Pamela Sontz and Natalie Muren of the Jackie Barton Lab; and Joe Nemanick and Lauren Webb of the Nate Lewis lab. The wonderful times at Caltech began with my Lumberjacks: Brad Cenko, Robert Ward, and Dan Busby.
A Preface to this Dissertation
Introduction
Methods
Samples were then removed from solution, washed in ethanol and water, and then dried under argon. Distilled, deionized water was added to the syringe and water droplets were gently added to the Au substrate.
Results and Discussion
Water is repelled from the surface and the resulting water droplets have a higher contact angle. In general, contact angle measurements are informative for determining the wettability of chips at the macroscale, but only provide qualitative insight.
Scanning Electron Microscopy
Methods
Substrate samples were then rinsed in ethanol, dried under an argon stream, and mounted on an SEM drill holder.
Results and Discussion
Although the SEM technique succeeds in producing high-resolution topographic images for substrates, it is not suitable for imaging individual proteins or molecules.
X-Ray Photoelectron Microscopy
Methods
Samples were mounted on chucks, loaded into the instrument loading arm and placed under vacuum for 1-2 hours before transport to the analysis chamber.
Results and Discussion
Similarly, the XPS spectrum for the oligo SAM sample exhibits peaks for Au, C, O and N, but not S or P. The low ionization potential of Au makes the atomic sensitivity factor for Au relatively high Table 2.2).
Electrochemisty
Methods
As illustrated above (Figure 2.5), the three-electrode cell consisted of Au-coated Si as the working electrode. PBS and 30 mM potassium ferrocyanide III (K3Fe(CN)6, Aldrich) served as the electrolyte and redox active species, respectively.
Results and Discussion
Samples treated with TEG-disulfide, TEG and mercaptoundecanol give CV traces with very different shapes (Figure 2.7d-f). The BAT SAM sample has an asymmetric CV trace, albeit similar in shape to the bare Au control (Figure 2.7g).
Fluorescence Microscopy
The lifetime of the singlet excited state is greatly shortened due to the energy transfer from the excited dye molecule to the bulk metal [32–34]. Addition of the complementary target strand breaks the hairpin formation and the fluorophore is removed approximately 80 ˚A from the Au surface. The fluorescence levels are increased 600 times in this scenario. Au distances were quantified by TEM and monitored by UV–Vis spectroscopy. Dependent quenching was measured by recording emission intensities before and after dissolving the Au with cyanide, which left the fluorophore and polymer shells intact.
In the case of beads and cells, the fluorophores are abundant and far enough from the Au that signals can remain easily detected.
Conclusions
The use of aqueous media for the formation of protein-binding alkyl thiolate monolayers on Au surfaces results in accelerated formation of alkyl thiolate monolayers and improvement in monolayer integrity, as visualized by fluorescence microscopy and CV techniques. Although each of these methods yields clean Au surfaces that enable high-quality SAM formation, they each present major drawbacks for cleaning integrated biosensors. Ozone plasma effectively oxidizes and degrades Au-bound contaminants, but it is difficult to clean the Au surfaces packed under PDMS layers with ozone.
I show that TEG and BAT insulating monolayers on Au surfaces adsorbed water at faster rates and with fewer monolayer holes than ethanol adsorbed SAMs.
Materials and Methods
- Reagents
- Preparation of Substrates and Monolayers
- Electrochemical Methods
- Protein-Binding Assays
- Fluorescence Microscopy
- Synthetic Methods
However, synthesis of 3 proceeds by adding the PEG group to biotin, followed by multi-step attachment of the alkyl chain [50]. Reaction of this compound with excess 1,12 dibromododecane resulted in attachment of the azotriethylene glycol to the alkyl chain to give 12-(azottriethyleneoxy)-1-bromododecane (7) in low yield (24% yield). Removal of the tert-butoxycarbonate group by trifluoroacetic acid (TFA) in CH2Cl2 gave ammonium triethylene oxidodecane disulfide 10 (80% yield).
Introduction of biotin into the linker by reaction of the appropriate amine with the activated biotin derivative4 was straightforward.
Results and Discussion
- Electrochemical Cleaning of Au Surfaces Improves Monolayer
- Effect of Adsorption Solvent on SAM Formation
- Specific Binding of Fluorescent Proteins to SAMs
- Electrocleaning to Recycle Au Substrates
Contamination of Au surfaces severely limits our ability to consistently generate quality alkylthiolate monolayers. The relative fluorescent intensity of streptavidin-Cy3 protein binding to the BAT:TEG monolayer was determined using fluorescence microscopy. We used CV to evaluate the quality of alkylthiolate monolayer coverage of Au substrates and protein binding to evaluate the biological functionality of the formed alkylthiolate monolayers.
The solubility of the thiol molecule in a given solvent plays a key role in the formation of SAMs [56–58].
Materials and Methods
Reagents
A positive potential is applied to the array and accelerates hybridization of the negatively charged complementary strand, similar to electrophoresis. This increases the sensitivity of the array as diffusion-limited transport of oligos to the array is overcome by electrostatic forces. Once a layer of cell-specific antibodies is bound, cells can be panned out of solution onto the 2-D array.
Furthermore, once the oligos are hybridized to the array, they can be further modified and manipulated with enzymes such as Klenow [ 4 ], T4 DNA ligase [ 7 ] and restriction enzymes [ 4 , 7 ].
Oligo Labeling Antibody
Oligo Hybridization and Cleavage
Synthesis of Dimethoxy Trityl-Modified Coarse Porous Glass
Synthetic Methods
The solution was then concentrated to a yellow oil which was loaded onto silica gel in hexane. The organic fraction was then washed with NaHCO3 and brine, then dried with Na2SO4 and concentrated to give a dark brown oil which was loaded onto a silica column in hexane. This organic phase was washed with brine, dried with Na2SO4 and concentrated to give a pale pink oil which was loaded onto a silica column in hexane.
The product was concentrated to yield a white powder and loaded onto a silicon column in hexane.
Synthesis of Oligo-Modified Disulfide
The crude material is resuspended in 600 µl of 50 mM NH4OAc and filtered by pooling HPLC fractions.
Results and Discussion
Titrating Oligo Hybridization on Au Substrates
According to their calculations, the maximum hybridization efficiency is achieved at a ss-oligo probe density of 2.0 × 1012 cm−2. In the case of oligo-alkylthiols, the flexibility and conformation of the molecule can play a role in both incorporation of the oligo into the SAM and complement hybridization. If we theorize a spacing of 5 ˚A for high-density packed oligo-alkylthiols and a diameter of 20 ˚A for double-stranded (ds)-oligos, this leaves very little room for 1:1 hybridization events.
It is possible that a single Cy3-labeled complement strand can bind to two surface-bound oligos, resulting in reduced hybridization capacity, or that some surface-bound oligos simply remain inaccessible to complement strands due to molecular crowding.
Temperature Effects and Enzyme Reactivity
We follow the intensity levels of samples (10% oligo) incubated with 4 units of BamHI over time (Figure 4.4) and observe greater signal losses at longer incubation times. To address steric hindrance as a source of reduced activity, SAMs generated from 0.1% oligo-alkylthiol solutions were incubated with BamHI for 60 min. Au substrates were treated with 0.1% oligo-alkylthiol in TEG, then hybridized with biotinylated complementary strands and then with Cy3-labeled streptavidin.
SAMs generated from 0.1% oligo-thiol in TEG hybridized with biotin-labeled complement strands bind with Cy3-labeled streptavidin.
Conclusions
Polystyrene plates are the standard for cell culture, but alternative substrates such as gold have been explored. In vivo and in vitro studies show that activation of endothelial cells by VEGF can result in changes in cell proliferation, migration and differentiation [14–16]. Since cellular mobilization is a precursor to blood vessel formation, in vitro studies of surface-bound VEGF can provide insight into receptor signaling and endothelial cell motility.
Increased cell proliferation of human umbilical vein endothelial cells (HUVEC) on these substrates was found to be dose dependent of VEGF incorporation into the polymer [16].
Materials and Methods
Substrates were then incubated in streptavidin (60 nM) in PBS for 30 min, washed with PBS, and soaked in biotinylated α-B220 IgG antibody (20 nM) for 30 min. Substrates were then soaked in streptavidin (60 nM) in PBS for 30 min, washed with PBS, then soaked in bioinactivated VEGF (20 nM) for 30 min. Slides were carefully rinsed in PBS, then soaked in 5 ug ml-1 streptavidin in PBS for 30 minutes, then incubated in bioinstilled VEGF for 30 minutes.
YSE2 cells were plated on Au at 1 x 105 cells ml−1 for 60 min in IMDM supplemented with body ceramide, and nonadherent cells were gently washed away.
Results/Discussion
- Cell Panning
- YSE2 Cell Morphology
- YSE2 Cell Migration on SAMs
- YSE2-Specific Response
Compared to Au substrates, cells on VEGF will spread to 80–85% of the surface area. VEGF is a known stimulator of endothelial cell migration during angiogenesis and vasculogenesis, so we used time-lapse microscopy to study the cell response to our modified substrates. A one-way ANOVA test showed that cell migration on 1.0 VEGF (substrates treated with 1BAT:0TEG, streptavidin, and biotinylated VEGF) was statistically different from the migratory activity on FN, 0.01 VEGF, or 0.25 VEGF.
No significant increases in mean cell displacement are calculated here, and in these studies VEGF alone does not stimulate the same levels of cell migration.
Conclusion
However, we note that the slope of the plot for YSE cells on VEGF shows a notable break after 110 min and cannot be described by Othmer's model equation. One-way ANOVA test shows that YSE2 cells on VEGF migrate significantly longer distances compared to YSE2 cells on Au or 3T3 cells on the same substrates. A one-way statistical ANOVA test was performed on cell displacements for YSE2 and 3T3 cells plated on TEG and plain Au substrates.
YSE2 cell migration was significantly greater on VEGF surfaces compared to Au and compared to 3T3 fibroblasts on both substrates.
Outstanding Issues
Next Steps
In the recent paper by Watanabe et al., researchers report a new technique for dividing stem cell colonies in vitro by applying an inhibitor of Rho-related kinase (ROCK) which greatly increases cell viability after trypsinization [11] . The ability to gently and effectively control and separate stem cells is still lacking in the stem cell arena. Many of the tools developed within my thesis can be applied towards the separation and purification of stem cells.
In parallel, high-throughput platforms can allow researchers to assess which proteins and reagents stimulate stem cell activity, or identify those that allow cells to continue to grow undifferentiated.
Generic alkylthiol structure and coordination on Au surfaces
Stages of alkylthiolate SAM formation on Au
Mixed SAMs on Au
Contact angle measurements by sessile water drop
Images were generated with auto contrast and auto brightness settings. the highest magnification used, individual Au colloid islands are visible, and even the boundary between the two alkanethiols is visible, but individual molecules cannot be resolved. In Figure 2.6 and the remaining CV traces given here, the X-axis runs from positive to negative. As the alkyl thiol concentration is lowered, longer time scales are required to achieve comparable electrode coverage (Figure 2.9b– .c).
From Figure 2.10, we find that the SAMs are stable on the electrodes after being left overnight in air at room temperature. The efficiency of electrochemical treatments of Au electrodes and subsequent SAM formation was tested by comparing electrically connected Au substrates with electrically isolated Au substrates on a single chip (Figure 3.2a). In these samples, electrically isolated regions consistently showed half the fluorescence intensity compared to electrically connected regions (Figure 3.2c–f).
Using both CV and fluorescence microscopy, Au substrates were characterized throughout the process, which included the following steps (Figure 3.7): 1) Au surface electrical cleaning (anodic sweep. Fluorescent images of the precleaned Au (Figure 3.7c) and post- SAM purified Au (Figures 3.7e and 3.7g) gave background fluorescence intensities.
TEG, hydroquinone, and oligo alkylthiol structures
SEM images of Au substrates
ESCA spectra for Au and Si surfaces
Electrochemical cell setup
Cyclic voltammetry curves
CV traces of various SAMs on Au
CV trace time course for BAT SAM formation
TEG concentration effects on SAM formation
Temperature effects on SAM stability
Sample fluorescent microscope setup
Alkylthiol chemical structures
Photolithography mask for Au samples
Synthesis of tri(ethylene glycol) dodecylthiol (TEG)
Biotinylated alkylthiol molecule
Synthesis of biotinylated alkylthiol
Relative FL intensities for SAMs on Au
CV traces and corresponding fluorescence microscopy images for Au
Synthesis scheme of DMT-modified CPG
Synthesis scheme of oligo-modified alkylthiols
FL intensities for Cy3-oligo SAMs on Au
Temporal tracking of ds-oligo cleavage by BamHI
Low-density ds-oligo cleavage by BamHI
Enzymatic cleavage of proteins from SAMs
Oligo-mediated adhesion of beads on Au
Structure of BamHI protein coordinated to DNA
Scheme for immuno-specific cell attachment to SAMs via streptavidin
