NANO LETTERS
Nanoscale Site-Specific Immobilization of Proteins through Electroactivated Disulfide Exchange
2003 Vol. 3, No. 6 779-781
Elisabeth Pavlovic,*,† Sven Oscarsson,†,‡ and Arjan P. Quist‡,§ Department of Surface Biotechnology, Uppsala UniVersity, Box 577, S75123 Uppsala, Sweden, Department of Biology and Chemical Engineering, Ma¨lardalen UniVersity, Box 325, S63105 Eskilstuna, Sweden, Department of Physics, Uppsala UniVersity, Box 530, S75121 Uppsala, Sweden Received March 31, 2003; Revised Manuscript Received April 24, 2003
ABSTRACT A new method based on the electrochemical oxidation and activation of thiols was used to generate nanoscale patterns on thiol-derivatized silicon oxide surfaces. By application of a potential difference between an AFM tip and a thiolated silicon surface, surface thiols were activated into thiolsulfinates/thiolsulfonates, which are reactive to thiol groups on biomolecules in solution. Thiol-rich protein, β-galactosidase, was immobilized onto the patterns.
In addition to being one of the main imaging1-3 and analytical4,5 techniques in surface science in general and in surface molecular nanotechnology in particular, atomic force microscopy (AFM) has also proven to be a versatile tool for nanometer scale chemical modifications6-8 or site-specific deposition of atoms or molecules9,10 on various surfaces. Several of these methods involve an electrochemical process resulting from applying a potential difference between the AFM tip and the sample surface. Air relative humidity (RH) has been shown to be an essential parameter in these experiments since condensation of atmospheric water between the AFM tip and the sample surface generates a meniscus that acts as a transfer medium to obtain a nanosized electrochemical cell. In our previous work,11 we described the activation of surface thiols to thiolsulfinates (Scheme 1A) and thiolsulfonates (Scheme 1B) by electrochemical oxidation, with the possibility of a reversible immobilization12,13 of thiolated peptides onto the activated surfaces through covalent disulfide bonds. The prime interest of this activation method is its potential to achieve site-specific activation of surface thiols, in sizes ranging from the millimeter to the nanometer scale, the resolution of the oxidized areas depending directly on the size of the cathode used for the electrochemical process. In this work, we show the miniaturization of this electrochemical activation process to the nanometer scale. As * Corresponding author. Phone +46 18 4713530. Fax +46 18 4713611. E-mail
[email protected]. † Department of Surface Biotechnology, Uppsala University. ‡ Ma ¨ lardalen University. § Department of Physics, Uppsala University. 10.1021/nl034191q CCC: $25.00 Published on Web 05/23/2003
© 2003 American Chemical Society
Scheme 1. Schematic Drawing of the Electrochemical and Chemical Reactions Expected to Occur on the Thiolated Surfaces
R is a thiol-rich molecule, in our experiments βGal.
described in Scheme 1, thiols are electrochemically oxidized to thiolsulfinates/thiolsulfonates (1-2) by applying a potential difference between the AFM tip and the thiol-derivatized surface. Immobilization of a thiol-rich protein, β-galactosidase (βGal), onto the localized patterns of oxidized thiols (3-4), and its subsequent release by treatment with dithiothreitol (DTT)14 (5-6) were tested. The presence of the electrochemically modified patterns was analyzed using lateral force microscopy (LFM). Immobilization and release of the protein from the surfaces were imaged using tapping mode AFM. p-Doped silicon surfaces (Silicon Sense Inc., Nashua, NH) were washed with “piranha” solution (H2SO4/H2O2 30% (v/ v) 2:1) and subsequently rinsed with ultrapure deionized water (18 MΩ, low carbon content). This procedure was repeated four times, after which the surfaces were dried in an argon flow inside the silanization reactor. The surfaces were then derivatized with 3-mercaptopropyltrimethoxysilane (3-MPTMS, ABCR, Karlsruhe, Germany) as described previously15 using a gas-phase reaction. In this method a 20
µL droplet of 3-MPTMS is placed inside the reactor next to the argon flow inlet, which enhances the evaporation rate of the reagent. The process was allowed to take place for 60 min. Subsequently, the surfaces were sonicated 10 min in 99% pure ethanol, followed by 10 min in ultrapure water. Electrochemical oxidation of the thiolated surfaces was achieved under ambient conditions or in an artificially humidified atmosphere to investigate the effect of air relative humidity (RH) from 28 to 43%, using a Nanoscope IIIa Multimode AFM (Digital Instruments, Santa Barbara, CA) in contact mode. To optimize the activating potential, different voltages, ranging from 1.0 to 5.0 V, were applied between the tip (Ultrasharp, spring constant 0.03 N/m, tip curvature radius
