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Langmuir 1993,9, 1574-1581
Total Internal Reflection Fluorescence with Energy Transfer: A Method for Analyzing IgG Adsorption on Nylon Thin Films Edith S . Grabbet Biotechnology Division, National Institute of Standards and Technology, CARB, 9600 Gudelsky Drive, Rockville, Maryland 20850 Received February 20, 1992. In Final Form: January 6, 1993
Total internal reflectionfluorescencewas used to analyze rabbit immunoglobulin G (IgG)nonspecifically adsorbedon thin nylon f i i . Fluorescenceenergytransfermeasurementswere recorded for two comparative systems: (1) adsorbed fluorescein isothiocyanate(FITC) tagged IgG and tetramethylrhodamine isothiocyanate (TRITC) tagged nylon; (2) FITC labeled IgG and TRITC labeled anti-rabbit IgG (anti-IgG). These experiments were used to examine the nylon/IgG and IgG/solution interfaces, respectively, as a function of the surrounding buffer pH and washing conditions. Results were correlated with IgG mass loading measured by the bicinchoninic acid (BCA) method and anti-IgG binding activity assessed using TRITC-taggedanti-IgG. When washed with pH 6.9 phosphate buffer,the amountof adsorbed IgG increased with the concentrationof IgG used in the adsorption step. Fluorescence data were consistent with an IgG bilayer model, where the upper layer did not interact with the TRITC labeled nylon surface but bound to and exchanged with solution borne anti-IgG. After washing with carbonate buffer at pH 8.7,only a tightly adsorbed, unexchangeable layer of IgG remained on the nylon surface, with mass corresponding to less than a monolayer. In a pH 8.7buffer, no energy transfer took place between adsorbed FITC labeled IgG and the TRITC tagged anti-IgG although anti-IgG did adhere to the nylon, suggesting coadsorption of anti-IgG rather than immunochemicalbinding. When the buffer was replaced by a pH 6.9 phosphate buffer, energy transfer was observed, indicating that binding had occurred. Introduction One of the most significant problems that limit the production of reliable biosensors is the inability to control nonspecific adsorption of proteins to surfaces. Considerable effort has been expended in examining adsorption isotherms of proteins on various polymer and inorganic surfaces.'-13 Adsorption has been recorded using Fourier transform infrared spectroscopy, isotope labeling, ellipsometry, and fluorescence, both in situ and after washing loosely bound proteins from surfaces.= Total internal reflection fluorescence (TIRF) has also been used to study protein adsorption to polymers.lk21 Data often have been fit to monolayer isotherms, although multilayer5have been t Present address: 7400San Francisco Rd.NE, Albuquerque, NM 87109. (1)Andrade, J. D. Surface and Interfacial Aspects of Biomedical Polymers; Plenum Press: New York, 1985;Vol. 2. ( 2 )Arnebrant, T.; Ivareson,B.; Lareson, K.; Lundstrdm, I.; Nylander, T.h o g . Colloid Polym. Sci. 1985,70,62. (3)Soderquiet,M. E.; Walton,A. G. J. Colloid Interface Sci. 1980,75, 386. (4)Young, B. R.; Pitt, W. G.; Cooper, S. L. J. Colloid Interface Sci. 1988,125,226. (5)Mandeniue, C. F.; Ljunggren, L. Biomuteriak 1991,12,369. (6)Wahlmen. M.: Amebrant. T. Trends Biotechnol. 1991.9.201. i7j Orosdan, P.;Blanco, R.; Lu,X.-M.; Yarmush, D.; Karger,'B. L. J. Chromatogr. 1990,500,481. (8) Mura-Galelli, M. J.; Voegel, J. C.; Behr, S.; Bres, E. F.; Schaaf,P. R o c . Natl. Acad. Scr. U.S.A. 1991,88,5667. (9) Andrade, J. D.; Hlady, V. Adu. Polym. Sci. 1986,79,1. (10)LundsMm, I.; Elwing, H. J.Colloid Interface Sci. 1990,136,68. (11)Lundstr6m, I. Rog. Colloid Polymer Sci. 1985,70,76. (12)Nygren, H.; Stenberg, M. Biophys. Chem. 1990,38,67. (13)Nygren, H.; Stenberg, M. Biophys. Chem. 1990,38,77. (14)Robertaon, C.R.; Darst, S. A. In Spectroscopy in the Biomedical Sciences;Gendreau,R. M., Ed.; CRC Press: Boca Raton, FL, 1986;p 175. (15)Horeley,D.; Herron,J.; Hlady,V.; Andrade, J. D. Langmuir 1991, 7 , 218. (16)Darst, S.A,; Robertson,C. R.; Berzofsky, J. A. Biophys. J. 1988, 53,533. (17)Lok, B. K.;Cheng, Y.-L.; Robertson, C. R. J. Colloid Interface Sci. 1983,91,87. (18)Lok, B. K.;Cheng, Y.-L.; Robertson, C. R. J. Colloid Interface Sci. 1989,91,104. (19)Burghardt, T.P. J. Chem. Phys. 1983,78,6913.
observed on metals and with proteins known to form matrices, such as fibrin~gen.~*~p~ Recent studies using microscopy have revealed that adsorbed proteins can form in clusters on surface~.'~J~ While more intricate theories describing irreversible adsorption with protein conformational changes have been de~cribed,~*'-~l methods for directly analyzingthe structures of adsorbed protein layers still need to be developed. Determining protein film structures would allow questions of bioinactivity, adsorption mechanisms, and evenness of coverage to be directly addressed. The purpose of this work was to use the capabilities of TIRF to yield more detailed information about the structure of protein layers formed by adsorption under similar conditions to those used in biosensor construction. To accomplish this, a series of energy transfer experiments was developed to probe both the adsorbate/substrate and the adsorbate/solution interfaces. A model system was developed using a thin nylon 6,6 film as a substrate deposited on a glass slide, which served as the reflection element. Nylon has been used previously as a substrate for immobilized enzyme reactor columns in our group.22 Rabbit immunoglobulin G, labeled with the fluorescence donor fluorescein isothiocyanate (IgG-FITC), was adsorbed onto the nylon from phosphate buffer. Since nylon was known to adsorb protein strongly and since our goal was to look at low levelsof nonspecificallyadsorbed protein, as might be found under limiting conditions in working biosensors, very low concentrations of IgG were used, in the range of 0.01-0.04mg/mL. The nylon film was labeled with the energy acceptor tetramethylrhodamine isothiocyanate (TRITC)in experiments which probed the nylon/ protein interface. This donor/acceptor pair was chosen due the accessibility of commercially labeled proteins and to match of the donor's excitation spectrum with our laser (20) Burghardt, T. P.; Axelrod, D. Biochemistry 1983,22,979. (21)Tilton, R. D.;Gast, A. P.; Robertson, C. R. Biophys. J. 1990,58,
1321. (22)Sundaram, P.V. Ann. N.Y. Acad. Sci. 1988,542,166.
This article not subject to U.S.Copyright. Published 1993 by the American Chemical Society
source. In other experiments designed to evaluate the activity of the adsorbed IgG, anti-rabbit IgG tagged with TRITC was employed as a fluorescence acceptor. Since our goal was to simulate a sensor environment, rather than to measure adsorption isotherms, washing steps were included after every adsorption step. Two buffers were used for washing, pH 6.9 phosphate or pH 8.7 carbonate, since dye binding measurementshad revealed that protein adsorptionwas dependent on the pH ofthe washing buffer. Similar results have been obtained by Bagchi, who monitored adsorption of rabbit IgG on poly(vinylto1uene) as a function of pH using UV absorbance.B These two systems, known to have different adsorbed protein layers, served as a model to test the ability of TIRF experiments to distinguish between them. Experimental Section Rabbit IgG, untagged and FITC conjugate (Sigma Chemicals, St. Louis, MO), was purified as described below. Anti-rabbit IgG (whole molecule),untagged and TRITC conjugate (Sigma), was used without further purification. Pure TRITC label was obtained from MolecularProbes (Eugene,OR). Phosphate buffer (0.05 M), pH 6.9 was prepared from mono- and dibasic sodium phosphate. Carbonate buffer was prepared at concentrations of 0.05 and 0.1 M, with a pH of 8.7. All water used was from an 18-MQ deionization purification system. Purification of IgG-FITC. IgG and IgG-FITC were purified by two-step isoelectric focusing (IEF) on a Rotofor separator (BioRad, Richmond, CA) at constant power (12 W) for 3 to 4 h using 15-30 mg of protein, 3.0 mL of 3-10 ampholyte solution, and 1.0 mL of 5-7 ampholyte solution. Cuts from the separation ) remove were dialyzed (dialysis tubing MWCO 1 2 ~ 1 4 0 0 0 to ampholytes, then tested for purity by analytical IEF on rehydrateable gels. Those cuts passing purity checkswere evaporated to near dryness with a vacuum centrifuge and stored at -20 OC. Adsorptionsolutionswere made fresh by dissolving these samples in 0.05 M phosphate buffer. Protein concentrations were determined before and after every adsorption experiment by UV absorbance at 280 nm. From ratios of absorbances, it was calculated that there was an average of 1.2 FITC tags per IgG molecule.26 Nylon Film Preparation. Thin, clear, hydrophilic nylon films were deposited on glass microscope coverslips in the following manner. Slips were rinsed with ethanol, soaked in hot 33% HNOs, rinsed with water, soaked in hot 50% N&OH and 0.1% H202, rinsed, and dried. To ensure that the nylon f i h would adhere to the glass surfaces, p-nitrophenylchloroformate (NPCF) was used to cross-linkthe glass to the amine end groups on the nylon in the manner of Sundaram." After reaction, the slips were rinsed with acetone and then water. Nylon f i i were spun onto the activated glass from 1.5% nylon 6,6 solutions in formic acid using a spin coater operating at 2500 rpm. This method was a modification of that used by Baier and Zisman.n The films had one edge removed by dissolution in formic acid and for more efficient coupling of light into the coverslip. TRITC labeling of the nylon consisted of pumping a solution of 0.002 mg/mL TRITC in 0.1 M carbonate buffer over the nylon surfaces in the TIRF flow cell for 30 min,followed by r i n s i i with carbonate buffer for 15 min. The amount of TRITC bound to the nylon was quantitated by immersing the whole nylon coated coverslipsin 0.002 mg/mL TRITC solutions in 0.1 M carbonate buffer for 30 min and then rinsing with buffer. After drying, these f i i s were dissolvedwith (23) Bagchi, P.; Birnbaum, S. M. J. Colloid Interface Sci. 1980, 83,
460.
(24) Identification of any commercial product does not imply recommendation or endonrementby the National Institute of Standards and Technology, nor that the material or equipment identified ia n e c e d y the beet available for this purpose. (25) Wells,A. F.; Miller, C. E.; Nadel, M. K. Appl. Microbiol. 1966,14,
271.
(26) Willinmaon, M. L.; Atha, D. H.;Reeder, D. J.; Sundaram, P. V. Anal. Lett. 1989, 22, 803. (27) Baier, R. E.; Zisman, W.A. Macromolecules 1970,3, 462.
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Langmuir, Vol. 9, No. 6, 1993 1575
IgG Adsorption on Nylon Films
TIRF Cell
Fiber Optic Bundle ,
+-lens
I
Cut-on' F i l t e r
I I
I
' I
SLM 4888 Spectrometer
4 A r + Laser
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Figure 1. TIRF experimental configuration.
1.5 mL of formic acid; visible absorbance spectra were recorded and calibrated with standard TRITC solutions in formic acid. Absorbance spectra were used to compute the overlap integral for FBrster distance calculations (see calculations section). Quantitation of Adsorbed IgG. Mass of adsorbed IgG was measured using similar adsorption solution compositions,timing, and washing conditions to those used in fluorescence analyses. A microversion of the bicinchoninic acid (BCA) assay= was employed, which uaea BCA to chelate Cu+formed by the reduction of Cu2+with proteins, causing an absorption of light at 562 nm. Larger area, 2.2 cm2,coverslips coated with nylon were used to generate measurable signals. This method was used because the colorometricreaction product was formed in solution, unlike dye binding assays, and because of ita high sensitivity. BCA working solution was prepared daily from stock solutions according to the method of Smith et al.% IgG-coated slips or aliquots of standard solutions were incubated in 1.50 mL of Ha0 plus 1.50 mL of working solution for 2 h at 60 OC. The absorbance of the samples was compared to a set of solution standards and nyloncoated blank coverslips. Spectroscopic Measurements. The TIRF experimental system used, pictured in Figure 1, consisted of an external argon laser excitation source, operated at 457 nm to minimize direct TRITC excitation, coupled into the coverslip at a fixed angle near the critical angle (approximately 60°) via 60° acute angle Schott SF2 glass prisms (n = 1.648 at 632.8 nm). The reflected The TIRF light had a spacingof approximately2 reflections/". cell consisted of a Lucite block with a flow channel cut into one side with dimensions 1.91 cm long by 0.13 cm wide, for an adsorption area0.24 cm2. Fluorescent light was collected through a 10 mm diameter optical fiber bundle placed normal to the slide and passed into the sample compartment of an existing commercial fluorometer (SLM 4800). The emissionmonochromator was removed for energy transfer experiments so that low excitation power (
