Synthesis and characterization of DNA-modified silicon (111) surfaces

Article abstract of DOI:10.1021/ja9936161

Hydrogen-terminated Si(111) surfaces are modified by attachment of oligodeoxynucleotides and characterized with respect to DNA surface density, chemical stability, and DNA hybridization binding specificity. Surface functionalization employs the reaction of ω-unsaturated alkyl esters with the Si(111) surface using UV irraaiation. Cleavage of the ester using potassium tert-butoxide yields a carboxyl-modified surface, which serves as a substrate for the attachment of DNA by means of an electrostatically adsorbed layer of polylysine and attachment of thiol-modified DNA using a heterobifunctional cross-linker. The resultant DNA-modified surfaces are shown to exhibit excellent specificity and chemical stability under the conditions of DNA hybridization. This work provides an avenue for the development of devices in which the exquisite binding specificity of biomolecular recognition is directly coupled to semiconductor devices.

Full text of DOI:10.1021/ja9936161

J. Am. Chem. Soc. 2000, 122, 1205-1209
1205
Synthesis and Characterization of DNA-Modified Silicon (111)
Surfaces
†
†
‡
†
,†
Todd Strother, Wei Cai, Xinsheng Zhao, Robert J. Hamers, and Lloyd M. Smith*
Contribution from the Departments of Chemistry, 1101 UniVersity AVenue, UniVersity of Wisconsin,
Madison, Wisconsin 53706-1396, and Peking UniVersity, Beijing 100871, China
ReceiVed October 8, 1999
Abstract: Hydrogen-terminated Si(111) surfaces are modified by attachment of oligodeoxynucleotides and
characterized with respect to DNA surface density, chemical stability, and DNA hybridization binding specificity.
Surface functionalization employs the reaction of ω-unsaturated alkyl esters with the Si(111) surface using
UV irradiation. Cleavage of the ester using potassium tert-butoxide yields a carboxyl-modified surface, which
serves as a substrate for the attachment of DNA by means of an electrostatically adsorbed layer of polylysine
and attachment of thiol-modified DNA using a heterobifunctional cross-linker. The resultant DNA-modified
surfaces are shown to exhibit excellent specificity and chemical stability under the conditions of DNA
hybridization. This work provides an avenue for the development of devices in which the exquisite binding
specificity of biomolecular recognition is directly coupled to semiconductor devices.
Introduction
composite materials is far from optimum, and their surface
chemistry remains poorly characterized. Desired attributes of
DNA-modified surfaces include the following: (a) surface
flatness and chemical homogeneity; (b) ability to control surface
chemical properties such as polarity or hydrophobicity, which
impact strongly upon nonspecific binding properties; (c) ame-
nability to DNA hybridization (duplex formation) and enzymatic
manipulation with DNA-modifying enzymes such as ligase,
polymerase, and restriction enzymes; (d) ability to control DNA
surface density; (e) thermal and chemical stability; and (f)
reproducibility of preparation. Few if any of these criteria are
met by the surface chemistries presently in use.
DNA-modified surfaces are the subject of considerable current
activity in the field of biotechnology.
importance, several aspects of the performance of these novel
1-25
Despite their growing
†
University of Wisconsin.
Peking University.
‡
(
1) Linford, M. R.; Fenter, P.; Eisenberger, P. M.; Chidsey, C. E. D. J.
Am. Chem. Soc. 1995, 117, 3145-3155.
2) Chrisey, L. A.; Lee, G.; O’Ferrall, C. E. Nucleic Acids Res. 1996,
5, 3031-3039.
3) Frutos, A. G.; Liu, Q.; Thiel, A. J.; Sanner, A. M. W.; Condon, A.
E.; Smith, L. M.; Corn, R. M. Nucleic Acids Res. 1997, 25, 4748-4757.
4) Shchepinov, M. S.; Case-Green, S. C.; Southern, E. M. Nucleic Acids
Res. 1997, 25, 1155-1161.
5) Wagner, P.; Nock, S.; Spudich, J. A.; Volkmuth, W. D.; Chu, S.;
(
2
(
(
Remarkable advances have been made in microelectronics
technology over the last 20 years, primarily due to increasingly
powerful capabilities for the parallel fabrication of transistors
and other microelectronic devices on small length scales. A
similar trend has become evident in the fields of biology/
biotechnology, where arrays of tens of thousands of distinct
DNA molecules on planar substrates have proven useful for
the parallel analysis of genetic variation and gene expression
levels. The development of robust, well-characterized surfaces
and surface attachment strategies for biological analyses could
benefit greatly from the well-developed infrastructure that exists
in microelectronics. Previous researchers have successfully
(
Cicero, R. L.; Wade, C. P.; Linford, M. R.; Chidsey, C. E. D. J. Struct.
Biol. 1997, 119, 189-201.
(
6) Arnold, B. A.; Hepler, R. W.; Keller, P. M. BioTechniques 1998,
2
5, 106-110.
(
7) Bamdad, S. Biophys. J. 1998, 75, 1997-2003.
(8) Livache, T.; Fouque, B.; Roget, A.; Marchand, J.; Bidan, G.; Teoule,
R.; Mathis, G. Anal. Biochem. 1998, 255, 188-194.
9) Sieval, A. B.; Demirel, A. L.; Nissink, J. W. M.; Linford, M. R.;
van der Mass, J. H.; de Jeu, W. H.; Zuilhof, H.; Sudholter, E. J. R. Langmuir
998, 14, 1759-1768.
10) Smith, L. M.; Corn, R. M.; Condon, A. E.; Lagally, M. G.; Frutos,
A. G.; Liu, Q.; Thiel, A. J. J. Comput. Biol. 1998, 5, 255-267.
11) Yang, M.; Kong, R. Y. C.; Kazmi, M.; Leung, K. C. Chem. Lett.
998, 257-258.
12) Rasmussen, S. R.; Larsen, M. R.; Rasmussen, S. E. Anal. Biochem.
991, 30, 138-142.
13) Kremski, J. N.; Wooters, J. L.; Dougherty, J. P.; Meyers, R. E.;
Collins, M.; Brown, E. L. Nucleic Acids Res. 1987, 15, 2891-2909.
14) Liu, Q.; Frutos, A. G.; Thiel, A. J.; Corn, R. M.; Smith, L. M. J.
Comput. Biol. 1998, 5, 269-278.
15) Lund, V.; Schmid, R.; Rickwood, D.; Hornes, E. Nucleic Acids Res.
988, 16, 10860-10878.
16) Pan, S.; Castner, D. G.; Ratner, B. D. Langmuir 1998, 14, 3545-
550.
(
1
(
(
1
1
1
3
12
(
attached DNA to substrates such as latex beads, polystyrene,
24,26
19,22,23,27
3,20-22,28
optical fibers,
carbon electrodes,
gold,
and
(
2,11,25,29
oxidized silicon.
Although largely unexplored to date,
(
(21) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chem. 1994, 66, 3830-
3833.
(22) Pang, D.; Zhang, M.; Wang, Z.; Qi, Y.; Cheng, J.; Liu, Z. J.
Electroanal. Chem. 1996, 403, 183-188.
(23) Wang, J.; Fernandes, J. R.; Kubota, L. T. Anal. Chem. 1998, 70,
3699-3702.
(24) Piunno, P. A. E.; Krull, U. J.; Hudson, R. H. E.; Damha, M. J.;
Cohen, H. Anal. Chim. Acta 1994, 288, 205-214.
(25) Souteyrand, E.; Cloarec, J. P.; Martin, J. R.; Wilson, C.; Lawrence,
I.; Mikkelsen, S.; Lawrence, M. F. J. Phys. Chem. B 1997, 101, 2980-
2985.
(26) Henke, L.; Piunno, P. A. E.; McClure, A. C.; Krull, U. J. Anal.
Chim. Acta 1997, 344, 201-213.
(27) Millan, K. M.; Mikkelsen, S. R. Anal. Chem. 1993, 65, 2317-2323.
(
1
3
6
(
(17) Huang, J.; Dahlgren, D. A.; Hemminger, J. C. Langmuir 1994, 10,
26-628.
(18) Bain, C. D.; Troughton, B. E.; Tao, Y.; Evall, J.; Whitesides, G.
M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335.
19) Millan, K. M.; Spurmanis, A. J.; Mikkelsen, S. R. Electroanalysis
992, 4, 929-932.
20) Zhao, Y.; Pang, D.; Wang, Z.; Cheng, J.; Qi, Y. J. Electroanal.
Chem. 1997, 431, 203-209.
(
1
(
1
0.1021/ja9936161 CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/28/2000

1206 J. Am. Chem. Soc., Vol. 122, No. 6, 2000
Strother et al.
crystalline silicon is a particularly attractive alternative substrate
for DNA immobilization because of its purity and well-defined
structure, and because crystalline silicon substrates provide the
opportunity to take full advantage of existing technologies in
microelectronics processing.
Chemical functionalization of silicon is complicated by the
fact that silicon readily oxidizes in air to produce an oxide that
is chemically similar to glass. Unfortunately, the use of glass
or oxidized silicon substrates for highly parallel DNA attachment
presents some problems that are a direct consequence of the
fact that glass and oxidized silicon surfaces are amorphous, and
that the relative number of Si-O-Si and Si-OH linkages
exposed at the surface is highly dependent on the past history
of the sample. This irreproducibility in surface chemistry leads
to difficulties in the control, reproducibility, homogeneity, and
stability of subsequent DNA attachment. Planar glass and
oxidized silicon substrates can be modified using methoxysilane
or chlorosilane reagents2
,11,30-33
to attach organic functional
groups such as amines or thiols to serve as DNA attachment
sites. Silane reagents containing only a single surface reactive
functionality (e.g. monochloro- or monomethoxysilanes) produce
poorly stable films.34 The stability can be improved by using
multiple reactive groups (e.g. trichloro- or trimethoxysilanes)
to form more Si-O-Si linkages, but such polyfunctional
reagents also lead to poorly controlled surface polymerization
reactions, compromising surface homogeneity and reproduc-
3
5
ibility.
A solution to this problem would be to develop strategies
for direct attachment of DNA to silicon surfaces without an
intervening oxide layer. In recent years, new attachment methods
for the organic functionalization of silicon surfaces through
formation of direct silicon-carbon bonds have been
1
,5,9,36-38
reported.
In this report we utilize these reactions as a
Figure 1. Drawings illustrating the chemistry employed for preparation
of the modified Si(111) surface. A layer of 10-undecylenic acid (UDA)
is bound to the surface by attachment and subsequent hydrolysis of
the ester. A layer of electrostatically bound polylysine (PL) and a layer
of sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate
(SSMCC) follow the UDA. Thiol-modified DNA is subsequently bound
to the SSMCC.
route for the attachment and hybridization of DNA to crystalline
silicon substrates. The resultant DNA-modified silicon surfaces
are reproducibly prepared, stable to the conditions of DNA
hybridization, and show no detectable nonspecific binding. The
high fidelity of these surfaces promises substantial benefit to
the emerging technology of large-scale biological analysis using
nucleic acid arrays.
surface.5,37 Similar reactions of ω-alkenes have previously been
1
Results and Discussion
described, mediated either by diacyl peroxides or by direct
9
thermal activation. Hydrolysis of the ester by treatment with
Overview of Surface Attachment Chemistry. The chemistry
employed for DNA attachment to Si(111) surfaces is dia-
grammed in Figure 1. A hydrogen-terminated Si(111) surface
is reacted with an ω-undecylenic acid methyl or trifluoroethyl
ester by UV irradiation of a thin film of the ester applied to the
39,40
potassium tert-butoxide in DMSO
yields a carboxylic acid-
modified surface. Subsequent addition of poly-L-lysine (PL) and
reaction of the lysine ꢀ-amino groups with the heterobifunctional
cross-linker SSMCC results in a maleimide-activated surface
that may then be coupled in aqueous solution with a thiol-
modified oligodeoxynucleotide to yield the DNA-modified
(
28) Frutos, A. G.; Smith, L. M.; Corn, R. M. J. Am. Chem. Soc. 1998,
20, 10277-10282.
29) Lamture, J. B.; Beattie, K. L.; Burke, B. E.; Eggers, M. D.; Ehrlich,
3
1
surface.
(
Surface Characterization by XPS. X-ray photoelectron
spectroscopy (XPS) was used to follow the surface coupling
reaction and ester hydrolysis, as well as providing a means of
monitoring the degree of oxidation occurring on the silicon
surface. In initial work the methyl ester of ω-undecylenic acid
was employed; however, it was found that the XPS signal from
the methyl group was too poorly resolved from the signal
corresponding to the undecylenic acid alkyl chain to permit
hydrolysis of the ester to be monitored. To address this, the
trifluoroethyl ester of ω-undecylenic acid was synthesized and
reacted with the surface; the fluorine 1s signal from the
trifluoroethyl group is strong and well resolved from other XPS
D. J.; Fowler, R.; Hollis, M. A.; Kosicki, B. B.; Reich, R. K.; Smith, S. R.;
Varma, R. S.; Hogan, M. E. Nucleic Acids Res. 1994, 22, 2121-2125.
(30) Guo, Z.; Guilfoyle, R. A.; Thiel, A. J.; Wang, R.; Smith, L. M.
Nucleic Acids Res. 1994, 22, 5456-5465.
31) O’Donnell, M. J.; Tang, K.; Doster, H.; Smith, D. L.; Cantor, C. R.
Anal. Chem. 1997, 69, 2438-2443.
32) Southern, E. M.; Case-Green, S. C.; Johnson, M.; Mir, K. U.; Wang,
L.; Williams, J. C. Nucleic Acids Res. 1994, 22, 1368-1373.
33) Rogers, Y.; Jiang-Baucom, P.; Huang, Z.; Bogdanov, V.; Anderson,
S.; Boyce-Jacino, M. T. Anal. Biochem. 1999, 266, 23-30.
34) Waddell, T. G.; Leyden, D. E.; DeBello, M. T. J. Am. Chem. Soc.
(
(
(
(
1
981, 103, 5303-5307.
(
35) Ulman, A. Chem. ReV. 1996, 96, 1533-1554.
(36) Terry, J.; Linford, M. R.; Wigren, C.; Cao, R.; Pianetta, P.; Chidsey,
C. E. D. J. Appl. Phys. 1999, 85, 213-221.
(37) Zhao, X.; Hamers, R. In preparation, 1999.
(
38) Hamers, R. J.; Hovis, J. S.; Seung, L.; Liu, H.; Shan, J. J. Phys.
(39) Chang, F. C.; Wood, N. F. Tetrahedron Lett. 1964, 40, 2969-2973.
(40) Gassman, P. G.; Schenk, W. N. J. Org. Chem. 1976, 42, 918-920.
Chem. B 1997, 101, 1489-1492.

DNA-Modified Silicon (111) Surfaces
J. Am. Chem. Soc., Vol. 122, No. 6, 2000 1207
Figure 2. X-ray photoelectron spectra of the trifluoroethyl ester (a). The fluorine spectrum shows a strong peak at 688.5 eV. The carbon spectra
shows peaks at 287.5 and 293.0 eV assigned to the carbons found in the trifluoroethyl ester moiety. The carbonyl carbon and the bulk carbons have
been assigned the peaks at 289.3 and 284.4 eV, respectively. Additionally, the silicon spectrum shows no oxidation of the surfaces as evidenced
by no signal in the 101-103 eV region. After treatment in 250 mM potassium tert-butoxide in DMSO for 30 s (b) the fluorine peak disappears,
as do the peaks at 287.5 and 293.0 eV in the carbon spectrum. After treatment the silicon spectrum shows little oxidation.
signals, providing a clear means of following the hydrolysis
reaction. Typical XPS results for this system are shown in Figure
2
1
. The upper (panel a) three spectra show the fluorine 1s, carbon
s, and silicon 2p signals from the Si(111) surface modified by
attachment of the ω-undecylenic acid ester. The lower three
spectra (panel b) show the same surface after hydrolysis of the
ester by treatment with potassium tert-butoxide in DMSO. A
comparison of the upper and lower panels reveals several
important points. First, the fluorine 1s signal is completely
removed by the hydrolysis procedure, proving that the ester was
efficiently cleaved from the surface. Second, four carbon 1s
signals are evident in the upper panel, corresponding to the alkyl
chain carbons, the carbonyl carbon, and the two trifluorethyl
group carbons. The two signals associated with the trifluoroethyl
ester are absent in the lower panel, again confirming the
complete hydrolysis of the ester; in contrast, the XPS signals
corresponding to the alkyl chain and carboxylic acid group
remain, demonstrating that the linkage between the alkyl chain
and the surface was not disrupted by the hydrolysis reaction.
Finally, comparison of panels a and b for the silicon 2p signal
shows very little signal corresponding to oxidized silicon atoms
on the surface, indicating that the overall integrity of the
Si(111) surface is not adversely affected by the hydrolysis
process.
Figure 3. Images of DNA-modified Si(111) hybridized with fluores-
cent complements. Two spots, approximately 2 mm across, of different
oligonucleotides are attached to the surface. Hybridization with
fluorescent complement of the lower spot shows a clear image (a).
Denaturation and hybridization to the upper spot with its fluorescent
complement also shows a clear image (b). Denaturation and hybridiza-
tion with both complements allow visualization of both spots (c).
subsequent hybridization using the alternate fluorescent oligo-
nucleotide revealed only the second individual spot (Figure 3b).
Denaturation and hybridization with both fluorescently tagged
oligonucleotides showed two distinct spots, as expected (Figure
3c). These results indicate that the thiol oligonucleotides are
indeed attached to the surface and are accessible to specific
hybridization with their respective complements. In control
experiments, a series of samples were prepared to determine if
the thiol oligonucleotides could attach to the silicon surface in
unexpected ways. Thiol oligonucleotides were spotted on the
silicon surfaces in various stages of preparation. Only those
samples with the hydrolyzed ester, polylysine, and SSMCC gave
significant signal when hybridized with the fluorescent comple-
ments. Those samples with no ester, just hydrolyzed ester, or
hydrolyzed ester with polylysine gave no signal. Similar controls
with unhydrolyzed ester were also performed. Unexpectedly,
significant fluorescent signal was seen with the unhydrolyzed
ester that had been treated with polylysine and SSMCC. This
DNA Hybridization Binding Specificity. The performance
of these DNA-modified Si(111) surfaces in DNA hybridization
was evaluated with respect to binding specificity, surface
density, and chemical stability to the conditions of DNA
hybridization. To evaluate binding specificity, two different thiol
oligonucleotides were attached to a piece of a silicon wafer
(
approximately 2 cm × 2 cm) in approximately 2 mm diameter
spots. A solution containing a fluorescent oligonucleotide
complementary to one of the attached oligonucleotides was
placed on the surface and hybridization was allowed to occur.
After washing the fluorescence image was acquired, and
revealed only a single spot (Figure 3a). Denaturation and

1208 J. Am. Chem. Soc., Vol. 122, No. 6, 2000
Strother et al.
a Millipore system and used to rinse the surfaces when scrupulously
clean conditions were required. Distilled water was used in other cases.
Other reagents were obtained as follows: 11-Mercaptoundecanoic
acid (MUA) (Aldrich), 10-undecylenic acid (UDA) (Fisher), 2,2,2-
trifluoroethanol (Aldrich), methanol (Fisher), potassium tert-butoxide
(Fisher), poly(L-lysine) hydrobromide (PL) (Aldrich), sulfo-succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SSMCC) (Pierce),
silicon (111) wafers (Virginia Semiconductor). Gold surfaces were
obtained from Evaporated Metal Films (Ithica, NY) with 50 Å of
chromium covered by 1000 Å of gold. Oligonucleotides were synthe-
sized by the University of Wisconsin Biotechnology Center. The
oligonucleotides were modified with either Glen Research 5′-Thiol-
Modifier C6 or 5′-Fluorescein (6-FAM) modifier to produce thiol-
modified oligonucleotides or the fluorescein-modified complements.
The thiol-modified oligonucleotides were deprotected according to
guidelines provided by Glen Research Corp42 then purified using
reverse-phase HPLC with a binary gradient elution. Fluorescent
oligonucleotides were also purified using HPLC. The thiol oligonucle-
otides were used at approximately 1 mM concentration and their
fluorescent complements were used at approximately 2 µM. The
hybridization and rinsing buffer was 300 mM NaCl, 20 mM sodium
phosphate, 2 mM EDTA, and 7 mM sodium dodecyl sulfate, pH 7.4,
referred to as “2×SSPE/0.2% SDS.”
Figure 4. Long-term stability showing behavior of the silicon surface
during 30 hybridization and denaturation cycles. Fluorescent signal from
the hybridized DNA decreases slightly with each hybridization until
the 23rd hybridization. A slight increase in fluorescent signal occurs
with later hybridizations. The wafers were imaged after each denatur-
ation to demonstrate the complete removal of the fluorescent comple-
ment. The fluorescent signal of the denatured DNA was negligible.
is consistent with binding of polylysine to the ester by polar
interactions and subsequent binding of the SSMCC to the
polylysine.
Synthesis of Esters. (a) Methyl Ester. The methyl ester of 10-
9
undecylenic acid was prepared similarly to the method used by Sieval.
Number Density. The density of fluorescent oligonucleotides
that hybridized to the surface was determined according to a
previously described method.28 In this procedure the surface
area of the spots is determined by fluorescence imaging, and
the amount of hybridized oligonucleotide is measured by
quantitative elution from the surface followed by gel electro-
phoretic analysis (see the Experimental Section). The surface
density determined in this manner was found to be ap-
The 10-undecylenic acid was dissolved in methanol and allowed to
reflux for 3 h with a small amount of sulfuric acid. Excess methanol
was removed by vacuum. The crude product was dissolved in ether,
washed with a saturated sodium bicarbonate solution, water, then a
saturated NaCl solution, and dried over magnesium sulfate. The identity
of the vacuum distilled product was confirmed using proton NMR.
1
H NMR: δ 5.73-5.89 (m, 1H), 4.89-5.04 (m, 2H), 3.67 (s, 3H),
2
1
.25-2.35 (t, 2H), 1.99-2.09 (m, 2H), 1.54-1.69 (m, 2H), 1.23-
.44 (m, 10H).
12
2
proximately 5.3 × 10 oligonucleotides per cm , comparable
(b) 2,2,2-Trifluoroethyl Ester. The fluorinated ethyl ester of 10-
to densities reported on other substrates.2
,29,31,41
Control experi-
undecylenic acid was prepared similarly to the methyl ester. To 30 g
of 10-undecylenic acid was added 22 g of 2,2,2-trifluoroethanol
dissolved in 40 mL of toluene. This mixture was allowed to reflux
with a small amount of sulfuric acid for 3 h using a Dean-Stark
apparatus. Workup similar to the methyl ester produced a crude product
that was further purified by vacuum distillation. Proton, COSY, and
fluorine NMR confirmed the identity of the product.
2
8
ments performed on similarly modified gold surfaces yielded
comparable results.
Chemical Stability Under the Conditions of DNA Hybrid-
ization. The stability of these DNA-modified Si(111) surfaces
to the conditions of DNA hybridization was determined by
performing a series of 30 successive cycles of oligonucleotide
hybridization, washing, fluorescence imaging, and denaturation
to regenerate the original surface (see the Experimental Section
for details). The fluorescence intensities obtained after hybrid-
ization in each cycle are plotted in Figure 4. The total
fluorescence intensity of the hybridized DNA decreases after
1
H NMR: δ 5.70-5.90 (m, 1H), 4.86-5.06 (m, 2H), 4.38-4.51
(
1
q, 2H), 2.35-2.45 (t, 2H), 1.98-2.10 (m, 2H), 1.55-1.75 (m, 2H),
.20-1.50 (m, 10H). Fluorine NMR: single triplet.
Preparation of Silicon Surfaces. The silicon (111) wafers were
treated similar to the method described by Sieval with some modifica-
9,37
tion. The wafers were sonicated in acetone for 5 min then methanol
3
0 cycles to approximately 60% of the initial value, corre-
for 5 min. They were then soaked in a hydrogen peroxide-ammonia-
water bath (1:1:4) for 5 min at 75 °C. After a brief dip in 2%
hydrofluoric acid to hydrogen terminate the silicon atoms, the wafers
were placed in a chamber. The chamber had a quartz window to allow
passage of ultraviolet light and was designed to have nitrogen
continually passed through at atmospheric pressure. Thin layers of the
esters were placed on the hydrofluoric acid etched wafers and they
were sealed in the chamber. The wafers were then subjected to
ultraviolet light from a low-pressure mercury vapor lamp for 2 h. The
wafers were subsequently sonicated in chloroform for 10 min then in
methanol for a further 10 min. The modified ester surface was then
converted to the carboxylic acid. Sieval and others have used acid baths
to hydrolyze the ester though there is evidence that such treatment does
sponding to a loss of approximately 2% per cycle. Control
3
experiments performed on similarly modified gold surfaces
showed comparable stability; however, the fluorescence back-
ground on the gold surfaces was roughly 75% higher than that
on the Si surfaces (possibly due to the greater reflectivity of
the gold surface), causing a higher fluorescence signal-to-
background ratio for the Si surfaces. The fluorescence intensity
of the DNA was measured after the denaturation step of each
of the 30 cycles and was found to be negligible compared to
background (Figure 4). This background fluorescence is also
plotted in Figure 4. It may be noted that the fluorescence
intensity actually appears to increase during the later hybridiza-
tion cycles. The cause of this unexpected increase is currently
under investigation.
9
,43
not adequately convert the ester to the carboxylic acid. We found
the neat ω-undecylenic esters did not completely hydrolyze even in
boiling concentrated HCl for several hours when measured with thin-
layer chromatography (TLC). An alternate method of hydrolysis using
potassium tert-butoxide in DMSO was explored;3 it was found that
dipping the surfaces in a 250 mM solution of potassium tert-butoxide
9,40
Experimental Section
Materials. All chemicals were reagent grade or higher and used as
received unless otherwise specified. Ultrapure water was obtained from
(42) User Guide to DNA Modification; Glen Research Corporation:
Sterling, VA, 1996.
(43) Wasserman, S. R.; Tao, Y.; Whitesides, G. M. Langmuir 1989, 5,
1074-1087.
(41) Jordan, C. E.; Frutos, A. G.; Thiel, A. J.; Corn, R. M. Anal. Chem.
1
997, 4939-4947.

DNA-Modified Silicon (111) Surfaces
J. Am. Chem. Soc., Vol. 122, No. 6, 2000 1209
in DMSO for 3 min at room temperatures followed by rinsing in
acidified water (100mM HCl) successfully hydrolyzed the esters. TLC
analysis of hydrolysis products confirmed the effectiveness of this
procedure. In addition, XPS measurements and contact angle measure-
ments of the surfaces indicated successful surface hydrolysis. Contact
angle measurements: methyl ester, 70.8 ( 1.6°, after hydrolysis, 52.2
Determination of Number Density. The method used to determine
the number of oligonucleotides that have hybridized to a gold surface
was previously reported by Frutos.28 A similar method was employed
here. Thiol oligonucleotides are immobilized on the silicon wafer
surface in a 5 mm diameter spot; complementary fluorescent oligo-
nucleotides are hybridized, the wafer is washed thoroughly, and the
fluorescent images are acquired, providing values for the exact surface
area of the spots. The silicon wafer is then heated at 90 °C for 15 min
in a microfuge tube with 800 µL of water. The wafer is removed and
rinsed twice with 200 µL water, which was added to the original 800
µL. After the fluorescent complements are quantitatively eluted into
the water, the surface is rescanned on the FluorImager to check for
complete denaturation (removal of fluorescence signal). The volume
of water containing the fluorescent complements is reduced by vacuum
centrifugation to about 10 µL and loaded on one lane of an acrylamide
gel, while known amounts of fluorescent oligonucleotide standards are
loaded in the other gel lanes. A 20% polyacrylamide gel containing
urea is prepared using standard techniques.44 Electrophoresis is
performed and the gel is imaged; analysis of the image permits
quantification of the unknown by reference to a standard curve prepared
from the known samples.
(
2.6°; fluorinated ethyl ester, 84.5 ( 1.2°, after hydrolysis, 58.2 (
1
.5°.
After being rinsed in water, the acid terminated surface was soaked
for 40 min in a 0.15 mg/mL PL solution in a 5mM borate buffer at pH
.5. The surfaces were then rinsed in ethanol and water. The final layer
8
was added by soaking the surfaces for 15 min in 1.5 mM SSMCC in
a 100 mM triethanolamine hydrochloride buffer at pH 7. A final rinse
with water was performed and the thiol-modified oligonucleotides were
spotted on the surface and left overnight in a humid chamber. The thiol
DNA used was approximately 0.5 µL of a 1 mM solution. It may be
noted that alternate methods of ester attachment have yielded similar
results. These include heating the ester with the cleaned silicon surface
at 200 °C for 2 h, use of a propyl ester, and use of silicon (001).9
Hybridization and Denaturation Conditions. The detailed proce-
dures employed for hybridization of the fluorescein-labeled comple-
ments to the surface-immobilized DNA and their subsequent denatur-
2
8
ation has been described elsewhere. Imaging of the surface-bound
oligonucleotides on silicon surfaces was done by scanning with a
Molecular Dynamics FluorImager 575 after hybridizing the immobilized
oligonucleotides with their fluorescent complements. Briefly, 5-10 µL
of 2 µM 5′-fluorescein-labeled complement in 2×SSPE/0.2% SDS
buffer was placed on a microscope cover slip in a humid chamber.
The DNA-modified silicon surfaces were placed in this droplet of
fluorescent complement for 20 min at room temperature to allow for
hybridization. The surfaces were then removed and soaked twice for 5
min in 2×SSPE/0.2% SDS buffer to remove any unhybridized
complement. The silicon wafers were then placed face down in a droplet
of 2×SSPE/0.2% SDS buffer on the FluorImager tray and scanned.
Denaturation was accomplished by placing the samples in an 8.3 M
urea solution for 15 min at 37 °C followed by rinsing with water.
Subsequent hybridizations could then be performed using the same
procedure.
Acknowledgment. The authors would like to thank Dr.
Robert Corn and Dr. Anthony Frutos for their assistance with
the surface chemistry. We also gratefully acknowledge Thomas
Ferris for his assistance with the NMR spectra. This work was
supported by NSF grant CCR-9613799 and NIH grant
1
R21-HG02101-01.
JA9936161
(44) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning A
laboratory Manual; Cold Spring Harbor Laboratory Press: Plainview, New
York, 1989; Vol. 2.