Fabrication of Micropatterns of Aligned Collagen Fibrils.

Many tissues in vivo contain aligned structures such as filaments, fibrils, and fibers, which expose cells to anisotropic structural and topographical cues that range from the nanometer to micrometer scales. Understanding how cell behavior is regulated by these cues during physiological and pathological processes (e.g., wound healing, cancer invasion) requires substrates that can expose cells to anisotropic cues over several length scales. In this study, we developed a novel method of fabricating micropatterns of aligned collagen fibrils of different geometry onto PDMS-coated glass coverslips that allowed us to investigate the roles of topography and confinement on corneal cell behavior. When corneal cells were cultured on micropatterns of aligned collagen fibrils in the absence of confinement, the degree of cell alignment increased from 40 ± 14 to 82 ± 5% as the size of the micropattern width decreased from 750 to 50 µm. Although the cell area (∼2500 µm2), cell length (∼160 µm), and projected nuclear area (∼175 µm2) were relatively constant on the different micropattern widths, cells displayed an increased aspect ratio as the width of the aligned collagen fibril micropatterns decreased. We also observed that the morphology of cells adhering to the surrounding uncoated PDMS was dependent upon both the size of the aligned collagen fibril micropattern and the distance from the micropatterns. When corneal cells were confined to the micropatterns of aligned collagen fibrils by a Pluronic coating to passivate the surrounding area, a similar trend in increasing cell alignment was observed (35 ± 10 to 89 ± 2%). However, the projected nuclear area decreased significantly (∼210 to 130 µm2) as the micropattern width decreased from 750 to 50 µm. The development of this method allows for the deposition of aligned collagen fibril micropatterns of different geometries on a transparent and elastic substrate and provides an excellent model system to investigate the role of anisotropic cues in cell behavior.

ECM stiffness modulates the proliferation but not the motility of primary corneal keratocytes in response to PDGF-BB.

During corneal wound healing, keratocytes present within the corneal stroma become activated into a repair phenotype upon the release of growth factors, such as transforming growth factor-beta 1 (TGF-ß1) and platelet-derived growth factor-BB (PDGF-BB). The process of injury and repair can lead to changes in the mechanical properties of the tissue, and previous work has shown that the TGF-ß1-mediated myofibroblast differentiation of corneal keratocytes depends on substratum stiffness. It is still unclear, however, if changes in stiffness can modulate keratocyte behavior in response to other growth factors, such as PDGF-BB. Here, we used a polyacrylamide (PA) gel system to determine whether changes in stiffness influence the proliferation and motility of primary corneal keratocytes treated with PDGF-BB. In the presence of PDGF-BB, cells on stiffer substrata exhibited a more elongated morphology and had higher rates of proliferation than cells in a more compliant microenvironment. Using a freeze-injury to assay cell motility, however, we did not observe any stiffness-dependent differences in the migration of keratocytes treated with PDGF-BB. Taken together, these data highlight the importance of biophysical cues during corneal wound healing and suggest that keratocytes respond differently to changes in ECM stiffness in the presence of different growth factors.

Production of erythrocyte microparticles in a sub-hemolytic environment.

Microparticles are produced by various cells due to a number of different stimuli in the circulatory system. Shear stress has been shown to injure red blood cells resulting in hemolysis or non-reversible sub-hemolytic damage. We hypothesized that, in the sub-hemolytic shear range, there exist sufficient mechanical stimuli for red blood cells to respond with production of microparticles. Red blood cells isolated from blood of healthy volunteers were exposed to high shear stress in a microfluidic channel to mimic mechanical trauma similar to that occurring in ventricular assist devices. Utilizing flow cytometry techniques, both an increase of shear rate and exposure time showed higher concentrations of red blood cell microparticles. Controlled shear rate exposure shows that red blood cell microparticle concentration may be indicative of sub-hemolytic damage to red blood cells. In addition, properties of these red blood cell microparticles produced by shear suggest that mechanical trauma may underlie some complications for cardiovascular patients.

ECM Stiffness Controls the Activation and Contractility of Corneal Keratocytes in Response to TGF-ß1.

After surgery or traumatic injury, corneal wound healing can cause a scarring response that stiffens the tissue and impairs ocular function. This fibrosis is caused in part by the activation of corneal keratocytes from a native mechanically quiescent state to an activated myofibroblastic state. This transformation is tied to signaling downstream of transforming growth factor-ß1 (TGF-ß1). Here, to better understand how biochemical and biophysical cues interact to regulate keratocyte activation and contractility, we cultured primary rabbit corneal keratocytes on flexible substrata of varying stiffness in the presence (or absence) of TGF-ß1. Time-lapse fluorescence microscopy was used to assess changes in keratocyte morphology, as well as to quantify the dynamic traction stresses exerted by cells under different experimental conditions. In other experiments, keratocytes were fixed after 5 days of culture and stained for markers of both contractility and myofibroblastic activation. Treatment with TGF-ß1 elicited distinct phenotypes on substrata of different stiffnesses. Cells on soft (1 kPa) gels formed fewer stress fibers and retained a more dendritic morphology, indicative of a quiescent keratocyte phenotype. Keratocytes cultured on stiff (10 kPa) gels or collagen-coated glass coverslips, however, had broad morphologies, formed abundant stress fibers, exhibited greater levels of α-smooth muscle actin (α-SMA) expression, and exerted larger traction forces. Confocal images of phospho-myosin light chain (pMLC) immunofluorescence, moreover, revealed stiffness-dependent differences in the subcellular distribution of actomyosin contractility, with pMLC localized at the tips of thin cellular processes in mechanically quiescent cells. Importantly, keratocytes cultured in the absence of TGF-ß1 showed no stiffness-dependent differences in α-SMA immunofluorescence, suggesting that a stiff microenvironment alone is insufficient to induce myofibroblastic activation. Taken together, these data suggest that changes in ECM stiffness can modulate the morphology, cytoskeletal organization, and subcellular pattern of force generation in corneal keratocytes treated with TGF-ß1.

Keratocyte mechanobiology.

In vivo, corneal keratocytes reside within a complex 3D extracellular matrix (ECM) consisting of highly aligned collagen lamellae, growth factors, and other extracellular matrix components, and are subjected to various mechanical stimuli during developmental morphogenesis, fluctuations in intraocular pressure, and wound healing. The process by which keratocytes convert changes in mechanical stimuli (e.g. local topography, applied force, ECM stiffness) into biochemical signaling is known as mechanotransduction. Activation of the various mechanotransductive pathways can produce changes in cell migration, proliferation, and differentiation. Here we review how corneal keratocytes respond to and integrate different biochemical and biophysical factors. We first highlight how growth factors and other cytokines regulate the activity of Rho GTPases, cytoskeletal remodeling, and ultimately the mechanical phenotype of keratocytes. We then discuss how changes in the mechanical properties of the ECM have been shown to regulate keratocyte behavior in sophisticated 2D and 3D experimental models of the corneal microenvironment. Finally, we discuss how ECM topography and protein composition can modulate cell phenotypes, and review the different methods of fabricating in vitro mimics of corneal ECM topography, novel approaches for examining topographical effects in vivo, and the impact of different ECM glycoproteins and proteoglycans on keratocyte behavior.

A high-throughput microfluidic method for fabricating aligned collagen fibrils to study Keratocyte behavior.

In vivo, keratocytes are surrounded by aligned type I collagen fibrils that are organized into lamellae. A growing body of literature suggests that the unique topography of the corneal stroma is an important regulator of keratocyte behavior. In this study we describe a microfluidic method to deposit aligned fibrils of type I collagen onto glass coverslips. This high-throughput method allowed for the simultaneous coating of up to eight substrates with aligned collagen fibrils. When these substrates were integrated into a PDMS microwell culture system they provided a platform for high-resolution imaging of keratocyte behavior. Through the use of wide-field fluorescence and differential interference contrast microscopy, we observed that the density of collagen fibrils deposited was dependent upon both the perfusion shear rate of collagen and the time of perfusion. In contrast, a similar degree of fibril alignment was observed over a range of shear rates. When primary normal rabbit keratocytes (NRK) were seeded on substrates with a high density of aligned collagen fibrils and cultured in the presence of platelet derived growth factor (PDGF) the keratocytes displayed an elongated cell body that was co-aligned with the underlying collagen fibrils. In contrast, when NRK were cultured on substrates with a low density of aligned collagen fibrils, the cells showed no preferential orientation. These results suggest that this simple and inexpensive method can provide a general platform to study how simultaneous exposure to topographical and soluble cues influence cell behavior.

Functional cargo delivery into mouse and human fibroblasts using a versatile microfluidic device.

Efficient intracellular cargo delivery is a key hurdle for the translation of many emerging stem cell and cellular reprogramming therapies. Recently, a microfluidic-based device constructed from silicon was shown to transduce macromolecules into cells via shear-induced formation of plasma membrane pores. However, the scalability and widespread application of the current platform is limited since physical deformation-mediated delivery must be optimized for each therapeutic application. Therefore, we sought to create a low-cost, versatile device that could facilitate rapid prototyping and application-specific optimization in most academic research labs. Here we describe the design and implementation of a microfluidic device constructed from Polydimethylsiloxane (PDMS) that we call Cyto-PDMS (Cytoplasmic PDMS-based Delivery and Modification System). Using a systematic Cyto-PDMS workflow, we demonstrate intracellular cargo delivery with minimal effects on cellular viability. We identify specific flow rates at which a wide range of cargo sizes (1-70 kDa) can be delivered to the cell interior. As a proof-of-principle for the biological utility of Cyto-PDMS, we show (i) F-actin labeling in live human fibroblasts and (ii) intracellular delivery of recombinant Cre protein with appropriate genomic recombination in recipient fibroblasts. Taken together, our results demonstrate that Cyto-PDMS can deliver small-molecules to the cytoplasm and biologically active cargo to the nucleus without major effects on viability. We anticipate that the cost and versatility of PDMS can be leveraged to optimize delivery to a broad array of possible cell types and thus expand the potential impact of cellular therapies.

Improved Performance of Glucose Bioanodes Using Composites of (7,6) Single-Walled Carbon Nanotubes and a Ferrocene-LPEI Redox Polymer.

The effect of incorporating different types of carbon nanotubes into composite films of a redox polymer (FcMe2-C3-LPEI) and glucose oxidase (GOX) was investigated. The composite films were constructed by first forming a high-surface area network film of either single-walled carbon nanotubes (SWNTs) or multiwalled carbon nanotubes (MWNTs) on a glassy carbon electrode (GCE) by solution casting of a suspension of Triton-X-100 dispersed SWNTs. Next a glucose responsive redox hydrogel was formed on top of the nanotube-modified electrode by cross-linking FcMe2-C3-LPEI with glucose oxidase via ethylene glycol diglycidyl ether (EGDGE). Electrochemical and enzymatic measurements showed that composite films made with (7,6) SWNTs produced a higher response (3.3 mA/cm2) to glucose than films made with (6,5) SWNTs (1.8 mA/cm2) or MWNTs (1.2 mA/cm2) or films made without SWNTs (0.7 mA/cm2). We also show that the response of the composite films could be systematically varied by fabricating SWNT films with different weight ratios of (7,6) and (6,5) SWNTs. Optimization of the (7,6) SWNTs loading and the redox polymer-enzyme film produced a glucose response of 11.2 mA/cm2. Combining the optimized glucose films with a platinum oxygen breathing cathode into a biofuel cell produced a maximum power density output of 343 µW/cm2.

Electrochemical Characterization of Layer-By-Layer Assembled Ferrocene-Modified Linear Poly(ethylenimine)/Enzyme Bioanodes for Glucose Sensor and Biofuel Cell Applications.

Ferrocenylhexyl- and ferrocenylpropyl-modified linear poly(ethylenimine) (Fc-C6-LPEI, Fc-C3-LPEI) were used with periodate-modified glucose oxidase (p-GOX) in the layer-by-layer assembly of enzymatic bioanodes on gold. Fc-C6-LPEI/p-GOX and Fc-C3-LPEI/p-GOX films of 16 bilayers were capable of generating up to 381 ± 3 and 1417 ± 63 µA cm(-2), respectively, in response to glucose. These responses are greater than those of analogous bioanodes fabricated using conventional cross-linking techniques and are extremely high for planar, low surface area, single-enzyme electrodes. (Fc-C3-LPEI/p-GOX)8 films generated 86 ± 3 µW cm(-2) at pH 7.0 and 149 ± 7 µW cm(-2) at pH 5.0, when poised against an air-breathing platinum cathode in a compartment-less biofuel cell. An increase in power output with decreasing pH was shown to be a result of increases in the platinum cathode performance, indicating it is the rate-limiting electrode in the biofuel cells. The effect of fabrication wash time on the buildup of material at the electrode's surface was probed using cyclic voltammetry (CV) and constant potential amperometry. The use of electrochemical techniques as a diagnostic tool for studying the material deposition process is discussed. CV peak separation (ΔE), surface coverage of the electroactive ferrocene (ΓFc), and amperometric sensitivity of the enzyme to glucose (Jmax), studied as a function of numbers of bilayers, showed that physisorption of materials onto the surface results from initial patchy deposition, rather than in distinctly uniform layers.

Platelets lacking PIP5KIγ have normal integrin activation but impaired cytoskeletal-membrane integrity and adhesion.

Three isoforms of phosphatidylinositol-4-phosphate 5-kinase (PIP5KIα, PIP5KIß, and PIP5KIγ) can each catalyze the final step in the synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn can be either converted to second messengers or bind directly to and thereby regulate proteins such as talin. A widely quoted model speculates that only p90, a longer splice form of platelet-specific PIP5KIγ, but not the shorter p87 PIP5KIγ, regulates the ligand-binding activity of integrins via talin. However, when we used mice genetically engineered to lack only p90 PIP5KIγ, we found that p90 PIP5KIγ is not critical for integrin activation or platelet adhesion on collagen. However, p90 PIP5KIγ-null platelets do have impaired anchoring of their integrins to the underlying cytoskeleton. Platelets lacking both the p90 and p87 PIP5KIγ isoforms had normal integrin activation and actin dynamics, but impaired anchoring of their integrins to the cytoskeleton. Most importantly, they formed weak shear-resistant adhesions ex vivo and unstable vascular occlusions in vivo. Together, our studies demonstrate that, although PIP5KIγ is essential for normal platelet function, individual isoforms of PIP5KIγ fulfill unique roles for the integrin-dependent integrity of the membrane cytoskeleton and for the stabilization of platelet adhesion.

Treatment with both TGF-ß1 and PDGF-BB disrupts the stiffness-dependent myofibroblast differentiation of corneal keratocytes.

During corneal wound healing, stromal keratocytes transform into a repair phenotype that is driven by the release of cytokines, like transforming growth factor-beta 1 (TGF-ß1) and platelet-derived growth factor-BB (PDGF-BB). Previous work has shown that TGF-ß1 promotes the myofibroblast differentiation of corneal keratocytes in a manner that depends on PDGF signaling. In addition, changes in mechanical properties are known to regulate the TGF-ß1-mediated differentiation of cultured keratocytes. While PDGF signaling acts synergistically with TGF-ß1 during myofibroblast differentiation, how treatment with multiple growth factors affects stiffness-dependent differences in keratocyte behavior is unknown. Here, we treated primary corneal keratocytes with PDGF-BB and TGF-ß1 and cultured them on polyacrylamide (PA) substrata of different stiffnesses. In the presence of TGF-ß1 alone, the cells underwent stiffness-dependent myofibroblast differentiation. On stiff substrata, the cells developed robust stress fibers, exhibited high levels of ⍺-SMA staining, formed large focal adhesions (FAs), and exerted elevated contractile forces, whereas cells in a compliant microenvironment showed low levels of ⍺-SMA immunofluorescence, formed smaller focal adhesions, and exerted decreased contractile forces. When the cultured keratocytes were treated simultaneously with PDGF-BB however, increased levels of ⍺-SMA staining and stress fiber formation were observed on compliant substrata, even though the cells did not exhibit elevated contractility or focal adhesion size. Pharmacological inhibition of PDGF signaling disrupted the myofibroblast differentiation of cells cultured on substrata of all stiffnesses. These results indicate that treatment with PDGF-BB can decouple molecular markers of myofibroblast differentiation from the elevated contractile phenotype otherwise associated with these cells, suggesting that crosstalk in the mechanotransductive signaling pathways downstream of TGF-ß1 and PDGF-BB can regulate the stiffness-dependent differentiation of cultured keratocytes. Statement of Significance: In vitro experiments have shown that changes in ECM stiffness can regulate the differentiation of myofibroblasts. Typically, these assays involve the use of individual growth factors, but it is unclear how stiffness-dependent differences in cell behavior are affected by multiple cytokines. Here, we used primary corneal keratocytes to show that treatment with both TGF-ß1 and PDGF-BB disrupts the dependency of myofibroblast differentiation on substratum stiffness. In the presence of both growth factors, keratocytes on soft substrates exhibited elevated ⍺-SMA immunofluorescence without a corresponding increase in contractility or focal adhesion formation. This result suggests that molecular markers of myofibroblast differentiation can be dissociated from the elevated contractile behavior associated with the myofibroblast phenotype, suggesting potential crosstalk in mechanotransductive signaling pathways downstream of TGF-ß1 and PDGF-BB.

Corneal keratocytes, fibroblasts, and myofibroblasts exhibit distinct transcriptional profiles in vitro.

Purpose: After stromal injury to the cornea, the release of growth factors and pro-inflammatory cytokines promotes the activation of quiescent keratocytes into a migratory fibroblast and/or fibrotic myofibroblast phenotype. Persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. This study aims to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which these phenotypic changes occur. Methods: Primary rabbit corneal keratocytes were cultured in either defined serum-free media (SF), fetal bovine serum (FBS) containing media, or in the presence of TGF-ß1 to induce keratocyte, fibroblast, or myofibroblast phenotypes, respectively. Bulk RNA sequencing followed by bioinformatic analyses was performed to identify significant differentially expressed genes (DEGs) and enriched biological pathways for each phenotype. Results: Genes commonly associated with keratocytes, fibroblasts, or myofibroblasts showed high relative expression in SF, FBS, or TGF-ß1 culture conditions, respectively. Differential expression and functional analyses revealed novel DEGs for each cell type, as well as enriched pathways indicative of differences in proliferation, apoptosis, extracellular matrix (ECM) synthesis, cell-ECM interactions, cytokine signaling, and cell mechanics. Conclusions: Overall, these data demonstrate distinct transcriptional differences among cultured corneal keratocytes, fibroblasts, and myofibroblasts. We have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to mechanotransduction and ECM biology. Our findings have revealed novel molecular markers for each cell type, as well as possible targets for modulating cell behavior and promoting physiological corneal wound healing.

Quantifying neutrophil extracellular trap release in a combined infection-inflammation NET-array device.

Excessive release of neutrophil extracellular traps (NETs) has been reported in various human pathologies, including COVID-19 patients. Elevated NET levels serve as a biomarker, indicating increased coagulopathy and immunothrombosis risks in these patients. Traditional immunoassays employed to quantify NET release focus on bulk measurements of released chromatin in simplified microenvironments. In this study, we fabricated a novel NET-array device to quantify NET release from primary human neutrophils with single-cell resolution in the presence of the motile bacteria Pseudomonas aeruginosa PAO1 and inflammatory mediators. The device was engineered to have wide chambers and constricted loops to measure NET release in variably confined spaces. Our open NET-array device enabled immunofluorescent labeling of citrullinated histone H3, a NET release marker. We took time-lapse images of primary healthy human neutrophils releasing NETs in clinically relevant infection and inflammation-rich microenvironments. We then developed a computer-vision-based image processing method to automate the quantification of individual NETs. We showed a significant increase in NET release to Pseudomonas aeruginosa PAO1 when challenged with inflammatory mediators tumor necrosis factor-α [20 ng mL-1] and interleukin-6 [50 ng mL-1], but not leukotriene B4 [20 nM], compared to the infection alone. We also quantified the temporal dynamics of NET release and differences in the relative areas of NETs, showing a high percentage of variable size NET release with combined PAO1 - inflammatory mediator treatment, in the device chambers. Importantly, we demonstrated reduced NET release in the confined loops of our combined infection-inflammation microsystem. Ultimately, our NET-array device stands as a valuable tool, facilitating experiments that enhance our comprehension of the spatiotemporal dynamics of NET release in response to infection within a defined microenvironment. In the future, our system can be used for high throughput and cost-effective screening of novel immunotherapies on human neutrophils in view of the importance of fine-tuning NET release in controlling pathological neutrophil-driven inflammation.

Signal-dependent slow leukocyte rolling does not require cytoskeletal anchorage of P-selectin glycoprotein ligand-1 (PSGL-1) or integrin αLß2.

In inflamed venules, neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin α(L)ß(2) in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate α(L)ß(2), leading to arrest on ICAM-1. Cytoskeletal anchorage of α(L)ß(2) has been linked to chemokine-triggered extension and force-regulated conversion to the high affinity state. We asked whether PSGL-1 must interact with the cytoskeleton to initiate signaling and whether α(L)ß(2) must interact with the cytoskeleton to extend. Fluorescence recovery after photobleaching of transfected cells documented cytoskeletal restraint of PSGL-1. The lateral mobility of PSGL-1 similarly increased by depolymerizing actin filaments with latrunculin B or by mutating the cytoplasmic tail to impair binding to the cytoskeleton. Converting dimeric PSGL-1 to a monomer by replacing its transmembrane domain did not alter its mobility. By transducing retroviruses expressing WT or mutant PSGL-1 into bone marrow-derived macrophages from PSGL-1-deficient mice, we show that PSGL-1 required neither dimerization nor cytoskeletal anchorage to signal ß(2) integrin-dependent slow rolling on P-selectin and ICAM-1. Depolymerizing actin filaments or decreasing actomyosin tension in neutrophils did not impair PSGL-1- or chemokine-mediated integrin extension. Unlike chemokines, PSGL-1 did not signal cytoskeleton-dependent swing out of the ß(2)-hybrid domain associated with the high affinity state. The cytoskeletal independence of PSGL-1-initiated, α(L)ß(2)-mediated slow rolling differs markedly from the cytoskeletal dependence of chemokine-initiated, α(L)ß(2)-mediated arrest.

Effects of microfluidic channel geometry on leukocyte rolling assays.

Microfluidic cell adhesion assays have emerged as a means to increase throughput as well as reduce the amount of costly reagents. However as dimensions of the flow chamber are reduced and approach the diameter of a cell (D(c)), theoretical models have predicted that mechanical stress, force, and torque on a cell will be amplified. We fabricated a series of microfluidic devices that have a constant width:height ratio (10:1) but with varying heights. The smallest microfluidic device (200 µm ×20 µm) requires perfusion rates as low as 40 nL/min to generate wall shear stresses of 0.5 dynes/cm(2). When neutrophils were perfused through P-selectin coated chambers at equivalent wall shear stress, rolling velocities decreased by approximately 70 % as the ratio of cell diameter to chamber height (D(c)/H) increased from 0.08 (H = 100 µm) to 0.40 (H = 20 µm). Three-dimensional numerical simulations of neutrophil rolling in channels of different heights showed a similar trend. Complementary studies with PSGL-1 coated microspheres and paraformaldehyde-fixed neutrophils suggested that changes in rolling velocity were related to cell deformability. Using interference reflection microscopy, we observed increases in neutrophil contact area with increasing chamber height (9-33 %) and increasing wall shear stress (28-56 %). Our results suggest that rolling velocity is dependent not only on wall shear stress but also on the shear stress gradient experienced by the rolling cell. These results point to the D(c)/H ratio as an important design parameter of leukocyte microfluidic assays, and should be applicable to rolling assays that involve other cell types such as platelets or cancer cells.

Layer-by-layer assembly of ferrocene-modified linear polyethylenimine redox polymer films.

Herein, both electrostatic and covalent layer-by-layer assembly were used for the construction of multicomposite thin films using a ferrocene-modified linear poly(ethylenimine) redox polymer (Fc-C6-LPEI) as the cationic polyelectrolye, and poly(acrylic acid) (PAA), poly(glutamic acid) (PGA), or glucose oxidase (GOX) as the negative polyelectrolyte. The assembly of the multilayer films was characterized by cyclic voltammetry (CV), UV/Vis spectroscopy, and ellipsometry with the enzymatic response of the films containing GOX being characterized via constant potential amperometry. CV measurements suggested that the successful buildup of multilayer films was dependent upon the nature of the anionic polyelectrolyte used. Electrostatic assembly of films composed of Fc-C6-LPEI and either PAA or PGA produced large oxidation peak current densities of 630 and 670 µA cm(-2), respectively, during cyclic voltammetry. Increased measured absorbance by UV/Vis spectroscopy and increased measured film thicknesses (400-600 nm) by ellipsometry provided additional evidence of successful film formation. In contrast, the films incorporating GOX that were electrostatically assembled surprisingly produced significantly lower electrochemical responses (12 µA cm(-2)), low absorbance values, and reduced film thicknesses (~15 nm), and glucose electro-oxidation current densities less than 1 µA cm(-2), which all suggested unstable or minimal film formation. Subsequently, we developed a covalent layer-by-layer approach to fabricate films of Fc-C6-LPEI/GOX by covalently linking the amine groups of Fc-C6-LPEI to the aldehyde groups of periodate-oxidized glucose oxidase. Covalent assembly of the Fc-C6-LPEI/GOX films produced oxidation peak current densities during cyclic voltammetry of 40 µA cm(-2) and glucose electro-oxidation current densities of 220 µA cm(-2). These films also showed an increase in their thicknesses (~140 nm) relative to the electrostatic GOX films. For the films containing either PAA or PGA, the pH of the polymer solutions used for construction was found to have a significant effect on the response of the multilayer films, and the electrochemical response of the Fc-C6-LPEI/PAA, Fc-C6-LPEI/PGA, or covalently assembled Fc-C6-LPEI/GOX films could be tuned by varying the number of bilayers (n=1-16) in the film. These results are important because this is the first report of the use of the novel Fc-C6-LPEI redox polymer in the successful development of multicomposite layer-by-layer films. The electrochemical response achieved with the covalently assembled Fc-C6-LPEI/GOX films demonstrates that this redox polymer and layer-by-layer assembly technique can be used for possible biosensor and biofuel applications, and the success of multiple anionic polyelectrolytes could lead to additional applications with other enzyme systems.

Effect of surfactant type and redox polymer type on single-walled carbon nanotube modified electrodes.

Electrodes modified with single-walled carbon nanotubes (SWNTs) offer a number of attractive properties for developing novel electrochemical sensors. A common method to immobilize SWNTs onto the electrode surface is by placing a droplet of a SWNT suspension onto the electrode surface and allowing the solvent to evaporate. In order to maximize the properties of individual SWNTs, surfactants are normally present in these suspensions to provide stable and homogeneous SWNT dispersions. In this study we investigated the effect of different surfactants on the electrochemical and enzymatic performance of SWNT modified glassy carbon electrodes (GCEs). Amperometic biosensors for glucose were fabricated by a two-step procedure. In the first step, SWNT films were deposited onto GCEs by solution casting suspensions of SWNTs in water, Triton X-100, Tween 20, sodium cholate or sodium dodecylbenzenesulfonate (NaDDBS). In the second step, hydrogels containing a redox polymer and the enzyme, glucose oxidase (GOX), were deposited and cross-linked onto the SWNT-modified GCE. Three different redox polymers were tested: 3-ferrocenylpropyl-modified LPEI, (Fc-C3-LPEI), 6-ferrocenylhexyl-modified LPEI, (Fc-C6-LPEI), and poly[(vinylpyridine)Os(bipyridyl)2Cl](2+/3+)(PVP-Os). Biosensors constructed with SWNT films from suspensions of Triton X-100 or Tween 20 generally produced the highest electrochemical and enzymatic responses, with Triton X-100 films producing current densities of ~1.7-2.1 mA/cm(2) for the three different redox polymers. In contrast, biosensors constructed with SWNT films from sodium cholate suspensions resulted in significant decreases in the electrochemical and enzymatic response and in some cases showed no enzymatic activity. The results with SWNT films from NaDDBS suspensions were dependent upon the specific redox polymer used, but in general gave reduced enzymatic responses (~0.05-0.4 mA/cm(2)). These results demonstrate the importance of surfactant type in fabricating SWNT-modified electrode films.

Label free impedance based acetylcholinesterase enzymatic biosensors for the detection of acetylcholine.

Realtime monitoring of neurotransmitters is of great interest for understanding their fundamental role in a wide range of biological processes in the central and peripheral nervous system, as well as their role, in several degenerative brain diseases. The measurement of acetylcholine in the brain is particularly challenging due to the complex environment of the brain and the low concentration and short lifetime of acetylcholine. In this paper, we demonstrated a novel, label-free biosensor for the detection of Ach using a single enzyme, acetylcholinesterase (ACHE), and electrochemical impedance spectroscopy (EIS). Acetylcholinesterase was covalently immobilized onto the surface of gold microelectrodes through an amine-reactive crosslinker dithiobis(succinimidyl propionate) (DSP). Passivation of the gold electrode with SuperBlock eliminated or reduced any non-specific response to other major interfering neurotransmitter molecules such as dopamine (DA), norepinephrine (NE) and epinephrine (EH). The sensors were able to detect acetylcholine over a wide concentration range (5.5-550 µM) in sample volumes as small as 300 µL by applying a 10 mV AC voltage at a frequency of 500 Hz. The sensors showed a linear relationship between Ach concentration and ΔZmod(R2 = 0.99) in PBS. The sensor responded to acetylcholine not only when evaluated in a simple buffer (PBS buffer) but in several more complex environments such as rat brain slurry and rat whole blood. The sensor remained responsive to acetylcholine after being implanted ex vivo in rat brain tissue. These results bode well for the future application of these novel sensors for real time in vivo monitoring of acetylcholine.

Effects of Topography and PDGF on the Response of Corneal Keratocytes to Fibronectin-Coated Surfaces.

During corneal wound healing, corneal keratocytes are exposed to both biophysical and soluble cues that cause them to transform from a quiescent state to a repair phenotype. How keratocytes integrate these multiple cues simultaneously is not well understood. To investigate this process, primary rabbit corneal keratocytes were cultured on substrates patterned with aligned collagen fibrils and coated with adsorbed fibronectin. After 2 or 5 days of culture, keratocytes were fixed and stained to assess changes in cell morphology and markers of myofibroblastic activation by fluorescence microscopy. Initially, adsorbed fibronectin had an activating effect on the keratocytes as evidenced by changes in cell shape, stress fiber formation, and expression of alpha-smooth muscle actin (α-SMA). The magnitude of these effects depended upon substrate topography (i.e., flat substrate vs aligned collagen fibrils) and decreased with culture time. When keratocytes were simultaneously exposed to adsorbed fibronectin and soluble platelet-derived growth factor-BB (PDGF-BB), the cells elongated and had reduced expression of stress fibers and α-SMA. In the presence of PDGF-BB, keratocytes plated on the aligned collagen fibrils elongated in the direction of the fibrils. These results provide new information on how keratocytes respond to multiple simultaneous cues and how the anisotropic topography of aligned collagen fibrils influences keratocyte behavior.

Independently controlling protein dot size and spacing in particle lithography.

Particle lithography is a relatively simple, inexpensive technique used to pattern inorganics, metals, polymers, and biological molecules on the micro- and nanometer scales. Previously, we used particle lithography to create hexagonal patterns of protein dots in a protein resistant background of methoxy-poly(ethylene glycol)-silane (mPEG-sil). In this work, we describe a simple heating procedure to overcome a potential limitation of particle lithography: the simultaneous change in feature size and center-to-center spacing as the diameter of the spheres used in the lithographic mask is changed. Uniform heating was used to make single-diameter protein patterns with dot sizes of approximately 2-4 or 2-8 µm, depending on the diameter of the spheres used in the lithographic mask, while differential heating was used to make a continuous gradient of dot sizes of approximately 1-9 µm on a single surface. We demonstrate the applicability of these substrates by observing the differences in neutrophil spreading on patterned and unpatterned protein coated surfaces.