Technical Notes Anal. Chem. 1995, 67,3605-3607
Patterning and Regeneration of Surfaces with David J. Pritchard, Hywel Morgan, and Jonathan M. Cooper* Bioelecfronics Research Centre, Department of Electronics & Electrical Engineering, Univetsily of Glasgow, Glasgow, G12 8QQ UK
We describe the application of a new technique for the site-specificimmobilization of six Merent proteinswithin an area of 200 pm x 50 pm. The procedure involves coating the surface with deglycosylated avidin, which binds a photoactivebiotin analogue, so providing a lightaddressable surface onto which proteins can be immobilized. This is the h t time that consecutive patteming of a number of merent proteins has been described, and we demonstrate this with the fabrication of an immunospot for the qualitative visualization of the binding of multiple antigens. We also show that such a structured surface can be readily regenerated and subsequentty repattemed. Over the last decade, there has been great interest in sitespecific deposition of biological molecules and in constructing complex patterns of immobilized biological molecules on surface~.l-~ A central requirement for incorporating different molecules into such structures has been the development of an immobilization method for their selective deposition at designated sites, while preventing their nonspecific binding (NSB) at areas where they are not required. In addition, where patterning of biomolecules exploits their functionality (e.g., molecular recognition through ligand binding), then there has been considerable interest in being able to regenerate the surface.6 Previous methods for patterning proteins have been adapted either from conventional technologies in the printing*,2or in the electronics ind~stries.~ For example, screen printing' and ink-jet deposition2have both been used to place proteins at distinct sites. Alternatively, site-specific immobilization of some proteins has been achieved by using photoresists and lift-off techniques3 Photoactivation and photodeactivation of functionalized surfaces have also been used to select regions where proteins are to be b ~ u n d . ~ To J date, these technique~l-~have suffered from (1) Hart, J. P.; Wring, S. A Electroanalysis 1994,6, 617. (2) Newman, J. D.; Turner, A P. F.; Marrazza, G. Anal. Chim. Acta. 1992, 262, 13. (3) Nakamoto, S.; Ito, N.; Kuriyama, T.; Kimura, J. Sens. Actuators 1988,13, 165. (4) Lowe, C. R; Earley F. G. P. U S . Patent 4562157, 1985. (5) Bhatia, S. IC;Teixeira, J. L.; Anderson, M.; Shriver-Lake, L. C.; Calvert, J. M.; Georger, J. H.; Hickman, J. J.; Dulcey, C. S.; Schoen, P. E.; Ligler, F. S. Anal. Biochem. 1993,208, 197. (6) Boitiew, J. L.; Biron, M. P.; Desmet, G.; Thomas, D. Clin. Chem. 1989, 35,1026.
0003-2700/95/0367-3605$9.00/0 0 1995 American Chemical Society
problems associated with the resolution obtainable, the number of proteins that can be deposited, and/or large amounts of NSB and have not, therefore, proved applicable for use in the formation of complex patterns or in multianalyte sensing for immunodiagnostics. Recently, we described a process that is capable of immobilizing proteins at micrometer-scale resolution with minimal NSB.7 We now demonstrate this technique as a method for the sitespecific attachment of six different proteins (including five functional antibodies) within an area of 200 x 50 pm. We also show, for the first time, that such surfaces can be regenerated and subsequently repatterned. The procedure we use exploits the fact that coating a surface with streptavidin or with deglycosylated forms of avidin will reduce the NSB of protein^.^^^ Such a functionalized surface may be used to specifically bind a photoactive ligand of biotin, called photobiotin, providing a light-addressable surface onto which proteins can be immobilized. This can be achieved since the photobiotin we used contains an aryl azide, which is activated with UV light. As a result, a reactive nitrene is formedg which is capable of binding any protein present in the solution above it. Photoactivation of the surface, through a photolithographic mask, can be repeated sequentially using a number of different proteins, forming high-resolution patterns of proteins. EXPERIMENTAL SECTION
A Si02 wafer was silanized with 2%1,3-(trimethylsilyl)propylethylenediamine in 95% ethanol/5% water for 2 min at room temperature. The wafer was rinsed in 95%ethanol/5%water and heated to 120 "C for 30 min prior to being incubated in 2% glutaraldehyde in phosphate-buffered saline [ 10 mM phosphate buffer, 2.7 mM KCl, and 137 mM NaCl, pH 7.4 (PBS)] for 15 min at room temperature and then in 0.2 mg mL-' Neutravidin (Fierce and Warriner, Cheshire, U.KJ in PBS containing 40 mM sodium cyanoborohydride at 4 "C for 16 h. The wafer was then incubated in 0.2 mg mL-I casein in PBS at room temperature for 1 h and subsequently in 10pg mL-' long-arm photobiotin (Vector, Peter(7) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Angew. Chem., Int. Ed. E q l . 1995,34,91. (8) Hiller, Y.; Gershoni, J. M.; Bayer, E. A; Wilchek, M. Biochem. 1. 1987, 248,167. (9) Smith, P. A. S. In Azides and Nitrenes. Reactivity and Utility; Scriven, E. F. V.,Ed.; Academic Press: London, 1984; pp 95-204.
Analytical Chemisfty, Vol. 67, No. 19, October 1, 1995 3605
A
B
I
Figure i. A series of fluorescence micrographs showing the Sequential patteming of different antibodies on a Si02 surface (see text for details).
borough, UK) in PBS for 20 min in the dark. All reagents, unless othewise stated were from BDH, (Poole, Dorset. UK) and all proteins were from Sigma (Poole. Dorset. UK). Five different antibodies were sequentiallybound to the surface by selective light activation of distinct areas using a 100 W highpressure Hg lamp (irradiance, 9 mW cm-2) and a series of five photolithographicmasks, each with a common registration mark (+) and a different number between 1 and 5 (50 p m x 25 p m in size). Light, A. 2) proteins. The method involves integrating surface chemistries with biological self-assembly in a fashion that is appropriate for the development of a multianalyte sensing device. Figure 2A shows an equal mark-space pattern of goat antirabbit antibody on an avidin-photobiotin-modified Si02 substrate, which has subsequently been “developed” by incubation in the presence of TRITC-labeled rabbit anti-rat. Following treatment with guanidine, no pattern was visible, as shown in Figure 2B. Figure 2C, however, shows that, following a subsequentincubation in a solution of photobiotin, a protein pattern (in this case, immobilized goat anti-rat IgG “developed” with FITC-labeled rat anti-rabbit IgG) can be regenerated on the surface. In this latter case, the mask had been rotated through 90”. From this result, (11) Green, N. M. Adv. Protein Chem. 1975,29, 85. 1321k (12) Heinemann, W. R; Halsall, H. B.A n d . Chem. 1985,57, (13) Cooper, J. M.; Greenough, IC;McNeil, C. J. 1.Elecfroanal. Chem. 1993, 357,267. (14) Volkenshtein, M. S. Biophysics; Mir Publishers: Moscow, 1983; pp 158160.
it is possible to conclude that treatment of the original (’WTC) pattern with guanidine broke the avidin-biotin bond, otherwise repatterning would not have been possible. At low pH in the presence of guanidine hydrochloride (e.g., pH 1.5), there is a full dissociation of the binding complex. At higher pH, the avidin separates into four monomeric subunits and may subsequently unfold depending upon guanidine concentration. The process is, however, further complicated by the number of occupied biotin binding sites.” We have also demonstrated that patterning and repatterning can be achieved on gold surfaces, so providing potential application of the general technique to electrochemical bioassay.12 In this latter work (data not shown), the avidin-modified surface was constructed on a thiol monolayer, formed with N-acetylcysteine and carbodiiide activation.13 The repatterning procedure was carried out, as described above. The fluorophore was situated on the construct suf6ciently distant from the gold surface that quenching of the signal was not a pr0b1em.I~ In conclusion, this is the first paper describing the micrometerscale patterning and repatterning of a number of different antibodies. The technique is applicable to device miniaturization, with potential uses in bioassay development and immunodiagnostics. To this end, we have demonstrated the application of these methods to the fabrication of a sensor element for the visualization of an immunospot for different antigens. ACKNOWLEWMENT This work was supported by ESPRIT as Project 7282 (TOPFIT)
and Biotechnology and Biosciences Research Council GR/H 31967. Received for review April 3, 1995. Accepted June 14,
1995.B AC950324J e. Abstract published in Advance ACS Absfructs, August 1, 1995.
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