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Langmuir 2005, 21, 4257-4261
4257
Nanoscale-Controlled Spacing Provides DNA
Microarrays with the SNP Discrimination Efficiency in
Solution Phase
Bong Jin Hong, Soon Jin Oh, Tae One Youn, Sung Hong Kwon, and
Joon Won Park*
Center for Integrated Molecular Systems, Department of Chemistry, Division of Molecular and
Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea
Received December 13, 2004. In Final Form: March 21, 2005
We have prepared solid substrates modified with a cone-shaped dendron that generates mesospacing
(3.2 nm on average) on the surface. This nanoscale-controlled surface provided an ideal DNA microarray
in which each probe DNA strand was given ample space for the incoming target DNA, resulting in selectivity
as high as that in solution (100:<1). In addition, high hybridization yield confirms that DNA probes on
the mesospaced surface are sterically unhindered for the hybridization.
Since the first report,1 DNA microarrays have attracted
a great deal of attention because they allow highly parallel
analysis of the DNA sequence, genetic variations, and
gene expression. It is known that this methodology
requires improvements in terms of fidelity and reproducibility that are essential for the standardization and
application to human gene diagnosis.2 To improve the
performance of DNA microarrays, several issues including
probe design, reaction conditions during spotting, hybridization and washing conditions, suppression of nonspecific binding, distance between the biomolecules and
the surface, and the space between the immobilized
biomolecules should be considered. Because most of these
factors are associated with the nature of the microarray
surface, and many problems are caused mainly by the
variations in the nature of the surface and molecular
interlayer structures that are far from ideal, surface
optimization has become one of the major goals in
microarray research. Several organic surfaces such as selfassembled monolayer (SAM),3 agarose film,4 polyacrylamide gel,5 and polyamidoamine (PAMAM) dendrimer6
have been tested for better performance of a DNA microarray. However, no organic surfaces have yet provided a
DNA microarray in which the characteristics of probe
oligonucleotides are the same as those of oligonucleotides
in solution. Even though the gel-type surface of agarose
and polyacrylamide films showed enhanced performance
including high immobilization capacity and sensitivity,
the observed selectivity was far inferior to the one in
solution.4,5 Poorly controlled distance between probe
DNAs immobilized on the gel surface seems to be
problematic. Therefore, it will be interesting to examine
whether ample spacing between each probe DNA gives
“freedom in solution phase”. For this investigation, a cone* Corresponding author. E-mail: [email protected].
(1) (a) Fodor, S. P. A.; et al. Nature 1993, 364, 555-556. (b) Saiki,
R. K.; Walsh, P. S.; Levenson, C. H.; Erlich, H. A. Proc. Natl. Acad. Sci.
U.S.A. 1989, 86, 6230-6234.
(2) Hackett, J. L.; Lesko, L. J. Nat. Biotechnol. 2003, 21, 742-743.
(3) (a) Oh, S. J.; Cho, S. J.; Kim, C. O.; Park, J. W. Langmuir 2002,
18, 1764-1769. (b) Guo, A.; Guilfoyle, R. A.; Thiel, A. J.; Wang, R.;
Smith, L. M. Nucleic Acids Res. 1994, 22, 2121-2125. (c) Kim, I.; et al.
Clin. Cancer Res. 2002, 8, 457-463. (d) Hou, P.; Ji, M.; He, N.; Lu, Z.
Anal. Biochem. 2003, 317, 276-279.
(4) (a) Afanassiev, V.; Hanemann, V.; Wölfl, S. Nucleic Acids Res.
2000, 28, e66. (b) Wang H.; et al. Nucleic Acids Res. 2002, 30, e61.
(5) Guschin, D.; et al. Anal. Biochem. 1997, 250, 2203-211.
(6) Benters, R.; Niemeyer, C. M.; Drutschmann, D.; Blohm, D.;
Wöhrle, D. Nucleic Acids Res. 2002, 30, e10.
shaped dendron molecule is a good candidate to begin
with. Because a dendron has a uniform size as well as a
well-defined three-dimensional structure, it has been
widely utilized.7 For example, when the monodisperse
molecule is assembled on a surface, a rather regular
spacing is generated between them. Our recent study
showed that a conical-shaped dendron provided primary
amino groups on a surface that are separated from each
other 3 nm on average.8 While a common approach, a
mixed monolayer approach, reduces the density of a
specific functional group on a surface, the regular spacing
realized by the dendron approach is not accessible due to
statistical distribution and the tendency to aggregate.9
We now report that a cone-shaped dendron was successfully introduced on various oxide substrates to obtain
relatively uniform mesospacing between apexes of the
dendron molecules, and the dendron-modified substrate
provided DNA microarrays with single nucleotide polymorphism (SNP) discrimination efficiency equivalent to
the solution value while concurrently suppressing
DNA nonspecific binding effectively. For comparison, an
APDES-modified surface10 which has a high density of
the amine functionality was used for DNA microarray
fabrication (Scheme 1).
A dendron was introduced to the substrates including
oxidized silicon wafer, fused silica, and glass slide through
covalent bond formation (Scheme 2). The dendron molecule
was designed for the efficient immobilization, facile
deprotection, intact reactivity of the amine at the apex,
and low nonspecific binding of oligonucleotide. Considering
diameter of duplex DNA, we chose the second generation
with triple branching. For facile conjugation with bioactive
molecules, a spacer was inserted to extend the amino
(7) (a) Newkome, G. R.; Moorefield, C. N.; Vögtle, F. Dendritic
Molecules: Concepts Synthesis, Perspectives; VCH: Weinheim, Germany, 1996. (b) Rahman, K. M. A.; Durning, C. J.; Turro, N. J.; Tomalia,
D. A. Langmuir 2000, 16, 10154-10160. (c) Zeng, F.; Zimmerman, S.
C. Chem. Rev. 1997, 97, 1681-1712. (d) Bosman, A. W.; Janssen, H. M.;
Meijer, E. W. Chem. Rev. 1999, 99, 1665-1688.
(8) Hong, B. J.; Shim, J. Y.; Oh, S. J.; Park, J. W. Langmuir 2003,
19, 2357-2365.
(9) (a) Roberts, C.; Chen, C. S.; Mrksich, M.; Martichonok, V.; Ingber,
D. E.; Whitesides, G. M. J. Am. Chem. Soc. 1998, 120, 6548-6555. (b)
Heise, A.; Stamm, M.; Rauscher, H. Duschner, H.; Menzel, H. Thin
Solid Films 1998, 327, 199-203. (c) Heise, A.; Menzel, M.; Yim, A.;
Foster, M. D.; Wieringa, R. H.; Schouten, A. J.; Erb, V.; Stamm, M.
Langmuir 1997, 13, 723-728. (d) Peterson, A. W.; Wolf, L. K.; Georgiadis,
R. M. J. Am. Chem. Soc. 2002, 124, 14601-14607.
(10) APDES: (3-aminopropyl)diethoxymethylsilane.
10.1021/la046951y CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/08/2005
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Langmuir, Vol. 21, No. 10, 2005
Letters
Scheme 1. Schematic Diagram Showing DNA Hybridization (a) on the Dendron-Modified Substrate and (b) on
the APDES-Modified Substrate
Scheme 2. Preparing the Dendon-Modified Substrate and the Structure of the Dendron
group. In our previous work, the dendron without the
spacer suffered from low coupling efficiency due to the
severe steric hindrance. In addition, covalent bonding
between the dendron’s carboxylic acid group and the
substrate’s hydroxyl group was employed to enhance
thermal and chemical stability of the final layer. Previously, an amine-modified surface was used to immobilize
the dendron,8,11 and it turned out that surface amino
groups gave rise to significant physisorption of DNAs. In
this scheme, the ethylene glycol sublayer was employed
to suppress nonspecific adsorption of DNA.
Self-assembly driven by the ester bond formation
increases the thickness of the molecular layer by 11 ( 2
Å. An absorption peak arising from the anthracene moiety
of the dendron was observed at 257 nm (Figure 1a), and
the layer was stable enough to show constancy in terms
of thickness and absorption characteristics upon stirring
in dimethylformamide (DMF) for 1 day (Figure 1b). After
the deprotection in 1.0 M trifluoroacetic acid (TFA), the
peak disappeared without any other detrimental change
in the UV spectrum (Figure 1a). This observation showed
that the protecting group was removed successfully
without chemically damaging the layer, while the thickness was slightly decreased due to the elimination of the
protecting group.
Mesospacing between the dendrons on the surface was
directly observed by using a high resolution scanning
electron microscope (HRSEM). To enhance the image, gold
nanoparticles (average diameter 1.4 nm) were introduced
on each apex of the immobilized dendrons. In this HRSEM
image, gold nanoparticles were identified clearly and did
not show any sign of aggregation. Counting the distance
of each particle to the nearest neighboring particle showed
(11) Choi, Y.; Yoon, C. W.; Lee, H. D., Park, M.; Park, J. W. Chem.
Commun. 2004, 1316-1317.
Letters
Langmuir, Vol. 21, No. 10, 2005 4259
Figure 2. (a) Schematic diagram for introducing gold nanoparticles into the dendron-modified substrate. (b) HRSEM
image after tethering gold nanoparticles at the apex of the
dendron on the surface. (c) Distribution graph of distance
between the nearest gold nanoparticles. The HRSEM image
size is 50 nm × 50 nm.
Table 1. Probe and Target Oligonucleotide Sequences
and Nomenclature
Figure 1. UV spectroscopic analysis (a) after each surface
modification step and (b) for stability test. “EG/GPDES” and
“Dendron” were recorded before and after the introduction of
the dendron on the ethylene glycol-modified substrate, respectively. “Deblocking” corresponds to the spectrum after the
deprotection step. “Washing” was recorded after stirring in DMF
at room temperature for 1 day.
that the average separation was 3.2 nm (Figure 2). Also,
in all of the cases, the separation was larger than 2 nm.
Topographical images obtained by tapping mode atomic
force microscopy (AFM) also showed that the resulting
layer was smooth and homogeneous without any aggregates or holes in macroscopic scale (for the detailed
experimental procedure and data, please see the Supporting Information).
Amine-tethered probe oligonucleotides (probes 1 and
2) were immobilized to evaluate the discrimination
efficiency between a complementary pair (A:T) and three
internal single-base mismatched pairs (T:T, G:T, and C:T).
Spotting was performed onto the activated surface of a
glass slide using a Microsys 5100 microarrayer (Cartesian
Technologies, Inc.) in a class 10 000 clean room. For
hybridization, a 15-base oligonucleotide (target 1) or a
45-base oligonucleotide (target 2) was used. The probe
and target oligonucleotides utilized in this work are listed
in Table 1.
Fluorescence images show that there is a dramatic
difference in the intensity between the matched and the
internal mismatched pairs (Figure 3a). The single point
mutation selectivity (100:<1) for the internal mismatches
designation
1a
probe
probe 2a
probe 3b
probe 4b
target 1
target 2
target 3
target 4
sequence
5′-NH2-C6-CATTCCGXGTGTCCA-3′
5′-NH2-C6-(T)30-CATTCCGXGTGTCCA-3′
5′-NH2-C6-GTGAGGCXCTGCCCC-3′
5′-NH2-C6-CGACATAXTGTGGTG-3′
5′-Cy3-TGGACACTCGGAATG-3′
5′-Cy3-CCTACGAAATCTACTGGAACG
AAATCTACTTGGACACTCGGAATG-3′
5′-Cy3-GGGGCAGCGCCTCAC-3′
5′-Cy3-CACCACACTATGTCG-3′
a X in the sequence of probe oligonucleotides is A (complementary), T, G, and C (mismatched). b X in the sequence of probe
oligonucleotides is G (complementary), A, T, and C (mismatched).
is unprecedented, and an increase of the selectivity
(43-82 times) is recorded in comparison with DNA
microarrays fabricated on an APDES-modified surface
(Table 2). The outstanding selectivity also holds even for
the longer 45-base target oligonucleotide (Figure 3b and
Table 2). It is believed that the efficacy of this DNA
microarray should be attributed to the peculiarity of the
dendron-modified surface, mesospacing between immobilized DNA strands. Previously, we also observed a
selectivity factor of 100:19-57 for microarrays fabricated
on various amine surfaces including a mixed self-assembled monolayer (i.e., mixed SAM).3a In addition, other
investigators improved the performance of DNA microarrays by modifying their surface or inventing a better
detection process, but none have reached this recordbreaking ratio as far as the fluorescence detection method
is concerned.4b,5,6,12 For example, a successful discrimina-
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Letters
Figure 3. Fluorescence images after the hybridization between
probe and target oligonucleotides. Images a and b were obtained
from the dendron-modified microarray, and images c and d,
from the APDES-modified microarray. Fluorescence images
were obtained after hybridization between (a) probe 1 and target
1, (b) probe 1 and target 2, (c) probe 1 and target 1, and (d)
probe 2 and target 1. PM signifies perfect match and IMM
one-base internal mismatch. The characters in parentheses
imply matched or mismatched base pairs of DNA duplexes.
Laser power and photomultiplier tube (PMT) gain of a laser
scanner were adjusted to record reasonable IMM signal while
avoiding saturation of PM signal.
Table 2. Single Nucleotide Polymorphism
Discrimination Ratio
normalized fluorescence
signal ratio
15-mera
dendron-modified surface,
dendron-modified surface, 45-merb
APDES-modified surface, C6 spacera
APDES-modified surface, (T)30 spacerc
matched
mismatched
A:T
T:T
G:T
C:T
100 0.5
100 0.6
100 41
100 17
0.8
0.9
38
18
0.6
0.9
26
12
a Hybridization between probe 1 and target 1. b Hybridization
between probe 1 and target 2. c Hybridization between probe 2 and
target 1.
tion ratio of 100:7 was reported for a three-component
hybridization/detection system (capture/target/probe).12a
Even when peptide nucleic acids (PNAs) capable of
increasing the selectivity were used, the selectivity on the
gold thin film and the gold nanoparticle was 100:14 and
100:7, respectively.12b In particular, a previous study in
solution phase with a molecular beacon showed that the
SNP discrimination ratio was as low as 100:∼1.4b The
result strongly supports that our dendron-modified surface
provides a solution-like environment for DNA duplex
formation.
For a comparison, a DNA mircroarray was fabricated
on the substrate modified with APDES,3a which was one
of the typical substrates for DNA or protein microarrays. Its selectivity was tested using the same procedure
and oligonucleotides as those for the dendron-modified
DNA microarray, except a 1,4-phenylenediisothiocyanate (PDITC) linker was used. Use of the di(N-succinimidyl)carbonate (DSC) linker on the APDES-modified
substrate resulted in very low and nonuniform florescence
Figure 4. Fluorescence images after the hybridization between
probe and target oligonucleotides including hot mutations of
the p53 gene. The images were obtained after hybridization
between (a and b) probe 3 and target 3 and (c) probe 4 and
target 4. Images a and b were acquired from a microarray
fabricated on a single slide. In image b, high laser power and
PMT gain were employed to visualize signal intensity at
mismatched sites, and spots in blue color are barely identifiable
for the A:C mismatched case.
intensity within each spot. On the other hand, the PDITC
linker assured strong and homogeneous fluorescence
intensity within a single spot like those of the dendronmodified substrate with the DSC linker (see the Supporting Information). Also, the linker-modified surface
gave rise to insignificant nonspecific binding of DNA as
low as that of the dendron-modified surface. Aminetethered oligonucleotides were employed as described by
Guo.3b The observed selectivities for T:T, G:T, and C:T
were 100:41:38:26 (Figure 3c and Table 2). For an
additional comparison, oligonucleotides (probe 2) having
an extra (T)30 spacer at the 5′ end of the oligomer on an
APDES-modified surface were utilized for the SNP
discrimination test. It has been known that introduction
of a vertical spacer to probe DNA enhances DNA hybridization efficiency.13 The observed selectivities for T:T, G:T,
and C:T were 100:17:18:12 (Figure 3d and Table 2). The
selectivity was obviously enhanced in comparison with
the case of probe DNA with a C6 spacer but still was largely
inferior to the dendron-modified DNA microarray.
To verify that the selectivity holds for other cases,
oligonucleotide sequences from the p53 gene (probe 3,
probe 4, target 3, and target 4) were tested using the
microarray. It has already been known that many cancer
patients have a single mutation in the above sequence
regions of the p53 gene.14 Probe 3 with 80% GC content
was chosen to examine the selectivity at the high GC
content. It has been believed generally that higher GC
contents show worse SNP discrimination efficiency. The
selectivity for A:C, T:C, and C:C internal mismatches were
100:0.2:0.1:0.5 for probe 3/target 3 (Figure 4a and b) and
100:0.4:0.3:0.4 for probe 4/target 4 (Figure 4c). The result
revealed at the high discrimination efficiency invariably
holds for various oligonucleotide sequences.
(12) (a) Zhao, X.; Tapec-Dytioco, R.; Tan, W. J. Am. Chem. Soc. 2003,
125, 11474-11475. (b) Chakrabarti, R.; Klibanov, A. M. J. Am. Chem.
Soc. 2003, 125, 12531-12540. (c) Taton, T. A.; Mirkin, C. A.; Letsinger,
R. L. Science 2000, 289, 1757-1760.
(13) (a) Chen, D.; Yan, Z.; Cole, D. L.; Srivasta, G. S. Nucleic Acids
Res. 1999, 27, 389-395. (b) Afanassiev, V.; Hanemann, V.; Wölfl, S.
Nucleic Acids Res. 2000, 28, e66. (c) Shchepinov, M. S.; Case-Green, S.
C.; Southern, E. M. Nucleic Acids Res. 1997, 25, 1155-1161.
(14) (a) Akyüz, N.; et al. Mol. Cell. Biol. 2002, 22, 6306-6317. (b)
Kang, M.; et al. Int. J. Cancer 1996, 67, 898-902. (c) Park J.; et al. Int.
J. Cancer 1997, 70, 443-449.
Letters
In our work, the dendron-modified surface also resulted
in 80-100% hybridization yield, whereas the APDESmodified surface gave only a hybridization yield lower
than 20%.15 It has already been found that a DNAimmobilized surface with sufficient distance between each
probe DNA could ensure high hybridization yield.9d,13c This
result confirms that probe DNA strands on the mesospaced
surface are unhindered for the hybridization.
We fabricated successfully a DNA microarray of the
highest fidelity by guaranteeing mesospacing among the
probe DNAs. The enhanced characteristics of DNA microarrays guarantee more reliable gene analysis and will
expedite wider acceptance of the analytical method.
Moreover, the approach is not confined to the particular
area but applicable to many analytical methods involving
(15) Hybridization efficiency was calculated by measuring the
fluorescence intensity in comparison with that of probe DNA that has
a Cy-3. In other words, the fluorophore-tagged probe DNA was spotted
and the intensity after washing was used for the reference value.
Langmuir, Vol. 21, No. 10, 2005 4261
immobilized molecules of the relevant size. Mesospacing
on a surface is expected to allow various immobilized
biomolecules to keep their inherent properties in solution
and will find numerous applications.
Acknowledgment. Dedicated on the occasion of
Professor Dong Han Kim’s retirement (Department of
Chemistry, Pohang University of Science and Technology).
Student fellowships of Brain Korea 21 are gratefully
acknowledged, and this work is also supported by the
Korea Science and Engineering Foundation through the
Center for Integrated Molecular Systems.
Supporting Information Available: Synthesis and
characterization of the dendron and the dendron-modified
substrate and experimental procedure for preparing the DNA
microarray and hybridization. This material is available free of
charge via the Internet at http://pubs.acs.org.
LA046951Y