Langmuir 1993,9, 2430-2435
2430
Ellipsomet ric Characterization of Streptavidin Binding to Biotin-FunctionalizedLipid Monolayers at the Water/Air Interface R.Reiter, H.Motschmann, and W . Knoll"9t Max-Planck-Institut fiir Polymerforschung, Ackermannweg 10,0-5512%Mainz, FRG Received March 9,1993. In Final Form: June I , 1993
Ellipsometricstudies of the streptavidin binding to a biotin-functionalized phospholipid monolayer at the water/air interface have been performed as a functiop of the lateral pressure of the monolayer. It is found that the protein monolayer formation upon specific binding is characterizedby a "spontaneous" (diffusion-limited)thickness increase followed by a slow reorganization process with a further thickness increase. The kinetic p a r w t e r s as well as the final thicknesses are stronglydependent on the phase state of the lipid monolayer. Strong evidence is given that the binding is completely blocked if the lipid is in a solid-condensed phase. 1. Introduction
The specific binding of the tetrameric protein streptavidin to various surfaces functionalized by biotin (vitamin H)is currently studied in many laboratorie~l-~ as a model reaction between a receptor bound to a planar substrate and a ligand diffusing from the adjacent aqueous medium to the solid/solution interfa~e.89~In addition to the fundamentalinterest in such highly specificbiorecognition reactions and the elucidation of the parameters that control their effectiveness,1°there is a practical aspectto this work, arising from the currently very active efforts to develop sensitive and specific biosensor configurations. As far asplanarmodelmembranesurfacesareconcerned, functionalized by biotin moieties, there are basically three different model systemsavailable: (i) monomolecular lipid layers a t the water/air interface, (ii) Langmuir-BlodgettKuhn multilayer assemblies1' built up by transferring these layers to solid substrates, and (iii) self-assembly monolayers formed,12 e.g., by the adsorption of long-chain o-functionalized thiols on gold or silanes on glass substrates. The two latter systems are particularly relevant for the optimization of sensor applications. These model mem*To whom correspondence should be addressed at the MaxPlanck-Institut ftir Polymerforschuug. + W.K.ia alsowithTheInstitute of Physical and ChemicalResearch (RIKEN),Frontier ResearchProgram,2-1 Hirosawa, Wako,Saitama 351-01, Japan. (1) Blankenburg, R.; Meller, P. H.; Ringsdorf, H.; Salesse, C. Biochemistry 1989,28,8214. (2) Schmidt, F.-J.; Knoll, W. Biophys. J. 1991, 60, 716.
U.S.A. (3) Helm,C.A.;Knoll,W.;Israelachvili,J.A.oc.Natl.Acad.Sci. 1991,88,8169. (4) Morgan, H.; Taylor, D. M.; DSilva, C.; Fukuehima, H. Thin Solid Films 1992, 2101211 , 773. (5) Vaknin, D.; Als-Nielsen, J.; Piepenstock, M.; Lbche, M. Biophys. J. 1991, 60, 1646. (6)Herron, J. N.; Miiller, W.; Paudler, M.; Riegler, H.; Ringsdorf, H.; Suci, P. A. Langmuir 1992,8, 1413. (7) Ku, A. C.; Darst. S. A.; Kornberg, R. D.; Robertaon, C. R.; Gast, A. P. Langmuir 1992,8,2357. ( 8 ) HBusslina, - L.: Rinmdorf, H.: Schmitt, F.-J.: Knoll, W. Langmuir
1991, 7, 1837. (9) Schmidt,A.;Spinke,J.;Bayerl, T.; S a c k " , E.; Knoll,W. Biophys. J. 1992, 63, 1385. (10) Spinke, J.; Liley, M.; Schmitt, F.-J.;Guder, H.-J.; Angermaier, L.;
Knoll, W. J. Chem. Phys., in press. (11) Kuhn, H.; Mbbius, D.; BBcher, H. In Physical Methods of Chemistry; Weissberger, A,, Rossiter, B. W., Eds.; Wiley: New York,
1972. (12) Ulman, A. An Introduction to Ultrathin Organic Films from Langmuil-Blodgett to Self-Assembly;Academic Press: San Diego, 1991.
branes suffer, however, from the fact that the molecules carrying the recognition labels are in an amorphous state, not allowing for a fluidlike response of the lipid matrix. Attempts are therefore necessary to restore some mobility by linkingthe biotin moiety to the lipid via a flexiblespacer grOUP. If one is primarily interested in the physicochemical principles of these interfacial recognition and binding reactions at a biomembrane-like surface, the Langmuir monolayers prepared a t a water/air interface offer the unique experimental features that the lateral packing density and hence the monolayer phase behavior can be externally controlled by the moving barrier. This allows for the investigation of the question of how the physical state of the lipid matrix determines the binding characteristics of incorporated receptors. Moreover, the water surface is perfectly flat (except for thermally excited capillary waves) which ensures that the observed binding reactions are not disturbed by the roughness of the surface of most solid substrates used in the studiesa t solid/solution interfaces. In passing we note that only mica substrates offer the same advantages as the water surface which has been employed in studies with the surface forces apparat~s.~J~ In this paper we present ellipsometric data on the binding of streptavidin to a lipid monolayer doped with 5 mol % biotinylated lipid. In particular, the kinetics and the equilibrium protein layer formation are studied as a function of the lateral pressure of the lipid matrix. 2. Experimental Section 2.1. Ellipsometer. Ellipsometric measurements were performed using a home-built automatically recording Null ellip someter in a vertical PCSA arrangement. The ellipsometer ie
described in detail elsewhere." A 5-m W HeNe laser (A = 633 nm) with an effective beam diameter of about 1 mm2served as the light source. According to standard procedure^,^^ the ellipsometric angles A and were deduced from two zone measurements (zoneI and zone 111)of the polarizer and analyzer extinction settings. The difference of the readings in the two zones was less than 5 / 1 ~ 0 proving , that the optical components were well aligned. The angleof incidencewas chosento be slightly below 50' (Brewsterangle - 5 3 O ) to obtain an adequatesensitivity (13) Leckband, D. E.;Israelachvili,J.;Schmitt, F.-J.;Knoll, W. Science 1992,255,1419. (14) Motechmann, H.; S t a " , H.; Toprakcioghlu, C. Macromolecules 1991,24, 3681. (15) Azzam, R. M.; Bashara, N. M. Ellipsometry and PolarizedLight; North Holland Publication: Amsterdam, 1979.
0743-7463/93/2409-2430$04.00/0 0 1993 American Chemical Society
Langmuir, Vol. 9, No. 9, 1993 2431
Streptavidin Binding to Lipid Monolayers in the angle A, which is the only exploitable quantity when investigatingtypical lipid monolayers. An increasein the optical contrast of the f i i with respect to the subphaea due to protein adsorption also leads to sensitivity in the ellipsometric angle 9. In this case it is poesible to determine a range of f i i parameters (nl,dl) by analyzing both measured quantities via Fresnel's formulas.'6 2.2. Langmuir Trough. Monolayers were prepared on a home-built computer-controlledLangmuir trough with special
featuresfor the integrationin the ellip~meter.~~ The all-Tefloncoated trough was placed into an acrylic glass box with a slit in the top cover to allow for the unhindered passage of the laser beam. The temperature of the subphase was regulated by a thermostat. The relative humidity was independentlymeasured to reach 95-99 % . A Wilhelmy system was used to measure the surface pressure. The whole setup was mounted on a vibrationisolated table to provide a smooth surface. 2.3. Materials and Method. Biotinylated phospholipid dipalmitoylphosphatidylethanolamine(DPPE) (5mol % ) with an e-aminocaproylspacer was cospread with dimyristoylphosphatidylethanolamine (DMPE) on an aqueous subphase containing 0.5 M NaC1. The refractive index of the subphase was determined with an AbM refractometer to be 1.3365. All experiments were carried out at a temperature of 20 f 0.1 O C . After evaporation of the delivering solvent (chloroform) the barrier was moved to producethe desired area per lipid molecule. For this particular phase of the monolayer the ellipsometricangles were measured several times to define the reference for the followingadsorption process. The protein solutionwas injected using a microsyringewith an &cm-longbent drain tube. It was inserted behind the barrier and pushed underneath the monolayer. In order to produce a homogeneous distribution of the protein, the microsyringe was moved carefully back and forth while injecting. The final protein concentration in the entire trough was 5 X lo-' M for most experiments. After a delay of about 2 min given by the duration of the injection process and the relaxation of the perturbed surface, the ellipsometric measurement was started. A set of ellipsometricangles A and was automatically recorded every 20 s up to several hours.
*
3. Results 3.1. Biotin-FunctionalizedLipid Monolayer. Prior to any binding study we characterized the phase behavior of a DMPE monolayer doped with 5 mol % biotinylated lipid. Figure l a shows the pressure-area (*-A) isotherm taken at T = 20 "C with the subphase containing 0.5 M NaC1. As reported for the pure DMPE this mixture, too, exhibits an extended range of a fluid analogue phase (2) with a sharp change of the compressibility at ca. a = 6 mN/m, indicating the onset of the first-order phase transition to the condensed state. The coexistence region (3) is well-pronounced and turns into the condensed state (4 and 5 ) with no clear evidence for any further transition. The ellipsometricisotherm (A-A curve) is given in Figure lb. The different phase regions seem to be characterized by distinctly different slopes, dAld.4 (thin broken lines), with a clear break a t the onset of the fluid-ordered phase transition. The three last A points in Figure l b may actually indicate the end of the phase coexistence. The pure DMPE monolayers exhibited virtually identical A-A isotherms; i.e., the relatively dilute biotin labels are not visible in the ellipsometric measurement. The following adsorption studies were performed at various monolayer densities as indicated by the arrows in Figure la. However, since we neither were interested in a characterization of the 2D streptavidin crystal formation typically performed in the gas analogue phase nor considered the heterogeneous two-phase coexistence range (16) Born, M.; Wolf, E. Principles oj0ptics;PergamonPress: Oxford, 1970. (17) Motachmann, H.; biter, R.; Lawall, R.; Duda, G.; Stamm,M.; Wagner, G.;Knoll, W. Langmuir 1991, 7, 2743.
.40
30
.80
1 .oo
molecular area A /nm2
180.0
\ 2.
4
I
I
\.;
40
.60
.80
1 .oo
molecular area A /nmz
Figure 1. (a) Preasure-area (PA) isotherm of a DMPE monolayerdoped with 5 mol % biotinylatad DPPE at T = 20 O C . The subphase contained 0.5 M NaC1. The arrow with the numbere refer to the streptavidin binding experiments. (b) Ellipsometric isotherm (A-A curve) of the same monolayer. particularly suitable for ellipsometric studies, we concentrated the kinetic binding experiments to the homogeneous phases, i.e., to the fluid state at ?r = 5 mN/m and to the condensed state at two different lateral pressures, a = 15 and 40 mN/m. The monolayer at the lower pressure actually still showed some remaining highly fluorescing areas between the ordered domains covering, however, less than 10% of the trough area. 3.2. Binding of Streptavidin to a Biotinylated Monolayer in Different Phase States. Figures 2 and 3 summarize the ellipsometric data taken from the monolayer-covered water surface for up to 5 h after the streptavidin had been injected (and distributed) into the Langmuir trough at t = 0 to give a final concentration of c = 5 X le7 M. Figure 2 gives the data obtained with the lipid layer being in a fluid state at a = 5 mN/m (cf. Figure la). Plotted are the differences of the A values (Figure 2a) and of the 9 values (Figure 2b), observed after the protein injection,relative to the values found prior to that. The open circles in both plots were obtained for the biotinylated lipid layer (labeled + biotin); the full triangles were for the pure DMPE layer (labeled- biotin), indicating a negligible unspecific adsorption to the lipid headgroup/ solution interface. A few observations should be emphasized (i) Within the delay in data recording due to the injection process and its disturbance of the water surface by waves which took about 2-3 min, there is an instant change of both ellipsometric angles. (ii) This fast rise is followed by a second process with a much slower change of the optical data. Ita relative contribution to the final equilibrium state which is reached only after 4-5 h amounts to ca. 30% of the total change upon protein binding. (iii) The time course and the relative amplitude of this second process as inferred from the A values are different from those obtained from the 9 data. Figure 3 presents the data monitored for the condeneed phase. The open circles (curves labeled 4, cf. Figure la)
Reiter et al.
2432 Langmuir, Vol. 9, No. 9, 1993
*I k
j I
1L
0 0
1
k
3
2
,
4
,, i
, ,5 , , , ,
time t Ih
I
I-
.15g (b)
.,a .05
I
.E
0
1
2
3
4
5
timet I h
Figure 2. Change of ellipsometric angles 6A (a) and 6 q (b) relative to the bare lipid monolayerafterinjectionof streptavidin (to give a find concentration c = 5 X le7M) at t = 0. Open circles (labeled + biotin) correspond to a monolayer in the expanded state at K = 5 mN/m (labeled 2, cf. Figure la); full triangles (labeled - biotin) were from a biotin-free DMPE monolayer. 5
f
lo-' M)are monitored. Again, two distinctly different processes contribute to the final changes. All parameters characterizing these changes (time course, final equilibrium) are different from the data taken in the fluid phase and, obviously, depend on the lateral pressure of the monolayers.
(a)
4F
4. Discussion 4.1. Lipid Monolayer Characterization. As we had discussed before the ellipsometric analysis of the optical thickness of a lipid monolayer at the water/air interface can be based only on the measurement of A because the change in \k (relativeto the bare water surface) for a layer with a thickness of typically d = 2 nm is too small to be reliably measured within the usual resolution of a com0.004'). Hence, it is pensating ellipsometer (d\k principallyimpossibleto resolve the layer's refractiveindex and its geometrical thickness independently. Nevertheless, even the recording of only the sensitive parameter A, e.g., as a function of the lateral pressure or while scanning across the trough surface, can yield valuable information about the monolayer properties. For the purpose of our study here the monolayer data presented in Figure 1serve only as referencestates for the phase-dependentbinding studies with streptavidin. The pressurearea isotherm (Figure la) and the fluorescence microscopic control of the phase transition (not shown) indicated the typical behavior of DMPE even if doped with biotinylated DPPE molecules. The A-area isotherm (Figure lb) shows some interesting features: (1)Given the good linear approximation of the dependence of A with d,18one would expect a hyperbolic increase of A with decreasing area according to
-
(1) provided the molecular volume V = dA is a constant. Any compressibilityof the lipids change in Veither by the (3D) or during the phase transition results in deviations from this simple estimate. Moreover, any changes of the anisotropy of the refractive index of the monolayer associated, e.g., with a reorientation of the lipids has the same consequence. The break of the A-A curve at A = 0.70 nm2is certainlydue to such changes. (ii) The overall change of A along the full isotherm is rather small. This indicates already a relatively low refractive index of the monolayer. (iii)A quantitativeevaluationof the refractive index (in an isotropic approximation) is only possible if the geometrical thickness as obtained from X-ray reflectometry is used. This is demonstrated in Figure 4. The full curve gives all pairs of n and d values compatiblewith the measured ellipsometric angle A = 181.01O. The thickness determination by X-ray reflectometry of d = 1.5 nm19 (thin solid line) then gives the appropriate index of refraction. (Notethat the ellipsometricdata taken from a lipid monolayer on the water surface are not compatible with the assumption A a nd). The result of this analysis for the liquid analoguephase with do = 1.5 nm then gives a refractive index of no = 1.427 (cf. thin straight lines that intersectat the A = 181.01O curve). All other values for the different phases of the monolayer are summarized in Table I for five different positions along the ?r-A area (cf. arrows in Figure la). These data are then used in the following evaluation of A a A-'
3 time t Ih
4
5
3
4
5
0
1
2
0
1
2
timet Ih
Figure 3. Change of ellipsometric angles 6A (a) and 6 9 (b) relative tothe bare lipid monolayerafter injectionof streptavidin (find concentration c = 5 X le7M) at t = 0. Open circles correspond to data taken from the monolayer in the condensed state at T = 15 mN/m (labeled 4, cf. Figure la), crosses were taken at T = 40 mN/m (5), and full triangles were from a pure DMPE monolayer (at T = 40 mN/m).
were found for a monolayer with 5 % biotinylated lipid at T = 15mN/m. The crosses (curves labeled 5 ) were taken a t T = 40 mN/m. The full triangles were again taken with no biotin labelspresent (datataken at ?r = 15and 40 mN/m were virtually identical). In (a) the change in A and in (b) the change in \k after protein injection at t = 0 (c = 5 X
(18) Reiter, R.; Motachmann, H.; Orendi, H.; Nemetz, A; Knoll, W. Langmuir 1992,8,1784. (19) Helm, C. A.; M6hwald, H.; Kjaer, K.; Als-Nieleen, J. Europhys. Lett. 1987,4,697.
Langmuir, Vol. 9, No. 9, 1993 2433
Streptauidin Binding to Lipid Monolayers 10.0
\
0.1
‘
, 0
I
2
3
tlh
Figure 5. Semilogarithmicplot of the kinetic data from Figures 2a and 3a. Labels are taken from Figure 1.
1.40 1.45 1.50 refractive index no, nl
1.35
Figure 4. Layer thicknesses (do, d l ) and refractive indices (no, nl) compatiblewiththe ellipsometricangle A = 181.01’ measured for a lipid monolayer of DMPE in the fluid analogue state (thick full curve),and after binding of a streptavidin layer (A = 186.48O; thick broken curve). In the latter case also the other ellipsometric angle 4 = 5.497O 0 . 0 0 4 O (thick dashed-dotted curve) gives some information as to a range of possible nl and dl values. The meaning of the various straight lines is discussed in the text.
*
Table I. Thickness and Refractive Index of the Lipid Monolayer and Streptavidin Layer As Deduced from Ellipsometric Data Taken at Various Monolayer Phase states
region no.a
d (mNm-l)
1 2
0 5 8 15 40
phase gasanalogue liquidanalogue phasecoexistence condensed1 condensed I1
ddmb 1.35 1.50 1.75 1.85 1.90
1.462 1.427 1.426 1.460 1.461
niC 1.466 1.472 1.475 1.431 1.370
3 4 5 DTaken from Figure la. Data taken from Helm (ref 19). Aasuming a layer thickness dl = 4.5 nm.
the protein adsorption and binding measurements as reference values. 4.2. Streptavidin Binding to a Biotinylated Monolayer in Different Phases. The binding curvespresented in Figures 2 and 3 are characterized by four distinct features: (i) the fast rise of 6A and 6*, (ii) a slower process observed as a change in the ellipsometricangles over several hours, (iii) the final equilibrium thickness of the protein layer bound to the functionalized lipid layer, and (iv) the dependenceof these processes and properties on the phase state of the monolayer. 4.2.1. Fast Process. The binding experiments were all performed with a (final) streptavidin concentration in the subphaseof c = 5 X le7 M. The correspondingnumber density of ca. 3 X 1014cm3 has to be compared with the maximum area density of a protein monolayer. Given the known dimensions for streptavidin of ca. 4.5 X 4.5 X 5.0 111113, this interfacialdensity is ca. 5 X 10l2cm-2at maximum packing. This means that for the formation of one monolayer the proteins from a solution layer of only ca. 150-bm thickness have to reach the interface. This thickness which is in the range of the value for the unstirred layer can be crossed by mere diffusion within ca. 1 min
(assuming a diffusion constant of streptavidin D = 5 X lo” cm2/s). This process, therefore, is outside the time resolution of our experimental procedure for applying the protein to the subphase, and it is hence understandable that the initial increase of 6A and 6Q appears “instantaneously”. In order to check for the diffusion control of the fast process, we reduced in one experiment the protein concentration to 8 X 1o-S M, expecting a resulting slowing down by a factor of ca. 40. The observed kinetic behavior, indeed, showed a reduced adsorption velocity. However, the quantitative analysis of the data in terms of a diffusioncontrolled process (by plotting the adsorbed amount as a resulted in an unreasonably high diffusion function of t1J2) constant. We conclude, therefore, that our experimental setup does not allow for a depiled evaluation of the fast early changes of the ellipsometric angles which are either transporbcontrolled (by convection)or determinedby local concentration fluctuations of the protein due to a slow redistribution of the applied solution to give the final homogeneous concentrations. 4.2.2. Slow Process. A remarkable result of the binding studies is the evidence for a second “rearrangement” process following the first instantaneous binding. Depending on the molecular nature of this process, complicated kinetics could be expected,of course. A simple description used in the following for a more quantitative evaluation of this process assumes a finite layer thickness at long times, dl,, and a thickness increase 6dl(t)/6twhich is given by the difference between the final thickness and the thickness &(t) at time t : ad,/& a d,,
- d,(t)
(2)
The resulting expression is then d,(t) = (dim - dlo)(l- e-t/‘)
+ d,,
(3) with dlo being the instantaneous thickness increase (at t = 0). In Figure 5 we have replotted the data from Figures 2 and 3 in a semilogarithmic way. It can be seen that after the fast rise (and a certain crossover region) the adsorptions in the expanded phase (2) and in the highly condensed phase (5), indeed, seem to follow the time dependence given by eq 3. The time constants Ti for the different phases as obtained from the slope of the straight lines are summarized in Table 11. It is tempting to associate the differences in these time constants to differences in the membranefluidity which determineslateral diffusion and rotational rearrangement processes. A detailed model of
Reiter et al.
2434 Langmuir, Vol. 9, No. 9, 1993 Table 11. Relaxation Times As Obtained from the Kinetic Data of Figure 5 region no."
phase
r/h
2
fluid condensed ( X = 15 mN/m) condensed ( X = 4OmN/m)
1-1.5 TI = 1 . 5 , = ~ ~3-4
4
5 a
3.5
Taken from Figure la.
the relevant molecular steps of the slowthickness increase, however, is not possible on the basis of ellipsometricdata. The %ondensed" state at a = 15 mN/m exhibits a biphasic behavior for the slow thickness increase with time constants very similar to those of the expanded and to the highly condensed state, respectively. We conclude, therefore, that the time dependence of this thickness increase mirrors the residual phase coexistence also seen in the fluorescence microscopic pictures at this lateral pressure. 4.2.3. Streptaddin Layer Thiokness. One of the key problems in the optical thickness determination of (ultrathin, dl < 5 nm) adsorbed (bio)polymer layers at solid/ solution interfaces still unsolved even in the isotropic box model approximation is the differentiation between the geometrical thickness and the (effective) refractive index of such a layer. For a growing monolayer two extreme approachescould be taken: the first assumesa constant index and describes the adsorption process by a time-dependent thickness increase. The other procedure defines the geometrical thickness of the layer by the independently known dimensions of the individual adsorbing units and describes the adsorption as an increase of the layer's effective index of refraction (by replacing more and more water in this film by protein). For the binding of streptavidin to a biotinylated lipid layer the change in \k even for high coverages is still too small to give any reliable solution to this problem. This is, once again, demonstrated in Figure 4. The thick broken curve corresponds to those pairs of streptavidin layer thicknesses dl and refractive indices nl compatible with the ellipsometric angle A = 186.48O measured for the fluid analogue monolayer (cf. Figures l a and 2) after long adsorption (and reorientation) for t > 5h. This curve was calculated with a two-layer model assuming for the lipid = 1.427 (cf. monolayer a thickness do = 1.5 nm and Table I). The measured \k value with its estimated error \k = 5.497O f 0.004' reduces the range of possible dl and nl values to a limit which is reasonable given the streptavidin's dimensions. However, a precise independent thickness determination is not possible. Nevertheless, these ellipsometric data give interesting estimates of the layer's refractive index helpful for other studies, e.g., surface plasmon spectroscopic investigations. Given the molecular architecture of the protein bound to the monolayer by incorporating within its binding pocket the biotin group and most of the spacer that links it to the lipid headgroup, we may estimate the geometrical thickness of a single protein monolayer to a range between 4.3 and 4.8 nm.5 This assumption based on a 3D X-ray structuredetermination2021was experimentally confirmed by X-ray and neutron reflectometrygand by data obtained with the surface forces a ~ p a r a t u s .The ~ reliable A values then allow for a refractive index range of 1.463 < nl < 1.476 (see shaded area in Figure 4). For the most likely thickness of dl = 4.5 nm we propose, therefore, a refractive (20) Hendrickean, W. A.; Pirhler, A.; Smith, J. L.; Satow, Y.;Merritt, E. A.; Phizackerley, R. P. Roc. Natl. Acad. Sci. U.S.A. 1989,86,2190. (21) Weber, P. C.; Ohlendorf, D. H.; Wendoloski, J. J.; Salemme, F. R. Science 1989,243,86.
index of nl = 1.470 for a high-coverage monolayer. According to
(4) the adsorbed amount of protein Q can be calculated from the monolayer parameters dl, nl, and the refractive index of the subphase, n,, provided the index increment dnlldc for the protein solution is known. The latter was determined with an Abbe refractometer and amounts to dnddc = 0.212 d / g . For the above high-coverage monolayer the adsorbed amount of protein is then found to be Q = 0.283 f 0.003 pg/cm2. Given the protein's mass density p = 0.96g/cm3,this corresponds to a coverage of ca. 65% . In view of the random distribution of the biotin moieties this high value certainly can be achieved only by the reorganization process discussed above. T w o aspecta are interesting in this context: (i) From the electron diffraction work on 2D streptavidin single crystals an area coverage of only 66% was estimated.22 Our data indicate that the random binding to biotin lipids dissolved in a fluid monolayer yields about the same coverage of the monolayers as that found in 2D crystals. Paudler and co-workers found a maximum thickness of dl = 5.0 nm calculated with a refractive index of nl = 1.45.s If we assume the same index (see dotted line in Figure 41, our monolayer would have to be described by a thickness dl = 5.5 nm which would point to a higher protein density of their streptavidin layer than found in 2D crystals. (ii) Assuming an index of n = 1.45,our surface plasmon spectroscopic data obtained for a streptavidin monolayer bound from solution to the biotin groups (of similar lateral density) at a functionalized lipid monolayer transferred in the fluid state to a solid substrate indicated a maximum layer thickness of only dl = 4.0nm. It is intriguing to note that this thickness increase corresponds quantitatively to the "instantaneous" thickness increase found for a Langmuir monolayer in the fluid phase (cf. Figure 2). We conclude, therefore, that the binding of the protein to the transferred and then amorphousmonolayer can occur only to the same instantaneously available binding sites. The reorganization, however, which requires a fluid matrix is frozen in. Any attempts to prepare a fluid layer on a solid support hence should allow one to optimize the binding capacity for ligand-receptor pairs. Assuming a layer thickness of dl = 4.5 nm the A values taken for the monolayer in other phase states were used to calculate the corresponding effective indices of refraction. The results are also given in Table I. 4.2.4. Monolayer Phase Dependence of Streptavidin Binding. As we have discussed so far all parameters characterizing the streptavidin binding to the biotinfunctionalizedmonolayer are dependent on the phase state of the lipid molecules in the matrix film. In order to summarize and directly compare the different data, we have plotted in Figure 6the ellipsometricangle change 6A (relative to the bare lipid layer, scaled to the maximum angle change observed, ),A6 as it was found at different areas per lipid or biotin label for different times after injection of the protein. For the sake of completeness we have also added the data taken in the gas analogue state (1,cf. Figure la) aswell as those from the phase-coexistence region (3). The complete monolayer formation is observed only for the gas analogue state and the fluid-expanded state and (22) Darst,S.A.;Ahlera,M.;Meller,P.H.;Kubalek,E. W.;Blankenburg, R.; Ribi, H. 0.; Ringsdorf, H.; Kornberg, R. D. Biophys. J. 1991,69,387.
Langmuir, Vol. 9, No. 9, 1993 2435
Streptavidin Binding to Lipid Monolayers
data233' indicated for the condensed domains in the phasecoexistence range a complete loss of streptavidin binding to integrated biotin labels. It is very conceivable that the transfer process again is accompanied by a reduction in molecular flexibility as it was discussed above in the fluid phase. On the water surface such as passivated film is only found in the extremely compressed case.
L
!
a' 9 .4
'
A
I h
x
3h
5. Concluding Remarks
5h .4
.5
.7
.6
.a
.9
A/nm
Figure 6. Ellipsometricdata (given as the change 6A,scaled to the maximum value 6 A A as obtained at various times (as indicated) as a function of the lipid area per molecule, A. Numbers correspond to those of Figure la.
also shortly after the onset of the phase coexistence. The high coverage in the gas analogue phase is remarkable given the known tendency of streptavidin to form 2D crystalline arrangements when bound to lipid monolayers in the gas analoguephase. Of course,ellipsometryprovides no direct indication for crystal formation, and also the usual preparation routine for crystal growth at fully biotinylated lipid layers at low density is very different from our experimental protocol. We may conclude, however, that our streptavidin monolayer formed by specific recognition is also organized with lateral periodicity, yielding a maximum packing density which is independent of the phase states but requires some time to fully develop. Both the instantaneous layer and the final equilibrium thickness of streptavidin bound to a condensed monolayer are largely reduced compared to the highly fluid phases. Most remarkable is the fact that the pressure increase from ?r = 15 mN/m to ?r = 40 mN/m again results in a 2-fold decrease of the protein layer thicknesses. If we extrapolate the data points in Figure 6 to zero thickness, we cut the abscissa at A = 0.38 nm2 per lipid molecule. This value corresponds to the highest compression possible in DMPE monolayers with the lowest translational and rotational flexibility of the individuallipid molecules.This provides strong evidence that the recognition and binding of streptavidin to a biotin linked to a lipid molecule (even if this linkage is via a flexible spacer) require some molecular flexibility which is strongly reduced if the matrix lipid is completely frozen in. Again, it is interesting to compare this finding with results obtained with transferred layers. Our AFM
The above presented and analyzed data provide strong evidence that the binding of streptavidin to a biotinylated lipid monolayer is extremely sensitive to the phase state of the lipid film. Maximum protein monolayer formation upon binding is observed only for fluid phases with a complete blocking of the binding sites upon compression of the monolayer into a highly condensed state. Given the possible control of a membrane bilayer's phase state by external chemical stimuli (in addition to the physiologically less interesting temperature dependence), these results can be taken as an example for a general mechanism for the control of the binding activity of membrane-integral receptors by the phase behavior of the lipid matrix. Finally, the finding of a complete insensitivity of the pure DMPE monolayer to unspecific adsorption of streptavidin has interesting implications for the optimization of biosensor configurations. This shows that the weak adsorption of the protein to nonfunctionalized monolayers found after transfer at the solid/solution interface2is not an intrinsic property of the interaction between streptavidin and the lipid headgroups but rather an artifact presumably inducedby the high defect density introduced during the transfer of the monolayer to the typically rough solid support. Recent attempts to decouple the selforganizing bilayer membrane from the substrate by a polymeric buffer layer should reduce the undesired unspecific a d s o r p t i ~ n . ~ ~
Acknowledgment. Helpful discussions with L. Angermaier, H. J. Guder, M. Liley, W. Miiller, H. Ringsdorf, A. Schmidt, F.-J.Schmitt, and J. Spinke are gratefully acknowledged. This work was supported by Boehringer Mannheim, Werk Tutzing. (23) Schmitt, F.-J.;Weisenhorn, A. L.; Hansma,P. K.; Knoll, W. Thin Solid Films 1992,2101211, 666. (24) Weirrenhorn, A. L.; Schmitt, F.-J.: Knoll.. W.:. Hansma. P. K. Ultramicroscopy 1992,4%44, 1125. (26) Spinke, J.; Yang, J.; Wolf, H.; Liley, M.; Ringsdorf, H.; Knoll, W. Biophys. J. 1993,63,1667.
