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A Noncovalent Approach to the Construction of Tween 20‐Based Protein Microarrays

In the development of efficient chips capable of recognizing biospecific interactions, such as DNA hybridization and peptide–antibody and protein–protein interactions, one of the most critical issues yet to be improved is how to prevent nonspecific binding (NSB) of incoming target biomolecules, while maximizing biospecific bindings of targets to the surface-immobilized probes. In the cases of protein and peptide chips, the minimization of NSBs of target proteins has generally been attempted by coating chip surfaces with bovine serum albumin (BSA). Although BSA protects the surface from nonspecifically binding proteins reasonably well, this approach still causes several undesirable problems, especially those involving the sizes of proteins. The efficiency of biospecific binding becomes quite low when the sizes of probe molecules are small. For example, when FLAG peptides were immobilized as a probe and the chip surface was covered with BSA, the large BSA spatially shielded the much smaller FLAG peptides from the incoming counterpart, anti-FLAG, resulting in inefficient biospecific binding. As an alternative approach, strategically designed, small organic molecules have been developed. These are coated as a monolayer onto various solid substrates in order to provide biologically “inert” proteinor cell-resistant surfaces. Compounds containing oligoor poly(ethylene glycol) have been widely used for this purpose. Recently, commercially available Tween 20 has drawn much attention as a potential coating material for hydrophobic surfaces, since it contains a long alkyl chain of twelve carbons (dodecyl chain), through which Tween 20 can effectively interact with other hydrophobic surfaces, along with twenty ethylene glycol groups and three terminal hydroxy ( OH) groups that should provide effective nonbiofouling properties and gateways for introducing bioactive probe molecules, respectively. Thanks to its structural characteristics, especially the dodecyl chain, Tween 20 has been utilized for carbon nanotube-based (CNT-based) protein sensors, in which CNTs were coated with Tween 20 through hydrophobic interactions between the surface of the CNTs and the ACHTUNGTRENNUNGdodecyl chain of Tween 20. More recently, we have also shown that single-walled CNT films effectively coated with Tween 20 were suitable for the highly efficient discrimination of antigen–antibody and small peptide–antibody pair recognition. Although Tween 20 is stably adsorbed onto CNTs through parallel hydrophobic interactions along the CNT growth axis, it is still a challenging task to apply Tween 20 to more common chip platforms such as Au and SiO2, due to the instability of immobilized Tween 20 on flat surfaces. Whitesides and coworkers studied the stability of various detergents, including Tween 20, adsorbed onto the self-assembled monolayer (SAM) of hexadecanethiol. They found that Tween 20 was eliminated easily from the hydrophobic SAM surface upon simple washing with water, which indicated that the attractive interactions between Tween 20 and the hydrophobic methyl-terminated surface were not sufficient for the stable immobilization of Tween 20. In this article we report a simple but versatile method for the formation of stable Tween 20 layers on flat surfaces. Because simple hydrophobic interactions were not strong enough to maintain the adsorbed Tween 20 on hydrophobic surfaces, we introduced additional attractive interactions—van der Waals interactions—into the system in order to increase the stability of the adsorbed Tween 20. Specifically, we designed the substrate surface in such a way as to present a SAM of loosely packed long alkyl chain molecules, through which the dodecyl chain of Tween 20 could be spontaneously guided and squeezed into the SAM through van der Waals interactions. The key strategy for the formation of the stable Tween 20 layer began with the generation of a smart molecular layer on a solid substrate, composed of a loosely packed SAM of long alkyl chains. We reasoned that the loosely packed SAM should provide adequate spaces for the dodecyl chain of Tween 20 to be squeezed and accommodated through van der Waals interactions (Scheme 1). The construction of the loosely packed SAM on gold, without phase segregation, has been rather challenging, and the reported examples are scarce. Langer and coworkers ingeniously designed a precursor of self-assembling thiols, in which a bulky (2-chlorophenyl)diphenylmethyl group was coupled with 16-mercaptohexadecanoic acid (MHDA) through the ester linkage. After the formation of SAMs of the MHDA derivative, the selective cleavage of the bulky group generated the loosely packed SAM of MHDA. Recently, by adopting a similar strategy, Jennings and a co-worker reported that loosely packed, hydroxyl-terminated SAMs on gold could be prepared by the base-mediated cleavage of the large fluorocarbon terminal group. We devised a rather simpler approach for the formation of the loosely packed SAM. The loosely packed SAM was prepared by allowing long alkyl amines to react with an interchain carboxylic anhydride-terminated (ICA-terminated) SAM on gold. 28] The SAM of MHDA was first prepared by the reported procedure, and the resulting SAM was characterized by el[a] Y. S. Chi, Prof. I. S. Choi Department of Chemistry and School of Molecular Science (BK21), KAIST Daejeon 305-701 (Korea) Fax: (+82)42-869-2810 E-mail : ischoi@kaist.ac.kr [b] H. R. Byon, Prof. H. C. Choi Department of Chemistry, Pohang University of Science and Technology (POSTECH) San 31, Hyoja-Dong, Nam-Gu, Pohang 790-784 (Korea) Fax: (+82)54-279-3399 E-mail : choihc@postech.edu Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.