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“Dip-Pen” Nanolithography in Registry with Photolithography for Biosensor Development David J. Pena,* Marc P. Raphael, and Jeff M. Byers Materials Physics Branch, Code 6340, Naval Research Lab, Washington, D.C. 20375 Received May 8, 2003. In Final Form: July 1, 2003 “Dip-pen” nanolithography (DPN) was used to deposit molecules of interest onto silicon oxide surfaces grown by a number of different deposition techniques and prepared by photolithography and wet-chemical etching. Using this method, layers of fluorescently tagged silazanes were placed within 50 nm of micrometerscale features with a 50 nm resolution on oxide substrates. It was also shown that these distances are even smaller when thiol molecules are used with Au substrates. Furthermore, proteins are immobilized on the silicon oxide substrates with sub-micrometer resolution.
Introduction Given the recent growth in the fields of proteomics and genomics, the development of a general biosensor capable of detecting a number of interactions is important. Among recently developed methods, two recurring themes are the drive for smaller samples and the capability to detect a greater number of binding events.1,2 To accomplish these goals, a number of microsensor-based methods have been developed, leading to microarray and “lab on a chip” devices.1,3-6 These chips are typically fabricated by dropping a small amount of DNA sequences onto a modified substrate, resulting in spot sizes as small as 200 nm but usually closer to 1-2 µm in diameter. In most cases, detection is performed by measuring the ratio of fluorescently tagged analytes on the surface. Normally, stringency is assured by washing with salt solutions or increasing the temperature. However, a new idea in chip design uses electronics to control binding of proteins and DNA. Platinum electrodes below the surface have been used to control the binding above them using the charges typically found on biomolecules.7,8 Although the ability to tailor the binding of molecules on the surface through the use of electric fields is a step in a new direction, this method, like the microarray methods above, still relies on fluorescence for detection. A new breed of sensor is being developed that uses electronic devices patterned below the test-bed surface to sense and actuate binding events. For example, magnetic-label-based bioassays that incorporate microarrays of giant magnetoresistive sensors have been developed.9,10 Unlike fluorescence-based techniques, these (1) Schena, M., Ed. Microarray Biochip Technology; Eaton Publishing: Natick, MA, 2000. (2) Southern, E.; Mir, K.; Shchepinov, M. Nat. Genet. Suppl. 1999, 21, 5-9. (3) Huang, J. X.; Mehrens, D.; Wiese, R.; Lee, S.; Tam, S. W.; Daniel, S.; Gilmore, J.; Shi, M.; Lashkari, D. Clin. Chem. 2001, 47, 1912-1916. (4) Schena, M.; Heller, R. A.; Theriault, T. P.; Konrad, K.; Lachenmeier, E.; Davis, R. W. Trends Biotechnol. 1998, 16, 301-306. (5) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767-773. (6) Templin, M. F.; Stoll, D.; Schrenk, M.; Traub, P. C.; Vohringer, C. F.; Joos, T. O. Trends Biotechnol. 2002, 20, 160-166. (7) Edman, C. F.; Raymond, D. E.; Wu, D. J.; Tu, E.; Sosnowski, R. G.; Butler, W. F.; Nerenberg, M.; Heller, M. J. Nucleic Acids Res. 1997, 25, 4907-4914. (8) Sosnowski, R. G.; Tu, E.; Butler, W. F.; O’Connell, J. P.; Heller, M. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1119-1123. (9) Graham, D. L.; Ferreira, H.; Bernardo, J.; Freitas, P. P.; Cabral, J. M. S. J. Appl. Phys. 2002, 91, 7786-7788.
devices do not degrade over the time necessary for a longer experiment. Nonetheless, whether these methods are used in conjunction with fluorescence or as an alternative, it is important to be able to bind biological molecules in registry with devices on the submicron scale. “Dip-pen” nanolithography (DPN)11-39 and nanografting (NG)40-50 have demonstrated their potential for patterning (10) Miller, M. M.; Sheehan, P. E.; Edelstein, R. L.; Tamanaha, C. R.; Zhong, L.; Bonnak, S.; Whitman, L. J.; Colton, R. J. J. Magn. Magn. Mater. 2001, 225, 138. (11) Hyun, J.; Ahn, S. J.; Lee, W. K.; Chilkoti, A.; Zauscher, S. Nano Lett. 2002, 2, 1203-1207. (12) Lee, K.-B.; Park, S.-J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002. (13) Wei, L.; Hong, X.; Guo, W.; Bai, Y. B.; Li, T. J. Chem. J. Chin. Univ.-Chin. 2002, 23, 1386-1388. (14) Weinberger, D. A.; Hong, S.; Mirkin, C. A.; Wessels, B. W.; Higgins, T. B. Adv. Mater. 2000, 12, 1600-1603. (15) Su, M.; Dravid, V. Appl. Phys. Lett. 2002, 80, 4434-4436. (16) Su, M.; Liu, X.; Li, S.-Y.; Dravid, V.; Mirkin, C. A. J. Am. Chem. Soc. 2002, 124, 1560-1561. (17) Ivanisevic, A.; Mirkin, C. A. J. Am. Chem. Soc. 2001, 123, 78877889. (18) Schwartz, P. V. Langmuir 2002, 18, 4041-4046. (19) Piner, R. D.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. Science 1999, 283, 661-663. (20) Hong, S.; Mirkin, C. A. Science 2000, 288, 1808-1811. (21) Hong, S.; Zhu, J.; Mirkin, C. A. Science 1999, 286, 523-525. (22) Hong, X.; Wei, L.; Guo, W.; Li, J.; Song, W. L.; Bai, Y. B.; Li, T. J. Chem. J. Chin. Univ. 2002, 23, 1778-1780. (23) Noy, A.; Miller, A. E.; Klare, J. E.; Weeks, B. L.; Woods, B. W.; DeYoreo, J. J. Nano Lett. 2002, 2, 109-112. (24) Mirkin, C. A.; Hong, S.; Demers, L. Chem. Phys. Chem. 2001, 2, 37-39. (25) Ivanisevic, A.; McCumber, K. V.; Mirkin, C. A. J. Am. Chem. Soc. 2002, 124, 11997-12001. (26) Lim, J. H.; Mirkin, C. A. Adv. Mater. 2002, 14, 1474. (27) Demers, L. M.; Park, S.-J.; Taton, T. A.; Li, Z.; Mirkin, C. A. Angew. Chem., Int. Ed. 2001, 40, 3071-3073. (28) Demers, L. M.; Ginger, D. S.; Park, S. J.; Li, Z.; Chung, S. W.; Mirkin, C. A. Science 2002, 296, 1836-1838. (29) Demers, L. M.; Mirkin, C. A. Angew. Chem., Int. Ed. 2001, 40, 3069-3071. (30) Zhang, H.; Li, Z.; Mirkin, C. A. Adv. Mater. 2002, 14, 1472. (31) McKendry, R.; Huck, W. T. S.; Weeks, B.; Florini, M.; Abell, C.; Rayment, T. Nano Lett. 2002, 2, 713-716. (32) Weeks, B.; Noy, A.; Miller, A. E.; DeYoreo, J. J. Phys. Rev. Lett. 2002, 88. (33) Wilson, D. L.; Martin, R.; Hong, S.; Cronin-Golomb, M.; Mirkin, C. A.; Kaplan, D. L. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1366013664. (34) Liu, X. G.; Fu, L.; Hong, S. H.; Dravid, V.; Mirkin, C. A. Adv. Mater. 2002, 14, 231-235. (35) Li, Y.; Maynor, B. W.; Liu, J. J. Am. Chem. Soc. 2001, 123, 2105-2106. (36) Maynor, B. W.; Li, Y.; Liu, J. Langmuir 2001, 17, 2575-2578. (37) Jang, J. Y.; Schatz, G. C.; Ratner, M. A. J. Chem. Phys. 2002, 116, 3875-3886.
10.1021/la034787t CCC: $25.00 © 2003 American Chemical Society Published on Web 09/16/2003
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molecules on surfaces with sub-100 nm precision. Both of these methods have shown that proteins can be patterned on Au surfaces via ω functionalized thiols.11,12,40,42,43 However, Au surfaces are not ideal in all cases. An oxide dielectric layer is needed to protect the embedded electronics from electrochemical processes in buffer solutions, while keeping them electrically isolated. In addition, Au has been shown to quench fluorescence at short distances, thereby limiting the accuracy of an experiment that uses a method like fluorescence resonance energy transfer (FRET), which monitors subtle changes in the fluorescence intensity or the lifetime of a closely spaced pair of fluorophores.51 By removing the fluorophore from contact with the metal substrate with the addition of a SiO2 layer, a fluorescence enhancement factor of 20 was observed.52 Therefore, it would be advantageous to be able to immobilize the proteins on an oxide layer for these types of assays. Recently, a variety of molecules have been patterned onto SiO2 and other nonmetallic surfaces via DPN.17,23,26,28,31 Herein, the feasibility of using DPN to create molecular nanostructures capable of binding biological molecules near devices is demonstrated. This work presents a general method to direct the assembly of proteins on an insulating SiO2 surface by first pretreating the surface with a polymer. Although it has previously been demonstrated that DPN can have sub-100 nm registry with other molecular features,17,20,24,28 this study shows the ability of DPN to work with micrometer-scale features produced by typical photolithographic methods. More specifically, it is shown that biotin and avidin, which can be used for the further binding of proteins, can be placed with submicrometer resolution near current lines and voltage pads in electronic devices.
biotin) was obtained from Pierce Biotechnology. All chemicals were used as received without further purification. A Thermomicroscopes CP-Research AFM was used for all lithography and non-fluoresence-based imaging. The nanolithography software Version 1.7 provided by Thermomicroscopes was used to create the patterns on the substrate, and image processing was carried out using the manufacturers software Version 2.1.15. Two types of AFM cantilevers were used: Si3N4 sharpened Microlevers with a nominal tip radius of
