Lateral diffusion of bovine serum albumin adsorbed at the solid-liquid interface

Lateral Diffusion of Bovine Serum Albumin Adsorbed at the Solid-Liquid Interface ROBERT D. TILTON, CHANNING R. ROBERTSON, AND ALICE P. GAST 1 Department o f Chemical Engineering, Stanford University, Stanford, California 94305 Received August 3, 1989; accepted October 16, 1989 The lateral mobility of eosin isothiocyanate-labeled bovine serum albumin irreversibly adsorbed to poly ( methylmethacrylate ) ( P M M A ) and poly ( dimethylsiloxane ) ( PDMS ) surfaces from aqueous solution was measured by a combination of total internal reflection fluorescence and fluorescence recovery after pattern photobleaching techniques. Lateral mobility over distances of several micrometers was probed by photobleaching and monitoring fluorescence with the fringe pattern formed by two intersecting coherent laser beams in total internal reflection. The period of the fringes determined the characteristic length for transport on the surface and was varied by changing the angle of intersection. The dependence of the fluorescence recovery time on the period of the fringes indicates that lateral migration of the adsorbed protein on P M M A is by surface diffusion with D = (1.2 + 0.3) X 10 -9 cm2/s, while the extent of fluorescence recovery reveals the coexistence of a mobile population and a population that is apparently immobile over 20 rain. For comparison, D = (2.6 _+ 0.1) × 10 -9 c m 2 / s on PDMS. © 1990 Academic Press,Inc.

INTRODUCTION

Extensive research has revealed much about the macroscopic aspects of protein adsorption at solid-liquid interfaces ( 1), including surface concentrations on various materials (2-8), adsorption reversibility (8-11 ), rates of adsorption and desorption ( 11-13), and relevant transport phenomena (8, 11, 14, 15). The importance of protein adsorption in the realm of biomaterials is well known ( 16, 17), and the surface activity of proteins is likely to significantly affect the behavior of cultured cells in biotechnology ( 18, 19). Technological advances in these fields, and in solid-phase immunoassay and affinity separation techniques, are likely to follow conceptual advances concerning the fundamental nature of the adsorption process. Investigations of these macroscopic issues have revealed the complexity of the protein adsorption process, yet a fundamental understanding of the adsorption l To w h o m all correspondence should be addressed.

mechanism has not been realized. In spite of the value of the considerable protein adsorption data base and the intuition it affords, only a detailed fundamental understanding of the process will take the design of biocompatible, or any protein-contacting, materials beyond empiricism. Tailoring of material properties to afford the most desirable protein surface behavior--even defining desirable behavior-will be facilitated by systematic molecularscale investigations. To date, relatively little information concerning the behavior of adsorbed proteins on a molecular scale is available, and the issues of conformation, orientation, ordering, and lateral mobility present many challenges for continued investigation. Lateral mobility, conformation, orientation, and ordering are likely to be associated in a complex manner. For example, a conformational change after adsorption may alter the lateral mobility of a protein. This, in turn, may alter its ability to interact with its neighbors, thereby affecting the saturation value of surface coverage. The kinetics of reactions cata-

192 0021-9797/90 $3.00 Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.

Journalof Colloidand InterfaceScience, VoL 137,No. I, June 1990

ADSORBED

BSA L A T E R A L D I F F U S I O N

lyzed by surface bound enzymes, or other protein-mediated processes confined to surfaces, may be influenced or perhaps controlled by each of these physical aspects of adsorbed reacting macromolecules. In this work, we report on protein lateral mobility after adsorption. The lateral mobility of adsorbed proteins has not been fully characterized, and much of the evidence in favor of mobility subsequent to adsorption is circumstantial. Since the ability of adsorbed proteins to form organized layers may be determined by their lateral mobility, evidence for the ordering of proteins on the surface (2, 20-22) may be extrapolated to arguments in favor of lateral mobility. On the basis of measurements of the mass of protein adsorbed, protein molecular dimensions, and available surface area, there is speculation that adsorbed proteins can arrange, in some cases, into closest packed monolayers (2, 20, 22), although very little direct information about the ordering of protein surface layers is available. Further, adsorbed proteins may aggregate to form "islands" (21 ). Bimodal "isotherms" (22-24) and shoulders in kinetic plots of protein adsorption (7, 24) are often interpreted in terms of phase changes on the surface. Apparent isotherms of bovine serum albumin (BSA) and -y-globulin on polystyrene latices are bimodal (22). Fair and Jamieson (22) attribute this behavior to a cooperative mode of adsorption, where nucleation and growth of surface crystals occurs above a threshold bulk concentration without any requirement for postadsorption lateral mobility. Without ruling out this mechanism, it is possible that phase changes may occur in a layer formed by random adsorption followed by surface migration. In the first attempt to quantify the lateral mobility of adsorbed proteins, Burghardt and Axelrod (25) estimated the diffusion coefficient of BSA on a quartz surface by fluorescence recovery after photobleaching. It is not clear whether this estimate represents diffusion of directly bound BSA or of proteins loosely associated with the adsorbed layer. The goal of the current work is to determine the lateral

193

mobility of BSA irreversibly adsorbed to a polymeric surface under carefully defined conditions. We report the surface diffusion coefficient and the fraction of mobile eosin isothiocyanate-labeled BSA (EITC-BSA) after adsorption at the poly ( methylmethacrylate)phosphate buffered saline interface (PMMA/ PBS) and the poly(dimethylsiloxane)-phosphate buffered saline interface (PDMS/PBS) measured by a combination of total internal reflection fluorescence (TIRF) and fluorescence recovery after pattern photobleaching (FRAPP).

Fluorescence Recovery after Pattern Photobleaching Variations on the fluorescence photobleaching technique most commonly are employed to investigate slow self-diffusion of proteins or lipids in cell membranes (26-28) or in model membranes (29). The technique has also been applied to dye diffusion in polymeric films (30) and to protein adsorption dynamics (25). The primary requirement for the technique is that the mobile species bear either an intrinsic fluorescent moiety or a tightly bound extrinsic fluorophore. In general, to determine rates of molecular transport, a gradient of fluorescent and nonfluorescent molecules is created with a photobleaching pulse of high intensity laser illumination. The gradient is not one of protein concentration, rather it is one of fluorescent labels; hence, self-diffusion is measurable. Relaxation of the gradient by molecular redistribution is monitored to calculate the diffusion coefficient of the mobile species. When a greatly attenuated laser beam illuminates the previously bleached sample, a recovery of fluorescence intensity may be recorded as the gradient relaxes, and there is a net flux of fluorescent molecules into the illuminated region. Use of periodic patterns for photobleaching allows simple analysis of the fluorescence recovery data. In the current investigation, the mobility of surface-bound EITC-BSA over distances on the order of several micrometers was probed Journal of Colloid and Interface Science, Vol. 137, No. i, June 1990

194

TILTON, ROBERTSON,

by FRAPP. Periodic patterns for photobleaching with characteristic length scales between 3 and 8 ~m were created by the interference of two coherent laser beams in total internal reflection. By intersecting the beams at the point of total internal reflection in a T I R F cell (15), the evanescent wave that probes the adsorbed layer bears the interference fringes. Thus, there is a sinusoidal intensity profile in the evanescent wave. A similar method was employed by McConnell and coworkers to photobleach fluorescently labeled antibodies bound to lipid haptens in model membranes (29). In the current work, both photobleaching and continuous monitoring of fluorescence were performed with the same laser illumination pattern. The fringe period determines the characteristic length for transport in the experiment and is easily varied by changing the angle of intersection according to

x0

2w - - 2n sin 0

[11

where Xo is the wavelength of the light in a vacuum, n is the index of refraction of the solid material, 0 is the half-angle between the incident beams, and w, the characteristic length for transport, is one-half the period of the fringe pattern, corresponding roughly to the width of a band on the surface. Figure 1 illustrates the sinusoidal intensity profile of the evanescent fringe pattern and the distribution of unbleached fluorophores created by photobleaching in this configuration. Assuming that the extent of photobleaching is a linear function of laser intensity (31 ), the fluorophore distribution is sinusoidal, 180 ° out o f phase with the laser fringes. Redistribution of the fluorophores by lateral transport of adsorbed proteins decreases the amplitude of the distribution. By subsequently monitoring fluorescence with the same evanescent fringes, greatly reduced in intensity to avoid further photobleaching, an increase of fluorescence is observed as the concentration of unbleached fluorophores near the fringe inJournal of Colloid andlnterface Science, VoL 137, No. 1, June 1990

AND GAST

FRINGE INTENSITY

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- - -

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.

em~....-.-~ X'

FIG. 1. Laser intensity in the evanescent field varies sinusoidally in the plane of the interface, and photobleaching in this configuration results in a sinusoidal distribution of unbleached fluorophore concentration 180° out of phase with the fringes. Distance x' is normalized by w, one-half the fringe period. Before photobleaching, fluorophores are uniformly distributed at the interface at concentration C, and immediately after photobleaching, the minimum local concentration is Cmi,. As time progresses, the amplitude of the distribution decays until a new uniform concentration of( d + Cmin)/2is reached in the limit of 100% mobile proteins. tensity m a x i m a increases and that near the m i n i m a decreases. The evanescent wave illuminated a 1 × 0.3 m m region of the surface. The fringes spanned the length of this region, and w was always less than 8 #m. Thus, the aspect ratio of the fringes was very large, and only transport in the direction normal to the fringes contributed significantly to the fluorescence recovery. Accordingly, the redistribution of adsorbed proteins was treated as diffusion in one dimension as

OC'

02C '

[2]

Ot' - Ox '2

and the appropriate boundary conditions, initially, C ' ( x ' , O) = ½ sin(Trx')

[3]

while for all t' > 0 at x ' = O, 1, 2 . . . . . n (fringe boundaries), C ' ( x ' , t') = O,

[41

where

C-C c'

-

-

C-

-

Cmin

1 2

.

[51

195

ADSORBED BSA LATERAL DIFFUSION

As indicated in Fig. 1, C is the concentration of adsorbed fluorescent proteins immediately before photobleaching, Cmin is the minimum local concentration immediately after photobleaching, and C is the position- and timedependent concentration after photobleaching. The distance normal to the fringes, x', is scaled on the fringe spacing x~ w, and the time after photobleaching, t', is scaled by the diffusion coefficient and fringe spacing t D / w 2. We allow for two species of adsorbed proteins, a fraction f that is mobile (m) and a fraction ( 1 - f ) that is immobile (im). Thus, Cm --

C i m - - (1 - f ) C Cmin,i m = (1 - f ) C m i n C i m - - (1 - f ) C .

[61

Solution of Eqs. [2] to [4] provides the time-dependent concentration distribution of mobile proteins as C'm(X', t') = ½e-~2t'sin0rx'),

[7]

while the dimensional concentration distribution is C + Cmin C m ( X t, t') = f 2 [8]

Equation [8] describes the concentration distribution of mobile fluorescent proteins, while immobile fluorescent proteins remain in the initial distribution created by the photobleaching pulse. Thus, the dimensional concentration distribution of immobile fluorescent proteins is

-

(1 - f )

C-

2

Cmin

2

× [fe-~2t'sinOrx ') + (1 - f ) s i n O r x ' ) ] . [10] We assume the measured fluorescence intensity to be directly proportional to the product of fluorophore concentration and incident light intensity (30, 31 ). The incident light intensity distribution is 180 ° out of phase with the concentration distribution, [11]

where/t is the total incident intensity, and Ii is the intensity of the laser beam. The fluorescence intensity profiles before, immediately after, and as a function of time after photobleaching ([, I0, and I(t')) are calculated by multiplying this intensity profile by the appropriate concentration distributions of both mobile and immobile fluorescent proteins (C, C( x', 0), and C(x', t')). The measured intensities are the integrals of these products over one full period (2w) of the fringe pattern. Finally, the normalized fluorescence intensity recovery becomes, [ - I(t) _ 1 [-io

Cmi, e_~t,sinOrx,).

C i m ( X t, l') = (1 - - f )

"~- Cmin

It = Ii + Iisin0rx'),

C== fC

2

C(x', t') -

fC

Cmin, m = f C m i n

f C-

while the total concentration distribution of fluorescent molecules, both mobile and immobile, is

f -

f e -~2Dt/w2 + -5

[12] •

The diffusion coefficient, D, and the fraction of mobile proteins, f, are determined by nonlinear least-squares regression of the experimental data. Fluorescence recovery was treated as a diffusive process in this work. Before applying this analysis, it was necessary to confirm the diffusive nature of the process, as is discussed later. APPARATUS

C + Cmin 2 C - Cmin sinOrx'), 2

[9]

Figure 2 illustrates the optical elements of the experimental setup. The 514.5 nm line of a frequency stabilized argon ion laser (Lexel 95-4) first enters a device to select between Journalof Colloidand InterfaceScience,Vol. 137,No. 1,June 1990

196

TILTON, ROBERTSON, AND GAST

LASER

P SF

L

M1

PR~M

FIG. 2. The optical train for total internal reflection

fluorescence recoveryafter pattern photobleaching: S is a shutter device to select between high intensity photobleaching and low intensity fluorescence monitoring beams; P verticallypolarizesthe laser beam; SF is a spatial filter; L is a 700-mm focal length converginglens; 50-50 beamsplitter BS splits the beam into two equal intensity beams; mirror M 1 redirects one of the beams while M2 reflects both beams upward into the prism of a TIRF cell to interfere at the point of total internal reflection.

high and low laser intensity for photobleaching and fluorescence monitoring, respectively. This device is similar to that described by Koppel (32). Exploiting the back reflections of light passing through a flat glass substrate, the beam is split into a high intensity beam and one that is attenuated by four orders of magnitude. The intense and attenuated beams remain parallel to one another until they are recombined by back reflections in a second glass substrate. Colinearity of the beams was ensured by passing the recombined beams through two (removable) pinholes. During fluorescence monitoring, a shutter blocks the intense beam, allowing the attenuated b e a m to enter the T I R F cell. Opening the shutter for a fraction of a second allows the colinear intense b e a m to enter the T I R F cell to photobleach the protein-bound fluorophores. Next in the optical train, the beam is vertically polarized and spatially filtered to remove the diffraction tings that typically degrade the laser beam profile. A 700-mm focal length lens focuses the light on the solid-liquid interface to provide approximately 0.8 M W / m 2 for photobleaching. Before entering the dove prism, the focused b e a m is split by a 50-50 cube beamsplitter. One b e a m passes directly through the beamsplitter while the other is redirected by a mirror, causing the paths of both beams to converge toward a second mirror that reflects them upward into the dove prism. The beams interfere at the point of total internal reflection. The path lengths of the two beams Journal of Colloid and Interface Science, Vol. 137,No. 1, June 1990

differ by only approximately 2%, so that both are focused to essentially the same degree and have the same intensity at the point of reflection/intersection. Beams of equal intensity provide m a x i m u m contrast in the fringes. A long focal length lens was selected to provide an elongated beam waist to further minimize the effect of the unequal path lengths on the degree of focus. To vary w, the beamsplitter and redirecting mirror are adjusted to change the angle of laser beam intersection. The entire apparatus is mounted on a vibration isolation table to eliminate fringe fluctuations. The heart of the apparatus is the T I R F setup, illustrated schematically in Fig. 3. A detailed discussion of T I R F theory and practice may be found in previous publications (8, 15 ). Briefly, the T I R F cell consists of a fused silica (Dynasil 4100 grade) 70 ° dove prism optically coupled by an index matching fluid, cyclohexanol, to a P M M A or PDMS spin-coated microscope slide. The polymer film serves as the adsorption substrate. The microscope slide is held in place by a Teflon flow cell with a slit measuring 63.5 × 12.7 × 0.6 m m that allows

~ O P E

I

PRISM

°wcELL

FIG. 3. Schematic diagram of the TIRF apparatus. A polymer-coated microscope slide is optically coupled to the prism so two equal intensity laser beams can be interfered and totally internally reflected at the solid-liquid interface. Fluorescent light passing through the prism is collected and focused on the cathode ofa thermoelectrically cooled photomultiplier tube. Detection of stray light is minimized by enclosing the prism and detection optics in a darkened chamber, and a sharp cutoff filter blocks scattered light while transmitting fluorescent light. PMT current is converted to a voltage signal by an operational amplifier, monitored by a digitizing oscilloscope, and transferred to a computer for data acquisition.

ADSORBED BSA LATERAL DIFFUSION

solutions to contact the polymer film in fully developed laminar flow. The flow cell may be oriented with the flow direction perpendicular or parallel to the long dimension of the fringes, allowing us to determine the importance of bulk flow in the transport of adsorbed proteins. Two laser beams enter the prism from below and intersect at the point of total internal reflection at the film-solution interface. Fluorescence excited by the evanescent wave is focused onto the cooled ( - 1 5 ° C ) cathode of a photomultiplier tube (Hamamatsu R1477), while scattered incident light is blocked by a glass sharp cutoff filter (Schott OG550). The photomultiplier current is converted to a voltage signal (Pacific Digital Photometer 124), monitored with a digitizing oscilloscope (Hewlett-Packard 54200A), and stored by a computer (Hewlett-Packard 310). For alignment, the instrument is designed to accommodate a microscope. The Teflon flow cell is replaced by a Teflon m o u n t that holds a slide in place against the prism and allows the microscope to focus on the slide surface. A dry fluorescent film is coated on this slide by evaporating a solution of EITCBSA. This slide is placed in the TIRF cell, and the beams are directed to intersect at the point of reflection. The instrument is properly aligned when fringes are seen in focus through the microscope. Since the emission from the fluorescent film is red-shifted relative to the laser excitation wavelength, fringes formed a t the surface will appear yellow. If the fringes were to form at some other point, inside the prism for example, they would appear green. Wearing laser safety glasses, we directly measure w with a reticle in the microscope eyepiece. This value is more reliable than that calculated from an estimate of the angle of intersection in our apparatus. Once alignment is complete, the flow cell and a fresh polymercoated slide are placed in the TIRF cell for the experiment. With the microscope, we verified that the position of the fringes is stable over time and that this position is identical for both the photobleaching and attenuated laser beams.

197

MATERIALS AND METHODS

Fluorescently Labeled BSA Solutions Bovine serum albumin (essentially fatty acid-free, Sigma A-7511 ) was covalently labeled with eosin-5-isothiocyanate (Molecular Probes) at lysine residues (33). Protein conjugates with different EITC/BSA labeling ratios were prepared by allowing stoichiometric quantities of BSA and EITC to react to completion in pH 9 sodium borate solution. The reaction mixture was passed through a BioGel P6 (Bio-Rad) size exclusion chromatography column to separate trace amounts of unreacted EITC from the EITC-BSA solution. Labeling ratios and stock protein solution concentrations were verified spectrophotometrically (Hewlett-Packard 8452A). The EITC-BSA conjugate has an absorption maximum at 528 nm, providing a good match for excitation with the 514.5 nm argon ion laser line. Fresh EITC-BSA solutions with an average of 2 EITC moieties per BSA molecule were used for all but two experiments when EITC/BSA equaled 1.0 and 1.2. All protein solutions were made in phosphate buffered saline (PBS), pH 7.4, [phosphate] = 10 m M , [NaC1] = 150 r a M , prepared with Milli-Q filtered, deionized water. PBS was degassed by a helium purge before addition of protein. In order to qualify as an appropriate system for study by the FRAPP technique, photobleaching of the fluorophore must be irreversible on the time scale of the experiment. Typically, this requirement is assumed to be satisfied (31, 32). We monitored dissolved EITC-BSA absorbance at 514 n m before and after photobleaching and found no reversibility over a 1 h period, an appropriate time scale for a study of slow surface diffusion.

Polymer Surfaces PMMA films were spin-coated from toluene solutions and PDMS films from heptane solutions on cleaned microscope slides as described in a previous publication ( 11 ). PMMA is a slightly hydrophobic, uncharged polymer with weak hydrogen bonding capability, and Journal of Colloid and Interface Science, Vol. 137, No, 1, June 1990

198

TILTON, ROBERTSON, AND GAST

it is one of the few investigated materials having a sufficiently low affinity for BSA so that kinetically limited adsorption can be measured (11 ). PDMS is a hydrophobic, uncharged polymer with no hydrogen bonding capability.

FRAPP Protocol All F R A P P experiments were conducted with layers of EITC-BSA adsorbed from 50 /~g/ml solutions ( 10/ag/ml for PDMS) flowing at a wall shear rate of 102 s -1 at 37°C. The flow path is shown in Fig. 4. In all but two of the P M M A experiments, solutions were flowing in the direction normal to the fringes, i.e., in the direction of measured protein migration. Mobility after 1 h adsorption time was measured in the following manner: First, deTO RECYCLE [.[~-]

~

-~.-. . . . . .

TIRF CELL

HEAT EXCHANGER TO

WASTE

PULSE DAMPENER

WATER BATH FIG. 4. PBS or protein solutions are continuously pumped through the T I R F ceK Labeled (P1) or unlabeled

(P2) protein solutions can be directed to the pump without an interruption of flow with valve 1. Similarly, valve 2 passes either PBS or the protein solution to the pump.

Valve 3 allowssolutionsto be divertedto a wastecontainer while the system is flushed and bubbles are purged from the Teflontubing systemprior to an experiment. Solutions are held at 37°C in a water bath, and a circulating water bath maintains the TIRF cell at 37°C. This second bath also provides the heating in a shell-and-tube solution preheater at the entrance of the TIRF cell. At steady state, solutions are recycledto the feed containers. Journal of Colloid and Interface Science,

Vol. 137,No. 1, June 1990

gassed PBS flowed through the T I R F cell for 30 min to allow the cell to reach thermal equilibrium and to eliminate protein-denaturing air-liquid interfaces that would alter the adsorption process. Next, flowing EITC-BSA solution was introduced to the T I R F cell. Forty minutes later, the flowing EITC-BSA solution was replaced without interruption of flow by unlabeled BSA solution at the same concentration and shear rate. This step has two goals. Exchange of any loosely associated, reversibly bound EITC-BSA for the nonfluorescent BSA assures that only the mobility of irreversibly adsorbed proteins is measured. Replacement of dissolved EITC-BSA by BSA removes complications arising from fluorescence recovery due to simultaneous molecular mobility and adsorption/exchange of fluorescent EITCBSA. Without this exchange step, a fluorescence recovery due only to exchange or adsorption of dissolved EITC-BSA could be falsely interpreted as a result of surface diffusion. Since postphotobleaching exchange of adsorbed unbleached EITC-BSA for dissolved unlabeled BSA would result in a loss of fluorescence and a lower estimate of D, it is necessary to either correct the fluorescence recovery data for this exchange process or to photobleach only after the exchange process has become immeasurably slow. Twenty minutes were allowed for the fluorescence intensity to reach a new steady level after switching protein solutions, before photobleaching the adsorbed molecules and recording the recovery. By waiting for the fluorescence intensity to reach a steady value, we eliminated the need to correct the fluorescence recovery data, as exchange had become negligibly slow over the time scale of the measurement. This was verified by independent experiments to monitor the slow exchange of unlabeled BSA for adsorbed labeled BSA on PMMA. The combination of T I R F and FRAPP was particularly advantageous, in that it allowed continuous monitoring of the adsorption process to assure reproducibility of the starting conditions for the F R A P P measurement.

199

ADSORBED BSA L A T E R A L DIFFUSION

The same physical conditions and protocol were maintained for the experiments with EITC-BSA adsorbed to PDMS in order to validate the technique and to allow comparison of two systems. During the PDMS experiments, flow was in the direction normal to the direction of measured migration.

Determination of Surface Coverage

2:

The concentration of adsorbed EITC-BSA molecules with the same adsorption time and physical conditions as for the F R A P P experiments was determined by the measurement of surface radioactivity of adsorbed tritiated BSA by a method described previously (7). The results are shown in Fig. 5. Surface concentrations of BSA adsorbed to PDMS have been reported (8). RESULTS

We have quantified EITC-BSA mobility after adsorption from 50 tzg/ml solutions at the P M M A / P B S interface for 1 h, at a surface concentration of0.16 ~zg/cm 2. Figure 6 shows that approximately 50% of the fluorescence signal is eliminated by photobleaching, followed by an exponential increase in intensity. It is important not to overbleach the adsorbed layer, or the assumption of a sinusoidal initial 1.0 0.25 0.8 0.20

% 0

g

o.e

"8

0.10

0.4

,~

0.05

0.2

0.15

eo

i

lo-2

iiiirJJI

,

lo,

, iifllfl

i

iiiii.]

1

,

lO

illll.I

k illll

lO2

g

';3

Co, p.g/ml

FIG. 5. Surface coverages of EITC-BSA on P M M A were

obtained by the surface radioactivitytechnique with area fractions calculated assuming side-on adsorption of BSA molecules with ellipsoidal dimensions of 140 × 38 × 38 A (22).

I 0

, ~ ~ ~ I ~~ 20

~ ,I 40

, ~ ~ ~ [ ~ ~ ~ ~ I ~ ~ ~ , I ~ ~ ~ ~ 60 time,

80

100

120

min

FIG. 6. After 30 min equilibration time with PBS flowing through the TIRF cell, EITC-BSA adsorption to the polymer surface was started. Forty minutes later, EITC-BSA was replaced by unlabeled BSA. A new steady intensity (D had been achieved by 60 m i n after the start of adsorption when the fluorophores of irreversibly adsorbed EITCBSA were photobleached, resulting in a rapid decrease in

fluorescence intensity (to Io) followedby an exponential recovery. concentration distribution for fluorescence recovery fails. Since our protocol assured that there would be no source of fluorescent molecules other than the layer of nonexchangeable EITC-BSA, the observed fluorescence recovery was attributed entirely to the mobility of irreversibly adsorbed EITC-BSA, and the diffusion coefficient and fractional mobility were determined unambiguously. The same data are plotted in Fig. 7 in terms of the dimensionless fluorescence recovery expression (Eq. [12]) along with the nonlinear regression curve. The characteristic recovery time, r, will scale with w 2 for a diffusive process, and with w if a convective process dominates. In order to verify the diffusive nature of the lateral redistribution of EITC-BSA, we varied w, the characteristic length in the experiments, from 3.3 to 7.7 ~zm by changing the angle of intersection of the interfering beams. Linear dependence of r on w 2 is shown in Fig. 8. The average diffusion coefficient for these experiments is D = ( 1.2 _+ 0.3) × 10 .9 cm2/s and the fractional mobility is f = 0.37 + 0.05 (valJournal of Colloid and Interface Science, Vol. 137,No. 1, June 1990

200

TILTON, ROBERTSON, AND GAST 1.00'

0.95

T-I T- I0 0.90

0.85 •

•

•

° •

°°

•

0.80

0

100

200

300

time after photobleaching, sec

FIG. 7. The exponential fluorescencerecoverydata from Fig. 6 are plotted in dimensionless form accordingto Eq. [12], with time zero the instant of photobteaching. This form represents the decayingamplitude of the fluorophore concentration distribution as proteins redistribute on the surface. The solid curve is Eq. [12] with f = 0.42 and D = 1.1 X 10 -9 c m 2 / s determined by least-squares regression of the data. ues + 1 standard deviation). The fractional mobility does not depend on w. The slope of the line in Fig. 8 equals 1/7c2D, corresponding to D = 1.1 X 10 -9 cm2/s. The second order dependence of r on w, along with the quantitative consistency of this slope and the average diffusion coefficient, confirms that lateral transport of adsorbed proteins occurs by surface diffusion. To verify this finding in another manner, we compared the lateral mobilities measured in directions parallel and perpendicular to the flow. Since migration is measured in the direction normal to the fringes, the role of convection is isolated by simply orienting the flow parallel to the fringes in one experiment and normal to the fringes in another. If there were a convective contribution to transport of adsorbed EITC-BSA, a fluorescence recovery measured with the fringes normal to the flow should be completed more rapidly than a recovery measured with flow parallel to the fringes. In fact, there was no such dependence on flow orientation. The diffusion coefficient from two experiments with migration measured normal to the flow (fringes parallel to the flow), D = 1.3 X 10 -9 cm2/s, was well Journal of Colloid and Interface Science, Vol. 137, No. 1, June 1990

within the limits of experimental uncertainty of the reported average diffusion coefficient. Having identified surface diffusion as the mechanism of migration, we sought to determine whether this process was a manifestation of the dynamics of adsorbed proteins alone or whether the transfer of kinetic energy by interactions with highly mobile dissolved proteins was required. Two experiments were conducted according to the protocol described earlier, except that after 40 min adsorption time, unlabeled BSA was introduced to the flow cell to displace dissolved and reversibly bound EITC-BSA for only 10 min. After 10 min the fluorescence signal had reached a new steady value, and the BSA solution was then replaced by flowing protein-free PBS. The adsorbed EITC-BSA layer was photobleached after 60 min total adsorption time, as usual. In the absence of any dissolved proteins, lateral diffusion with D = 1.1 X 10 -9 c m 2 / s was measured. Again, within experimental uncertainty, this is indistinguishable from the average diffusion coefficient and indicates that surface diffusion is a property of the adsorbed layer itself, independent of interactions with dissolved proteins. When adsorbed to PDMS surfaces at a concentration of 0.14/zg / cm 2 (8), EITC-BSA exhibits a smaller fraction of mobile proteins, f 100

75

v

25

20

40

60

w 2 (l~m 2)

FIG.8. Fluorescencerecoverytimes scalewith the square of the fringe half-period, consistentwith a diffusivemechanism of lateral mobility. The slope of the line predicts D = 1.1 X 10 -9 c m 2 / s . / x : EITC/BSA = 2.0, q): EITC/BSA = 1.2, X: EITC/BSA = 1.0.

ADSORBED

BSA L A T E R A L D I F F U S I O N

= 0.28 + 0.02, but a larger diffusion coefficient, D = (2.6 + 0.1) × 10 9 cm2/s, than when adsorbed to PMMA at a similar surface coverage. DISCUSSION

By eliminating processes other than adsorbed protein redistribution, we determined the diffusion coefficient and fractional mobility of irreversibly adsorbed EITC-BSA at the P M M A / P B S and PDMS/PBS interfaces in an unambiguous fashion with no additional parameters for curve fitting. The mobile fraction, f , could be verified independently by an examination of the long-time asymptote of the fluorescence recovery: f=

311

-

f - iI(t--~ oo)1 • ---T0

[131

A concern that must be addressed in TIRF experiments with extrinsic fluorescent labels is whether the label affects the behavior of adsorbing proteins. Fluorescei~ isothiocyanate labels do not affect the adsorbed amount of BSA on PDMS surfaces (8). Since EITC is a structural derivative of FITC, it also should not affect the adsorption process. Two FRAPP experiments with EITC/BSA = 1.0 and 1.2 were performed to investigate the effect of labeling on lateral mobility. It is apparent in Fig. 8 that the diffusion coefficients for these conjugates are indistinguishable from that of the doubly labeled protein. The fractional mobilities were also indistinguishable. Eosin labeling does not appear to alter the lateral mobility of adsorbed BSA. Generation of singlet oxygen during the photobleaching pulse can cause oxidative cross-linking of fluorescently labeled proteins, but there is evidence that this process is not significant under the conditions of typical FRAPP experiments ( 34, 35 ). The likelihood of protein cross-linking in the present work was further reduced both by the reduction of the dissolved oxygen concentration via the helium purge and by the convective transport of photobleaching by-products away from the il-

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luminated surface region. The absence of photo-induced artifacts was confirmed by performing repeated photobleachings of the same surface region in four of the experiments. The average ratio of the diffusion coefficients measured after the second and first photobleachings, was 1.1, with values both greater and less than unity. The second order dependence of the fluorescence recovery time on the characteristic length for transport confirmed that diffusion, not convection, is reponsible for EITC-BSA lateral mobility on the PMMA surface. It is possible to envision a mechanism for fluorescence recovery due to rotational motion of laterally restricted molecules (36). If photobleaching were sufficiently rapid such that only fluorophores with their transition dipoles aligned with the electric vector of the excitation light were bleached, a fluorescence recovery would be observed as rotation of the adsorbed proteins or of the eosin labels themselves caused their transition dipoles to align with the electric vector, become excited, and fluoresce. This recovery could take place without lateral mobility of adsorbed proteins. In contrast with our results, however, rotational fluorescence recovery times would be independent of the length scale in the experiment. The second order dependence of r on w rules out contributions to the fluorescence recoveries from rotational motions of otherwise stationary molecules. The diffusion coefficient is a measure of the dynamics of adsorbed BSA molecules, while the fractional mobility provides insight into the distribution of dynamical states. Fractional mobilities less than unity indicate nonuniformity of the adsorption states of EITC-BSA. As f = 0.37 + 0.05, different populations of EITC-BSA, characterized by different mobilities, prevail on the PMMA surface. Since, in all FRAPP measurements with various values of w, the fluorescence intensity did not increase beyond the initial exponential recovery over a 15 to 20 min period, the "immobile" adsorbed proteins must have a diffusion coefficient no greater than approximately 1 0 -11

D2/D1,

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TILTON,

ROBERTSON,

cm2/s. This nonuniform lateral mobility is consistent with the coexistence of tightly packed adsorbed protein aggregates and isolated adsorbed proteins. Self-diffusion is hindered by lateral interactions (37), so proteins in aggregates may be less mobile than isolated molecules, perhaps appearing immobile over the course of these experiments. It is interesting to note in Fig. 5 that coverages in excess of the 0.55 area fraction random parking limit are possible for EITC-BSA adsorbed to PMMA. This is consistent with the notion of a coexistence ofpr0tein aggregates and isolated proteins, and some degree of aggregation may be present in the 0.6 area fraction protein layer studied in this work. While a distribution of lateral mobilities may be a consequence of an ordering phenomenon, lateral mobility itself may be a prerequisite for the formation of such ordered arrangements. This nonuniformity may also be due to a distribution of BSA conformational states. We have detected mobility of irreversibly bound BSA, and not of BSA molecules only loosely associated with the adsorbed layer. We note that exchange of adsorbed proteins is typically a very slow process that continues over days (8, 11 ), and the term "irreversible" in this work refers only to the time scale of the fluorescence recovery. During this time, the surface concentration is constant within measurable limits, and adsorbed proteins are tightly bound. The irreversibility of adsorption may render the detection of lateral mobility somewhat surprising. C o m m o n observations of irreversibly adsorbed proteins and the observation that the rate-determining step in exchange of adsorbed and dissolved proteins is the slow desorption of the protein ( 11 ) indicate that the interaction between a protein and the surface is very strong. This interaction is likely to be the sum of many weak "bonds" between parts of the protein molecule and the surface, each having an adsorption energy on the order of k T (38 ). We propose a model of surface diffusion that is consistent with this construct of an adsorbed protein and does not require interactions with dissolved proteins. At Journal of Colloid and Interface Science, Vol. 137, No. 1, June 1990

AND

GAST

any time, some fraction of t h e weak bonds between different protein segments and the surface may be broken, while a large number remain intact. The protein remains adsorbed. Since protein molecules are dynamic, flexible structures, surface migration may occur as some segments of the molecule break free from the surface, flex, and reattach close to the original site. The net result is random lateral motion of the entire molecule by a sum of tiny steps. BSA adsorption to the hydrophobic PDMS surface occurs at a higher rate than adsorption to the less hydrophobic PMMA surface ( 11 ), suggesting a greater affinity for the protein. A surface with a greater affinity for a protein might be expected to exhibit a smaller diffusion coefficient due to the tight binding of the protein to that surface. We find, however, that the diffusion coefficient of EITC-BSA adsorbed to PDMS exceeds that of the protein adsorbed to PMMA at a similar surface coverage. Perhaps the formation of hydrogen bonds after BSA has adsorbed to PMMA render the binding of BSA to PMMA stronger than the binding to PDMS, while the relatively long-range effect of the hydrophobic attraction accounts for the greater rate of adsorption to the more hydrophobic, but nonhydrogen bonding PDMS. The hydrogen bonding capability of PMMA may result in an inhibition of the detachment of protein segments from their surface binding sites, the measurable manifestation of this inhibition being a reduced diffusion coefficient. CONCLUSIONS

We present the lateral mobility of proteins irreversibly bound to the surface, free from the complicating effects of dissolved fluorescent protein molecules, and verify the existence of post adsorption self-diffusion of proteins. The mobile population of EITC-BSA irreversibly adsorbed at the P M M A / P B S interface for 1 h at a surface coverage of 0.16 # g / c m 2 has a diffusion coefficient D = (1.2 + 0.3) × 10 -9 cmZ/s. These proteins are able

ADSORBED BSA LATERAL DIFFUSION

to migrate over distances of several micrometers by a diffusive process that does not involve collisions with dissolved protein molecules. The existence of EITC-BSA in more than one state, possibly with different ordering or conformation, after adsorption to P M M A results in a fraction of 0.37 + 0.05 of mobile molecules. After adsorption at the PDMS / PBS interface for 1 h at 0.14 , g / c m 2, a fraction of 0.28 + 0.02 of EITC-BSA molecules diffuse with D = (2.6 + 0.1 ) × 10-9 cm2/s, indicating that lateral mobility can be influenced by differences in surface properties. These results show that lateral mobility must be considered a possibility in any molecular model of protein adsorption at a solid-liquid interface that attempts to explain protein ordering or surface coverage. ACKNOWLEDGMENTS We are grateful to A. B. Anderson and Dr. M. R. Munch for sharing their expertise and insights over the course of this work. This research was initiated with support from the Center for Materials Research at Stanford University under the NSF-MRL Program and the National Science Foundation under Grant CBT-8552495 and continued under NSF Grant CBT-8813517. R.D.T. was supported in part by a Chevron graduate fellowship. REFERENCES 1. Andrade, J. D., in "Surface and Interfacial Aspects of Biomedical Polymers" (J. D. Andrade, Ed.), Vol. 2, pp. 1-80. Plenum Press, New York, 1985. 2. Brash, J. L., and Lyman, D. J., J. Biomed. Mater. Res. 3, 175 (1969). 3. Young, B. R., Pitt, W. G., and Cooper, S. L., J. Colloid Interface Sci. 124, 28 (1988). 4. J6nsson, U., Ivarsson, B., Lundstrrm, I., and Berghem, L., J. Colloid Interface Sci. 90, 148 (1982). 5. Sandwick, R. K., and Schray, K. J., J. Colloidlnterface Sci. 121, 1 (1988). 6. Norde, W., and Lyklema, J., J. Colloid Interface Sci. 66, 257 (1978). 7. Darst, S. A., Robertson, C. R., and Berzofsky, J. A., J. Colloid lnterface Sci. 111, 466 (1986). 8. Lok, B. K., Cheng, Y. L., and Robertson, C. R., J. Colloid Interface Sci. 91, 104 (1983). 9. Chan, B. M. C., and Brash, J. L., J. Colloid Interface Sci. 82, 217 (1981). 10. Norde, W., MacRitchie, F., Nowicka, G., and Lyklema, J., J. Colloid Interface Sci. 112, 447 (1986). 11. Cheng, Y. L., Darst, S. A., and Robertson, C. R., J. Colloid Interface Sci. 118, 212 (1987).

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