NANO LETTERS
Patterned Fluorescent Particles as Nanoprobes for the Investigation of Molecular Interactions
2003 Vol. 3, No. 8 995-1000
Jaehyuck Choi,*,†,‡ Yihua Zhao,‡,§ Deying Zhang,† Shu Chien,§ and Y.-H. Lo† Department of Electrical Engineering and The Whitaker Institute of Biomedical Engineering and Department of Bioengineering, UniVersity of California at San Diego, 9500 Gilman DriVe, La Jolla, California 92093-0407 Received February 23, 2003; Revised Manuscript Received May 14, 2003
ABSTRACT Fluorescent colloidal particles, fabricated by depositing a thin gold film on one side of the particle, show intensity fluctuation (blinking) under an optical microscope because of their rotational Brownian motion. Interactions between molecules immobilized on the surfaces of the particle and the solid substrate restrict the rotation of the particle, thus modulating the intensity fluctuations. Because we can obtain the timedependent rotational angle from the fluorescent intensity, we can use the intensity signals to investigate the weak interactions between unlabeled molecules by analyzing the angular distribution of the particle. To the best of our knowledge, this is the first demonstration of an optical measurement of the rotations of nanoparticles and the first attempt at using such a technique to study molecular interactions.
Fluorescent colloidal particles have found many applications in biosensors1 and fundamental biological research2 because of their ease of fabrication and versatility as a substrate for molecular attachment. So far, most of the studies using these particles have involved the observation of the motions of the particles under a fluorescent microscope or a confocal scanning microscope, and the resolution is limited by the diffraction of light. Laser tweezers overcome the diffraction limit to allow the detection of particle motions as small as a few nanometers.3 However, laser tweezers require a very sophisticated experimental setup, and laser heating through light absorption remains an issue. We propose and demonstrate a new concept of using patterned fluorescent particles as transducers to convert the intermolecular interactions into optical signals. The variations of fluorescent intensity with time seen under the microscope depend only on the rotations of the patterned particles and are independent of their translations. For a particle with a 100-nm diameter and an angular detection resolution of 2° (limited by the noise of the CCD rather than the spatial resolution of the microscope), we can achieve a sensitivity of 1 nm. This technique requires neither laser trapping/excitation nor sophisticated optics but can achieve resolution comparable to that of laser tweezers. * Corresponding author. E-mail:
[email protected]. Phone: (858) 822-2777. † Department of Electrical Engineering. ‡ These authors contributed equally to this work. § The Whitaker Institute of Biomedical Engineering and Department of Bioengineering. 10.1021/nl034106e CCC: $25.00 Published on Web 07/11/2003
© 2003 American Chemical Society
In this paper, we present the first results of using such patterned particles for the study of intermolecular interactions. The fabrication process of patterned particles has been reported4 and is shown schematically in Figure 1a. A monolayer of particles5 was at first spin-coated onto the surface of the solid substrate, followed by the deposition of a thin layer of gold by metal evaporation. The gold thin film was deposited only on one hemisphere of the particle as shown in Figure 1a. The half-coated particles were then detached from the solid substrate by sonication for 2-3 h. Figure 1b shows the SEM photograph of a half-coated polystyrene particle. The particle diameter is 960 nm, and the film thickness is 75 nm. The film thickness can vary from a few to several hundred nanometers by controlling the metal deposition rate and time. The patterned particles emit fluorescent light only from the uncoated hemisphere because the opaque metal coating blocks the light emission from the other half. When such half-coated fluorescent particles are suspended in a liquid medium and examined under a fluorescence microscope, they blink constantly because of the rotational Brownian motions of the particle. The blinking frequency depends on factors such as the particle size, the viscosity, and the temperature of the medium and the interaction between the particles and their surroundings.6 Figure 2 shows the intensity fluctuation of a half-coated particle in water. The intensity was measured from images recorded using an inverted microscope equipped with a CCD
Figure 2. (a) Intensity fluctuation of a half-coated particle in water at room temperature. The polystyrene particle has a diameter of 960 nm and is coated with a 75-nm gold layer on one side. (b) Particle images taken at different times (in seconds).
Figure 1. (a) Schematic description of the fabrication process of the half-coated particle. (b) SEM (scanning electron microscopy) picture of a half-coated particle. The particle is made of polystyrene and has a diameter of 960 nm. The gold film has a thickness of 75 nm.
camera. When the uncoated side of the particle faces down toward the objective lens, the particle appears bright, and a high fluorescence intensity is recorded. Conversely, the particle appears dark and a low intensity is recorded when the coated side of the particle faces down. We used such particles as active probes to investigate the interactions between different kinds of molecules coated on the surfaces of the particle and the solid substrate. Interactions between surface-bound molecules are abundant in biology as well as in chemistry, and such interactions are generally very different from the interactions of the same molecules in solution. There has been a great deal of effort to develop effective and efficient methods to analyze the interactions between surface-bound molecules.7 To demonstrate that the interactions between surface-bound molecules can be transduced in real time into optoelectronic signals through the rotations of the nanoparticles, we had the particles in contact 996
with three differently treated glass surfaces that present, respectively, hydroxyl, thiol, and amino functional groups.8 The intermolecular binding forces slow or stop the linear and rotational Brownian motions of the particles on the substrate surface. Although under the microscope we are not able to resolve the linear motions much better than 1 µm, we can clearly detect the effect of angular motions down to a few nanometers through the intensity variation. Because the polystyrene particle we used is carboxylic-functionalized on its surface, the patterned particle has two different surface functional groups: a carboxylic group on the uncoated side and gold on the coated side. The reason that we chose three differently functionalized glass surfaces is the following: the hydroxyl group does not specifically interact with either the carboxylic group or gold under our experimental conditions.9 Therefore, we expect a relatively unconstrained Brownian rotation of particles on the hydroxyl-presenting glass surface. In contrast, the thiol group is known to have a strong interaction with gold10 but no specific interaction with the carboxylic group.11 Therefore, we can expect a preferred interaction between the gold-coated side of the particle and the thiol-presenting substrate surface. In the third case, the amino group is known to have some binding affinity for gold12 but a much stronger electrostatic interaction with the carboxylic group,13 so we expect a preferred interaction between the uncoated side of the particle and the substrate surface. As shown in Figure 3, the intensity modulations clearly reflect such different interaction characteristics for the three types of surfaces. Figure 3a shows the intensity modulation of a patterned particle on the hydroxyl-presenting glass surface. The magnitude of the maximum intensity fluctuation is about 40 units. The minimum intensity was Nano Lett., Vol. 3, No. 8, 2003
Figure 3. Intensity fluctuations of a patterned fluorescent particle (a) on the hydroxyl-presenting glass surface, (b) on the thiol-presenting glass surface, and (c) on the amino-presenting glass surface in the medium of water at room temperature. The arrow illustrates the amount of rotation of the particle on the glass surface.
obtained when the gold-coated semihemisphere was facing the objective lens with its central axis normal to the substrate (i.e., at an angle of 180°). Conversely, the maximum intensity was obtained when the uncoated semihemisphere was facing the objective lens with its central axis normal to the substrate (i.e., at an angle of 0°). Therefore, an intensity fluctuation of 40 units between the maxima and minima suggests that the nanoparticle went through a rotation of 180° through Brownian motion. Figure 3b shows the intensity fluctuation of a particle on the thiol-presenting glass surface. The maximal intensity fluctuation is about 20 units, and the average intensity is less than 10 units, suggesting that the particle spent most of the time having its gold-coated side in contact with the substrate and facing the microscope objective. Figure 3c, which shows the result of particle rotation on the amino-presenting glass surface, exhibits very different intensity fluctuation characteristics in comparison Nano Lett., Vol. 3, No. 8, 2003
to the previous two cases. A nearly constant intensity at the maximal value of 40 units indicates that the uncoated side of the particle was in contact with the amino-presenting substrate and that the particle was nearly immobilized on the surface with minimum Brownian rotational motions. The above discussion of the intensity and orientation of the particle is rather qualitative. Two approaches can be used to establish a quantitative relation between the intensity and orientation of the particle. The first is to solve the electromagnetic radiation pattern given the properties of the Au film and assuming that the fluorescence is generated from an incoherent array of oscillating dipoles with random polarization states. The radiation within the numerical aperture of the objective lens is proportional to the detected intensity. The second approach is to use the ray-tracing program by assuming a proper reflectivity of the Au film. The total number of emitting rays within the numerical 997
Figure 5. Histogram of the rotational angle of a particle on the hydroxyl-presenting surface (white bars) and the thiol-presenting surface (black bars). Note that the black bars end at about 90°, suggesting that thiol-Au bonding is much preferred over thiolhydroxyl bonding.
Figure 4. (a) Intensity fluctuation of a blinking particle as a function of rotational angle. (b) Rotational angle profile of a particle in the hydroxyl-presenting surface as a function of time. (c) Rotational angle profile of a particle in the thiol-presenting surface as a function of time.
aperture of the lens is proportional to the intensity. For both methods, we need to consider not only the fluorescence emission but also the excitation, which also depends on the relative directions between the excitation light and the orientation of the patterned particle. Because we adopted a confocal configuration in our experiment, the emission light and excitation light follow basically the same path. Although the full-wave electromagnetic analysis is more accurate than the ray-tracing method that neglects diffraction effects, we chose to use the ray-tracing method because the computation is much simpler and the result is quite accurate because of the large numerical aperture used in our experiment. Figure 4a shows the calculated relation between the intensity and the orientation of the particle from the ray-tracing analysis. 998
One of the noticeable characteristics is that the intensity is nearly constant between 0 and 40° and between 140 and 180° because the current setup cannot tell the exact angle but can give an angular range when the intensity readings reach saturation levels at its maximum or minimum.14 We have converted the intensity data on the glass surfaces with hydroxyl and thiol groups shown in Figure 3b and c into angles, as shown in Figure 4b and c, respectively. It should be noted that only angles in the range between 40 and 140° represent the real angles of the particle. When the data reads 140° in Figure 4b and c, the actual angles can be anywhere between 140 and 180°. Similarly, a reading of 40° can be anywhere between 0 and 40°. Also note that the transition point between the gold-hemisphere and the carboxylhemisphere occurs at 90°, perhaps with 5° uncertainty due to the fact that the gold- coated surface may not be perfectly hemispherical. For the study on amino-presenting surface in Figure 3(c), the intensity readings are essentially constant at 40 units. Hence, we are not able to convert the data into precise angles other than concluding that the particle has been oriented between 0 and 40° over the entire period of measurement. Figure 5 shows the histogram of the rotational angle of a particle on the hydroxyl- and thiol-presenting glass surfaces. The two most notable features in Figure 5 are that the slopes for both types of surfaces rise monotonically with the angle and that the slope is steeper for the thiol-presenting surface than for the hydroxyl-presenting surface. The shapes of the histograms (Figure 5) and the data in Figure 4b and c contain rich information about the details of the molecular interactions and deserve further discussions. The factor contributing the most to the characteristic of the angular distribution functions shown in Figure 5 is the relative bonding strength of two sides of the particle surfaces (gold and carboxylic) to specific substrate surfaces (hydroxyl or thiol). Angles larger than 90° represent interactions between gold and the hydroxyl or thiol functional groups, and angles less than 90° represent interactions between the carboxyl and the hydroxyl or thiol functional groups. The fact that a much higher frequency of occurrence was detected between gold and hydroxyl as well as thiol groups suggests Nano Lett., Vol. 3, No. 8, 2003
Figure 6. (a) Intensity fluctuations and (b) rotational profile of a patterned fluorescent particle on the amino-presenting glass surface in the medium of water at room temperature. (i), (ii), and (iii) correspond to pH 3, 7, and 12, respectively.
that gold forms stronger bonds with hydroxyl and thiol groups than carboxyl does. Our finding agrees with the established result that strong bonds form between gold atoms and the thiol group,10 and this provides evidence in support of the effectiveness of our method. However, it has not been as clear from previous studies regarding the relative strength between gold-hydroxyl bonding and carboxyl-hydroxyl bonding. Our study suggests that the former may have greater bonding energy, although gold-hydroxyl bonding appears to be weaker than gold-thiol bonding, judging from the different slopes of the histograms in Figure 5. To confirm further that the experimental setup was not biased with respect to the gold surface, we did the experiment with the amino-presenting substrate where the strongest interaction exists between the carboxyl group and the amine group. The result in Figure 3c indeed shows that the intensity remains almost constant at the maximal (40 units) level, indicating that the amino group was bonded to the carboxyl group but not to the gold surface. This experiment also rules out the possibility of any major contribution due to the gravitational force of gold, which would shift the center of mass of the particle toward the gold side from the geometric center of the particle. Furthermore, the gravitational force due to the gold film is only on the order of 10 fN, which is too small to play a significant role here. To verify further the ability of the technique to characterize molecular interactions, we applied this method to the study Nano Lett., Vol. 3, No. 8, 2003
of the pH dependence of the interaction strength between different functional groups. At pH 7, Figure 6 shows that a strong interaction exists between the amino group and the carboxylic group, yielding a nearly constant maximum signal intensity of 40 units. This is not surprising because the electrostatic interactions between COO- and NH3+ are expected at pH 7, which is greater than the pKa (4.3-5) of the carboxylic group immobilized on the particle surface but less than the pKa (9-10) of the amino group immobilized on the glass substrate. When we reduced the pH value of the solution to 3.0 so that the predominant interaction occured between neutral COOH and NH3+, we observed intensity fluctuations caused by the rolling of the particle, suggesting a weaker interaction between these two groups (Figure 6). When we increased the pH value to 12 so that predominant interaction occured between COO- and neutral NH2, we observed even larger and faster intensity fluctuations (Figure 6). These results clearly indicate that the molecular interaction strength is ordered as COO-/NH3+ (pH 7) > COOH/ NH3+ (pH 3) > COO-/NH2 (pH 12). These experimental results provide another demonstration of how our technique can be used to characterize molecular interactions. To summarize, we have developed a new technique of using patterned fluorescent nanoparticles as a tool for the investigation of intermolecular interactions. This method can detect a very small rotational distance on the order of nanometers through the change in fluorescence intensity without the complicated setup of a laser tweezers system. Studies on the angular distribution of the particle under Brownian forces revealed interesting information on molecular interactions. The patterned fluorescent particles may be used as nanoprobes for the investigation of molecular interactions and as biological and chemical sensors. References (1) Neumann, T.; Knoll, W. AdV. Funct. 2002, 12, 575-586. Han, M.; Nie, S. Nature Biotech. 2001, 19, 631-635. Velev, O. D.; Kaler, E. W. Langmuir 1999, 15, 3693-3698. Mawell, D. J.; Nie, S. J. Am. Chem. Soc. 2002, 124, 9606-9612. Retter, R.; Bakker, E. Anal. Chem. 2002, 74, 5420-5425. (2) Brunchez, M.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 20132016. Chan, W. C. W.; Nie, S. Science 1998, 281, 2016-2018. Kuriyama, S.; Mitoro, A.; Fukui, H. Gene Therapy 2000, 7, 11321136. Florin, E.-L.; Stelzer, H. K. J. Struct. Biol. 1997, 119, 202211 (3) Laser Tweezers in Cell Biology; Sheetz, M. P., Ed.; Methods in Cell Biology; Academic Press: San Diego, CA, 1998; Vol. 55. Ashkin, A. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4853-4860. Paterson, L.; MacDonald, M. P.; Dholakia, K. Science 2001, 292, 912-914. Tsuda, Y.; Yasutake, H.; Yanagida, T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12937-12942. Kellermayer, M. S. Z.; Smith, S. B.; Bustamante, C. Science 1997, 276, 1112-1116. Hirano, K.; Mizuno, A. Appl. Phys. Lett. 2002, 80, 515-517. (4) Love, J. C.; Gates, B. D.; Wolfe, D. B.; Paul, K. E.; Whitesides, G. M. Nano Lett. 2002, 2, 891-894. (5) The particle was purchased from Bangslabs and used directly after dilution with DI water. The particle is made of polystyrene and is carboxylic-functionalized on the surface. The particle has FITC dispersed inside it as a fluorescent dye. (6) Probstein, R. F. Physicochemical Hydrodynamics; Wiley-Interscience: 1994. Raynaud, H.; Strzelecki, L. Phys. ReV. Lett. 1984, 52, 1180-1183. (7) Yang, T.; Simanek, E. E.; Cremer, P. Anal. Chem. 2000, 72, 25872589. Liphardt, Y.; Onoa, B.; Smith, S. B.; Bustamante, C. Science 2001, 292, 733-737. Rief, M.; Gautel, M.; Gaub, H. E. Science 1997, 999
276, 1109-1112. Morjani, H.; Manfait, M. Biospectroscopy 1998, 4, 297-302. (8) The original cover glass we used has Si-OH groups exposed on the surface. We treated the cover glass with a mixture of (3-mercaptopropyl)-trimethoxysilane and acetone (1:9 by volume) overnight to make the thiol-presenting glass surface. Similarly, we used a mixture of 10% 3-(aminopropyl)triethoxysilane and 90% acetone by volume to make the amino-presenting glass surface. After silanization, the glasses were washed with acetone, acetone/ethanol, ethanol, and DI water. Then the glasses were dried overnight. The treated glasses were used within 48 h for this experiment. (9) In this experiment, the particles were suspended in DI water of pH 7. It requires the presence of a strong acid or base catalyst for carboxylic and hydroxyl groups to react and form esters. Usually, heating is required for acid-catalyzed esterification.
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(10) Aizenberg, J.; Black, A. J.; Whitesides, G. M. J. Am. Chem. Soc. 1999, 121, 4500-4509. Lahiri, J.; Ostuni, E.; Whitesides, G. M. Langmuir 1999, 15, 2055-2060. Fleming, M. S.; Walt, D. R. Langmuir 2001, 17, 4836-4843. (11) Zhang, H.; Grim, P. C. M.; Liu, D.; De Schryver, F. C. Langmuir 2002, 18, 1801-1810. (12) Doran, A.; Katz, E.; Willner, I. Langmuir 1995, 11, 1313-1317. Petri, D. F. S.; Schimmel, T. Langmuir 1999, 15, 4520-4523. (13) Gole, A.; Sainkar, S. R.; Sastry, M. Chem. Mater. 2000, 12, 12341239. (14) This ambiguity can be clarified by adjusting the experimental setup. For example, an objective lens with higher NA can be substituted into the optical system.
NL034106E
Nano Lett., Vol. 3, No. 8, 2003
