Copyright 1998 American Chemical Society

APRIL 28, 1998 VOLUME 14, NUMBER 9

Letters
Printing Patterns of Proteins
Andre Bernard,, Emmanuel Delamarche, Heinz Schmid, Bruno Michel, Hans Rudolf Bosshard, and Hans Biebuyck*,
IBM Research Division, Zurich Research Laboratory, CH-8803 Ruschlikon, Switzerland, and Biochemisches Institut der Universitat Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland Received January 5, 1998. In Final Form: March 2, 1998
Microcontact printing of proteins proves to be an excellent means of directly patterning biomolecules on solid substrates. Monolayer quantities of protein equilibrated on the surface of a hydrophobic, elastomeric stamp are immobilized there to rinses with buffer. These biomolecules can nevertheless transfer with >99% efficiency from the stamp to a substrate after just 1 s of contact. This capability allows the simple creation of functional patterns of proteins at scales that involve the placement of <1000 molecules in well-defined locations on a surface. The method is suited for the transfer of proteins of many different types onto hydrophilic or hydrophobic substrates.

1. Introduction What is a biological assay? Typically, it comprises a series of operations to check for the presence or activity of a biologically relevant analyte in a liquid sample. Often, the assay takes place at a solid surface. The surface allows separation of bound and free species by washing and concentrates the available signal in a two-dimensional layer, thereby minimizing the use of reagents and facilitating the manipulation of diminishing quantities of material. When screening complex solutions of targetss known or soughtsis the goal, a facility to place many putative ligands at well-defined locations on a surface is an enabling capability that allows entirely new types of studies.1-3 These compelling examples based on the formation of matrixes of DNA oligomers have sparked the imaginations of many and virtually launched an industry. In contrast to short peptides and oligonucle

otides, proteins cannot be synthesized on a solid surface but can be patterned, albeit somewhat indirectly, by their selective adsorption onto surfaces with the right chemistry: diffusion brings the protein to the surface, where it immobilizes by specific or nonspecific adsorption.4-8 An alternative is to use a structured, elastomeric stamp to print monolayer quantities of proteins directly onto the areas of a substrate it contacts. This method allows the useful attachment of biomolecules in a few seconds on a variety of surfaces with submicrometer resolution and control. The print provides high contrast in the placement of proteins while maintaining a sufficient amount of their structure to permit immunoassays or to preserve enzymatic activity. Thus, complex biomolecules are handled and transferred to substrates with an ease familiar to users of a common office stamp.
(4) Singhvi, R.; et al. Science 1994, 264, 696. (5) Tender, L. M.; Worley, R. L.; Fan, H.; Lopez, G. P. Langmuir 1996, 12, 5515. (6) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714. (7) Delamarche, E.; et al. Science 1997, 276, 779. (8) Mrksich, M.; Whitesides, G. M. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 55.

IBM Research Division, Zurich Research Laboratory. Biochemisches Institut der Universitat Zurich.

(1) Chee, M.; et al. Science 1996, 274, 610. (2) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467. (3) Southern, E. M.; et al. Nucleic Acids Res. 1994, 22, 1368.

S0743-7463(98)00037-7 CCC: $15.00 1998 American Chemical Society Published on Web 04/11/1998

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2. Experimental Section
2.1. Formation of Stamps. Poly(dimethylsiloxane) (PDMS) stamps resulted from curing Sylgard 184 (Dow Corning, Midland, MI) on fluorinated silicon masters having etched features 300 nm deep on their surface or, when flat stamps were needed, they were formed on polystyrene dishes.9 2.2. Proteins. Biomolecules and assay reagents were obtained from Sigma unless indicated otherwise. Polyclonal chicken IgGs were purified with a 0.22-m Millipore filter and dialyzed with a 30 kDa cutoff (Slide-a-lyze, Pierce, Rockford, IL) in 200 mL of phosphate-buffered saline (PBS, pH 7.4, Sigma) for 2 h to eliminate protein fragments and other low-molecularweight species. The mouse monoclonal IgG was developed at the University of Zurich (clone X7B3), and purified cell adhesion molecules were a gift from the group of Professor P. Sonderegger (University of Zurich). 2.3. Preparation of Substrates. Stamps and other substrates (glass slides, Menzel-Glaeser, Germany; polystyrene dishes, Falcon, Optilux 1001, Becton Dickinson) were sonicated in a 2:1 solution of water and ethanol for 5 min and dried under a stream of nitrogen prior to their use. Hydrophobic silicon resulted by dissolving the native oxide from silicon wafers (1525 /cm, Siltronix, Geneva, Switzerland) with a 0.5% solution of hydrofluoric acid (the HF solution was handled with gloves and appropriate protection in a well-ventilated hood). Substrates prepared in this way had an excellent reproducibility regarding their physical and chemical characteristics, had a mean surface roughness of <0.2 nm, and allowed fluorescence detection of tagged species with little quenching of the emission. Monolayers on gold were prepared as described previously.6 Stamps could be reused, if necessary, after sonication in a 2:1 solution of water and ethanol for 5 min or, alternatively, after exposure to an oxygen plasma (for 20 s at 100 W) and leaving the stamp at ambient air for an additional 20 min. 2.4. Printing Proteins. Stamps were equilibrated for <40 min with a solution of the targeted IgGs (50 g/mL in PBS) or enzyme (alkaline phosphatase diluted 1:200 in PBS), rinsed with PBS (3 10 mL) and deionized water (3 10 mL), and dried under a stream of nitrogen. The stamp was positioned over the surface and brought into contact with the substrate under the forces of its weight and interfacial adhesion, where it remained for 1 s prior to its removal. 2.5. Detection Assays. Fluorescent images were acquired with a microscope (Nikon Labophot-2) equipped with a chargecoupled camera cooled to 0 C (ST-8, SBIG, Santa Barbara, CA). The images were captured and analyzed using software for this camera (SkyPro, Software Bisque, Golden, CO). Red and greenlabeled IgGs were functionalized by tetramethylrhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC), respectively. The atomic force microscope (AFM) scans were performed in air using a noncontact mode on a Dimension 3000 (Nanoscope, Santa Barbara, CA) with TESP silicon cantilevers (Nanoscope, Santa Barbara, CA) coated by a monolayer of dodecyltrichlorosilane (ABCR, Karlsruhe, Germany). The antichicken antibodies were developed in rabbit and used in a 1:300 dilution in PBS of the stock solution received from Sigma. The sample was rinsed with buffer and dried under a stream of nitrogen after a 40-min immunocomplexation period. Absorbance assays were carried out on an HP 8452 spectrophotometer equipped with a photodiode array detector. The enzymatic assays used 50-L droplets of standard substrate solutions placed on each tested region of the stamped or solution-adsorbed surface, e.g., in the case of phosphatase a droplet of p-nitrophenyl phosphate (100 g) in buffer (100 mM glycine, 1 mM MgCl2, 1 mM ZnCl2, pH 10.4) was used. This chromogenic substrate was cleaved by enzymatic action of the phosphatase, producing a nitrophenylate that absorbed at 405 nm (yellow).

Figure 1. Procedure for directly patterning proteins on a substrate using elastomeric stamps. The stamp is fashioned by molding it from a master having a series of surface reliefs that will define areas of contact between the stamp and a substrate. (a) Equilibration of the surface of the stamp with a buffered solution of protein results, after a thorough rinsing in buffer and drying of the stamp under a stream of nitrogen, in (b) a monolayer of IgG on its surface. (c) Placement of the stamp under its own weight on a substrate and its removal after 1 s leaves a monolayer of IgG on the substrate wherever contact occurred. (d) Example of a PDMS stamp supported on a rigid backplane (glass) to facilitate alignment. The PDMS layer was 100 m thick and had a relief pattern approximately 1 m deep (evidenced here by defracting ambient light).

3. Results and Discussion Figure 1 summarizes our approach to protein patterning. We used microcontact printing (CP) to place a monolayer (see below) of protein on a substrate. Micro(9) Delamarche, E.; et al. Adv. Mater. 1997, 9, 741.

contact printing10 is a well-established technique to deliver molecules that form self-assembled monolayers on substrates. In CP a stamp, typically formed of an elastomeric polymer based on poly(dimethylsiloxane) (PDMS), is soaked with the target molecule, which diffuses into the rubbery bulk, the latter providing a reservoir of reactant.11 Conformal contact between the stamp and the substrate causes local delivery of these molecules, allowing their reaction with the surface. We find that CP is similarly capable of transferring monolayer quantities of proteins present on the surface of a stamp to a substrate after 1 s of their contact. A crucial, but simple, step in carrying out this procedure is inking the surface of the stamp with protein. Its equilibration (for times of 10-60 min) with dilute (20-200 g/mL) solutions of the biomolecule, much as one prepares polystyrene (PS) titer plates for conventional assays, allowed direct control over the quantity of protein transferred to the printed substrate. The surface of PDMS is hydrophobic with a wettability similar to that of silanized glass or PS. Proteins adsorb nonspecifically from solution on all three surfaces at approximately similar rates: typically one monolayer per hour from 50 g/mL solutions of protein.12 A subsequent, thorough rinse of the surface of the stamp with phosphate buffer removed excess material and left a monolayer quantity of protein, again as occurred when silanized glass or PS was the substrate. An important difference between these surfaces, and a key to the utility of CP for bioassays, is that many proteins readily transfer from PDMS to silanized glass or PS but not the other way (Figure 2). Remarkably, this transfer from the stamp to the substrate occurred after just seconds of contact and appeared quantitative (>99%) for most of the cases we investigated (see below). This fact is critical to the success of the technique because only a monomolecular layer of protein was present initially on the surface of the stamp. The
(10) Kumar, A.; Whitesides, G. M. Appl. Phys. Lett. 1993, 63, 2002. Xia, Y.; Zhao, X.-M.; Whitesides, G. M. Microelectron. Eng. 1996, 32, 255. (11) Larsen, N. B.; Biebuyck, H. A.; Delamarche, E.; Michel, B. J. Am. Chem. Soc. 1997, 119, 3017. (12) Bernard, A.; Bosshard, H. R. Eur. J. Biochem. 1995, 230, 416.

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Figure 2. Monolayer of TRITC-tagged IgG transferred from a stamp to a glass substrate only where the two made contact. The fluorescence image (ex ) 552 nm) of the inked stamp appeared homogeneous prior to printing (top left image) with an intensity consistent with that for a monolayer of protein. The stamp had a series of patterned surface reliefs 300 nm deep, not visible in the image because tagged IgGs were present everywhere on the surface of the PDMS after the inking step (Figure 1a). Fluorescence was absent from raised regions in the same area of the stamp (top right image) after transfer of tagged IgGs from these areas by 1 s of contact between the stamp and a glass slide that initially had no emission at this wavelength (bottom left image). The complementary pattern in fluorescence appeared after printing on the glass slide (lower right image) that confirmed this transfer of tagged IgGs (a mirror operation was applied to this image to facilitate comparison with its upper counterpart). Quantifying the fluorescence signals showed that delivery of IgG from the stamp to the glass was >99% efficient in areas of their contact and that outside of these areas no changes to either surface occurred.

result stresses the role of the elastomer and its ability to concentrate proteins on its surface without irreversibly binding them, allowing their release on contact with another substrate. How and why the transfer of proteins from stamps occurred is not investigated here in detail. We note that printing worked with high efficiency on a variety of hydrophilic and hydrophobic substrates including PS, hydrophobic silicon, and monolayers on gold terminated by hydroxyl, acid, or ethylene glycol functionalities. We tracked the location of printed biomolecules using light (fluorescence, ellipsometry), radioactivity, enzymatic activity, cell attachment, and atomic force microscopy (AFM). AFM assessment of the surface required no additional label when an immunoglobin (IgG) was used and was thus particularly convenient, in addition to its well-known sensitivity and spatial resolution. Figure 3 shows a typical sequence of patterning IgGs onto hydrophobic silicon by CP followed by an assay for their presence. This example shows, first, that the process of CP with IgGs delivered protein-derived material to the silicon substrate in a way that reproduced the dimensions of features of the stamp. The change in the topology of the surface after the print was striking. Edges in the pattern were sharp on a 20-nm scale and the appearance of silicon outside the regions of contact with the stamp remained virtually unaltered, underscoring the contrast resulting from this method. Likewise the printed pattern of protein was accurate ((50 nm) to the dimensions of the stamp, suggesting that the proteins did not diffuse on the substrate. Control studies of stamped surfaces inked with buffer without IgG showed no change in the appearance of the surface, eliminating the possibility that salts or polymer from the stamp accounted for the additional topology on the substrate after CP (data not shown). Second, the coverage of the silicon surface appeared high (>85% of closest packing). We used unstructured stamps to deliver IgG to 4 cm2 regions on silicon substrates. Ellipsometry measured the average thickness (3-3.5 nm using a refractive index of 1.45 for the protein) of the printed film over this area and showed that it was indistinguishable from a similar film of IgG adsorbed onto silicon from a solution (50 g/mL) of the protein. Higher

or lower coverage of the surface is possible under different conditions to equilibrate the solution with the surface of the stamp, but this parameter was not explored systematically here. We emphasize that the coverage of the substrate with protein-derived material reflected the occupation of the stamp prior to CP and that all (>99%) of this material left the stamp after the print. Many of the individual features within stamped regions on the silicon surface had dimensions, in both their spatial extent and height, like those of individual IgGs (20 25 4 nm3). The latter, in particular, reinforced our conclusion that only a single layer of IgG was present on the printed regions. Third, a large fraction of the printed surface was available for specific recognition by polyclonal antibodies having a nanomolar solution avidity for the immobilized antibody. Furthermore, a sandwich-type assay was possible. A third antibody in solution recognized the bound secondaries with excellent specificity as judged by AFM or fluorescence measurements (data not shown). Other related polyclonal IgGs without specificity for the bound antibodies caused no observable changes in the appearance, fluorescence, or activity of the surface at either the first or second stage of recognition. We did not attempt in this work to correlate individual features of the AFM scans with their capacity to bind antibodies, although we generally found a high level of specific binding everywhere (estimates obtained by counting the features in our AFM scans) on the printed parts of the surface. Each of the patterned features in Figure 3 had no more than 1500 IgGs, as could be determined by their direct count. Detection of their placement and immunocompetence was nevertheless easily explored by a number of techniques, even on areas (1 0.5 m2) that corresponded to <500 IgGs. Our observations are representative for several combinations of IgGs printed on glass, PS, and silicon. Figure 4 shows a typical case that uses fluorescence from tagged secondary antibodies and their recognition of the surface-bound antibodies to demonstrate the compatibility of two very different deposition schemes for proteins, namely, CP and selective adsorption. The first IgG was printed onto the surface of PS in 2 s. A second IgG was equilibrated in 1 h onto sites that remained open on the

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Figure 3. Detection by AFM of printed polyclonal chicken IgGs on hydrophobic silicon and their availability for specific recognition by secondary antibodies. (a) The image shows increases in height (3-4 nm) on the printed, and only the printed, regions of the substrate that are consistent with the localization of individual IgGs on the surface. (b) The same sample as in (a) was incubated in a solution of BSA to prevent nonspecific deposition of proteins on the substrate. The image, taken at the same location as in (a), shows a loss of topological contrast because BSA filled unoccupied sites on the surface. (c) The sample was removed from the AFM and incubated in a solution of anti-chicken antibodies. The sample was repositioned in the AFM to the area scanned in (a). The majority (70% by our count) of the printed IgGs remained sufficiently intact to allow their specific recognition by the secondary antibody. No significant presence of these secondary antibodies was evident anywhere outside the printed regions. The three schemes correlate the topographical information and the objects evident in the AFM images with the different types of proteins brought to the surface by the successive stamping, blocking, and recognition steps of the assay.

Figure 4. Secondary antibody recognition of printed or selectively adsorbed antibodies on a PS substrate showed similar levels of recognition using either preparation method. A stamp prepared with a polyclonal chicken IgG was used to print a pattern onto a PS culture dish. In a second step, monoclonal mouse IgGs were adsorbed from solution onto nonstamped areas of this polystyrene surface. The immunoassay was carried out with a solution of recognition IgGs: TRITC labeled anti-chicken IgGs (red), fluorescein isothiocyanate-conjugated antibody to mouse IgG (green), and R-phycoerythrin-conjugated antibody to bovine IgG (red-orange). Areas having stamped IgGs consequently appeared red and those with adsorbed IgG appeared green as a result of their specific recognition. No antibovine antibodies, providing a control for nonspecific deposition, were detected on the dish. Exchanging the identities of the stamped and solution-adsorbed IgG yielded the expected inversion in color in the image but did not otherwise affect the intensities of the fluorescence from either type of area (data not shown). Each color channel was collected independently (individual filters) so that emission resulting from crossreactivity between the antibodies or their uncontrolled deposition would have been easily detected. All three images were then combined to form a digital composite of the result.

substrate. We found that specific recognition by antispecies antibodies occurred with similar efficiency in each area when the entire substrate was exposed to a solution containing equimolar amounts of several different types of these recognition IgGs, each tagged by a different fluorophore to allow specific (differential) mapping of their location. In this case the density of the printed IgGs was evidently sufficient to prevent nonspecific deposition onto the patterned parts of the substrate. The use of polyclonal IgGs in all the preceding examples was particularly helpful, of course, because of the multiplicity of possible sites available for recognition on the otherwise unoriented antibodies. Table 1 generalizes the observations in Figure 4. We took a limited survey of additional and representa-

tive cases to compare printed and solution-adsorbed proteins using a number of different assays. We used unstructured stamps, that is, having no pattern, to facilitate the direct interpretation of these experiments. Our basic experience was that the CP method maintained a sufficiently high level of protein functionality to prove useful in many cases. How does CP-based patterning of proteins compare with the available alternatives? Several methods are distinguishable. Placing the molecules by means of an electrospray technology appears to be one possibility,2 although the application of this procedure would be more suitable for the formation of oligonucleotide arrays than for proteins. The resulting surface coverage and the degree to which proteins thus deposited remain viable in an assay are still unexplored, to the best of our knowledge. The apparatus for performing this type of patterning is certainly not common or even readily obtainable for high-resolution (<100 m) printing. Optical lithography onto photoactivatable substrates is probably

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Table 1. Comparison of Printed and Solution-adsorbed Proteins protein on substrate assay of functionality immuno staining direct fluorescence p-nitrophenylphosphate (pNPP) ellipsometry immuno staining ellipsometry, ELISA-block biotin-phosphatase/pNPP fluorescently labeled IgG protein as substrate/Ninhydrinb H2O2, ABTS (chromogenic substrate) N-benzoyl-L-tyrosinethylester/phenol red cell attachment/cell growthe % rel performance stamped vs coateda 100 100 90 100 100 100 80 100 50 60c 70d 70

polyclonal chicken IgG polyclonal rabbit IgG-TRITC phosphatase (calf intestine) cytochrome c (horse heart) monoclonal mouse IgG bovine serum albumin (BSA) streptavidin, avidin protein A (staphylococcus aureus) proteinase K (tritirachium album) peroxidase (horseradish) chymotrypsin (bovine pancreas) cell adhesion molecule (NgCAM)

a Here, 100% means that the microcontact-printed protein or enzyme is indistinguishable in its functionality or activity, respectively, from the solution-adsorbed preparation. b Sameijama, K.; et al. Anal. Biochem. 1971, 42, 237. c Remaining 40% activity was found on the stamp. d 20% of the remaining activity was found on the stamp. e Qualitative estimate based on the observation of growth on chick embryo dorsal root ganglia supported on coated substrates. Stoeckli, E.; et al. Develop. Biol. 1996, 177, 15.

the most appropriate of the light-based methods for the capture of proteins on surfaces13 and is somewhat more readily available, although it too requires special equipment. This approach is limited to surfaces capable of photochemistry, of course. More important is the issue of nonspecific adsorption onto these types of surfaces: cross-interference with proteins targeted to adjacent areas poses quite a difficulty.14 Building up a matrix having n different proteins requires n masks and process steps, each needing sufficient time (up to 1 h in the case of passive deposition) for proteins to diffuse to the solid interface. Similarly, techniques that rely on other forms of surface modification to bind proteins4,5 share this inefficiency in building up combinatorial arrays of biomaterial. The golden standard in bioassay development on surfaces is the passive adsorption of proteins onto hydrophobic titer plates. We demonstrated that directly printing proteins using elastomeric stamps proved indistinguishable from this standard in terms of the level of measured recognition or the activity of the protein per unit area, an area that CP effectively controls and can make very small. Stamps allow amortization of the duration of protein deposition, or inking, on their surface: they can be inked in parallel, allowing a comparatively fast conveyance of their monolayer cargo in a sequence of iterated steps, each lasting just a few seconds. Targeted areas on a substrate are physically and chemically decoupled from each other during this procedure, greatly reducing the probability of contamination and interference of the bound proteins. Stamps are very easy to replicate from a master in materials that cost but a few cents per square millimeter and require no special expertise to make or use. Although virtually disposable, we found that individual stamps could be used for five cycles (and probably many more) of inking
(13) Rozsnyai, L. F.; Benson, D. R.; Fodor, S. P. A.; Schultz, P. G. Angew. Chem., Int. Ed. Engl. 1992, 31, 759. (14) Delamarche, E.; et al. Langmuir 1996, 12, 1997.

and printing without compromising the printed patterns of protein. The solution used to ink the surface of the stamp can be almost entirely recovered without taking extraordinary precautions, and the stamp can be reused in successive cycles of inking and printing. Microcontact printing proteins on glass, silicon, and PS proved readily possible and suggests a pathway for protein placement on active regions of electronic devices. It is complementary in many senses to biomolecule patterning with microfluidic networks (FNs).7 The latter are more general concerning the range and type of biomaterial that can be transferred to a surface, in particular because the molecules always remain in well-defined solutions. Microfluidic networks, however, have considerable restrictions regarding the type and scale of patterns that can be formed, are relatively slow in effecting pattern formation, and are generally more difficult to use than stamps. Stamps expose biomolecules to additional nonsolution environments that may be too compromising for some applications, although the speed of transfer, range of possible patterns, and shear simplicity of the method all favor this approach as a first choice for many studies. We think CP-based patterning of proteins promises to expand the tool box of practical techniques available for placing and manipulating biomolecules on surfaces at scales from millimeters to micrometers and below. Acknowledgment. A.B. acknowledges support from the Swiss National Science Foundation NFP36 project. We thank A. Bietsch and H. Rothuizen for their help and discussions, B. Kunz (University of Zurich) for supplying the monoclonal antibodies, and P. Gueret (IBM) for his support. Note Added in Proof. A publication using a similar approach recently appeared in Langmuir. See: James, C. D.; et al. Langmuir 1998, 14, 741.
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