2.3 Attaching DNA to a surface
2.3.1 Surface treatment
Figure 2.4: Functionalization of a glass surface with streptavidin and anti-digoxigenin.
In the last step R denotes either streptavidin or anti-digoxigenin.
Immobilization of short single-stranded DNA (ssDNA) on a surface is used in biotechnology for rapid sequencing of DNA [40, 41, 42]. Attaching of longer DNA by one end is used for example in combing experiments of DNA [43, 44, 45] which provides a new method for optical restriction mapping of genes on a single molecule level. Most of the protocols uses the formation of self assembled monolayers (SAM) of silane on OH-groups at glass surfaces. The principle of the used chemistry is shown in Fig.2.4. The protocol in detail consists of the following steps.
Cleaning of the coverslips The coverslips are normally made from borosilicate glass (D263). If the glass is very dirty and dust is visible, it is rinsed with isopropanol, acetone and water (in this succession), and if necessary ultrasoni-cated in water. Then the coverslips are cleaned in Piranha water, which consists of 50% concentrated sulfuric acid (H2SO4) and of 50% H2O2 (25%), four 1 hour.
The mixture is not only a very strong acid but mixing of the two ingredients results in a strong exothermal reaction, and care has to be taken for the heat during handling the mixture and a temperature stable container is needed. The coverslips are thoroughly rinsed with water and then dried with pure nitrogen.
Silanization The dry coverslips are silanized for 1 hour in a solution of 2% (v/v) 3-amino-propyltriethoxysilane (APTES) in pure ethanol (p.a.). One should avoid water to get into contact with the dissolved APTES, because it induces polymerization of the APTES and formation of SiO2 particles. If this happens a white precipitate is visible on the coverslips, and we discard the coverslips.
The excess silane is removed by extensively washing with ethanol (p.a.). At this stage a batch of coverslips is stored dry or in ethanol.
Glutaraldehyde To the amino group of the silane glutaraldehyde is covalently bound, which is commonly used in biology for fixation of biomolecules and cells for microscopy purpose. Therefore a drop of glutaraldehyde (used as sup-plied) is put onto the slide for 30 minutes. To avoid evaporation the incubation is done in a wet chamber at room temperature. The sample is then washed several times with PBS buffer.
Coverage with Streptavidin/anti-digoxigenin For covering the surface with strep-tavidin a droplet of strepstrep-tavidin solution is put onto the surface. The standard concentration is 0.1 mg/ml in PBS. For functionalization with anti-digoxigenin a standard concentration of 0.05 mg/ml in PBS is used. The incubation time is 1 hour in a wet chamber. The sample is washed several times with 1×TBE buffer (pH∼8.6), and ready for use. The protein coverage causes a strong wet-ting of water and therefore one can assess the success of coverage by watching the wetting behavior of water on the functionalized spot.
From literature it is known that streptavidin can be visualized by AFM if it is ad-sorbed on mica [46, 47]. We therefore tried to measure the degree of coverage of streptavidin on glass by AFM. In Fig. 2.5 a few typical AFM images acquired in tap-ping mode are shown. Because of its better contrast the phase signal, which reflects a change in the interaction between the AFM tip and the substrate, is shown. In gen-eral the AFM images without streptavidin appears flat (see Fig. 2.5(a)). Streptavidin appears as discrete spots or as aggregates (see Fig. 2.5.b and 2.5(c) ). The concentra-tion of the inserted strepatvidin soluconcentra-tion in Fig.2.5(c) is less than in Fig.2.5.b but it
Figure 2.5: A set of Afm Images of strepatavidin covered glas surfaces. The phase signal of the AFM is shown. a) Pure glass. The bright spot is probably dirt. b) Strepativdin concentration 10µg/mul. Drift degraded the image. And the strepta-vidin is visible as bright stripesc)Strepativdin concentration0.5µg/µl. Streptavidin appears as bright spots and aggregates.
seems as if the surface coverage is higher in Fig. 2.5(c), which is not expected. It was not possible to get quantitative and consistent results, with respect to the inserted streptavidin concentrations. The goal to characterize the streptavidin coverage by AFM imaging was therefore abandoned.
For coupling DNA onto a gold surface we evaporated first few nanometers of chromium, as an adhesion agent onto a coverslip, because gold does not adhere to glass directly.
Then gold is evaporated. The exact thickness depends on the experiment one wants to perform. For fluorescence microscopy on an inverted microscope such as it it is described in Sec.2.3.2 one needs very thin layers to be able to detect fluorescence signal through the layer. Typically 1 nm of chromium and 14 nm of gold are sufficient to get a fluorescence signal of thiol-linked, fluorescently labelled DNA. Above a gold thickness of ∼ 25 nm the gold film is not transparent anymore. For force experi-ments as they are described in Chapter 4 we evaporated typically a layer thickness of
∼70 nm gold. After evaporation the surfaces are ready to use for attaching DNA. It is very important to do this immediately after breaking the vacuum of the evapora-tion chamber, because the gold layer loses very rapidly its capability to bind to thiol groups. The reason of this inhibition of the gold surface is not clear. It could be due to water as it comes from the humidity of air and which inhibits the binding sites of
Figure 2.6: a) Fluorescence microscope image of a DNA carpet on a strepatvidin surface. The DNA appears as bright spots. The field of view is around30µm×20µm.
b) DNA stretched by an electrical field parallel to the surface. The length of the stretched DNA is roughly16µm. Obviously only one end of DNA is attached to the surface as it is predetermined from the used surface chemistry. Note that image a) and image b) shows two different samples.
the gold for the thiol.