VOLUME 3, NUMBER 6, JUNE 2003 © Copyright 2003 by the American Chemical Society
A Strategy for the Sequential Patterning of Proteins: Catalytically Active Multiprotein Nanofabrication Chang-Hyun Jang,† Benjamin D. Stevens,‡ Ryan Phillips,‡ Michael A. Calter,*,‡ and William A. Ducker*,† Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061, and Department of Chemistry, UniVersity of Rochester, Rochester, New York 14627 Received December 20, 2002; Revised Manuscript Received April 24, 2003
ABSTRACT We describe a new and simple method for immobilizing enzymes that will allow the preparation of thin films containing several different catalytically active enzymes. We use this method to immobilize acetylcholine esterase, then we show that its catalytic activity is retained after immobilizing a different protein on the same film.
The preparation of arrays of immobilized proteins is important for the preparation of assays and sensors and is useful for the study of protein activity.1 Recently, there has been some interest in the preparation of nanoscale protein arrays using the nanografting technique of atomic force microscopy (AFM).2 This technique may allow the fabrication of small and high-density sensors. For most applications, an array of proteins finds use when the array contains a number of different proteins, but to the best of our knowledge there is no established technique for the preparation of an array of different active proteins on the nanoscale. In a serial approach to producing heterogeneous arrays, difficulty can arise because the immobilization steps for the subsequent proteins can adversely affect the activity of the previously immobilized enzymes. For example, it is simple to immobilize proteins by reaction with aldehyde groups,3 but aldehyde † ‡
Virginia Tech. University of Rochester.
10.1021/nl0259636 CCC: $25.00 Published on Web 05/10/2003
© 2003 American Chemical Society
introduced into a solution that bathes an immobilized enzyme will react with amine groups on the immobilized enzyme and could inactivate the enzyme (see Figure 1A). In this letter, we describe a method for the serial immobilization of nanoscale protein patches without inactivating enzymes. We demonstrate the method by first immobilizing acetylcholine esterase, then immobilizing insulin, and then showing that the acetylcholine esterase (AChE) remains catalytically active. In our method, we rely on the oxidative selectivity of periodate for the diol group.4 None of the 20 or so common amino acids contain the diol group, so proteins are relatively immune to oxidation by periodate. The diol group does not react with proteins, but oxidation of the diol with periodate proceeds only as far as the aldehyde, which can then undergo reaction with the amine groups in proteins (see Figure 1B). In addition, as we show, it is facile to incorporate diols into self-assembled monolayers (SAMS). Therefore, the diol can be introduced into a SAM as a “latent aldehyde” ready to
Figure 1. Immobilization of a series of enzymes. (A) Reagents that can immobilize an enzyme can also modify enzymes that are already in the film. (B) We expose the enzyme to two reagents, neither of which react with the enzyme, but which react together to form an immobilization site.
be switched into an immobilizing site. The switch does not inactivate enzymes that are already immobilized on the surface. In fact, the in situ preparation of aldehyde films from diols is so convenient that the technique might also be useful for preparing homogeneous films of immobilized molecules (e.g., for surface plasmon resonance (SPR) studies.) The most common type of SAM is produced through reaction of thiol groups with gold, so our diol SAMs were produced using long chain thiols with an ω-diol. Our synthesis of diol thiol 1 began with the alkylation of polyethylene(ethyleneglycol) diol 2 with ω-bromoundecene (Scheme 1). Dihydroxylation of this compound using catalytic osmium tetroxide and stoichiometric N-methyl morpholine-N-oxide (NMO) afforded a mixture of the singly and doubly dihydroxylated compounds. After separation, the singly dihydroxylated compound was converted into 1 by addition of thioacetic acid followed thioester hydrolysis. Scheme 1
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As a first check of our hypothesis that oxidation with NaIO4 does not adversely affect the catalytic activity of enzymes, we examined the activity of the two-component nitrogenase system in buffer solution in the absence and then in the presence of periodate solution.5 We made an aqueous mixture of ATP Fe-P, MoFe-P, and creatinephosphate kinase in 0.1 mM NaIO4. At the same time, the same enzyme assay solution was prepared without periodate as a reference. After 20 min, the enzyme activities of each were measured by analyzing the amount of product gas (H2) using gas chromatography. There was only a slight reduction in activity (13%) in the presence of periodate, thereby supporting the hypothesis that periodate does not inactivate the enzyme. Note that this test is more harsh than the conditions for the immobilization strategy. The periodate is actually present during the catalysis, whereas in our strategy the periodate is a wash prior to any step requiring catalysis. In the latter case, any reversible reaction of the enzyme with periodate could potentially be undone to recover activity. For our immobilization experiments, we first produced a very smooth gold sphere by melting the end of a gold wire with a H2/O2 flame.6 A smooth surface facilitates observation of surface structures that are only a few nanometers high. After cooling, we then deposited a monolayer of HS(CH2)11(OCH2CH2)3OH7 (EOthiol) by deposition from ethanol solution for 24 h. The ω-ethylene oxide groups are known to resist the adsorption of protein molecules.8 We then used nanografting to create a small patch of 1,2-diol groups on the surface by deposition of HS(CH2)11(OCH2CH2)6O(CH2)11CH(OH)CH2OH (diol). Nanografting is a procedure in which a SAM is scraped with an AFM tip in a solution of a different SAM-forming molecule. The molecules from solution are incorporated into the film after the original molecules are scraped out.2 We used Si3N4 cantilever/tips (TM Microscopes) with a nominal spring constant of 0.5 Nm-1 and a radius of
