Cells do not live in static surroundings, they exist in highly evolving dynamic environments. During cell adhesion and migration, cells adapt and communicate to their environment by numerous methods ranging from differentiation, gene expression, growth and apoptosis. How and when cells determine to adhere, polarize and migrate is important to a number of fundamental biological processes such as wound healing, metastasis, inflammation and development.
In order to elucidate the spatial and temporal mechanisms of these important complex processes on a molecular basis, the Yousaf Group has generated model substrates that can present nanopatterned ligands, well defined gradients of ligands and surfaces that can be dynamically modulated where the interaction between cell and material is defined at the molecular level.
Indium tin oxide (ITO) is a transparent conductor used for applications ranging from solar cells to neurobiology. Tailoring ITO surfaces with a range of functional groups is challenging due to the difficulty in synthesizing phosphonate or siloxane terminated molecules. As reported in Advanced Materials, the Yousaf Group has developed a chemoselective immobilization strategy to tailor ITO surfaces by selectively oxidizing hydroxyl-terminated phosphonate SAMs to aldehydes, using microfluidics, followed by reaction with oxyamine-containing ligands. This rapid, inexpensive, and selective, on-chip activation allows for a wide range of ligands to be tethered onto ITO. Electrochemistry, contact angle, and XPS characterize the alcohol oxidation and subsequent reactivity. They also show control of ligand density and patterned cells on the newly generated aldehyde-terminated ITO surface.
This methodology allows for the generation of patterned complex surface chemistry on ITO surfaces from a simple hydroxyl-terminated SAM surface. The ability to generate complex surfaces with simple starting materials and minimal to no synthesis may have wide-ranging utility for numerous applications in molecular electronics and biotechnology including co-culture and cell arrays.
Members of the Yousaf Group have recently developed a new rapid and inexpensive strategy to generate two chemistries from one surface composition for dual-ligand immobilization using microfluidic oxidation on indium tin oxide surface. Through chemical oxidation of an alcohol-terminated self-assembled monolayer, aldehyde and carboxylic acid groups are formed with density and spatial control. These surface groups may then react with oxyamine- and amine-tethered ligands to generate covalent oxime and amide linkages, respectively.
This patterning strategy circumvents multi-step syntheses and is applicable to tailoring a variety of other materials. Ongoing research includes exploring multiple ligand immobilization for co-culture studies, cell migration studies, and for generating high-throughput ligand microarrays on other metal-oxide surfaces.
As reported in Angewandte Chemie International Edition, Devin Barrett of the Yousaf Group reports a new methodology that combines surface chemistry patterning with soft lithography and centrifugation for the rapid, inexpensive, and complete patterning of cells and cell co-cultures on surfaces with spatial and temporal control.
Although, the patterning of one cell type to a range of materials has become routine, the ability to pattern multiple cell lines with spatial and temporal control of cell population interactions remains technically challenging and impractical for the larger biological community to access. Until now, a simple, fast and inexpensive strategy to generate multiple cell-patterned arrays would greatly expand the current scope of cell biological research and generate new co-culture array screens, tissue patterning materials and cell based devices.
By employing micro-contact printing in conjunction with poly(dimethylsiloxane) (PDMS) masks and centrifugation, we report the patterning of co-cultures on substrates with feature sizes as small as 30 μm. This strategy can routinely and rapidly immobilize single cells that are separated by as little as 100 μm, which provides exquisite spatial control for autocrine and paracrine signalling studies. In addition, we show the patterning of co-cultures of fibroblast cells and GFP transfected Drosophila cells to show the generality of the method for cell biological applications.
As published in Molecular BioSystems, the Yousaf Group reports a combined photochemical and electrochemical method to pattern ligands and cells in complex geometries and gradients on inert surfaces. Their work demonstrates: (1) the control of density of immobilized ligands within overlapping photopatterns, and (2) the attached cell culture patterned onto ligand defined gradients for studies of directional cell polarity. Our approach is based on the photochemical activation of benzoquinonealkanethiols. Immobilization of aminooxy terminated ligands in selected region of the quinone monolayer resulted in patterns on the surface.
This approach is unique in that the extent of photochemical deprotection, as well as ligand immobilization can be monitored and quantified by cyclic voltammetry in situ. Furthermore, complex photochemical patterns of single or multiple ligands can be routinely generated using photolithographic masks. Finally, this methodology is completely compatible with attached cell culture and we show how the subtle interplay between cell-cell interactions and underlying peptide gradient influences cell polarization. The combined use of photochemistry, electrochemistry and well defined surface chemistry provides molecular level control of patterned ligands and gradients on surfaces.
As reported in Langmuir, Brian Lamb, Devin Barrett, and Nathan Westcott of the Yousaf Group report a straightforward, flexible, and inexpensive method to create patterned self-assembled monolayers (SAMs) on gold using microfluidics – microfluidic lithography. Using a microfluidic cassette, alkanethiols were rapidly patterned on gold surfaces to generate monolayers and mixed monolayers. The patterning methodology is flexible and, by controlling solvent conditions and thiol concentration, permeation of alkanethiols into the surrounding PDMS microfluidic cassette can be advantageously used to create different patterned feature sizes and to generate well-defined SAM surface gradients.
To demonstrate the utility of microfluidic lithography, multiple cell experiments were conducted. By patterning cell adhesive regions in an inert background, a combination of selective surface masking and centrifugation achieved spatial and temporal control of patterned cells, enabling the design of both dynamic surfaces for directed cell migration and contiguous co-cultures. Cellular division and motility resulted in directed, dynamic migration, while the centrifugation-aided seeding of a second cell line produced contiguous co-cultures with multiple sites for heterogeneous cell-cell interactions.
The Yousaf lab has recently generated a straightforward and inexpensive method to synthesize and etch biodegradable poly(1,2,6-hexanetriol £\-ketoglutarate) films for tissue engineering applications, Langmuir, 9861–9867, 2008. Microfluidic delivery of the etchant, a solution of NaOH, can create micron-scale channels through local hydrolysis of the polyester film. In addition, the presence of a ketone in the repeat unit allows for prior or post chemoselective modifications, enabling the design of functionalized microchannels.
Delivery of oxyamine tethered ligands react with ketone groups on the polyketoester to generate covalent oxime linkages. By thermally sealing an etched film to a second flat surface, poly(1,2,6-hexanetriol £\-ketoglutarate) can be used to create biodegradable microfluidic devices. In order to determine the versatility of the microfluidic etch technique, poly(£'-caprolactone) was etched with acetone. This strategy provides a facile method for the direct patterning of biodegradable materials, both through etching and chemoselective ligand immobilization.
To generate model substrates for cell adhesion, the Yousaf Group has developed two different biocompatible strategies based on self-assembled monolayers (SAMs) of alkanethiolates on gold terminated with latent ketones and aldehydes. Under spatial control, the hydroquinone and alcohol terminated SAMs can be oxidized to allow for oxyamine ligand patterning on the surface with microfluidic cassettes. These immobilization strategies were characterized by electrochemistry and fluorescence microscopy. By utilizing a cell adhesive peptide, cell patterns were also generated.
These methods are of broad utility to the research community as an easily accessible chemoselective strategy to immobilize ligands to surfaces. Previous immobilization strategies require multistep synthesis to generate the reactive head group on the surface. The Yousaf method requires either a simple synthesis or commercially available materials. The many different functional groups compatible with carbonyl chemistry allow for a range of ligands to be immobilized. In the future, the ability to oxidize hydroxy terminated SAMs may be extended to tailor a broad range of materials for molecular electronic and biological sensing applications.
The Yousaf Group reports a combined photochemical and electroactive self-assembled monolayer (SAM)-based substrate strategy to generate a co-culture platform with spatial and temporal control of cell-cell interactions. These dynamic substrates possess the ability to present a variety of ligands on the surface for biospecific interactions between the ligands and cell surface receptors.
Furthermore, the photo-patterning step enables the ligands to be immobilized in patterns and even gradients. This feature provides additional flexibility to study the role of ligand pattern (geometry) and presentation (gradient) on co-culture interactions on complex dynamic surfaces.
As published in Analytical Chemistry, Nathan Westcott and Brian Lamb in the Yousaf Group report a combined microfluidics and electrochemical approach to generate complex surfaces for studies of mechanistic cell adhesion and cell migration. A variety of surfaces were generated including gradients of gold height, completely etched gold/glass hybrids, and partially etched gold surfaces for pattern visualization.
The integration of microfluidics and the electrochemical and chemical etching methods allow for the creation of gold/glass hybrids surfaces for numerous biointerfacial studies. In particular, the gradient surfaces allow for the study of cell migration on varying slopes of gold height with tailored surface chemistry. In the future, the etched gold surfaces will be used to simulate the varying nanotopology experienced by migrating cells in vivo.
The Yousaf Group reports a strategy for the fabrication of tailored electroactive nanorod substrates for biospecific studies of cell adhesion and stem cell differentiation. To control the interfacial properties of the nanorods they formed self-assembled monolayers of an electroactive hydroquinone group that is able to chemoselectively immobilize oxyamine tethered ligands.
These tailored nanorods may be used to study how topology and surface chemistry influence cell behavior.
The National Science Foundation has awarded Carolina Chemistry Assistant Professor Muhammad Yousaf its prestigious CAREER Award, in the amount of $600,000 over five years. The award recognizes his well known pioneering work in applying surface chemistry to cell biology for studies of cell adhesion, polarization and migration. Yousaf’s group will develop and integrate new surface chemistries, live cell high resolution fluorescence microscopy techniques and microfluidic lithography based approaches to generate new cell based microarrays and a class of dynamic surfaces to study the internal and external cues that are critical for cell polarization and directed cell migration.
The Faculty Early Career Development Program is a foundation-wide activity that offers the National Science Foundation's most prestigious awards in support of junior faculty who exemplify the role of teacher-scholars through outstanding research, excellent education and the integration of education and research within the context of the mission of their organizations.
As reported in Macromolecules, researchers in the Yousaf Group report the design of several elastomers based on the thermal polycondensation of α-ketoglutaric acid and one of three triols: glycerol, 1,2,4-butanetriol, or 1,2,6-hexanetriol. By varying the curing temperature and the duration of the curing process, a wide range of mechanical properties was achieved.
The values of the Young’s modulus (0.1 - 657.4 MPa), ultimate stress (0.2 – 30.8 MPa), and ultimate strain (22 - 583 %) encompass the mechanical properties of many biological materials, allowing for potential use of poly(triol α-ketoglutarate) as a biomaterial. Furthermore, the poly(triol α-ketoglutarate) series hydrolytically degraded in as fast as 2 days and as long as 28 days in phosphate-buffered saline solutions. For post-polymerization modifications, the repeat units contain ketones, which are capable of reacting with a variety of oxyamine-terminated molecules to generate stable oxime linkages. Finally, the versatility and utility of these elastomers were demonstrated by creating micro-patterned structures and films for biospecific cell scaffold supports.
The Yousaf Group has developed an electroactive and catalytic dynamic substrate strategy that captures and subsequently releases ligands and cells in-situ via an electrochemical potential. The surface is catalytic for multiple rounds of immobilization and release with a quantitative functional group transformation from an oxyamine group to a primary alcohol upon mild electrochemical potential that is pH dependent.
Combining this strategy with a photochemical approach, the Yousaf Group shows the capture and release of peptide ligands that mediate biospecific cell attachment on defined surface gradient patterns.