The results for all cases in Figure 3 are the same for scattering and luminescence measurements. This demonstrates that no obvious difference exists between u(h) constructed from Brownian height excursions of levitated QDPS measured using either scattering or luminescence intensities in eq 1. Fitting the data with the theoretical potential in eqs 4-6 produces values of colloid sizes (eq 5) and Debye lengths (eq 6) in excellent agreement with independent measurements as reported in Table 1. Colloid size is estimated using the manufacturer reported density for PS as 1055 kg/m3, which assumes that a relatively low concentration of QDs within a < 100 nm surface shell (∼1014% of colloid volume) does not significantly alter the net QDPS
colloid density. This assumption appears justified by the agreement between measured and manufacturer reported colloid sizes in Table 1. In addition to verifying eq 1 for describing the height-dependent luminescence of EW excited QDPS colloids, the agreement between experiment and theory in Figure 3 also indicates that QDPS core-shell optical properties do not invalidate EW scattering measurements from such colloids. All single and ensemble u(h) for each case in Figure 3 are also in excellent agreement for both scattering and luminescence measurements. This agreement indicates the absence of significant effects of physical nonuniformities (e.g., colloid size polydispersity, surface charge heterogeneity)7,20,21,47 or optical/spectral nonuniformities (e.g., QD aggregates) that could produce different u(h) for different QDPS colloids. Simultaneous measurements of multiple colloids in each experiment in Figure 3 demonstrate (47) Pangburn, T. O.; Bevan, M. A. J. Chem. Phys. 2005, 123, 174904.
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Table 1. Fitted Parameters Found for Potential Energy Profiles Given in Figure 3 2a/µma C/mMb κ -1/nmc signald numbere κ -1/nmf 2a/µmg
4.0 ( 0.17 µm 0.1 28.9 sca 3 31.2 4.30
5.2 ( 0.29 µm 1.0 9.6
lum 3 30.1 4.34
sca 4 11.2 4.02
0.1 31.2 lum 4 10.7 4.18
sca 3 31.9 5.02
5.9 ( 0.58 µm 1.0 9.7
lum 3 32.5 5.18
sca 3 10.1 5.04
0.1 30.0 lum 4 10.6 4.88
sca 7 28.9 5.68
0.5 13.4 lum 5 30.7 5.72
sca 10 12.4 5.56
lum 10 13.5 5.72
a Manufacturer reported diameters. b Nominal electrolyte concentrations as prepared. c Debye lengths from conductivity measurements. d Signal measured; scattering (sca) or luminescence (lum). e Number of single particles measured to construct each average potential profile. f Debye lengths from curve fits to ensemble profiles in Figure 3 using eq 4. g Diameters from curve fits to ensemble profiles in Figure 3 using eqs 4 and 5.
the robust nature of these measurements in terms of obtaining identical results for different colloids and different surface positions. In contrast, presenting only single colloid results could be misleading by not being representative of average statistical properties. Finally, with knowledge of the QDPS colloid-surface u(h), the extent of EW exposure of QDPS colloids can be more quantitatively estimated, which can be used to revisit the discussion of time-variant luminescence in Figure 2c,d. The average exposure of particles, 〈I〉, can be estimated as
〈I〉 )
∫I(h)n(h) dh/∫n(h) dh
(7)
where eq 1 is used for I(h), and n(h) can be obtained from either the measured histogram or u(h) by inverting eq 3. Equation 7 provides a relative weighting of the EW intensities sampled by levitated colloids having the same size and observation time but with different u(h). For the 4-6 µm QDPS colloids investigated in Figure 3, eq 7 indicates that deposited colloids have an EW exposure ∼40-80 times that for colloids levitated in 0.1 mM media and ∼10 times for colloids in 1 mM media. This ∼10100 factor in average exposure could explain why the timevariant luminescence for deposited QDPS in Figure 2d differs from levitated QDPS in Figure 2c over the same observation period. When allowing for the additional effect of Brownian rotation on QD residence time within the EW for levitated QDPS colloids, it is possible that the time-variant luminescence in Figure 2c may correspond to only the initial “charging up” portion of the temporal response for deposited QDPS colloids in Figure 2d.
Conclusions We have demonstrated quantitative measurements of colloidsurface u(h) by monitoring the EW excited luminescent intensity of QD-modified PS colloids. Agreement of u(h) determined from scattering and luminescence measurements with theoretical predictions and independently measured parameters indirectly confirms an exponential relationship between height and luminescence intensity. Luminescence intensities of levitated QDPS colloids do not display obvious time-dependent behavior, although this is not trivial to confirm directly for levitated colloids.
Time-variant luminescence is observed for irreversibly deposited colloids that might occur as a result of the significantly greater EW exposure compared to levitated colloids. In future studies, care should be taken to check for the relative importance of time-dependent luminescence. From CSLM, spectral, and FC results, QDs are uniformly distributed within each colloid and among all colloids, and QDPS colloids retain the luminescent emission properties of single QDs. On the basis of our findings, EW excited luminescence of QDPS colloids can be used to quantitatively measure nanometerscale colloid-surface interactions. Such luminescence measurements do not appear to offer any disadvantages compared to scattering measurements, beyond an additional synthesis effort. In some cases, luminescence intensity measurements of QDPS colloids in EWs could offer several advantages including lower noise, contrast in index matched media, and spectral multiplexing capabilities. Examples of measurements in interfacial colloidal systems that might exploit QDPS luminescence could include combinatorial measurements of different-colored diffusing probes, each bearing different biomacromolecules,22 or measurements of interfacial self-assembly of index-matched colloids using integrated evanescent wave-video-confocal microscopy measurements.48 Acknowledgment. We acknowledge financial support for this work by the National Science Foundation (CTS-0346473, CTS-0553286) and the Robert A. Welch Foundation (A-1567). We thank Shankarapandian Muthukumar for help synthesizing quantum dots and Roger Smith in the Texas A&M College of Veterinary Medicine and Biomedical Sciences for help with FC. We acknowledge use of the Texas A&M University Materials Characterization Facility. Supporting Information Available: Movie 1 shows scattering at the incident wavelength (488 nm) from three 6 µm QDPS colloids diffusing in an EW (as in Figure 2c). Movie 2 shows luminescence from the same three particles (as in Figure 2d), where the incident wavelength has been blocked with a high-pass filter (>500 nm). Both movies have been artificially colored according to realistic spectral properties. This material is available free of charge via the Internet at http://pubs.acs.org. LA701012J (48) Beckham, R. E.; Bevan, M. A. 2007, submitted.
