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TMOS-APTMS films for immobilization of biomolecules

By Denise Fox,2014-05-07 19:31
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TMOS-APTMS films for immobilization of biomolecules

     Aminopropyl embedded silica films as potent substrates in DNA

    microarray applications

    a,b;a,bbacabbK. Saal, T. Tätte, I. Tulp, I. Kink, A. Kurg, R. Lõhmus, U. Mäeorg, A. Rinken and

    a A. Lõhmus

    aInstitute of Physics, University of Tartu, 142 Riia St.,51014 Tartu, Estonia bInstitute of Organic and Bioorganic Chemistry, University of Tartu, 2 Jakobi St., 51014

    Tartu, Estonia

    cInstitute of Molecular and Cell Biology, Estonian Biocentre, 23 Riia St., 51010 Tartu,

    Estonia

Abstract

    Sol-gel derived (3-aminopropyl)trimethoxysilanetetramethoxysilane (APTMS,

    (CHO)SiCHCHCHNHTMOS, Si(OCH)) hybrid films are shown to have properties 33222234

    that make the films suitable for DNA microarray applications. The detailed characteristics are studied using aminated 25-mer oligonucleotide DNA and 1,4-phenylenediisothiocyanate 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.

    Keywords: sol-gel hybrid films, uniform surface, atomic force microscopy, FTIR spectroscopy, DNA immobilization.

    PACS: 81.20.-n, 81.20.Fw, 87.15.By, 61.16.Ch.

     ; 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: type@fi.tartu.ee

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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 the 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, silane-treated 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

    -(CH)-SiCl) on alkyltrichlorosilanes, particularly octadecyltrichlorosilane (OTS, CH32173

    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].

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    Since introduction [7] as a reliable route for immobilization of DNA for SPM studies the silanization of mica or glass using trialkoxyaminopropylsilanes, particularly (3-aminopropyl)triethoxysilane (APTES, HNCHCHCH-Si(OCH)) has become a common 2222253

    12,13,14]. However, self-assembling and polymerisation procedure in similar investigations [

    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 self-assembling tendency in terms of the

    17]. Furthermore, introduction of a polar amino group at orientation of the chains than OTS [

    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.

     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

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    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.

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2. Experimental procedures

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

    O)SiCHCHCHNH)and TMOS APTMS ((3-aminopropyl)trimethoxysilane, (CH332222

    (tetramethoxysilane, Si(OCH)) (both Sigma-Aldrich Co) were mixed at molar ratios 0:1, 34

    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)/HO/MeOH 2

    was kept as 1:2:2. In the case of pure TMOS (APTMS-TMOS 0:1) the mixture of water/methanol was acidified with concentrated HCl, so that the final molar ratio of TMOS/HO/MeOH/HCl was 1:2:2:0.005. The mixtures were stirred till they turned to highly 2

    viscous spinnable matter (ca 30 min). Then, the polymerisation reaction was suppressed by introducing cold dry methanol to the mixture of silanes, thus making up the final molar ratio

    oof (APTMS+TMOS)/MeOH 1:7. The final product was kept sealed at 4C as stock solutions.

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    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

    oo140C (0.3C/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.

    o The spotted slides were incubated in humid air at 37C 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.

    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).

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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 1,8,9-trihydroxyanthracene (dithranol) as matrix (Fig. 2).

2.5. AFM measurements

    The topographic features of APTMS-TMOS-films were investigated with an atomic force

    oC, microscope SMENA-B (NT-MDT, Russia) working in semi-contact mode in air (at 20relative 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

    ocamera through an optical microscope gazing at 90 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.

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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. The ability of the films to bind 25-mer oligonucleotide DNA was measured comparative to commercial slides. The results are presented in 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 film. Consequently, it means that the non-specific binding of aminated DNA was very low. The binding of DNA to APTMS-TMOS 1:10 and 1:5 films was on the level of 10% of the SAL film. Further increase in the ratio of APTMS (APTMS-TMOS 1:3 film) gave considerable rise in the amount of DNA immobilised, but the signal still remained below 50 % level of SAL-glass. Additional increase of APTMS content in the film (APTMS-TMOS 1:1 and 3:1 films) led to substantially higher binding efficiencies (140 % and 135 % of SAL, respectively). Further increase in the relative amount of APTMS in the film (APTMS-TMOS 5:1, 10:1 and 0:1 films) led to scattered DNA spots and thus, no comparative binding efficiencies could be obtained. The scattering can be explained by dissolution of the films in aqueous environment due to lower rate of cross-linking between aminosiloxane oligomers. The dimensions of the DNA spots decreased with the increase of the amount of APTMS in the films (Fig. 1., inset). This can be explained by the decrease in wettability, caused by additional amount of hydrophobic aminopropyl groups. The dimensions of the spots on APTMS-TMOS 1:3, 1:1 and 3:1 films were close to the spot sizes on SAL-glass. Starting from APTMS-TMOS 5:1 film the spots were not clearly outlined due to the dissolving of silane coating in aqueous medium.

    The layer of DNA on SAL-slide and on APTMS-TMOS 1:1 film was uniform and the edges of the spots were well-defined, but the nanoscale surface roughness was higher on the SAL-

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    slide both inside and outside the spot area (Fig. 2), which can originate either from topographical features of the glass surface or immobilized siloxane clusters.

3.2. Wettability of APTMS-TMOS films

    The contact angle measurements indicated that APTMS-TMOS 0:1 and 1:10 films were completely wettable, e.g. no water drops formed on their surfaces (Table 1). The contact angle of APTMS-TMOS 1:5 film was 14 degrees and abrupt jump to 40 and further on to 60 degrees was observed in the case of APTMS-TMOS 1:3 and 1:1 film, respectively. 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 APTMS-TMOS 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 cross-linking between individual siloxane oligomers, which means that the “building blocks” 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-

    -1, a band that corresponds to OH stretching of SiOH, CHOH and HO (Fig. 3). After 3420 cm32

    oheating at 140 C the intensity of this absorption decreased substantially and starting from the

    -1APTMS-TMOS 1:1 film two well defined signals appeared at 3366 and 3284 cm. These

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     group, respectively. bands correspond to the antisymmetric and symmetric stretching of NH2

    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 NH and SiOH groups. This supports the proposed mechanism of formation of 2

    23]. APTMS layer on silica [

    -1The most intriguing region is between 1700 and 900 cm. The spectra of unbaked films

    -1clearly showed strong signals at 1580 cm that corresponds to N-H scissoring vibration and at

    -1+1486 cm that is believed to correspond to symmetric NH deformation mode partly 3

    superimposed by CH bending [24]. After heating of the precursor films the strong signal at 2

    -1-11486 cm disappeared and the medium bending signal of CH of the usual value of 1476 cm 2

    was detected. The change of absorption bands corresponding to N-H and CH vibrations at 2

    -11580 and 1486 cm, respectively (Fig. 3), is supposedly caused by decomposition of the relatively labile H-bonding network between SiOH and NH groups. As a result of this 2

    -1process the degree of polymerization increases and 3D structure forms. At 1630 cm 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 NH deformation as it was proposed 3

    24]. earlier [

    It is interesting to point out that the pure TMOS based precursor film had Si-O-Si main

    -1-1absorption at 1060-1070 cm and only a weak shoulder was seen at 1130-1150 cm.

    -1However, with the increase of APTMS content the shoulder at 1150 cm increased until the

    -1situation was reversed and the absorption at 1060 cm appeared much weaker than at 1150

    -1cm. 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.

    -1-1As it can be seen in Fig. 3 there appear two well-defined bands at 1034 cm and 1122 cm in

    the spectra of baked APTMS-TMOS films (0:1,1:1, 1:0). Similar structure at 1055 and 1088

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