3228
Langmuir 2006, 22, 3228-3234
Deposition of Patterned Glycosaminoglycans on Silanized Glass Surfaces Antonio Peramo, Ashley Albritton, and Garrett Matthews* Department of Physics, UniVersity of South Florida, Tampa, Florida 33620 ReceiVed April 30, 2005. In Final Form: January 27, 2006 We report the robust attachment of glycosaminoglycans (GAGs) on silanized glass surfaces. Depositions were performed both by immersion and by application of a pattern by means of microcontact printing. Immunofluorescence assays were performed to verify the deposition and the quality of the patterns. In addition, AFM studies of the coated surfaces were performed in order to study some physical characteristics of the deposited GAGs layers. These results may prove useful for the characterization of the mechanical properties of GAGs in the glycocalyx and its relation with cellular migration.
Introduction Patterning of biomolecules onto solid substrates has been extensively used in previous years and is of interest for several biological applications, including controlled adhesion and growth of cells, surface functionalization, biosensors, chromatography, and immunoassays.2-4 Here we report the robust attachment of glycosaminoglycan (GAG) polysaccharides, both in a continuous layer by immersion and in a pattern produced by microcontact printing (µCP). The resulting surfaces should prove useful for a variety of studies, including those on protein/GAG interactions and cell adhesion and migration.5-8 µCP9 has been established as a patterning technique delivering self-assembled monolayers onto substrates in a simple, rapid, and reproducible manner. In the past, patterns of proteins,10 lipids,11 and amino acids12 have been created by this method. Along with these works, there are extensive studies on the adsorption of proteins at surfaces13,14 giving clear indication that, to date, efforts have been focused primarily on developing patterned protein surfaces. However, to our knowledge, there are no reports in the literature on reactive µCP of mucopolysaccharides as reported here. Given the increasing importance of the study of the biological processes of polysaccharides, we herein introduce a surface functionalization technique that will further the field of “glycomics”, a term that reflects, much in the sense of the term proteomics, the scientific and economical opportunities of their expanded study. * To whom correspondence should be addressed. Email: gmatthew@ cas.usf.edu. (1) Abbreviations: µCP, microcontact printing; GAGs, glycosaminoglycans; APTES, 3-aminopropyltriethoxy-silane; PDMS, poly(dimethylsiloxane); AFM, atomic force microscope. (2) Dontha, N.; Nowall, W. B.; Kuhr, W. G. Anal. Chem. 1997, 69, 2619. (3) Homola, H. B. Lu; Yee, S. S. Electron. Lett. 1999, 35, 1105. (4) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Langmuir 1998, 14, 2225. (5) Wu, Y. J.; La Pierre, D. P.; Wu, J.; Yee, A. J.; Yang, B. B. Cell Res. 2005, 15, 483. (6) Handel, T. M.; Johnson, Z.; Crown, S. E.; Lau, E. K.; Sweeney, M.; Proudfoot, A. E. Annu. ReV. Biochem. 2005, 74, 385. (7) Nomura, K. Trends Glycosci. Glyc. 2004, 16, 125. (8) Kalluri, R. Nat. ReV. Cancer 2003, 3, 422. (9) Kumar, A.; Whitesides, G. N. Appl. Phys. Lett. 1993, 63, 2002. (10) James, C. D.; Davis, R. C.; Kam, L.; Craighead, H. G.; Isaacson, M.; Turner, J. N.; Shain, W. Langmuir 1998, 14, 741. (11) Hovis, J. S.; Boxer, S. G. Langmuir 2000, 16, 894. (12) Branch, D. W.; Corey, J. M.; Weyhnmeyer, J. A.; Brewer, G. J.; Wheeler, B. C. Med. Bio. Eng. Comput. 1998, 36, 135. (13) Hlady, V.; Buijs, J. Curr. Opin. Biotechnol. 1996, 7, 72. (14) Ortega-Vinuesa, J. L.; Tengvall, P.; Lundstrom, I. Thin Solid Films 1998, 324, 257.
Our choice of biomolecules is explained easily. The functionalized surfaces will contain a layer of molecules that are expressed in the outer area of the cell surface, known as glycocalyx. This is a periodic brushlike structure located on the surface of endothelial cells that only recently has been recognized to be of great biological importance. The glycocalyx contains, among other molecules, various GAGs in the form of proteoglycans, including heparan sulfate proteoglycan and chondroitin sulfate proteoglycan. These extracellular matrix GAGs provide structural links between fibrous and cellular elements, contribute to the viscoelasticity of the glycocalyx, and regulate the permeability of plasma elements within the matrix.15 Chemically, keratan sulfate is primarily composed of the disaccharide repeat D-galactose and D-glucosamine, heparan sulfate of D- or L-glucuronic acids, and D-galactosamine and chondroitin sulfates mainly of D-glucuronic acid and D-glucosamine.16 As reported by Weinbaum,17 little was known about the glycocalyx until recently. Squire18 and co-workers showed that the glycocalyx in essence consists of a fibrous meshwork with a 20 nm characteristic spacing with brushes of size ∼10-12 nm. Models of the glycocalyx structure based solely on lengths of GAGs side chains proposed a matrix of 7 or 8 nm gap spacing, which was associated with the disaccharide repeat of the GAGs chains. In our simplified process of simulating this structure, we will initially focus our attention on a 2D view that, observed from a zenithal position, provides a meshwork of the protein terminal-chain GAGs. Within this simplification, deposition of GAGs monolayers with chains separated by 10-12 nm would be the target of experimental procedures such as the GAG submersion deposition presented here. Although we will focus primarily on the study of GAGs, we have carried out a parallel work with heparan sulfate proteoglycan, known as perlecan, which represents a proteinacious component of the glycocalyx. Perlecan expression is found primarily in mature tissues and is prominent in the endothelial cell basement membrane of all vascularized organs, specifically liver, lung, pancreas, and kidney. Perlecan contains three GAG chains, heparan, and chondroitin sulfate chains in different proportions, (15) Wight, T. N. Atherosclerosis 1989, 9, 1. (16) Lindahl, U.; Hook, M. Annu. ReV. Biochem. 1978, 47, 385. (17) Weinbaum, S.; Zhang, X.; Han, Y.; Vink, H.; Cowin, S. Proc. Nat. Acad. Sci. 2003, 100, 7988. (18) Squire, J. M.; Chew, M.; Nneji, G.; Neal, C.; Barry, J.; Michel, C. J. Struct. Biol. 2001, 136, 239.
10.1021/la051166m CCC: $33.50 © 2006 American Chemical Society Published on Web 03/02/2006
Deposition of Patterned Glycosaminoglycans
Langmuir, Vol. 22, No. 7, 2006 3229
Figure 1. Simplified scheme of the procedure of surface modification. APTES is first fixed to glass in ethanol forming an amino-terminated layer that is further modified with different GAGs for 24 h in the presence of cyanoborohydride.
Figure 2. Simplified scheme of the procedure of surface modification for heparan sulfate proteoglycan. First, the cross-linker and heparan sulfate proteoglycan were reacted and immediately brought into conformal contact for 5 min with the APTES-functionalized glass and then separated. The procedure yields two molecules of N-hydroxysuccinimide (NHS).
located at one end of the molecule. A deposition of heparan sulfate proteoglycan on glass will give us an understanding of the orientation of the GAG chains and will provide some insights into the characteristics of the depositon of glycoproteins vs GAGs alone. However, it is important to emphasize that heparan sulfate proteoglycan exposes for attachment to the APTES surface additional amino terminal positions not belonging to the GAG chains. The utility of surface attachment of biomolecules for the various applications outlined above requires that the molecules retain their activity. Additionally, the attachment should be stable over long times under various environmental conditions. In our case, that means that GAGs will be functional for long periods after the deposition on glass; thus, we have developed a means by which to robustly attach the polysaccharides through their reducing ends, producing an orientation that mimics that of the GAGs bound to the protein backbone of the proteoglycan. While attempting to select a particular orientation of the molecules bound to the surface may not guarantee that reactivity is retained, the combination of the simplicity of the polysaccharide structure with a deposition technique that mimics the native orientation of the GAGs on the protein backbone of the proteoglycan should in principle increase this possibility. The attachment of GAGs to the glass surface is produced by reductive amination in a reaction mediated by sodium cyanoborohydride (NaBH3CN) in which GAGs are bonded to the amino-terminated monolayer 3-aminopropyltriethoxysilane (APTES). Two different procedures were used. (1) GAGs were transferred to surfaces by µCP and bound to an amino-terminated monolayer on glass and (2) GAGs were bound to similar surfaces in continuous layers by performing all reactions by complete immersion, without using the polymeric stamp to transfer the polysaccharides. Demonstration of successful attachment of GAGs and heparan sulfate proteoglycan was done through fluorescence microscopy. A biotinylated primary monoclonal antibody was used with streptavidin tagged with a fluorophore as the secondary marker, allowing for rapid visualization of the quality and extent of the depositions. Further characterization and analysis of these nanoscale-modified surfaces was done by means of AFM: surface images were taken, and the thickness of the deposited layers was measured.
Materials and Methods Materials. Glass cover slips (Corning 0211) used for deposition were cleaned with a plasma cleaner (Harrick Scientific), rinsed with Nanopure water, and dried under a filtered N2 stream. Heparan Sulfate (Seikagaku America, MW ) 11 kDa), Keratan Sulfate (Seikagaku America, MW ) 13 kDa), Chondroitin Sulfate C (Seikagaku America, MW ) 60 kDa) and Chondroitin Sulfate A (Sigma Aldrich, MW ) 25 kDa) were the mucopolysaccharides used, along with the proteoglycan Heparan Sulfate Proteoglycan (Sigma Aldrich, MW > 400 kDa). Surface Modification. Silanization of the glass surface with APTES was performed as previously described,19 with minor modifications. Briefly, cover slips were rinsed with ethanol and Nanopure water and placed in a plasma cleaner for 45 s. Immediately after, the cover slips were incubated in freshly made 0.86 mM solutions of APTES in ethanol for 15 min at room temperature. Afterward, they were rinsed in ethanol and water (twice). Samples were used immediately. This modification produces an NH2terminated submonolayer or monolayer, as is shown in Figure 1. GAG Submersion Deposition. The APTES substrates were incubated for 24 h at room temperature in solutions of 0.1 µg/mL heparan sulfate, keratan sulfate, chondroitin sulfate A, and chondroitin sulfate C in PBS with NaBH3CN (Acros Organics) at a concentration of 3 µg/mL.20 The proposed reaction is shown schematically in Figure 1. After incubation, samples were rinsed copiously with water, followed by ethanol and water rinses, and dried under a nitrogen stream. In the case of heparan sulfate proteoglycan, additional APTES surfaces were modified by first reacting 0.1 µg/mL solutions of the proteoglycan with the cross-linker bis[sulfosuccinimidyl] suberate, (Pierce Biotechnology) for 30 min followed by quenching for 15 min with 1 M Tris, following the protocol described by the manufacturer. This reaction links primary amines found on the protein core backbone of the heparan sulfate proteoglycan through bis[sulfosuccinimidyl] suberate to the primary amine bound to the substrate (see Figure 2). At this point, all samples were ready for characterization by immunofluorescence or AFM. GAG Patterning by µCP. In this experiment, GAGs were transferred to the glass surface using a PDMS stamp that had been cast from a calibration grid with a ∼1.5 µm spacing between ∼1.5 µm wide parallel lines 500 nm in height (pitch ≈ 3 µm). Silicon gratings (TGZ03, MikroMasch) were used to cast the PDMS stamp, (19) Weiping Q.; Bin, X.; Lei, W.; Chunxiao, W.; Danfeng, Y.; Fang, Y.; Chunwei, Y.; Yu, W. J. Colloid Interface Sci. 1999, 214, 16. (20) Jentoft, N.; Dearborn, D. G. J. Biol. Chem. 1979, 254, 4359.
3230 Langmuir, Vol. 22, No. 7, 2006
Peramo et al.
Table 1. List of Primary Antibodies and Streptavidin Secondary Antibodies List of Primary Antibodies antigen recognized
label
dilution
heparan sulfate keratan sulfate chondroitin sulfate 6 heparan sulfate proteoglycan
biotin biotin biotin biotin
1:50 1:100 1:100 1:50
host
source
mouse mouse mouse rat
USBiological Seikagaiku America Seikagaiku America LabVision
List of Streptavidin Secondary Antibodies antigen recognized
fluorophore
concentration/dilution
source
keratan sulfate-biotin heparan sulfate-biotin chondroitin sulfate C-biotin chondroitin sulfate A-biotin heparan sulfate proteoglycan-biotin
DTAF Qdot 525 Qdot 605 Qdot 605 Qdot 655
4.5 µg/mL 1:50 1:50 1:50 1:50
Jackson ImmunoResearch Qdot Corp. Qdot Corp. Qdot Corp. Qdot Corp.
as described elsewhere.21 Briefly, PDMS (Sylgard 184, Dow Corning) was prepared with a 10:1 mass ratio of base to curing agent. The mixture was poured into the glass container in which the silicon grating was placed and allowed to degas overnight in a fume hood. Curing was performed at 65 °C for at least 2 h. The patterning of the GAGs on the glass surface takes place in the following manner. First, an APTES surface is produced as described in the surface modification method. Then, and separately, equal volume amounts of the GAG solutions of 0.1 µg/mL and NaBH3CN are mixed, and 20 µL of these solutions are deposited on the patterned surface of the PDMS stamp for 15 min. During this time, to avoid possible evaporation, the stamp was covered with a humidified soft wiper. Later, the solution on the PDMS was removed with a stream of nitrogen and immediately put into conformal contact with the APTESfunctionalized glass surface, briefly exerting low pressure to help in the contact. The transfer of the patterned biomolecules from the PDMS to the glass took place overnight, and afterward, experiments on the detection and analysis of the deposition were performed. When necessary, the patterned cover slips were stored before use at 4 °C in 0.2 µm filtered Nanopure water. Samples that were used for AFM measurements were dried for measurements in air. In the case of heparan sulfate proteoglycan, the procedure for pattern transfer necessarily was modified. The method of transfer uses a bifunctional N-hydroxysuccinimide ester (bis[sulfosuccinimidyl] suberate or BS3, Pierce Biotechnology, Inc.) that is primary amine reactive to perform the cross-linking of the protein. The reaction is shown in Figure 2. With this reactant, only the amine of the amino acid lysine will give a stable product after reaction, although nitrogen is present on the side chains of other amino acids. First, the crosslinker and heparan sulfate proteoglycan were reacted on the surface of a patterned hydrophilic PDMS for 30 min followed by incubation with the quenching agent Tris (1 M) for 15 min. Excess solution was quickly blown off with a N2 stream and immediately brought into conformal contact for 5 min with the APTES-functionalized glass and then separated. The sample was ready for AFM measurements or immunofluorescence detection. This procedure works because the secondary reaction between APTES and the cross-linker can be performed at any time after reaction with the heparan sulfate proteoglycan. The following procedure was also tried to deposit heparan sulfate proteoglycan, but different attempts failed to materialize the pattern on the glass surface. First, cross-linker was reacted on the APTESfunctionalized glass for 30 min, while at the same time, heparan sulfate proteoglycan was adsorbed on the surface of hydrophilic PDMS for 1 min. Short times are required for the deposition of a monolayer of product. Cross-linker excess and heparan sulfate proteoglycan were both blown off of the surfaces by a N2 stream and immediately brought into conformal contact for 1 min and then separated. This procedure was tried both with and without the addition of the quenching agent. (21) Harrison, C.; Cabral, J. T.; Stafford, C. M.; Karim, A.; Amis, E. J. J. Micromech. Microeng. 2004, 14, 153.
Antibodies and Fluorescence Microscopy. Glass cover slips functionalized by µCP with APTES and GAGs or heparan sulfate proteoglycan were subjected to indirect immunofluorescence detection. Table 1 lists the antibodies used. Before the addition of primary antibodies, cover slips were subjected to streptavidin/biotin blocking (streptavidin/biotin blocking kit, Vector Labs) as follows. First, cover slips were rinsed with PBS and then incubated with streptavidin solution for 15 min and rinsed with PBS and then incubated for 15 min with the biotin solution, as recommended by the manufacturer. Biotinylated primary antibodies were applied to the surfaces as described in Table 1 for 1 h. After incubation, samples were rinsed with cold PBS (3 × 5 min). After the deposition of primary antibodies, fluorescence labeling was performed for keratan sulfate with DTAF streptavidin-labeled secondary antibody (Jackson ImmunoResearch) for 30 min; for heparan sulfate, Qdot 655 Streptavidin Conjugate; for chondroitin sulfate C and chondroitin sulfate A, Qdot 605 Streptavidin Conjugate; and for heparan sulfate proteoglycan, Qdot 525 Streptavidin Conjugate were used. Qdots were incubated for 1 h. After being rinsed with cold PBS (3 × 5 min), samples were taken to an inverted epifluorescence microscope (Nikon TE2000) equipped with a QImaging Retiga EX Monochrome 12-bit digital camera for fluorescence imaging of the reaction between GAGs and antibodies. Samples with DTAF fluorophore were mounted with Vector Labs Hard Mounting Media H1500, while samples with Qdots were mounted with a solution of 90% glycerol in PBS. AFM Surface Measurements. The commercial AFM used for surface measurements and imaging was an Asylum Research MFP 3D. The silicon cantilevers used (NSC36, MikroMasch) had a nominal spring constant of 1.75 N/m and resonant frequency of 155 kHz. During each experiment, the spring constant and resonance frequency were calculated using the built-in software according to the thermal response method.22 Measurements were taken in air. Samples were dried under N2, and images were recorded immediately. All experiments were performed with bare cantilever tips at room temperature.
Results and Discussion Surface Modification and GAGs Deposition. We chose silanization in ethanol because the same procedure in aqueous media yields a low surface concentration of amines.23 This low concentration of APTES produces a monolayer or submonolayer of product. The thickness of the layers of silane depositions and the number of reactive -NH2 groups present on the surface have been quantified previously.24 To avoid APTES polymerization, the concentration of APTES was kept at the low level used. APTES aggregates were not observed over the glass surface (data not shown). APTES multilayer formation could result in (22) Cleveland J. ReV. Sci. Instrum. 1996, 67, 3583. (23) Xiao, S.-J.; Textor, N.; Spencer, N. D. J. Mater. Sci. Mater. Med. 1997, 8, 867. (24) Weiping Q.; Bin, X.; Lei, W.; Chunxiao, W.; Danfeng, Y.; Fang, Y.; Chunwei, Y.; Yu, W. J. Colloid Interface Sci. 1999, 214, 16.
Deposition of Patterned Glycosaminoglycans
undesirable aggregates, giving unreliable results in force measurements. Work with increasing concentrations of APTES is ongoing in order to assess the effects of APTES multilayers on the deposition of GAGs. µCP. The use of 1.5 µm gratings is justified because resolving features at this length scale is more than sufficient for most applications in cell biology and biosensing. PDMS stamps were plasma-cleaned before deposition of biomolecule solutions to make them hydrophilic and to increase wettability. This is an important step in cases where aqueous solutions of biomolecules are used and that provides a more efficient transfer of the GAGs to the functionalized glass surface. The polymeric stamp makes good conformal contact with the APTES-glass substrate, resulting in the relief pattern of its surface being translated to the glass as a pattern of GAG molecules. In all cases, GAGs have been bound strongly enough to provide long term (over several weeks of storage) fixation to the surface. It must be noted that the adsorption of GAGs to PDMS has not been analyzed. While we do not have direct evidence of covalent binding, the control experiments in which the stamping process was carried out in the absence of either the APTES monolayer or the catalyst cyanoborohydride suggests the possibility. It is important to note that the GAG pattern generated remains even after (1) sonication in saturated salt solution for 24 h and (2) after contact mode and lateral force microscopy are performed over several hours of work, abrading the patterned surface with the cantilever tip. In the experiments, the biological activity of the molecules was partially tested by recognition by monoclonal antibodies in immunofluorescence assays. The case of the reaction of heparan sulfate proteoglycan requires additional commentary. Initially, only one ester group reacts with the amine on the protein, releasing just one succinimide. PBS is used to avoid excessive hydrolysis of the cross-linker at basic pH. The reaction is arrested by adding Tris, which contains amino groups, thus exhausting the remaining cross-linker. Instead of Tris, other quenching agents may be used, for instance lysine, which may be added in excess to the reaction. After the reactants are blown from the PDMS surface, the heparan sulfate proteoglycan linked to half of the cross-linker reacts with the primary amine on the APTES substrate, and there is no need for additional whole cross-linker, given that the ester is already linked to the protein. In addition, there is no need for additional quenching agent, given that the interest is in having all proteins containing half of the cross-linker linked to the aminoterminated APTES surface at the end of the reaction. Given that the cross-linker is in huge molar excess, almost all proteins are linked with one-half of the cross-linker and produce the pattern on the surface by being able to find the amino terminal groups. Next, we explain why the failed method does not work. In the case of depositing the cross-linker directly over the aminoterminated glass surface, after the cross-linker is blown from the surface, the remaining concentration is very low and the amount of ester available for reaction is, at a maximum, the number of amino groups, which is much lower than the number of proteins available. Finally, while in the first reaction, the ester is free to move in solution to interact with the lysine, in the second, there is a clear steric restriction for the lysine and ester to find each other. It is possible that at least some quenching of the reaction after the cross-linker has been bound to the proteoglycan. The formation of covalent bonds has not been tested; therefore, we cannot rule out physisorption as the mechanism by which these molecules are bound to the surface. In the end, the proteoglycan pattern was produced as shown in Figure 3D. Because the pattern was not
Langmuir, Vol. 22, No. 7, 2006 3231
produced when the procedure was modified by reacting BS3 with the APTES monolayer first, which should not alter substantially the adsorption process, the proposed means by which the proteoglycan was transferred to the surface in the successful method is by being covalently bound to the amino group of the APTES monolayer. Immunofluorescence. Figure 3 shows fluorescence images that reveal the pattern of the GAGs by the fluorophore DTAF (for keratan sulfate) or Quantum Dots (in all other cases). In all cases, the pattern formed shows high contrast and resolution. The ∼1.5 µm wide lines were transferred over distances of many tens to hundreds of micrometers. No diffusion of the molecules into the open spaces of the pattern is observed, and the edges of the pattern are very well defined at this scale. The use of biotinylated monoclonal antibody and the use of DTAF-labeled streptavidin provided an efficient method for immunofluorescence detection of the pattern on the basis of the strong affinity of the biotin-streptavidin binding. Streptavidin-conjugated Quantum Dots provided also a variation of the method that allowed photographic imaging of the detection at different wavelengths. In one of the cases (chondroitin sulfate A, Figure 3E), it is possible to observe a small increase in the thickness of the patterned lines. This is due to the process of exerting low pressure between the glass and the patterned PDMS stamp when in conformal contact. Further experimentation using defined forces will give a standard procedure to overcome this difficulty, and an example of the initial attemps is included in the Supporting Information. To avoid dropouts in the pattern, PDMS stamps were cleaned in a sonicator for 30 min in ethanol, while the silicon gratings were also sonicated in pure acetone for 30 min after their use. However, defects can be found in the pattern, possibly indicating incomplete cleaning of the stamp prior to use. Both the PDMS stamps and silicon gratings were reused several times, and that may have caused some of the observed defects in the pattern. In another case (heparan sulfate proteoglycan, Figure 3D), it is possible to observe some bleeding. This situation could have been caused by incomplete removal of the liquid from the PDMS surface, which may be easily solved by longer exposure of the sample to the N2 stream. Immunofluorescence control experiments were performed with the following sample types: clean glass with no deposition of GAGs, glass functionalized with APTES with no deposition of GAGs, and APTES functionalized glass with a deposition of GAGs without the reactive NaBH3CN. All cases tested negative to the presence of patterned GAGs by immunofluorescence detection, as can be see in the accompanying Supporting Information. AFM Imaging. AFM images of cover slip surfaces of one the GAGs, chondroitin sulfate A, were collected, and the patterned structures are shown in Figure 4. Some large particulates are observable in the AFM images, along with the desired pattern. They do not appear in depositions of APTES alone, so these particles are most likely either aggregates of GAGs or are contaminants that arrived on the surface during storage. As mentioned, the PDMS stamps were used several times. Over time, the surfaces of the stamps may become contaminated by excess sugar molecules despite the cleaning efforts. This would account for the presence of GAG aggregates. However, the particulates do not interfere substantially with the analysis of the heights of the patterned GAGs, which are easily distinguishable. Table 2 shows a theoretical calculation of the contour lengths of the GAGs, where estimated values of the molecular mass of the products indicated by the manufacturers were used. For the number of sulfates per disaccharide unit, ref 12 was used as a
3232 Langmuir, Vol. 22, No. 7, 2006
Peramo et al.
Figure 3. Fluorescence micrographs. In all cases, products were deposited on a monolayer of APTES-functionalized glass cover slip and reacted with specific biotinylated monoclonal primary antibodies (see text). All images are accompanied by optical intensity profiles produced using ImageJ Image Processing and Analysis software. (A) 40× epifluorescence microscopy image of keratan sulfate with DTAF-labeled streptavidin. DTAF is a fluorescein derivative with the same emission and excitation wavelengths. (B) 40× epifluorescence microscopy image of heparan sulfate with Quantum Dot 655-labeled streptavidin. (C) 40× epifluorescence microscopy image of chondroitin sulfate C with Quantum Dot 605-labeled streptavidin. (D) 63× epifluorescence microscopy image of heparan sulfate proteoglycan with Quantum Dot 525-labeled streptavidin. (E) 63× epifluorescence microscopy image of chondroitin sulfate A with Quantum Dot 605-labeled streptavidin.
Deposition of Patterned Glycosaminoglycans
Langmuir, Vol. 22, No. 7, 2006 3233
Figure 4. AFM surface images. Selected tapping-mode topographic AFM images in air of deposited layers of chondroitin sulfate A on APTES-functionalized surfaces, with cantilevers described in the text. (A) 20 µm × 20 µm scan and 6 nm z scale, scan rate 1 kHz and (B) 10 µm × 10 µm scan. The free amplitude of the cantilever was chosen to be about 100 nm. Two samples were analyzed. For each sample, measurements were taken at two different positions. (C) Plot of the cross-sectional line marked in image (B). Table 2. GAG Characteristic Dataa
primary source to calculate molecular mass of dimers. The estimation assumes a mean disaccharide monomer length ranging from 1 nm, using values from ref 14, to 1.28 nm,25 reported in the chondroitin sulfate-GAGs present in aggrecan in the cartilage, although calculations made using data reported by Arnott and Scott26 and from Rees27 give values between 0.92 and 1.16 nm,
more in line with the 1 nm estimate. We defer the discussion of the conformation of these biopolymers to a future publication. Analysis of cross-sections for chondroitin sulfate A, as shown in Figure 4, gave a mean deposited height of 2.1 ( 0.6 nm. For measurements in air, this height can be considered the thickness in the dry state for chondroitin sulfate A. Given our contour lengths and GAG chain molecular weights, our results are within the range of values reported by Seog and co-workers who used chondroitin sulfate chains with contour lengths of 35 nm and indicated an estimated value of 1.5 nm for the incompressible layer thickness of the GAG in air using AFM isoforce imaging, while reporting a value of 3.18 nm by ellipsometry. Discussion. Two types of surface treatments were perfomed in these experiments: coverage of the entire surface with GAGs by simple submersion and patterning of the molecules on selected areas using µCP via polymeric stamps cast from a commercial calibration grid. The patterned structures are not constructed to provide a perfect simulation of the structure of the GAGs in the glycocalyx. Instead, the patterning procedure has been used in these experiments primarily for the purpose of demonstration of the successful attachment of the molecules to the glass substrates.
(25) Seog, J.; Dean, D.; Plaas, A. H. K.; Wong-Palms, S.; Grodzinsky, A. J.; Ortiz, C. Macromolecules 2002, 35, 5601.
(26) Arnott, S.; Scott, W. E, J. Chem. Soc., Perkin Trans. 1972, 2, 324. (27) Rees, D. A. J. Chem. Soc. B 1969, 217.
keratan sulfate chondroitin sulfate C chondroitin sulfate A heparan sulfate
MM (kDa)
estimated dimer MM (Da)
no. disaccharides per chain
Lc (nm)
13 60 25 11
403 456 456 496
32 131 54 22
32-42 131-170 54-70 22-29
a MM is molecular mass in kiloDaltons (kDa) or Daltons (Da). L c shown is the range of values of the contour lengths. The disaccharides per chain are calculated by dividing the total mass by the mass per dimer. The range of values in contour lengths is calculated by multiplying the number of disaccharides per chain by the mean disaccharide monomer lengths, as reported in the text.
3234 Langmuir, Vol. 22, No. 7, 2006
The demonstration was carried out by means of immunofluorescence and AFM experiments, techniques that cannot be implemented when the entire surface of the glass cover slip is completely covered with the molecules, without any patterned feature. The patterning experiments, while useful in this context as a control for the successful deposition of GAGs, are of further interest to those studying systems in which the spatial extent of GAGs may play an important role. The experiments in this study are intended to describe surface deposition techniques for general use with polysaccharides. Examples of work in which surfaces prepared using these methods may prove useful include the study of the adhesive properties of cells and proteins and for studies of cell migration. Because the point of attachment mimics that of the GAGs bound to the protein backbone, these surfaces are particularly well suited for the study of interactions with the sugars of proteoglycans. The submersion procedure provides the basis for the development of a model system to study cell adhesion to GAGs and a broader list of polysaccharides beyond GAGs. The substrates as prepared simply expose a layer of isolated GAGs whose interaction with cells or other isolated molecules can be analyzed. Previous methods for the deposition of isolated biomolecules on substrates frequently have consisted of culturing cells on the surfaces and then eliminating part of the biological material via lysis.28 However, these methods cannot study interactions between cells and single molecular species of choice, because the lysis procedure cannot eliminate all but one single molecule. As another example of experiments where these surfaces are of interest, an AFM cantilever tip can be functionalized with GAGs or any other molecule and brougth into contact with the substrates to study their interaction and possibly to analyze the conformational properties of the molecule on the substrates. Thus, the surfaces may prove useful, for instance, in individualized studies of GAGGAG or GAG-protein interactions. As mentioned in the Introduction, there are no known reports in the literature on reactive µCP of mucopolysaccharides. The (28) Miao, H.-Q.; Elkin, M.; Aingorn, E.; Ishai-Michaeli, R.; Stein, C. A.; Vlodavsky, I. Int. J. Cancer 1999, 83, 424.
Peramo et al.
method presented for the production of GAG-patterned surfaces also may prove useful in microarray technology or in carbohydrate affinity screening. Finally, as an additional example of an experiment of interest that makes use of the patterned GAG surfaces, investigation of cell migration into regions where the lateral extent of the GAGs changes would give interesting information on the dependence of cell adhesion on the physical dimensions of the surface to which they are adhering.29
Conclusions The deposition and robust attachment of mucopolysaccharides on glass substrates functionalized with a silane agent, exposing an amino-terminated monolayer as functional substrate, has been performed, and confirmation of patterning was demonstrated with fluorescence imaging. A representative patterned surface was characterized by AFM. The outcomes of this study are useful for investigations of glycobiology in an environment exhibiting controlled carbohydrate composition, organization, and orientation. Given the importance of the process and biomolecules involved, additional analysis of other GAG surfaces is under way in other work taking place in our laboratory, including surface analysis by ellipsometry to further characterize the deposition. Acknowledgment. We thank the Analytic Microscopy Core Facility at the Moffit Cancer Center, University of South Florida, for access to its facilities. Moffit Cancer Center is a nationally recognized comprehensive Cancer Research Center. We gratefully acknowledge funding provided for this work by the American Cancer Society Institutional Research Grant No. 032. Supporting Information Available: Control fluorescence micrographs and additional fluorescence micrographs. This material is available free of charge via the Internet at http://pubs.acs.org. LA051166M (29) Li, S.; Guan, J.-L.; Chien, S. Annu. ReV. Biomed. Eng. 2005, 7, 105.
