As explained before, during imaging, a Gaussian fit is performed for determining the exact place of emission after detection of a single fluorophore. Subsequently, many of such localizations can be combined to reconstruct a final image of high resolution.[15] Labeling BTA-monomers with a fluorophore like Cy5 thereby gives us the ability to look at BTA-polymer fibers. A comparison in achieved resolution between conventional and super-resolution techniques can be seen in figure 1.12, in which BTA-fibers imaged using conventional techniques look blurred compared to the super-resolution images.
In previous experiments, it was already found that monomers dynamically exchange position between different BTA-polymers, as shown schematically in figure 1.13, in contrast to covalently bonded polymers.[20] STORM was used to further examine this process. Experiments showed a homogeneous exchange of monomers along the polymer backbone.[19] By imaging the migration of Cy3-labeled and Cy5-labeled monomers between BTA-fibers, dynamic exchange between solvent and polymer-backbones was demonstrated. This investigation was performed using
1.5. SURFACE CHEMISTRY
FIGURE1.12. Difference in conventional imaging (top) and super-resolution imaging (down) of BTA-fibers. Figure reproduced from [19]
snapshot images at different time points after mixing, as can be seen in figure 1.14. Limitations in sample preparation prevent real-time imaging of these dynamics. The properties of the microscope glass coverslips favour fiber attachment and thus prevent us from imaging kinetic behaviour. Modification of these surfaces using surface chemistry could prove useful for looking at these dynamics, since anti-fouling coatings can be used to prevent settlement of organic residues.[21][22][23]
1.5 Surface chemistry
1.5.1 Basic concepts of silanization
Modification of the chemical composition of surfaces can be performed to obtain surface coatings with altered physical properties. This can be done by incorporating specifically selected functional molecules.
One type of organic molecules that can chemisorb to silicon oxide substrates (glass), are silanes. They contain a chemical group that can bind spontaneously and covalently to the surface and an organic rest group, schematically shown in figure 1.15. The silane part consists of a silicon atom with three leaving groups Xiand a linker part, which often consists of a long hydrophobic alkyl chain. Leaving groups are often hydroxy (−OH), chlorine (−Cl), methoxy (−OCH3) or ethoxy (−OCH2CH3).
Silanes can react with silanol groups ( −SiOH) on silicate substrates like glass surfaces.[ 25]
FIGURE1.13. Monomer exchange after mixing different BTA-fibers. Figure reproduced from [20]
This reaction is called silanization and is shown in figure 1.16. It contains a crucial condensation step, in which small molecules are released. This release prevents the reverse reaction. For example, when a trifunctional silane binds to the silanol groups on the surface, it releases three HX molecules per silane. When X is a hydroxyl group, three molecules of water are released.
The process of silanization consists of several underlying steps. To react, the molecules have to diffuse towards the substrate and at a certain point adsorb to the surface. After adsorption the molecules migrate laterally until they either desorb again or form a chemical bond with the substrate by binding in a potential minimum. The groups X of the silane will first react with water to form silanol groups (SiOH). This is called hydrolysis and it determines the overall reaction speed. The reaction rate depends on the identity of groups X and increases in the series OEt < OMe < Cl. After hydrolysis, the second step is a condensation reaction where the silanol group of the reactant binds to the silanol group on the surface, releasing a water molecule. The quality of deposition is strongly influenced by migration, possible nucleation to the substrate and desorption of the reactant.[24]
These processes are heavily influenced by the experimental steps of the functionalization protocol. Silane chemistry applied to silicate surfaces is usually performed using either vapor phase deposition or solution phase deposition of reactant silanes. Reactions take place at the gas-solid interface with deposition of thin films using chemical vapor deposition.[26] Especially at low pressure, surfaces can be coated with a homogeneous layer. A stable coating is obtained when a gas can react with the substrate surface, also forming gaseous by-products. Using this method, layer thicknesses of the order of 5 - 10µm can be obtained, depending on the exact system and
1.5. SURFACE CHEMISTRY
FIGURE1.14. STORM-imaging of fiber-exchange at different time points. Figure repro-duced from [19]
FIGURE1.15. Structure of a silane. Figure reproduced from [24].
protocol used.[24] The reaction can also be performed in solution, using solution phase deposition, in which we look at the liquid-solid interface. Surface coating can be obtained by immersing glass slides in a solution of reactant silanes. An incubation period of 10 - 60 minutes allows the dissolved silanes to react with the surface, resulting in a more densely functionalized surface.
1.5.2 Surface structure
The surfaces that are formed after reaction of silanes with solid substrates are often so-called monolayers. Because the binding of these molecules occurs spontaneously, these layers are called self-assembled monolayers (SAM).[27]
The growth of a self-assembled monolayer can take place in multiple ways and the quality of
FIGURE1.16. Reaction scheme for silanization. Figure reproduced from [24].
a SAM is highly dependent on the formation mechanism. Different growth mechanisms can be envisioned for these SAMs, because the formation of a SAM is governed by the aforementioned diffusion and adsorption processes. Those two processes occur at different rates, causing SAM formation to be either diffusion or adsorption controlled.[24] Different possible scenarios of monolayer growth are shown in figure 1.17. One is that reactants prefer to adsorb to aggregates already present on the substrate surface until a closed film is formed, scenario A. Another scenario is that first a closed film is formed, either ordered or disordered, scenarios B and C, after which the molecules in the film reorganize to form the final SAM in a second step.[24]
FIGURE1.17. Schematic view of growth of a monolayer by different scenarios. Figure reproduced from [24].
1.6. AIM AND OUTLINE OF THIS THESIS
The final structure of this monolayer is determined by three types of interaction.[24] The first one is the strength of the bond between the reactive head group and the substrate surface.
Further interactions are the attractive van der Waals forces between the linker groups of the silanes. The third and final interaction is the often repulsive interaction between the tail groups of the silanes due to, for example, steric hindrance.
A perfect monolayer is very difficult to form, because of the complex interplay between aforementioned interactions. It is found that it is sterically impossible to form a 1:1 stoichiometric saturation of all silanol groups on the substrate surface.[ 24] The hypothesis is that the surface layer will consist of a polysiloxane network anchored to the surface on some points, where approximately one in five silanes will bind to the surface and the other four are somewhat cross-linked above it, following scenario A, as can be seen schematically in figure 1.18.
FIGURE1.18. Schematic drawing of silanes bound to a silicon oxide surface. Figure reproduced from [24].
To prevent polymerization, water needs to be excluded from the reaction, hence the fact that usually this reaction is performed in dry organic solvents. However, a trace of water is needed for activating both the surface and the silanes. Water can react to the silanes in solution, forming polysiloxane networks that will precipitate, because the water molecules can compete with the hydroxyl groups on the substrate.[24] This could result in a rough surface, while the polysiloxanes can still, partly, bind to the substrate surface. Suitable silane molecules and reaction conditions should therefore be chosen to obtain the desired surface properties.
1.6 Aim and outline of this thesis
In this project, we aim to use surface chemistry approaches to create functionalized glass coverslips suitable for dynamic STORM experiments. The exchange of monomers between solution and aggregate state of a BTA-fiber in an aqueous environment can then be investigated.
In earlier experiments, BTA-fibers were physically adsorbed to glass microscope coverslips before imaging.[19][20] Once the fibers become physisorbed, no dynamic behaviour and monomer
exchange can be studied. This inhibits us from looking in real-time at changes in molecular composition of BTA-fibers. In order to understand how this exchange occurs, physisorption of BTA-fibers needs to be prevented. The approach towards imaging dynamic exchange of monomers consists of several experiments.
The first step is modification of the glass coverslip surface to prevent physisorption of BTA-fibers. The second step involves mixing in a bi-functional linker molecule, that at certain points links the BTA-fiber to the surface to prevent drift, while still maintaining its dynamic properties.
A schematic visualization of this approach is shown in figure 1.19. It shows that BTA-fibers are not completely bonded to the surface, but merely a loose flexible connection via linker molecules is formed, resulting in a fiber that still has the capacity to exchange monomers between solution and aggregate-state. The final step then consists of studying real-time dynamic exchange. In order to study this mechanism at the single-aggregate level, the super-resolution optical microscopy technique STORM is utilized.
FIGURE 1.19. Schematic model of BTA-fibers attached to the surface via linker molecules.
Modification of glass surfaces will be performed using silane chemistry. For anti-fouling behaviour, perfluorinated molecules will be used to create glass with a very high surface energy, and pegylated compounds will be used for their known anti-fouling characteristics in biomimetic anti-fouling coatings. After functionalization of the glass surfaces, the anti-fouling properties will be analysed using fluorescence microscopy and quantitative STORM measurements.
Subsequently, a commercially available silane linker molecule with an NHS-ester functional-ized end-group will be incorporated in the obtained anti-fouling surface for reactivation of this surface. Analysis using fluorescence microscopy will show if BTA-fibers indeed bind to these linker molecules, while maintaining their fiber-like shape and dynamic behaviour.
The final goal is to come up with a general method of producing glass coverslips that can be used for real-time imaging of the dynamics of supramolecular fibers in solution. If surface treatment is promising and successful, also other compounds and structures can be studied via selective binding to these glass coverslips.
C
HAPTER2
BTA-
SYNTHESIS AND DYE LABELINGL
ately, state-of-the-art experiments aim to investigate the dynamics of BTA-fibers using fluorescence microscopy, and for that it is necessary to acquire BTA-fibers with fluorescent properties. These are obtained by assembling fibers in which a small percentage of the monomers are labeled with a fluorescent dye molecule. This chapter describes how these BTA-molecules are obtained after reduction of BTA-triazide and subsequent coupling of BTA-triamine to a fluorescent cyanine dye (Cy3 and Cy5).2.1 Reduction of azide
A BTA-monomer has three arms with functional end-groups, schematically depicted in figure 2.1. The commercially available fluorescent dye molecule we use to couple with the BTA contains an NHS-ester reactive group. To react with our BTA, we need to obtain a BTA-monomer with primary amines at the ends of the three arms.
The triamine cannot be stored for too long, because it is unstable over time. The BTA-triazide does not degrade appreciably over time when stored at minus 20 degrees Celsius, and was therefore used as the starting material for the synthesis. Thus, the first reaction step is a reduction from precursor BTA-triazide to BTA-triamine. The reaction scheme of this reduction is shown in figure 2.2.
We used Staudinger conditions for the reduction that was performed. This reaction was named after Hermann Staudinger, who in 1919 reported that azides could react with triphenylphosphine to form phosphazide.[28] The reaction produces a high phosphazide yield and is nowadays commonly referred to as the Staudinger reduction. The exact reaction mechanism is shown in figure 2.3. It entails a nucleophilic attack of the phosphine at the terminal nitrogen atom of the azide, that eventually produces an iminophosphorane intermediate. Nitrogen gas is evolved
FIGURE2.1. Different end-groups of a BTA-monomer.
FIGURE2.2. Reaction scheme of reduction of BTA-triazide to BTA-triamine.
during this reaction, making it irreversible. In the second step, this intermediate is hydrolysed to form the amine and triphenylphosphine oxide. The reaction conditions are very mild and are here preferred over hydrogenation using palladium on carbon.
Exact experimental details can be found in the materials and methods section at the end of this chapter. Analysis using1H-NMR and IR-spectroscopy showed complete reduction of the starting material. The spectra can be found in appendix A. The desired product was obtained with a yield of 70 mg (55µmol), or 74%.
2.2. COUPLING WITH FLUORESCENT DYE
FIGURE2.3. Reaction mechanism of Staudinger reduction.
2.2 Coupling with fluorescent dye
Next step was coupling the obtained BTA-triamine with a fluorescent dye. Commercially available NHS-esters of the dye were available for this. In this case, two different dyes were used, namely the Cy3 and Cy5 cyanine dyes, because of their high extinction coefficient and their fluorophore emission maximum in the green and red region. These dyes have an activated ester that can react with primary amines.
The coupling of this dye with an amine is strongly pH-dependent reaction. At a low pH-value, the amino group is protonated, so that no reaction takes place. The optimal pH-value for reaction is around 8.3-8.5. At a higher value, NHS-ester hydrolysis is fast, so that modification yield diminishes.
The coupling reaction is performed as a statistical reaction, because the starting material is a BTA-monomer with three primary amines, that can all react with the dye-ester. The coupling reaction is shown in figure 2.4 and figure 2.5 for the case of Cy5. The preferred product is the mono-labeled BTA-dye, but during the reaction also the di- and tri-labeled BTA-dye molecules are formed. A stoichiometric ratio of 0.8 was used in the reaction, maximizing the theoretical relative yield of the mono-labeled BTA-dye.
2.3 Separation of desired product from statistical mixture
The desired product is the mono-substituted BTA-dye, because for detecting a BTA-monomer, only a single fluorophore is required. Thus, it was necessary to separate it from the mixture with unreacted BTA-triamine, di-substituted and tri-substituted BTA-dye. Separation of the multiple components has been performed using reversed-phase high-pressure liquid chromatography. The crude mixture of dry product was dissolved in a 30:70 mixture of acetonitrile and demineralized water. Next, the solution mixture was injected into the column and then run with a gradient of
FIGURE2.4. Reaction scheme of coupling of the BTA-triamine with a cyanine dye.
(a) (b) (c)
FIGURE2.5. Coupling reaction of BTA-triamine (a) after adding 0.8 eq of Cy5-NHS (b) and dissolving in DMF (c).
acetonitrile (MeCN) and demineralized water with 0.1% TFA from 40% MeCN to 50% MeCN in 10 minutes.
After performing these steps, multiple compound fractions were found, separated and charac-terized by mass. The mass-spectra can be found in appendix A. Some unidentified and unwanted side products were present, which are indicated by the multiple peaks in the spectra. These other compounds competed with the BTA-triamine in the coupling reaction, resulting in a mixture of different based compounds, each with or without the dye-molecule. The desired BTA-diamine-monoCy3 and BTA-diamine-monoCy5 were identified in the spectra and separated from the mixture.
2.4. ASSEMBLING BTA-FIBERS WITH FLUORESCENTLY ACTIVE MONOMERS
Each fraction with the mono-labelled BTA-dye was lyophilized and subsequently redissolved in a small volume of 30:70 acetonitrile and demineralized water. After recombination of these small solutions and another lyophilization step, the final product was obtained. The final yield was determined to be 1.6 mg (5%) for BTA-Cy3 and 1.2 mg (4%) for BTA-Cy5 as a rough estimate after weighing. The dried products obtained after final purification were dissolved as a stock solution in DMSO to an approximate concentration of 1 mM.
2.4 Assembling BTA-fibers with fluorescently active monomers
2.4.1 Calculating exact yield using calibration curve
After obtaining BTA-Cy3 and BTA-Cy5 monomers, the exact yield was determined by measuring absorbance spectra and constructing a calibration curve. Dilution series of both the Cy3-NHS and Cy5-NHS ester dyes in DMSO were made (shown in figure 2.6), after which absorption spectra were recorded. The absorption coefficient was determined by a linear fit through the origin. The absorption values at 554 nm for the Cy3-NHS ester solutions and values at 649 nm for the Cy5-NHS ester solutions were used for plotting the calibration curve, which are shown in figure 2.7. The absorption coefficient of Cy3-NHS ester in DMSO was found to be 101·103 M−1·cm−1 and the absorption coefficient of Cy5-NHS ester in DMSO was calculated at 158·103 M−1·cm−1.
(a) (b)
FIGURE 2.6. Dilution series of Cy3-NHS ester (a) and Cy5-NHS ester (b) in DMSO, series range: 10 - 5 - 2.5 - 1.25 - 0.625µM.
Subsequently, the absorption of diluted samples of the synthesized BTA-Cy3 and BTA-Cy5 stock solutions were determined. These measurements showed that the exact concentrations of these solutions were 0.69 mM for the BTA-Cy3 solution and 0.43 mM for the BTA-Cy5 solution.
Using these values, a more precise estimation of the reaction yield could be calculated than weighing. It was found that the exact yield of the BTA-Cy3 was 0.35 mg and of the BTA-Cy5
(a) (b)
FIGURE2.7. Calibration curve for Cy3-NHS ester (a) and Cy5-NHS ester (b) in DMSO.
was 0.22 mg. After this final determination, the stock-solutions could be used for assembling BTA-fibers to desired specification.
2.4.2 Stock solutions and stack-assembly
Two stock solutions of mono-labelled BTA-Cy3 and BTA-Cy5 in DMSO were prepared and used for the assembly of supramolecular polymer samples. The mono-labelled Cy3 and BTA-Cy5 were mixed with BTA-3OH monomers (see figure 2.1, R = OH), after which milliQ water was added to obtain two stock-solutions of 25µM total BTA concentration. The ratio between BTA-Cy3/BTA-Cy5 and BTA-3OH was chosen, so that 2% of the monomers were labeled with a dye molecule. The samples were then equilibrated for 24 hours before experiments, crucial for reliable self-assembly of BTA-fibers. Exact experimental details can be found in the materials and methods section at the end of this chapter. The obtained stock-solutions in milliQ are used throughout all further experiments mentioned in this thesis.
2.5 Conclusion
BTA-triamine was synthesized using the Staudinger reaction. The reduction from BTA-triazide towards BTA-triamine was successful, shown by analysis using1H-NMR and IR-spectroscopy.
The desired BTA-triamine was obtained with a yield of 70 mg (55µmol), or 74%.
After a coupling reaction, both the mono-labeled BTA-Cy3 and BTA-Cy5 were obtained. The final yield turned out to be relatively low, because of undesired side-products that competed in the coupling reaction. We managed to separate and purify the final product, resulting in a yield of 0.35 mg of the BTA-diamine-monoCy3 and 0.22 mg of the BTA-diamine-monoCy5.
2.5. CONCLUSION
Even though the yield was low, it was enough for assembling the desired BTA-fibers, for which the mono-labelled BTA-Cy3 and BTA-Cy5 were mixed with BTA-3OH monomers. Finally, two stock-solutions of 25µM were obtained in which 2% of the monomers were labeled with a dye molecule.
2.6 Materials and methods
2.6.1 Materials
All commercial reagents were purchased from Aldrich and used as received unless stated otherwise. All solvents were purchased from Biosolve and used without further purification unless stated otherwise. Water was purified on an
All commercial reagents were purchased from Aldrich and used as received unless stated otherwise. All solvents were purchased from Biosolve and used without further purification unless stated otherwise. Water was purified on an