Langmuir 1991, 7,995-999
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In Situ Quantitation of Protein Adsorption Density by Integrated Optical Waveguide Attenuated Total Reflection Spectrometry S. S. Saavedra; and W. M. Reichert Department of Biomedical Engineering and Center for Emerging Cardiovascular Technologies, Duke University, Durham, North Carolina 27706 Received August 20, 1990. In Final Form: November 13,1990 The adsorption of hemoglobin to a polystyrene (PS)thin film has been examined by integrated optical waveguide attenuated total reflection (IOW-ATR) spectrometry. Protein adsorption densities were determined by measuring the evanescent attenuation of propagating modes that were prism coupled into and out of the PS film. Knowledge of the electric field intensity distribution of the waveguide structure permitted adsorbed protein to be quantitated in the presence of bulk dissolved protein. The Langmuir adsorption model was used to fit the isotherm data and indicates that Hb binds with high affinity (K, > 10s M-1) to PS and forms a complete monolayer at bulk concentrationsgreater than 3 pM. The results demonstrate that the IOW-ATR technique is particularly well suited to in situ measurements of protein adsorption density at the polymer/solution interface.
Introduction The adsorption of dissolved proteins to solid substrates is a subject of considerable interest in numerous fields, including biocompatible materials engineering, biomolecule separations, controlled drug delivery, and bioanalytical One of the most studied aspects of the problem is the adsorption density, or surface excess, of the protein(s) as a function of bulk solution composition. Radiolabeling is widely used to measure protein adsorption densities but cannot be employed in situ because a rinse step is required. Consequently loosely bound protein molecules that are removed from the surface by washing are not quantitated. In addition, generation of an isotherm requires that multiple substrates be used for the several bulk protein concentrations. Other techniques that have been used to measure protein adsorption include Fourier transform infrared attenuated total reflectance (FT-IR/ ATR) spectroscopy, total internal reflection fluorescence (TIRF) spectroscopy, solution depletion, ellipsometry, X-ray photoelectron spectroscopy, and immunological techniques. For in situ, kinetic studies, FT-IR/ATR and TIRF have received considerable attention but are difficult to employ for quantitative measurement^.^-' The major limitation of TIRF spectroscopy is that scattered incident light excites significant fluorescence emission from beyond the evanescent depth of penetration. Quantitative determination of adsorbed amount from fluorescence emission intensity therefore requires that the cell be flushed of bulk protein before each measurement. TIRF spectroscopy at the more biomedically relevant polymer/solution interface (1) Andrade, J. D. In Surface and Interfacial Aspects of Biomedical Polymers: Protein Adsorption; Andrade, J. D., Ed.; Plenum Press: New York, 1986; Vol. 2, Chapter 1. (2) Horbett, T. A. In Biomaterials: Interfacial Phenomena and Applications; Cooper, S. L., Peppas, N. A., Eds.;Advances in Chemistry Series; American Chemical Society: Washington, D.C., 1982; Vol. 199, p 233. (3) Andrade, J. D.; Hlady, V. Adu. Polym. Sci. 1986, 79, 1. (4) Hlady, V.; Van Wagenen, R. A.; Andrade, J. D. In Surface and Interfacial Aspects of Biomedical Polymers: Protein Adsorption; Andrade, J. D., E Plenum Press: New York, 1985; Vol. 2, Chapter 2. (5) Hlady, V.; Reinecke, D. R.; Andrade, J. D. J.Colloid Interface Sci. 1986.111.656. - - - -, - - -,- - -. (6) Chittur,K. K.; Fink, D. J.;Leininger, R. I.; Hutaon,T. B. J.Colloid Interface Sci. 1986, 111, 419. (7) Reichert, W. M. CRC Crit. Reo. Biocompat. 1989, 5, 173.
'.;
is possible by coating the internal reflection element (IRE) with a polymer film but is complicated by background fluorescence and increased scattering from the film. The major disadvantage of FT-IR/ATR is that water absorbs strongly in the IR and, even with spectral subtraction, can overwhelm the adsorbed protein signal. Furthermore, the bulk contribution is enhanced because the evanescent depth of penetration is greater in the IR than at the shorter wavelengths characteristic of TIRF. FT-IR/ATR studies at polymer/solution interfaces can also be performed by coating the IRE with a thin film but this creates an additional source of strong background absorbance. Integrated optical waveguide (IOW) spectroscopy of thin solid films, in which the film itself functions as the IRE, is an established technique for studying polymer thin film and interfacial behavior.*1° Recently the theory and application of IOW-ATR spectrometry for characterizing adsorbates at polymer/liquid interfaces has been reported.11J2 Here IOW-ATR spectrometry is used to examine protein adsorption on polystyrene films. The high number of internal reflections over the sensing path length of IO waveguides yields the required submonolayer sensitivity. Knowledge of the electric field intensity distribution of the waveguide structure permits adsorbed protein to be quantitated in the presence of bulk dissolved protein. The results demonstrate that the IOW-ATR technique is particularly well suited to in situ measurements of protein adsorption density at the biomedically relevant polymer/solution interface. Preliminary linear dichroism measurements indicate that chromophore orientation in weakly absorbing monolayers can be studied.
Experimental Section Waveguide Fabrication and Characterization. Polystyrene (PS)IO waveguides were fabricated accordingto previously published methods.l*J4 Waveguides were initially prism coupled in air in order to qualitatively judge their optical quality.
Typically only 20-40% of the waveguides in a batch exhibited (8) Rabolt, J. F.; Santo, R.; Schlotter, N. E.; Swalen, J. D. IBM J. Res.
Deu. 1982.26. . .. _-, -. , 209. - .
(9)Schlotter, N. E.; Rabolt, J. F. J. Phys. Chem. 1984,88, 2062.
(io) Bohn, P. W. Anal. Chem. 1985,57, 1203.
(11) Saavedra, S. S.; Reichert, W. M. Appl. Spectrosc. 1990,44,1420. (12) Saavedra, S. S.; Reichert, W. M. Anal. Chem. 1990,62,2251. (13) Saavedra, S. S.; Reichert, W. M. Appl. Spectrosc. 1990,44,1210. (14) Ive18, J. T.; Reichert, W. M. J. Appl Polym. Sci. 1988,36, 429.
0743-7463/91/2407-0995$02.50/0 0 1991 American Chemical Society
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Table I. Physical and Optical Parameters of PS Waveguides waveguide no. of refractive thickness, Pm designation polarization obsd modes index 1 2 3 3
TE TE TE TM
4 4 5 5
1.595 1.598 1.600 1.596
2.226 2.044 2.291 2.255
sufficiently low scattering losses and intermodal crosstalk to be suitable for liquid phase IOW-ATR experiments. These highquality waveguides were subsequently prism coupled under a distilled water superstrate using a flow cell that contains a pair of 45-45-90 LaSFb coupling prisms mounted in its upper housing. The construction and operation of this flow cell, which permits IO waveguide experimenta to be conducted in liquid superstrates, have been described previoualy.ll The flow cell assembly was mounted on a high-resolution goniometer with the right angle corner of the incoupling prism placed at the center of the goniometer stage. Rotation of the stage with respect to a stationary laser beam (linearly polarized Ar+ laser line, 488 or 514.5 nm) allowed the incoupling angles at which guided modes were launched in a waveguide to be measured. From the observed angles, waveguide thickness and refractive index were determined by using a convergence technique.lg These properties are listed in Table I for the three waveguides used in this study and were employed in conjunction with the measured incouplingangles to calculate theoretical evanescent path length parameters, employing the equations and treatment detailed in ref 12. No further characterization of PS waveguides was performed. Protein Solutions. Bovine hemoglobin (Hb, Sigma, 75% methemoglobin,remainder primarily oxyhemoglobin) and sperm whale myoglobin (Mb, Sigma,primarily metmyoglobin) were used as received. Dilution series were prepared in 50 mM NaHzPO,, pH 7.2, containing 100 mM NaC1. Mb solutions were passed through a 0.45-rm filter before use. Mb concentration in the filtrate was determined by visible spectrophotometry using €410 = 1.79 X 10 M-1 cm-1 and a molecular weight of 18 oo0.15 IOW-ATRSpectrometry. The optical configuration used to perform IOW-ATR spectrometry has been described previously.11-18 Rotation of the goniometer stage allowed the incoupling mode angleto be selected. The outcoupledmode pattern was directed onto a paper screen and photographed with a chargecoupled device (CCD) array detector that was cooled to -110 O C , yielding a two-dimensionalpixel image of the pattern. The CCD was binned 10-fold in the vertical direction. Protein solutions were infused into the flow cell at approximately 3 mL/min. After allowing adsorption to proceed for 20 (for Hb) or 30 min (for Mb), outcoupled.mode pattern images, corrected for dark count, were recorded at multiple incoupling mode angles. The process was then repeated for the next highest protein concentration. TE polarization was used in experiments with Hb, whereas with Mb both T E and TM polarizations were used. For statistical purposes, three mode pattern images were recorded at each incoupling angle, protein concentration, and polarization. Integrated absorption of guided mode evanescent energy by adsorbed protein was assessedby analysisof the outcoupledmode pattern images.12 The absolute intensity of an outcoupled mode was taken as the average pixel intensity within a user-defined graphica window centered over the appropriate region of the mode pattern image. The background (stray light) intensity was estimated from the average pixel intensity in off-mode regions of the recorded images (regionswhere modes were not observed). Under the assumption that the stray light was constant over the entire image area, the background-corrected, outcoupled mode intensity was obtained by subtracting the off-mode background intensity from the absolute mode intensity. In experiments with Hb, raw absorbance measurements were not corrected for evanescent absorption by bulk dissolved protein since, at the concentrations employed, the contribution of bulk absorbance to total absorbance was calculated to be on the order of 4% or (15) Harrison, S. C.; Blout, E. R. J . Biol. Chem. 1966, 240, 299.
Table 11. Calculated Hb Adsorption Densities on Waveguide 1 adsorption density,' mol cm-2 X 10l2 bulk Hb concn, IM m=2 m=3 0.10 1.9 f 0.14 2.8 0.07 0.20 0.41 1.09 1.86 3.10 6.20 12.4 24.8
3.2 f 0.12 4.0 f 0.14 4.9 0.16 5.4 0.06 5.5 0.16 5.9 0.07 6.1 0.12 6.2 0.03
*
* * * * **
3.9 0.10 4.6 0.08 5.0 0.02 5.1 i 0.07 5.2 0.05 5.6 0.11 5.6 0.07 6.0 0.14
Error limits denote standard deviation of three measurements. less (see below). Mb absorbance data were corrected for the bulk contribution.
Results Hb Adsorption on Polystyrene. Hb adsorption on PS waveguide #l was examined by measuring the absorbance at 514.5 nm of outcoupled TE modes as a function of bulk Hb concentration. Adsorption densities of Hb (in mol/cm2) at t h e waveguide/solution interface were calculated from
dFf = Af[lOOON(Ze/Ii)4-l (1) where df is the thickness of t h e adsorbed film (in cm), cy is the concentration of protein in the film (in mol/cms), Af is the integrated absorption of evanescent energy by t h e protein film, N is the number of reflections between t h e coupling prisms, Ze/Zi is the evanescent transmitted interfacial intensity per unit incident intensity, and q is t h e molar absorptivity of the protein film. The derivation of this equation and an explanation of the pertinent parameters are provided in ref 12. N and Ze/Z., were calculated from knowledge of the waveguide thickness, the refractive indices of the waveguide stack, and the coupling angles. It was assumed that (1)the protein film molar absorptivity was equal to the bulk value of 24 400 M-l cm-l at 514.5 nm (our measurement, using a molecular weight of 64 500) and (2) the orientation of adsorbed molecules with respect to the surface was isotropic. Since t h e bulk molar absorptivity was measured with unpolarized light while IOW-ATR measurements were performed with TE polarized light, a correction factor (2X) was applied to t h e calculated adsorption densities to account for the zero electric field intensity parallel to the direction of mode propagation. Listed in Table I1are the calculatedadsorption densities as a function of bulk Hb concentration for the m = 2 and m = 3 modes. Although measurements were also made in the m = 1 and m = 0 modes, only the higher order modes were sufficiently sensitive to submonolayer densities. Good agreement is observed between the two sets of data, especially at the higher concentrations, which is consistent with previous work showing that the use of a ray optics approach t o treat modal differences in evanescent path length is valid.12J6 The only comparable study of Hb adsorption known to these authors was published by Chen e t al.,17 who used a radiolabeling technique to examine adsorption of oxyHb and deoxyHb to planar PS substrates. Employing a constant bulk protein concentration of 3.9 pM a n d incubation times varying from 1to 60 min, they reported adsorption densities in the range of (6 to 11) X (16)DeGrandpre, M.D.;Burgeee, L.W.;White, P.L.;Goldman, D. S. A d . Chem. 1990,62, 2012. (17)Chen, J.; Andrade, J. D.; Van Wagenen, R. A. Biomateriab 1986,
6,231.
Langmuir, Vol. 7, No. 5,1991 997 6.5 i
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n,
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-
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-
a
3.5-
x
m 2
9
2.5-
-
1.5-,
I I
I1
6.53
1
Bulk Hb Concentmtlon (M x 105)
Figure 1. Adsorptiondensity of Hb on waveguide 1 aa a function of bulk protein concentration. Data were calculated by using eq 1: squares, m = 2; circles, m = 3. Solid lines represent the best fit of the experimental data to the Langmuir model and were calculated by using eq 3 and the K,and a values listed in Table 111. Table 111. Parameters of Langmuir Model Reciprocal Fits to Hb Adsorption Isotherms on Waveguide 1 parameter m52 m=3 4.4 x 108 1.0 x 107 K., M-1 a,Alladsorption site 2700 3000 corr coeff 0.99 0.99
10-l2 mol cm-2. Chen's data are in excellent agreement with those reported here, particularly when considering (1)differences in the two techniques employed, (2)that the oxidation state of Hb may affect its adsorption behavior, and (3)that the molar absorptivity of adsorbed Hb is not known. The adsorption density data (plotted in Figure 1)show initial high affinity binding at low bulk concentrations followed by a plateau indicating apparent saturation coverage at bulk concentrations greater than 3 pM. The simple Langmuir model for surface adsorption18 states that
where 19 is the ratio of occupied adsorption sites to total sites, K, is the adsorption equilibrium constant, and c b is the solute molarity approximatingits activity in solution. If a is defined as the substrate surface area per adsorption site, then 19 = &rN,a, where Na is Avagadro's number. Introducing these parameters into eq 2 yields Langmuir's expression in terms of the adsorbed film thickness and concentration Kacb
(3) dFf = aN, + aN,KaCb The adsorption density data were fit to the reciprocal form of eq 3 using a linear regression model. The estimates obtained for a and Ka for each mode are listed in Table 111. These estimated values were then used in eq 3 to calculate the solid lines plotted in Figure 1, representing the best fit of the experimental data to the Langmuir (18) Hiemenz, P. C. Principle8 of Colloid and Surface Chemistry, 2nd ed.; Marcel Dekker: New York, 1986; Chapter 7.
model. The correlation coefficientsare also listed in Table I11 and suggest that the Langmuir model adequately describes these isotherms. Despite the apparent goodness of fit, it is unlikely that Hb adsorption to PS obeys ideal Langmuir behavior. Specifically, the inherent assumptions that only one type of surface site is present, that lateral interactionsbetween adsorbed molecules are absent, and that the adsorption process is reversible are probably not valid. The calculated a and K , values should therefore be treated as empirical parameters that describe the adsorption isotherms. The K , values calculated for modes m = 2 and m = 3 are 4.4 X 106 and 1.0 X lo7M-l, respectively, and indicate that Hb binds strongly to the PS surface. Although it is widely accepted that proteins adsorb with high affinity to hydrophobic surfaces, few quantitative measurements of binding have been reported. In the only example known to these authors, a binding constant of 6 X 106 M-' was measured for BSA adsorption to PS beads using a radiolabeling technique.lg The calculated values for substrate surface area/adsorption site (a,Table 111)are 2700 A2for m = 2 and 3000A2 for m = 3. These values are consistent with the average cross section of the Hb molecule, approximately 3200 A2, which was estimated from the dimensions of crystallized Hb (50 X 55 X 64 A).20 Hb adsorbed to PS thus occupies a surface area per molecule in the interfacial plane that is approximately equal to the cross section of the native protein. These data therefore indicate that Hb forms a complete monomolecular layer at bulk concentrations greater than 3 r M and multilayer adsorption does not occur, at least up to 25 pM bulk concentration. Since the polymer film is removed from the glass substrate upon disassembly of the flow cell, only one IOWATR experiment per waveguide is possible with our technique. Even if the waveguide could be recovered intact, cleaning the adsorbed layer from the surface without damaging the polymer film would be difficult. Consequently, only one set of measurements were performed to obtain the data in Table 11. However, the results obtained with waveguide 1 can be compared with the results of a previous experiment (data not shown) in which Hb adsorption to waveguide 2 was measured in the m = 3 mode under a different set of experimentalconditions (absorbance measured at 488 nm after 15 min incubation time using seven bulk protein concentrations ranging from 0.31 to 31.0 rM). The calculated adsorption densities ranged from (2.1to 5.5)X 10-12 mol/cm2 at bulk concentrations of 0.31-31.0rM, respectively. A least-squares fit of the isotherm data to the reciprocal form of eq 3 yielded a = 3600 A2 and K , = 2.6 X lo7 M-l with a correlation coefficient of 0.98. Considering the different conditions under which the two sets of data were acquired, the agreement between the results obtained with waveguides 1 and 2 is quite good. An additional concern is that Hb adsorption was allowed to proceed for only 20min prior to each ATR measurement, which may not have been sufficient to reach equilibrium. Chen's data show that the amount of Hb adsorbed to PS increased between 10 and 60 min incubation time (Le., equilibrium was not established at 10 min).17 We did not examine the time course of Hb adsorption to PS. However, we did observe that Mb adsorption to waveguide 3 from a 0.37 pM bulk solution reached apparent equilibrium (19) Dezelic, G.; Dezelic, N.; Teliaman, Z. Eur. J. Biochem. 1971,29, 575. (20)Lehninger, A. L. Biochemistry,2nd ed.; Worth: New York, 1976; Chapter 6.
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998 Langmuir, Vol. 7, No. 5, 1991 5.0 0
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i 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Bulk Mb Concentration (M x l o 5 ) Figure 2. Ratio of experimental TM to T E absorbances in m = 4 (circles, (Am,4/Am,4).) for Mb adsorbed to waveguide 3 as a function of bulk Mb concentration. The dashed line is the theoretical thin film absorbance ratio, (Am,4/AmJt,calculated for an isotropic distribution of heme orientations in the adsorbed protein film.
quite rapidly. Between 0 and 2 min incubation time, the thin film absorbance (At) increased to 0.070; between 2 and 30 min incubation time, At was 0.073 f 0.014 (n = 8, data not shown). Considering the structural similarity between the Hb subunit and Mb and the similar diffusion coefficients expected for these proteins,20the use of the Langmuir model to fit the data in Table I1 appears valid, although the Mb data certainly do not rule out a further increase in adsorption after 30 min. It should also be noted that the rinse step necessary in a radiolabeling assay removes loosely adsorbed protein. This loosely adsorbed protein may become more strongly bound after long incubation times' and, hence, less easily removed by rinsing, leading to an apparent increase in adsorbed amount. Thus a comparison of protein adsorption kinetics by radiolabeling and IOW-ATR is probably not valid. Linear Dichroismof Adsorbed Mb. The polarization control inherent in IO waveguide spectroscopy suggests the possibility of examining orientational anisotropy in adsorbed molecular layers, as recently discussed by Cropek and Bohn.21 The electronic transitions of the heme group in Hb and Mb arex-y polarized in the heme plane.22 Since the four heme groups in Hb are out of plane with respect to each other,20 it is unlikely that linear dichroism measurements would be capable of detecting an anisotropic orientation of heme groups in an adsorbed Hb layer. However, Mb contains a single heme whose orientation in the protein is known,20 raising the possibility that the occurrence of oriented adsorption could be detected. Consequently, linear dichroic IOW-ATR measurements of Mb adsorbed to waveguide 3 were performed by measuring the differential absorbance of the TE and TM m = 4 modes. Raw absorbance data were corrected for absorbance by bulk dissolved protein.12 The experimental dichroic ratio of TM to TE absorbances for m = 4, (ATM,~/ ATE,^)^, is plotted in Figure 2 as a function of bulk Mb concentration (note that saturation coverage was observed at the upper end of the plotted concentration range). Also plotted in Figure 2 is a dashed line representing the theoretical thin film dichroic ratio, (ATM,~/ATE,&, that would be expected for an isotropic distribution of heme plane orientations with respect to the interface. Such a distribution would correspond to an average angle of 4 5 O between the heme plane and the surface normal.21 (ATM,I/ ATE,& and the average orientation angle were calculated from the theoretical treatment detailed in refs 12 and 21, ~
~~
~ ~ _ _ _ _ _ _ _ _
(21)Cropek, D.M.; Bohn, P. W. J. Phys. Chem. 1990,94,6452. (22)Makinen, M. W.; C h u g , A. K. In Iron Rophyrins; Lever, A. B. P., Gray, H. B., Eds.; Addison-Wesley: Reading, MA, 1983;Part 1,p 141.
taking into account the modal differences in waveguiding angle, film refractive index, Ie/Ii, and N. Figure 2 shows that the esperimental dichroic ratio is greater than the theoretical ratio at the three bulk Mb concentrations investigated, meaning that the absorbance is stronger for radiation polarized normal to the interface than in the interfacial plane. Although these data are preliminary, they indicate that the average orientation of the heme plane in Mb molecules adsorbed to PS lies between Oo and 4 5 O from the surface normal. More importantly, these data demonstrate the potential of IOWATR for measuring linear dichroism in thin, weakly absorbing layers, Due to the inherently low reflection densities characteristic of conventional internal reflection geometries, linear dichroic ATR studies have to date been largely restricted to strongly absorbing layers containing a high density of chromophore^.^^*^^^^^ Further characterization of the observed anisotropy in adsorbed Mb layers and its dependence on experimental parameters will be the subject of future investigations.
Discussion Since the polymer film itself functions as the IRE in IOW-ATR spectrometry, this technique is well suited for analysis of protein adsorption at the polymer/solution interface. Polymer IO waveguides can be fabricated from materials that have a refractive index greater than the substrate material (usually quartz or glass), can be cast or coated as a smooth, thin films, and are transparent a t the desired wavelength.26 Many polymers that are currently being investigated as biocompatible materials satisfy the first two requirements and are transparent in the visible spectrum. Operation at a visiblewavelength has the added advantage that interferences from water, buffering agents, and naturally occurring chromophores are minimized. In this study, Hb was used as a model protein because of its strong, intrinsic absorbance at visible wavelengths. However, adsorption of any protein can be examined with IOWATR spectrometry by first labeling the protein with an extrinsic chromophore that absorbs at the intended laser line. This is a common practice in visible TIRF spectroscopy of protein adsorption where fluorescein and rhodamine labels are frequently e m p l ~ y e d . ~ ~ ~ The internal angle of a propagating mode in an IO waveguide is highly dependent on wavelength.12J3 Consequently, monochromatic sources are usually employed in IO waveguide experiments, which precludes measuring absorbance spectra of adsorbed molecular layers. This inability toacquire spectrais a major disadvantage of IOWATR in comparison to FT-IR/ ATR spectroscopy. Conformational changes that may occur in proteins upon surface adsorption can be studied by comparison of FTIR/ATR spectra of absorbed proteins with solution phase spectra.26 The results presented here show that quantitation of protein adsorption density by IOW-ATR is possible if the molar absorptivity of the protein film is known. In these experiments the molar absorptivity of adsorbed Hb was assumed equal to the bulk dissolved value. In future work, we intend to measure adsorbed protein molar absorptivities by validating the IOW-ATR technique through (23)Mirabella, F. M.; Harrick, N. J. InternalReflectionSpectroscopy: Reoiew and Supplement; Harrick Scientific: Ossining, NY,1985;p M. (24)Yacynych, A. M.; Mark, H. B., Jr.; Giles, C. H. J. PhYS. Chem. 1976,80,839. (25)Swalen, J. D.;Santo, R.; Tacke, M.; Fischer, J. IBM J. Res. Deu. 1977,2I,168. (26)Gendreau, R. M. In Spectroscopy in the Biomedical Sciences; Gendreau, R. M., Ed.; CRC Press: Boca Rnton, FL, 1985; Chapter 2.
Quantitation of Protein Adsorption Density comparison with a radioisotopic assay. Protein adsorption density can be quantitated in the presence of bulk dissolved protein by subtracting the calculated bulk contribution from the measured (raw) absorbance. The bulk absorbance contribution is calculated through application of the necessary equations as detailed in ref 12. Sperline et al. have validated this correction method in a FT-IR/ ATR study of cetylpyridinium adsorption onto zinc selenide.27 Thus with IOW-ATR, it is possible to measure ‘loosely bound” protein that may be removed by a rinsing step. Alternately,if the protein binds to the waveguide surface with high affinity, then the use of a sufficiently thick, multimode waveguide makes it possible to ignore the bulk absorbancecontribution altogether. This is a consequence of the inverse dependence of sensitivity on waveguide thickness, meaning that a relatively thick waveguide will be insensitive to low bulk concentrations.12 For example, although the higher order modes supported by waveguide 1 exhibited sufficient sensitivity to monitor a submonolayer of adsorbed Hb, the integrated bulk evanescentpathlengths for m = 2 and m = 3 were calculated to be only 0.015and 0.027 cm, respectively. Consequently, the bulk absorbance contributions to total absorbance were 14% over the entire range of bulk concentrations investigated and were therefore ignored in the adsorption density calculations. An additional advantage of using a multimode waveguide is that the differencein the refractive indices of the protein film and the bulk solution will minimally perturb the internal waveguidingangles in comparison to a singlemode waveguide. Listed in Table IV are the prism incoupling angles for waveguide 1measured before and after the Hb adsorption isotherm data were acquired (7.5h elapsed between measurements), the experimental angular shifts, and the respective confidenceintervalsthat were calculated from the Student’s t distribution table. The confidence intervals are greater than the experimental angular shifts for m = 0, 1, and 2, indicating that these shifts are statistically insignificant. Also listed in Table IV are the theoretically predicted shifts in the incoupling angles calculated for adsorption of a 100 A thick protein film with a refractive index of 1.50 onto waveguide 1. The theoretical angular shifts are less than the confidence (27)Sperline,R. P.;Muralidharan,S.; Freiser, H.Langmuir 1987,3, 198.
Langmuir, Vol. 7, No. 5, 1991 999 Table IV. Prism Coupling Angles for Waveguide 1 measured prism incoupling anglesasb pt-Hb angular shift pre-Hb adsorption adsorption experiment* theow 0 28.065 f 0.035 28.020 f 0.040 -0.045 f 0.049 +0.002 1 26.835 f 0.023 26.805 f 0.032 -0.030 f 0.036 +0.008 2 24.810f 0.037 24.825 f 0.040 +0.015 i 0.050 +0.017 3 22.150 f 0.065 +0.028 a Incoupling angles measured before and after acquisition of Hb adsorption isotherm data listed in Table I1 (elapsed period of 7.5 h). The postadsorption, m = 3 mode intensity was too weak to permit accurate measurement of the incoupling angle. Error limita denote the 95% confidence interval calculated from the student’s t distribution table (n = 3 in all trials). Theoretical shift in prism coupling angles calculated for adsorption of a 100 A thick film of refractive index 1.50 on waveguide 1. mode (TE)
interval of the angular measurement for each mode. Thus, both theory and experiment are in agreement that the two-phase approximation7J2employed here has no measurable effect on the computed protein adsorption densities. In summary, the results presented here demonstrate that IOW-ATR spectrometry can be used for quantitative, in situ studies of protein adsorption at the waveguide/ solution interface. The extremely high internal reflection densities characteristic of 10waveguiding makes possible submonolayer sensitivity. IOW-ATR is particularly well suited to the study of protein adsorption on polymers, which is difficult to investigate by more commonly employed spectroscopic techniques. In future work, we intend to investigatekinetic aspects of competitiveprotein adsorption, using simultaneous mode coupling of two or more laser lines to assay for proteins individually labeled with spectrally resolved chromophores. Another promising application for IOW-ATR spectrometry, based upon the Mb results, is determination of chromophore orientation in weakly absorbing, monomolecular films at the solid/liquid interface via linear dichroic ATR measurements.
Acknowledgment. This research was supported by NIH Grant HL32132 and biomedical research grants from the Whitaker Foundation. S.S.S. acknowledges fellowship support from the NSF Engineering Research Center for Emerging Cardiovascular Technologies.
