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Protein interactions in covalently attached dextran layers
Colloids and Surfaces B: Biointerfaces 13 (1999) 325 – 336
Protein interactions in covalently attached dextran layers Jacob Piehler *, Andreas Brecht, Karin Hehl, Gu¨nter Gauglitz Institut fu¨r Physikalische Chemie, Auf der Morgenstelle 8, D-72076 Tu¨bingen, Germany Accepted 21 April 1999
Abstract Protein interactions with polymeric carbohydrates play an important role for application in chromatography, biomaterials and biophysics. In this study, we present a detailed morphological and functional characterization of covalently side-bound dextran layers by spectroscopic ellipsometry (SE) and reflectometric interference spectroscopy (RIfS). The surface chemistry was monitored step-by-step by ellipsometric characterization of the surface loading. Dextrans of various molecular masses (10–2000 kD) were immobilized leading to surface loadings between 3 and 8 ng mm − 2. The refractive indices of the covalently attached dextran layers under atmospheric conditions (nD =1.51) were very close to the refractive index of a spin-coated dextran layer (nD = 1.52) indicating dense and homogeneous coverage achieved by the coupling chemistry. Under buffer solution, refractive indices between 1.34 and 1.365 and thicknesses between 20 and 40 nm of these dextran layers were determined. A dextran concentration in the hydrated layers of 0.05–0.21 g cm − 3 was estimated from the refractive index. The density and the thickness of the hydrated layers increased with molecular mass of the dextran. Non-specific binding was strongly reduced by the dextran layers and decreased with increasing thickness and density of the layer. Specific antibody binding to haptens immobilized in the dextran layer lead to an increase of both the density and the thickness of the layers. Time resolved detection by RIfS indicated significant decrease of protein mobility in the dextran layer. From these results we conclude that the functional properties of dextran layers with respect to protein interactions are determined by their effective pore-size, which is controlled by the number of bonds, the surface loading and the concentration of charged groups in the polymer. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Dextran layer; Affinity interaction; Immobilization; Spectroscopic ellipsometry; RIfS (reflectometric interference spectroscopy)
1. Introduction
* Corresponding author. Present address: Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel. Tel.: + 972-8-934-2727; fax: + 972-8-9344118. E-mail address: [email protected] (J. Piehler)
Dextrans are hydrophilic and non-charged natural polymeric carbohydrates which are soluble in water in any proportion and form highly hydrated hydrogels. For these properties, dextrans show very low non-specific interactions with proteins. Owing to the high concentration of hydroxyl
0927-7765/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 0 4 6 - 6
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groups in the dextran molecule, chemical modification of these polymers is possible without significantly affecting their hydrophilicity. The essentially non-branched polymer chains are highly flexible and ligands immobilized in dextran matrices are well accessible for proteins. Hence, dextran provide optimum conditions for controlling protein interactions at interfaces and dextran-based polymers have found application in several fields of biochemistry and biotechnology. In particular in affinity and size exclusion chromatography, dextrans have been applied very successfully [1,2]. Cross-linked dextran polymer beads [3] and dextran coated solid supports [4–6] have been used. Dextrans are also frequently used for biomedical and biophysical applications: In the field of biomaterials, dextran coatings for reducing protein adsorption at surfaces to enhance biocompatibility were investigated [7–9]. For heterogeneous phase affinity detection, covalently attached dextran layers have become a standard modification for various transducer materials [10 – 12]. The extraordinary hydrophilicity of dextran coated surfaces made them attractive for preparing supported lipid bilayers [13]. Dextran matrices have also proved to provide suitable conditions for the investigation of inter- and intramolecular forces by atomic force spectroscopy [14,15]. Furthermore, dextran coatings have found interest for masking surface charges and thereby reducing electrokinetic effects [9]. The functional properties of dextran coating are mostly optimized empirically and informations about them are rather confusing. Surface loadings of B 1 to 8 ng mm − 2 have been obtained on different substrate materials by various preparation methods [5,7,13,16]. By scanning force microscopy (SFM) on silica surfaces in air, clustered distribution of dextran molecules attached by non-aqueous coupling chemistry was observed [17]. SFM measurements in aqueous environment have indicated high extension (140 – 190 nm) of covalently attached carboxymethylated dextran layers on gold surfaces, strongly depending on the pH and the ionic strength of the ambient buffer [18]. Using reflection interference contrast microscopy, ex-
tensions of 80–100 nm were found for covalently attached, non-functionalized dextran layers in aqueous medium [13]. Attachment of the dextran molecules site-directed in side-on or end-on configuration led to significantly different functional properties [9]. Investigating the kinetics of protein interaction in dextran layers by label-free optical detection, influence of the polymer matrix on the interaction rates was observed [19–21]. Different accessibility of the binding sites within the dextran layer [20,21] and different protein mobility in the dextran matrix [22] have been proposed to be responsible for these effects. These results indicate that the morphology of dextran coatings and their functional properties with respect to protein interactions are correlated and strongly depend on the preparation technique. Hence, for a systematic design of the functional properties of dextran coated surfaces, a more profound understanding of processes occurring in dextran layers is required. In this study we present a detailed investigation of the morphological and functional properties of interfacial dextran layers. Dextrans of various molecular mass were covalently attached onto planar silica surfaces in a side-on configuration. Spectroscopic ellipsometry (SE) was used for physical characterization of attached layers. This technique allows for an absolute and non-destructive assessment of the thickness and the dielectric function of thin organic and biochemical layers in atmospheric and aqueous environment [23]. These parameters gave information about the surface loading and the morphology of the layers. Protein binding at the modified surfaces was investigated time-resolved by reflectometric interference spectroscopy (RIfS), a very sensitive technique for monitoring binding events at interfaces [24,25]. Protein surface loadings as well as binding kinetics during specific and non-specific interactions at different dextran layers were investigated. We correlate the functional properties and the morphology revealed by these investigations and draw principal conclusions on how to control these properties by the preparation method.
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2. Experimental
2.1. Materials Common organic compounds were purchased either from Fluka, Neu-Ulm, Germany or Sigma, Deisenhofen/Germany. 4Aminobutyldimethylethoxysilane (ABDMS) was purchased from ABCR, Karlsruhe, Germany. 4Chloro-6-(isopropylamino) ×-1,3,5-triazine-2-(6%-amino)caproic acid (CTCA) × and affinity-purified, polyclonal sheep anti-triazine antibodies and Fab fragments were kindly supplied by Dr Ram Abuknesha from GEC Research, Borehamwood, UK. Dextrans of various average molecular mass (9.3, 38, 298 and 2000 kD) were purchased from Sigma. Partially aminofunctionalized dextrans (AMD) were obtained from these dextrans (AMD10, AMD40, AMD300 and AMD2000 according to the average molecular mass of the dextran) by diol oxidation and reductive amination as described recently [12]. By this method, approximately 10% of the anhydroglucose units were functionalized.
2.2. Preparation of dextran layers AMD was covalently attached in a side-on configuration by amide-based chemistry as detailed before [12]. The silica surface was activated by silanization with an amino-functionalized silane (ABDMS). After conversion of the surface amino groups with succinic anhydride (SA) into carboxyl groups, AMD was coupled at concentrations of 0.2 – 0.5 mg ml − 1 water via its amino groups using EDC. This selective coupling technique allows limiting the number of bonds to the surface by the number of amino groups introduced along the dextran chains. The hapten CTCA was coupled to the remaining amino groups of the AMD in DMF in presence of diisopropylcarbodiimide. As a reference surface, CTCA was directly coupled to the amino groups of an ABDMS-silanized surface. Owing to a high excess of the immobilized CTAC and a high affinity of the anti-CTCA-antibodies, the antibody bound to the surface are essentially trapped
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by rebinding and only very low dissociation is observed [12]. Dense dextran coatings of several 100 nm thickness were prepared by spin-coating (Convac 1001, Convac, Wiernsheim, Germany) aqueous dextran solutions (0.1–1 mg ml − 1) onto silicon wafer for 50 s at 5000–7000 rpm and drying at 60°C.
2.3. Spectroscopic ellipsometry (SE) The experimental set-up for the ellipsometric investigation of the attached layers with a spectroellipsometer (ES4G, Sopra, Paris, France) was discussed in detail before [26,27]. Oxidized silicon wafers (silica layers of approximately 170 and 250 nm in thickness, a gift from Wacker Chemitronic, Burghausen, Germany) were used as substrates for the surface chemistry. Each step of surface chemistry was characterized under atmospheric conditions. For characterizing the morphology of the dextran layer, the layer properties before and after maximum loading with an antibody binding specifically to the CTCA immobilized in the dextran layer were investigated under aqueous conditions. The antibody (50 mg ml − 1 in PBS) was incubated on the wafer (100 ml cm − 2) for 30 min. After the incubation, the samples were quickly rinsed with PBS and put back in the measurement chamber containing PBS with 5 mg ml − 1 antibody to reduce dissociation by providing steady state conditions during the measurement. The antibody was selectively removed from the surface by alternate rinsing with 2 mg ml − 1 pepsin pH 2 and a mixture of water, acetonitrile and propionic acid (50:50:1) according to reference [12].
2.4. E6aluation Refractive index and thickness of the attached layers were determined by fitting a model layer system to the ellipsometric angles using a non-linear least squares algorithm. The substrate was parameterized assuming a thin layer of amorphous silicon between the crystalline silicon substrate and the silica layer on top as detailed before [26,27]. Silanization with ABDMS and conversion by SA were assumed as an increase of the silica layer since refractive index and layer thickness
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could not be determined independently because of the small changes of the signal. The dextran layers were described as a distinct layer on top of the previously characterized silica layer. They were assumed to be homogeneous, isotropic and nonabsorbing and the real part of the dielectric functions was described by a two-parameter Cauchy model n(l)= A+
B l2
(1)
The surface loading for each step of chemistry was determined by estimating the density from the refractive index of the layer under atmospheric conditions. A typical refractive index nD of 1.43 was assumed for an organic layer with a density r of 1.0 g cm − 3 and an increment dn/dr of 0.24 g cm − 3. These values were estimated from : 25 different liquid aliphatic organic compounds. Dextran layers, protein layers and mixed dextran/ protein layers under buffer solution were assumed to form one single isotropic and homogeneous layer. The refractive indices were again parametrized by a two parameter Cauchy model. All other parameters were kept constant during the fitting procedure. The dextran concentrations in the hydrated layers cdex were estimated from the refractive index nD of the layer nlayer and the ambient buffer nambient by assuming linearity of the refractive index increment dn/dc according to de Feijter [28]: cdex =
nlayer − nambient (dn/dc)
(2)
For mixed dextran/protein layers, the refractive index was assumed to be a linear combination from the refractive index increments of the components of the concentration ci: nlayer = namb +% i
dn ·ci dc i
(3)
monitoring binding events at interfaces was discussed in detail before [24,29]. The change in apparent optical thickness of a thin transparent layer upon protein binding is detected by interference of white light reflected at the interfaces of the layers using a diode array spectrometer. Binding curves S(t) were recorded as apparent optical thickness versus time. A change of the surface loading of 1 ng mm − 2 protein leads to a signal S:1.7 nm (theoretical calculations). The characterization of the functional properties of dextran layers was carried out as published before [12]: non-specific adsorption was investigated by incubating 1 mg ml − 1 ovalbumin or 100 ml ml − 1 calf serum. Maximum loading by specific binding to the immobilized CTCA was determined by incubating 50 mg ml − 1 antibody. The protein solutions were incubated for approximately 500 s. The total cycle including baseline measurement before and after the binding curve took approximately 14 min. The surface was regenerated on-line by incubation of 2 mg ml − 1 pepsin pH 2 for several minutes and a short pulse (30 s) of a mixture of water, acetonitrile and propionic acid (50:50:1).
2.6. Biomolecular interaction analysis The binding kinetics was determined from the binding curve S(t) using a model described in detail before [30,31]: bimolecular interaction of the receptor R in solution with the immobilized ligand L was assumed. The slope of the binding curve dS(t)/dt is then given as dS(t) = ka·cR·(Smax − S(t))− kdS(t). dt
(4)
S(t) is the response with time, Smax is the maximum loading response, cR is the receptor concentration in solution, ka is the association rate constant and kd is the dissociation rate constant, or
2.5. Reflectometric interference spectroscopy (RIFS)
dS(t)dt = ka·cR·Smax − (ka·c+ kd)·S(t) dt
The interaction of proteins with the surface was monitored time-resolved by RIfS. The principles and the experimental set-up of this technique for
Binding curves where analyzed by plotting the slope of the binding curve dS/dt versus the response S(t) [31]. From the slope of the curves the rate constant ks
(5)
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ks = ka·c+ kd
(6)
was derived which was taken as a measure to compare the kinetics at different dextran layers. For a profound and reliable evaluation of the kinetics at different layers, the influence of mass transport contributions has to be negligible [32]. For the flow system used for these investigations, a slope dS(t)/dt of 3 – 4 pm s − 1 per mg ml − 1 protein was found for mass transport limited binding of antibodies. Undisturbed binding kinetics were assumed at slopes below 2 pm s − 1 per mg ml − 1 protein.
3. Results and discussion
3.1. Characterization of the dextran layer under atmospheric conditions The surface chemistry was stepwise characterized by using ellipsometry under atmospheric conditions. The layer density r was estimated from the refractive index of the layer. The loading of the surface after each step of surface chemistry was calculated from the thickness and the refractive index (Table 1). For the ABDMS- and for the SA-layer, a density of 1.15 g cm − 3 was estimated assuming the same refractive index as the silica layer (1.466). The molar surface loading of 2.6 pmol mm − 2 corresponds to a surface coverage of about 1012 ABDMS molecules mm − 2. This figure is in good agreement with results from measurements using fluorescamine for determination of the amine group surface density (data not published). The molar loading of 2.5 pmol mm − 2 by
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SA indicates that most of the amino groups ( \ 90%) were converted by this reaction. Hence, the concentration of reactive groups on the surface for the attachment of the AMD was approximately 1012 mm − 2. After coupling the AMD, determination of both the thickness and the refractive index of the dextran layer from the ellipsometric angles was possible with good accuracy. A refractive index nD of :1.51 was found and a layer thickness of 5.3 nm for AMD300 coupled at a concentration of 0.2 mg ml − 1 water (cf. Table 1). A density of 1.3 g cm − 3 was estimated for this refractive index (other authors have assumed 1.56 g cm − 3 [7,13] which seems to be too high compared to other organic compounds of this refractive index). From these parameters, a surface loading of 6.9 ng mm − 2 was determined for this dextran layer. In case of maximum coupling efficiency (the dextran is attached to all active groups available at the surface), about 6% of the monomer units are linked to the surface. Hence, the minimum average loop length is 10 nm assuming a monomer size of 0.65 nm. It is not probable that the monomer units attached to the surface are homogeneously distributed along the polymer chain. However, the partial functionalization of the dextran (10% of the monomer units) limits the minimum average loop length to 6.5 nm. The results found for various layers using dextrans of different molecular masses coupled at a concentration of 0.5 mg ml − 1 water are summarized in Table 2. For a comparison of the surface density, the refractive indices of compact dextran layers of several 100 nm thickness prepared by spin-coating were determined. The refractive in-
Table 1 Characterization of the surface chemistry by spectroscopic ellipsometrya Compound
nD
r (g cm−3)b
d (nm)
DG (ng mm−2)
DG (pmol mm−2)
ABDMS SA AMD300
1.466c 1.466c 1.507
1.15 1.15 1.3
0.38 9 0.07 0.22 90.02 5.3 90.3
0.44 9 0.08 0.25 9 0.02 6.9 9 0.4
2.690.5 2.5 9 0.2 43 9 2d
Refractive index at 589 nm nD, density r, layer thickness d and surface loading DG estimated from d and r. Estimated from the refractive index of the layer. c Refractive index of the silica layer assumed. d With respect to a monomer unit (162 g mol−1). a
b
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Table 2 Summary of the some properties of various dextran layers under atmospheric conditions and in buffer solutiona AMD
dair (nm)
nD (air)
G (ng mm−2)b
fbond (%) dPBS (nm)
AMD10 AMD40 AMD300 Spin-coated
2.5 9 0.3 4.1 9 0.3 6.2 9 0.2 500–5000
1.5190.02 1.5290.02 1.5090.02 1.5290.004
3.39 0.4 5.3 9 0.4 8.19 0.3 700–7000
10 6 4 –
19 90.1 28 98 36 9 6 –
dPBS/dair nD (in PBS) 7.5 6.8 5.8 –
1.341 90.003 1.351 90.002 1.365 90.006 –
cdex (g cm−3)c 0.05 9 0.02 0.12 9 0.01 0.21 9 0.04 –
a
Parameters determined from measurements in air: layer thickness dair, refractive index nair and the same parameters from measurements under PBS (index PBS), surface loading G, maximum fraction of bonded monomer units fbond, swelling dPBS/dair and the concentration of dextran cdex in the hydrated layer. b Estimated assuming a density of 1.3 g cm−3. c Estimated from the refractive index increment.
dices of all covalently attached dextran layers were in the range of the refractive index of the densely packed, spin-coated layers, indicating that a high coverage and homogeneous distribution is achieved by the immobilization technique. A higher surface loading was obtained when coupling at a higher dextran concentration (cf. AMD300 in Tables 1 and 2). The thickness of the dry layers and the surface loading increased with molecular mass of the AMD used for coupling. Since the same percentage of monomer units was functionalized in all AMDs, the average, maximum possible fraction of monomer units bonded to the surface decreased with molecular mass of the AMD. At the same time, the distribution of bonds along the chain length becomes more heterogeneous due to the increasing folding of the molecules with increasing molecular mass.
3.2. Characterization of the dextran layer under buffer solution For understanding protein interaction with the dextran layers, investigations were carried out by SE under aqueous medium (PBS). The properties of the dextran layers found under these conditions are summarized in Table 2. Strong swelling of the layers was observed for all layers, up to thicknesses of 20 – 40 nm. The relative increase of the layer thickness (dPBS/dair) decreased with molecular mass of the dextran from 7.5 to 5.8. Hence, the lowest refractive index of the hydrated polymer layer was found for AMD10. The dextran contents in the hydrated layer was estimated from
the refractive index increment of 0.15 ml g − 1 for dextrans in water [33] by using Eq. (2). The dextran concentration increased with molecular mass of the dextran from 0.05 g cm − 3 for the AMD10-layer to 0.21 g cm − 3 for the AMD300 layer. Two reasons may be responsible for these differences: (i) the coupling chemistry becomes more efficient with increasing molecular mass of the dextran due to an increasing number of functional groups per molecule leading to more bonds between each AMD molecule and the surface; (ii) folding entropy leads to a lower density of the lower molecular mass dextran molecules. However, the extensions of all hydrated layers were significantly lower (and the density much higher) than those reported from other investigations of dextran layers (80–190 nm) [13,18]. These strong differences cannot be explained solely by the fact that spectral ellipsometry has a lower sensitivity to dilute polymer tails than the techniques used for these investigations [34]. Two further reasons are probably responsible for the low thickness observed for the AMD layers: (i) the coupling chemistry: the higher extended dextran layers were attached by the reaction of the carbohydrate hydroxyl groups with an epoxy-activated surface in aqueous solution. Coupling of AMD by the more efficient and site-directed amide based chemistry provides more bonds to the surface and a more homogeneous distribution of these bonds along the polymer chain. Furthermore, non-covalent interaction of the dextran molecules with the highly carboxy-functionalized silica surface is expected; (ii) low repulsive inter- and intramolecular
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interaction due to the low concentrations of charged groups in the dextran layer. In particular for the highly carboxymethylated dextran used for protein immobilization in label-free biosensors [10,11], much stronger inter- and intramolecular repulsion is expected due to the high concentration of carboxyl groups which are negatively charged above pH 4 – 5. These layers show the highest extension ever reported [18] and could not at all be detected by ellipsometry [35].
3.3. Layer properties after protein binding For a further characterization of the layer morphology, ‘filling’ the dextran matrices with protein was investigated. For this purpose, anti-triazine antibodies, which bound with high affinity to the triazine derivative CTAC immobilized in the dextran layer, were incubated at the surface. The layer thickness and refractive index after maximum loading of the surface with the antibody were determined by SE. High concentration of the CTCA in the layer guaranteed that binding of the antibody was limited only by the flexibility of the dextran layer. When using IgG, cross-linking in the dextran layer due to the two binding sites per molecule was observed. To avoid such interference with the binding event, Fab-fragments with a single binding site were used for characterizing specific binding into the dextran layers. At all dextran layers investigated, both an increase in refractive index and a further strong increase of the layer thickness were observed. A typical change of the real part of the dielectric function of the dextran layer is shown in Fig. 1. This swelling of the layers during antibody binding was found to be reversible and reproducible as demonstrated in Fig. 2 for three cycles of maximum loading with the antibody. These results prove that: (i) the protein was bound into the dextran hydrogel; and (ii) the hydrogel further swelled during the binding event. The properties of the different dextran layers after maximum loading with a specific antibody are summarized in Table 3. For comparison, the results obtained with the ligand directly attached to the silanized surface are added. In this case, formation of a dense protein layer was observed with a refractive
Fig. 1. Real part of the dielectric function of an AMD2000 layer in buffer solution before (A) and after (B) specific antibody binding (average from three measurements, the CV is given by the two dotted lines adjacent to the curves). The refractive index of the buffer solution (C) is plotted for comparison.
index of 1.391 and a thickness of approx. 10 nm. This layer thickness corresponds well to the size of an antibody molecule. The density of the protein saturated dextran hydrogels increased with the molecular mass of the dextran. The refractive index of the AMD300-protein layer is already close to the refractive index of approximately 1.39 found for the pure protein layer. The changes of the layer thickness and refractive index are visualized in Fig. 3. The relative swelling of the dextran layer during protein binding
Fig. 2. change of the thickness d (() and the refractive index nD ((2) of a dextran layer (AMD2000) during three times of maximum loading by specific antibody binding (steps 2, 4 and 6) and regeneration of the surface (1, 3 and 5).
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Table 3 Properties of dextran layers using various AMDs after maximum specific binding of the antibody to the ligand immobilized in the dextran comparison with the protein binding to the directly attached liganda AMD
dprot (nm)
Nd
G (ng mm−2)
dprot/dPBS
dprot/dair
Gprot/ Gdex
–b AMD10 AMD40 AMD300
10.1 90.4c 299 1 469 1 609 4
1.3919 0.001c 1.36990.007 1.3829 0.003 1.3889 0.002
3.5 90.3c 2.9 9 0.3d 7.6 90.3d 11.0 9 1.0d
– 1.58 1.61 1.67
– 12 11 10
– 1.2 1.6 1.4
Thickness dprot and refractive index nD of the layer, protein loading G, relative increase during protein binding dprot/dPBS, overall increase dprot/dair and relative protein loading Gprot/Gdex). b CTCA directly attached to the amino-silanized surface. c Maximum loading by anti-triazine IgG; including considerable non-specific binding. d Calculated from the total d and nD assuming the dextran loading determined from the dry layers. a
(dprot/dPBS) slightly increased with molecular mass of the AMD. However, the overall swelling from the dry dextran layers to the protein loaded layers (dprot/dair) is nearly constant for all layers, indicating only negligible differences in the capacity of the layers. This result is confirmed by the fact that the protein– dextran ratio of the layers does not differ significantly for all layers. In contrast to the swelling, the increase of the layer refractive index relative to the buffer refractive index strongly decreased with increasing molecular mass (Fig. 3). Hence, binding of proteins into the dextran layer changes from ‘filling the pores’ at lower molecular mass to ‘expatiate the layer’ at higher molecular mass of the dextran. However, the maximum extension reached even at very high density of the layer was still significantly below the extension reported for dextran layers attached by epoxy-activation [13,18]. These differences confirm that the
Fig. 3. Increase of thickness d and refractive index n during antibody binding compared for three different AMDs.
coupling chemistry plays an important role for the flexibility of the attached layer.
3.4. Time resol6ed measurement of protein binding Specific and non-specific interaction of proteins with the AMD layers was carried out by RIfS. A typical characterization cycle of protein binding including regeneration of the surface is shown in Fig. 4. The results of the functional characterization by RIfS are compared in Table 4. High and fast non-specific binding of ovalbumin was observed when the ligand was directly attached to the silanized surface [12]. At dextran-modified
Fig. 4. Time resolved characterization of protein interaction at an AMD2000 layer and repeated regeneration of the surface with pepsin and acetonitrile.
J. Piehler et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 325–336 Table 4 Maximum signal (increase in optical thickness) during nonspecific binding of 1 mg ml−1 ovalbumin Gnon and specific binding of a Fab-fragment Gspec at dextran modified surfacesa AMD
Gnon (nm)
Gspec (nm)
ks
–b AMD10 AMD40 AMD300 AMD2000
2.2 0.25 0.05 B0.01 B0.01
2.9 4.5 14 20 25
:0.1 0.067 0.022 0.016 0.009
a Determined by RIfS (mean value from four measurements), as well as the rate constant ks. b CTCA directly attached to the silanized surface.
surfaces, much lower non-specific protein adsorption was observed (B 15%). The amount of nonspecifically bound ovalbumin decreased with the molecular mass of the dextran. At dextran loadings above 5 ng mm − 2 (AMD300), no significant non-specific protein binding was detectable (response of less than 10 pm optical thickness within the incubation period). The binding curves revealed that not only the absolute amount of adsorbed protein, but also the adsorption rate constants were strongly reduced by the dextran layers. Apparently, the dextran layers effectively shielded the surface against ovalbumin, a 60 kD protein with a diameter of : 6 – 7 nm. The shielding by these layer against other proteins was less effective: when incubating diluted serum —a mixture of proteins of various molecular mass— much stronger non-specific adsorption was observed (Fig. 5). However, again the binding rate was significantly reduced by the dextran coating. Proteins of lower size can apparently permeate the layer more easily. These results indicate that nonspecific binding at the dextran modified surfaces mainly occurs at the original (silanized) substrate surface underneath the dextran layer. Shielding of the surface is apparently achieved by the dextran meshwork, sterically hindering proteins to approach the substrate surface. The observation that shielding of dextran layers is much less effective when the dextran molecules are coupled in end-on configuration [9] corroborates this mechanism. Hence, the shielding efficiency is determined by an ‘effective pore size’ of the dextran layer, the prop-
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erty which is utilized for size-exclusion chromatography. In contrast to this shielding principle, ‘entropic’ shielding is achieved by coatings of high exclusion volume polymers such as poly(ethylene glycole) (PEG) [36]. Characterizing the kinetics of specific antibody binding to the immobilized CTCA gave further insight in the properties of the AMD-layers. The maximum signal reached during maximum loading of the surface increased with the molecular mass of the AMD (Fig. 6(A)) in linear correlation with the protein loadings determined by ellipsometry (1.990.1 nm signal per ng mm − 2 protein). Furthermore, significant differences in the binding kinetics at different AMD-layers were observed. This becomes evident from the kinetic evaluation of the binding curves during incubation of 50 mg ml − 1 Fab at three different layers (Fig. 6(A)). Plotting the binding rate versus the surface loading, the slope of the curves corresponds to the ks of the binding event (Fig. 6(B)). At binding rates below 0.1 nm s − 1, the contribution by mass-transport to the surface is negligible and only the binding kinetics of the hapten–antibody interaction is detected. In case of binding kinetics independent of the layer properties, the same rate constants ks are expected, i.e. parallel curves in Fig. 6(B). However, the rate constants determined from the linear region below 0.1 nm s − 1 strongly decreased with increasing molecular mass of the AMD over one order of magnitude
Fig. 5. Comparison of non-specific binding during incubation of 100 ml ml − 1 calf serum in PBS at a silanized surface (—) and at an AMD300 layer (--- ).
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Fig. 6. Binding kinetics at three different dextran layers (50 mg ml − 1 Fab-fragment). (A) binding curves, twice for each dextran. The marks indicate the first point taken into account for the evaluation; (B) slope of the binding curve versus loading ( , AMD10; , AMD300; 2, AMD2000).
(last column in Table 4). Since the binding rate decreases with increasing thickness and density of the layer, again steric hindrance of the protein binding into the dextran layer is most probably responsible for this effect. The protein already bound into the dextran layer could contribute to this steric hindrance, but the effect was also observed when using reduced CTCA concentrations (cf. [12]). Furthermore, binding of the proteins into the layer requires additional free enthalpy for compensating the entropic forces required for the swelling of the dextran. Thus, the layer morphology considerably affects the kinetics of protein binding into dextran hydrogels. These results confirm that reduced mobility of proteins in the dextran matrix can play an important role for the functional properties of the layers as postulated by Schuck [22].
4. Summary and conclusions In this study, we have thoroughly characterized the morphological and functional properties of dextran layers covalently attached at planar surfaces in a side-on configuration. Monitoring of each step of surface chemistry by ellipsometry confirmed a high functionalization of the surface.
Non-charged dextran layers with surface loadings between 3 and 8 ng mm − 2 were obtained with AMD of different molecular masses by using a selective amide-based coupling chemistry. The density of the dry mono-molecular layers was similar to the density macroscopic dextran layers, suggesting that homogeneous layers were formed. The functional properties of these dextran in aqueous environment were investigated by monitoring protein interactions at the interface. Increasing density and thickness of the dextran layer reduced the kinetics of both specific and non-specific binding. Our results suggest that both types of interactions are dominantly affected by a decreased mobility within the interfacial hydrogel. This mobility can be described as an effective pore size, which is determined by the density and the thickness of the layer, and the number of attachments per molecule (i.e. the loop size). In the absence of charged groups, the dextran–dextran interaction is comparable to the water–dextran interaction and the conformational entropy determines the layer density. The essentially nonbranched polymer chains are therefore not fully extended in this state, leading to a smaller effective pore size compared to the maximum extended dextran. Since the folding entropy increases with the chain length, the density of dextran layer
J. Piehler et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 325–336
increases with molecular mass of the dextran. Hence, the mobility of proteins is reduced by steric hindrance within the matrix. In case of highly charged dextran layers (e.g. carboxymethylated dextran layers used for protein immobilization in biosensor systems), the layer density is mainly determined by inter-chain repulsion of the charged groups, leading to a high extension in aqueous solution, modulated by the ionic strength [15]. Protein uptake and mobility in these these layers is predominantly affected by electrostatic interactions. The functional properties of dextran can be controlled by following parameters: 1. The number of bonds to the surface and their distribution along the polymer chain. These parameters depend on the concentration and distribution of functional groups along the polymer chains and on the surface, as well as on the coupling efficiency. 2. The molecular mass of the attached molecules and the solvent used for the coupling reaction controlling the surface loading and the density of the layer. 3. The concentration of charged groups under the conditions of application (pH, ionic strength) determining the thermodynamics of the arrangements of the polymer chains and their mode of interaction with proteins. In particular for the biophysical application of dextran layers, possible effects of the mesh properties of such interfacial layers can be critical. Efficient shielding of non-specific binding requires high-density layers, which at the same time strongly affect the dynamics of specific interactions. PEGs shield surfaces against macromolecules at much lower molecular mass (500–5000 g mol − 1) by their high exclusion volume [36]. The pore size of these layers is not determined by the polymer conformation, but by the distance of the PEG molecules on the surface [37], allowing for very thin layers with much more defined interfacial arrangement. Dextrans are more suitable as flexible, low-density hydrogels on top of such a protective layer, providing a quasihomogeneous environment for studying protein interactions.
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Acknowledgements The triazine derivative CTCA and polyclonal anti-triazine antibodies and Fab fragments were kindly supplied by Dr Ram Abuknesha, Hirst Research Center, Borehamwood, UK. Oxidized silicon wafers were a gift from Wacker Chemitronic, Burghausen, Germany. K.H. was supported by the Graduiertenkolleg ‘Analytische Chemie’ funded by the Deutsche Forschungsgemeinschaft.
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[21] P.R. Edwards, A. Gill, D.V. Pollard-Knight, M. Hoare, P.E. Buckle, P.A. Lowe, R.J. Leatherbarrow, Anal. Biochem. 231 (1995) 210–217. [22] P. Schuck, Biophys. J. 70 (1996) 1230–1249. [23] R.M.A. Azzam, N.M. Bashara, Ellipsometry and Polarized Light, North-Holland, Amsterdam, 1988. [24] A. Brecht, G. Gauglitz, J. Polster, Biosens. Bioelectron. 8 (1993) 387–392. [25] J. Piehler, A. Brecht, G. Gauglitz, Anal. Chem. 68 (1996) 139 – 143. [26] C.H. Striebel, A. Brecht, G. Gauglitz, Biosens. Bioelectron. 9 (1994) 139–146. [27] K. Spaeth, A. Brecht, G. Gauglitz, J. Colloid Interface Sci. 196 (1997) 128–135. [28] J.A. De Feijter, J. Benjamins, F.A. Veer, Biopolymers 17 (1978) 1759–1772. [29] G. Gauglitz, A. Brecht, G. Kraus, W. Nahm, Sens. Actuators B 11 (1993) 21–27.
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Protein interactions in covalently attached dextran layers Jacob Piehler *, Andreas Brecht, Karin Hehl, Gu¨nter Gauglitz Institut fu¨r Physikalische Chemie, Auf der Morgenstelle 8, D-72076 Tu¨bingen, Germany Accepted 21 April 1999
Abstract Protein interactions with polymeric carbohydrates play an important role for application in chromatography, biomaterials and biophysics. In this study, we present a detailed morphological and functional characterization of covalently side-bound dextran layers by spectroscopic ellipsometry (SE) and reflectometric interference spectroscopy (RIfS). The surface chemistry was monitored step-by-step by ellipsometric characterization of the surface loading. Dextrans of various molecular masses (10–2000 kD) were immobilized leading to surface loadings between 3 and 8 ng mm − 2. The refractive indices of the covalently attached dextran layers under atmospheric conditions (nD =1.51) were very close to the refractive index of a spin-coated dextran layer (nD = 1.52) indicating dense and homogeneous coverage achieved by the coupling chemistry. Under buffer solution, refractive indices between 1.34 and 1.365 and thicknesses between 20 and 40 nm of these dextran layers were determined. A dextran concentration in the hydrated layers of 0.05–0.21 g cm − 3 was estimated from the refractive index. The density and the thickness of the hydrated layers increased with molecular mass of the dextran. Non-specific binding was strongly reduced by the dextran layers and decreased with increasing thickness and density of the layer. Specific antibody binding to haptens immobilized in the dextran layer lead to an increase of both the density and the thickness of the layers. Time resolved detection by RIfS indicated significant decrease of protein mobility in the dextran layer. From these results we conclude that the functional properties of dextran layers with respect to protein interactions are determined by their effective pore-size, which is controlled by the number of bonds, the surface loading and the concentration of charged groups in the polymer. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Dextran layer; Affinity interaction; Immobilization; Spectroscopic ellipsometry; RIfS (reflectometric interference spectroscopy)
1. Introduction
* Corresponding author. Present address: Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel. Tel.: + 972-8-934-2727; fax: + 972-8-9344118. E-mail address: [email protected] (J. Piehler)
Dextrans are hydrophilic and non-charged natural polymeric carbohydrates which are soluble in water in any proportion and form highly hydrated hydrogels. For these properties, dextrans show very low non-specific interactions with proteins. Owing to the high concentration of hydroxyl
0927-7765/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 0 4 6 - 6
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groups in the dextran molecule, chemical modification of these polymers is possible without significantly affecting their hydrophilicity. The essentially non-branched polymer chains are highly flexible and ligands immobilized in dextran matrices are well accessible for proteins. Hence, dextran provide optimum conditions for controlling protein interactions at interfaces and dextran-based polymers have found application in several fields of biochemistry and biotechnology. In particular in affinity and size exclusion chromatography, dextrans have been applied very successfully [1,2]. Cross-linked dextran polymer beads [3] and dextran coated solid supports [4–6] have been used. Dextrans are also frequently used for biomedical and biophysical applications: In the field of biomaterials, dextran coatings for reducing protein adsorption at surfaces to enhance biocompatibility were investigated [7–9]. For heterogeneous phase affinity detection, covalently attached dextran layers have become a standard modification for various transducer materials [10 – 12]. The extraordinary hydrophilicity of dextran coated surfaces made them attractive for preparing supported lipid bilayers [13]. Dextran matrices have also proved to provide suitable conditions for the investigation of inter- and intramolecular forces by atomic force spectroscopy [14,15]. Furthermore, dextran coatings have found interest for masking surface charges and thereby reducing electrokinetic effects [9]. The functional properties of dextran coating are mostly optimized empirically and informations about them are rather confusing. Surface loadings of B 1 to 8 ng mm − 2 have been obtained on different substrate materials by various preparation methods [5,7,13,16]. By scanning force microscopy (SFM) on silica surfaces in air, clustered distribution of dextran molecules attached by non-aqueous coupling chemistry was observed [17]. SFM measurements in aqueous environment have indicated high extension (140 – 190 nm) of covalently attached carboxymethylated dextran layers on gold surfaces, strongly depending on the pH and the ionic strength of the ambient buffer [18]. Using reflection interference contrast microscopy, ex-
tensions of 80–100 nm were found for covalently attached, non-functionalized dextran layers in aqueous medium [13]. Attachment of the dextran molecules site-directed in side-on or end-on configuration led to significantly different functional properties [9]. Investigating the kinetics of protein interaction in dextran layers by label-free optical detection, influence of the polymer matrix on the interaction rates was observed [19–21]. Different accessibility of the binding sites within the dextran layer [20,21] and different protein mobility in the dextran matrix [22] have been proposed to be responsible for these effects. These results indicate that the morphology of dextran coatings and their functional properties with respect to protein interactions are correlated and strongly depend on the preparation technique. Hence, for a systematic design of the functional properties of dextran coated surfaces, a more profound understanding of processes occurring in dextran layers is required. In this study we present a detailed investigation of the morphological and functional properties of interfacial dextran layers. Dextrans of various molecular mass were covalently attached onto planar silica surfaces in a side-on configuration. Spectroscopic ellipsometry (SE) was used for physical characterization of attached layers. This technique allows for an absolute and non-destructive assessment of the thickness and the dielectric function of thin organic and biochemical layers in atmospheric and aqueous environment [23]. These parameters gave information about the surface loading and the morphology of the layers. Protein binding at the modified surfaces was investigated time-resolved by reflectometric interference spectroscopy (RIfS), a very sensitive technique for monitoring binding events at interfaces [24,25]. Protein surface loadings as well as binding kinetics during specific and non-specific interactions at different dextran layers were investigated. We correlate the functional properties and the morphology revealed by these investigations and draw principal conclusions on how to control these properties by the preparation method.
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2. Experimental
2.1. Materials Common organic compounds were purchased either from Fluka, Neu-Ulm, Germany or Sigma, Deisenhofen/Germany. 4Aminobutyldimethylethoxysilane (ABDMS) was purchased from ABCR, Karlsruhe, Germany. 4Chloro-6-(isopropylamino) ×-1,3,5-triazine-2-(6%-amino)caproic acid (CTCA) × and affinity-purified, polyclonal sheep anti-triazine antibodies and Fab fragments were kindly supplied by Dr Ram Abuknesha from GEC Research, Borehamwood, UK. Dextrans of various average molecular mass (9.3, 38, 298 and 2000 kD) were purchased from Sigma. Partially aminofunctionalized dextrans (AMD) were obtained from these dextrans (AMD10, AMD40, AMD300 and AMD2000 according to the average molecular mass of the dextran) by diol oxidation and reductive amination as described recently [12]. By this method, approximately 10% of the anhydroglucose units were functionalized.
2.2. Preparation of dextran layers AMD was covalently attached in a side-on configuration by amide-based chemistry as detailed before [12]. The silica surface was activated by silanization with an amino-functionalized silane (ABDMS). After conversion of the surface amino groups with succinic anhydride (SA) into carboxyl groups, AMD was coupled at concentrations of 0.2 – 0.5 mg ml − 1 water via its amino groups using EDC. This selective coupling technique allows limiting the number of bonds to the surface by the number of amino groups introduced along the dextran chains. The hapten CTCA was coupled to the remaining amino groups of the AMD in DMF in presence of diisopropylcarbodiimide. As a reference surface, CTCA was directly coupled to the amino groups of an ABDMS-silanized surface. Owing to a high excess of the immobilized CTAC and a high affinity of the anti-CTCA-antibodies, the antibody bound to the surface are essentially trapped
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by rebinding and only very low dissociation is observed [12]. Dense dextran coatings of several 100 nm thickness were prepared by spin-coating (Convac 1001, Convac, Wiernsheim, Germany) aqueous dextran solutions (0.1–1 mg ml − 1) onto silicon wafer for 50 s at 5000–7000 rpm and drying at 60°C.
2.3. Spectroscopic ellipsometry (SE) The experimental set-up for the ellipsometric investigation of the attached layers with a spectroellipsometer (ES4G, Sopra, Paris, France) was discussed in detail before [26,27]. Oxidized silicon wafers (silica layers of approximately 170 and 250 nm in thickness, a gift from Wacker Chemitronic, Burghausen, Germany) were used as substrates for the surface chemistry. Each step of surface chemistry was characterized under atmospheric conditions. For characterizing the morphology of the dextran layer, the layer properties before and after maximum loading with an antibody binding specifically to the CTCA immobilized in the dextran layer were investigated under aqueous conditions. The antibody (50 mg ml − 1 in PBS) was incubated on the wafer (100 ml cm − 2) for 30 min. After the incubation, the samples were quickly rinsed with PBS and put back in the measurement chamber containing PBS with 5 mg ml − 1 antibody to reduce dissociation by providing steady state conditions during the measurement. The antibody was selectively removed from the surface by alternate rinsing with 2 mg ml − 1 pepsin pH 2 and a mixture of water, acetonitrile and propionic acid (50:50:1) according to reference [12].
2.4. E6aluation Refractive index and thickness of the attached layers were determined by fitting a model layer system to the ellipsometric angles using a non-linear least squares algorithm. The substrate was parameterized assuming a thin layer of amorphous silicon between the crystalline silicon substrate and the silica layer on top as detailed before [26,27]. Silanization with ABDMS and conversion by SA were assumed as an increase of the silica layer since refractive index and layer thickness
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could not be determined independently because of the small changes of the signal. The dextran layers were described as a distinct layer on top of the previously characterized silica layer. They were assumed to be homogeneous, isotropic and nonabsorbing and the real part of the dielectric functions was described by a two-parameter Cauchy model n(l)= A+
B l2
(1)
The surface loading for each step of chemistry was determined by estimating the density from the refractive index of the layer under atmospheric conditions. A typical refractive index nD of 1.43 was assumed for an organic layer with a density r of 1.0 g cm − 3 and an increment dn/dr of 0.24 g cm − 3. These values were estimated from : 25 different liquid aliphatic organic compounds. Dextran layers, protein layers and mixed dextran/ protein layers under buffer solution were assumed to form one single isotropic and homogeneous layer. The refractive indices were again parametrized by a two parameter Cauchy model. All other parameters were kept constant during the fitting procedure. The dextran concentrations in the hydrated layers cdex were estimated from the refractive index nD of the layer nlayer and the ambient buffer nambient by assuming linearity of the refractive index increment dn/dc according to de Feijter [28]: cdex =
nlayer − nambient (dn/dc)
(2)
For mixed dextran/protein layers, the refractive index was assumed to be a linear combination from the refractive index increments of the components of the concentration ci: nlayer = namb +% i
dn ·ci dc i
(3)
monitoring binding events at interfaces was discussed in detail before [24,29]. The change in apparent optical thickness of a thin transparent layer upon protein binding is detected by interference of white light reflected at the interfaces of the layers using a diode array spectrometer. Binding curves S(t) were recorded as apparent optical thickness versus time. A change of the surface loading of 1 ng mm − 2 protein leads to a signal S:1.7 nm (theoretical calculations). The characterization of the functional properties of dextran layers was carried out as published before [12]: non-specific adsorption was investigated by incubating 1 mg ml − 1 ovalbumin or 100 ml ml − 1 calf serum. Maximum loading by specific binding to the immobilized CTCA was determined by incubating 50 mg ml − 1 antibody. The protein solutions were incubated for approximately 500 s. The total cycle including baseline measurement before and after the binding curve took approximately 14 min. The surface was regenerated on-line by incubation of 2 mg ml − 1 pepsin pH 2 for several minutes and a short pulse (30 s) of a mixture of water, acetonitrile and propionic acid (50:50:1).
2.6. Biomolecular interaction analysis The binding kinetics was determined from the binding curve S(t) using a model described in detail before [30,31]: bimolecular interaction of the receptor R in solution with the immobilized ligand L was assumed. The slope of the binding curve dS(t)/dt is then given as dS(t) = ka·cR·(Smax − S(t))− kdS(t). dt
(4)
S(t) is the response with time, Smax is the maximum loading response, cR is the receptor concentration in solution, ka is the association rate constant and kd is the dissociation rate constant, or
2.5. Reflectometric interference spectroscopy (RIFS)
dS(t)dt = ka·cR·Smax − (ka·c+ kd)·S(t) dt
The interaction of proteins with the surface was monitored time-resolved by RIfS. The principles and the experimental set-up of this technique for
Binding curves where analyzed by plotting the slope of the binding curve dS/dt versus the response S(t) [31]. From the slope of the curves the rate constant ks
(5)
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ks = ka·c+ kd
(6)
was derived which was taken as a measure to compare the kinetics at different dextran layers. For a profound and reliable evaluation of the kinetics at different layers, the influence of mass transport contributions has to be negligible [32]. For the flow system used for these investigations, a slope dS(t)/dt of 3 – 4 pm s − 1 per mg ml − 1 protein was found for mass transport limited binding of antibodies. Undisturbed binding kinetics were assumed at slopes below 2 pm s − 1 per mg ml − 1 protein.
3. Results and discussion
3.1. Characterization of the dextran layer under atmospheric conditions The surface chemistry was stepwise characterized by using ellipsometry under atmospheric conditions. The layer density r was estimated from the refractive index of the layer. The loading of the surface after each step of surface chemistry was calculated from the thickness and the refractive index (Table 1). For the ABDMS- and for the SA-layer, a density of 1.15 g cm − 3 was estimated assuming the same refractive index as the silica layer (1.466). The molar surface loading of 2.6 pmol mm − 2 corresponds to a surface coverage of about 1012 ABDMS molecules mm − 2. This figure is in good agreement with results from measurements using fluorescamine for determination of the amine group surface density (data not published). The molar loading of 2.5 pmol mm − 2 by
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SA indicates that most of the amino groups ( \ 90%) were converted by this reaction. Hence, the concentration of reactive groups on the surface for the attachment of the AMD was approximately 1012 mm − 2. After coupling the AMD, determination of both the thickness and the refractive index of the dextran layer from the ellipsometric angles was possible with good accuracy. A refractive index nD of :1.51 was found and a layer thickness of 5.3 nm for AMD300 coupled at a concentration of 0.2 mg ml − 1 water (cf. Table 1). A density of 1.3 g cm − 3 was estimated for this refractive index (other authors have assumed 1.56 g cm − 3 [7,13] which seems to be too high compared to other organic compounds of this refractive index). From these parameters, a surface loading of 6.9 ng mm − 2 was determined for this dextran layer. In case of maximum coupling efficiency (the dextran is attached to all active groups available at the surface), about 6% of the monomer units are linked to the surface. Hence, the minimum average loop length is 10 nm assuming a monomer size of 0.65 nm. It is not probable that the monomer units attached to the surface are homogeneously distributed along the polymer chain. However, the partial functionalization of the dextran (10% of the monomer units) limits the minimum average loop length to 6.5 nm. The results found for various layers using dextrans of different molecular masses coupled at a concentration of 0.5 mg ml − 1 water are summarized in Table 2. For a comparison of the surface density, the refractive indices of compact dextran layers of several 100 nm thickness prepared by spin-coating were determined. The refractive in-
Table 1 Characterization of the surface chemistry by spectroscopic ellipsometrya Compound
nD
r (g cm−3)b
d (nm)
DG (ng mm−2)
DG (pmol mm−2)
ABDMS SA AMD300
1.466c 1.466c 1.507
1.15 1.15 1.3
0.38 9 0.07 0.22 90.02 5.3 90.3
0.44 9 0.08 0.25 9 0.02 6.9 9 0.4
2.690.5 2.5 9 0.2 43 9 2d
Refractive index at 589 nm nD, density r, layer thickness d and surface loading DG estimated from d and r. Estimated from the refractive index of the layer. c Refractive index of the silica layer assumed. d With respect to a monomer unit (162 g mol−1). a
b
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Table 2 Summary of the some properties of various dextran layers under atmospheric conditions and in buffer solutiona AMD
dair (nm)
nD (air)
G (ng mm−2)b
fbond (%) dPBS (nm)
AMD10 AMD40 AMD300 Spin-coated
2.5 9 0.3 4.1 9 0.3 6.2 9 0.2 500–5000
1.5190.02 1.5290.02 1.5090.02 1.5290.004
3.39 0.4 5.3 9 0.4 8.19 0.3 700–7000
10 6 4 –
19 90.1 28 98 36 9 6 –
dPBS/dair nD (in PBS) 7.5 6.8 5.8 –
1.341 90.003 1.351 90.002 1.365 90.006 –
cdex (g cm−3)c 0.05 9 0.02 0.12 9 0.01 0.21 9 0.04 –
a
Parameters determined from measurements in air: layer thickness dair, refractive index nair and the same parameters from measurements under PBS (index PBS), surface loading G, maximum fraction of bonded monomer units fbond, swelling dPBS/dair and the concentration of dextran cdex in the hydrated layer. b Estimated assuming a density of 1.3 g cm−3. c Estimated from the refractive index increment.
dices of all covalently attached dextran layers were in the range of the refractive index of the densely packed, spin-coated layers, indicating that a high coverage and homogeneous distribution is achieved by the immobilization technique. A higher surface loading was obtained when coupling at a higher dextran concentration (cf. AMD300 in Tables 1 and 2). The thickness of the dry layers and the surface loading increased with molecular mass of the AMD used for coupling. Since the same percentage of monomer units was functionalized in all AMDs, the average, maximum possible fraction of monomer units bonded to the surface decreased with molecular mass of the AMD. At the same time, the distribution of bonds along the chain length becomes more heterogeneous due to the increasing folding of the molecules with increasing molecular mass.
3.2. Characterization of the dextran layer under buffer solution For understanding protein interaction with the dextran layers, investigations were carried out by SE under aqueous medium (PBS). The properties of the dextran layers found under these conditions are summarized in Table 2. Strong swelling of the layers was observed for all layers, up to thicknesses of 20 – 40 nm. The relative increase of the layer thickness (dPBS/dair) decreased with molecular mass of the dextran from 7.5 to 5.8. Hence, the lowest refractive index of the hydrated polymer layer was found for AMD10. The dextran contents in the hydrated layer was estimated from
the refractive index increment of 0.15 ml g − 1 for dextrans in water [33] by using Eq. (2). The dextran concentration increased with molecular mass of the dextran from 0.05 g cm − 3 for the AMD10-layer to 0.21 g cm − 3 for the AMD300 layer. Two reasons may be responsible for these differences: (i) the coupling chemistry becomes more efficient with increasing molecular mass of the dextran due to an increasing number of functional groups per molecule leading to more bonds between each AMD molecule and the surface; (ii) folding entropy leads to a lower density of the lower molecular mass dextran molecules. However, the extensions of all hydrated layers were significantly lower (and the density much higher) than those reported from other investigations of dextran layers (80–190 nm) [13,18]. These strong differences cannot be explained solely by the fact that spectral ellipsometry has a lower sensitivity to dilute polymer tails than the techniques used for these investigations [34]. Two further reasons are probably responsible for the low thickness observed for the AMD layers: (i) the coupling chemistry: the higher extended dextran layers were attached by the reaction of the carbohydrate hydroxyl groups with an epoxy-activated surface in aqueous solution. Coupling of AMD by the more efficient and site-directed amide based chemistry provides more bonds to the surface and a more homogeneous distribution of these bonds along the polymer chain. Furthermore, non-covalent interaction of the dextran molecules with the highly carboxy-functionalized silica surface is expected; (ii) low repulsive inter- and intramolecular
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interaction due to the low concentrations of charged groups in the dextran layer. In particular for the highly carboxymethylated dextran used for protein immobilization in label-free biosensors [10,11], much stronger inter- and intramolecular repulsion is expected due to the high concentration of carboxyl groups which are negatively charged above pH 4 – 5. These layers show the highest extension ever reported [18] and could not at all be detected by ellipsometry [35].
3.3. Layer properties after protein binding For a further characterization of the layer morphology, ‘filling’ the dextran matrices with protein was investigated. For this purpose, anti-triazine antibodies, which bound with high affinity to the triazine derivative CTAC immobilized in the dextran layer, were incubated at the surface. The layer thickness and refractive index after maximum loading of the surface with the antibody were determined by SE. High concentration of the CTCA in the layer guaranteed that binding of the antibody was limited only by the flexibility of the dextran layer. When using IgG, cross-linking in the dextran layer due to the two binding sites per molecule was observed. To avoid such interference with the binding event, Fab-fragments with a single binding site were used for characterizing specific binding into the dextran layers. At all dextran layers investigated, both an increase in refractive index and a further strong increase of the layer thickness were observed. A typical change of the real part of the dielectric function of the dextran layer is shown in Fig. 1. This swelling of the layers during antibody binding was found to be reversible and reproducible as demonstrated in Fig. 2 for three cycles of maximum loading with the antibody. These results prove that: (i) the protein was bound into the dextran hydrogel; and (ii) the hydrogel further swelled during the binding event. The properties of the different dextran layers after maximum loading with a specific antibody are summarized in Table 3. For comparison, the results obtained with the ligand directly attached to the silanized surface are added. In this case, formation of a dense protein layer was observed with a refractive
Fig. 1. Real part of the dielectric function of an AMD2000 layer in buffer solution before (A) and after (B) specific antibody binding (average from three measurements, the CV is given by the two dotted lines adjacent to the curves). The refractive index of the buffer solution (C) is plotted for comparison.
index of 1.391 and a thickness of approx. 10 nm. This layer thickness corresponds well to the size of an antibody molecule. The density of the protein saturated dextran hydrogels increased with the molecular mass of the dextran. The refractive index of the AMD300-protein layer is already close to the refractive index of approximately 1.39 found for the pure protein layer. The changes of the layer thickness and refractive index are visualized in Fig. 3. The relative swelling of the dextran layer during protein binding
Fig. 2. change of the thickness d (() and the refractive index nD ((2) of a dextran layer (AMD2000) during three times of maximum loading by specific antibody binding (steps 2, 4 and 6) and regeneration of the surface (1, 3 and 5).
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Table 3 Properties of dextran layers using various AMDs after maximum specific binding of the antibody to the ligand immobilized in the dextran comparison with the protein binding to the directly attached liganda AMD
dprot (nm)
Nd
G (ng mm−2)
dprot/dPBS
dprot/dair
Gprot/ Gdex
–b AMD10 AMD40 AMD300
10.1 90.4c 299 1 469 1 609 4
1.3919 0.001c 1.36990.007 1.3829 0.003 1.3889 0.002
3.5 90.3c 2.9 9 0.3d 7.6 90.3d 11.0 9 1.0d
– 1.58 1.61 1.67
– 12 11 10
– 1.2 1.6 1.4
Thickness dprot and refractive index nD of the layer, protein loading G, relative increase during protein binding dprot/dPBS, overall increase dprot/dair and relative protein loading Gprot/Gdex). b CTCA directly attached to the amino-silanized surface. c Maximum loading by anti-triazine IgG; including considerable non-specific binding. d Calculated from the total d and nD assuming the dextran loading determined from the dry layers. a
(dprot/dPBS) slightly increased with molecular mass of the AMD. However, the overall swelling from the dry dextran layers to the protein loaded layers (dprot/dair) is nearly constant for all layers, indicating only negligible differences in the capacity of the layers. This result is confirmed by the fact that the protein– dextran ratio of the layers does not differ significantly for all layers. In contrast to the swelling, the increase of the layer refractive index relative to the buffer refractive index strongly decreased with increasing molecular mass (Fig. 3). Hence, binding of proteins into the dextran layer changes from ‘filling the pores’ at lower molecular mass to ‘expatiate the layer’ at higher molecular mass of the dextran. However, the maximum extension reached even at very high density of the layer was still significantly below the extension reported for dextran layers attached by epoxy-activation [13,18]. These differences confirm that the
Fig. 3. Increase of thickness d and refractive index n during antibody binding compared for three different AMDs.
coupling chemistry plays an important role for the flexibility of the attached layer.
3.4. Time resol6ed measurement of protein binding Specific and non-specific interaction of proteins with the AMD layers was carried out by RIfS. A typical characterization cycle of protein binding including regeneration of the surface is shown in Fig. 4. The results of the functional characterization by RIfS are compared in Table 4. High and fast non-specific binding of ovalbumin was observed when the ligand was directly attached to the silanized surface [12]. At dextran-modified
Fig. 4. Time resolved characterization of protein interaction at an AMD2000 layer and repeated regeneration of the surface with pepsin and acetonitrile.
J. Piehler et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 325–336 Table 4 Maximum signal (increase in optical thickness) during nonspecific binding of 1 mg ml−1 ovalbumin Gnon and specific binding of a Fab-fragment Gspec at dextran modified surfacesa AMD
Gnon (nm)
Gspec (nm)
ks
–b AMD10 AMD40 AMD300 AMD2000
2.2 0.25 0.05 B0.01 B0.01
2.9 4.5 14 20 25
:0.1 0.067 0.022 0.016 0.009
a Determined by RIfS (mean value from four measurements), as well as the rate constant ks. b CTCA directly attached to the silanized surface.
surfaces, much lower non-specific protein adsorption was observed (B 15%). The amount of nonspecifically bound ovalbumin decreased with the molecular mass of the dextran. At dextran loadings above 5 ng mm − 2 (AMD300), no significant non-specific protein binding was detectable (response of less than 10 pm optical thickness within the incubation period). The binding curves revealed that not only the absolute amount of adsorbed protein, but also the adsorption rate constants were strongly reduced by the dextran layers. Apparently, the dextran layers effectively shielded the surface against ovalbumin, a 60 kD protein with a diameter of : 6 – 7 nm. The shielding by these layer against other proteins was less effective: when incubating diluted serum —a mixture of proteins of various molecular mass— much stronger non-specific adsorption was observed (Fig. 5). However, again the binding rate was significantly reduced by the dextran coating. Proteins of lower size can apparently permeate the layer more easily. These results indicate that nonspecific binding at the dextran modified surfaces mainly occurs at the original (silanized) substrate surface underneath the dextran layer. Shielding of the surface is apparently achieved by the dextran meshwork, sterically hindering proteins to approach the substrate surface. The observation that shielding of dextran layers is much less effective when the dextran molecules are coupled in end-on configuration [9] corroborates this mechanism. Hence, the shielding efficiency is determined by an ‘effective pore size’ of the dextran layer, the prop-
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erty which is utilized for size-exclusion chromatography. In contrast to this shielding principle, ‘entropic’ shielding is achieved by coatings of high exclusion volume polymers such as poly(ethylene glycole) (PEG) [36]. Characterizing the kinetics of specific antibody binding to the immobilized CTCA gave further insight in the properties of the AMD-layers. The maximum signal reached during maximum loading of the surface increased with the molecular mass of the AMD (Fig. 6(A)) in linear correlation with the protein loadings determined by ellipsometry (1.990.1 nm signal per ng mm − 2 protein). Furthermore, significant differences in the binding kinetics at different AMD-layers were observed. This becomes evident from the kinetic evaluation of the binding curves during incubation of 50 mg ml − 1 Fab at three different layers (Fig. 6(A)). Plotting the binding rate versus the surface loading, the slope of the curves corresponds to the ks of the binding event (Fig. 6(B)). At binding rates below 0.1 nm s − 1, the contribution by mass-transport to the surface is negligible and only the binding kinetics of the hapten–antibody interaction is detected. In case of binding kinetics independent of the layer properties, the same rate constants ks are expected, i.e. parallel curves in Fig. 6(B). However, the rate constants determined from the linear region below 0.1 nm s − 1 strongly decreased with increasing molecular mass of the AMD over one order of magnitude
Fig. 5. Comparison of non-specific binding during incubation of 100 ml ml − 1 calf serum in PBS at a silanized surface (—) and at an AMD300 layer (--- ).
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Fig. 6. Binding kinetics at three different dextran layers (50 mg ml − 1 Fab-fragment). (A) binding curves, twice for each dextran. The marks indicate the first point taken into account for the evaluation; (B) slope of the binding curve versus loading ( , AMD10; , AMD300; 2, AMD2000).
(last column in Table 4). Since the binding rate decreases with increasing thickness and density of the layer, again steric hindrance of the protein binding into the dextran layer is most probably responsible for this effect. The protein already bound into the dextran layer could contribute to this steric hindrance, but the effect was also observed when using reduced CTCA concentrations (cf. [12]). Furthermore, binding of the proteins into the layer requires additional free enthalpy for compensating the entropic forces required for the swelling of the dextran. Thus, the layer morphology considerably affects the kinetics of protein binding into dextran hydrogels. These results confirm that reduced mobility of proteins in the dextran matrix can play an important role for the functional properties of the layers as postulated by Schuck [22].
4. Summary and conclusions In this study, we have thoroughly characterized the morphological and functional properties of dextran layers covalently attached at planar surfaces in a side-on configuration. Monitoring of each step of surface chemistry by ellipsometry confirmed a high functionalization of the surface.
Non-charged dextran layers with surface loadings between 3 and 8 ng mm − 2 were obtained with AMD of different molecular masses by using a selective amide-based coupling chemistry. The density of the dry mono-molecular layers was similar to the density macroscopic dextran layers, suggesting that homogeneous layers were formed. The functional properties of these dextran in aqueous environment were investigated by monitoring protein interactions at the interface. Increasing density and thickness of the dextran layer reduced the kinetics of both specific and non-specific binding. Our results suggest that both types of interactions are dominantly affected by a decreased mobility within the interfacial hydrogel. This mobility can be described as an effective pore size, which is determined by the density and the thickness of the layer, and the number of attachments per molecule (i.e. the loop size). In the absence of charged groups, the dextran–dextran interaction is comparable to the water–dextran interaction and the conformational entropy determines the layer density. The essentially nonbranched polymer chains are therefore not fully extended in this state, leading to a smaller effective pore size compared to the maximum extended dextran. Since the folding entropy increases with the chain length, the density of dextran layer
J. Piehler et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 325–336
increases with molecular mass of the dextran. Hence, the mobility of proteins is reduced by steric hindrance within the matrix. In case of highly charged dextran layers (e.g. carboxymethylated dextran layers used for protein immobilization in biosensor systems), the layer density is mainly determined by inter-chain repulsion of the charged groups, leading to a high extension in aqueous solution, modulated by the ionic strength [15]. Protein uptake and mobility in these these layers is predominantly affected by electrostatic interactions. The functional properties of dextran can be controlled by following parameters: 1. The number of bonds to the surface and their distribution along the polymer chain. These parameters depend on the concentration and distribution of functional groups along the polymer chains and on the surface, as well as on the coupling efficiency. 2. The molecular mass of the attached molecules and the solvent used for the coupling reaction controlling the surface loading and the density of the layer. 3. The concentration of charged groups under the conditions of application (pH, ionic strength) determining the thermodynamics of the arrangements of the polymer chains and their mode of interaction with proteins. In particular for the biophysical application of dextran layers, possible effects of the mesh properties of such interfacial layers can be critical. Efficient shielding of non-specific binding requires high-density layers, which at the same time strongly affect the dynamics of specific interactions. PEGs shield surfaces against macromolecules at much lower molecular mass (500–5000 g mol − 1) by their high exclusion volume [36]. The pore size of these layers is not determined by the polymer conformation, but by the distance of the PEG molecules on the surface [37], allowing for very thin layers with much more defined interfacial arrangement. Dextrans are more suitable as flexible, low-density hydrogels on top of such a protective layer, providing a quasihomogeneous environment for studying protein interactions.
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Acknowledgements The triazine derivative CTCA and polyclonal anti-triazine antibodies and Fab fragments were kindly supplied by Dr Ram Abuknesha, Hirst Research Center, Borehamwood, UK. Oxidized silicon wafers were a gift from Wacker Chemitronic, Burghausen, Germany. K.H. was supported by the Graduiertenkolleg ‘Analytische Chemie’ funded by the Deutsche Forschungsgemeinschaft.
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