Aminopropyl embedded silica films as potent substrates in DNA microarray
applications
K. Saala,b, T. Tättea,b, I. Tulpb, I. Kinka, A. Kurgc, R. Lõhmusa, U. Mäeorgb, A. Rinkenb
and A. Lõhmusa
a
b
Institute of Physics, University of Tartu, 142 Riia St.,51014 Tartu, Estonia
Institute of Organic and Bioorganic Chemistry, University of Tartu, 2 Jakobi St., 51014
Tartu, Estonia
c
Institute of Molecular and Cell Biology, Estonian Biocentre, 23 Riia St., 51010 Tartu,
Estonia
Abstract
Sol-gel derived (3-aminopropyl)trimethoxysilane–tetramethoxysilane (APTMS,
(CH3O)3SiCH2CH2CH2NH2–TMOS, Si(OCH3)4) hybrid films are shown to have
properties that make the films suitable for DNA microarray applications. The detailed
characteristics are studied using aminated 25-mer oligonucleotide DNA and 1,4phenylenediisothiocyanate linker. The binding of DNA onto the films is shown to
depend on films’ composition having an optimum where the binding is substantially
superior compared to commercial analogues. The essential properties of the films are
characterized by AFM, FTIR and wettability measurements.

Kristjan Saal, Institute of Physics, University of Tartu, 142 Riia St, 51014 Tartu, Estonia, Phone: +372 7
383037, Fax: +372 7 383 033, E-mail: saal@fi.tartu.ee
1
Keywords: Thin films; Amorphous materials; Silicon oxide; Glass; Infrared
spectroscopy
2
1. Introduction
DNA microarrays are devices displaying specific oligonucleotides or longer DNA
fragments attached in two-dimensional order onto activated solid surface [1]. DNA
microarrays permit the analysis of gene expression and DNA sequence variation in a
massively parallel format. The physical and chemical nature of the substrate on which
the reactions are performed is one of the key factors influencing the quality and
reproducibility of the results. Among many different types of substrates for DNA
microarray analysis, the most common chemical treatments provide chemically reactive
amine or aldehyde groups prepared by silanization. Despite being widely used, silanetreated slides lack the desired reproducibility – a fact that has driven a constant search
for chemically alternative techniques rather than improvements of silanization
protocols.
From the general point of view, silanization of hydroxyl-terminated substrates is an
effective and frequently used procedure for modification of chemical and physical
properties of the substrate as well as for covalent immobilization of a variety of
compounds. Silane coatings serve a number of applications such as protective coatings
or adhesion promoters on metal surfaces [e.g. 2,3], adhesives in industrial paints [e.g.
4], selectively binding surfaces for tethering biological molecules in biosensor and
DNA chip design [5,6], in scanning probe microscopy (SPM) studies of biomolecules
[7], and in chemical force microscopy studies as probe functionalizing agents [8].
Recently, several new technological applications have given rise to growing attention to
studies on self-assembling silane monolayers. Focus has mainly been on formation of
uniform monolayers of long-chained organosilanes, where alkyltrichlorosilanes,
3
particularly octadecyltrichlorosilane (OTS, CH3-(CH2)17-SiCl3) on different
hydroxylated surfaces such as oxidized silicon or mica [9,10] are among the most
studied systems. In contrast, alkyltrialkoxysilanes bearing short tail group have been
studied only in a limited number cases [e.g. 11].
Since introduction [7] as a reliable route for immobilization of DNA for SPM studies
the silanization of mica or glass using trialkoxyaminopropylsilanes, particularly (3aminopropyl)triethoxysilane (APTES, H2NCH2CH2CH2-Si(OC2H5)3) has become a
common procedure in similar investigations [12,13,14]. However, self-assembling and
polymerisation of silanizing agent depend strongly on reaction conditions like humidity,
used solvents, temperature, etc. Nevertheless, these factors are often neglected which, in
turn, has led many authors to point to poor reproducibility in formation of homogeneous
APTES layer and to its instability in aqueous medium [13, 15, 16]. It follows from
earlier studies that regardless of the chain length n-alkylsilanes exhibit much lower selfassembling tendency in terms of the orientation of the chains than OTS [17].
Furthermore, introduction of a polar amino group at the chain terminus hinders
formation of ordered monolayers due to hydrogen bonds between the amino group and
the surface silanols (SiOH) [17,18]. The heterogeneity of formed layers can also be
caused by self-polymerisation of silane, initiated by the trace quantities of water in
reaction medium [19]. From the other side, APTES and APTMS are widely used in
several scientific and commercial applications mainly because of their availability, low
cost, and simplicity of processing.
4
Formation of a perfect monolayer of short-tailed functionalised silanes can be achieved
if precisely control concentrations of silanizing agent and traces of water in the reaction
medium and on the substrate [19]. For instance, optimal formation of closely packed
monolayers of OTS occurs at 1.5 ppm water content in the solvent [20]. However, the
structure of formed monolayer depends also on the chemical nature of the silanizing
agent, the density of silanols on the substrate and its nanoscale surface structure. In
search for a more robust procedure, which is less dependent on humidity and nature of
the substrate for reproducible fabrication of silane coatings we have proposed an
alternative silanization technique that substantially improved homogeneity and
smoothness of the surfaces [21]. This was achieved by dip coating mica substrate with
partially pre-polymerized APTMS sol, followed by its gelation in humid air. Still, the
films did not feature prolonged stability in water, which is probably caused by low rate
of cross-linkage between individual siloxane molecules. In the present study we focus
on fabrication of APTMS-TMOS hybrid films in search of new and improved substrates
for DNA microarray analyses. The potential of the films for immobilization of 25-mer
oligonucleotide DNA is discussed in comparison with their commercial analogues
(SAL-1 slides, Asper Biotech Ltd. 22). The characteristics of the films are investigated
by infrared (FTIR) spectroscopy, wettability and atomic force microscopy (AFM)
measurements.
5
2. Experimental
2.1. Cleaning of glass slides before silanization
In order to exclude the possible effects of impurities on reproducibility of coupling of
silane and subsequently DNA to glass surface the slides were subjected to cleaning
procedure developed in Asper Biotech Ltd. Glass slides (75x25x1 mm, Waldemar
Knittel Glasbearbeitungs GmbH & Co KG) were sonicated for 10 min in 0.5 % aqueous
Alconox solution (Sigma-Aldrich Co), washed thoroughly with distilled water and
sonicated for 10 min in acetone (Naxo Ltd, analytical grade). Thereafter the slides were
gently shaken for 1 h in 3 M NaOH solution in 1:1 v/v mixture of water/95 % ethanol
(Naxo Ltd, analytical grade) and thoroughly washed with distilled water. Finally, the
water was expelled by centrifugation of slides at 280 g (Jouan CR422) for 3 min and the
slides were stored in clean box until usage.
2.2. Preparation of APTMS-TMOS films
APTMS ((3-aminopropyl)trimethoxysilane, (CH3O)3SiCH2CH2CH2NH2) and TMOS
(tetramethoxysilane, Si(OCH3)4) (both Sigma-Aldrich Co) were mixed at molar ratios
0:1, 1:10, 1:5, 1:3, 1:1, 3:1, 5:1, 10:1, and 1:0, respectively. Then, at room temperature
and constant stirring, a mixture of water/methanol (Naxo Ltd, analytical grade) was
added dropwise to the mixture of silanes. The final molar ratio of
(APTMS+TMOS)/H2O/MeOH was kept as 1:2:2. In the case of pure TMOS (APTMSTMOS 0:1) the mixture of water/methanol was acidified with concentrated HCl, so that
the final molar ratio of TMOS/H2O/MeOH/HCl was 1:2:2:0.005. The mixtures were
stirred till they turned to highly viscous spinnable matter (ca 30 min). Then, the
polymerisation reaction was suppressed by introducing cold dry methanol to the mixture
6
of silanes, thus making up the final molar ratio of (APTMS+TMOS)/MeOH 1:7. The
final product was kept sealed at 4oC as stock solutions.
For silanization of glass slides the stock solutions were diluted 40 times with dry
methanol and subsequently the slides were dipped in these solutions. Thereafter the
slides were kept in open air (relative humidity 30%) for 48 h, and subsequently the
temperature was raised to 140oC (0.3oC/min) for 12 h.
2.3. Immobilization of 25-mer oligonucleotide DNA onto silanenized slides and DNA
spot analysis
The silanized slides were gently shaken in 0.2 % w/w 1,4-phenylenediisothiocyanate
(Sigma-Aldrich Co) solution in 10 % w/w pyridine/dimethylformamide (Fluka,
analytical grade) for 2 h, which activated the slides for immobilization of DNA. Then
the slides were thoroughly washed with acetone, methanol and ethanol (Naxo Ltd) and
centrifuged at 280 g for 3 min.
1 part of Cy3 3’labelled 5’-aminomodified 25-mer oligonucleotide DNA (a type of
fluorescent-labelled oligonucleotide DNA, MWG-Biotech) was mixed with 100 parts of
unlabelled 25-mer oligonucleotide DNA (MWG-Biotech). The mixture was spotted to
glass slides with a spotter (Virtek CWP) in 100, 80, 50, 30, 10, 3, 1, 0.4 and 0.1
micromolar series, respectively. For dilutions Genorama Spotting Solution Type I
(Asper Biotech Ltd) was used. The spotted slides were incubated in humid air at 37oC
for 2 h and subsequently treated with ammonia vapour for 1 h, washed thoroughly with
hot distilled water and wiped dry by centrifugation at 280 g for 3 min.
7
For comparison, SAL-slides (prepared by incubation of cleaned glass slides in 2% w/w
APTMS solution in 95% w/w acetone/water for 2 min) were processed in the similar
manner.
The fluorescence of DNA spots was detected using ScanArray 5000 (Perkin-Elmer Inc.)
microarray scanner. The spots were analyzed with Genorama Genotyping Software 4.0
(Asper Biotech Ltd).
2.4. Spectroscopic measurements
IR spectra were measured with Perkin-Elmer PC 16 Fourier transform infrared (FTIR)
spectrometer. A conventional Perkin-Elmer equipment was used for preparation of KBr
pellets (ø 12 mm) by compressing spectroscopically pure KBr powder under 10 tons of
pressure. Freshly prepared pellets were coated with solutions of pre-polymerized
precursor of pure (or mixtures thereof) APTMS and TMOS in methanol. Thereafter the
coated pellets were left to hydrolyze at room temperature and 30% of relative humidity
for 48 h and finally they were heated at 140°C for 12 h.
MALDI TOF MS measurements were performed with instrument designed at the
National Institute of Chemical and Biological Physics of Estonia using 1,8,9trihydroxyanthracene (dithranol) as matrix (Fig. 2).
2.5. AFM measurements
The topographic features of APTMS-TMOS-films were investigated with an atomic
force microscope SMENA-B (NT-MDT, Russia) working in semi-contact mode in air
8
(at 20oC, relative humidity 30%) using ultrasharp non-contact “Golden” silicon
cantilevers NSG11 (NT-MDT). Different locations typically spanning over several
square cm were scanned with different resolutions on each sample for reliable
characterization of a sample.
2.6. Contact angle measurements
Five 3 l drops of distilled water were placed on a slide in a glass chamber saturated
with water vapour and let to stabilize for 15 min. The drops were photographed with a
digital camera through an optical microscope gazing at 90o relative to surface normal.
The contact angles of the drops were estimated directly from the photographs by fitting
surface profile of the drops with segment of a ring.
9
3. Results and discussion
3.1. DNA immobilised to APTMS-TMOS films
A series of APTMS-TMOS films were prepared by variation of the relative content of
two silanes and measured their ability to bind 25-mer oligonucleotide DNA (Fig. 1). In
the case of APTMS-TMOS 0:1 film no binding was detected, which is because of the
absence of isothiocyanate groups on the surface. It indicates also that the non-specific
binding of aminated DNA to the film was very low. The binding of DNA to APTMSTMOS 1:10 and 1:5 films was also low remaining on the level of 10% of the
commercial SAL film binding. Further increase of the content of APTMS in the mixture
gave considerable rise in the amount of DNA immobilised, and with the equimolar
mixture the signal achieved 140% level of the SAL-glass (Fig.1). Similar high binding
was achieved also in the case of APTMS-TMOS 3:1 films, but increase of APTMS
excess to 5 fold or higher led to diffuse DNA spots, which binding efficiencies could
not be obtained. The latter conforms with expectations that low fraction of TMOS in the
mixture causes slower and lower cross-linking between aminosiloxane oligomers and
therefore the formed film is not stable for immobilization of biomolecules.
It is important to note that the dimensions of the DNA spots decreased with the increase
of the amount of APTMS used for the films (Fig. 1., inset). The size of the spots
correlates with the wettability of the surface, which is determined by the amount of
hydrophobic aminopropyl groups. Therefore, for the best practical conditions - maximal
florescence signal within minimal area - an optimal mixture should be selected.
10
Fig. 1.
The layer of DNA on APTMS-TMOS 1:1 film was uniform showing some roughness
only on nanometer scale, which allowed also clearly visualize the edge of the DNA spot
(Fig. 2a). Smoothness of the surface, its stability and presence of sufficient reactive
groups for the immobilization would be a very valuable starting point for more detailed
visualization of biomolecules. The surface of the SAL-slide had higher surface
roughness on both inside and outside the spot area (Fig. 2b), which can originate either
from topographical features of the glass surface or immobilized siloxane clusters.
11
Fig. 2.
3.2. Wettability of APTMS-TMOS films
The contact angle measurements indicated that at very low APTMS concentrations the
formed films were completely wettable, e.g. no water drops formed on their surfaces
(Table 1). The hydrophobicity of the surface could be determined starting from the
APTMS-TMOS 1:5 film, where the contact angle was 14 degrees. Increase of APTMS
concentration decreased proportionally the surface wettability, reaching the plateau of
contact angle close to 60 degrees in the case of APTMS-TMOS 1:1 film (Table 1). The
contact angles of APTMS-TMOS 3:1, 5:1 and 10:1 films also remained in proximity of
60 degrees, whereas in the case of APTMS-TMOS 1:0 film the contact angle dropped to
50 degrees.
The increase of the contact angle with increasing the amount of APTMS in APTMSTMOS hybrid film is due to the additional amount of hydrophobic aminopropyl groups.
On the other hand, the increase of the amount of APTMS decreases the rate of crosslinking between individual siloxane oligomers, which means that the “building blocks”
12
of the film become more loosely bound and as a consequence, the film dissolves when
exposed to water. The drop-down of contact angle correlates with the scattering of DNA
spots starting from APTMS content 5:1 (see 3.1.).
3.3. Spectroscopic data of APTMS-TMOS precursors and films
The formation of precursors and films was studied by FTIR and MALDI TOF mass
spectrometry. It was observed that unbaked films have relatively strong absorption at
3342-3420 cm-1, a band that corresponds to OH stretching of SiOH, CH3OH and H2O
(Fig. 3). After heating at 140C the intensity of this absorption decreased substantially
and starting from the APTMS-TMOS 1:1 film two well defined signals appeared at
3366 and 3284 cm-1. These bands correspond to the antisymmetric and symmetric
stretching of NH2 group, respectively. Surprisingly, these two absorptions were not
detected even in pure APTMS precursor. The reason could be an overlap with strong
vibration of OH bond or formation of hydrogen bond between NH2 and SiOH groups
before the film formation, which is in agreement with the proposed mechanism of
formation of APTMS layer on silica [23].
The most intriguing region is between 1700 and 900 cm-1. The spectra of unbaked films
clearly showed strong signals at 1580 cm-1 that corresponds to N-H scissoring vibration
and at 1486 cm-1 that is believed to correspond to symmetric NH3+ deformation mode
partly superimposed by CH2 bending [24]. After heating of the precursor films the
strong signal at 1486 cm-1 disappeared and the medium bending signal of CH2 of the
usual value of 1476 cm-1 was detected. The change of absorption bands corresponding
to N-H and CH2 vibrations at 1580 and 1486 cm-1, respectively (Fig. 3), is supposedly
13
caused by decomposition of the relatively labile H-bonding network between SiOH and
NH2 groups. As a result of this process the degree of polymerization increases and 3D
structure forms. At 1630 cm-1 only a weak shoulder in spectra of precursors as well as
baked films was detected. This signal did not change during heating and we can not
assign this for NH3+ deformation as it was proposed earlier [24].
APTMS-TMOS
APTMS-TMOS film s before
heating
0:1
1034
1122
1486
1580
1630
1:0
3420
3342
Transmittance
1:1
0
90
00
10
00
11
00
12
00
13
00
14
00
15
00
16
00
17
00
28
00
29
00
30
00
31
00
32
00
33
00
34
00
35
cm -1
0:1
1034
1122
1476
1630
1580
0
90
00
10
00
11
00
12
00
13
00
14
00
15
00
16
00
17
00
28
00
29
00
30
00
31
00
32
00
33
00
34
00
35
Fig. 3.
1:1
1:0
3366
3284
Transmittance
APTMS-TMOS film s after
heating
cm -1
It is interesting to point out that the pure TMOS based precursor film had Si-O-Si main
absorption at 1060-1070 cm-1 and only a weak shoulder was seen at 1130-1150 cm-1.
However, with the increase of APTMS content the shoulder at 1150 cm-1 increased until
14
the situation was reversed and the absorption at 1060 cm-1 appeared much weaker than
at 1150 cm-1. The absorption of C-N stretching vibration that appear in this region is
usually much weaker compared to Si-O vibrations and can not be distinguished.
As it can be seen in Fig. 3, two well-defined bands at 1034 cm-1 and 1122 cm-1 appeared
in the spectra of baked APTMS-TMOS films (0:1,1:1, 1:0). Similar structure at 1055
and 1088 cm-1 in the spectra of (CH3)2Si(OCH2CH3)2 has been assigned to the linear and
cyclic forms of siloxane polymer, respectively [25 and refs. 15-19 cited therein]. The
relative shift can be also explained by structural differences of initial monomers.
MALDI TOF mass spectra revealed that co-polymerized APTMS-TMOS hybrid
materials had molar masses in range of up to 1500 amu (Fig. 4). The mass range
detected did not significantly depend on the relative compositions of APTMS/TMOS in
the mixtures. As expected, the spectra had very complicated structure that contained
different “families“ of oligomers. For pure pre-polymerized APTMS it was estimated
that such mass distribution corresponds to the oligomers containing approximately up to
12 monomers.
15
100
Intensity, %
80
60
40
20
0
-200
0
200
400
600
800 1000 1200 1400 1600 1800 2000
Mass (m/z)
Fig. 4.
3.2. The surface of APTMS -TMOS films
APTMS-TMOS 0:1 film exhibited a uniform and smooth surface (average vertical
difference 5 nm per 1 μm scan) (Fig. 5a). APTMS/TMOS 1:10 film showed surface
consisting of grains with several to a hundred nanometers in diameter and average
height distribution of 20 nm/μm (Fig. 5b). The surfaces of APTMS/TMOS 1:5 and 1:3
films were similar to 1:10 film, but the surface line profiles indicated to substantially
lower deviations in height (Fig. 5c and 5d). Starting from APTMS/TMOS 1:1 film the
surface profiles ranged between two nanometers, thus showing practically featureless
topography in 1 μm2 scale (Fig. 5e-i).
16
Fig. 5.
It has been shown that in basic medium alkoxysilanes polymerise to three-dimensional
nanosized siloxane particles, whereas in acidic medium linear molecules are formed
[26]. This result is confirmed since the polymerisation of TMOS was carried out in
acidic medium where spinnable viscous material was formed and the corresponding
film showed relatively uniform surface with no evidence of grainy texture (Fig. 5a).
When no acid as catalyst was used white powder-like material precipitated, indicating to
the formation of branched siloxane particles. On the other hand, the polymerisation of
the mixture of APTMS/TMOS can be considered as base-catalysed process due to
17
amino groups of APTMS. Thus, in great excess of TMOS silane polymerises to
nanosized granules that are clearly evident in Fig. 5b-d. At higher APTMS content the
granules appear smaller probably because of the steric hindrance of aminopropyl groups
that diminishes the condensation to progress in all directions (see inset in Fig. 5g). In
between these extremes of APTMS/TMOS ratios observed we believe this steric
hindrance to remain sufficient for disabling the growth of siloxane particles big enough
to precipitate, which makes them use of as precursors for film making. Consequently,
the APTMS/TMOS hybrid films can be thought of as consisting of densely packed
nanosized siloxane particles, and the dimensions as well as the rate of cross-linking
between individual “building blocks” are determined by the ratio of APTMS/TMOS.
18
4. Conclusions
1. It was shown that APTMS-TMOS hybrid films have potential as substrates for
immobilisation of aminated DNA via 1,4-phenylenediisothiocyanate linkering.
2. The ratio of APTMS to TMOS chosen in precursor synthesis determines the number
of amino groups on the films’ surfaces available for immobilization of DNA and
shape of the formed spots. The maximal binding of DNA was achieved on the
APTMS-TMOS 1:1 and 3:1 films.
3. Films with higher APTMS molar content (starting from APTMS-TMOS 5:1 film)
were not stable in aqueous medium. The instability was probably caused by
relatively low content of cross-linking agent TMOS.
19
Acknowledgements
This work was supported by the Estonian Science Foundation grants no. 5015, 5545 and
4603, and by European Science Foundation Nanotribology Programme. I.K.
acknowledges support by EC FW5 “Centres of Excellence” programme (ICA1-199970086). The authors acknowledge Asper Biotech Ltd. for helpful cooperation.
20
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21
Figure Captions
Fig.1. The binding curves of 25-mer oligonucleotide DNA to APTMS-TMOS hybrid
films, normalized to the signal of 80 μM DNA spot on the SAL-slide. Each data point
corresponds to an average fluorescence intensity of 16 independent spots. The inset next
to legends on the plot shows the typical fluorescence images (300x300 μm2) of the 50
μM spots on the corresponding films.
Fig.2. Semi-contact mode AFM images of DNA spots on APTMS-TMOS 1:1 film (a)
and on SAL slide (b); scan range 8.5x8.5 μm2. The scale bars on the right correspond to
the line profiles drawn on the images to illustrate sharp edges of DNA spots. Bright
white spots are experimental artifacts.
Fig.3. FTIR spectra of APTMS-TMOS hybrid films at 3 characteristic APTMS/TMOS
mixtures. The pre-polymerized APTMS-TMOS mixtures were gelled on KBr pellets
and spectra recorded before and after baking the pellets at 140 oC. Several well-known
absorption peaks are marked. Intensity is in arbitrary units.
Fig.4. MALDI TOF mass spectrum of APTMS-TMOS 1:1 hybrid material.
Fig.5. Semi-contact mode AFM images of APTMS-TMOS hybrid films; scan range 1x1
μm2. The scale bar on the right corresponds to the line profiles drawn in the middle of
the scan. AVE represents the average height difference in scan profile. High-resolution
inset (200x200 nm2) in Fig.5g illustrates granule-like structure of the surface (see text).
22
Tables
Table 1. The contact angles of 3 μl water drops on APTMS-TMOS hybrid films. The
results are presented as an average of contact angles of five independent drops.
APTMS/TMOS
Contact angle, degrees
0:1
-
1:10
-
1:5
14 ± 2
1:3
40 ± 1
1:1
61 ± 2
3:1
61 ± 3
5:1
55 ± 2
10:1
58 ± 2
1:0
51 ±2
23