Direct-Write Fabrication of Functional Protein Matrixes Using a Low

Anal. Chem. 2006, 78, 3198-3202

Direct-Write Fabrication of Functional Protein Matrixes Using a Low-Cost Q-Switched Laser Bryan Kaehr,† Nusret Ertas¸,† Rex Nielson, Richard Allen, Ryan T. Hill, Matthew Plenert, and Jason B. Shear*

Department of Chemistry & Biochemistry and the Institute for Cellular & Molecular Biology, 1 University Station A5300, University of Texas, Austin, Texas 78712

We report the use of an inexpensive, small, and “turnkey” Q-switched 532-nm Nd:YAG laser as a source for nonlinear, direct-write protein microfabrication. In this approach, microJoule pulses (pulse widths, ∼600 ps) are focused using high numerical aperture optics to submicrometer focal spots, creating instantaneous intensities great enough to promote multiphoton excitation of a photosensitizer and subsequent intermolecular crosslinking of protein molecules. By scanning the femtoliter focal volume through reagent solution, extended proteinbased structures can be fabricated with precise, threedimensional topographies. As with earlier studies using a femtosecond titanium:sapphire laser costing more than $100K, physically robust and chemically responsive microstructures can be fashioned rapidly with feature sizes smaller than 0.5 µm, and cross-linking can be achieved using both biologically benign sensitizers (e.g., flavins) and by using the proteins themselves to sensitize cross-linking. We demonstrate in situ fabrication to corral neurite outgrowth and show the ability to functionalize avidin structures with biotinylated reagents, an approach that enables chemical sensing to be performed in specified microenvironments. Characterization of this inexpensive, low-power source will greatly broaden access to directwrite protein microfabrication. Reductionist studies of biological systems routinely exploit technologies for immobilizing macromolecules on surfaces in their functional states. Such capabilities have been integral to fabrication of sensor array chips,1 single-molecule force measurements,2,3 and adhesion and stimulation of cells in culture.4-6 A strategy for extending macromolecular patterning so that functional matrixes * To whom correspondence should be addressed. E-mail: jshear@ mail.utexas.edu. † These authors contributed equally to this work. (1) Min, D.-H.; Mrksich, M. Curr. Opin. Chem. Biol. 2004, 8, 554-558. (2) Frederix, P. L. T. M.; Akiyama, T.; Staufer, U.; Gerber, C.; Fotiadis, D.; Muller, D. J.; Engel, A. Curr. Opin. Chem. Biol. 2003, 7, 641-647. (3) Clausen-Schaumann, H.; Seitz, M.; Krautbauer, R.; Gaub, H. E. Curr. Opin. Chem. Biol. 2000, 4, 524-530. (4) Jung, D. R.; Kapur, R.; Adams, T.; Giuliano, K. A.; Mrksich, M.; Craighead, H. G.; Taylor, D. L. Crit. Rev. Biotechnol. 2001, 21, 111-154. (5) Cukierman, E.; Pankov, R.; Yamada, K. M. Curr. Opin. Cell Biol. 2002, 14, 633-640. (6) Silva, G. A.; Czeisler, C.; Niece, K. L.; Beniash, E.; Harrington, D. A.; Kessler, J. A.; Stupp, S. I. Science 2004, 303, 1352-1355.

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could be constructed in the presence of living cells with precise three-dimensional control would offer powerful opportunities for influencing and probing the activity of model cell systems in real time. In this biocompatible microfabrication approach, biological macromolecules would serve as building blocks for creating microstructured materials in aqueous environments, provide functionality to structures based on their intrinsic catalytic or binding properties, and allow biomaterials to be fashioned into three-dimensional (3D) landscapes that optimize mass-transfer rates of analytes and molecular stimulants both into and away from matrixes. Campagnola and co-workers first reported the use of multiphoton excitation to promote protein cross-linking in defined 3D voxels, allowing extended protein-based matrixes to be created in aqueous environments by scanning a laser focus through a reagent solution containing protein and photosensitizer. Several reports by the Campagnola and Shear groups have described the use of such materials for small-molecule storage and release, mediation of cellular interactions, and chemical microsensing.7-10 Critical to our interest in maintaining cell viability during and after fabrication, we have evaluated the biocompatibility of various photosensitizers. A number of biologically based, relatively benign sensitizers, including flavins and nicotinamides, were found to promote nonlinear photo-cross-linking of proteins.9,10 In direct-write protein microfabrication, intermolecular crosslinks are formed between residue side chains (e.g., tyrosine, cysteine, histidine, and lysine) via nonlinear excitation of a photosensitizer using multiple, low-energy photons. As in twophoton microscopy, the superlinear dependence of excitation rate on laser intensity allows one to confine photochemistry not only in the two radial dimensions but also axially (along the laser propagation axis). By tightly focusing a high peak-power laser beam to a submicrometer focal spot, it is feasible to generate photochemical voxels smaller than 1 µm3, allowing complex threedimensional protein matrixes to be created with feature sizes that range from several hundred nanometers to several millimeters. (7) Pitts, J. D.; Campagnola, P. J.; Epling, G. A.; Goodman, S. L. Macromolecules 2000, 33, 1514-1523. (8) Basu, S.; Wolgemuth, C. W.; Campagnola, P. J. Biomacromolecules 2004, 5, 2347-2357. (9) Kaehr, B.; Allen, R.; Javier, D. J.; Currie, J.; Shear, J. B. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16104-16108. (10) Allen, R.; Nielson, R.; Wise, D. D.; Shear, J. B. Anal. Chem. 2005, 77, 5089-5095. 10.1021/ac052267s CCC: $33.50

© 2006 American Chemical Society Published on Web 03/24/2006

The ability to fabricate functional 3D biomaterials could have a broad impact on patterning and analysis of model cell culture systems. Widespread adoption of protein microfabrication by biological scientists, however, depends on the availability of inexpensive, “turn-key” and preferably small lasers that are capable of promoting protein cross-linking via nonlinear excitation. Unfortunately, efficient nonresonant multiphoton excitation requires extremely high peak intensity laser pulses (Ipeak ∼ 1010 - 1012 W cm-2), a condition that has been met primarily by focusing the output of femtosecond laser sources using high numerical aperture optics. Although the accessibility of femtosecond laser systems has improved markedly in the last 15 years (fueled in part by the popularization of two-photon microscopy), the cost of these lasers remains prohibitive for most individual users. In the current studies, we investigate the practicality for overcoming this important hurdle to the dissemination of directwrite protein microfabrication by evaluating the capacity of an inexpensive laser to promote multiphoton protein photo-crosslinking. The sourcesa small-footprint, Q-switched, frequencydoubled (532-nm) Nd:YAG lasersproduces subnanosecond (∼600 ps) pulses with energies of up to several microJoules and peak powers comparable to those needed to cross-link proteins with femtosecond titanium:sapphire (Ti:S) laser light. We demonstrate the possibility for promoting protein photo-cross-linking using low average laser powers (25 mW, a pulse width of ∼600 ps, and a repetition rate of 7.65 kHz. These values correspond to a peak power of ∼7 kW and a pulse energy of ∼3.5 µJ. The laser dimensions are approximately 3 × 4 × 15 cm (height × width × length), and the cost of the system, including power supply, was less than $6K. The laser output was adjusted to approximately fill the back aperture of a high-power objective (Zeiss 100× Fluar, 1.3 numerical aperture, oil immersion) situated on a Zeiss Axiovert inverted microscope system. As a point of comparison, structures

also were fabricated using the output of a mode-locked Ti:S laser (Tsunami; Spectra Physics, Mountain View, CA) operating at 740 nm. Powers used to cross-link were measured immediately before the objective and were generally between 0.5 and 3 mW. Desired powers were obtained by attenuating the laser beam using a halfwave plate/polarizing beam splitter pair. All structures were fabricated by scanning the sample at a rate of 5 µm s-1 using a motorized X-Y stage (ProScan; Prior Scientific, Cambridge, U.K.). In cases where structures extended along the z-dimension (i.e., along the optical axis), the position of the laser focus was translated manually within sample solution using the microscope’s fine focus adjustment. Microstructures composed of photo-cross-linked BSA were fabricated from solutions containing protein at 100-400 mg mL-1, in some cases with 1-4 mM Rose Bengal added as a photosensitizer. Avidin matrixes were fabricated using a solution containing 400 mg mL-1 protein with no additional sensitizer. In biotinbinding studies, BSA and avidin microstructures were incubated in 2 µM fluorescein biotin for 2 min; dishes were washed 10 times using 500 µL of Hepes buffer (pH 7.4) and were imaged in a pH 7 phosphate buffer. As a control to determine the specificity of fluorophore labeling, in some experiments structures were pretreated with unlabeled D-biotin (25 µM) as a blocking agent and then rinsed with biocytin-TMR (tetramethyl rhodamine) or fluorescein biotin. Fluorescence Microscopy. Wide-field fluorescence imaging was performed on the Axiovert microscope, which was equipped with a mercury arc lamp and standard “red” and “green” filter sets (Chroma, Rockingham, VT). Fluorescence emission was collected using the Fluar 100× objective and detected using a 12bit 1392 × 1040 element CCD (Cool Snap HQ; Photometrics, Tucson, AZ). Data were processed using Image J and Metamorph (Universal Imaging, Sunnyvale, CA) image analysis software. Cell Culture. Rat brain cortical cells (embryonic days 1819) were harvested by QBM Cell Science (Ottawa, Canada) and cultured according to standard procedures as previously described.9 Briefly, cryopreserved neurons were transferred to flametreated, poly(L-lysine)-coated coverslips and incubated in neurobasal medium (Invitrogen, Carlsbad, CA) with L-glutamine, 1 unit mL-1 penicillin-streptomycin, and 2% B27 serum-free culture supplement. Experiments were performed 1-2 days after plating. In the periods immediately before and after fabrication, cells were maintained in a supplemented pH 7.4 Hepes buffer (10 mM sodium Hepes, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM D-glucose). Scanning Electron Microscopy (SEM) Preparation. Samples were fixed in 3.5% gluteraldehyde solution for 20 min, dehydrated by using 10-min sequential washes (2:1 ethanol/H2O; twice in 100% ethanol; 1:1 ethanol/methanol; 100% methanol; all solutions stated as v/v), allowed to air-dry for 3 h, and sputter-coated to a nominal thickness of 15 nm with Au/Pd. RESULTS AND DISCUSSION Nonlinear Photofabrication of Protein Microstructures. We previously used a femtosecond Ti:S laser to create protein microstructures that function as physical barriers for developing Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

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Figure 1. SEMs of microstructures fabricated from BSA using multiphoton-excited cross-linking. (a) An intersection of two lines fabricated from BSA (200 mg mL-1) and Rose Bengal (4 mM) using the Q-switched YAG laser (average power, 0.5 mW). Scale bar, 1 µm. (b) A structure fabricated in the same manner as that described in (a) except that a femtosecond Ti:S was used as the photofabrication source (average power, 11 mW). Scale bar, 2 µm. (c) Intersection of BSA matrixes fabricated using the YAG laser at 2.0 (thick bent line) and 1.0 mW (narrow straight line). Lines were fabricated from a solution containing 400 mg mL-1 BSA and no additional photosensitizer. Scale bar, 5 µm. (d) Higher magnification image of lines fabricated using the same conditions as in (c). Scale bar, 1 µm.

neurites,9 scaffolds for metal-nanoparticle wires,11 and enzymebased biosensors for neurotransmitters.10 Excitation of UV- and visible-absorbing photosensitizers (e.g., flavins, methylene blue) was accomplished via nonresonant absorption of multiple quanta of near-infrared Ti:S laser light. Recently, we have directly crosslinked proteins without use of an additional photosensitizer by using Ti:S wavelengths capable of exciting aromatic amino acids via three-photon excitation. In the current work, we have evaluated the feasibility for fabricating protein microstructures using an inexpensive 532-nm Q-switched Nd:YAG laser. Because this source previously has been shown to be a useful two-photon fluorescence excitation source for aromatic amino acids,12 it presented a rational choice for promoting both direct and photosensitized protein cross-linking. We found the YAG laser to be highly efficient at photo-crosslinking BSA using Rose Bengal. Figure 1 compares BSA structures fabricated from a solution of 200 mg mL-1 BSA and 4 mM Rose Bengal using the YAG laser (Figure 1a) and a Ti:S source (Figure 1b). Although differences in grain characteristics of the structures is evident from these SEM images, in both cases, protein matrixes could be fabricated with similar gross morphologies and high structural integrity. Structures produced using both lasers could be repeatedly rinsed, although greater care was required in the case of those written using the YAG laser as adherence of the matrix to the untreated borosilicate coverslips was less robust. (11) Hill, R. T.; Lyon, J. L.; Allen, R.; Stevenson, K. J.; Shear, J. B. J. Am. Chem. Soc. 2005, 127, 10707-10711. (12) Paul, U. P.; Li, L.; Lee, M. L.; Farnsworth, P. B. Anal. Chem. 2005, 77, 3690-3693.

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The dimensions of surface-adherent protein lines generally increase with reagent concentrations and decrease at faster scan rates of the laser focus. As shown in Figure 1c,d, at a constant reagent concentration and scan rate, feature sizes also can be modified by varying laser power. The width of protein lines generated using 1 mW of 532-nm light typically was less than 0.5 µm and increased by ∼50% at twice the laser power. To evaluate the degree to which nonlinear excitation contributes to the fabrication process when using the Q-switched laser, we also attempted to photo-cross-link protein matrixes using a continuous-wave 543.5-nm HeNe laser (05-LGR-193, Melles Griot, Carlsbad, CA). Several protein/photosensitizer solutions were evaluated, including BSA/Rose Bengal (200 mg mL-1/4 mM), BSA/FAD (200 mg mL-1/6 mM), and cyt c/FAD (100 mg mL-1/ 10 mM). At powers up to 3 mW, no photo-cross-linking could be observed for any combination of protein and photosensitizer. In contrast, the YAG laser provided the capacity to cross-link proteins using each of the test solutions. (At the objective back aperture, the HeNe laser beam had a somewhat smaller (1/e2) diameter than the Q-switched YAG and did not fully fill the objective back aperture. To ensure that both beams were focused to similar (Gaussian) focal spot sizes, 1- or 2-mm pinholes were placed in the beam paths before the objective to define beam diameters.) It is worth noting that while Rose Bengal does maintain significant one-photon absorption at 543.5 nm,13 the photosensitization action cross section apparently is insufficient at these powers to promote observable cross-linking. By tightly focusing a pulsed laser beam, nonlinear photochemistry can be contained within a three-dimensionally defined femtoliter focal volume, enabling complex topographical structures to be fashioned with submicrometer control. Several groups have explored the use of Ti:S laser light to promote polymerization of acrylates and other polymers, demonstrating the ability to construct plastic ruminants,14 interlocking buildings, and microscopic words.15 The possibility for photofabricating 3D-defined matrixes from biomaterials such as proteins opens opportunities for creating sophisticated cellular interfaces that could guide cell growth in three dimensions or restrict diffusion of analytes secreted from subcellular regions of interest. In earlier studies, we used a Ti:S-based direct-write system to fabricate 3D protein microstructures, including low-profile arches and long-aspect-ratio cables.9-11 We now demonstrate that a similar level of 3D control can be obtained using the inexpensive Q-switched YAG laser. Figure 2 shows a sequence of images tracking the construction of an interlocking chain link composed of cross-linked BSA (see movie, S-1, Supporting Information). Here, the chain is fabricated entirely in a concentrated protein solution (i.e., with no mooring to a surface). Brownian displacements of the growing structures approach the resolution of the direct-write procedure on time scales of tens of seconds, making it necessary to rapidly scan the beam focus through its entire path before the nascent structure undergoes significant drift. In these studies, fabrication times were 8.5 s for the first chain link and 21 s for the second, interlocking link. In addition, the laser beam (13) Neckers, D. C. J. Photochem. Photobiol., A: Chem. 1989, 47, 1-29. (14) Kawata, S.; Sun, H. B.; Tanaka, T.; Takada, K. Nature 2001, 412, 697698. (15) Baldacchini, T.; LaFratta, C. N.; Farrer, R. A.; Teich, M. C.; Saleh, B. E. A.; Naughton, M. J.; Fourkas, J. T. J. Appl. Phys. 2004, 95, 6072-6076.

Figure 2. Direct-write of a free-floating 3D object using the Q-switched YAG laser (average power, 1.7 mW). (a) A DIC image sequence acquired over ∼40 s demonstrating fabrication of interlocking chain links from a solution of BSA (400 mg mL-1 protein with no additional photosensitizer). Arrows in the first, third, and fifth panels indicate the direction of protein structure fabrication in planes orthogonal to the optical axis; asterisks in the second and fourth panels identify protein matrix as it is fabricated along the optical axis. Scale bar, 5 µm. (b) Schematic representation of the interlocking links.

Figure 3. Confinement of neurite outgrowth from a cultured rat cortical neuron by in situ fabrication of a BSA corral. The low-profile (less than 1 µm high) lines were fabricated from a solution of 200 mg mL-1 BSA using an average laser power of 1.5 mW. DIC micrographs (a) immediately before microstructure fabrication, (b) 10 min after fabrication, (c) 26 min after fabrication, and (d) 95 min after fabrication. Scale bars are 5 µm; the bar in (a) applies to the two upper panels, while that in (d) applies to the lower panels.

was shuttered for ∼10 s between the continuous scans used to cross-link the two independent links of the interlocking structure. In the final (right-most) panel, a schematic depicts the approximate appearance of the three-dimensional structure. Biocompatible Microfabricaton. Our previous Ti:S work has shown it is feasible to fabricate protein microstructures within cultures of primary cortical neurons and neuroblastoma-glioma cells, results that open the possibility for affecting development and growth of cells in real time. By using FAD to photosensitize BSA and laminin cross-linking, low-profile lines could be fabricated that redirected and confined axonal outgrowth with no observable compromise in cell viability.9 Similar capabilities can be obtained using the Q-switched YAG laser (Figure 3). Here, a low-profile protein fence was constructed in the presence of a cultured rat cortical neuron using a solution of BSA (200 mg mL-1, pH 7.4) containing no additional sensitizer, rinsed, and observed over a period of more than 1.5 h. The neurite extended to the protein

microstructure within ∼17 min but did not scale the barrier for the duration of the experiment (∼78 additional min). Such axon corralling represents a basic but important milestone toward more sophisticated cellular manipulations such as fabrication of neuronal networks. Retention of Avidin Functionality. We previously have shown that microfabricated matrixes composed of avidin retain the ability to bind biotinylated molecules, offering a means to localize enzymes, sensors, and cellular effectors with high spatial resolution.9,10 Here we demonstrate that biotin-binding capacity is retained by avidin matrixes fabricated using the YAG laser. Figure 4 compares the binding of fluorescein biotin by square spiral structures fabricated from avidin (left) and BSA (right). The intensity along a horizontal line from the arrow across the fluorescence image (Figure 4a, lower panel) shows that emission from labeled avidin is generally at least 10-fold more intense than that observed from BSA (i.e., where no specific binding should occur). Figure 4b, a surface intensity plot of the fluorescent avidin spiral, reveals variability in intensity that is qualitatively similar from line to line. These variations may arise as a result of inconsistencies in the scan process, such as the short breaks that can be seen in the differential interference contrast (DIC) image (Figure 4a, top panel). To further assess the specificity of biotin binding by avidin structures, protein matrixes were incubated either with or without unlabeled (nonfluorescent) biotin and then washed with a biocytin-TMR. Intense fluorescence was observed only for structures that were not blocked with unlabeled biotin (data not shown). Avidin structures fabricated with the YAG laser were useful as sensors for local chemical environments. Figure 5 shows the results of cycling bath pH in a dish containing an avidin structure decorated with fluorescein biotin. As with dissolved fluorescein, emission from immobilized fluorescein is pH-dependent (maintaining a larger fluorescence quantum yield at higher pH values) and, unfortunately, subject to significant photobleaching. Consistent pH responsivity was maintained over 20 pH cycles (10 measurements at pH 7.4 interlaced with 10 measurements at pH 4.0), although the data were superimposed on a monotonic decay caused by degradation of the dye. This rudimentary demonstration of chemical sensing should be readily extended to more sophisticated applications, provided appropriate chemical receptors can be immobilized to avidin microstructures via biotinylation. Analytical Chemistry, Vol. 78, No. 9, May 1, 2006

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Figure 5. pH sensing by a fluorescein biotin-labeled avidin microstructure. The structure was subjected to 20 alternating solutions of pH 7.4 and 4.0 buffers. Emission from the fluorophore demonstrated the expected sensitivity to hydrogen ion activity while also undergoing continual photobleaching throughout the sequence of measurements. The avidin structure was a spiral similar to that shown in Figure 4 and was fabricated using similar conditions. Data points represent an average of 600 pixels from the fluorescence image on a region corresponding to the protein structure; error bars represent the 1σ error in the pixel values.

micrometer feature sizes that are physically robust and chemically active. Moreover, conditions can be identified for microfabricating these structures using either benign (minimally toxic) photosensitizers (e.g., FAD) or without an exogenous sensitizer, making it possible to create defined biomaterials within cellular microenvironments to effect cell development and activity. Importantly, the current work shows that these attributes do not require use of a mode-locked femtosecond light source, but rather can be obtained by using an inexpensive, small, and turn-key Q-switched Nd:YAG laser. This advance will facilitate dissemination of protein microfabrication techniques to a broad cross section of analytical and biological researchers.

Figure 4. Retention of biotin binding by photo-cross-linked avidin. Structures were fabricated from solutions of 400 mg mL-1 protein (with no additional photosensitizer) using 1.7 mW average power from the YAG laser. (a) Spiral structures composed of cross-linked avidin (left images) and BSA (right images) after application of 2 µM fluorescein biotin and extended washout. The upper and lower images were acquired using DIC and fluorescence microscopy, respectively. Fluorescence intensity versus horizonal position (from the arrow) is plotted on the upper portion of the fluorescence image. Scale bar, 5 µm. (b) Fluorescence intensity plot of the avidin structure from (a).

CONCLUSIONS As with our earlier studies, the current experiments demonstrate that nonlinear excitation can be used to photo-cross-link proteins into well-defined three-dimensional matrixes with sub-

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ACKNOWLEDGMENT J.B.S. is a fellow at the Institute for Cellular and Molecular Biology. Funding from the Robert A. Welch Foundation (Grant F-1331) and the National Science Foundation (Grant 0317032) is gratefully acknowledged. SUPPORTING INFORMATION AVAILABLE Video clip of chain-link fabrication depicted in Figure 2 of the main text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 22, 2005. Accepted February 28, 2006. AC052267S