© Copyright 1997 American Chemical Society
APRIL 2, 1997 VOLUME 13, NUMBER 7
Letters Gradient Sensitive Microscopic Probes Prepared by Gold Evaporation and Chemisorption on Latex Spheres H. Takei* and N. Shimizu Advanced Research Laboratory, Hitachi, Ltd., Hatoyama, Saitama 350-03 Japan Received December 16, 1996. In Final Form: February 3, 1997X We describe a simple yet general method for preparing microscopic probes that are gradient sensitive. Gold was first evaporated on one side of latex spheres, which were then chemically modified by a thiol. As one example, by using thiols with ionizable groups, we prepared microscopic spheres with pH dependent electric dipole moments. Under an electric field such spheres reoriented themselves depending on the pH value of the environment. This method can be naturally extended to other thiol-modified molecules such as dyes and antibodies to prepare various probes that are sensitive to the gradient of the physicochemical microenvironment or that are directional in binding.
Introduction Microspheres such as latex spheres are widely used for biochemical purposes. Many forms of immunoassay utilize latex spheres bearing an antibody, and presence of the antigen leads to agglutination of the spheres or change in fluorescence signals. Another application is cell sorting; latex spheres bind to specific type of cells, and paramagnetism or fluorescence of the spheres is used to sort out desired cells. These applications naturally require different spheres, but common in all cases, the surface of spheres is more or less uniformly modified. In this report, however, we describe a simple yet general method for preparing latex spheres that are chemically modified on one side only. Such chemical modifications produce spheres that are sensitive to the gradient of the physicochemical microenvironment or are directional in binding. The method consists of thermal evaporation of gold on latex spheres on one side and subsequent chemisorption of a thiol-modified molecule from a liquid phase (Figure 1). Spheres have been used as a mask for lithography,1,2 but to our knowledge, this is the first report of gold evaporation on spheres with the intent of specifically * To whom correspondence should be addressed. X Abstract published in Advance ACS Abstracts, March 15, 1997. (1) (a) Fischer, U. Ch.; Zingsheim, H. P. J. Vac. Sci. Technol. 1981, 19, 882. (b) Deckman, H. W.; Dunsmuir, J. H. Appl. Phys. Lett. 1982, 41, 377. (c) Hulteen, J. C.; Van Duyne, R. P. J. Vac. Sci. Technol., A 1995, 13 (3), 1553.
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Figure 1. Preparation of gradient sensitive microscopic probes. Gold evaporated on one side of latex spheres was chemically modified with a thiol-modified molecule, X.
modifying the characteristics of spheres themselves by subsequent chemisorption. In our example, we chose thiols with ionizable groups, which resulted in spheres having pH dependent electric dipole moments. These spheres orient themselves under an electric field only in a certain pH range.3 (2) Musil, C. R.; Jeggle, D.; Lehmann, H. W.; Scandella, L.; Gobrecht, J.; Doebeli, M. J. Vac. Sci. Technol., B 1995, 13 (6), 2781. (3) Electrophoretic rotation has been studied previously with a slightly similar system of colloidal doublets consisting of two different spheres. Refer to: Velegol, D.; Anderson, J. L.; Garoff, S. Langmuir 1996, 12, 675.
© 1997 American Chemical Society
1866 Langmuir, Vol. 13, No. 7, 1997
Letters
Experimental Section A 30 µL portion of a latex sphere suspension (Dynospheres from Dyno Particles A.S., Norway, and Fluoresbrite TM Plain Microspheres from Polyscience, Warrington, PA) was centrifuged in a 1.5 mL polypropylene centrifuge microtube at 2000g for 10 s. Supernatant was removed, and spheres were resuspended in 10 µL of ethanol (99.5 vol %, Kanto Chemical Co., Inc., Tokyo, Japan). Spheres were then deposited without overlapping on a 7 × 10 mm polystyrene piece (cut out from the cover of a Falcon tissue culture dish 3002, Becton Dickinson Co., Ltd.) by allowing the suspension to dry. The substrate was placed in the vacuum chamber of an evaporator (Model EBV-6DA, ULVAC Japan, Ltd., Tokyo, Japan), 30 cm away from the source. Under a vacuum of 10-6 Torr, gold was evaporated at a rate of 0.5 Å/s, monitored by a crystal thickness monitor (Model CRTM-5000, ULVAC Japan, Ltd., Tokyo, Japan). A polystyrene substrate was used because evaporated gold adhered well to it even in water, thus later facilitating removal of spheres from the substrate without gold film flaking off. Within 1 min of removal from the evaporation chamber, the substrate with deposited spheres was placed in a 1.5 mL polypropylene centrifuge microtube, and 1.0 mL of a freshly prepared 10 mM thiol solution of 2-aminoethanethiol (2-AET), HSCH2CH2NH2HCl, or thioglycholate (TG), HSCH2COONa (2AET and TG, Kanto Chemical Co., Inc., Tokyo, Japan), was added. Later (24 h) the substrate was carefully removed from the microtube and transferred to another microtube containing ethanol. Sonication (10 s) with a ultrasonic cleaner dislodged spheres from the substrate. After spheres were placed on a fine 5 × 5 mm Cu mesh (1000 mesh copper screen with 16 × 16 µm square openings, Ted Pella, Inc., Redding, CA) and rinsed with water a few times, they were then resuspended in 5 µL of water in a 1.5 mL polypropylene centrifuge microtube. To demonstrate that spheres modified with 2-AET or TG possess pH dependent electric dipole moments, spheres were subjected to an alternating electric field in various buffer solutions. A pair of parallel gold wires with a 3 mm gap were used as electrodes. A function generator (Model HP 3310A, Hewlett-Packard Co.) supplied square waves, 1-10 V (peak to peak), 0.1-1 Hz. The buffers used were as follows: pH 3, 10 mM citric acid or glycine; pH 5, 10 mM acetic acid; pH 7, 10 mM HEPES and phosphate; pH 9, 10 mM boric acid or glycine; pH 11, 10 mM phosphate. For the purpose of facilitating observation of rotation, spheres impregnated with a fluorescence dye (excitation maximum at 458 nm and emission maximum at 540 nm) were used; evaporated gold blocked light sufficiently that rotation of a sphere resulted in variations in fluorescence observed under a fluorescence microscope, Model IMT-2 inverted microscope from Olympus Optical Co. Ltd., Tokyo, Japan, equipped with a IMT2-DMB dichroic mirror.
Results and Discussion Gold adhered only on the side of spheres facing the evaporation source. We have evidence that this holds true for spheres as small as 100 nm (to be published elsewhere). Gold adhered sufficiently strongly to spheres that application of mild ultrasound did not remove evaporated gold. In contrast to the well-documented chemisorption procedures and characterizations of a thiol on planar gold4,5 or a gold nanoparticle,6 there is little published on chemisorption of a thiol on gold evaporated on microscopic latex spheres. Two issues are of concern: does gold evaporated on polystyrene allow chemisorption, and in the presence of an electrostatically charged and exposed polystyrene surface in close vicinity, does a thiol chemisorb selectively to gold surface? These questions required careful characterization of prepared spheres. (4) Whitesides, G. M.; Laibinis, P. E. Langmuir 1990, 6, 87. (5) Ulman. A. An Introduction to Ultrathin Organic Films from Langmuir-Blodgett to Self-Assembly; Academic Press, Inc.: San Diego, CA, 1991. (6) (a) Weisbecker, C. S.; Merritt, M. V.; Whitesides, G. M. Langmuir 1996, 12, 3763. (b) Badia, A.; Gao, W.; Singh, S.; Demers, L.; Cuccia, L.; Reven, L. Langmuir 1996, 12, 1262.
Figure 2. Three 20 µm latex spheres. 100 nm gold colloid particles adhered to the chemically modified surface of spheres.
To make selective chemisorption on a latex sphere visible under electron microscopy, we further modified spheres with another thiol to form an exposed SH group; if the spheres were initially modified by 2-AET, then TG was attached to chemisorbed 2-AET via a peptide bond.7 The exposed SH group was then used to bind 100 nm gold colloid particles,8 thus allowing observation of selective chemisorption with a scanning electron microscope. Figure 2 is an electron micrograph of three 20 µm spheres.9 With the sphere on top, the surface labeled with gold colloid particles is in full view, whereas with the sphere at bottom right, the labeled surface faces away toward the substrate. The third sphere at bottom left displays the boundary between the labeled and none-labeled surfaces. We observed no difference whether spheres were first modified with 2-AET and then TG or the order of modifications was reversed. To eliminate the possibility that gold colloid particles adsorbed to the exposed polystyrene surface of latex spheres rather than on the evaporated gold surface, we prepared a test gold pattern on a flat polystyrene substrate by using a regular array of 5 µm latex spheres as a mask for gold evaporation.10 The substrate displayed quasitriangular regions where gold was evaporated and circular (7) We describe here the procedure for attaching TG to surface bound 2-AET; attaching 2-AET first and then TG was no different. Spheres already modified by 2-AET were placed in a 1.5 mL microtube, to which 100 µL of 10 mM TG and 100 µL of 50 mM carbodiimide solution (1ethyl-3-(3-(dimethylamino)propyl)carbodiimide, Dojindo Laboratories, Kumamoto, Japan) were added. The tube was mounted on a rotary shaker and incubated for 24 h while rotating at 10 rpm. After 1 mL of ethanol was added, spheres were collected under centrifugation at 2000g and placed on a 5 × 5 mm Cu screen. Rinsing consisted of a gentle wash with ethanol. (8) Spheres were placed in a 1.5 mL microtube containing 0.5 mL of a 100 nm gold colloid suspension (Gold Colloid 100 nm electron microscope grade, British BioCell International, Cardiff, England) and an equal volume of ethanol. Spheres and gold colloids were cocentrifuged at 2000g for 5 min and resuspended. This process was repeated a few times, and spheres were rinsed with water on a 5 × 5 mm Cu screen. Refer to the following for an earlier study on adsorption of gold colloid particles on a chemically modified surface: Freeman, R. G.; Grabar, K. C.; Allison, K. J.; Bright, R. M.; Davis, J. A.; Guthrie, A. P.; Hommer, M. B.; Jackson, M. A.; Smith, P. C.; Walter, D. G.; Natan, M. J. Science 1995, 267, 1629. (9) After spheres were dried on a polystyrene substrate, the sample was given a 5 nm coat of Pt with a sputterer (Model E102, Hitachi, Ltd., Tokyo, Japan) and observed with a scanning electron microscope (Model S-800, Hitachi, Ltd., Tokyo, Japan). (10) A latex suspension from Dyno Particles A.S. was simply allowed to dry on a substrate; without further manipulation, spheres tended to form a regular array. Refer to ref 1a for earlier works on formation of a regular array of latex spheres.
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Figure 4. Electrophoretic rotation of a sphere with localized chemisorption of a charged thiol.
Figure 3. Gold patterns formed with a 5 µm latex sphere array: (A, top) control without any chemical modification; (B, bottom) sequential chemical modifications with 2-AET and TG lead to preferential adsorption of 100 nm gold colloid particles to the gold pattern.
regions where polystyrene was exposed. With a procedure similar for spheres, the gold pattern was chemically modified with 2-AET and TG, and 100 nm gold colloid particles were deposited.11 Figure 3 shows electron micrographs of such polystyrene substrates. (A) is a control where gold colloid particles were deposited on a pattern not chemically modified. (B) is a substrate that was doubly modified with 2-AET and TG, thus exposing an SH group. It is clear that the gold colloid particles adsorbed preferentially to the gold surface. Although not shown, depositing gold colloid particles on a gold surface modified with 2-AET or TG alone yielded results similar to (A), showing that gold colloid particles were not attached merely by electrostatic forces. As shown in Figure 4, localized chemisorption of a charged thiol on a charged latex sphere would be expected to result in electrophoretic rotation of the sphere. Figure (11) This procedure required a modified centrifuge tube with a flat bottom; 0.3 g of silicone rubber was placed in a 5 mL centrifuge tube and allowed to solidify while the tube rotated at 10 000 rpm in a swing rotor (Model RPS65T, Hitachi Koki Co., Ltd., Ibaraki, Japan). The polystyrene substrate was placed at the bottom of the centrifuge tube, and 1 mL of 100 nm gold colloid suspension was added. The tube was centrifuged at 15 000 rpm for 15 min, resulting in even deposition of colloid particles on the substrate. The substrate was then rinsed with water gently but thoroughly before observation with the scanning electron microscope.
5 shows spheres modified with 2-AET actually undergoing electrophoretic rotation as the polarity of the electric field was reversed (5-10 V (peak to peak), 0.5 Hz). Sides of spheres coated with gold appeared dark. In the top photograph, therefore, modified surfaces of three spheres were attracted to the right, whereas in bottom photograph, the spheres rotated 180°; simultaneously spheres as a whole were attracted to the left and a fourth sphere appeared at the right edge. Upon application of an electric field, some small bubbles formed on the electrodes in some buffers, but there was apparently no adverse effect on electrophoretic rotation of spheres in terms of either hydrodynamic disturbance from an ionic current or changing the pH value significantly.12 Table 1 summarizes pH dependence of electrophoretic orientation.13 First, control spheres did not orient themselves upon application of an alternating field whereas spheres modified with 2-AET or TG showed pH dependent reorientation. Secondly, spheres modified with 2-AET exhibit strong response at pH 3, 9, and 11, pointing the modified gold surface toward the negative electrode. On the other hand, with spheres modified with TG, it is only at pH 9 and 11 that spheres showed similarly strong responses. Moreover, the modified gold surface was attracted to the negative electrode, rather than to the positive electrode as expected from the negative ionization state of TG. The behavior of 2-AET modified spheres was consistent with an ionized form of the primary amine in the entire pH range we examined.14 On the other hands, behavior of TG modified spheres could not be easily accounted for. A previous study with contact angle titration15 showed that at least for long chain alkanethiols with ionizable carboxyl groups bound to planar gold surface, the extent of ionization changes in the pH range we used. Obviously, (12) Spheres away from the electrodes were observed though spheres did not exhibit peculiar behavior unless in the close vicinity of the electrode (
