Nathan P. Westcott

Synergistic microfluidic and electrochemical strategy to activate and pattern surfaces selectively with ligands and cells.

We show a straightforward, flexible synergistic approach that combines microfluidics, electrochemistry, and a general immobilization strategy to activate regions of a substrate selectively for the precise immobilization of ligands and cells in patterns for a variety of cell-based assays and cell migration and cell adhesion studies. We develop microfluidic microchips to control the delivery of electrolyte solution to select regions of an electroactive hydroquinone SAM. Once an electrical potential is applied to the substrate, only the hydroquinone exposed to electrolyte solution within the microfluidic channels oxidizes to the corresponding quinone. The quinone form can then react chemoselectively with oxyamine-tethered ligands to pattern the surface. Therefore, this microfluidic/electrochemistry strategy selectively activates the surface for ligand patterning that exactly matches the channel design of the microfluidic channel. We demonstrate the ease of this system by first quantitatively characterizing the electrochemical activation and immobilization of ligands on the surface. Second, we immobilize a fluorescent dye to show the fidelity of the methodology, and third, we show the immobilization of biospecific cell adhesive peptide ligands to pattern cells. This is the first report that combines microfluidics/electrochemistry and a general electroactive immobilization strategy to pattern ligands and cells. We believe that this strategy will be of broad utility for applications ranging from fundamental studies of cell behavior to patterning molecules on a variety of materials for molecular electronic devices.

Rapid in Situ Generation of Two Patterned Chemoselective Surface Chemistries from a Single Hydroxy-Terminated Surface Using Controlled Microfluidic Oxidation

In this work, we develop a new, rapid and inexpensive method to generate spatially controlled aldehyde and carboxylic acid surface groups by microfluidic oxidation of 11-hydroxyundecylphosphonic acid self-assembled monolayers (SAMs) on indium tin oxide (ITO) surfaces. SAMs are activated and patterned using a reversibly sealable, elastomeric polydimethylsiloxane cassette, fabricated with preformed micropatterns by soft lithography. By flowing the mild oxidant pyridinium chlorochromate through the microchannels, only selected areas of the SAM are chemically altered. This microfluidic oxidation strategy allows for ligand immobilization by two chemistries originating from a single SAM composition. ITO is robust, conductive, and transparent, making it an ideal platform for studying interfacial interactions. We display spatial control over the immobilization of a variety of ligands on ITO and characterize the resulting oxime and amide linkages by electrochemistry, X-ray photoelectron spectroscopy, contact angle, fluorescence microscopy, and atomic force microscopy. This general method may be used with many other materials to rapidly generate patterned and tailored surfaces for studies ranging from molecular electronics to biospecific cell-based assays and biomolecular microarrays.

Renewable and Optically Transparent Electroactive Indium Tin Oxide Surfaces for Chemoselective Ligand Immobilization and Biospecific Cell Adhesion

In this report, we show the successful transfer of a sophisticated electroactive immobilization and release strategy to an indium tin oxide (ITO) surface to generate (1) optically transparent, robust, and renewable surfaces, (2) inert surfaces that resist nonspecific protein adsorption and cell attachment, and (3) tailored biospecific surfaces for live-cell high-resolution fluorescence microscopy of cell culture. By comparing the surface chemistry properties on both ITO and gold surfaces, we demonstrate the ITO surfaces are superior to gold as a renewable surface, in robustness (durability), and as an optically transparent material for live-cell fluorescence microscopy studies of cell behavior. These advantages will make ITO surfaces a desired platform for numerous biosensor and microarray applications and as model substrates for various cell biological studies.

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.

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.

General chemoselective and redox-responsive ligation and release strategy.

We report a switchable redox click and cleave reaction strategy for conjugating and releasing a range of molecules on demand. This chemoselective redox-responsive ligation (CRRL) and release strategy is based on a redox switchable oxime linkage that is controlled by mild chemical or electrochemical redox signals and can be performed at physiological conditions without the use of a catalyst. Both conjugation and release reactions are kinetically well behaved and quantitative. The CRRL strategy is synthetically modular and easily monitored and characterized by routine analytical techniques. We demonstrate how the CRRL strategy can be used for the dynamic generation of cyclic peptides and the ligation of two different peptides that are stable but can be selectively cleaved upon changes in the redox environment. We also demonstrate a new redox based delivery of cargoes to live cells strategy via the CRRL methodology by synthesizing a FRET redox-responsive probe that is selectively activated within a cellular environment. We believe the ease of the CRRL strategy should find wide use in a range of applications in biology, tissue engineering, nanoscience, synthetic chemistry, and material science and will expand the suite of current conjugation and release strategies.

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.

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.

A Dual Receptor and Reporter for Multi-Modal Cell Surface Engineering.

The rapid development of new small molecule drugs, nanomaterials, and genetic tools to modulate cellular function through cell surface manipulation has revolutionized the diagnosis, study, and treatment of disorders in human health. Since the cell membrane is a selective gateway barrier that serves as the first line of defense/offense and communication to its environment, new approaches that molecularly engineer or tailor cell membrane surfaces would allow for a new era in therapeutic design, therapeutic delivery, complex coculture tissue construction, and in situ imaging probe tracking technologies. In order to develop the next generation of multimodal therapies, cell behavior studies, and biotechnologies that focus on cell membrane biology, new tools that intersect the fields of chemistry, biology, and engineering are required. Herein, we develop a liposome fusion and delivery strategy to present a novel dual receptor and reporter system at cell surfaces without the use of molecular biology or metabolic biosynthesis. The cell surface receptor is based on bio-orthogonal functional groups that can conjugate a range of ligands while simultaneously reporting the conjugation through the emission of fluorescence. We demonstrate this dual receptor and reporter system by conjugating and tracking various cell surface ligands for temporal control of cell fluorescent signaling, cell-cell interaction, and tissue assembly construction.