J. BIOMED. MATER. RES.
VOL. 11, PP. 915-938 (1977)
A Total Internal-Reflection Technique for the Examination of Protein Adsorption ROBERT W. WATKINS* and CHANNING R. ROBERTSON, Department of Chemical Engineering, Stanford University, Stanford, California 94305
Summary A total internal-reflection fluorescence (TIRF) technique for examining protein adsorption a t solid-liquid interfaces is described. For the representative case of adsorption of bovine 7-globulin onto silicone rubber, the applicability of the technique to several important aspects of protein adsorption phenomena is demonstrated. Specifically, in addition to the tightly adsorbed protein layer observed herein and previously by many ot,hers, the presence of a loosely adsorbed protein layer was also noted under conditions of either static or flowing protein solutions. Taking advantage of the continuous real-time measurements possible with TIRF, a rate constant for the protein adsorption step during laminar flow was estimated a t both 25 and 37°C. These results serve to demonstrate that the T I R F technique represents a significant and versatile new tool for the study of protein adsorption phenomena.
INTRODUCTION When blood is contacted with an artificial surface, a proteinaceous layer is deposited within a matter of seconds. Although the amount of adsorbed protein is small (0.2-2 pg/cm2),’r2 the subsequent initiation of thrombus formation on such a surface may often be correlated with the amount and composition of protein initially dep o ~ i t e d . ~Accordingly, .~ it may well be the case that a clearer understanding of the way in which proteins adsorb onto surfaces will permit a more systematic identification of the material properties which a n implanted prosthetic device must possess to remain non-
* Present address: Research and Development Department, Atlantic Richfield Company, Plano, Texas 75074. Author to whom correspondence should be addressed. 915 @ 1977 by John Wiley & Sons, Inc.
916
WATKINS AND ROBERTSON
thrombogenic. As a result, important areas to be considered are: 1) the types and amounts of proteins adsorbed by various surfaces; 2) the kinetics of the diffusion-adsorption process; and 3) the extent t o which fluid flow as well as the presence of suspended particlessuch as erythrocytes-aff ect the adsorption process. I n the past, a number of techniques have been used to investigate the adsorption of protein from water or buffered saline solutions onto a variety of materials. One of the simplest techniques involves the depletion of protein from solution following the addition of a high specific surface-area Unfortunately, this method lacks the sensitivity required to examine the large class of synthetic materials available in sheet or membrane forms which possess inherently low specific surface areas, and thus its use has been limited primarily to carbon, silica, and modified silica surfaces. Several investigators have obtained the necessary sensitivity with the use of wet chemical techniques. Included among these are the modified ninhydrin assay and the amido-black procedure applied to membranes or films having relatively large surface areas ( > 100 cm2).1-8 Although these techniques have proven useful for the study of equilibrium adsorption, they are inappropriate for the study of kinetic or competitive adsorption behavior. Like the chemical techniques just mentioned, multiple internalreflection infrared spectroscopy (IRS) has proven to be a sensitive tool for the study of protein adsorption.9J0 I n contrast to these chemical assay techniques, however, being a n optical technique, IRS is more straightforward to employ. I n I R S spectroscopy, the amount of adsorbed protein is determined by measuring the infrared absorption near 1650 cm-l and 1550 cm-I, these frequencies being characteristic of the amide bonds in all proteinaceous material. However, since water also absorbs strongly in this region of the infrared spectrum, use of IRS is limited t o studies where care has been taken t o exclude water, either through substitution of DzO for HzO in preparing the protein solutions or by first rinsing the adsorbed film t o remove hydroscopic salts followed by oven drying. An optical technique which overcomes this difficulty with water is ellipsometry. As discussed more fully elsewhere, ellipsometry permits a n estimation of the thickness and density of a n adsorbed film.6 This tool has been used by several groups of investigators for the study of protein adsorption onto a variety of materials.2.6J1 Al-
INTERNAL-REFLECTION EXAMINATION O F ADSORPTION 917
though the interpretation of ellipsometric data requires the solution of a transcendental equation and as such makes difficult the continuous collection of data, an investigation of time-dependent desorption phenomena using a recording ellipsometer has been reported." I n addition, ellipsometry has proven useful for the qualitative study of interactions between proteins in solution and those preadsorbed onto a surface.12 While each of the above-mentioned techniques has certain advantages, none of them is able to directly distinguish any one particular protein in a n adsorbed film consisting of a mixture of proteins. Thus, to date, direct quantitative measurements of competition among adsorbing proteins have been made exclusively using radioactive tracer methods. Although the discrete manner in which data are gathered makes difficult the direct observation of the adsorption kinetics, the sensitivity and specificity of the radiotracer technique has resulted in its widespread use.'^^.^ It should be noted, however, recent studies indicate that the incorporation of a radioactive element, such as the commonly used isotopes lZ5Iand 1311, into the adsorbing proteins may introduce serious artifacts into the r e ~ u l t s . ~ J ~ The present work describes a total internal-reflection fluorescence (TIRF) technique which incorporates many of the advantages of the above methods into one instrument. T I R P is unique in that it combines the required sensitivity with the capability to conveniently observe the adsorption of a single fluorescently labeled protein alone or from a mixture of proteins in solution. Furthermore, by continuously collecting data in real time, the T I R F technique is particularly well suited to the examination of adsorption kinetics. As described herein, this technique has been validated for the adsorption of fluorescein-labeled bovine yglobulin onto a silicone rubber surface.
TOTAL INTERNAL-REFLECTION FLUORESCENCE (TIRF) When light leaves a medium characterized by a n index of refraction, nl, and enters a second less optically dense medium with a n index of refraction n2 (nz < nl) a t an angle with the surface normal, 01, exceeding the critical angle, aC,total reflection within the more optically dense medium occurs. Owing to the wave nature of light, however, a portion of the incident beam will penetrate into the medium of lower refractive index, giving rise to a n evanescent
918
WATKINS AND ROBERTSON
wave.14 It is the interaction of this evanescent wave with an adsorbed protein layer which is utilized in IRS14 and the internalreflection fluorescence techniques used herein and by 0thers.’~J6 I n T I RF , the extent to which the evanescent wave interacts with fluorescently labelled protein is proportional to the square of the magnitude of the electric field vector, given by:l4 E2
=
E&-~/dp
(1)
where
and
a,
=
X1
4n(sin2a - [n2/n1]2)1’2
(3)
Accordingly, the light energy available for interaction decreases exponentially with distance from the interface with a characteristic length, d,, of the order of 1000 A. As such, photon interaction with the fluorescently labeled protein is limited to the immediate proximity of the surface. With the proper selection of the incident wavelength (Al), the presence of a fluorescent probe a t the surface in very low concentrations (-0.5 ng/cm2 for fluorescein in the system described herein) may be easily detected.
APPARATUS The apparatus constructed to utilize T I R F is shown schematically in Figure 1. A beam of light from a high intensity mercury arc lamp (HBO 200W, Osram, West Germany) is directed into the otherwise light-tight housing containing the remainder of the apparatus through a polarizing filter (F,) (Optical Industries, Inc., Santa Ana, Calif.) which selects the component of light perpendicular to the plane of reflection, and a blue filter (F2) (#47B wrattan, Eastman Kodak, Co., Rochester, N.Y.). Once inside, the beam passes through a 75mm focal length lens (L,) and is separated into two portions by a beam splitter (BS). One portion is directed onto the photodiode (PD) surface (PIN-lODP, United Detector Technology, Inc., Santa Monica, Calif.) to monitor the arc intensity while
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 919
Fig. 1. Block diagram of T I R F apparatus.
(a) Fig. 2.
(b)
(a) Fused silica prism. (b) Optical coupling between prism and fused silica plate is provided by a thin layer of cyclohexanol.
the remainder passes through another 75mm focal length lens (Lz), a 0.07cm diameter aperture (AJ, and onto the internal-reflection prism. The trapezoidal prism is made of high grade fused silica, nl = 1.467 (Spectra Physics, Inc., Mountain View, Calif.) and as shown in Figure Za, each end face intersects the plane of the base at a n angle of 70". The top, bottom, and angular end faces are polished to a 60-40 finish to minimize light scattering by surface imperfections. The prism is held in place by two machined aluminum blocks containing channels for circulating constant temperature fluid as shown in Figure 3.
920
Fig. 3.
WATKINS AND ROBERTSON
Thermostat4 aluminum supports used to hold fused silica prism. I
/’
FUSED SILICA PRISM
/
CHANNEL FOR CIRCULATION OF CON STAN T TEM P E R ATU RE F L u I D
Fig. 4. Schematic diagram of the flow channel assembly.
Protein solutions may be circulated through the rectangular channel (0.20 cm X 1.43 cm X 7.0 cm) in the Teflon base shown assembled within its thermostated aluminum housing in Figure 4. Flow ports connected to medical-grade silicone rubber tubing permit the introduction and withdrawal of solution during each experiment. I n practice, the boundary of the flow channel on the side of the prism
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 921
face is formed by a polished fused silica plate (0.1 cm X 2.5 cm X 7.5 em) placed directly on top of the Teflon base. An O-ring is used to give a fluid-tight seal. As indicated in Figure 2b, a thin layer of cyclohexanol (n = 1.465, within 0.2% of the refractive index of fused silica) provides optical coupling between the prism and the removable fused silica plate when assembled. I n this study, the fused silica plates were coated with silicone rubber. However, a wide range of polymeric coatings may be studied without further modification of the apparatus. The entire prism-channel assembly is clamped to the face of the photomultiplier tube (PMT) (9558B, Gencom Div., Emitronics, Inc., Plainview, N.Y.) as shown schematically in Figures 1 and 5. That portion of the fluorescence emitted in the direction of the photodetector reaches the PMT after first passing through the green blocking filter (Fa) (if55 wrattan, Eastman Kodak Co., Rochester, N.Y.) The filters Fz and F3are chosen to maximize the sensitivity of the system to fluorescein, the dye used in these experiments. As shown in Figure 6, the desired sensitivity is achieved by the significant overlap of the Fz transmission spectrum with the absorption peak for fluorescein (shaded region). At the same time, the blocking filter, Fa, essentially prohibits incident light from reaching the PMT, as evidenced by the minimal overlap of the Fz and F3 transmission spectra, while allowing the passage of the green fluorescent signal (A* = -520 nm). The processing of the electronic signals from both the PMT and P D is shown in Figure 7. (Further details may be found elsewhere.'*)
/-
FLOW CHANNEL /PRISM &SSEMBLY ASSEMBLY
Fig. 5. Attachment of t.he flow channel assembly to the PMT.
WATKINS AND ROBERTSON
922 7
5
7
-
1
WAVELENGTH,
X
(nm)
Fig. 6 . Transmission spectra for the Fz and Fa filters17relative to the adsorbance spectrum for fluorescein.
PMT INPUT PRE-AMP
OUTPUT TO STRIP CHART
RECORDER
+-
I
P 3 INPUT
I
SUMMING AMP
I OFFSET NULL
PRE-AMP
Fig. 7. Functional diagram of the electronic circuit.
The P D signal, proportional to the arc intensity, is amplified and used as the divisor in the analog divider (AD 530, Analog Devices, Inc., Norwood, Mass.). Similarly, the PMT current, proportional to the fluorescence intensity, is converted to a voltage by the picoammeter (4145, Keithly Instruments, Inc., Cleveland, Ohio), amplified, and used as the dividend in the analog divider. Consequently, the resulting output is normalized with respect to arc intensity and
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 92
therefore is directly proportional to the fluorescent signal. Sucl normalization is necessary because the arc output varies with age line voltage, temperature, and other determinants. Finally, a sum ming amplifier is used to subtract the background offset prior tc displaying the result on a chart recorder. This last step serves t c remove that portion of the signal generated by scattered light whicl is not absorbed by the blocking filter, F3, as well as the small flu. orescence emitted by the prism and plate coatings themselves.
MATERIALS AND METHODS Bovine y-globulins were obtained (Calbiochem, San Diego, Calif.) with an electrophoretic purity of 9801, and used without further purification. Fluorescein isothiocyanate (FITC) (10% on Celite, Calbiochem, San Diego, Calif .) was covalently bonded to the protein molecules by the method of Goldman.'g Unreacted dye was removed by passing the reaction mixture over a Bio Gel P6 chromatography column (Bio-Rad Laboratories, Richmond, Calif.) Protein concentrations and fluorescein-to-protein ratios (F/P), ranging from 2.5 to 7 pg fluorescein per mg y-globulin, were measured spectrophotometrically by the method of Wells et a1.20 The protein-fluorescein conjugate was found to have a broad absorption band in the visible region displaying a maximum at X = 493 nm with the corresponding emission maximum near X = 520 nm, denoted as X* in Figure 6. The surfaces used in all experiments were prepared by applying a thin coating of silicone rubber (SR) to the fused silica plates in the following manner. After cleaning with detergent (Alconox, Inc., N.Y.), each plate was immersed in chromic acid for a t least 1 hr. After removal, each plate was rinsed in distilled water and then in reagent-grade isopropanol. The isopropanol was removed from the surface with a stream of dry filtered nitrogen. The silicone rubber coating was applied by immersing and slowly withdrawing (-10 cm/min) the cleaned plate from an approximately 0.05% (w/v) solution of silicone rubber (Dow Corning 891 Medical Grade adhesive, Dow Corning Corp., Midland, Mich.) dissolved in ligroine. After the plates had been triply coated and cured for at least 4 hr a t 100°C, they were stored under distilled water or isopropanol until used.
WATKINS AND ROBERTSON
924
The critical surface tension of surfaces prepared in the above manner was estimated by the method of Zisman.21 The approximately linear relationship between liquid surface tension and the cosine of the contact angle on the prepared silicone rubber surface is shown in Figure 8. The critical surface tension, defined to be the intercept at cos p = 1,has been shown to be a characteristic property of surfaces. The experimentally determined value of the critical surface tension found hercin, 22 dyne/cm, is in good agreement with the published value of 24 dyne/cm for silicone rubber.22 Accordingly, the silicone rubber surfaces prepared in the above-described manner possess surface free energies similar to those of SR surfaces employed by other investigators. All adsorption experiments were carried out a t either 25 f 0.1"C or 37 f 0.1"C using protein solutions diluted to the desired concentration with 0.01 M phosphate buffered 0.85% NaCl (pH 7.4) (PBS). At the start of each measurement, the rectangular flow channel was filled with PBS to prevent the formation of a n airsolution interface following the introduction of the protein solution. The experiments were continued until the level of fluorescence emitted by the adsorbed protein reached an apparent plateau level, a t which point a 5 min PBS rinse was initiated to remove loosely bound protein and the unadsorbed protein contained within the evanescent region. 10-
I
I
1-
40
50
0 8 .~
72 8 63 4
cos
43 0 40 9
28 9
0
10
20'
30
60
70
80
SURFACE TENSION (dynes/cm)
Fig. 8. Cosine of SR-liquid contact angle, p , as a function of liquid surface tension.
INTERNAL-REFLECTION EXAMINATION O F ADSORPTION 925
Estimates of the surface concentration of adsorbed protein were obtained by comparing the output voltage levels from the T I R F system for a particular experiment with those obtained from calibration measurements performed in a manner similar to that of Brash and Lyman.g For the 7-globulin-FITC system, the fluorescence due to a given protein surface concentration was found to be directly proportional to the measured F/P ratio. Direct comparison between the experimental response a t a known F/P value and the calibration performed a t a different F/P value was possible by normalizing all experimental measurements to the calibration F/P of 4.1 pg/mg. For such calibration experiments, surfaces of known protein surface concentration were prepared by evaporating salt-free solutions of fluorescently labeled protein a t various concentrations in isopropanol on fused silica plates. Using these specially prepared plates, the output voltage levels from the T I R F system with air in the sample channel were measured for surface concentrations between 0 and 1.6 pg/cm2. Adjusting for the increased sensitivity with PBS (n, = 1.334) over that with air (n2 = 1.00) in the sample compartment according to eq. (2), the relationship between normalized system response and surface concentration of 7-globulin is shown in Figure 9. I n this context, the normalized system response is simply the ratio of the output voltage levels for the T I R F system I
I
0
0.5
1
1
I
I
I .o
1.5
20
C, (pqlcrn') Fig. 9. Calibration curve showing normalized system response (F/P = 4.1 pg FITC/mg protein) as a function of protein surface concentration.
926
WATKINS AND ROBERTSON
to that voltage which corresponds to a y-globulin surface coverage of 2.0 pg/cm2. Although the choice of the normalization voltage is arbitrary, this approach permits a convenient means for obtaining estimates of absolute yglobulin surface coverage directly from the T I R F system output. Use of the T I R F technique with other fluorescent protein systems would require similar calibration techniques for each protein to be studied.
RESULTS AND DISCUSSION Using the methods described above, the adsorption of bovine yglobulin onto silicone rubber surfaces was examined for bulk protein concentrations between 0.5 and 40 mg % at 25 and 37°C under both static and laminar flow conditions, the latter being at shear rates, y, ranging from 9.7 =t1.0 to 17.5 =t 2.0 sec-I. Steady-state surface concentrations of protein obtained after times ranging from 15 min at the higher bulk protein concentrations to 240 min a t the lower protein concentrations for both 25 and 37°C at the indicated values of F/P and shear rate are presented in Figures 10a and lob, respectively. I n agreement with other investig a t o r ~ , the ~ J ~surface protein concentration of the PBS rinsed surfaces was found to have reached a maximum at bulk protein concentrations of about 15 mg %. No influence of either F/P or flow on plateau levels was observed in the range of conditions studied. The associated double reciprocal plots (Fig. 11) are also in agreement with results reported by other investigators9 in that the adsorption data obtained in this manner may be described by a relationship of the form: l/c,
=
l/C,*(l
+ l/Kco)
(4)
where c, is the observed steady-state surface protein concentration at a given bulk protein concentration, co, and cs* is the limiting value of c, at high bulk protein concentrations. Both cs* and K were found to be somewhat temperature-sensitive, c,* decreasing from 0.25 pg/cm2 to 0.22 pg/cm2 and K decreasing from 0.33 cm3/pg to 0.27 cm3/pg as the temperature was increased from 25°C to 37°C. Matching the data to such a relation [eq. (4)] or the equivalent expression : c,
=
c,*Kco/(l
+ Kco)
(5)
INTERNAL-REFLECTION EXAMINATION O F ADSORPTION 927 I
I
025-
1
I
'
1
I
C'l - F/P(pglmg)
-5 In 0
I
"
X
o x
.
I
0.15
V
2.8 2.8 4.6 5.3 6.1
a
dl
x 0
0
0.10
0
.
*
0 17.5 17.5 17.5 17.5 Q7
.
F/P lpg /mq)
475 475 58 58 58 63 63
x 0
0
0 05 .-
0 V
I
I
1
I
I -
r(5')
-
0 17 5
17 5
-
I7 5
17 5 175 175 175
63
I
y
I
I
I
(b) Fig. 10. Protein surface concentration as a function of bulk protein concentration: (a) 25°C; (b) 37°C; the solid line is given by eq. (5).
as in Figure 10, both characteristic of the Langmuir has led other investigators to conclude that the adsorption process is of the Langmuir type with K being an equilibrium constant and cs* the surface concentration for a monolayer surface coverage of protein. The fact that the observed maximum surface concentration, cs*, was
928
WATKINS AND ROBERTSON
0
I
I
I
.5
1.0
1.5
1/c0
I 2.0
( m g yo-’)
(a)
Fig. 11. Inverse protein surface concentration as a function of inverse bulk protein concentration: (a) 25°C; (b) 37°C; the solid line is given by eq. (4). Same symbols as in Fig. 10.
found t o be in good agreement with the value of cs* predicted using the molecular dimensions of bovine yglobulin assuming a close packed side-on arrangement of about 0.25 pg/cm2 lends support to this theory. 8 , 9 However, the applicability of the Langmuir model together with its implication of dynamic equilibrium and the consequent reversibility of the adsorption process is not necessarily justified by the agreement of the data with a relation having the form of eq. (4). Although the apparent equilibrium behavior of the protein adsorption process observed in this study agrees with prior investigations, these findings remain to be reconciled with those studies wherein this process has been reported to be either partially reversible or irreversible. I n addition to examining the relatively tightly adsorbed protein layer just described, the in situ measurement capability of TIRF
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 929
- 7.0 W
:4.0r 3.O
I
I
-
.o 0
.5
1.0
1 /c, ( m g
1.5
2.0
YO-')
(b)
Fig. 11. (continued)
makes possible the study of protein more loosely bound to the adsorbing surface. Attributing the difference in the amount of adsorbed protein before the PBS rinse and that remaining after the PBS rinse to this loosely associated layer, it was possible to estimate the amount of protein held by the surface in this highly reversible form as shown in Figure 12. At both 25 and 37"C, the amount of loosely held protein was found to be approximately proportional to bulk protein concentration over the range studied. Fitting the data in Figure 12 to a relation of the form: GL =
KLCO
(6)
where csL is the amount of loosely held protein and KL the partition coefficient, K L was estimated from a linear least-squares analysis constrained to pass through the origin to be approximately 4.0 X cm and 2.0 X lo-* cm at 25 and 37"C, respectively. Thus, a t 25"C,
930
WATKINS AND ROBERTSON
C, ( m g Yo)
(b) Fig. 12. Net amount of loosely held protein, C ~ L ,as a function of bulk protein concentration, cg: (a) 25°C; (b) 37°C.
for example, the fraction of total protein reversibly associated with the surface was found to be approximately 20% for co = 15 mg % increasing to approximately 40% for co = 40 mg %, the maximum bulk protein concentration employed in this study. While extrapolation of a relation having the form of eq. (6) to physiologic protein concentrations may not be justified, it is evident that this loosely associated protein layer would be a highly significant component of the total protein associated with any artificial surface placed in contact with blood or plasma. Under these circumstances, the ac-
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 931
tual surface exposed to the blood is not the artificial material itself or even the tightly adsorbed protein layer but rather this more loosely associated proteinaceous layer. It is precisely the fact that this layer is loost y associated with the surface which has prevented its study in the pa, L. Techniques based on IRS or radiotracers, as discussed previously, require the surface to be dried and/or rinsed before measurements can be made. This pretreatment is certain to strip away most, if not all, of the loosely held protein. Thus, techniques which allow in situ measurements, such as TIRF and ellipsometry, are required for the study of such loosely bound layers. It is noted that the presence of a loosely held y-globulin layer observed herein is qualitatively similar to that observed by Cuypers for fibrinogen using a special automated ellip~ometer.~~ I n addition to its applicability to the study of steady-state adsorption phenomena as described above, T I R F is well suited for examining adsorption rate behavior owing to the continuous, realtime collection of data. For example, the data presented in Figure 13 depict the timedependent protein adsorption process for a silicone rubber surface prior to rinsing with PBS. Taking advantage of this capability, it was possible to estimate a second-order rate constant for the protein adsorption step under laminar flow conditions by comparing the experimentally determined adsorption data a t short times to the solution of eq. (7):
~ ( b c l b s= ) D(d2c/by2)
(7)
subject to ats = 0
c = co
(8)
y = b/2
c
(9)
y = o
D ( ~ C / ~=Yklc(i )
= CO
- e)
(10)
where
Assuming fully developed laminar flow and recognizing that under the conditions of this study a diffusion boundary layer is formed characterized by a thickness, 6 , where
WATKINS AND ROBERTSON
932
C.=154mg%
03
TIME
(mint
Fig. 13. Typical total protein surface concentrations (unrinsed) vs. time for indicated bulk protein concentrations.
with Pe = @/D mated as
= 0(106),the velocity in eq.
v = YY
( 7 ) may be approxi(13)
Time-dependent terms, which would account for the fact that the flow channel is initially filled with PBS, have been neglected in eq. ( 7 ) . As such, the only time dependence of any concern is that which enters parametrically through the time-dependent surface coverage, 0, in eq. (10). The effect of neglecting the time-dependent terms in eq. ( 7 ) has been examined in greater detail e l s e ~ h e r e . ~ I*n~these ~~ studies, it was shown that the primary difference between the solution of eq. (7) and the fully time-dependent system is that the latter includes a time lag relative to the former prior to reaching the steadystate adsorption rate. The time delay under experimental conditions was estimated to be approximately 90 sec at the observation point which was located 2.38 cm from the flow channel inlet. The numerical solution of the above system [eqs. (7)-(ll)] was obtained either by applying the standard Crank-Nicholson technique,26 or as was found to be computationally more efficient, by casting the problem in the form of a Volterra integral equation prior to solving.ls Recognizing that all parameters of the model system
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 933 TABLE I Range of Experimental Conditions Parameter Shear rate, y Bulk protein concentration, Diffusion coefficient, D
Value
cg
Maximum surface protein concentration, c.*
0, 9.7 f 1.0, and 17.5 f 2.0 sec-1 0.5-40.0 mg % 4.0 X ~rn~/sec~~ 0.2.5 pg/cm2 at 25°C 10.22 pg/cm2 a t 37°C
[eqs. (7)-(11)], with the exception of the second-order rate constant, k,, are either available in the literature or may be measured independently (Table I), it was possible to compare the numerical predictions with the experimental data and thus obtain a unique estimate of kl. This was accomplished using a least-squares technique to match the predicted and experimental adsorption data for e < -0.7. I n this manner, the second-order rate constant for shear rates of 9.7 sec-I and 17.5 sec-I and bulk protein concentrations ranging from 0.5 to 2.5 mg yowas found to be ( N = 30): kl = (1.3 f 0.45) X
cm/sec
where the uncertainty represents the standard deviation. No significant difference was observed between results a t the two temperatures studied. Inasmuch as the adsorption rate constant, kl, may be an important characteristic of the protein-surfaee interaction, the TIRF technique represents a significant improvement over techniques previously employed for the measurement of this quantity. I n fact, with the exception of the work of Lee and Kimlo using IRS, studies of timedependent adsorption phenomena to solid surfaces are notably lacking. Unfortunately, the work of Lee and Kim'O is difficult to interpret owing to the lack of detail regarding their quantitative analysis of the protein diffusion process which was used to estimate the adsorption rate constant. Thus, while the model presented for the initial deposition of protein at the solid surface characterized by the second-order reaction a t the surface [eq. (lo)] is in no way unique, the usefulness of TIRF in the study of kinetic phenomena has been demonstrated.
WATKINS AND ROBERTSON
934
As is customary for nearly all techniques utilizing tracer-labeled proteins, it was assumed a t the outset that the presence of the fluorescein moiety (MW = 389) would have no influence on the adsorption-diffusion behavior of the much larger protein molecule (MW = 160,000). The validity of this assumption was confirmed by measuring the adsorption behavior at 37°C of a series of protein solutions prepared by diluting a solution of FITC-labeled yglobulin with a solution of unlabeled yglobulin at the same concentration. The results, shown in Table 11, demonstrate that the amount of protein adsorbed is not affected by the average F/P ratio. In reviewing the recent literature concerning protein adsorption, it is apparent that a wide range of values for the limiting surface concentration, c.*, has been reported for bovine 7-globulin on silicone rubber. Values ranging (at 37°C) from 0.8l to 1.8 pg/cm* may be found, these values apparently being dependent on the buffering salts, ionic strength, and even the measurement techniques. The value of cs* presented herein (cs* = 0.22 pg/cm2 a t 37°C) falls within the same order of magnitude as, although less than, previously reported findings for similar experimental conditions. As discussed by Fenstermaker et a1.,6 however, consideration of surface roughness in computation of the adsorbing surface area is notably lacking. Since it is likely that a very uniform silicone rubber surface prepared by the dipping technique employed herein would be smoother than the relatively rough commercial preparations used by other investigators, quantitative comparison of protein surface concentrations between the two types of surfaces may be misleading. It is apparent from comparing photomicrographs (Fig. 14) of the SR-coated surface used in this study with the commercial preparation used by others (5 mil medical-grade Silastic sheet, Dow Corning Corp., Midland, Mich.) that the commercial material has a larger TABLE I1 Protein Surface Concentrations as a Function of F/P Ratio (T co (mg
%)
27.5 27.5 27.5
=
F/P (rglmg)
c. bg/cm2)
4.75 2.38 1.19
0.21 0.24 0.24
37°C)
INTERNALREFLECTION EXAMINATION OF ADSORPTION 935
(b) Fig. 14. Photomicrographs of silicone rubber surface: (a) dip-coated surface used in this study, 160X (bright field); Silastic sheet (5 mil, Dow Corning Corp.); (b) smooth side, 160X (oblique illumination) ; (c) dull side, 160X (Normarski) ; interference contrast.
936
WATKINS AND ROBERTSON
(c)
Fig. 14
(continued from previous page.)
surface area for adsorption than that estimated simply from the geometric surface area. Proper consideration of this variable could well account for the discrepancy between the results of this work and those previously reported. It is apparent that the T I R F instrument described herein is well suited to the study of a variety of problems relating to protein adsorption from static and flowing solutions previously not possible with any single piece of experimental apparatus. Inasmuch as thc solution properties, with the exception of the refractive index, have no effect on the total internal-reflection phenomena, it appears the technique may be extended to the study of protein adsorption from flowing model suspensions or even blood containing fluorescently labeled proteins. The only restriction on the polymeric surface to be studied is that the material must either be transparent or thin enough t o be rendered transparent, as was the case for silicone rubber employed in the described investigation.
CONCLUSIONS Using T I RF, the adsorption of bovine y-globulin onto silicone rubber was examined in detail. As has been previously observed,
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 937
the adsorption isotherm for tightly bound protein was described by a relation characteristic of the Langmuir isotherm [eq. ( 5 ) ] at both 25 and 37°C. Moreover, the in situ nature of TIRF measurements permitted for the first time the detection of a loosely held protein layer reversibly associated with the solid surface. The loosely bound protein was shown to constitute a significant fraction of the total protein associated with the SR surface within the range of bulk protein concentrations studied. This loosely held and previously neglected protein layer could be of considerable importance in relating to the biocompatibility of any artificial surface. I n addition, the ease with which real-time data may be collected makes it possible to gain further insight into the kinetic behavior of the protein adsorption process. The second-order rate constant for the protein adsorption step under controlled laminar flow conditions was measured. Inasmuch as this parameter may also be an important characteristic of the protein-surface interaction, the relative ease with which it may now be obtained represents a significant improvement over previously used techniques. This new technique, based on total internal reflection, has been demonstrated to be a versatile tool for the investigation of protein adsorption onto polymer surfaces. T I R F combines into a single instrument many of the advantages of methods currently used to examine protein adsorption, in particular, ellipsometry, IRS, and radiotracers. Like ellipsometry, T I R F measurements may be made in situ and continuously. Moreover, as with IRS and radiotracer methods, the data may be interpreted with relative ease. This work was supported by a grant from the National Science Foundation, NSF-ENG-14744. The authors acknowledge Prof. Andreas Acrivos and Mr. Brent K. Lok for helpful comments and suggestions prior to and throughout the preparation of this manuscript.
References 1. T. A. Horbett and A. S. Hoffman, Advan. Chem. Ser., 145, 230 (1975). 2. R. R. Stromberg, B. W. Morrissey, L. E. Smith, W. H. Grant, and R. E.
Dehl, National Bureau of Standards Report NBSIR 76-1017 (1976). 3. M. A. Packham, G . Evans, M. F. Glynn and J. F. Mustard, J. Lab. Clin. Med., 73(4), 686 (1969). 4. S. W. Kim, R. G . Lee, H. Oster, D. Coleman, J. D. Andrade, D. J. Lentz, and D. Olsen, Trans. '4SA10, 20, 20 (1974). 5. F. MacRitchie, J . Colloid Interface Sci., 38(2), 484 (1972).
938
WATKINS AND ROBERTSON
6. C. A. Fenstermaker, W. H. Grant, B. W. Morrissey, L. E. Smith, and R. R. Stromberg, National Bureau of Standards Report NBSIR 7 4 4 7 0 (1974). 7. S. Puszkin, S. Kochwa, E. G. Puszkin, and R. E. Rosenfield, J. Biol. Chem., 250(6), 2085 (1975). 8. W. J. Dillman, Jr. and I. F. Miller, J. Colloid Interjuce Sci., 44(2), 221 (1973). 9. J. L. Brash and D. J. Lyman, J. Biomed. Mater. Res., 3, 175 (1969). 10. R. G. Lee and S. W. Kim, J. Biomed. Mater. Res., 8, 251 (1974). 11. L. Vroman and A. L. Adams, Thromb. Diath. Haemorrh., 18, 510 (1967). 12. L. Vroman, A. I,. Adams, and M. Klings, Fed. Proc., 30(5), 1494 (1971). 13. W. H. Grant, L. E. Smith, anf R. It. Stromberg, J. Biomed. Mater. Res. Symposium No. 8, 33 (1977). 14. N. J. Harrick, Internal Rejection Spectroscopy, Wiley-Interscience, New York (1967). 15. N. J. Harrick and G. I. Loeb, Anal. Chem., 45(4), 687 (1973). 16. M. N. Kronick and W. A. Little, J . Immunol. Methods, 8. 235 (1975). 17. Kodak Filters for Scientific and Technical Uses, Eastman Kodak Co., Rochester, N.Y., 1973. 18. R. W. Watkins, Ph.D. Thesis, Stanford University (1977). 19. M. Goldman, Fluorescent Antibody Techniques, Academic Press, New York, 1968, pp. 123-125. 20. A. F. Wells, C. E. Miller, and M. K. Nadel, Appl. Mierobiol., 14(2), 272 (1966). 21. W. A. Zisman, Advan. Chem. Ser., 43, 1 (1964). 22. J. D. Andrade, Med. Instrum., 7(2), 110 (1973). 23. A. W. Adamson, Ed., Physical Chemistry of Surfaces, Interscience, NewYork, 1967. 24. Cuypers, P. A., Ann. N . Y . Acad. Sci., in press. 25. Y . A. Butruille, S; R. Savitz, E. F. Leonard, and R. S. Litwak, J . Biomed. Mater. Res., 10, 145 (1976). 26. W. G. Ames, Numerical Methods for Partial Differential Equations, Barnes and Noble, New York, 1969. 27. L. J. Gosting, Advan. Protein Chem., 11, 429 (1956).
Received November 11, 1976 Revised April 6, 1977
VOL. 11, PP. 915-938 (1977)
A Total Internal-Reflection Technique for the Examination of Protein Adsorption ROBERT W. WATKINS* and CHANNING R. ROBERTSON, Department of Chemical Engineering, Stanford University, Stanford, California 94305
Summary A total internal-reflection fluorescence (TIRF) technique for examining protein adsorption a t solid-liquid interfaces is described. For the representative case of adsorption of bovine 7-globulin onto silicone rubber, the applicability of the technique to several important aspects of protein adsorption phenomena is demonstrated. Specifically, in addition to the tightly adsorbed protein layer observed herein and previously by many ot,hers, the presence of a loosely adsorbed protein layer was also noted under conditions of either static or flowing protein solutions. Taking advantage of the continuous real-time measurements possible with TIRF, a rate constant for the protein adsorption step during laminar flow was estimated a t both 25 and 37°C. These results serve to demonstrate that the T I R F technique represents a significant and versatile new tool for the study of protein adsorption phenomena.
INTRODUCTION When blood is contacted with an artificial surface, a proteinaceous layer is deposited within a matter of seconds. Although the amount of adsorbed protein is small (0.2-2 pg/cm2),’r2 the subsequent initiation of thrombus formation on such a surface may often be correlated with the amount and composition of protein initially dep o ~ i t e d . ~Accordingly, .~ it may well be the case that a clearer understanding of the way in which proteins adsorb onto surfaces will permit a more systematic identification of the material properties which a n implanted prosthetic device must possess to remain non-
* Present address: Research and Development Department, Atlantic Richfield Company, Plano, Texas 75074. Author to whom correspondence should be addressed. 915 @ 1977 by John Wiley & Sons, Inc.
916
WATKINS AND ROBERTSON
thrombogenic. As a result, important areas to be considered are: 1) the types and amounts of proteins adsorbed by various surfaces; 2) the kinetics of the diffusion-adsorption process; and 3) the extent t o which fluid flow as well as the presence of suspended particlessuch as erythrocytes-aff ect the adsorption process. I n the past, a number of techniques have been used to investigate the adsorption of protein from water or buffered saline solutions onto a variety of materials. One of the simplest techniques involves the depletion of protein from solution following the addition of a high specific surface-area Unfortunately, this method lacks the sensitivity required to examine the large class of synthetic materials available in sheet or membrane forms which possess inherently low specific surface areas, and thus its use has been limited primarily to carbon, silica, and modified silica surfaces. Several investigators have obtained the necessary sensitivity with the use of wet chemical techniques. Included among these are the modified ninhydrin assay and the amido-black procedure applied to membranes or films having relatively large surface areas ( > 100 cm2).1-8 Although these techniques have proven useful for the study of equilibrium adsorption, they are inappropriate for the study of kinetic or competitive adsorption behavior. Like the chemical techniques just mentioned, multiple internalreflection infrared spectroscopy (IRS) has proven to be a sensitive tool for the study of protein adsorption.9J0 I n contrast to these chemical assay techniques, however, being a n optical technique, IRS is more straightforward to employ. I n I R S spectroscopy, the amount of adsorbed protein is determined by measuring the infrared absorption near 1650 cm-l and 1550 cm-I, these frequencies being characteristic of the amide bonds in all proteinaceous material. However, since water also absorbs strongly in this region of the infrared spectrum, use of IRS is limited t o studies where care has been taken t o exclude water, either through substitution of DzO for HzO in preparing the protein solutions or by first rinsing the adsorbed film t o remove hydroscopic salts followed by oven drying. An optical technique which overcomes this difficulty with water is ellipsometry. As discussed more fully elsewhere, ellipsometry permits a n estimation of the thickness and density of a n adsorbed film.6 This tool has been used by several groups of investigators for the study of protein adsorption onto a variety of materials.2.6J1 Al-
INTERNAL-REFLECTION EXAMINATION O F ADSORPTION 917
though the interpretation of ellipsometric data requires the solution of a transcendental equation and as such makes difficult the continuous collection of data, an investigation of time-dependent desorption phenomena using a recording ellipsometer has been reported." I n addition, ellipsometry has proven useful for the qualitative study of interactions between proteins in solution and those preadsorbed onto a surface.12 While each of the above-mentioned techniques has certain advantages, none of them is able to directly distinguish any one particular protein in a n adsorbed film consisting of a mixture of proteins. Thus, to date, direct quantitative measurements of competition among adsorbing proteins have been made exclusively using radioactive tracer methods. Although the discrete manner in which data are gathered makes difficult the direct observation of the adsorption kinetics, the sensitivity and specificity of the radiotracer technique has resulted in its widespread use.'^^.^ It should be noted, however, recent studies indicate that the incorporation of a radioactive element, such as the commonly used isotopes lZ5Iand 1311, into the adsorbing proteins may introduce serious artifacts into the r e ~ u l t s . ~ J ~ The present work describes a total internal-reflection fluorescence (TIRF) technique which incorporates many of the advantages of the above methods into one instrument. T I R P is unique in that it combines the required sensitivity with the capability to conveniently observe the adsorption of a single fluorescently labeled protein alone or from a mixture of proteins in solution. Furthermore, by continuously collecting data in real time, the T I R F technique is particularly well suited to the examination of adsorption kinetics. As described herein, this technique has been validated for the adsorption of fluorescein-labeled bovine yglobulin onto a silicone rubber surface.
TOTAL INTERNAL-REFLECTION FLUORESCENCE (TIRF) When light leaves a medium characterized by a n index of refraction, nl, and enters a second less optically dense medium with a n index of refraction n2 (nz < nl) a t an angle with the surface normal, 01, exceeding the critical angle, aC,total reflection within the more optically dense medium occurs. Owing to the wave nature of light, however, a portion of the incident beam will penetrate into the medium of lower refractive index, giving rise to a n evanescent
918
WATKINS AND ROBERTSON
wave.14 It is the interaction of this evanescent wave with an adsorbed protein layer which is utilized in IRS14 and the internalreflection fluorescence techniques used herein and by 0thers.’~J6 I n T I RF , the extent to which the evanescent wave interacts with fluorescently labelled protein is proportional to the square of the magnitude of the electric field vector, given by:l4 E2
=
E&-~/dp
(1)
where
and
a,
=
X1
4n(sin2a - [n2/n1]2)1’2
(3)
Accordingly, the light energy available for interaction decreases exponentially with distance from the interface with a characteristic length, d,, of the order of 1000 A. As such, photon interaction with the fluorescently labeled protein is limited to the immediate proximity of the surface. With the proper selection of the incident wavelength (Al), the presence of a fluorescent probe a t the surface in very low concentrations (-0.5 ng/cm2 for fluorescein in the system described herein) may be easily detected.
APPARATUS The apparatus constructed to utilize T I R F is shown schematically in Figure 1. A beam of light from a high intensity mercury arc lamp (HBO 200W, Osram, West Germany) is directed into the otherwise light-tight housing containing the remainder of the apparatus through a polarizing filter (F,) (Optical Industries, Inc., Santa Ana, Calif.) which selects the component of light perpendicular to the plane of reflection, and a blue filter (F2) (#47B wrattan, Eastman Kodak, Co., Rochester, N.Y.). Once inside, the beam passes through a 75mm focal length lens (L,) and is separated into two portions by a beam splitter (BS). One portion is directed onto the photodiode (PD) surface (PIN-lODP, United Detector Technology, Inc., Santa Monica, Calif.) to monitor the arc intensity while
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 919
Fig. 1. Block diagram of T I R F apparatus.
(a) Fig. 2.
(b)
(a) Fused silica prism. (b) Optical coupling between prism and fused silica plate is provided by a thin layer of cyclohexanol.
the remainder passes through another 75mm focal length lens (Lz), a 0.07cm diameter aperture (AJ, and onto the internal-reflection prism. The trapezoidal prism is made of high grade fused silica, nl = 1.467 (Spectra Physics, Inc., Mountain View, Calif.) and as shown in Figure Za, each end face intersects the plane of the base at a n angle of 70". The top, bottom, and angular end faces are polished to a 60-40 finish to minimize light scattering by surface imperfections. The prism is held in place by two machined aluminum blocks containing channels for circulating constant temperature fluid as shown in Figure 3.
920
Fig. 3.
WATKINS AND ROBERTSON
Thermostat4 aluminum supports used to hold fused silica prism. I
/’
FUSED SILICA PRISM
/
CHANNEL FOR CIRCULATION OF CON STAN T TEM P E R ATU RE F L u I D
Fig. 4. Schematic diagram of the flow channel assembly.
Protein solutions may be circulated through the rectangular channel (0.20 cm X 1.43 cm X 7.0 cm) in the Teflon base shown assembled within its thermostated aluminum housing in Figure 4. Flow ports connected to medical-grade silicone rubber tubing permit the introduction and withdrawal of solution during each experiment. I n practice, the boundary of the flow channel on the side of the prism
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 921
face is formed by a polished fused silica plate (0.1 cm X 2.5 cm X 7.5 em) placed directly on top of the Teflon base. An O-ring is used to give a fluid-tight seal. As indicated in Figure 2b, a thin layer of cyclohexanol (n = 1.465, within 0.2% of the refractive index of fused silica) provides optical coupling between the prism and the removable fused silica plate when assembled. I n this study, the fused silica plates were coated with silicone rubber. However, a wide range of polymeric coatings may be studied without further modification of the apparatus. The entire prism-channel assembly is clamped to the face of the photomultiplier tube (PMT) (9558B, Gencom Div., Emitronics, Inc., Plainview, N.Y.) as shown schematically in Figures 1 and 5. That portion of the fluorescence emitted in the direction of the photodetector reaches the PMT after first passing through the green blocking filter (Fa) (if55 wrattan, Eastman Kodak Co., Rochester, N.Y.) The filters Fz and F3are chosen to maximize the sensitivity of the system to fluorescein, the dye used in these experiments. As shown in Figure 6, the desired sensitivity is achieved by the significant overlap of the Fz transmission spectrum with the absorption peak for fluorescein (shaded region). At the same time, the blocking filter, Fa, essentially prohibits incident light from reaching the PMT, as evidenced by the minimal overlap of the Fz and F3 transmission spectra, while allowing the passage of the green fluorescent signal (A* = -520 nm). The processing of the electronic signals from both the PMT and P D is shown in Figure 7. (Further details may be found elsewhere.'*)
/-
FLOW CHANNEL /PRISM &SSEMBLY ASSEMBLY
Fig. 5. Attachment of t.he flow channel assembly to the PMT.
WATKINS AND ROBERTSON
922 7
5
7
-
1
WAVELENGTH,
X
(nm)
Fig. 6 . Transmission spectra for the Fz and Fa filters17relative to the adsorbance spectrum for fluorescein.
PMT INPUT PRE-AMP
OUTPUT TO STRIP CHART
RECORDER
+-
I
P 3 INPUT
I
SUMMING AMP
I OFFSET NULL
PRE-AMP
Fig. 7. Functional diagram of the electronic circuit.
The P D signal, proportional to the arc intensity, is amplified and used as the divisor in the analog divider (AD 530, Analog Devices, Inc., Norwood, Mass.). Similarly, the PMT current, proportional to the fluorescence intensity, is converted to a voltage by the picoammeter (4145, Keithly Instruments, Inc., Cleveland, Ohio), amplified, and used as the dividend in the analog divider. Consequently, the resulting output is normalized with respect to arc intensity and
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 92
therefore is directly proportional to the fluorescent signal. Sucl normalization is necessary because the arc output varies with age line voltage, temperature, and other determinants. Finally, a sum ming amplifier is used to subtract the background offset prior tc displaying the result on a chart recorder. This last step serves t c remove that portion of the signal generated by scattered light whicl is not absorbed by the blocking filter, F3, as well as the small flu. orescence emitted by the prism and plate coatings themselves.
MATERIALS AND METHODS Bovine y-globulins were obtained (Calbiochem, San Diego, Calif.) with an electrophoretic purity of 9801, and used without further purification. Fluorescein isothiocyanate (FITC) (10% on Celite, Calbiochem, San Diego, Calif .) was covalently bonded to the protein molecules by the method of Goldman.'g Unreacted dye was removed by passing the reaction mixture over a Bio Gel P6 chromatography column (Bio-Rad Laboratories, Richmond, Calif.) Protein concentrations and fluorescein-to-protein ratios (F/P), ranging from 2.5 to 7 pg fluorescein per mg y-globulin, were measured spectrophotometrically by the method of Wells et a1.20 The protein-fluorescein conjugate was found to have a broad absorption band in the visible region displaying a maximum at X = 493 nm with the corresponding emission maximum near X = 520 nm, denoted as X* in Figure 6. The surfaces used in all experiments were prepared by applying a thin coating of silicone rubber (SR) to the fused silica plates in the following manner. After cleaning with detergent (Alconox, Inc., N.Y.), each plate was immersed in chromic acid for a t least 1 hr. After removal, each plate was rinsed in distilled water and then in reagent-grade isopropanol. The isopropanol was removed from the surface with a stream of dry filtered nitrogen. The silicone rubber coating was applied by immersing and slowly withdrawing (-10 cm/min) the cleaned plate from an approximately 0.05% (w/v) solution of silicone rubber (Dow Corning 891 Medical Grade adhesive, Dow Corning Corp., Midland, Mich.) dissolved in ligroine. After the plates had been triply coated and cured for at least 4 hr a t 100°C, they were stored under distilled water or isopropanol until used.
WATKINS AND ROBERTSON
924
The critical surface tension of surfaces prepared in the above manner was estimated by the method of Zisman.21 The approximately linear relationship between liquid surface tension and the cosine of the contact angle on the prepared silicone rubber surface is shown in Figure 8. The critical surface tension, defined to be the intercept at cos p = 1,has been shown to be a characteristic property of surfaces. The experimentally determined value of the critical surface tension found hercin, 22 dyne/cm, is in good agreement with the published value of 24 dyne/cm for silicone rubber.22 Accordingly, the silicone rubber surfaces prepared in the above-described manner possess surface free energies similar to those of SR surfaces employed by other investigators. All adsorption experiments were carried out a t either 25 f 0.1"C or 37 f 0.1"C using protein solutions diluted to the desired concentration with 0.01 M phosphate buffered 0.85% NaCl (pH 7.4) (PBS). At the start of each measurement, the rectangular flow channel was filled with PBS to prevent the formation of a n airsolution interface following the introduction of the protein solution. The experiments were continued until the level of fluorescence emitted by the adsorbed protein reached an apparent plateau level, a t which point a 5 min PBS rinse was initiated to remove loosely bound protein and the unadsorbed protein contained within the evanescent region. 10-
I
I
1-
40
50
0 8 .~
72 8 63 4
cos
43 0 40 9
28 9
0
10
20'
30
60
70
80
SURFACE TENSION (dynes/cm)
Fig. 8. Cosine of SR-liquid contact angle, p , as a function of liquid surface tension.
INTERNAL-REFLECTION EXAMINATION O F ADSORPTION 925
Estimates of the surface concentration of adsorbed protein were obtained by comparing the output voltage levels from the T I R F system for a particular experiment with those obtained from calibration measurements performed in a manner similar to that of Brash and Lyman.g For the 7-globulin-FITC system, the fluorescence due to a given protein surface concentration was found to be directly proportional to the measured F/P ratio. Direct comparison between the experimental response a t a known F/P value and the calibration performed a t a different F/P value was possible by normalizing all experimental measurements to the calibration F/P of 4.1 pg/mg. For such calibration experiments, surfaces of known protein surface concentration were prepared by evaporating salt-free solutions of fluorescently labeled protein a t various concentrations in isopropanol on fused silica plates. Using these specially prepared plates, the output voltage levels from the T I R F system with air in the sample channel were measured for surface concentrations between 0 and 1.6 pg/cm2. Adjusting for the increased sensitivity with PBS (n, = 1.334) over that with air (n2 = 1.00) in the sample compartment according to eq. (2), the relationship between normalized system response and surface concentration of 7-globulin is shown in Figure 9. I n this context, the normalized system response is simply the ratio of the output voltage levels for the T I R F system I
I
0
0.5
1
1
I
I
I .o
1.5
20
C, (pqlcrn') Fig. 9. Calibration curve showing normalized system response (F/P = 4.1 pg FITC/mg protein) as a function of protein surface concentration.
926
WATKINS AND ROBERTSON
to that voltage which corresponds to a y-globulin surface coverage of 2.0 pg/cm2. Although the choice of the normalization voltage is arbitrary, this approach permits a convenient means for obtaining estimates of absolute yglobulin surface coverage directly from the T I R F system output. Use of the T I R F technique with other fluorescent protein systems would require similar calibration techniques for each protein to be studied.
RESULTS AND DISCUSSION Using the methods described above, the adsorption of bovine yglobulin onto silicone rubber surfaces was examined for bulk protein concentrations between 0.5 and 40 mg % at 25 and 37°C under both static and laminar flow conditions, the latter being at shear rates, y, ranging from 9.7 =t1.0 to 17.5 =t 2.0 sec-I. Steady-state surface concentrations of protein obtained after times ranging from 15 min at the higher bulk protein concentrations to 240 min a t the lower protein concentrations for both 25 and 37°C at the indicated values of F/P and shear rate are presented in Figures 10a and lob, respectively. I n agreement with other investig a t o r ~ , the ~ J ~surface protein concentration of the PBS rinsed surfaces was found to have reached a maximum at bulk protein concentrations of about 15 mg %. No influence of either F/P or flow on plateau levels was observed in the range of conditions studied. The associated double reciprocal plots (Fig. 11) are also in agreement with results reported by other investigators9 in that the adsorption data obtained in this manner may be described by a relationship of the form: l/c,
=
l/C,*(l
+ l/Kco)
(4)
where c, is the observed steady-state surface protein concentration at a given bulk protein concentration, co, and cs* is the limiting value of c, at high bulk protein concentrations. Both cs* and K were found to be somewhat temperature-sensitive, c,* decreasing from 0.25 pg/cm2 to 0.22 pg/cm2 and K decreasing from 0.33 cm3/pg to 0.27 cm3/pg as the temperature was increased from 25°C to 37°C. Matching the data to such a relation [eq. (4)] or the equivalent expression : c,
=
c,*Kco/(l
+ Kco)
(5)
INTERNAL-REFLECTION EXAMINATION O F ADSORPTION 927 I
I
025-
1
I
'
1
I
C'l - F/P(pglmg)
-5 In 0
I
"
X
o x
.
I
0.15
V
2.8 2.8 4.6 5.3 6.1
a
dl
x 0
0
0.10
0
.
*
0 17.5 17.5 17.5 17.5 Q7
.
F/P lpg /mq)
475 475 58 58 58 63 63
x 0
0
0 05 .-
0 V
I
I
1
I
I -
r(5')
-
0 17 5
17 5
-
I7 5
17 5 175 175 175
63
I
y
I
I
I
(b) Fig. 10. Protein surface concentration as a function of bulk protein concentration: (a) 25°C; (b) 37°C; the solid line is given by eq. (5).
as in Figure 10, both characteristic of the Langmuir has led other investigators to conclude that the adsorption process is of the Langmuir type with K being an equilibrium constant and cs* the surface concentration for a monolayer surface coverage of protein. The fact that the observed maximum surface concentration, cs*, was
928
WATKINS AND ROBERTSON
0
I
I
I
.5
1.0
1.5
1/c0
I 2.0
( m g yo-’)
(a)
Fig. 11. Inverse protein surface concentration as a function of inverse bulk protein concentration: (a) 25°C; (b) 37°C; the solid line is given by eq. (4). Same symbols as in Fig. 10.
found t o be in good agreement with the value of cs* predicted using the molecular dimensions of bovine yglobulin assuming a close packed side-on arrangement of about 0.25 pg/cm2 lends support to this theory. 8 , 9 However, the applicability of the Langmuir model together with its implication of dynamic equilibrium and the consequent reversibility of the adsorption process is not necessarily justified by the agreement of the data with a relation having the form of eq. (4). Although the apparent equilibrium behavior of the protein adsorption process observed in this study agrees with prior investigations, these findings remain to be reconciled with those studies wherein this process has been reported to be either partially reversible or irreversible. I n addition to examining the relatively tightly adsorbed protein layer just described, the in situ measurement capability of TIRF
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 929
- 7.0 W
:4.0r 3.O
I
I
-
.o 0
.5
1.0
1 /c, ( m g
1.5
2.0
YO-')
(b)
Fig. 11. (continued)
makes possible the study of protein more loosely bound to the adsorbing surface. Attributing the difference in the amount of adsorbed protein before the PBS rinse and that remaining after the PBS rinse to this loosely associated layer, it was possible to estimate the amount of protein held by the surface in this highly reversible form as shown in Figure 12. At both 25 and 37"C, the amount of loosely held protein was found to be approximately proportional to bulk protein concentration over the range studied. Fitting the data in Figure 12 to a relation of the form: GL =
KLCO
(6)
where csL is the amount of loosely held protein and KL the partition coefficient, K L was estimated from a linear least-squares analysis constrained to pass through the origin to be approximately 4.0 X cm and 2.0 X lo-* cm at 25 and 37"C, respectively. Thus, a t 25"C,
930
WATKINS AND ROBERTSON
C, ( m g Yo)
(b) Fig. 12. Net amount of loosely held protein, C ~ L ,as a function of bulk protein concentration, cg: (a) 25°C; (b) 37°C.
for example, the fraction of total protein reversibly associated with the surface was found to be approximately 20% for co = 15 mg % increasing to approximately 40% for co = 40 mg %, the maximum bulk protein concentration employed in this study. While extrapolation of a relation having the form of eq. (6) to physiologic protein concentrations may not be justified, it is evident that this loosely associated protein layer would be a highly significant component of the total protein associated with any artificial surface placed in contact with blood or plasma. Under these circumstances, the ac-
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 931
tual surface exposed to the blood is not the artificial material itself or even the tightly adsorbed protein layer but rather this more loosely associated proteinaceous layer. It is precisely the fact that this layer is loost y associated with the surface which has prevented its study in the pa, L. Techniques based on IRS or radiotracers, as discussed previously, require the surface to be dried and/or rinsed before measurements can be made. This pretreatment is certain to strip away most, if not all, of the loosely held protein. Thus, techniques which allow in situ measurements, such as TIRF and ellipsometry, are required for the study of such loosely bound layers. It is noted that the presence of a loosely held y-globulin layer observed herein is qualitatively similar to that observed by Cuypers for fibrinogen using a special automated ellip~ometer.~~ I n addition to its applicability to the study of steady-state adsorption phenomena as described above, T I R F is well suited for examining adsorption rate behavior owing to the continuous, realtime collection of data. For example, the data presented in Figure 13 depict the timedependent protein adsorption process for a silicone rubber surface prior to rinsing with PBS. Taking advantage of this capability, it was possible to estimate a second-order rate constant for the protein adsorption step under laminar flow conditions by comparing the experimentally determined adsorption data a t short times to the solution of eq. (7):
~ ( b c l b s= ) D(d2c/by2)
(7)
subject to ats = 0
c = co
(8)
y = b/2
c
(9)
y = o
D ( ~ C / ~=Yklc(i )
= CO
- e)
(10)
where
Assuming fully developed laminar flow and recognizing that under the conditions of this study a diffusion boundary layer is formed characterized by a thickness, 6 , where
WATKINS AND ROBERTSON
932
C.=154mg%
03
TIME
(mint
Fig. 13. Typical total protein surface concentrations (unrinsed) vs. time for indicated bulk protein concentrations.
with Pe = @/D mated as
= 0(106),the velocity in eq.
v = YY
( 7 ) may be approxi(13)
Time-dependent terms, which would account for the fact that the flow channel is initially filled with PBS, have been neglected in eq. ( 7 ) . As such, the only time dependence of any concern is that which enters parametrically through the time-dependent surface coverage, 0, in eq. (10). The effect of neglecting the time-dependent terms in eq. ( 7 ) has been examined in greater detail e l s e ~ h e r e . ~ I*n~these ~~ studies, it was shown that the primary difference between the solution of eq. (7) and the fully time-dependent system is that the latter includes a time lag relative to the former prior to reaching the steadystate adsorption rate. The time delay under experimental conditions was estimated to be approximately 90 sec at the observation point which was located 2.38 cm from the flow channel inlet. The numerical solution of the above system [eqs. (7)-(ll)] was obtained either by applying the standard Crank-Nicholson technique,26 or as was found to be computationally more efficient, by casting the problem in the form of a Volterra integral equation prior to solving.ls Recognizing that all parameters of the model system
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 933 TABLE I Range of Experimental Conditions Parameter Shear rate, y Bulk protein concentration, Diffusion coefficient, D
Value
cg
Maximum surface protein concentration, c.*
0, 9.7 f 1.0, and 17.5 f 2.0 sec-1 0.5-40.0 mg % 4.0 X ~rn~/sec~~ 0.2.5 pg/cm2 at 25°C 10.22 pg/cm2 a t 37°C
[eqs. (7)-(11)], with the exception of the second-order rate constant, k,, are either available in the literature or may be measured independently (Table I), it was possible to compare the numerical predictions with the experimental data and thus obtain a unique estimate of kl. This was accomplished using a least-squares technique to match the predicted and experimental adsorption data for e < -0.7. I n this manner, the second-order rate constant for shear rates of 9.7 sec-I and 17.5 sec-I and bulk protein concentrations ranging from 0.5 to 2.5 mg yowas found to be ( N = 30): kl = (1.3 f 0.45) X
cm/sec
where the uncertainty represents the standard deviation. No significant difference was observed between results a t the two temperatures studied. Inasmuch as the adsorption rate constant, kl, may be an important characteristic of the protein-surfaee interaction, the TIRF technique represents a significant improvement over techniques previously employed for the measurement of this quantity. I n fact, with the exception of the work of Lee and Kimlo using IRS, studies of timedependent adsorption phenomena to solid surfaces are notably lacking. Unfortunately, the work of Lee and Kim'O is difficult to interpret owing to the lack of detail regarding their quantitative analysis of the protein diffusion process which was used to estimate the adsorption rate constant. Thus, while the model presented for the initial deposition of protein at the solid surface characterized by the second-order reaction a t the surface [eq. (lo)] is in no way unique, the usefulness of TIRF in the study of kinetic phenomena has been demonstrated.
WATKINS AND ROBERTSON
934
As is customary for nearly all techniques utilizing tracer-labeled proteins, it was assumed a t the outset that the presence of the fluorescein moiety (MW = 389) would have no influence on the adsorption-diffusion behavior of the much larger protein molecule (MW = 160,000). The validity of this assumption was confirmed by measuring the adsorption behavior at 37°C of a series of protein solutions prepared by diluting a solution of FITC-labeled yglobulin with a solution of unlabeled yglobulin at the same concentration. The results, shown in Table 11, demonstrate that the amount of protein adsorbed is not affected by the average F/P ratio. In reviewing the recent literature concerning protein adsorption, it is apparent that a wide range of values for the limiting surface concentration, c.*, has been reported for bovine 7-globulin on silicone rubber. Values ranging (at 37°C) from 0.8l to 1.8 pg/cm* may be found, these values apparently being dependent on the buffering salts, ionic strength, and even the measurement techniques. The value of cs* presented herein (cs* = 0.22 pg/cm2 a t 37°C) falls within the same order of magnitude as, although less than, previously reported findings for similar experimental conditions. As discussed by Fenstermaker et a1.,6 however, consideration of surface roughness in computation of the adsorbing surface area is notably lacking. Since it is likely that a very uniform silicone rubber surface prepared by the dipping technique employed herein would be smoother than the relatively rough commercial preparations used by other investigators, quantitative comparison of protein surface concentrations between the two types of surfaces may be misleading. It is apparent from comparing photomicrographs (Fig. 14) of the SR-coated surface used in this study with the commercial preparation used by others (5 mil medical-grade Silastic sheet, Dow Corning Corp., Midland, Mich.) that the commercial material has a larger TABLE I1 Protein Surface Concentrations as a Function of F/P Ratio (T co (mg
%)
27.5 27.5 27.5
=
F/P (rglmg)
c. bg/cm2)
4.75 2.38 1.19
0.21 0.24 0.24
37°C)
INTERNALREFLECTION EXAMINATION OF ADSORPTION 935
(b) Fig. 14. Photomicrographs of silicone rubber surface: (a) dip-coated surface used in this study, 160X (bright field); Silastic sheet (5 mil, Dow Corning Corp.); (b) smooth side, 160X (oblique illumination) ; (c) dull side, 160X (Normarski) ; interference contrast.
936
WATKINS AND ROBERTSON
(c)
Fig. 14
(continued from previous page.)
surface area for adsorption than that estimated simply from the geometric surface area. Proper consideration of this variable could well account for the discrepancy between the results of this work and those previously reported. It is apparent that the T I R F instrument described herein is well suited to the study of a variety of problems relating to protein adsorption from static and flowing solutions previously not possible with any single piece of experimental apparatus. Inasmuch as thc solution properties, with the exception of the refractive index, have no effect on the total internal-reflection phenomena, it appears the technique may be extended to the study of protein adsorption from flowing model suspensions or even blood containing fluorescently labeled proteins. The only restriction on the polymeric surface to be studied is that the material must either be transparent or thin enough t o be rendered transparent, as was the case for silicone rubber employed in the described investigation.
CONCLUSIONS Using T I RF, the adsorption of bovine y-globulin onto silicone rubber was examined in detail. As has been previously observed,
INTERNAL-REFLECTION EXAMINATION OF ADSORPTION 937
the adsorption isotherm for tightly bound protein was described by a relation characteristic of the Langmuir isotherm [eq. ( 5 ) ] at both 25 and 37°C. Moreover, the in situ nature of TIRF measurements permitted for the first time the detection of a loosely held protein layer reversibly associated with the solid surface. The loosely bound protein was shown to constitute a significant fraction of the total protein associated with the SR surface within the range of bulk protein concentrations studied. This loosely held and previously neglected protein layer could be of considerable importance in relating to the biocompatibility of any artificial surface. I n addition, the ease with which real-time data may be collected makes it possible to gain further insight into the kinetic behavior of the protein adsorption process. The second-order rate constant for the protein adsorption step under controlled laminar flow conditions was measured. Inasmuch as this parameter may also be an important characteristic of the protein-surface interaction, the relative ease with which it may now be obtained represents a significant improvement over previously used techniques. This new technique, based on total internal reflection, has been demonstrated to be a versatile tool for the investigation of protein adsorption onto polymer surfaces. T I R F combines into a single instrument many of the advantages of methods currently used to examine protein adsorption, in particular, ellipsometry, IRS, and radiotracers. Like ellipsometry, T I R F measurements may be made in situ and continuously. Moreover, as with IRS and radiotracer methods, the data may be interpreted with relative ease. This work was supported by a grant from the National Science Foundation, NSF-ENG-14744. The authors acknowledge Prof. Andreas Acrivos and Mr. Brent K. Lok for helpful comments and suggestions prior to and throughout the preparation of this manuscript.
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Received November 11, 1976 Revised April 6, 1977
