Lloyd Smith Research Group - The University of Wisconsin-Madison

Bio-chip surface chemistry

Surfaces for DNA immobilization have become very important. Key properties of ideal surfaces include surface flatness, homogeneity, ease of control of surface properties, thermal stability, chemical stability, reproducibility, ability to immobilize DNA, and ability to manipulate the biochemistry. In an effort to reach these goals, we have developed new surface chemistries for DNA attachment.

Amine-modified Si(001) surfaces for thiol-modified oligonucleotide attachment1

Si(001) wafers are prepared using previously described methods.2,3 These wafers are then treated with 2% HF in water for hydrogen termination of the surface. The surfaces are then covered with t-BOC protected 10-aminodec-1-ene and exposed to UV light for 2 hours. The surfaces are then treated with 25% trifluoroacetic acid (TFA) in methylene chloride and rinsed with 10% NH4OH for removal of the t-BOC group. This leaves an amine on the surface, which is reacted with sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SSMCC). The SSMCC can then react with thiol-modified DNA, which is bound to the surface (Figure 1). These surfaces retain high sequence specificity in hybridization reactions (Figure 2). They are also very stable to multiple hybridizations, showing ~1% loss per cycle over 15 hybridization cycles (Figure 3).

Carboxylic acid-modified Si (111) surfaces for thiol-modified oligonucleotide attachment4

First, ?-undecylenic acid methyl or trifluoroethyl ester are added to the surface as a thin film and activated by UV radiation. This generates a self-assembled monolayer (SAM) with an ester available for reaction. The ester is hydrolyzed by tert-butoxide in DMSO, which leaves a carboxylic acid group on the surface. Poly-L-lysine is then added to the surface, creating electrostatic interactions between the side chains and the carboxylic acid groups on the SAM. SSMCC is then added and will react with side chains that are not attached to the SAM. Thiol-modified DNA is then added, which will react with the SSMCC (Figure 4). Hybridization signal showed an average dropoff of ~2% per cycle to about 30 cycles (Figure 5).

Amine-modified Si (100) surfaces for thiol-modified oligonucleotide attachment5

Hydrogen-terminated Si (100) is deprotected with UV light and then functionalized with 1-amino-3-cyclopentene (ACP) (Figure 6a,d). This reveals a primary amine off of the surface, which can react with SSMCC. The SSMCC can then react with thiol-functionalized DNA (Figure 6b,c). An alternative approach uses N-1-BOC-amino-3-cyclopentene (BACP) rather than ACP. BACP is a protected amine and attachs to the surface with greater density than ACP (Figure 6e). Before SSMCC is added, the molecule must first be deprotected, however. Trifluoracetic acid (TFA) is added to the surface, causing acid hydrolysis and removal of the protecting group. This can leave an ammonium-like ion on the molecule, which is returned to a primary amine state by addition of ammonium hydroxide. Multiple hybridizations were performed on the surfaces and they showed ~2% drop off at each cycle (Figure 7a). These surfaces also retain high specificity under hybridization conditions.

Amine-modified diamond thin film surfaces for thiol-modified oligonucleotide attachment6

Two types of diamond films can be used, ultra nano-crystalline diamond (UNCD) and nano-crystalline diamond (NCD). Both types were grown by first placing a diamond powder on an n-type Si (100) surface and ultrasonicating to create nucleation positions. UNCD films used a plasma reactor with 1% methane gas (99% Ar) at 150 torr and 800oC for two hours, providing films 0.75 µm thick. NCD films were prepared with a plasma reactor with hydrogen gas and methane gas (99.999%) at 15 torr, providing films 0.50 µm thick. The samples were then cleaned with acid and placed in a hydrogen plasma chamber at 15 torr and 800oC for 20 minutes. The hydrogen plasma treatment etches away any graphitic carbon and hydrogen terminates the surface. The diamond thin films are reacted with 10-aminodec-1-ene, with a trifluoroacteamide (TFAAD) protecting group. The amine is then deprotected with HCl/methanol. The primary amine is reactive to SSMCC which will also react with thiol-modified DNA (Figure 8). The DNA on the surface retains its hybridization specificity (Figure 9a,b). The DNA-modified UNCD diamond is more stable to multplie hybridizations than other surfaces (Figure 9c).

Carboxylic acid-modified gold surfaces for thiol-modified oligonucleotide attachment7

A gold surface is reacted with 11-mercaptoundecanoic acid (MUA), which binds to the surface using a gold-thiol bond and creates a SAM. The MUA SAM reveals a carboxylic acid group, which can absorb a monolayer of poly-l-lysine (PL) with an electrostatic interaction. Much like with the Si (111) surface chemistry, the PL layer can be reacted with SSMCC, which can bind thiol-modified DNA (Figure 10).

Amine-modified gold surfaces for thiol-modified oligonucleotide attachment8

In a similar fashion, a gold surface can be reacted with 11-mercaptoundecanoic acid maleimide (MUAM) creating a SAM. The MUAM SAM reveals an amine, which can bind SSMCC to the surface directly. The SSMCC can then bind thiol-modified DNA (Figure 11).

Aldehyde-modified gold surfaces for amine-modified oligonucleotide attachment9

First, di(10-decanal) disulfide is added to the surface, forming a SAM with an aldehyde group sticking up from the surface. Amino-modified DNA can directly bind to the aldehyde, replacing the oxygen. This forms a Schiff base, which is then reduced with sodium cyanoborohydride.

1. Strother , T., Hamers , R.J. and Smith, L.M. Covalent attachment of oligodeoxyribonucleotides to amine-modified Si (001) surfaces. (2000) Nucleic Acids Res., 28 (18), 3535-3541.
2. Wagner,P., Nock,S., Spudich,J.A., Volkmuth,W.D., Chu,S., Cicero,R.L., Wade,C.P., Linford,M.R. and Chidsey,C.E.D. Bioreactive self-assembled monolayers on hydrogen-passivated Si(111) as a new class of atomically flat substrates for biological scanning probe microscopy. (1997) J. Struct. Biol., 119, 189?01.
3. Strother,T., Cai,W., Zhao,X., Hamers,R.J. and Smith,L.M. Synthesis and Characterization of DNA-Modified Silicon (111) Surfaces. (2000) J. Am. Chem. Soc., 122, 1205?209.
4. Strother , T., Cai , W., Zhao, X., Hamers , R.J. and Smith, L.M. 2000. Synthesis and characterization of DNA-modified silicon (111) surfaces. J. Am. Chem. Soc., 122 (6), 1205-1209.
5. Lin, Z., Strother , T., Cai , W., Cao , X., Smith, L.M. and Hamers , R.J. 2002. DNA attachment and hybridization at the silicon (100) surface. Langmuir , 18 , 788-796.
6. Yang, W., Auciello , O., Butler , J.E., Cai , W., Carlisle , J.A., Gerbi , J.E., Gruen , D., Knickerbocker , T., Lasseter , T.L., Russell, J.N., Smith, L.M. and Hamers , R.J. 2002. DNA-modified nanocrystalline diamond thin films as stable, biologically active substrates. Nature Materials , 1 (4), 253-257.
7. Emily A. Smith, Matt J. Wanat, Yufei Cheng, Sergio V. P. Barreira, Anthony G. Frutos and Robert M. Corn "Formation, Spectroscopic Characterization and Application of Sulfhydryl-Terminated Alkanethiol Monolayers for the Chemical Attachment of DNA onto Gold Surfaces," Langmuir, 17 2502-2507 (2001).
8. Jennifer M. Brockman, Anthony G. Frutos and Robert M. Corn, "A Multi-Step Chemical Modification Procedure to Create DNA Arrays on Gold Surfaces for the Study of Protein-DNA Interactions with Surface Plasmon Resonance Imaging," J. Am. Chem. Soc., 121 8044-8051 (1999).
9. Peelen D, Smith LM. Immobilization of amine-modified oligonucleotides on aldehyde-terminated alkanethiol monolayers on gold. Langmuir. 2005 Jan 4;21(1):266-71.