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Multiplexed Protein Patterns on a Photosensitive
Hydrophilic Polymer Matrix
By Parijat Bhatnagar,* George G. Malliaras, Il Kim, and Carl A. Batt*
Multiplexed functional proteins immobilized on microfabricated sensors and surfaces [1–3]^ have found applications in high- throughput screening [4]^ of drug molecules,[5]^ early disease detection, [6]^ organ printing, and complex tissue engineering. [7–10] Complex biological integrated patterns emulating physiological microenvironments have been used to engineer tissue junctions from stem cells by selective differentiation [2,11]^ and study interaction with the extracellular matrix (ECM). [11–13]^ Parallel developments in lab-on-a-chip (LOC) platform technologies have been identified for label-free biosensing [14]^ with faster analysis using less reagent and analyte volumes. [15–17]^ If LOC technology is to take advantage of the developments in the semiconductor industry,[16]^ efforts are needed to create biologically friendly microfabrication processes to allow integration of microelec- tronic circuitry with protein patterns. [17–19]^ Currently used methods for multiplexed protein patterns include soft- lithography,[1,20]^ inkjet printing, [21]^ and dip-pen nanolithogra- phy.[22]^ However, none of these have been integrated with complementary metal–oxide–semiconductor (CMOS) processing for high-volume manufacturing. [23]^ Soft-lithography and inkjet printing have proven to be versatile for protein patterning, however, resolution and hence alignment of the protein patterns with pre-existing features remains a challenge. [1,19]^ Dip-pen nanolithography, an analogue of scanning probe microscopy, can achieve high resolution but is extremely slow and has not been adopted by industry. Here we demonstrate a photolitho- graphic process [24,25]^ on hydrogel-based biomaterial [26]^ for patterning three different types of proteins. The technique is scalable and capable of patterning a multitude of proteins aligned with respect to each other and surface microstructures. UV light (365 nm), benign to proteins and DNA, [27,28]^ was used. This
strategy allowed us to integrate harsh upstream CMOS processing involving extreme pH, vacuum processes, and organic solvents, with downstream aqueous biomolecular processing at neutral pH. We have earlier demonstrated methods to array single oligonucleotides or proteins.[29,30]^ Lift-off-based photolithogra- phy [2]^ and oxygen-plasma-etch-based patterning of two pro- teins [31]^ has also been demonstrated and is capable of scaling up to more proteins, but due to the subtractive nature of these processes none can be adopted with multiple layers in 3D. [8,9] Bochet et al. have described solution-based photochemistry of orthogonal photolysis of inter- and intramolecular acid groups using two different photolabile protecting groups (PLPGs) with differential sensitivity to 254-nm and 420-nm UV light. [25]^ This was further developed by Campo et al. who illustrated photopatterning to create chemically diverse areas for patterning colloidal particles and different biomolecules. [24]^ Photogenerated functional groups have also been used for solid-phase synthesis of multiplexed gene chips [32]^ and peptide chips,[33]^ which utilizes synthetic nucleotides or amino acid residues, respectively, protected by PLPGs. Photochemical immobilization strategies can be categorized into two groups: photocatalyzed reactions and photodeprotection of reactive groups. [34]^ The former involves a single-step reaction by creating short-lived reactive groups on the surface by photoexposure. Although advantageous in facilitating a single- step reaction, this technique cannot be integrated with semiconductor industry equipment because it requires the substrate to be present in a liquid environment inside the photolithographic equipment. Due to the aforementioned limitations we resorted to a photodeprotection strategy to generate either an amine- (photogenerated base, PGB) or a carboxylic-acid- (photogenerated acid, PGA) functionalized sur- face [24,25]^ followed by subsequent immobilization of proteins. [35] Cr microstructures, which serve as alignment marks in downstream protein patterning, were first patterned on a wafer using electron-beam evaporation of Cr, standard projection photolithography, and subtractive wet-etching of Cr. A self- assembled monolayer (SAM) of [3-(methacryloyloxy)propyl]- trimethoxysilane with a polymerizable terminal group was formed on the wafer surface from solution-phase (MOP- SAM). [30]^ A functional-group-containing monomer (FGM) (amine or protected carboxylic acid) was then polymerized with a thin film of acrylamide (AAm)–methylenebisacrylamide (Bis) copolymer [poly(AAm–Bis–FGM)] [30]^ (Figure 1). 2-Nitrobenzyl succinimidyl carbonate (NBSC), a PLPG adduct prepared as described elsewhere, [36]^ was subsequently used to protect surface amine groups as 2-nitrobenzyl-derived carbamate (Scheme 1). In the case of 2-nitrobenzyl-derived ester groups (that yield surface
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[*] Dr. P. Bhatnagar [+] Dept. of Biomedical Engineering Cornell University, Ithaca, NY 14853 (USA) E-mail: pb96@cornell.edu Prof. C. A. Batt Dept. of Food Science Cornell University, Ithaca, NY 14853 (USA) E-mail: cab10@cornell.edu Prof. G. G. Malliaras Dept. of Materials Science & Engineering Cornell University, Ithaca, NY 14853 (USA) Prof. I. Kim Dept. of Polymer Science & Engineering Pusan National University, Busan 609-735 (Korea) [+] Present Address: Intel Corp., 5200 NE Elam Young Pkwy, RA3-355, Hillsboro, OR 97124, USA
DOI: 10.1002/adma.
Adv. Mater. 2009 , 21, 1–5 ß 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1
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carboxylic acid groups upon photoexposure), the monomer itself contained acid groups protected as 2-nitrobenzyl-derived esters. The photosensitive hydrogel surface was then exposed in selective regions to 365-nm UV light (730 mW cm^2 intensity) to create spatial patterns of PGB (Scheme 1) or PGA (Scheme 2) as a result of photochemical cleavage of 2-nitrobenzyl-derived carbamate or 2-nitrobenzyl-derived ester, respectively. Proteins were covalently immobilized on the PGB or PGA surface through stable amide linkages with primary amines available on lysine residues of protein molecules using bis(sulfosuccinimidyl)sube- rate (BS 3 ) (Scheme 1) or carbodiimide chemistry (Scheme 2), respectively.[35]^ After protecting the functional groups on the immobilized protein molecules (amine-group protection for PGB-based protocol and acid-group protection for PGA-based protocol), the process was sequentially repeated using different photomasks with a previously patterned Cr alignment mark to
create a multiplexed protein surface. It was necessary to wash the surface with mildly acidic (pH 4) sodium citrate buffer for 3 h after every protein immobilization step to promote positive charges on nonspecifically adhered protein molecules, resulting in their electro- static repulsion from the aminated surface. Our initial efforts used photogenerated surface amine groups for protein immobiliza- tion (Scheme 1). Figure 2 shows an early effort to immobilize IgG antibodies from rat, rabbit, and mouse separately on individual chips with PGB, however, nonspecific adhesion was a challenge. It has been reported that photo- cleavage of 2-nitrobenzyl protecting groups to generate amine groups is accompanied by generation of an aldehyde-based side product (2-nitrosobenzaldehyde) that can react with primary amines to form imines, [24,27,37,38]^ thus rendering photogenerated primary amine groups unavailable for further conjugation. Semicarbazide hydrochloride has been used as an aldehyde scavenger in solution, [24,27,38]^ but due to CMOS equipment compatibility issues we were unable to use any solution-phase protocol during photoexposure. This led us to explore photogenerated surface acid groups[24,25]^ (Scheme 2). We believed that this approach would be a better choice since the photochemistry of this system is similar to the widely used i-line photoresists in the semi- conductor industry, where acid functional groups are photogenerated by exposure to 365-nm UV light. Carbodiimide chemistry was then utilized for surface immobilization of each protein on PGA regions (Scheme 2). Carboxylic groups on attached proteins were transformed to primary hydroxyl groups using carbodiimide chemis- try. [35]^ This step prevented a chemical reaction between primary amines of subsequent pro- teins with carboxylic acid groups of proteins already immobilized on surface. Rat, rabbit and mouse IgG were immobilized sequentially as per Scheme 2. Proteins were detected using fluorescently labeled probe antibodies on a single chip and are shown in Figure 3a–c. Figure 3d shows an overlay image of the three images. Considerable nonspecific adhesion was still observed, which did not reduce after treatment with acidic or alkaline buffers. However, nonspecific physical adhesion is also an inherent property of IgG antibodies [39]^ and in fact this property of IgG molecules is utilized to block surfaces in biological assays (e.g., western blots) and eliminate nonspecific adhesion of protein- specific IgG molecules in subsequent steps. We argued that this property of IgG may have resulted in our observation of background fluorescence from nonspecific adhesion and may not interfere when we immobilize other proteins of interest. We might also be able to prevent nonspecific adhesion of labeled probe antibodies towards the protein of interest by blocking the
Figure 1. Fabrication of multiplexed protein patterns on a photosensitive surface. i) Electro- n-beam evaporation of Cr, projection photolithography (spin, 365-nm UV exposure, develop), subtractive wet etch of exposed Cr, MOP-SAM formation on SiO 2 surface; ii) formation of crosslinked polymer thin film containing FGM; iii) 365-nm UV exposure of selective areas, aligned to Cr patterns, on polymer thin film to cleave PLPG resulting into PGB or PGA; iv) use of Scheme 1 (for PGB) or Scheme 2 (for PGA) for covalent immobilization of proteins P1 on the exposed surface; v) chemical protection of functional groups on proteins P1 ((v-1) amine group protection for PGB based protocol and (v-2) acid group protection for PGA based protocol); vi) 365-nm UV exposure of selective areas on polymer thin film resulting into PGB or PGA; vii) immobilization of proteins P2 on newly exposed areas. Steps (vi–vii) are repetitions of steps (iii–v) for immobil- ization of second protein P2. viii) Steps (iii–v) are again repeated for immobilization of third protein P3. All three proteins are aligned to Cr patterns and hence also to each other.
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TBST). Finally, it was treated with anti-rabbit IgG probe antibodies labeled with Alexa 488 (0.004 mg mL^1 in TBST). Each treatment was 1 h. An Olympus BX50 upright epifluorescence microscope (Olympus America, Inc., Center Valley, PA) with a Retiga Exi cooled monochrome CCD camera (QImaging, Surrey, BC, Canada) and a Metamorph V6.1 (Universal Imaging Corp., Molecular Devices Corp., Downington, PA) was used for imaging.
Acknowledgements
PB thanks Prof. David Collum, Department of Chemistry and Chemical Biology, Cornell University for technical discussions. IK thanks LG Culture. The authors acknowledge the support of the National Science Foundation (NSF) (Grant ECS-0330110). This work was performed in part at the Cornell NanoScale Science and Technology Facility (CNF), a member of the National Nanotechnology Infrastructure Network, which is supported by the NSF (Grant ECS 03-35765). Additional work was performed at the Nanobiotechnology Center (NBTC) at Cornell University, an STC program of the NSF under Agreement No. ECS-9876771.
Received: September 23, 2009 Published online:
[1] J. Tien, C. M. Nelson, C. S. Chen, Proc. Natl. Acad. Sci. USA 2002 , 99 ,
[2] J. Doh, D. J. Irvine, Proc. Natl. Acad. Sci. U. S. A. 2006 , 103 , 5700. [3] Z. H. Nie, E. Kumacheva, Nat. Mater. 2008 , 7 , 277. [4] a) B. Kersten, E. E. Wanker, J. D. Hoheisel, P. Angenendt, Expert Rev. Proteomics 2005 , 2 , 499; b) H. Zhu, M. Snyder, Curr. Opin. Chem. Biol. 2003 , 7 , 55. [5] a) D. H. Yu, B. Blankert, J. C. Vire, J. M. Kauffmann, Anal. Lett. 2005 , 38 , 1687; b) B. Y. Feng, A. Shelat, T. N. Doman, R. K. Guy, B. K. Shoichet, Nat. Chem. Biol. 2005 , 1 , 146. [6] a) R. Etzioni, N. Urban, S. Ramsey, M. McIntosh, S. Schwartz, B. Reid, J. Radich, G. Anderson, L. Hartwell, Nat. Rev. Cancer 2003 , 3 , 243; b) D. Sidransky, Nat. Rev. Cancer 2002 , 2 , 210. [7] a) A. Khademhosseini, R. Langer, J. Borenstein, J. P. Vacanti, Proc. Natl. Acad. Sci. U. S. A. 2006 , 103 , 2480; b) S. J. Hollister, Nat. Mater. 2005 , 4 ,
[8] L. P. Cunningham, M. P. Veilleux, P. J. Campagnola, Opt. Express 2006 , 14 ,
[9] a) K. Arcaute, B. K. Mann, R. B. Wicker, Ann. Biomed. Eng. 2006 , 34 , 1429; b) S. J. Hollister, R. A. Levy, T. M. Chu, J. W. Halloran, S. E. Feinberg, Int. J. Oral Maxillofac. Surg. 2000 , 29 , 67. [10] a) A. G. Mikos, S. W. Herring, P. Ochareon, J. Elisseeff, H. H. Lu, R. Kandel, F. J. Schoen, M. Toner, D. Mooney, A. Atala, M. E. Van Dyke, D. Kaplan, G. Vunjak-Novakovic, Tissue Eng. 2006 , 12 , 3307; b) R. Langer, D. A. Tirrell, Nature 2004 , 428 , 487. [11] S. Raghavan, C. S. Chen, Adv. Mater. 2004 , 16 , 1303. [12] a) M. L. Dustin, D. R. Colman, Science 2002 , 298 , 785; b) A. Grakoui, S. K. Bromley, C. Sumen, M. M. Davis, A. S. Shaw, P. M. Allen, M. L. Dustin, Science 1999 , 285 , 221. [13] C. Werner, T. Pompe, K. Salchert, in: Polymers For Regenerative Medicine, Springer-Verlag Berlin, Berlin 2006 , pp. 63–93. [14] a) R. L. Rich, D. G. Myszka, Anal. Biochem. 2007 , 361 , 1; b) M. A. Cooper, Drug Discov. Today 2006 , 11 , 1068. [15] a) S. F. Kingsmore, Nat. Rev. Drug Discov. 2006 , 5 , 310; b) P. S. Dittrich, A. Manz, Nat. Rev. Drug Discov. 2006 , 5 , 210; c) H. Craighead, Nature 2006 , 442 , 387; d) D. Janasek, J. Franzke, A. Manz, Nature 2006 , 442 , 374; e) J. El-Ali, P. K. Sorger, K. F. Jensen, Nature 2006 , 442 , 403. [16] G. M. Whitesides, Nature 2006 , 442 , 368. [17] D. Psaltis, S. R. Quake, C. H. Yang, Nature 2006 , 442 , 381. [18] M. A. Cooper, Nat. Rev. Drug Discov. 2002 , 1 , 515. [19] E. Verpoorte, Lab Chip 2003 , 3 , 42N.
Figure 3. PGA groups (Scheme 2) were used to immobilize a) rat IgG, b) rabbit IgG, and c) mouse IgG. d) An overlay of (a–c) with a specific color attributed to each panel. Rat IgG: blue; rabbit IgG: red; mouse IgG: green. e) Bovine fibronectin immobilized on PGA groups (Scheme 2). Scale bar: 50 mm in all panels.
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[20] a) G. M. Whitesides, E. Ostuni, S. Takayama, X. Y. Jiang, D. E. Ingber, Annu. Rev. Biomed. Eng. 2001 , 3 , 335; b) B. Michel, A. Bernard, A. Bietsch, E. Delamarche, M. Geissler, D. Juncker, H. Kind, J. P. Renault, H. Rothuizen, H. Schmid, P. Schmidt-Winkel, R. Stutz, H. Wolf, IBM J. Res. Dev. 2001 , 45 , 697; c) Y. N. Xia, G. M. Whitesides, Annu. Rev. Mater. Sci. 1998 , 28 , 153. [21] a) M. J. Heller, Annu. Rev. Biomed. Eng. 2002 , 4 , 129; b) A. V. Lemmo, D. J. Rose, T. C. Tisone, Curr. Opin. Biotechnol. 1998 , 9 , 615. [22] a) D. S. Ginger, H. Zhang, C. A. Mirkin, Angew. Chem. Int. Ed. 2004 , 43 , 30; b) R. D. Piner, J. Zhu, F. Xu, S. H. Hong, C. A. Mirkin, Science 1999 , 283 , 661; c) K. Salaita, Y. H. Wang, J. Fragala, R. A. Vega, C. Liu, C. A. Mirkin, Angew. Chem. Int. Ed. 2006 , 45 , 7220. [23] E. Stern, J. F. Klemic, D. A. Routenberg, P. N. Wyrembak, D. B. Turner-Evans, A. D. Hamilton, D. A. LaVan, T. M. Fahmy, M. A. Reed, Nature 2007 , 445 , 519. [24] A. del Campo, D. Boos, H. W. Spiess, U. Jonas, Angew. Chem. Int. Ed. 2005 , 44 , 4707. [25] C. G. Bochet, Angew. Chem. Int. Ed. 2001 , 40 , 2071. [26] M. Zourob, J. E. Gough, R. V. Ulijn, Adv. Mater. 2006 , 18 , 665. [27] A. Patchornik, B. Amit, R. B. Woodward, J. Am. Chem. Soc. 1970 , 92 , 6333. [28] C. P. Holmes, J. Org. Chem. 1997 , 62 , 2370. [29] P. Bhatnagar, S. S. Mark, I. Kim, H. Y. Chen, B. Schmidt, M. Lipson, C. A. Batt, Adv. Mater. 2006 , 18 , 315.
[30] P. Bhatnagar, A. D. Strickland, I. Kim, G. G. Malliaras, C. A. Batt, Appl. Phys. Lett. 2007 , 90 , 144107. [31] L. M. Lee, R. L. Heimark, R. Guzman, J. C. Baygents, Y. Zohar, Lab Chip 2006 , 6 , 1080. [32] S. P. A. Fodor, J. L. Read, M. C. Pirrung, L. Stryer, A. T. Lu, D. Solas, Science 1991 , 251 , 767. [33] J. P. Pellois, X. C. Zhou, O. Srivannavit, T. C. Zhou, E. Gulari, X. L. Gao, Nat. Biotechnol. 2002 , 20 , 922. [34] G. Dorman, G. D. Prestwich, Trends Biotechnol. 2000 , 18 , 64. [35] G. T. Hermanson, Academic Press, San Diego, CA 1996 , pp. 173–176, 194-196, 218- [36] N. Nimura, K. Iwaki, T. Kinoshita, K. Takeda, H. Ogura, Anal. Chem. 1986 , 58 , 2372. [37] J. F. Cameron, J. M. J. Frechet, J. Am. Chem. Soc. 1991 , 113 , 4303. [38] B. Amit, U. Zehavi, A. Patchornik, J. Org. Chem. 1974 , 39 , 192. [39] a) P. Roach, D. Farrar, C. C. Perry, J. Am. Chem. Soc. 2005 , 127 , 8168; b) R. J. Marsh, R. A. L. Jones, M. Sferrazza, Colloids Surf. B 2002 , 23 , 31. [40] V. Luginbuehl, L. Meinel, H. P. Merkle, B. Gander, Eur. J. Pharm. Biopharm. 2004 , 58 , 197. [41] a) W. Y. Yeong, C. K. Chua, K. F. Leong, M. Chandrasekaran, Trends Biotechnol. 2004 , 22 , 643; b) D. W. Hutmacher, Biomaterials 2000 , 21 , 2529. [42] M. S. Sanford, P. T. Charles, S. M. Commisso, J. C. Roberts, D. W. Conrad, Chem. Mat. 1998 , 10 , 1510.
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