Brian M. Lamb

Microfluidic Permeation Printing of Self-Assembled Monolayer Gradients on Surfaces for Chemoselective Ligand Immobilization Applied to Cell Adhesion and Polarization

To study complex cell behavior on model surfaces requires biospecific interactions between the interfacing cell and material. Developing strategies to pattern well-defined molecular gradients on surfaces is difficult but critical for studying cell adhesion, polarization, and directed cell migration. We introduce a new strategy, microfluidic SPREAD (Solute PeRmeation Enhancement And Diffusion) for inking poly(dimethylsiloxane) (PDMS) microfluidic cassettes with a gradient of alkanethiol. Using SPREAD, an oxyamine-terminated alkanethiol is able to permeate into a PDMS microfluidic cassette, creating a chemical gradient, which can subsequently be transfer printed onto a gold surface to form the corresponding chemoselective gradient of oxyamine-alkanethiol self-assembled monolayer (SAM). By first patterning regions of the gold surface with a protective SAM using microfluidic lithography, directional gradients can be stamped exclusively onto unprotected bare gold regions to form single cell gradient microarrays. The microfluidic SPREAD strategy can also be extended to print micrometer-sized islands of radial SAM gradients with excellent geometric resolution. The immobilization of a cell adhesive Arg-Gly-Asp (RGD)-ketone peptide to the SPREAD stamped oxyamine-alkanethiol SAMs provides a stable interfacial oxime linkage for biospecific studies of cell adhesion, polarity, and migration.

Expedient generation of patterned surface aldehydes by microfluidic oxidation for chemoselective immobilization of ligands and cells.

An expedient and inexpensive method to generate patterned aldehydes on self-assembled monolayers (SAMs) of alkanethiolates on gold with control of density for subsequent chemoselective immobilization from commercially available starting materials has been developed. Utilizing microfluidic cassettes, primary alcohol oxidation of tetra(ethylene glycol) undecane thiol and 11-mercapto-1-undecanol SAMs was performed directly on the surface generating patterned aldehyde groups with pyridinium chlorochromate. The precise density of surface aldehydes generated can be controlled and characterized by electrochemistry. For biological applications, fibroblast cells were seeded on patterned surfaces presenting biospecifc cell adhesive (Arg-Glyc-Asp) RGD peptides.

Microfluidic Lithography of SAMs on Gold to Create Dynamic Surfaces for Directed Cell Migration and Contiguous Cell Cocultures

A straightforward, flexible, and inexpensive method to create patterned self-assembled monolayers (SAMs) on gold using microfluidics-microfluidic lithography-has been developed. 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 the 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 with a single microfluidic chip. 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 masking of the surface and centrifugation achieved spatial and temporal control of patterned cells, enabling the design of both dynamic surfaces for directed cell migration and contiguous cocultures. Cellular division and motility resulted in directed, dynamic migration, while the centrifugation-aided seeding of a second cell line produced contiguous cocultures with multiple sites for heterogeneous cell-cell interactions.

Remote Control of Tissue Interactions via Engineered Photo-switchable Cell Surfaces

We report a general cell surface molecular engineering strategy via liposome fusion delivery to create a dual photo-active and bio-orthogonal cell surface for remote controlled spatial and temporal manipulation of microtissue assembly and disassembly. Cell surface tailoring of chemoselective functional groups was achieved by a liposome fusion delivery method and quantified by flow cytometry and characterized by a new cell surface lipid pull down mass spectrometry strategy. Dynamic co-culture spheroid tissue assembly in solution and co-culture tissue multilayer assembly on materials was demonstrated by an intercellular photo-oxime ligation that could be remotely cleaved and disassembled on demand. Spatial and temporal control of microtissue structures containing multiple cell types was demonstrated by the generation of patterned multilayers for controlling stem cell differentiation. Remote control of cell interactions via cell surface engineering that allows for real-time manipulation of tissue dynamics may provide tools with the scope to answer fundamental questions of cell communication and initiate new biotechnologies ranging from imaging probes to drug delivery vehicles to regenerative medicine, inexpensive bioreactor technology and tissue engineering therapies.

Microfluidic Lithography to Create Dynamic Gradient SAM Surfaces for Spatio-temporal Control of Directed Cell Migration

Cells exist in a complex, dynamic environment and are able to compute and process a myriad of signals, which initiate a range of diverse activities including cell growth, migration, and apoptosis. Many extracellular signals are provided in either soluble or surface immobilized concentration gradients. Through these gradients, ligandand receptor-mediated interactions cause the cell to generate local asymmetry, resulting in activation of signaling or cytoskeletal components. This in turn leads to changes in the sub-cellular nanoarchitecture and initiates a range of cellular behavioral responses. In order to quantitatively and qualitatively understand the effects of surface-immobilized ligand gradients on development, directed migration (haptotaxis), inflammation, and wound healing, it is necessary to generate molecularly well-defined surface gradients integrated with dynamic surfaces for the spatial and temporal control of cell behavior. Although there are several strategies to generate switchable or dynamic surfaces for cell adhesion and migration, none are able to spatially and temporally control, at the molecular level, directed cell migration on dynamic gradients. In order to quantitatively study the effects of surface immobilized ligand gradients on cell migration, the surface composition of the material should ideally meet the following criteria. The surface is 1) molecularly well defined, 2) inert to non-specific protein adsorption and cell adhesion, 3) compatible with a quantitative and chemoselective immobilization strategy to tether a variety of molecules to the surface, 4) enables dynamic, switchable control of surface composition at the molecular level, and 5) compatible with live-cell high-resolution fluorescence microscopy. We believe the most flexible surfaces to study biointerfacial phenomena are based on self-assembled monolayers (SAMs) of alkanethiolates on gold. The synthetic flexibility of thiol chemistry with a variety of functional groups allows for a diverse set of orthogonal and chemoselective surface coupling strategies to be employed. Furthermore, several techniques that provide high resolution spatial control have been developed to pattern SAMs on gold, such as microcontact printing (mCP), dip-pen nanolithography (DPN), and photolithography. Herein, we introduce a new strategy—microfluidic lithography (mFL)—to generate patterns and gradients of electroactive SAMs that can subsequently react chemoselectively to immobilize ligands and can be dynamically controlled in the presence of cells to promote directional cell migration. We use these dynamic surfaces to show the migration of Swiss 3T3 fibroblasts towards the higher density regions of a biospecific cell adhesive peptide gradient. These gradients can be quantitatively characterized with both cyclic voltammetry (CV) and scanning electron microscopy (SEM), are straightforward to generate, and highly reproducible. This strategy can be generalized and used for a variety of biological studies in addition to its potential to pattern SAM gradients of any alkanethiol. mFL takes advantage of the intrinsic rapid rate of SAM formation of thiols on gold to create well-defined features and gradients. 15] We have shown previously that hydroquinone terminated SAMs are electroactive and provide both an activation method for ligand immobilization and a method to quantitate the density of ligands by in situ electrochemistry. The oxidized quinone form is able to chemoselectively react with oxyamine-containing ligands to generate covalent, stable oxime linkages. This synthetic scheme is especially advantageous due to the ease of solid-phase peptide and carbohydrate synthesis of oxyamine-containing molecules and the commercial availability of oxyamine terminated ligands, monomers, and biomolecules. Furthermore, the interfacial oxime reaction to install ligands on the surface is fast, kinetically wellbehaved, can be done at physiological conditions (37 8C, aqueous media) in the presence of cells, and does not cross react with proteins, nucleic acids, or other biopolymers. OxyACHTUNGTRENNUNGamine-ligand immobilization on quinone SAMs can be analytically monitored with CV, which allows for precise measurement of the SAM surface composition. The strategy of mFL is illustrated in Figure 1. This is a straightforward, simple, and effective method to pattern micrometer-sized SAM features and can be utilized for the creation of hydroquinone-terminated SAM gradients. A polydimethylsiloxane (PDMS) microfluidic cassette is placed on the surface of a gold substrate, and a small volume of hydroquinone-terminated tetra(ethylene glycol) undecanethiol (H2Q-TEG) solution is added to an access hole and slowly drawn into the microfluidic cassette through capillary action. The surface gradient is controlled by three parameters: 1) reactant depletion due to SAM formation on the gold surface as fluid flows through the channel, 2) differential surface exposure time due to the slow rate of capillary force driven fluid flow, and 3) thiol diffusion from the microfluidic channel into the PDMS cassette. By adjusting the thiol concentration and flow conditions with engineered patterns or syringe pumps, gradients of varying slopes can be produced. The gradient monolayer established by mFL is initially disordered and requires removal of the cassette and subsequent exposure to alkanethiolcontaining solutions to backfill the remaining regions. For biological applications, these surfaces are immersed in 1 mm tetra(ethylene glycol) undecanethiol (TEG) in ethanol for 12 h. [a] B. M. Lamb, N. P. Westcott, Prof. M. N. Yousaf Department of Chemistry and the Carolina Center for Genome Science University of North Carolina at Chapel Hill Chapel Hill, NC 27599–3290 (USA) Fax: (+1)919-962-2388 E-mail : mnyousaf@email.unc.edu Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Electrochemical and Chemical Microfluidic Gold Etching to Generate Patterned and Gradient Substrates for Cell Adhesion and Cell Migration

To generate patterned substrates of self-assembled monolayers (SAMs) for cell adhesion and migration studies, a variety of gold/glass hybrid substrates were fabricated from gold evaporated on glass. A variety of surfaces were generated including gradients of gold height, completely etched gold/glass hybrids, and partially etched gold surfaces for pattern visualization. Etch rates were controlled by the alkanethiol present on the surface. Gradients of gold height were created using an electrochemical etch with control over the position and slope of the gold height gradient. Cells were seeded to these surfaces, and their adhesion to the gold was controlled by the surface chemistry present in the channel regions. In the future, the etched gold surfaces will be used to simulate the varying nanotopology experienced by the migrating cell in vivo.

Microfluidic etching and oxime-based tailoring of biodegradable polyketoesters.

A straightforward, flexible, and inexpensive method to etch biodegradable poly(1,2,6-hexanetriol alpha-ketoglutarate) films is reported. 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 alpha-ketoglutarate) can be used to create biodegradable microfluidic devices. In order to determine the versatility of the microfluidic etch technique, poly(epsilon-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.

Live‐Cell Fluorescence Microscopy of Directed Cell Migration on Partially Etched Electroactive SAM Gold Surfaces

The ability of a cell to adhere, polarize, and migrate is influenced by the complex and dynamic extracellular matrix. The role of the extracellular matrixin directed cell migration has been under intense investigation and is important for a number of fundamental biological processes, including tissue repair, immune response, embryogenesis, and cancer metastasis. Before cells can migrate they must first polarize in response to external cues from the local microenvironment and establish spatial, temporal and functional asymmetry internally. Cell polarization involves the complex interplay of various signaling pathways that induce reorganization of the cytoskeleton and key organelles to initiate directed migration. The development of molecularly well defined model substrates that combine sophisticated surface chemistry, microfabrication technology, molecular biology, and live-cell fluorescence microscopy imaging would allow for further investigation into these complex cellular processes. We believe that the most flexible model surfaces for studying biointerfacial science are based on self-assembled monolayers (SAMs) of alkanethiolates on gold. These surfaces have four principle advantages over other surfaces for studying biospecific cell behavior: they are 1) synthetically amenable to a wide range of terminal groups to generate many different tailored surfaces, 2) inert to unspecific protein adsorption, 3) redox active monolayers, and 4) compatible with several surface spectroscopy techniques used for the characterization of interfacial interactions and reactions. However, until now, two major limitations have hindered the study of cell biology on SAMs. Due to gold’s fluorescent quenching, SAMs of alACHTUNGTRENNUNGkanethiolates on gold are incompatible with the live-cell high resolution fluorescence microscopy needed to visualize dynamic features within cells. Additionally, the directional path of cellular migration must be determined ex post facto because a ligand pattern cannot be independently visualized during ACHTUNGTRENNUNGmigration. To study complex cell behavior, especially directed cell polarity and migration, a molecularly well-defined surface that can be patterned with a chemoselective immobilization strategy would be of tremendous utility. Additionally, the surface should be compatible with live-cell high resolution microscopy and simultaneous visualization of the cell-path trajectory. Herein, we introduce three new, straightforward technologies that combine synergistically to provide complete spatial and visual control of directed cell polarity and migration. This strategy uses: 1) partially etched gold surfaces to determine the precise track or path of cell migration trajectory, 2) microfluidic lithography (mFL) to pattern gold surfaces rapidly with a variety of alkanethiols for biospecific cell interactions, and 3) live-cell high-resolution fluorescence microscopy on gold surfaces to visualize internal organelle dynamics during polarity and migration. To obtain ligand control over the surface and characterize the patterning, we utilize a chemoselective electroactive SAM immobilization methodology to pattern ligands on the partially etched gold surfaces. We show live-cell directed migration of a mammalian cell line in which the nucleus and Golgi have been fluorescently labeled to determine the role of cell polarity on motility. The strategy is based on the use of microfluidic cassettes to partially etch the gold surface and then install a monolayer on the etched regions for subACHTUNGTRENNUNGsequent immobilization of biospecific adhesive peptides. By using a slightly modified inverted microscopy set-up (see the Supporting Information), routine live cell fluorescence microscopy is now possible on gold surfaces. This study is the first demonstration of live-cell fluorescence microscopy of directed cell migration on tailored gold surfaces. The strategy to chemoselectively immobilize ligands, visualize the ligand patterns, and monitor directed cell migration through live-cell fluorescence microscopy is outlined in FigACHTUNGTRENNUNGure 1. First, a polydimethylsiloxane (PDMS) microfluidic cassette was reversibly sealed to a bare gold surface to achieve spatial control. To partially etch and pattern the gold surfaces, a mild tri-iodide solution (18 mm KI, 4.3 mm I2) was flowed into the microchannels and allowed to react for 10 s. Without removing the PDMS stamp, the microchannels were subsequently flushed with water and then ethanol by flowing each into the channels for 10 s. To form a patterned SAM on the etched ACHTUNGTRENNUNGregions, we developed a new technology termed mFL. To perform mFL, an ethanolic alkanethiol solution containing either a terminal ligand or reactive head group was flowed into the channels and allowed to self-assemble on the partially etched regions. By controlling the concentration of alkanethiol solution and duration of SAM formation, mixed SAMs and even gradients can be patterned. It should be noted that the mFL strategy can be used to pattern a variety of alkanethiols rapidly onto flat or etched gold surfaces (Supporting Information). Once the monolayer was installed onto the partially etched regions, the microfluidic PDMS cassette was removed from the gold surface. The etched and patterned gold surface was then immersed in a tetra(ethylene glycol) undecane thiol (EG4C11SH) solution to backfill the unetched and unreacted regions of the gold surface. The ethylene ACHTUNGTRENNUNG(glycol) group prevents unspecific [a] B. M. Lamb, N. P. Westcott, Prof. M. N. Yousaf Department of Chemistry and the Carolina Center for Genome Science University of North Carolina at Chapel Hill Chapel Hill, North Carolina 27599–3290 (USA) Fax: (+1)919-962-2388 E-mail : mnyousaf@email.unc.edu [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Cell division orientation on biospecific peptide gradients.

An assay was developed for determining cell division orientation on gradients. The methodology is based on permeating microfluidic devices with alkanethiols and subsequent printing of cell adhesive peptide gradient self-assembled monolayers (SAMs) for examining oriented cell divisions. To our knowledge, there has been no study examining the correlation between cell division orientations based on an underlying ligand gradient. These results implicate an important role for how the extracellular matrix may control cell division. These surfaces would allow for a range of cell behavior (polarization, migration, division, differentiation) studies on tailored biospecific gradients and as a potential biotechnological platform to assess small molecule perturbations of cell function.