Langmuir 1994,10, 1314-1318
1314
A Comparison of Different Approaches To Calculate Surface Free Energies of Protein-Coated Substrata from Measured Contact Angles of Liquids Wiesje van der Vegt,* Henny C. van der Mei, and Henk J. Busscher Laboratory for Materia Technica, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands Received March 17,1993. In Final Form: January 20,1994" Contact angle measurements constitute the most widely-used method to assess interfacial free energies between a solid and a liquid. Axisymmetric drop shape analysis by profile, ADSA-P, has been suggested as an alternative method, which might have, both on experimental and on fundamental grounds, major advantages especially for the determination of yel of biological substrata in particular. Here, solid-liquid interfacial free enegies ysl of bovine serum albumin coated substrata as derived from ADSA-P experiments are compared with those calculated from measured contact angles of liquids on the bare substrata and on the dried, adsorbed protein layers, while making use of the equation of state, the geometric-mean equation, both neglecting and accounting for spreading pressures, and the Lifshitz-van der Waals/acidbase approach to convert the contact angle data into surface free energies. During ADSA-P, the protein was adsorbed from a protein solution droplet placed on the substratum. The liquid surface tension ylv and the contact angle 0 were measured as a function of time after positioning of the droplet and used to calculate the correspondingvalues for y d t ) using the Young equation. For the contact angle measurementa on dried, adsorbed protein layers, the substrata were coated in advance and air-dried and sessile drop contact angles on both the bare and the coated substrata were measured with a variety of liquids and used for the calculation of yd according to one of the thermodynamicapproaches mentioned. Statistical analyses of yd and Aysl data showed that, considering both bare and protein-coated substrata together, the geometricmean method was internally consistent with ADSA-P and that the equation of state and the Lifshitz-van der Waals/acid-base approach corresponded well with ADSA-P on an independent basis (PS 0.05). This simultaneouslyindicatesthat contact angle measurements on dried, adsorbed protein films have a relevance with regard to polymer/water/protein interactions. Considering the t = 0 data as representative for the bare substratum surface shows that the internal consistency of the approach accounting for spreading pressures is higher on poly(methy1methacrylate) than on fluoroethylenepropylene-Teflon as compared with those of the other approaches. This emphasizes that accounting for spreading pressures becomes more important as the materials surface free energies increase.
Introduction The interaction of biological fluids including proteins with a surface is, a t least partly, determined by the interfacial free energies present in the system. Moreover, the adsorption of proteins to solid surfaces precedes many processes of industrial fouling as well as the interactions of cells and bacteria with biomaterials. Unfortunately, the interfacial free energies between a solid, even without an adsorbed protein layer, and a liquid are extremely difficult to assess reliably. Contact angle measurements in combination with one of the thermodynamic approaches available to extend Young's equation to an equation that can be solved using the contact angle data constitute the most widely-used method to estimate these interfacial free energies. One such extension has been described by Neumann et a1.l assuming the existence of an equation of state, which can be used to derive the interfacial free energies in a solid-liquid-vapor system. Conversion tables are published to yield the interfacial free energy a t the solidliquid and the solid-vapor interface, ynl and ysv,respectively, from the measured contact angle of a liquid with known liquid surface tension, ylv.2 Also it has been suggested to separate the interfacial free energy into a published in Advance ACS Abstracts, April 1, 1994. (1) Neumann, A. W.; Good, R. J.; Hope, C. J.; Sejpal, M. J. Colloid Interface Sci. 1974, 49, 291. ( 2 ) Neumann, A. W.; Absolom, D. R.; Francis, D. W.; Van Oss, C. J. Sep. Purif. Methods 1980,9,69. e Abstract
dispersion component and a polar component3 Using this concept of dispersion and polar components, Busscher et aL4proposed to take into account the spreading pressure, re(rebeing the difference between the solid surface free energy in air,ys,and that in the presence of vapor molecules adsorbed from the liquid used, ysv).Van Oss et al.smade the most extensive separation of surface free energies, recognizing that the dispersion component results from all three types of Lifshitz-van der Waals forces, and that the polar component results from the hydrogen-donating and hydrogen-accepting functions, i.e., the acid-base interactions. Once these approaches yield the solid surface free energy (ynor ysv),the same approach also allows one to calculate the interfacial free energy yd between the solid surface and a liquid. Whereas contact angle measurements are relatively easy on inert, solid substrata, their measurement becomes tedious in the case of protein-coated surfaces. First, adsorbed protein layers have to be dried in order to remove the excess of free water when the sessile drop technique is used for contact angle measurements. This may stimulate conformational changes and rearrangement of the adsorbed molecules.6 Second, when using the sessile drop or the captive bubble method, the positioning of a droplet may give rise to so-called stripping-off of the (3) Kaelble, D. H. J. Adhes. 1970,2, 66. (4) Busscher, H. J.; Van Pelt, A. W. J.; De Jong, H. P.; Arends, J. J. Colloid Interface Sci. 1983,95, 23. (5) Van Oss, C. J.; Chaudhury, M.K.; Good, R. J. Chem. Reu. 1988, 88, 927. (6) Absolom, D. R.; Van Oss, C. J.; Zingg, W.; Neumann, A. W. Biochim. Biophys. Acta 1981, 670,74.
0743-7463/94/2410-1314$04.50/00 1994 American Chemical Society
Surface Free Energies of Protein-Coated Substrata
adsorbed proteins? which subsequently adsorb at the liquid-vapor interface, affecting ylvof the probing liquid. Also spreading pressure effects may be completely different on protein-coated substrata as compared to the effects on bare substrata. However, several researchers have obtained useful data about adsorbed protein layers and other proteinaceous surfaces using this technique.'-11 Recently, another approach has been proposed, based on axisymmetric drop shape analysis by profile (ADSAP)12 to determine the interfacial free energy changes involved in protein adsorption to a solid directly.1S15 This technique is an in situ method, based on the relation between the interfacial free energies present in a system consisting of a protein solution droplet on a solid substratum and the shape of that droplet. By observing only the changes in the droplet profile, both the contact angle 0 and the liquid surface tension ylv can be obtained simultaneously. Subsequently, combination with the equation of Young makes it possible t o estimate ysl as a function of time according t o
This approach has been taken for the adsorption of fibrinogen13 and albumin,13J4 and its advantages and disadvantages have been discussed in detai1.'3Js Clearly, there are two important uncertainties involved in the measurement of interfacial free energies of proteincoated surfaces: (1)Is the interaction between a liquid and a dried, adsorbed protein film as during contact angle measurements energetically comparable with the one in a three-phase (polymer/water/protein) system? (2) Which is the best thermodynamic approach t o be employed for the conversion of measured contact angles into surface free energies? It is the aim of this paper to address these issues by comparing the interfacial free energies yd between two bare and albumin-coated materials with different wettabilities and an aqueous liquid as derived from ADSA-Pexperiments with those calculated from measured contact angles of liquids on bare substrata and on dried, adsorbed protein layers while making use of various methods to convert the contact angle data into surface free energies.
Materials and Methods Materials. Fluoroethylenepropylene-Teflon (FEP-Teflon; Fluorplast, The Netherlands) and poly(methy1 methacrylate) (PMMA;Vink Kunststoffen BV, The Netherlands) were used as solid substrata. The substrata were cleaned ultrasonically in ethanol,99-100 3' 6 ultra pure grade (Boom BV, The Netherlands), and air-dried overnight. For the present experiments, bovine serum albumin (BSA; Sigma, A-4503) was used without further purification. The proteins were dissolved in 0.01 M phosphate buffered saline, pH 7.0 (PBS), to concentrations of 0.005 and 1.00 mpmL-1. (7) Van der Scheer, A.; Smolders, C. A. J. Colloid Interface Sci. 1978, 63, 7. (8) Schakenraad, J. M.; Noordmans, J.; Wildevuur, Ch. R. H.; Arends, J.; Bueecher, H. J. Biofouling 1989, 1, 193. (9)Absolom, D. R.;Neumann, A. W. Colloids Surf. 1988, 30, 25. (10)Baszkin, A.; Lyman, D. J. J. Biomed. Mater. Res. 1980,14,393. (11)Amory, D. E.;Rouxhet, P. G. Biochim. Biophys. Acta 1988,938, 61. (121 Rotenbera, Y.:Boruvka,. L.:. Neumann. A. W. J. Colloid Interface Sei; 1983, 93, 166.. (13)Voigt,A.;Thiel,O.; Williis,D.;Policova,Z.;Zingg,W.;Neumann, A. W. Colloids Surf. 1991.58. 315. (14)Busscher, H.J.; Van der Vegt, W.; Noordmans, J.; Schakenraad, J. M.: Van der Mei. H. C. Colloids Surf. 1991.58. 229. (15)Van der Veh, W.; Van der Mei, H. C.; Buskher, H. J. J. Colloid Interface Sci. 1993, 156, 129. '
Langmuir, Vol. 10, No. 4, 1994 1315 Profilometry indicated that the stylus surface roughness RA of the substrata ranged from 0.3-0.5 pm, i.e., well in the submicrometer range. Thus, it is unlikelythat roughnesseffects will invalidatethe thermodynamicstatus of the measured contact angles.'e Protein Adsorption, Contact Angle Measurements and Interfacial Free Energy Calculations. For the contact angle measurements on dried, adsorbed protein layers, the protein was allowed to adsorb from a 35-mL protein solution in polystyrene Petri dishes (94/16mm). Two substrata (10 X 30 mm) were put in each Petri dish. After 5 min one substratum was removed, dip-rinsed twice in demineralized water, and dried at 30 O C for 2 h. After 60 min of adsorption, the other substratum waa taken out and the same procedure was repeated. These types of experiments were performed in triplicate at room temperature. After drying, so-called plateau contact angles" (reached after 2 h) were determined by the sessile drop technique at room temperature with 0.5-1.0-pL droplets of water, formamide, diiodomethane, a-bromonaphthalene, and a series of water/lpropanol mixtures. Droplets of each liquid were put on one substratum (bare as well as protein-coated) for contact angle measurements while care was taken not to wet the same spot twice. The data obtained were used for calculation of the solidliquid interfacial free energy ya according to the following approaches: (1)the equation of state,lV2(2a) the geometric-mean equation4 neglecting re, (2b) the geometric-mean equation' accounting for r e , and (3) the Lifshitz-van der Waals/acid-base approach.6 In the first method the interfacial free energies are related by
which, combined with the Young equation, yields
In the present application of this approach, only water contact angles were used to obtain ya and published computer tables2 were employed rather than calculating the data from eqs 2 and 3 ourselves. In the geometric-mean approach (method 2) the relation between the interfacial free energies is expressed as (4)
where yd and y p represent the dispersion and polar components of y,respectively. Combination with the Young equation gives
in which redenotes the spreading pressure, being the difference between the solid surfacefree energy in air and that in the presence of vapor molecules from the liquid used for contact angle measurements. In the case of neglecting re (method 2a), the contact angles of the pure liquids with known dispersion and polar surface free energy components were used to fiid 7," and y,Pby least-squaresfitting to eq 5 and ya was calculated from eq 4 and the known surface tension components of water.18 While assuming that uehas a fixed value independent of the type of liquid used (method 2b), y: was calculated with the aid of eq 5 and the contactangle of a-bromonaphthaleneon the solid surface, assuming that yf = 0 and re= 0 for a-bromonaphthalene (an almost completely apolar liquid). Subsequently,contact angles (16)Busscher, H.J.; Van Pelt, A. W. J.; De Boer, P.; De Jong, H. P.; Arenda, J. Colloids Surf. 1984,9, 319. (17)VanDijk, J.; HerkstrBter,F.;Bwher, H.; Weerkamp,A.;Jansen, H.; Arends, J. J. Clin. Periodontol. 1987, 14, 300.
1316 Langmuir, Vol. 10, No. 4, 1994
van der Vegt et al.
Table 1. Averaged Contact Angles (deg) with Standard Deviations over Three Separate Experiments for Bare Albumin-Coated Substrata.
(t
contact angle on dried adsorbed protein layers sub-
%?
stratum FEP 0.005 1.00
PMMA 0.005 1.00
t
(min)
0 5 60 0 5 60 0 5 60 0 5 60
form- diiodo- a-bromowater amide methane naphthalene 105f 1 93+1 94 f 2 105 f 1 91*3 82 + 6 67 i 1 68*2 66f0 67 + 1 71 + 1 70 f 2
78 f 2
89f3 85+1 6 8 + 1 85 + 0 71 f 1 89 3 78f2 77 f 3 68+6 82+1 68+2 52 + 2 36f2 46*4 3 6 a 3 4 8 t 3 35 f 2 52 + 2 36+2 33f1 40+ 1 31 + 1 38f2
*
71+1 62f2 61+2 71f1 54t6 52f2 15+1 22i1 19+0 15f1 26f1 25+1
water/l-propanol mixtures (%)
0.6 1.5 2.7 4.0 6.5 103f3 100+1 97f1 9 4 f 1 8 8 f 2 92+2 88f3 8 9 f 2 85+1 82+2 92fl 90f2 86f2 85f1 81f1 103f3 100f1 97f1 9 4 f 1 88+2 79f7 86+3 7 8 + 0 78f2 68f4 80f11 79+8 7 6 f 4 76f4 67f4 62f2 62fl 59f1 56f2 50f2 65f3 62f2 59fl 54fO 5 1 1 2 62f2 62f2 59f1 53+l 4 9 f 2 62f2 6 2 f l 5 9 f l 56f2 50f2 59f4 5 6 f 4 55i1 50f5 42f7 65f3 61f2 55f2 50*6 45+2
10.4
15
40
82t3 74+3 74fO 82+3 71f2 67f6 47f4 46+1 43i2 47f4 34f2 39+2
74f2 65fl 67+1 74f2 62f3 61f3 31f3 33fl 34f3 31+3 24+3 25f3
55+2 48f1 47+1 55f2 50+4 39+2
= 0) and
contact angle of a protein solution droplet by ADSA-P ~
Of0 Of0
Of0 OtO O+O Of0
10412 104f3 100+3 110 0 104+3 97 2 70 + 1 70fl 68+2 66*3 65 + 2 64fl
* *
The proteins were adsorbed from PBS solutions with two protein concentrations c during different adsorption periods t. of water and water/l-propanol mixtures were least-squares fitted to eq 5 to yield -yt and us,and hence yd could be calculated from eq 4. According to the third method, the interfacial free energies are related by
where y!iwdenotes the dispersion component of
ya1with
and -@ denotes the polar component of yakwith
Substituting eqs l a and l b into eq 6 results in
of the droplet. B ( t ) and yl&) were then calculated from the changes in the droplet profile and used to calculate y d ( t ) by eq 1.
Values for ylv were assumed to be constant and obtained from the contact angles on the clean substratum using the approach, 1,2a, 2b, or 3, under consideration. However, this last point is only important in case absolutevalues for Ya(t)are to be obtained, because the changes AT&) can be calculated on the basis of ADSA-P measurements,without any assumption concerning the value of yo" since Pya, = 0 and therefore
All experiments were done in triplicate at room temperature. Statistics. Both the 711and the Aya data were statistically analyzed for the two substrata together as well as for the hydrophobic and hydrophilic substratum separately. The data obtained by one approach were combined to form a group and were subsequentlycompared with the data in another group for the same experimental conditions but from another approach. All groups were analyzed to test the hypothesis of no difference between the approachesused to determineydand Aya by pairwise comparisons between the groups with Student's t test.
Finally, combining eq 8 with the Young equation and neglecting ue gives
Rssults
In this approach, contact angles of diiodomethane and a-bromonaphthalene were used to obtain ytWassuming that these liquids are completely apolar, whereas contact angles of water and formamide yielded yf. Again, yFWand y y were obtained from the literature18 to calculate ya from eq 8. In all approaches, contact angle data for the bare substrata were employed to calculate the intial value yd(0) so that the absolute value of yd of the dried, adsorbed protein layer as well as the change in yd caused by the adsorption of proteins can be calculated. Protein Adsorption, Adsymmetric Drop Shape Analysis by Profile, and Interfacial Free! Energy Calculations. ADSA-P was carried out as described by Noordmans and B u s s ~ h e r .The ~ ~ substratum was put on a plateau containing a reservoirfiied with water, and the protein was allowed to adsorb from 1OO-pLprotein solution droplets positioned on top of it. As soon as the droplet was placed, a small glass chamber (50 X 50 X 30 mm) was positionedaround the substratum and the reservoir to prevent evaporation. The first image that could be taken was assumed to represent t = 0. For all experiments, measurements were done as a function of time at 0,5, and 60minafter positioning
All contact angles measured are compiled in Table 1for completeness. Table 2 summarizes the calculated solid-liquid interfacial free energies yal for all the experimental combinations. Since calculation of absolute values of ysl by ADSA-P requires knowledge of yavtaken from one of the thermodynamic approaches described, four groups of ADSA-PIidata were obtained, in which the number i refers to one of the approaches 1-3. As can be seen in Table 2, the absolute values of yd calculated by ADSA-PIi are influenced by the approach chosen. Considering the t = 0 data in Table 2, representative for the bare polymeric surfaces, it can be seen that all approaches yield more or less similar ~~1 values for FEP-Teflon. On PMMA, the more polar material, the values for yalinvolving approach 2b, accounting for spreading pressures, gives significantly lower ysl values. Furthermore, it is obvious that the interfacial free energy ysl against an aqueous liquid is smaller for PMMA than for FEP-Teflon. Table 3 gives the relative changes of ysl due to the adsorption of proteins to the solid surface. As no knowledge of ysv is required to calculate Ayal, the thermodynamic approaches considered can be compared with one set of independent ADSA-P data now. Table 4 presents the results of the statistical analysis carried out. In each case, comparisons are made for all yel as well as all Ayal data for the FEP-Teflon and PMMA
(18)Bellon-Fontaine,M. N.; Mozes, N.; Van der Mei, H. C.; Sjollema, J.; Cerf, 0.;Rouxhet, P. G.;Buascher, H. J. Cell. Biophys. 1990,17,93. (19)Noordmans, J.; Buescher, H. J. Colloids Surf. 1991,58,239.
Langmuir, Vol. 10, No. 4,1994 1317
Surface Free Energies of Rotein-Coated Substrata
Table 2. Averaged Solid-Liquid Interfacial Free Energier r a with Standard Deviationr over Three Separate Expc”ntr for Albumin-Coatad Subrtrata A8 Derived from Adsymmetric Drop Shape Analyrir by Profile and from Conhat Angler Combined with One of the Thermodynamic Approach 1-3 (See Text) To Convert Mearured Contact A n g h on Solidr into Surface Free Enerrtido. ~
7.1 (mJ.m-9
subotratum c (mpmL-1) t (min) ADSA-P/1 ADSA-P12a ADSA-PI2b ADSA-PI3 1 FEP 0.005 0 36.8f 2.7 36.7 f 2.7 29.0f 2.7 36.6 f 2.7 38.8f0.8 6 34.6 f 3.2 34.4 f 3.2 27.7 f 3.2 34.3 f 3.2 30.8 f 0.6 60 28.4 f 4.2 28.3 f 4.2 21.6 f 4.2 28.2 f 4.2 31.8 f 1.0 1.00 0 37.6f 1.6 37.4f 1.6 30.7 f 1.6 37.3f 1.6 38.8f 0.8 6 32.1 f 1.6 32.0 f 1.6 26.3 i 1.6 31.9 f 1.6 29.6 f 2.1 60 26.6 f 1.6 26.6 f 1.6 18.7 f 1.6 26.4 f 1.6 23.9 f 3.7 PMMA 0.006 0 18.1 f 2.2 22.0 f 2.2 7.1 f 2.2 17.2 f 2.2 14.9 f 0.6 6 19.9 f 2.6 23.8 f 2.6 8.9 f 2.6 19.0 f 2.6 16.6 f 1.0 60 22.6f 0.7 26.4f 0.7 11.6f 0.7 21.6f 0.7 14.4f 0.2 1.00 0 19.1 f 1.7 23.0f 1.7 8.1 f 1.7 18.2f 1.7 14.9f 0.6 6 19.8 f 1.8 23.7 f 1.8 8.8 f 1.8 18.9 f 1.8 17.1 f 0.6 60 20.3 f 0.2 24.2 f 0.2 9.3 f 0.2 19.4 f 0.2 16.6 f 1.4
2a
39.8i 1.6 30.9 f 0.7 32.0 f 1.1 39.8f 1.6 30.2 f 1.2 23.0 f 4.8 21.8 i 0.2 22.0 f 1.1 21.0f0.2 21.8i0.2 22.6 i 0.7 22.4 f 1.7
2b 36.3f 4.9 21.3 1.6 22.7 f 0.9 36.3f 4.9 16.1 i 3.1 12.1 i 7.0 8.0 f 0.6 8.6 f 1.3 7.7 f 0 . 2 8.0f 0.5 8.2 f 1.1 9.4 f 1.6
*
3 37.3i 3.7 20.6 i 1.3 23.6 f 2.9 37.3a 9.7 26,s i 7.0 6.6 f 8.2 13.8 t 0.7 17.6 i 3.4 14.4f 1.3 13.6f0.7 22.6 0.7 22.2 2.0
*
a The proteins were adsorbed from PBS solutions with two protein concentrationsc during different adsorption periode t. The aboolute values for y&) by ADSA-P were obtained using the yrr calculated from approach i with which the ADSA-P data are to be compared, and hence them results are denotad aa being obtained by ADSA-PIi.
Table 8. Averaged Changer in the Solid-Liquid Interfacial Free Energier 7.1 with Standard Deviationr for Albumin-Cwtad Subrtrata As Derived from ADSA-P and from Contact Angler Combined with the Thermodynamic Approach- 1-1(See Test). Aya (mJ-m-2) substratum c (mpmL-9 t (min) ADSA-P 1 3a 2b 3 FEP 0.005 6 -1.3 f 1.3 -8.1 f 1.0 -8.9 f 1.7 -16.0 f 6.1 -16.8 8 9 60 -7.4 f 1.6 -7.0 f 1.3 -7.8 f 1.9 -13.6 f 4.9 -13.8 4.7 1.00 6 -6.4 f 2.7 -9.2 f 2.2 -9.6 f 2.0 -20.2 f 6.8 -11.8 i 7.9 60 -11.9 f 2.9 -14.9 f 3.8 -16.8 f 6.1 -24.2 f 8.4 -30.8 9.0 6 1.7 f 1.8 0.7 f 1.1 0.2 f 1.2 0.5 f 1.4 3.9 f 3.6 PMMA 0.005 60 4.3 f 2.8 -0.6 f 0.6 -0.8 f 0.2 -0.3 f 0.6 0.8 i 1.6 0.8 f 0.7 6 0.7 f 0.2 2.2 f 0.7 0.2 f 1.2 8.9 i 1.0 1.00 60 1.2 f 1.7 1.7 f 1.6 0.6 f 1.7 8.6 f 2.1 1.4 f 1.6 a The proteins were adsorbed from PBS solutions with two protein concentrationsc during different adsorption periode t. Note that the values for AT&) by ADSA-P are obtained independently from any theoretical approach.
** *
T8bb 4. t Valuer Obtained with a Paired t Teat To Test the Hypothesir of No Difference between the Various App-cheo To Convert Measured Contact Angler On solids into Surface Free Energier. approach ADSA-PIi 1 2a
2b 3
ADSA-P 2.23 2.88
6.12 3.69
1 FEPb
2a
0.01
-0.29 -1.14
3.02
2b 0.48 4.29 4*93
8.10
6.81
3.06
3.06
0.02
2.60 -26.71
0.98 22.00 68.94
3 2.15
2.86 3*38
PMMAb ADSA-Pli 1 2a
2b 3 ADSA-PIi 1 2a
2b 3
6.66 0.69 1.54 1.43 -1.32
3.22 1.18
-0.83
-3.30 -3.31 FEP + PMMAE 2.30 0.92 2.05 -3.61 3.03 4.67 3.04 2.86 2.33
-2.93
1.18
-1.31
0.70
0.39
0.72
8.30 9.76
1.01 -1.33
3.04 -6.86 2.27
1.44
4.48 -2*93
a The upper riehtpartofeach section p e m to the YII data while the bottom left part refere to the Aya data. T h e top section includes data for FEP-Teflon, the middle section is for PMMA only, a d the bottom section includes all data. Italic-font data represent t values for which p 0.06. b For yd there are 6 degrees of freedom (t0.W = 2.671), while for ~ y there a are 3 degrees of freedom (t0.W = 3.182). c For 7.1 there are 11degrees of freedom (t0.W = 2.201), while for Aya there are 7 degrees of freedom (t0.U = 2.366).
data separately and for the combination of the data on both substrata. Taking a good correspondence (P I0.05) between ~a data of ADSA-PIi and of any of the approaches
1-3 as an indication of internal consistency of the approach, we can see from Table 4 that all approaches perform satisfactorily on FEP-Teflon, but not on PMMA. For the combination of substratum data, only approaches 2a and 2b are internally consistent at a significance level of P I 0.05. Alternatively, a good correspondence (PIO.05) between A Y data ~ of ADSA-P and of any of the approaches 1-3 can be taken as an indication of a good performance of the approach on an independent basis. Such a satisfactory performance can be seen for all approaches on PMMA, but only for approaches 1 and 2a on FEPTeflon. On the combined Aradatafor the both substrata, a satisfactorycorrespondencebetween the data is observed for approaches 1 and 3.
Discussion In this paper, changes in interfacial free energies caused by the adsorption of proteinsto solid surfaces are measured directly by axisymmetric drop shape analysis by profile and by different indirect approaches involving dried, adsorbed protein fiisfor both a hydrophobic FEP-Teflon and a more hydrophilic PMMA substratum. Different adsorption times and protein concentrations were employed in order to create different typesofa&rw prohi,, layers. The data in Table 2 demonstrate that the surface energetics of the adsorbed protein layer vary at least between t = 5 min and t = 60 min and poesibly longer.’6 Furthermore, the interfacial free energy decreases due to protein adsorption are clearly larger on FEP-Teflon as the protein concentration is increased,but not on PMMA. This is probably because the changes in interfacial free energies due to protein adsorption on PMMA hover about zero (see also Table 3).
1318 Langmuir, Vol. 10, No. 4, 1994 Table 6. List of Approaches to Convert Measured Contact Angles into Surface Free Energies, Which Show Internal Consistency and a Good Independent Correspondence on the Basis of a Comparison with ADSA-P for the Bare and Protein-Coated Substrata Together. subtratum P I0.05 P I0.01 Internal Consistency on the Basis of yd Data FEP ADSA-P/i-l,2a, 2b, 3 ADSA-P/i-l,2a, 2b, 3 PMMA ADSA-P/i-2b, 3 ADSA-P/i-2a, 2b, 3 FEP + PMMA ADSA-P/i-2a, 2b ADSA-P/i-l,2a, 2b, 3 Independent Correspondence on the Basis of Ayd Data FEP ADSA-P-l,2a ADSA-P-l,2a, 3 PMMA ADSA-P-l,2a, 2b, 3 ADSA-P-l,2a, 2b, 3 ADSA-P-l,2a, 2b, 3 FEP + PMMA ADSA-P-1,3 "Two significance levels are included to base the degree of similarity upon.
ADSA-P as a technique to measure interfacial free energy changes due to protein adsorption has a number of advantages over indirect contact angle measurements: (1)ADSA-P makes it possible to do in situ measurements, while contact angle measurements imply distortion of the adsorbed layer through rinsing, drying, and the presence of the reference liquid itself. (2) Both the liquid surface tension and the contactangle are simultaneouslymeasured. (3) Contrary to contact angle methods, ADSA-P does not make use of any controversial thermodynamic theory. (4) ADSA-P is comparatively easy to perform and less time consumingthan indirect contact angle methods. However, as a severe disadvantage, ysl(0) is hard to determine. Previous analysesls have shown that this may give rise to deviations in yd0) of up to 5 mJ.m-2. However, since the indirect approaches used are all based on much more dubious theoretical assumptions and experimental procedures,we have decided to use the ADSA-P data obtained in this paper as a "gold standard" to compare the results of the independent approaches with. Table 5 lists the approaches which are internally consistent for bare and protein-coated substrata at the significance level P I0.05 (comparison of ysl data from ADSA-P/i)and which perform well at the same significance level on an independent basis (comparison of Ayd data from ADSA-P). Since, however, P I0.05 may be a very strict criterium, we have extended Table 5 with a similar comparison at a significancelevel of P I0.01 Noteworthy conclusions are that most approaches are internally consistent, except approach 1on PMMA at both P I0.05 as well as P I0.01, and approaches 1 and 3 considering both substrata at P I0.05. On an independent basis only
van der Vegt et al. Table 6. t Values Obtained with a Paired t Test To Test the Hypothesis of No Difference between ADSA-P/i and the Various Approaches i To Determine 7.1 of the Bare Polymeric Substrata (Le., the t = 0 Data) substratum 1 2a 2b 3 -2.53 -3.82 FEPa -7.59 -1.00 PMMAa 7.40 1.40 -0.80 8.20 0.45 -1.05 FEP + PMMAb -1.91 1.09 0 There is 1degree of freedom; t0.m = 12.706. There are 3 degrees of freedom; t0.m = 3.182.
approach 1performs well at a significancelevel of P I0.05 in all situations. At the lower significance level P I 0.01 all approaches perform well on an independent basis except approach 2b on FEP-Teflon. From the above comparisons,one may draw the general conclusion with regard to the first uncertainty mentioned in the Introduction of this paper that contact angle data and calculated surfacefree energiesfor biological substrata (protein-coatedmaterials,microorganisms,cells, etc.) have a definitive relevance for polymer/water/protein interactions, despite the fact that the substratum is often a dried, adsorbed protein film or similar type of substratum. A way to evaluate the merits of the different approaches to convertmeasured contact anglesinto surface free energy values, therewith addressing the second uncertainty mentioned, can be based on t = 0, Le., the bare polymeric surface data (see also Table 2). For all approaches, these ADSA-PIi results compare well with those of approach i as such ( P I 0.051,proving internal consistency (see Table 6). Although ysl values are different for the various approaches,approach 2b yields the most deviatingresults, especially on PMMA (see Table 2). This is due to the fact that only approach 2b accounts for spreading pressures. Accounting for spreading pressures becomes increasingly important as the material surface free energy is higher,m*21 which is probably the reason why the internal consistency of approach 2b is higher than that of the other approaches neglecting spreadingpressures on PMMA but not on FEPTeflon (see Table 6). Acknowledgment. The authors are greatly indebted to Dr. R. E. Stewart for his advise concerningthe statistics included in this paper and to Mrs. M. Schakenraad-Dolfing for assistance in the paper preparation. (20) Busscher, H. J.; Van Pelt, A. W. J. J. Mater. Sci. Lett. 1987, 6, 815. (21)Tadros, M.E.;Hu,P.;Adamaon, A. W. J. Colloid Interface Sci. 1974,49,184.
