A Nanoengineering Approach to Supramolecular Chemistry

Positioning protein molecules on surfaces: A nanoengineering approach to supramolecular chemistry

Gang-Yu Liu* and Nabil A. Amro

Department of Chemistry, University of California, Davis, CA 95616

Edited by Jack Halpern, University of Chicago, Chicago, IL, and approved February 4, 2002 (received for review December 21, 2001) We discuss a nanoengineering approach for supramolecular chem- such as scanning tunneling microscopy (STM) (16) and atomic istry and self assembly. The collective properties and biofunction- force microscopy (AFM) (17), are best known for their ability to alities of molecular ensembles depend not only on individual visualize surfaces of materials with the highest spatial resolution molecular building blocks but also on organization at the molecular (18–21). Taking advantage of the sharpness of the tips and strong or nanoscopic level. Complementary to ‘‘bottom-up’’ approaches, and localized tip–surface interactions, SPM has also been used which construct supramolecular ensembles by the design and to manipulate atoms on metal surfaces and to fabricate nan- synthesis of functionalized small molecular units or large molecular opatterns of metal and semiconductor surfaces (22–28). Various motifs, nanofabrication explores whether individual units, such as approaches to controlling the local interactions between the tip small molecular ligands, or large molecules, such as proteins, can be and surface molecules have also been reported. These methods positioned with nanometer precision. The separation and local include AFM-based lithography such as tip-catalyzed surface environment can be engineered to control subsequent intermo- reactions (29), dip-pen nanolithography (DPN) (30), tip- -lecular interactions. Feature sizes as small as 2 ؋ 4nm2 (32 directed formation of metal-oxide devices (31, 32), and STM alkanethiol molecules) are produced. Proteins may be aligned based lithography such as tip-assisted electrochemical etching along a 10-nm-wide line or within two-dimensional islands of and field-induced desorption (33, 34). Domains of collagen and desired geometry. These high-resolution engineering and imaging collagen-like molecules with dimensions of 30–50 nm were studies provide new and molecular-level insight into supramolecu- produced by using an AFM-based method, DPN (35). We focus lar chemistry and self-assembly processes in bioscience that are on control of the tip–molecule interaction to more precisely otherwise unobtainable, e.g., the inﬂuence of size, separation, position molecular units ranging from small molecular ligands to orientation, and local environment of reaction sites. This nanofab- proteins. As discussed below, nanoislands of only 32 alkanethiol rication methodology also offers a new strategy in construction of molecules are produced on metal surfaces. Individual proteins two- and three-dimensional supramolecular structures for cell, may be aligned on a 10 ϫ 150 nm2 line. This level of precise virus, and bacterial adhesion, as well as biomaterial and biodevice

control provides a new mechanism for studying supramolecular CHEMISTRY engineering. chemistry and for constructing complex structures, as one can preposition the molecular units to dictate the subsequent as- upramolecular chemistry has become an area of intense sembly or reactions. Sresearch (1–4), partly inspired by biological ensembles in nature, such as collagen and enzymes or protein assemblies in Materials and Methods general. In nature, the collective properties and biofunctionali- Preparation of Self-Assembled Monolayers (SAM). All alkanethiol ties of these ensembles depend not only on the individual compounds were purchased from Aldrich, with purity greater molecular units but also (perhaps even more importantly) on the than 95%. Compounds of 3-mercapto-1-propanal and 11- organization at the molecular or nanoscopic level (1, 2, 4, 5). mercapto-1-undecanal were synthesized by oxidation with pyr- SPECIAL FEATURE Such organization dependence can be attributed to polyvalent idium dichromate (36). Solutions of thiols in ethanol or 2- interactions in biological systems (6). ‘‘Bottom-up’’ approaches butanol were prepared by using 5- to 20-min sonication to have been taken to mimic nature and have resulted in creative accelerate dissolution. synthesis of small molecular units (7, 8) and large molecular Two types of gold thin films were used: high vacuum deposited motifs (9). These molecular building blocks contain the desired films and ultraflat gold films. Gold (Alfa Aesar, 99.999%) was charge, polarization, or chemical functionalities that will affect deposited in a high vacuum evaporator (Denton Vacuum, Model intermolecular interactions such as van der Waals forces, hy- DV502-A, Moorestown, NJ) at Ϸ10Ϫ6 torr (1 torr ϭ 133 Pa) onto ͞ drogen bonding, polar attractions, and or hydrophobic interac- freshly cleaved mica substrates (Mica, New York; clear ruby tions (7–9). These interactions dictate the subsequent assembly muscovite). To enhance the formation of terraced Au(111) into supramolecular structures (4, 9–11). Complementary to domains, the mica was preheated to 325°C before deposition by these synthetic approaches, we explore whether individual units using quartz lamps mounted behind the substrate. Typical such as small molecular ligands or large molecules such as evaporation rates were 3 Å͞s, and the thickness of the films was proteins can be positioned with nanometer precision by using 150 nm. The Au films were annealed at 325°C under vacuum for nanoengineering methodologies. The separation and local en- approximately 15 min after deposition. This procedure produced vironment can be engineered to influence subsequent intermo- samples with flat Au(111) terraces as large as 300 ϫ 300 nm2 in lecular interactions. dimension according to our AFM images. Ultraflat gold, 150 nm Micrometer-sized patterns of biomolecules can be produced in thickness, was prepared according to the method developed by using relatively well known microfabrication technologies. Without using templates, DNA micropatterns may be directly produced by using photolithography (12–14). Micropatterns of This paper was submitted directly (Track II) to the PNAS ofﬁce. proteins have also been created by first making patterned Abbreviations: AFM, atomic force microscopy; NPRW, nanopen reader and writer; SAM, surfaces as templates, followed by selective adsorption of pro- self-assembled monolayer; LYZ, lysozyme. teins (15). More precise positioning of biomolecules requires *To whom reprint requests should be addressed at: Department of Chemistry, University of new fabrication strategies. Scanning probe microscopy (SPM), California, One Shields Avenue, Davis, CA 95616. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.072695699 PNAS ͉ April 16, 2002 ͉ vol. 99 ͉ no. 8 ͉ 5165–5170 Downloaded by guest on September 30, 2021 by Hegner et al. (37) and Wagner et al. (38). The resulting gold surfaces have a mean roughness of 2–5 Å according to our AFM measurements. Thiol SAMs, were prepared by immersing the freshly prepared gold films into the corresponding thiol solutions (0.1–1.0 mM) for at least 18 h.

Preparation of Protein Solutions. Bovine serum albumin (BSA) (fraction V, which is essentially fatty acid free), lysozyme (LYZ, from hen egg, 95% purity), rabbit IgG (purity 95%), and mouse anti-rabbit IgG were purchased from Sigma and used immedi- ately as received. The proteins were diluted to the desired concentrations of 10 ␮g͞ml in Hepes buffer solutions before AFM experiments.

AFM Imaging and Fabrication. The AFM used for this study incorporates a home-constructed deflection-type scanner con- trolled by commercial electronics and software (RHK Technol- ogy, Troy, MI). The instrument allows simultaneous acquisition of multiple images such as topography, frictional force, and elasticity. The scanner may be operated under ambient labora- tory conditions, in vacuum, or in solution (39). The Si3N4 cantilevers were either sharpened microlevers from ThermoMi- croscopes (Sunnyvale, CA), with a force constant of 0.1 N͞m, or standard microlevers from Digital Instruments (Santa Barbara, CA) with a force constant of 0.38 N͞m. Images were acquired under contact mode with a typical load of 0.15 nN (measured from force–distance curves). The fabrication force was determined for individual systems by using procedures described previously (40). Results High-Resolution Imaging and Fabrication. Although scanning tunneling microscopy is known for its capability of atomic and molecular resolution (41–46), researchers have not been opti- mistic about the resolution of AFM, especially when imaging assemblies of organic molecules or biological or polymeric motifs because of the relatively large tip size and the ‘‘soft-and-sticky’’ nature of the sample. Through technical advances in the design and construction of scanning heads and the development of new imaging modes for organic and biosystems, molecular resolution images are obtained for SAMs by using AFM (47, 48), despite Fig. 1. High-resolution AFM topographs of organic thin ﬁlms and proteins. ϫ 2 ͞ the structural complexity of the molecules. Fig. 1A is a high- (A)A40 40 nm scan of CH3(CH2)17S Au(111) SAM imaged under 2-butanol. This image is taken within a nanofabricated region, thus contains newly resolution image of an alkane–thiol SAM on gold formed during formed Au(111) single atomic islands. Ordered domains are clearly visible. nanografting (48, 49). The structural features are visualized in Bright areas are 0.24 nm above the surrounding because of single atomic striking detail: (i) ordered structure within domains with a lattice Au(111) steps. Domain boundaries appear darker in contrast than the ordered constant of 0.5 nm and (ii) molecular level defects such as domains. (B) A 300 ϫ 400 nm2 scan of LYZ molecules immobilized on a domain boundaries and single atomic steps. For immobilized H(CO)(CH2)10S͞Au(111) SAM. proteins such as LYZ on a planar surface, individual molecules are visible from the AFM topograph in Fig. 1B, from which their heights are measured. The orientation of the proteins can be following the scanning trajectory of the tip. In NPRW, the tip is determined from the height measurements (36). precoated with the desired molecules by soaking the tip in the The fact that molecules can be resolved by using AFM desired solution, then drying it in nitrogen. These molecules are indicates that the tip–molecule interactions are localized to transferred under high force to the exposed substrate (51). molecular dimensions, especially in the case of SAMs. There- Nanostructures of thiol molecules serve as templates for attach- fore, in principle, by enhancing the local interactions, molecules ing proteins. Selectivity of protein adsorption can be achieved by may be moved, and chemical bonds may be selectively broken. using the variation in protein affinity toward different SAMs The detailed methodology in controlling these local interactions (52–58). is the key to positioning molecules precisely. The methods used in this report are nanografting (49, 50) and nanopen reader and Precise Positioning of Small Molecules and Proteins on Surfaces. writer (NPRW) (51). The surface structure of SAM resists is first Compared with other fabrication techniques, nanografting and characterized under a very low force or load. Fabrication NPRW have the highest precision and resolution (40, 51). An locations are then selected, normally in flat regions, e.g., edge resolution of 1 nm is routinely obtained. The smallest Au(111) plateau areas. Then nanopatterns are engineered under feature fabricated is 2 ϫ 4nm2 (shown in Fig. 2A), consisting of high forces. In nanografting, the SAM and the AFM cantilevers 32 thiol molecules (49). One-dimensional features, such as are immersed in a solution containing another thiol with protein 10-nm-wide lines (Fig. 2B) or two-dimensional nanoislands with adhesive terminal groups. As the AFM tip plows through the various shapes (40, 49), can also be produced. More importantly matrix, the thiol molecules in the solution adsorb on the newly for positioning biomolecular building blocks, bioadhesive ligands exposed gold surface. Protein adhesive thiols are positioned such as biotin, -COOH, and -CHO can be positioned with

5166 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.072695699 Liu and Amro Downloaded by guest on September 30, 2021 nanometer precision on surfaces (49). In the AFM image shown ϫ 2 Fig. 2C, an HS(CH2)17CH3 pattern (600 600 nm ) was first grafted within an HS(CH2)9CH3 matrix, resulting in a nanosquare of positive contrast. Then, two nanostructures of aldehyde-terminated thiols, a 70 ϫ 70 nm2 square and a 70 ϫ 300 nm2 rectangle, were produced within the HS(CH2)17CH3 nanosquare. The example shown in Fig. 2C demonstrates the capability of nanografting for producing nanostructures with various heights and functionalities. For research in supramolecular chemistry and self assembly, making arrays of nanostructures is essential. Fig. 2D is a proof-of-concept experiment, in which a 9 ϫ 8 array of nanostructures is created. Each element shaped similar to a ‘‘space invader’’ icon has a 150 ϫ 115 nm2 head attached to two small ‘‘legs’’ of 40 ϫ 45 nm2. These nanostructures consist of octadecanethiol, 0.8 nm taller than the decanethiol matrix. The examples in Fig. 2 demonstrate the precision of AFM-based lithography in positioning small molecules, as well as flexibility in producing various nanostructures and arrays. The nanostructures of these ligands exhibit little lateral diffusion, as they are organized into closely packed structures and are sur- rounded by matrix SAMs (40). The precision in organizing large molecular units depends on the quality of template nanostructures and the selectivity in pattern transfer reactions. With templating nanostructures precisely engineered, proteins can be selectively attached to the bioadhesive areas. As shown in Fig. 3A, a 300 ϫ 350 nm2 pattern of a small protein, BSA was successfully produced. The SAM- based template was first produced within a hexanethiol matrix using mercapto-propanal and washed thoroughly with deionized water. Next, the medium was replaced with a 20 mM Hepes buffer solution (pH 6.5) containing 10 ␮g͞ml of BSA. After about 3 min of immersion, a near monolayer of BSA was observed exclusively on the aldehyde-terminated area. BSA molecules may adsorb individually or as aggregates. The heights

measured for individual BSA are shown in the cursor plot (Fig. CHEMISTRY 3B) and are consistent with the known dimensions of its active conformation: approximately spherical in shape with a diameter of 4 nm (59). A less symmetric protein molecule, LYZ, can also be positioned, following a similar procedure. The template in this case contains two nanopatterns of mercapto-propanoic acid in a decanethiol SAM: a narrow line (10 ϫ 150 nm2) above a rectangle (100 ϫ 150 nm2). The two patterns are separated by

30 Ϯ 5 nm. The surface was rinsed thoroughly to completely SPECIAL FEATURE remove any residual thiols, first with deionized water, then with 20 mM Hepes buffer (pH 7.0). After rinsing, a 10 ␮g͞ml solution of LYZ was injected. Within 3 min, proteins adsorbed exclusively onto the two patterned areas, as shown in Fig. 3C. The high selectivity observed at pH 7 is mostly because of electrostatic interactions between the LYZ molecules and the carboxylate- terminated nanopatterns. Because the isoelectric point of LYZ is 11.1 (60), LYZ exhibits a net positive charge at pH 7. The pKa value of mercapto-propanoic acid SAM is 8 (61), thus at neutral pH, approximately 10% of the nanopatterned area has a net negative charge. Consequently, the selectivity of LYZ adsorption is mediated by electrostatic attraction. Under these conditions, little adsorption was observed at methyl-terminated areas within the time frame of the entire experiment (4 h). Further, the boundary between the two nanopatterns remained clearly visible. ͞ Fig. 2. Nanopatterns produced and imaged within a CH3(CH2)9S Au(111) Individual LYZ particles can be resolved in the AFM image ϫ monolayer. (A) Thirty-two CH3(CH2)17SH molecules are grafted, forming a 2 ϫ 2 of Fig. 3C. Three LYZ molecules are positioned along the 10 4nm ‘‘dot.’’ The ‘‘dot’’ is 8.8 Å higher than the surrounding monolayer, 150 nm2 nanoline, whereas eight protein particles are confined indicating that thiols are closely packed. (B)A10ϫ 100 nm2 line of within the 100 ϫ 150 nm2 nanorectangle. The corresponding CF3(CF2)11(CH2)2SH molecules produced using NPRW. (C) Nanostructures with multiple components may be produced. Two small patterns of aldehyde cursor profiles in Fig. 3D reveal that the immobilized protein 2 Ϯ Ϯ terminated SAMs are grafted in a 600 ϫ 600 nm square of CH3(CH2)17SH area. molecules exhibit two different heights: 4.3 0.2 nm and 3.0 (D) Fabrication of arrays of nanostructures. Each element consists of 0.2 nm. It is known that physical interactions are not specific, CH3(CH2)17SH molecules. This array was engineered within 4 min. therefore various orientations with respect to the surface are

Liu and Amro PNAS ͉ April 16, 2002 ͉ vol. 99 ͉ no. 8 ͉ 5167 Downloaded by guest on September 30, 2021 2 Fig. 3. Positioning of proteins on surfaces. (A) BSA molecules are assembled onto a 200 ϫ 250 nm pattern of H(CO)(CH2)2S͞Au(111) SAM. (B) Cursor profile as indicated in A.(C) Three LYZ molecules are aligned along a 10 ϫ 150 nm2 line (indicated by the small arrow). Eight LYZ molecules are confined within a 100 ϫ 2 150 nm rectangle of HO(CO)(CH2)2S͞Au SAM (large arrow). (D) Cursor profiles as indicated in C but taken as each step of nanofabrication. Black and shaded areas represent the matrix and patterned SAM regions, respectively, whereas the clear region corresponds to adsorbed protein molecules. (E) IgG molecules are 2 immobilized onto a 40 ϫ 40 nm nanopattern of H(CO)(CH2)2SH. Little adsorption is observed on the surrounding CH3(CH2)9S͞Au(111) matrix. (F) Cursor profile as indicated in E. In the cursor plots, the origin is the gold.

observed for the adsorbed proteins. Because LYZ molecules are immobilized within a 40 ϫ 40 nm2 square. IgG molecules within ellipsoidal with the approximate dimensions 4.5 ϫ 3.0 ϫ 3.0 the nanopattern are near close-packed and cover the entire nm3 from x-ray crystallographic studies (62), the observed nanopattern. At pH 6.5, the aldehyde groups react with primary heights correspond to side-on and end-on orientations of LYZ, amines (e.g., in lysine residues) in IgG, forming covalent imine respectively. bonds (63, 64). The hydrophobic adsorption of IgG on methyl- Large proteins such as IgG can also be assembled within terminated areas may be removed by washing with 1% Tween 20 nanometer confinement. In Fig. 3E, rabbit IgG molecules are surfactant solution, because the attachment is relatively weak.

5168 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.072695699 Liu and Amro Downloaded by guest on September 30, 2021 IgG nanopatterns in a solution of mouse anti-rabbit IgG resulted in a further increase in height, ranging from 2.8 to 7.5 nm, indicating the attachment of secondary antibodies. Such a wide height range is expected because the rabbit IgG molecules within the nanopatterns can have various orientations on surfaces. The end of the Fc fragment of each rabbit IgG serves as the binding site for the mouse anti-rabbit IgG. The ends of the Fab fragment of the Y-shaped mouse anti-rabbit IgG bind specifically to the rabbit IgG. Various orientations of the rabbit IgG lead to different configurations of the antibody–antigen-binding com- plexes. In contrast, injection of nonspecific antibodies did not result in any observable height increase. The spatial precision and specificity achieved by using this nanoengineering method provides an opportunity for three-dimensional construction of bioassemblies. Discussion A nanoengineering approach is used to position molecules on surfaces. The molecules range from small alkanethiols to larger biosystems such as proteins or DNA (M. Liu, N.A.A., J. C. Garno, C. Chow, and G.-Y.L., unpublished work; ref. 66). The nanofabrication method may be used as a generic approach to position other molecules such as enzymes, collagens, fibronectin, or synthetic motifs. The smallest features produced include a 2 ϫ 4nm2 dot (32 thiol molecules), and a 10 ϫ 150 nm2 line (containing three Fig. 4. Specific antibody–antigen recognition process for proteins immobi- proteins). These high-resolution engineering and imaging studies lized on nanopatterns. (A) Rabbit IgG are immobilized on a 300 ϫ 300 nm2 reveal nanoscopic and molecular level information for supramo- H(CO)(CH2)10SH nanosquare. (C) The same area after the introduction of mouse anti-rabbit IgG. Cursor profiles in B and D are taken from the same lecular chemistry and self-assembly processes in bioscience that location and at each step of nanofabrication. Black and shaded areas repre- are otherwise unobtainable, e.g., the influence of size, separa- sent the matrix and patterned SAM regions, respectively. The clear region tion, orientation, and the local environment of reaction sites. corresponds to adsorbed rabbit IgG, whereas the gray area represents the Work is in progress to explore the possible improvement in secondary IgG molecules. An increase in height (compare cursor B and D)is nanofabrication methods to reach single molecule precision and observed, which suggests that specific binding of antibody to antigen occurs. in protein immobilization chemistry to control both the lateral and perpendicular orientations of molecules. CHEMISTRY In terms of technique development, nanolithography is com- The cursor profile shown in Fig. 3F reveals heights ranging from plementary to the ‘‘bottom-up’’ material synthesis approach, in 4 to 6 nm for immobilized IgG molecules. The observed heights providing a means to fabricate ultra-small electronic compo- result from several possible orientations that the Y-shaped IgG nents, sensing elements, and scaffolds for biomaterial engineer- (65) can adopt, as the IgG has multiple lysine residues. ing. One concern with scanning probe lithography is the serial Reactivity of the Immobilized Proteins with Nanostructures. The nature of the process and low throughput. Two approaches are in progress to address these issues: automating the nanofabri- reactivity of each molecular unit is important for subsequent ͞ construction of supramolecular assemblies. In the case of protein cation (66–70) and or using parallel probes (70–73). Nanostruc- SPECIAL FEATURE nanostructures, the activity may be tested by the corresponding tures of SAMs are sufficiently stable, at least 40 h without biochemical reactions. Fig. 4 illustrates how the bioactivity of a observable desorption in pure solvent and8hwithout observable nanopattern of rabbit IgG was tested by reaction toward a exchange in thiol solutions (50). Protein nanopatterns can specific antibody, mouse anti-rabbit IgG. In Fig. 4A, rabbit IgG sustain washing with buffer and surfactant solutions, as well as molecules are positioned within a 300 ϫ 300 nm2 frame. The mechanical forces (64). Detachment or denaturation was not ligands under the IgG are 11-mercapto-1-undecanal grafted observed within the maximum duration of our experiments, i.e., within an octadecanethiol SAM. The weakly bound IgG mole- 48 h. The observed stability makes nanolithography a promising cules on the methyl-terminated areas were removed by washing technique for applications. Two specific areas of applications with 1% Tween 20 surfactant solution. Within 5 min of injecting include: (i) construction of two- or three-dimensional supra- 10 ␮g͞ml of mouse anti-rabbit IgG, recognition (or antibody- molecular structures with well positioned binding sites for cell, antigen binding) occurred, resulting in adsorption of the sec- virus, and bacterial adhesion, and for biomaterial engineering; ondary IgG onto the nanosquare. By comparing the topographic and (ii) construction of inorganic and biomolecular hybrid images before and after secondary IgG injection, a height systems in which functional building blocks are positioned with increase was observed, which indicates the binding of mouse molecular precision. anti-rabbit IgG. The height measurements are illustrated from the cursor profiles in Figs. 4 B and D. In Fig. 4B, the proteins We appreciate many helpful technical and scientific discussions with appear 3.0–4.4 nm taller than the surrounding matrix (or 3.8–5.2 Jayne Garno, Kapila Wadu-Mesthrige, Sylvain Cruchon-Dupeyrat, and Song Xu. We thank all four reviewers for their careful reading of nm above the nanopattern). Analysis of the entire pattern the manuscript, as well as their constructive and insightful comments. indicates that IgG molecules exhibit heights ranging from 3.8 to N.A. thanks the National Science Foundation Integrative Graduate 7.0 nm. The variation in height is consistent with the expectation Education and Research Traineeship program for a graduate research that each rabbit IgG has 57 lysine residues containing amines fellowship. This work is also supported by The National Science (PDB ID code 1IGT). Thus immobilized IgG may adopt various Foundation (Grant CHE-9733400), The Whitaker Foundation (Bio- configurations after reacting with aldehyde. Immersion of the medical Engineering Grant), and the University of California at Davis.

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