Protein Crystals as Scanned Probes for Recognition Atomic Force

NANO LETTERS

Protein Crystals as Scanned Probes for Recognition Atomic Force Microscopy

2005 Vol. 5, No. 12 2418-2421

Nissanka S. Wickremasinghe† and Jason H. Hafner*,†,‡ Department of Physics & Astronomy and Department of Chemistry, 6100 Main Street, Rice UniVersity, Houston, Texas Received August 22, 2005; Revised Manuscript Received October 15, 2005

ABSTRACT Lysozyme crystal growth has been localized at the tip of a conventional silicon nitride cantilever through seeded nucleation. After crosslinking with glutaraldehyde, lysozyme protein crystal tips image gold nanoparticles and grating standards with a resolution comparable to that of conventional tips. Force spectra between the lysozyme crystal tips and surfaces covered with antilysozyme reveal an adhesion force that drops significantly upon blocking with free lysozyme, thus confirming that lysozyme crystal tips can detect molecular recognition interactions.

Protein crystals are strongly associated with the determination of biomolecular structure by X-ray diffraction and to some extent with protein purification.1 Protein crystals are not traditionally thought of as an applied material because they are held together by weak interactions and are stable only under crystallization conditions. Cross-linked protein crystals (CLPCs) created by treatment with glutaraldehyde are much more robust.2 CLPCs can be handled under varying temperatures and solution conditions, and the individual molecules can retain their biological function.3 Cross-linked enzyme crystals (CLECs), for example, have been applied successfully as materials for biocatalysis.4-6 The crystalline state of the protein provides a high density of functional sites and, once cross-linked, resists degradation in a wide variety of environments including organic solvents.7,8 Because of their unique microporosity, CLPCs can act as a bioorganic zeolite for molecular separations based on size exclusion, adsorption, and chirality.9 Cross-linked antibody crystals (CLACs) have been applied as an immunoaffinity material for enantiomeric separation of a drug racemate,10 which demonstrates that antibody binding sites can remain active after cross-linking. Here we describe the first steps toward a microscopic device application of lysozyme CLPCs: their use as probe tips for biological atomic force microscopy (AFM). AFM enables nanometer-scale imaging of individual macromolecules and biological interfaces in fluids, suggesting broad application in molecular, cellular, and structural biology.11 However, AFM imaging provides only molecular topography with no ability to probe specific biomolecular interactions. Recognition AFM achieves specificity through ligands conjugated to the AFM tip.12-21 Several imaging schemes have been * Corresponding author. E-mail: [email protected]. † Department of Physics & Astronomy. ‡ Department of Chemistry. 10.1021/nl0516714 CCC: $30.25 Published on Web 10/28/2005

© 2005 American Chemical Society

described to provide simultaneous topographical and biomolecular contrast,22-28 but the problematic tip chemistry may limit the widespread application of this important technique.29,30 As an alternative tip technology for recognition AFM, we have grown protein crystals localized at the end of silicon and silicon nitride AFM cantilevers by seeded nucleation. AFM observations of molecular recognition have been reported for several complementary biomolecular pairs including biotin/avidin, polynucleotides, actin monomers/ fibers, and antibody/antigen. Ligands are typically immobilized onto tips via either nonspecific adsorption or a covalent linker to the tip surface.14,15,18-20 Antigen-antibody studies benefit from an extended poly(ethylene glycol) linker between the antibody and tip surface to increase conformational flexibility.16,17 Unbinding due to single antigenantibody pairs creates adhesion forces ranging from 50 to 240 pN, sufficient for generating recognition contrast while imaging.17 Recognition AFM is evolving as a critical technology for future biological applications of AFM;22,26,27 however, as in most forms of scanned probe microscopy, the tip technology is limiting. Covalent modification of silicon nitride tips through esterifcation or organosilane coupling can be difficult because the surface chemistry is highly sensitive to ambient conditions.29,30 Although tip functionalization via alkanethiol self-assembled monolayers (SAMs) is more robust, the deposition of chrome and gold layers on the tip reduces topographic resolution. Apart from these practical difficulties, protein conjugated tips suffer a fundamental limitation: there is no way to independently confirm that a ligand exists at the tip apex. One only knows a tip is “good” when the experiment yields the expected results. Although this has sufficed for demonstration experiments and will continue to serve well under certain condi-

Figure 1. Schematic of the fabrication CLPC tips. A standard silicon or silicon nitride tip is scratched against a preformed protein crystal to deposit small fragments (a). This tip is placed in crystallization solution (b), which causes the fragments to grow into a larger crystal, which is then cross-linked (c). The detail to c shows an idealized CLPC tip structure in which the ligand binding site (red) faces the sample.

tions, it is clearly problematic for future applications of recognition AFM in which one will study a complex, poorly characterized biomolecular system. With the lysozyme CLPC tips described here, the protein acts as both the tip material and the recognition element so that one can visually confirm its presence under a low-power optical microscope. To fabricate CLPC tips, 100 µm lysozyme crystals were grown by the sitting drop method in 5 mL culture microplates on PDMS pedestals. The reservoir solution was 600 µL of 8% (wt/vol) NaCl and 0.1 M sodium acetate buffer at pH 4.8. The protein solution consisted of 7.5 µL of reservoir solution and 7.5 µL of 50-100 mg/mL purified hen egg white lysozyme (Fisher Scientific) in 0.1 M sodium acetate buffer at pH 4.8. Protein crystallization was confirmed with Izit crystal dye (Hampton Research, see the Supporting Information). Because of the purity of the starting material, we are confident that the crystals consist of at least 94% lysozyme.31,32 If silicon nitride AFM tips were simply placed in the crystallization solution, then lysozyme crystals grew on the surface of the silicon nitride chip but with no preference for the cantilever. Therefore, a seeded crystallization approach was pursued as outlined in Figure 1. Silicon nitride AFM tips (NP, Veeco Probes) were incubated in 0.05% poly-L-lysine (Sigma) for a few hours, washed with water, and dried prior to seeding. The entire seeding process was conducted in a Petri dish of the reservoir solution that contained preformed lysozyme crystals and was observed at 5× magnification under dark-field illumination. The end of the cantilever was scraped against the preformed crystals until visible fragments were attached to the cantilever tip. The cantilever edge of the silicon nitride chip was placed in a 15 µL drop alongside 600 µL of reservoir solution as described above but in a Petri dish so that the tip growth could be monitored. Crystal growth began immediately from the seeds at the tip. As soon as the crystal grew to sufficient size, the tip was immersed in a 2 mL bath of fresh reservoir solution for 20 s to wash away noncrystalline protein from the crystal surface. Lysozyme crystal growth at the tip was Nano Lett., Vol. 5, No. 12, 2005

Figure 2. Lysozyme cross-linked protein crystal tips. The cantilever legs are 20-µm wide.

confirmed with crystal dye (see the Supporting Information). Figure 2 displays optical images of three such lysozyme crystal tips, including a side view to demonstrate that the crystals protrude much further from the cantilever than the pyramidal silicon nitride tip. We found that the yield of cantilevers with a useful lysozyme crystal at the end was approximately 80%. As grown, protein crystals tips were very fragile and dissolved when transferred to a simple buffer solution. To cross-link the tips, they were placed in a solution containing 50 µL of reservoir solution and 50 µL of a 2% grade 1 glutaraldehyde solution (Sigma-Aldrich), sealed, and left for 3.5 h. Subsequently, the cross-linked tips were taken out of the solution, washed by immersion in fresh reservoir solution for 20 s, and stored in reservoir solution at 4 °C. After cross-linking with glutaraldehyde, the CLPC tips were much more stable and could be transferred to air and various buffers. The optical images of Figure 2 portray the macroscopic structure of the tip to confirm that it will not interfere with the operation of the cantilever. These images make it clear that the CLPC tips do not have the micrometer-scale structural regularity of pyramidal silicon or silicon nitride tips. However, for AFM imaging only the tip structure up 2419

Figure 3. AFM images of sharp silicon spikes that provide an inverse image of the tip structure for a silicon tip (a) and a lysozyme CLPC tip (b). The scan size is 4 × 4 µm2 and the spikes are 700nm tall. This image demonstrates that the CLPC tips have an aspect ratio and cone angle similar to those of microfabricated tips. AFM images of 5-nm gold colloids recorded in water with a silicon nitride tip (c) and a CLPC tip (d) reveal a similar level of topographical resolution. The scan size is 1 µm and the height is represented by a 20-nm linear gray scale.

to the height of the tallest sample feature is important. To evaluate the CLPC tip structure up to a height relevant for cellular and biomolecular imaging, two imaging standards were tested. The first was a microfabricated array of 700nm tall spikes on silicon (TGT01, Silicon-MDT), which provide an inverse image of the tip apex structure. Figure 3a and b shows images of this array taken with a new silicon tip (FESP, Veeco Probes) and a CLPC tip (Veeco Metrology Nanoscope IV Multimode, tapping mode in air). Although the CLPC tip image is slightly asymmetric, it is clear that the general 0.1-1 µm scale structure of the CLPC tip is similar to that of standard silicon tips. More relevant to biological imaging is the resolution obtained on protein-sized structures in fluid. Five-nm diameter colloidal gold particles deposited on mica were employed as a high-resolution imaging standard.33 This standard was scanned with a silicon nitride tip (NP, Veeco Probes) and a CLPC tip by fluid tapping mode in buffer solution. The images in Figure 3c and d reveal a similar level of topographic resolution, although with a slight tip artifact for the CLPC tip. These results, which could be obtained with approximately 50% of the tips with a lysozyme crystal, suggest that the CLPCs are sufficiently sharp and stable for high-resolution topographical AFM of biological samples. Molecular recognition by lysozyme CLPC tips was tested by measurements similar to those used to test protein conjugated tips.16 A 10 mg/mL solution of polyclonal hen egg white antilysozyme IgG (Rockland) was incubated for 10 min on freshly cleaved mica in 0.02 M potassium phosphate buffer and 0.15 M NaCl at pH 7.2. The mica was then washed with 2 mL of water and imaged in PBS in tapping mode with a silicon nitride tip to confirm that a dense protein film was obtained. The images reveal a rough surface with dense features 2-10 nm tall, indicative of a layer that is several proteins thick. Several hundred force curves were 2420

Figure 4. Molecular recognition by CLPC tips. (a) Representative force curves for lysozyme CLPC tips over antilysozyme substrates before (red) and after (blue) blocking by exposure to free lysozyme. The vertical and horizontal scale bars are 2 and 10 nm, repsectively. Extension curves are dashed and retraction curves are solid. (b) Adhesion histograms for three lysozyme CLPC tips measured over polyclonal antilysozyme substrates before (red) and after (blue) blocking show a significant decrease in adhesion.

recorded with a lysozyme CLPC tip over the antilysozyme sample from at least five different locations at a loading rate of approximately 5 nN/sec. The adhesion force for each curve was measured as the minimum cantilever deflection of the extension curve minus the minimum cantilever deflection on the retraction curve. A nominal value of 0.06 N/m was used for the spring constant. The resulting adhesion forces were binned at 2.5 pN to create adhesion histograms. The same sample was then exposed to 100 mg/mL free lysozyme (with the tip removed) for 2 h to block the antibody binding sites. After rinsing with PBS, the force curves were repeated with the same CLPC tip. Typical force curves and adhesion histograms for three lysozyme CLPC tips are displayed in Figure 4. The occasional negative adhesion values are due to drift or unstable force curve measurements because no force curves were eliminated from the analysis. The CLPC tips clearly exhibit recognition. The adhesion force drops from values over 100 pN to a mean value below 50 pN upon blocking the binding sites with free lysozyme. The large adhesion forces and broad distribution suggest multivalent Nano Lett., Vol. 5, No. 12, 2005

binding, which is advantageous for recognition AFM contrast and lateral resolution because large adhesion forces are being generated by rigidly localized antibodies at the tip apex. We found that 60% of the lysozyme CLPC tips exhibit a recognition contrast similar to that shown in Figure 4. The tips were unavoidably dried for a few seconds upon transfer to a storage solution during the block experiment. To confirm that this did not inactivate the tip, we have repaeated the block experiment multiple times with the same tip. The middle panel of Figure 4b is the recognition contrast observed with a tip on its second block experiment several days after the first. The most significant obstacle to the realization of the ideal CLPC tip structure shown schematically in Figure 1 is control over the crystal orientation. The widespread use of CLPC tips will require the protein crystal to have a specific binding site facing the sample. From the method described here, there is no clear path to controlling the crystal orientation on the tip. Lysozyme was chosen as the CLPC tip material in part for this reason. Because lysozyme is 80% antigenic on its surface,34 most crystal orientations should experience a recognition interaction when brought against polyclonal antilysozyme. In future applications, one would prefer to crystallize antibody fragments onto the tip with the binding site facing down. This could perhaps be accomplished by orienting the deposited crystal fragments in a magnetic field,35 or using oriented protein monolayers as the seed to direct crystal growth.36 The conformational flexibility of biological samples, such as cell membranes or large protein complexes, should relax the precision required for this orientation by providing sufficient conformational freedom to bind the relatively rigid tip-bound protein crystal. Here we have demonstrated that CLPCs can be grown selectively onto AFM tips, have sufficient mechanical properties to act as scanned probes once cross-linked, and retain their molecular recognition capabilities. CLPC tips could serve as a powerful technology for broad applications of recognition AFM because of their stability37 and robustness, even after tip wear. Their rigid structure could provide both topographic and recognition resolution superior to that possible with tips bound nonspecifically or via a flexible linker. The recognition contrast demonstrated in Figure 4 is currently being studied as an imaging contrast mechanism. Thinking more broadly, these results demonstrate that CLPCs can be grown in selected locations and are sufficiently stable for device applications. This suggests other applications in areas such as microfluidic separations and cantilever-based sensing, which would benefit from the small size and high density of functional sites inherent to CLPCs. Acknowledgment. This work was supported by the Arnold and Mabel Beckman Foundation and the Nanoscale Science and Engineering Initiative of the National Science Foundation under NSF award no. EEC-0118007. Supporting Information Available: Images of the stained protein crystals. This material is available free of charge via the Internet at http://pubs.acs.org. Nano Lett., Vol. 5, No. 12, 2005

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