Faculty of Bioscience Engineering Academic Year 2015 – 2016
Self-assembly of synthetic polymer modified calcium carbonate particles
Jeroen Beheyt Promotor: Prof. dr. ir. Andre Skirtach Tutor: Bogdan Parakhonskiy
Masterproef voorgedragen tot het behalen van de graad van Master of Science in de industriële wetenschappen: biochemie
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Date: Signed:
Prof. Dr. Ir. Andre Skirtach
Prof. Dr. Bogdan Parakhonskiy
Promotor
Tutor
Jeroen Beheyt
Foreword During this academic year, I was fortunate enough to find colleagues, (post-)doctorates and professors that helped me with my thesis. In this foreword I want thank everyone for their (in)direct contribution to my work. Namely I’d like to thank my promotor, Ph. D. Andre Skirtach, who helped me a lot with understanding the basic and more advanced theoretical background. He was also very accessible for questions or discussing potential hypotheses on the results achieved. Furthermore, when things didn’t go the way they should go, he was always very understanding and helpful to look for a fast solution. Secondly, my tutor in the lab, Bogdan Parakhonskiy, greatly assisted me in achieving the results by aiding in setting up new protocols, analyzing results and always being available to give access to the lab. His immense patience surely motivated me to continue working on a daily base. Additionally, in the laboratory, at the rare times Bogdan was not available for questions, I could always count on the knowledge of Dmitry Khalenkow and Alexej Yashchenok. Especially at times Bogdan was out of office for a few days, they did a great job replacing him, with Dmitry also providing knowledge on microscopy and Alexej helping with the Raman spectrometer. I also want to thank Xavier Castellví and Isabel Soler, two fellow thesis students, for keeping a good atmosphere in the lab. During the long waiting times inherent to several methods used, they were always ready to have interesting conversations. When we had long, hard working days in the lab, we could always count on each other to stay motivated to keep going until the experiment was finished. For the writing part of the thesis, I would like to thank my friends and family for keeping my spirit high at all times during the semester. Without them, my motivation would be lower to work hard on the times it's needed. In particular I want to thank Agnieszka Kania as well, for designing some schematics in my thesis and my mother for proofreading the work. Last but not least, a thank you to the several professors at the University of Ghent and additionally at the University of Natural Resources and Life Sciences (Vienna) for providing me a good education on both the theoretical as the practical aspect of biochemistry and biotechnology. Science always fascinated me, but choosing for biological sciences, getting introduced to the different aspects, was the spark that ignited the fire of my interest in biotechnology. Without their contribution to my knowledge, it would not be possible to finish or even start this thesis. In conclusion, a big thank you to everyone that directly or indirectly helped me create this master thesis.
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Abstract Self-assembly of spherical vaterite calcium carbonate particles on glass and plastic surfaces. Both the particles as the surface can be modified with polymer coatings. Nanoparticle coatings have been used for several applications, however the self-assembly of a monolayer with these particles has not yet been researched, neither their (dis)advantages. This problem could be solved by investigating several parameters and their effect on the layer: cleaning protocol, polymer coating method, solution of particles and polymers, concentration, volume and interactions of the polymers. On RCA-treated objective glasses, best results were achieved for drops of 5 µL with a particle concentration of 17,5 mg/mL in water. The polymer coatings that achieved the highest quality had a PEI/PSS coating on the objective and a PDADMAC/PSS coating on the vaterite. This combination covered 86% of the surface. In an untreated 96-well, monolayer self-assembly was possible with a particle concentration of 10 mg/mL in a 50% ethanol solution and a volume of 30 µL. The polymer interactions gave the highest monolayer quality with PEI on the microwell and PDADMAC/PSS on the particles. Two applications were researched: Surface-Enhanced Raman Spectroscopy (SERS) and cell growth. For Raman spectroscopy, the signal could be enhanced by the order of two. Cell growth was inhibited by the polymer PEI on the well.
Keywords: Self-assembly, monolayer, calcium carbonate, SERS, Toxicity
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Table of Contents
Foreword .............................................................................................................................. 1 Abstract................................................................................................................................ 2 Table of Contents ................................................................................................................ 3 Abbreviations ...................................................................................................................... 7 List of Figure and Tables .................................................................................................... 8 Figures............................................................................................................................... 8 Tables ...............................................................................................................................10 1.
Introduction .................................................................................................................12
2.
Literature Review ........................................................................................................14 2.1.
Nano- and Microparticles........................................................................................14
2.1.1.
Mechanical properties .....................................................................................14
2.1.2.
Calcium Carbonate Particles ...........................................................................15
2.1.3.
Synthesis of Calcium Carbonate Particles .......................................................17
2.1.4.
Silver Nanoparticles ........................................................................................19
2.2.
Polymer Coatings ...................................................................................................21
2.2.1.
Structure and properties ..................................................................................21
2.2.2.
Polyethyleneimine (PEI) ..................................................................................21
2.2.3.
Polystyrene Sulfonate (PSS) ...........................................................................22
2.2.4.
Poly(Allylamine Hydrochloride) (PAH) .............................................................22
2.2.5.
Poly(Diallyldimethylammonium Chloride) (PDADMAC) ...................................23
2.2.6.
Polymer Coatings on Calcium Carbonate Particles .........................................24
2.3.
Surface...................................................................................................................27
2.3.1.
Objective Glass ...............................................................................................27
2.3.2.
Microwell .........................................................................................................28
2.3.3.
Hydrophobicity ................................................................................................28
2.4.
Self-Assembly of monolayer ...................................................................................29
2.5.
Raman Spectroscopy .............................................................................................30
2.6.
Toxicity ...................................................................................................................30
2.6.1.
HeLa ...............................................................................................................31
2.6.2.
alamarBlue assay............................................................................................31 3
3.
Goals ............................................................................................................................32
4.
Materials and Methods ................................................................................................33 4.1.
Materials ................................................................................................................33
4.2.
Polymer Coatings ...................................................................................................33
4.3.
Analysis..................................................................................................................33
4.4.
Objective Glass ......................................................................................................34
4.4.1.
Cleaning Procedure.........................................................................................34
4.4.2.
Dip Coating .....................................................................................................35
4.4.3.
Drop Coating ...................................................................................................35
4.5.
4.5.1.
Cleaning Procedure.........................................................................................35
4.5.2.
Coating of Microwell ........................................................................................35
4.6.
Particle Synthesis ...................................................................................................35
4.6.1.
Synthesis Big Spherical Vaterite Particles .......................................................35
4.6.2.
Coating of Particles with Polymer(s) ................................................................36
4.6.3.
Particle Coating: Coating of Particles with Silver .............................................36
4.6.4.
Layer Coating: Silver Staining of Particles .......................................................36
4.7.
Particle Concentration ............................................................................................36
4.7.1.
Objective glass ................................................................................................36
4.7.2.
Microwell .........................................................................................................36
4.8.
Interaction Particles and Objective Glass ...............................................................37
4.8.1.
Objective Glass Coatings: ...............................................................................37
4.8.2.
Particle Coatings: ............................................................................................37
4.9.
5.
Microwell ................................................................................................................35
Interaction Particles and Microwell .........................................................................37
4.9.1.
Microwell Coatings: .........................................................................................38
4.9.2.
Nanoparticle Coatings: ....................................................................................38
4.10.
Application: SERS ..............................................................................................38
4.11.
Application: Toxicity Test ....................................................................................39
Results .........................................................................................................................40 5.1.
Hydrophobicity Test................................................................................................40
5.1.1.
Objective Glass Cleaning Procedure ...............................................................40
5.1.2.
Coating of the Objective Glass ........................................................................40
5.1.3.
Solution of Particles.........................................................................................42 4
5.2.
5.2.1.
Objective Glass ...............................................................................................43
5.2.2.
Microwell .........................................................................................................44
5.3.
6.
Monolayer Creation ................................................................................................43
Polymer Interactions...............................................................................................45
5.3.1.
Polymer Coated Objective Glass .....................................................................45
5.3.2.
Polymer Coated Particles on Objective Glass .................................................46
5.3.3.
Polymer Interactions Objective Glass ..............................................................46
5.3.4.
Polymer Coated Microwell ...............................................................................48
5.3.5.
Polymer Coated Particles on Microwell ...........................................................48
5.3.6.
Polymer Interactions Microwell ........................................................................49
5.4.
Application: SERS ..................................................................................................50
5.5.
Application: Toxicity................................................................................................52
Discussion ...................................................................................................................54 6.1.
Hydrophobicity Test................................................................................................54
6.1.1.
Objective Glass Cleaning Procedure ...............................................................54
6.1.2.
Coating of the Objective Glass ........................................................................54
6.1.3.
Solution of Particles.........................................................................................56
6.2.
Monolayer Creation ................................................................................................57
6.2.1.
Objective Glass ...............................................................................................57
6.2.2.
Microwell .........................................................................................................59
6.3.
Polymer Interactions...............................................................................................61
6.3.1.
Polymer Coated Objective Glass .....................................................................61
6.3.2.
Polymer Coated Particles on Objective Glass .................................................61
6.3.3.
Polymer Interactions Objective Glass ..............................................................62
6.3.4.
Polymer Coated Microwell ...............................................................................63
6.3.5.
Polymer Coated Particles on Microwell ...........................................................63
6.3.6.
Polymer Interactions Microwell ........................................................................63
6.4.
Overview of monolayer self-assembly parameters .................................................65
6.5.
Application: SERS ..................................................................................................65
6.6.
Applications: Toxicity ..............................................................................................66
7.
Conclusion...................................................................................................................68
8.
Prospects .....................................................................................................................70
References ..........................................................................................................................71 5
Attachment 1: Cleaning Method ........................................................................................80 Attachment 2: PEI with(out) Salt .......................................................................................81 Attachment 3: Multilayer Formation ..................................................................................82 Attachment 4: Agglomeration ...........................................................................................83 Attachment 5: Area Fraction..............................................................................................84 Attachment 6: Concentration Determination Objective Glass ........................................86 Attachment 7: Concentration and Volume Determination Microwell ..............................87 Attachment 8: Interactions Polymers Objective ...............................................................89 Attachment 9: Interaction Polymers Microwell ................................................................90 Attachment 10: Raman spectra Rhodamine .....................................................................91 Attachment 11: Raman spectra 4-MBA .............................................................................92 Attachment 12: Toxicity Test .............................................................................................93
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Abbreviations 4-MBA EF LbL PAH PDADMAC PEI PEM PSS SERS
= = = = = = = = =
4-Mercaptobenzoic Acid Enhancement Factor Layer-by-Layer Poly(Allylamine Hydrochloride) Poly(Diallyldimethylammonium Chloride) Polyethyleneimine Polyelectrolyte Multilayers Polystyrene Sulfonate Surface-Enhanced Raman Spectroscopy
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List of Figure and Tables Figures Figure 1: Collection of spherical vaterite calcium carbonate microparticles, 400x zoom. Figure 2: Different crystal forms of calcium carbonate (ACC = Amorphous Calcium Carbonate) (Niedermayr and Immenhauser, 2016). Figure 3: Images of CaCO3 microparticles. a) spherical vaterite b) ellipsoid vaterite c) rhombohedral calcite. Scale: bar is 2 µm (Yashchenok et al., 2013) Figure 4: Image of star-like vaterite (Trushina et al., 2016) Figure 5: Image of needle-like aragonite (Hu and Deng, 2004) Figure 6: Polyelectrolyte capsule based on calcium carbonate with silver nanoparticles adsorbed. Time of silver-mirror reaction: a) 45 min b) 70 min (Bukreeva et al., 2009) Figure 7: Structure of PEI (Sigma-Aldrich, 2016d) Figure 8: Structure of PSS (Sigma-Aldrich, 2016c) Figure 9: Structure of PAH (Sigma-Aldrich, 2016a) Figure 10: Structure of PDADMAC (Sigma-Aldrich, 2016b) Figure 11: PEM assembly by adsorption of differently charged polymers, simplified (Decher et al., 1992) Figure 12: Distribution of particles in solution with different contact angles. The resulting sedimentation is different. The representation is simplified (Kania, 2016c). Figure 13: Example of angle measurement. Figure 14: Area fraction measurement protocol using ImageJ. Figure 15: Contact angle for different cleaning methods. RCA treatment and ethanol treatment with wash show similar results. 6 angles are measured out of 3 drops. Figure 16: Contact angle for different coating methods. There is a significant difference between the two coating methods. 6 measurements out of 3 samples are used for the drip coating, 12 contact angles out of 6 samples are used for the drop coating measurement. Figure 17: Contact angle for PEI with and without salt. For all 3 objective coatings, no significant difference between the two PEI solutions is noted. 12 contact angles are measured out of 6 drops. Figure 18: Water and 50% ethanol drops on objective glass and microwell. 6, 10 or 12 contact angles are measured out of 3, 5 or 6 samples. Figure 19: Concentration determination of 5 µL vaterite solution on objective glass. No significant differences from 35 to 20 mg/mL. 17,5 mg/mL and 15 mg/mL show a decrease. 18 area fraction measurements out of 6 samples. Figure 20: Microwells covered with a 5 mg/mL vaterite solution in 50% ethanol. Left: 20 µL of the solution is added. Right: 40 µL of the solution is added. Figure 21: Microwells covered with a 10 mg/mL vaterite solution in 50% ethanol. From left to right, respectively 20, 30 and 40 µL of this solution is added. Figure 22: Area fraction of vaterite particles covering (un)coated objective glass. 9 measurements out of 3 samples. 8
Figure 23: Area fraction of (un)coated vaterite particles on objective glass. 9 measurements out of 3 samples. Figure 24: Area fraction of (un)coated vaterite particles on (un)coated objective glass. Bottom scale is the polymer coating of the objective itself, whilst the blocks with the same colours contain the same polymer on the particles. 9 measurements out of 3 samples. Figure 25: Area fraction of vaterite particles on (un)coated microwells. 9 measurements out of 3 samples. Figure 26: Area fraction of (un)coated vaterite particles on microwells. 9 measurements out of 3 samples. Figure 27: Area fraction of (un)coated vaterite particles on (un)coated microwells. 9 measurements out of 3 samples. Figure 28: Raman spectrum for rhodamine. From bottom to top: Rhodamine reference; uncoated objective with silver coated vaterite; PEI/PSS coated objective with silver coated vaterite; uncoated objective with the vaterite monolayer coated with silver. Figure 29: Raman spectrum for 4-MBA. From bottom to top: 4-MBA reference; PEI coated objective with silver coated vaterite; PEI/PSS coated objective with silver coated vaterite; PEI/PSS/PDADMAC coated objective with silver coated vaterite. Figure 30: Schematic representation of the adsorption of PSS on dip/drop coated objective glasses. On the left, the dip coating is shown. On the right, the supposed effect is pictured. The contact angles are not according to their real value; the representation is simplified (Kania, 2016b). Figure 31: Crystal formation with different methods of adsorbing the polymers. Figure 32: Calcium carbonate particles assembled in 50% ethanol and water. Agglomeration and separation is higher in the 50% ethanol sample. Figure 33: Concentration determination of vaterite solution in different concentrations on objective glass. 20 mg/mL, 17,5 mg/mL and 15 mg/mL are shown. Figure 34: Schematic representation of origin of gaps between agglomerates. The representation is simplified (Kania, 2016a). Figure 35: Schematic representation of sedimentation of particles in a tube. The sedimentation happens almost instantly and can therefore not be avoided entirely. Samples are taken in the middle of the solution, where the concentration is most likely lower than the original one. Figure 36: Schematic representation of the two different evaporation effects. The representation is simplified. Figure 37: Optimal results for self-assembly of calcium carbonate particles on objective glasses. Figure 38: Self-assembly of calcium carbonate particles in microwells. Figure 39: Perfect monolayer self-assembly of PDADMAC/PSS coated calcium carbonate in PEI coated microwells. Figure 40: Examples of monolayer or multilayer assembly evaluation. Figure 41: Examples of agglomeration evaluation. Figure 42: Examples of area fractions.
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Figure 43: Concentration determination of vaterite on objective glass. A) 35 mg/mL B) 30 mg/mL C) 25 mg/mL D) 20 mg/mL E) 17,5 mg/mL and F) 15 mg/mL. Heavy multilayer formation in samples A-D. Gaps appearing in E, but much more present in F. Figure 44: Pictures of calcium carbonate samples with different concentrations and volume. Figure 45: Profile distributions of calcium carbonate samples with different concentrations and volume. Figure 46: Raman spectra of Rhodamine and particles coated with two methods on differently coated surfaces. Rhodamine is the bottom line (green). On top, in red, is PEI + BS + Silver, vaterite on top of PEI coated objective glass with silver nanoparticles on the multilayer. This is the control. The three blue lines above it are from bottom to top: None + BS-Silver (silver-coated vaterite on top of uncoated objective glass), PEI-PSS + BS-Silver (silver-coated vaterite on top of PEI/PSS coated objective glass) and None + BS + Silver (vaterite on top of uncoated objective glass with silver nanoparticles on the multilayer). Y-axis is a.u. (arbitrary unit). Figure 47: spectra of 4-MBA and particles coated with two methods on differently coated surfaces. From top to bottom: 4-MBA spectrum, PEI + BS-Silver (silver-coated vaterite on top of PEI coated objective glass), PEI-PSS + BS-Silver (silver-coated vaterite on top of PEI/PSS coated objective glass), PEI-PSS-PDADMAC + BS-Silver (silver-coated vaterite on top of PEI/PSS/PDADMAC coated objective glass) and None + BS-Silver (silver-coated vaterite on top of uncoated objective glass). The top spectrum is the control. Y-axis is a.u. (arbitrary unit). Figure 48: Results of alamarBlue assay on microwell.
Tables Table 1: Wavenumbers at which typical molecules for SERS show peaks. Table 2: Enhancement factors of the different coating combinations, with Rhodamine and 4MBA. Table 3: Fluorescence of polymer modified calcium carbonate particles on top of polymer modified microwells. A control without calcium carbonate is used. Fluorescence values are rounded up or down to the nearest value dividable by 500. Table 4: Overview of monolayer self-assembly parameters Table 5: Cleaning methods and their angle measurement and deviation. RCA treatment and ethanol treatment with wash show similar results. RCA treatment is preferred. 6 angles are measured out of 3 drops. Table 6: Comparison between using PEI with or without salt and water as a reference. No significant difference (p 8.5), the swelling of the bilayer was found to be discontinues. This is related to the discontinuous changes in the ionization degree of the PAH (Itano et al., 2005). The humidity of the air surrounding the PEM reversibly relates to the thickness of the PEM film. The higher the humidity, the thicker the layer becomes. This was shown in a multilayer of PAH and PSS (Wong et al., 2004). The influence of salt was also researched in Irigoyen et al. (2012). A PEM consisting of PSS and PDADMAC was assembled in the presence of either water either a 3 M NaCl solution. The PEM's thickness was reduced by 46% in the presence of water in comparison with the salt solution. This effect is reversible and therefore opens up applications, for example creating a controllable PEM barrier (Irigoyen et al., 2012). Another effect of salt on the coatings, is the agglomeration of the polymer on the particles. Unlike the swelling and deswelling of the microcapsule, this effect takes place at the coating part itself. When 0.005 mM was added to the polymer solution, the radius of the PDADMAC/PSS coated particles was the lowest (Starchenko et al., 2012). Another parameter that influences the size of the PEM, is the charge of the polymer itself. In this research, homogeneous PDADMAC was defined as having a degree of charge of 100% and homogeneous N-methyl-N-vinylacetamide (NMNV) having a degree of charge of 0%. Copolymerization of both polymers gave charges within the range of 0 to 100%. Steitz et al. (2001) found for polymers with a degree of charge lower than 50%, no changes in film thickness were found. When the degree of charge is higher than 50%, the film thickness has a linear correlation with the degree of charge. Swelling due to the increased salt concentration was not noticed in PEMs with a degree of charge lower than 50%. At 100% degree of charge, the thickness of the PEM increased with increasing salt concentration (Steitz et al., 2001). One of the advantages of using PEMs with calcium carbonate particles, is that they significantly inhibit the recrystallization of the vaterite particles into calcite. This was proven in Sergeeva et al. (2015), where vaterite calcium carbonate particles were coated with an LbLassembled PAH/PSS multilayer. The effect was thought to be related to the suppression of ion exchange of the particle (Sergeeva et al., 2015) Another advantage of LbL, is the attachment of silver nanoparticles on the polymeric shells. In Bukreeva et al. (2008), calcium carbonate particles were coated with four bilayers of PAH/PSS. The negative charges of the outer PSS layer, allowed the positively charged silver nanoparticles to be adsorbed (Bukreeva et al., 2008; de Dardel and Arden, 2008; Dondi et al., 2012). These silver nanoparticles could be released using a laser (Bukreeva et al., 2009).
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On the contrary, a disadvantage is that materials can diffuse through the PEM and destroying the precise structure of the multilayer. For instance, hydrogen bonds and electrostatic interactions could be changed by adding chitosan to a multilayer of poly(ethylene oxide) and poly(acrylic acid). However, this effect could be completely blocked by adding a layer of PAH (Gilbert et al., 2013). When particles are coated with a high amount of layers (n>20), the calcium carbonate at the centre of the porous sphere, can be dissolved again. This process will leave a hollow nanosphere, a shell of only the polyelectrolyte multilayer. This process was proven with gold nanoparticles and opens up possibilities for drug delivery purposes (Schneider and Decher, 2004). In Skirtach et al. (2008), silica nanoparticles were coated with PDADMAC and PSS, where the PDADMAC also contained golden nanoparticles. Using a near-infrared laser, the temperature of the gold nanoparticles increased, deforming the polymeric shells. This released the content of the microcapsule. The process was found to be reversible (Skirtach et al., 2008). Adding a polymer to the calcium carbonate, also means the overall charge of the surface of the molecule can be changed. In one research, the negative charged calcium carbonate was coated with a positive charged PAH polymer. This allowed the particles to be absorbed by human smooth muscle cells. This proved to have an effect on almost 2000 genes, influencing the cytoskeleton and the cell adhesion, along with some other effects (Wang et al., 2012a). 2.2.7. Applications of Polymer Coatings on (Calcium Carbonate) Particles The different properties of (calcium carbonate) particles coated with polymer and/or silver nanoparticles have proven their use in research. As mentioned earlier, microcapsules of a core and surrounding multilayer can release the core particles. This release can be stimulated chemically with certain substrates, by changing the pH, by changing the ionic strength, by laser, enzymes, temperature, … (Ariga and Hill, 2008; Delcea et al., 2011; Kiryukhin et al., 2013; Marchenko et al., 2012; Sergeeva et al., 2015; Skirtach et al., 2010; Wuytens et al., 2014). This particular property opens up possibilities for intracellular delivery and in particular drug release. For instance, golden nanoparticles were used to create a hollow capsule. The encapsulated material could be released by irradiating the particles (Bédard et al., 2009). In a different research, the content of calcium carbonate with a PAH and PSS multilayer coating, could be released by degrading the PEM with Pronase, an enzyme (Marchenko et al., 2012). A thick multilayer of several polymers, including chlorhexidine acetate, could be attached to a wound dressing. Chlorhexidine acetate has antibacterial properties, therefore improving wound recovery after application (Agarwal et al., 2012). Donath (2002) found a PSS/PAH multilayer permeable for proteins at an ionic strength of 100 mmol, therefore replacing the content of the shell and including these proteins. This creates potential applications such as "cages" for chemical reactions or containers for these proteins (Donath et al., 2002).
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An entirely different application using the same effect is within the field of membrane filtration. Since the structure of the polymer can be changed using a wide field of parameters (see earlier), a membrane coated with polymer could be functionalized. The parameters can then influence the permeability of the membrane, allowing a controlled membrane filtration (Bruening and Adusumilli, 2011). Further, metal hydrides are components that can be used to adsorb H2 gas, useful in cars. These components however were very reactive to air and moisture, which is disadvantageous for commercial use. To counter this effect, the metal hydride could be covered with a multilayer film, decreasing the effects of the moisture and the air significantly (Dobbins et al., 2007). In the field of immunodetection, cellulose fibre network could be coated with a PEM that can interact with a detection antibody. This allowed the researchers to find traces of a model virus in lower concentrations than with conventional techniques such as ELISA (Larsson et al., 2013).
2.3.
Surface
This thesis focuses on monolayer self-assembly of particles on top of two surfaces: objective glass and microwells. These surfaces can be modified with polymers using several methods (Merz and Bard, 1978). One of them is the dip coating method, where the surface is put in a bath of solution. Another one mentioned in the literature is the spin coating method. Here, the surface is spun around, with the polymer solution added in the middle of the surface. Centrifugal forces spread the solution. The higher the spinning speed, the thinner the coating will be (Scriven, 1988). A third method of polymer adsorption is the drop coating method. Here, a drop is put on the surface and gravitational forces will allow interactions between the polymers and the surface (Gowda et al., 2011). Polyelectrolyte multilayer (PEM) creation can be improved by using PEI as an anchoring polymer at the bottom of the PEM. This is due to the PEI forming a matrix which acts like a scaffold for the additional polymers. This results in a thicker polymer layer as well as an increased stability of the PEM (Kolasińska et al., 2007). 2.3.1. Objective Glass The objective glasses used are ThermoFisher Scientific Frosted Microscope slides. These slides are charged (Thermo Scientific, 2011). This may or may not influence adsorption of the polymers on the surface, as well as monolayer assembly of the (modified) calcium carbonate. The charge of the microscope slides can be influenced by cleaning the objective glass with an ethanol solution (Stonebraker and Wise, 1969). Another option of slide treatment is the RCA cleaning method, developed by Werner Kern in 1965 (Kern, 1970). This four step method is used in the industry to clean wafers before other processing steps are taken (Kern,
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1990). The first step of the RCA treatment, the Standard Clean 1 (SC-1) will get rid of organic residues as well as particles which may be left behind on the wafers. It is also proven to change the potential of the surface, which might result in different reactions with the polymers (Itano et al., 1993). This makes the treatment particularly interesting for this work. 2.3.2. Microwell The most commonly used microwells consist of polystyrene. Conventionally they are not treated before use in various research (Chu et al., 2005; Rasmussen et al., 1991; Sugimura et al., 2010). Therefore, the microwells will not be treated. PEI is however used as an anchoring polymer (Kolasińska et al., 2007; Vancha et al., 2004). 2.3.3. Hydrophobicity Measurement of the relevant surface properties can be done quite effectively by estimation of the hydrophobicity. Hydrophobicity is the lack of interaction between water and a molecule, which seemingly causes repulsion of the molecule (Ben-Naim, 1980). Hydrophobic particles prefer interaction with other hydrophobic molecules. As the molecules used in this work are all quite hydrophilic, the hydrophobicity should be as low as possible (Bronstein et al., 1998; Dautzenberg, 2000; Wang et al., 2009; Zamarreño et al., 2007). Hydrophobicity can be measured easily by the contact angle. The contact angle is the angle between the surface and the tangent of the drop that is put on it (Yuan and Lee, 2013). The lower the contact angle, the lower the hydrophobicity (Mozes and Rouxhet, 1987). Another benefit of a low contact angle is the improved distribution of particles suspended in the solution. This allows the particles to assemble more evenly over the surface (de Gennes, 1985). This is demonstrated in Figure 12.
Figure 12: Distribution of particles in solution with different contact angles. The resulting sedimentation is different. The representation is simplified (Kania, 2016c).
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Contact angles for the different polymer layers have been researched already. They are mostly defined by the polymer that is on top of the PEM and not so much influenced by the ones underneath (Wong et al., 2004). A PAH/PSS coating on top of glass showed a contact angle of 10° for PAH and an angle of 20° for PSS. This angle increased by the number of layers adsorbed (Palamà et al., 2013). Another PAH angle measurement also showed a contact angle of lower than 20° (Lingström et al., 2007). This contact angle can be influenced by adjusting the pH of the solution; it increases with increasing pH (Elzbieciak et al., 2008). The contact angle of PAH in a PAH/PSS multilayer is lower than the contact angle of PDADMAC in a PDADMAC/PSS multilayer (Koehler et al., 2014). Using an ethanol solution instead of water to measure contact angles, will also decrease it (Lundgren et al., 2002). This effect could be useful for choosing the optimal solution to suspend the particles in.
2.4.
Self-Assembly of monolayer
Particles have already been assembled in monolayer-like structures, or strictly aligned on a grid, however this process takes energy and resources (Karg et al., 2015). With selfassembly, expensive equipment or methods can be avoided, since this process is passive. The particles align themselves on the surface due to sedimentation, evaporation of the solution and electrostatic interactions (Brinker et al., 1999; Whitesides and Grzybowski, 2002; Whitesides et al., 1991). In order to self-assemble a monolayer of calcium carbonate particles, several parameters discussed previously need to be defined. The hydrophobicity of the solution will influence distribution of the particles (de Gennes, 1985). This can be measured by the contact angle (Mozes and Rouxhet, 1987). Treatment methods which change the potential of the surface, could also influence this contact angle (Itano et al., 1993; Kern, 1970; Stonebraker and Wise, 1969; Thermo Scientific, 2011). The solution the particles are suspended in, must be investigated as well for their hydrophobicity; ethanol and water are compared (Lundgren et al., 2002). The surface can be modified by polymer adsorption (Suetsugu and White, 1983). PEI is usually used as an anchoring polymer, improving the structure of the PEM (Kolasińska et al., 2007; Vancha et al., 2004). These polymers can be solved in salt solutions or in plain water, resulting in different thicknesses of the PEM (Irigoyen et al., 2012). The salinity of the polymer solution should however not influence the contact angle (de Gennes, 1985). The polymers can be adsorbed using different methods, each resulting in different PEMs (Gowda et al., 2011; Scriven, 1988). Particles might also be modified with polymers, using conventional coating methods.(Bukreeva et al., 2009). The interactions of both might influence sedimentation of the particles and therefore monolayer assembly.
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2.5.
Raman Spectroscopy
When a monolayer is established, the results are used for Surface-Enhanced Raman Spectroscopy (SERS). With Raman spectroscopy, a laser interacts with the a molecule giving a Raman signal, resulting in a Raman spectrum which can be used as a "fingerprint" to identify the molecule (Gardiner et al., 1989). This Raman signal was found to be abnormally increased on certain surfaces and was called SERS (Albrecht and Creighton, 1977; Dieringer et al., 2006; Jeanmaire and Van Duyne, 1977). With SERS, the signal is enhanced, resulting in the calculation of the enhancement factor (EF) (Severyukhina et al., 2015). This is calculated against a standard solution of typical molecules such as Rhodamine and 4 – mercaptobenzoic acid (Jensen and Schatz, 2006; Orendorff et al., 2005). Both contain typical Raman peaks at certain wavenumbers (1/cm). Using SERS, the Raman signal could be enhanced by a factor of the order of 5 until 11 (Le Ru et al., 2007). This allows the technique to be used for detection of single molecules (Blackie et al., 2009). Table 1: Wavenumbers at which typical molecules for SERS show peaks.
Rhodamine (Jensen and Schatz, 2006) 616 cm-1 1185 cm-1 1297 cm-1 1346 cm_1 1497 cm-1
4-MBA (Orendorff et al., 2005) 1073 cm-1 1575 cm-1
Calcium carbonate modified with silver nanoparticles can also be used as a platform for SERS (Parakhonskiy et al., 2010). These can be used for Raman spectroscopy (Wang et al., 2012b). Reportedly they can have an enhancement factor of the order of 6 (Parakhonskiy et al., 2014a). In combination with modifying the particles with polymers, a SERS signal was reached allowing the technique to be used in analysis of biological samples (Stetciura et al., 2013). In one research, the calcium carbonate particles were coated with a PEM and silver nanoparticles after which the calcium carbonate core was removed. The remaining hollow microsphere could be used as a SERS platform. This would allow quick identification of bacteria (Lengert et al., 2016).
2.6.
Toxicity
Nanoparticles have proven their use in cell growth systems as well as polyelectrolyte nanofilms on top of glass (Palamà et al., 2013; Yan et al., 2013). In another research, hollow PEM microcapsules, created by coating a particle with several layers then removing the core, could be internalized by the cells as well (Kastl et al., 2013). A combination of these properties allows the use of polymer coated surfaces with polymer modified particles on top to culture cells. In Parakhonskiy et al. (2015), different forms of calcium carbonate were first labelled with TRITC-Dextran for analysis and then coated with PAH/PSS (for cuboidal
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particles, the PEM consisted of PSS/PAH/PSS). The best internalisation of particles was reached with cuboid particles, but most other calcium carbonate forms reached the same level of internalisation (Parakhonskiy et al., 2015). These properties might make cell culture growth on a self-assembled monolayer particularly interesting. On the other hand, it must be noted that the anchoring PEI layer also has toxic properties, which may be disadvantageous for the cell growth (Boussif et al., 1995; Kolasińska et al., 2007; Vancha et al., 2004). 2.6.1. HeLa For this purpose, HeLa cells are used in the experiments. HeLa cells are cancer cells, retrieved from Henrietta Lacks who suffered from cervical cancer (Scherer et al., 1953). These cancer cells are the most used human cells in research (Rahbari et al., 2009). Like bacteria, they are proven to be able to internalise small particles (Gratton et al., 2008; Yan et al., 2013). The HeLa cells could be used to grow on calcium carbonate particles, justifying their use in this work (Parakhonskiy et al., 2015) 2.6.2. alamarBlue assay In order to evaluate cell culture growth, the alamarBlue assay will be used, developed by Alamar Biosciences Laboratory, Inc. With this assay, the fluorescence dye alamarBlue is added to the cells and internalised. Metabolically active cells convert the fluorescent dye from a blue and low fluorescence to a pink and high fluorescence (Lancaster and Fields, 1996). This makes the simple method good for research purposes (Voytik-Harbin et al., 1998). The disadvantage of the assay is that the fluorescent end product can be either accumulated leading to overestimation, either reduced further leading to underestimation of cellular activity (O’Brien et al., 2000). However, in an extensive research, the method was compared to another assay, BABTEC 460, and showed high similarities. This does make the alamarBlue assay a sensitive, simple and cheap method to screen cellular activity (Collins and Franzblau, 1997).
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3. Goals The main goal of this work is to determine the parameters which might influence the selfassembly of a calcium carbonate monolayer on two different surfaces: objective glasses and microwells. The particles and the surface can be modified with polymers, influencing the monolayer assembly. To demonstrate the purpose of the research, two practical applications are tested: using the calcium carbonate monolayer for SERS and for cell culture growth. To reach this goal, a logical step-by-step protocol is followed, starting from the treatment of the surface to the adsorption of polymers on the different materials. Following subgoals are defined for this protocol:
Studying the influence of different surface treatment methods on the solution distribution.
Finding optimal conditions to modify the surfaces with polymers: o
Coating methods are compared
o
Salinity effects are checked
o
The treatment of the polymer coating may influence particle distribution.
The effect of the particle suspension media is investigated.
Testing different volumes and concentrations of the particle suspension and finding the influences both parameters have on the monolayer assembly.
Studying the impact of charged polymers adsorbed on either surface either particle on the monolayer quality.
Applications: o
To investigate the ability of silver stained particles or silver stained monolayer to be used as a SERS platform
o
To study the different polymer coatings on particles and surface for toxicity.
After each subgoal is reached, optimal conditions are defined and used in the further tests. Determination of all subgoal results will allow us to reach the main goal of this work.
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4. Materials and Methods 4.1.
Materials
All used chemicals are from the purest grade commercially available. Unless specified, water means ddH2O. Unless specified, ethanol means a 70% ethanol solution. The objective glasses used are ThermoFisher Scientific Frosted Microscope slides.
4.2.
Polymer Coatings
PEI (no salt); polyethyleneimine (2 mg/mL). Average MW ~25000 by LS, Average MN 10000 by GPC, branched (Sigma-Aldrich).
PEI (salt); polyethyleneimine (2 mg/mL; 0,5M NaCl). Average MW ~25000 by LS, Average MN 10000 by GPC, branched (Sigma-Aldrich).
PSS; polystyrene sulfonate (2 mg/mL; 0,5 M NaCl). Average MW ~70000, powder (Sigma-Aldrich).
PAH; poly(allylamine hydrochloride) (2 mg/mL; 0,5 M NaCl). MW 450000 (SigmaAldrich).
PDADMAC; poly(diallyldimethylammonium chloride) (2 mg/mL; 0,5 M NaCl). Average MW 200000-350000 (medium molecular weight), 20 wt. % in H2O (Sigma-Aldrich).
4.3.
Analysis
Statistical analysis is performed using default statistical analysis functions of Microsoft Office Excel (AVERAGE; STDEV.P; ANOVA). The significance level is set at p ˂ 0.05. Every test has a repetition of at least 3 samples and often more.
Figure 13: Example of angle measurement.
Angle measurements are done with a CoolTech camera. Sideways pictures of drops are taken with this camera, after which the pictures are investigated using the analysis tool
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ImageJ (). The angle between the surface and the drop is measured, as is described in Fout! Verwijzingsbron niet gevonden..
Figure 14: Area fraction measurement protocol using ImageJ.
For area fraction calculations, 3 representable pictures are taken from each sample, after which the analysis tool ImageJ () is used. For this goal the images are binarized via setting up a treshold level in order to create a black (particles) and white (background) image of the sample. The area fraction is calculated as the amount of black pixels divided by the total amount of pixels (Severyukhina et al., 2015). Examples of different area fractions can be found in Attachment 5: Area Fraction. The distribution of the particles (multilayer formation and agglomeration) is visually determined with a microscope. Examples of how they were classified, can be found in Attachment 3: Multilayer Formation and Attachment 4: Agglomeration.
4.4.
Objective Glass
4.4.1. Cleaning Procedure Compare four different methods for washing the objective glasses used in following experiments (Kern, 1990). 1. Blank. No washing 2. Ethanol. Dip objective glass in ethanol for 5 minutes. 3. Ethanol with washing. Dip objective glass in ethanol for 5 minutes. Wash with water for 3 minutes 4. RCA clean (Standard clean 1). Mix 7 mL of NH4OH (28%) with 7 mL of H2O2 (30%). Add 35 mL of H2O. Dip objective glass in this solution for 5 minutes. Wash with water for 3 minutes. After washing is done, put a drop of water on the objective glass. Measure the angle between the objective glass and the edge of the droplet. This will provide information about the hydrophobicity of the objective glass. Repeat the experiment with a few drops.
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4.4.2. Dip Coating To coat the surface of the objective glass, put it in the polymer solution (2 mg/mL). Shake for 15 minutes. Wash in water for 3 minutes. Let the objective glass dry before use (Scriven, 1988). 4.4.3. Drop Coating Compare three different methods for coating the objective glass. Put 5 µL droplets of polymer solution on the objective glass 1. Drying. Wait until the droplet is dry. 2. No wash. After 15 minutes, remove excess polymer solution. 3. 3x wash. After 15 minutes, remove excess polymer solution. Wash by adding a droplet of water (5 µL) then removing the water. Repeat 3 times. If necessary, a new layer can be added after completion of these methods (drop or dip coating). Compare the different methods by examining the surface of the coatings with a microscopy as well as the hydrophobicity of adding a second layer of polymers.. Since PEI is well fit to form an anchor on the surface of the objective glass for the other polymers to attach to, it will be used as the first coating on the glass. However, salt might have an influence on the matrix formation. Compare PEI dissolved in a 0,5 M NaCl salt solution with one solved in water by calculating the hydrophobicity (see Fout! Verwijzingsbron niet gevonden.).
4.5.
Microwell
4.5.1. Cleaning Procedure The microwells used are already sterile. They will not be cleaned. However, measure the hydrophobicity of the 2 possible solutions that will be used to suspend the particles in: water and a 50% ethanol solution. 4.5.2. Coating of Microwell Add 40 µL of the polymer solution to the microwell and let it react for 15 minutes. Remove excess polymer solution. Wash 3 times with water; add 100 µL water to the well then remove it again 3 times (Scriven, 1988).
4.6.
Particle Synthesis
4.6.1. Synthesis Big Spherical Vaterite Particles Add 1 mL of 0,33 M Na2CO3 to 1 mL of 0,33 M CaCl2. Rapidly mix with magnetic stirrer for 60 seconds. After this, sediment the particles by centrifugation (1500 rpm; 180 s). Wash with ethanol by suspending the particles in ethanol, sedimentating them by centrifugation (1500
35
rpm; 180 s) and then removing the supernatant. Repeat the washing step three times. After this, dry the particles in the oven (60 °C) overnight (Parakhonskiy et al., 2014b). For direct use or usage within 2-3 days, the particles can be saved suspended in water without losing its properties. Ethanol will increase this time by a few days. Longer storage is possible by freezing the particles suspended in water or by drying them completely, however certain conditions can still cause the particles to change shape. Therefore, every time before use, the shape of the particle needs to be checked (Parakhonskiy et al., 2012b; Sergeeva et al., 2015; Svenskaya et al., 2013). 4.6.2. Coating of Particles with Polymer(s) Add an excess of polymeric solution (2 mg/mL) to the dry particles; for 10 mg of the particles, 500 µL will suffice. Shake for 12 minutes. Wash 3 times in the centrifuge (1500 rpm; 300 s) with ethanol (Bukreeva et al., 2009) 4.6.3. Particle Coating: Coating of Particles with Silver Mix 1 mL of 0,5 M AgNO3 with 1 mL of 0,5 M NH4OH to create Tollens' reagent. Add 0,1 mL of a 2% glucose solution as a catalyst for the reaction. Add this Tollens/glucose mix (1,1 mL) to 10 mg of the particles and shake for 1 hour. Wash with water by suspending the particles in water, sedimentating them by centrifugation (2000 rpm; 300 s) and then removing the supernatant. Repeat the washing step three times (Bukreeva et al., 2009; Dondi et al., 2012). 4.6.4. Layer Coating: Silver Staining of Particles Mix 1 mL of 0,5 M AgNO3 with 1 mL of 0,5 M NH4OH to create Tollens' reagent. Assemble a layer of particles on the objective glass. Now, add 5 µL of this Tollens' reagent to the particle spots, after which 5 µL of a 2% glucose solution is added as a catalyst. The reaction is completed after 1 hour (Clark, 2015; Dondi et al., 2012; Fontana-masson, 1980).
4.7.
Particle Concentration
4.7.1. Objective glass To cover an area the size of a 5 µL water drop (ø 2,5 mm on average) with particles (ø 3,5 µm; density = 1600 kg/m³; volume = 5 µL), theoretically a concentration of 14,66 mg/mL is needed.
Therefore testing is done with the concentrations 15 mg/mL, 17,5 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL and 35 mg/mL. The area fraction is calculated with the tool ImageJ as described in 4.3 as well as visually determining the level of multilayer formation and aggregation. A 50% ethanol solution is compared to water to suspend the particles in.
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4.7.2. Microwell For covering one well (ø 4 mm) with particles (ø 3,5 µm; density = 1600 kg/m³; concentration = 10 mg/mL), theoretically a volume of 18,76 µL is needed.
Therefore testing is done with a combination of two concentration, 5 mg/mL and 10 mg/mL, with three dilutions: 20 µL, 30 µL and 40 µL. The cross-section of each well is determined as well as the area fraction, both using the tool ImageJ as described in 4.3 as well as visually determining the level of multilayer formation and aggregation. The particles are suspended in a 50% ethanol solution for practical reasons.
4.8.
Interaction Particles and Objective Glass
Compare the interaction of four different coatings of objective glasses with three different coatings of nanoparticles. The PEI is without salt, due to the results of 4.4.3. 4.8.1. Objective Glass Coatings: 1. None 2. Dip coat with PEI (2 mg/mL). 3. Dip coat with PEI (2 mg/mL). Add a layer of PSS (2 mg/mL; 0,5 M NaCl) using the drop coat method. 4. Dip coat with PEI (2 mg/mL). Add a layer of PSS (2 mg/mL; 0,5 M NaCl) using the drop coat method. Add a layer of PAH/PDADMAC (2 mg/mL; 0,5 M NaCl) using the drop coat method. 4.8.2. Particle Coatings: 1. No coating. 2. Coated with PAH/PDADMAC (2 mg/mL; 0,5 M NaCl). 3. Coated firstly with PAH/PDADMAC (2 mg/mL; 0,5 M NaCl) and secondly with PSS (2 mg/mL; 0,5 M NaCl). After drying, weighing and coating of particles, suspend them in water (4.7.1) in order to get solutions with equal concentrations, determined in the previous experiment. Put 5 µL droplets of particle solution on the objective glass coatings and wait until they dried; the self-assembly process takes place. Using microscopy, measure the fraction area covered by the particles, the amount of layers and the aggregation of them. For this thesis, the image analyzing tool ImageJ was used to determine the area fraction. The amount of layers and the aggregation are visually concluded.
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4.9.
Interaction Particles and Microwell
Compare the interaction of four different coatings with three different nanoparticle coatings. To prepare the experiment, uncoated big spherical particles are used to determine the optimal concentration of particles, the optimal volume and the best solution for the particles, needed to create a monolayer. 4.9.1. Microwell Coatings: 1. 2. 3. 4.
No coating Coated with PEI (2 mg/mL) Coated with PEI (2 mg/mL). Add a second layer of PSS (2 mg/mL; 0,5 M NaCl). Coated with PEI (2 mg/mL). Add a second layer of PSS (2 mg/mL; 0,5 M NaCl) and then a third layer of PAH/PDADMAC (2 mg/mL; 0,5 M NaCl)
4.9.2. Nanoparticle Coatings: 1. No coating. 2. Coated with PAH/PDADMAC (2 mg/mL; 0,5 M NaCl). 3. Coated firstly with PAH/PDADMAC (2 mg/mL; 0,5 M NaCl) and secondly with PSS (2 mg/mL; 0,5 M NaCl). The particles are suspended in a 50% ethanol solution for practical reasons; experimentally determined, water dries too slow to be of practical use. The particles are dried, weighed and coated, then suspended in the ethanol (50%) in order to create equal concentrations, determined in the previous experiment. The volume of the drop is obtained as a result from the previous experiment. With the right combination of volume and concentration, put these drops in each microwell and wait until the ethanol evaporated; the self-assembly takes place. Using microscopy, measure the fraction area covered by the particles, the amount of layers and the distribution of them. For this thesis, the image analyzing tool ImageJ was used to determine the area fraction. The amount of layers and the aggregation are visually concluded.
4.10.
Application: SERS
Objective glasses are RCA treated and they adsorbed different polymers (uncoated, PEI, PEI/PSS, PEI/PSS/PDADMAC). Synthesize calcium carbonate particles and assemble a monolayer. The calcium carbonate is coated with silver nanoparticles using two different methods: particle coating (4.6.3) and monolayer coating (4.6.4). There are now two different sets of silver stained particles. In one, the silver nanoparticles are adsorbed on the particles themselves. In the other, the silver nanoparticles are adsorbed on the monolayer as a whole. To create a Raman spectrometry signal, add 5 µL of 4-mercaptobenzoic acid (4-MBA) and 5 µL of rhodamine to every sample. Measure the Raman signal and compare with a standard of 4-MBA and rhodamine. Calculate the enhancement factor (see 2.5) of every sample if there is a distinct signal enhancement. 38
The enhancement factor is calculated by the following formula:
with ISERS and INR the Raman intensities at the reference peaks respectively for the standards and the samples. cSERS and cRaman are the concentrations of respectively the standard solutions and the sample solutions (Severyukhina et al., 2015).
4.11.
Application: Toxicity Test
Create a microwell with a monolayer of particles as described in 4.9. The particles are either not coated, either coated with PAH/PDADMAC (2 mg/mL; 0,5 M NaCl) or first coated with PAH/PDADMAC (2 mg/mL; 0,5 M NaCl) and then with PSS (2 mg/mL; 0,5 M NaCl). The microwell itself is coated in 4 different ways. The first way is without a coating, the second one is coated with PEI (2 mg/mL), the third one is first coated with PEI (2 mg/mL) then with PSS (2 mg/mL; 0,5 M NaCl) and the final one is first coated with PEI (2 mg/mL) then with PSS (2mg/mL; 0,5 M NaCl) and eventually with PAH/PDADMAC (2 mg/mL; 0,5 M NaCl). Make combinations of every coating method, but also leave some wells without any particles and therefore only the coating of the microwell. These wells will be used as a reference. Test the toxicity of the different wells using HeLa cells. Seed the cells into a 24-well cellculture plate with a cell density of . Cultivate for 24 hours then add the samples to the microwell covered with polymers and particles. The microwell is sterilized shortly before the experiment by adding 100 µL of ethanol to each well and then letting the ethanol evaporate. Incubate the microwell with the particles, coatings and HeLa cells overnight at 37 °C under 5% CO2. After incubation, add 0,5 mL of fresh medium and 50 µL of fluorescent dye AlamarBlue (Sigma-Aldrich) to each well. Measure the intensity of the fluorescence with the spectrophotometer (Gemini XPS Microplate Reader, Molecular Devices) after 24 hours of incubation.
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5. Results 5.1.
Hydrophobicity Test
5.1.1. Objective Glass Cleaning Procedure Different methods for cleaning the objective glass are compared. After cleaning, a drop of water is added. The angle of the drop with the surface of the objective glass is determined. 6 angles are measured for each cleaning method, using 3 drops.
RCA Cleaning Treatment Contact Angle (°)
40 30 20 10 0 No treatment
Treatment RCA treatment
Ethanol treatment (wash)
Ethanol treatment (no wash)
Figure 15: Contact angle for different cleaning methods. RCA treatment and ethanol treatment with wash show similar results. 6 angles are measured out of 3 drops.
When the objective glass is untreated, it shows the highest hydrophobicity and therefore the highest contact angle compared to the washing methods. When the objective glass is treated with ethanol and then dried (no wash), the contact angle is lower. However, the lowest hydrophobicity is achieved by the RCA treatment or by cleaning with ethanol then washing with water. No significant difference between the two is found (p
