VOLUME 3, NUMBER 11, NOVEMBER 2003 © Copyright 2003 by the American Chemical Society
Lithographing of Biomolecules on a Substrate Surface Using an Enzyme-Immobilized AFM Tip Seiji Takeda,† Chikashi Nakamura,*,†,‡ Chie Miyamoto,‡ Noriyuki Nakamura,†,‡ Masami Kageshima,§ Hiroshi Tokumoto,§ and Jun Miyake†,‡ Tissue Engineering Research Center (TERC), National Institute of AdVanced Industrial Science and Technology (AIST), 3-11-46 Nakoji, Amagasaki, Hyogo 661-0974, Japan, Department of Biotechnology, Tokyo UniVersity of Agriculture and Technology (TUAT), 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan, and Nanotechnology Research Institute (NRI), National Institute of AdVanced Industrial Science and Technology (AIST), 1-1-4 Higashi, Tsukuba, Ibaraki 305-8562, Japan Received June 26, 2003; Revised Manuscript Received September 8, 2003
ABSTRACT A method of enzymatic lithography was successfully performed in a buffered solution using Staphylococcal serine V8 protease and atomic force microscopy (AFM). The retained activity of the protease immobilized on the AFM tip was confirmed by force measurement to rupture the enzyme−substrate complex. After contact scanning using the enzyme-immobilized tip to the substrate peptide layer, a square shape of the lithographed area was clearly observed by subsequent AFM imaging.
The controlled alignment of biomolecules on solid surfaces at the nanometer to micrometer scale is useful not only for fundamental studies examining the interactions between enzymes and substrates, ligands and receptors, and antigens and antibodies,1-4 but also for its potential role in the design of peptide-based sensors and biomaterials. Atomic force microscopy (AFM) is a technique that not only measures the topography of a surface but which can also be used for manipulating biomolecules.5-10 For these * Corresponding author. Phone +81-6-6494-7858; Fax +81-6-64947862; E-mail
[email protected] † TERC/AIST. ‡ TUAT. § NRI/AIST. 10.1021/nl034448k CCC: $25.00 Published on Web 10/01/2003
© 2003 American Chemical Society
reasons, together with the fact that the average diameter of an AFM tip is generally between 5 and 50 nm, AFM has become a useful tool for aligning or modifying biomolecules. A typical method for aligning biomaterials on substrate surfaces using AFM at nanometer levels is by dip pen nanolithography.11-12 Such methods are available for lithography of organic and inorganic materials. However, modification of biomolecules requires buffered conditions. Microcontact printing has also been used for lithography at the micrometer level, but it is not suitable for use with biomolecules. The nanostructure of the DNA surface was constructed by scratching a self-assembled monolayer of DNA with an AFM tip.13 This method could be performed in buffered solutions, but it is used only for simple detachment
Figure 1. Forces to stretch intermediates of enzyme immobilized on AFM tip and peptide immobilized on mica. (A) Forces observed in the tip contact to the peptide II layer. (B) Forces observed in the tip contact to the peptide I layer.
of molecules. A novel method for alignment and modification of biomolecules on a substrate would be a powerful tool for biomolecular fabrication. Recently, it has been reported that the enzyme shikimate kinase, when covalently immobilized to an AFM tip, retained its enzymatic activity.14 If enzymes such as proteases, polymerases, and the like could be covalently immobilized to an AFM tip without denaturation, such enzyme-immobilized tips would be powerful tools for modification of biomolecules on surfaces. In this study we chose the enzyme Staphylococcal serine V8 protease to be immobilized onto an AFM tip. This enzyme is a monomer and remains active even in the presence of 4 M urea or 0.2% sodium dodecyl sulfate. From this, we demonstrated that Staphylococcal serine V8 protease, when immobilized onto an AFM tip, is able to digest a peptide on a mica surface and that lithographing is possible by scanning the surface with an enzyme-immobilized AFM tip. Staphylococcal serine V8 protease recognizes either glutamic or aspartic acid residues in the peptide and digests the peptides carboxyl acid terminus. The enzyme was covalently immobilized to the tip via amide bonds using N-(6-maleimidocaproyloxy)succinimide (EMCS), a heterobifunctional reagent, after the tip was silanized using 3-mercaptoproplytrimethoxysilane. A Nanoscope IIIa (Digital Instruments, Santa Barbara, CA) with a fluid cell was employed to measure force curves and surface images. For the AFM measurements, an optical head is used to sense the tip deflection by sensing the change in position of a laser beam that is reflected off the back of the cantilever. The cantilevers used in this study had a nominal spring constant of 0.06 or 0.12 N/m. The retained activity of the protease immobilized on AFM tip was investigated by force measurement to rupture enzyme-substrate complex. 1472
Substrate peptides, peptide I, A(AEAAKA)6C, and peptide II, A(ASAAKA)6C, were synthesized using a solid-phase method with fluorenylmethoxycarbonyl chemistry. Whereas peptide I was designed for V8 protease digestion at the glutamic acid residues, peptide II was designed as a control peptide that contained no glutamic acid residues and was not digested by the protease. Each peptide was covalently immobilized via EMCS to a mica surface silanized using 3-aminoproplytrimethoxysilane. The force curve measurement was performed in 50 mM of phosphate buffer at pH 7.8. The tip was initially brought into contact with the layer of peptide and was then retracted from the peptides. The force curves are shown in Figure 1 and were recorded in succession after changing the sample position. The applied force on the AFM cantilever was plotted as a function of the sample displacement. During the retracting process, although less than 0.5 nN of the force was observed in the case of peptide II, A(ASAAKA)6C (Figure 1A), a larger force of over 2 nN was sometimes applied to the cantilever in the case of peptide I, A(AEAAKA)6C (Figure 1B). For this measurement, the contact period of the tip with the surface of the sample was less than 10 ms and the enzymatic turnover rate of the serine protease was about 1 ms. Since the density of the enzyme of the tip surface was not controlled in this experiment, several enzyme molecules (more than one) were probably involved in the interaction with peptide I in the force curve measurement. A possible explanation for the large force observed in the case of peptide I is that since some enzyme molecules recognized the peptide’s glutamic acid residues, intermediates were formed during the contact period. The large force might be due to stretch and the rupture of the intermediates. To confirm the hypothesis, we measured the force curves with an inactivated protease tip and peptide I. The inactivated Nano Lett., Vol. 3, No. 11, 2003
Figure 2. The surface topography images of the peptide layers measured by contact mode AFM. The black scale bars indicate 5 µm and blue square represents scanned (lithographed) area. (A) Surface image of the lithographed peptide layer using an enzyme-immobilized AFM tip before addition of streptavidin. (B) Same surface image as A after addition of streptavidin. (C) Surface image of the lithographed peptide layer using a cysteine-immobilized tip after addition of streptavidin. (D) Cross-section of image B indicated by a white line. Two red arrows in Figure 2B correspond to arrows in Figure 2D. A schematic drawing shows the lithography process.
enzyme-immobilized tip was prepared by immersing the tip in a phenylmethylsulfonyl fluoride (PMSF) solution, which irreversibly inactivated the serine proteases. A force of less than 0.4 nN was observed during the retracting period after contact with the surface of peptide I (data not shown). It would appear that the large force was therefore due to stretching of the intermediate. We then investigated whether the enzyme was able to digest the peptide after forming the intermediate. If digestion of peptide layer is possible, this technique could be applied for lithography of biomolecules. We tried to lithograph the peptide layer by contact scanning with the enzyme-immobilized tip. Before scanning we checked the activity of the immobilized enzyme by measuring the force curves and used only tips showing binding activity. N-Terminusbiotinylated peptide I was synthesized and used in this experiment to visualize an area of digested peptides using an abiding-biotin binding. The lithograph of the peptide layer was carried out by scanning over the surface of the peptide layer to form a square at a scanning rate of 0.05 µm/sec. The applied force during lithography was kept less than 50 pN. After lithographing, 1 mg/mL of streptavidin was injected into the AFM cell and incubated for 20 min. Thereafter, the surface height image was measured using the same tip at a scanning rate of 1 µm/sec (enough fast rate not to digest the peptides). After lithographing using the enzyme-immobilized tip, the surface topography of peptide layer did not show any obvious structure (Figure 2A) since the difference between the length of digested peptides and not-digested peptides is too small to make a clear height Nano Lett., Vol. 3, No. 11, 2003
difference in the AFM image. After addition of streptavidin, the lithographed area was clearly observed as a dark area shown in Figure 2B. As a no enzyme-immobilized tip control, a cysteineimmobilized tip was used for same test. The height image was also measured after streptavidin addition (Figure 2C). In this case, no dark area was observed after scanning. When N-terminus-biotinylated peptide II was used for lithography with the enzyme-immobilized tip, the dark area was also not observed after scanning (data not shown). The peptides in square shape dark area of Figure 2B were not scratched away by the contact scanning of the tip. Since the biotins at the N-terminus of the peptide I were released from the peptide layer by enzymatic digestion, no streptavidin could bind to the lithographed area as shown in the schematic of Figure 2. A cross-section of the sample is shown in Figure 2D, and the height of the dark area was found to be about 4 nm lower than the nonlithographed area. The height does not contradict to the theoretical size of streptavidin molecule. We also measured the force curves at the lithographed area using the enzyme-immobilized tip. A probability of the large force occurrence decreased obviously compared to Figure 1B. These results indicate that the enzyme immobilized on the AFM tip retained its digestion activity and that the peptide in the lithographed area was digested by the immobilized enzyme. In this paper we have demonstrated that digestion of a peptide layer on a substrate surface is possible using an enzyme immobilized onto an AFM tip in a buffered solution. This is the first report of direct lithography of a biomolecule. 1473
To apply this technique for other enzymes, more studies would be needed; however, the technique would be developed to the precision machining of biomolecules at the submicrometer to nanometer scale. It means that not only digestion molecules but also build-up of or modification of molecules would be possible using this technique. We strongly believe that our lithography method has potential to be used in a wide variety of applications, such as development of a DNA chip, bioMEMS, microTAS, microbiosensor, etc., especially in the field of biotechnology. Acknowledgment. This work was partially supported by Millennium Project of Ministry of Economy, Trade and Industry of Japan. References (1) Mckendry, R.; Theoclitou, M. E.; Rayment, T.; Abell, C. Nature 1998, 391, 566. (2) Kaasgaard, T.; Mouritsen, O. G.; Jorgensn, K. FEBS Lett. 2002, 515, 29.
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(3) Kienberger, F.; Kada, G.; Gruber, H. J.; Pastushenko, V. P.; Riener, C.; Trieb, M.; Knaus, H. G.; Schindler, H.; Hiterdorfer, P. Single Mol. 2000, 1, 59. (4) Hinterdorfer, P.; Schilcher, K.; Baumgartner, W.; Gruber, H. J.; Schidler, H. Nanobiology 1998, 4, 177. (5) Grandbois, M.; Beyer, M.; Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Science 1999, 283, 1727. (6) Mitsui, K.; Hara, M.; Ikai, A. FEBS Lett. 1996, 385, 29. (7) Oesterhelt, F.; Oesterhelt, D.; Pfeiffer, M.; Engel, A.; Gaub, H. E.; Muller, D. J. Science 2000, 288, 143. (8) Takeda, S.; Ptak, A.; Nakamura, C.; Miyake, J.; Kageshima, M.; Jarvis, S. P.; Tokumoto, H. Chem. Pharm. Bull. 2001, 49, 1512. (9) Ptak, A.; Takeda, S.; Nakamura, C.; Miyake, J.; Kageshima, M.; Jarvis, S. P.; Tokumoto, H. J. Appl. Phys. 2001, 90, 3095. (10) Kageshima, M.; Lantz, M. A.; Jarvis, S. P.; Tokumoto, H.; Takeda, S.; Ptak, A.; Nakamura, C.; Miyake, J. Chem. Phys. Lett. 2001, 343(1/ 2), 77. (11) Piner, R.; Zhu, J.; Xu, F.; Hong, S.; Mirkin, C. A. Science 1999, 283, 661. (12) Hyun, J.; Ahn, S. J.; Lee, W. K.; Chilkoti, A.; Zauscher, S. Nano Lett. 2002, 2, 1203. (13) Liu, M.; Amro, N. A.; Chow, C. S.; Liu, G. Nano Lett. 2002, 2, 863. (14) Fiorini, M.; Mckendry, R.; Cooper, M. A.; Rayment, T.; Abell, C. Biophy. J. 2001, 80, 2471.
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