Article

Laser Patterning of Self-Assembled Monolayers on PEDOT:PSS Films for Controlled Cell Adhesion

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Abstract

Conducting polymers have shown great potential as a means to interface electronics with living tissues, toward a plethora of different biological applications ranging from in vitro to in vivo systems. However, the development of effective functionalization approaches to render this interface biomimetic still remains rather challenging, due to the lack of inherent surface functionalities in such polymers. Here, a straightforward and versatile modification strategy of poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) surfaces is demonstrated for preferential and spatially confined cell adhesion and growth. By combining three simple surface modification steps, including chemical modification using self-assembled monolayers and their selective laser ablation, this study is able to design either cell-adhesive or cell-repulsive patterns of various shapes on PEDOT:PSS films. Studies using Madin–Darby canine kidney II epithelial cells reveal preferential cell adhesion and growth with good precision following the preformed patterns. The proposed surface modification approach can be extended to encompass a variety of polymeric biomaterials, without affecting their bulk properties.

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... On the other hand, the fabrication of precise PEDOT:PSS nanostructures have permitted the modification and enhancement of the resulting polymer properties [24][25][26][27], in turn, leading to boosted applications [28][29][30][31][32][33][34]. In this way, several groups have developed different nanostructured PEDOT:PSS surfaces by laser irradiation [27,31,33,35,36], nanoimprint lithography [25,29,37,38], and by using templates and molds [31,34,39]. ...
... On the other hand, the fabrication of precise PEDOT:PSS nanostructures have permitted the modification and enhancement of the resulting polymer properties [24][25][26][27], in turn, leading to boosted applications [28][29][30][31][32][33][34]. In this way, several groups have developed different nanostructured PEDOT:PSS surfaces by laser irradiation [27,31,33,35,36], nanoimprint lithography [25,29,37,38], and by using templates and molds [31,34,39]. In these works, it was possible to fabricate distinct surface features as linear nanostructures or nanogratings [25,27,34,35,37,39], nanocavities [36], nanopores/nanopillars [31], hemispherical patterns [29], with characteristics lengths ranging from 10 2 -10 4 nm. ...
... Also, it requires access to clean room facilities and the use of a nanopatterned stamp, introducing several conditions to fulfill prior the actual nanostructuring process. Moreover, the use of laser-based PEDOT:PSS nanostructuring has been mostly applied by ablating the polymer surface [33,35,36,41]. ...
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We present the preparation of nanostructured conducting PEDOT:PSS thin films by solvent vapor annealing (SVA), using the low boiling point solvent tetrahydrofuran (THF). An Atomic Force Microscopy (AFM) study allowed the observation of distinct nanostructure development as a function of solvent exposure time. Moreover, the nanostructures’ physical properties were evaluated by nanomechanical, nanoelectrical, and nano-FTIR measurements. In this way, we were able to differentiate the local response of the developed phases and to identify their chemical nature. The combination of these techniques allowed to demonstrate that exposure to THF is a facile method to effectively and selectively modify the surface nanostructure of PEDOT:PSS, and thereafter its final properties. Moreover, our nanoscale studies provided evidence about the molecular rearrangements that PEDOT:PSS suffers during nanostructure fabrication, a fundamental fact in order to expand the potential applications of this polymer in thermoelectric and optoelectronic devices.
... In this specific case, the streptavidin-biotin chemistry was adopted to functionalize the surface of the device electrodes with the selected antibody for the target analyte [42,43]. Figure 1 describes our functionalization strategy, which consists of 5 main steps: (1) PEDOT:PSS thin films were plasma-treated with low power intensity to introduce hydroxyl groups on the semiconducting polymer surface without etching it, nor affecting its conductivity [44]; (2) (3-Aminopropyl)triethoxysilane (APTES) molecules were covalently bound to the activated surfaces; (3) biotin units were linked to the APTES-functionalized polymer surfaces; (4) streptavidin molecules bound with high affinity to biotin; (5) a biotinylated, capture antibody (therefore binding to streptavidin) was used to impart specificity towards the target analyte/cytokine. ...
... The plasma treatment induced a reduction of the contact angle of the PEDOT:PSS film from 41.9°±0.2°to 0°, due to the introduction of hydroxyl groups [44]. Plasma-treated surfaces were functionalized with an APTES layer by means of either CVD or Soaking (S) process, as described in the Materials and Methods section. ...
... respectively). According to [44][45][46], a hydrophilic surface functionalized with APTES will display values of contact angle close to 45°. ...
... Drug release [13, 15-18, 39, 51-56] Surface functionalization [32][33][34][35][36][37] Composites/blends [12-25, 29, 30, 38, 40-50] Stretchability [22,[76][77][78][79][80][81][82] σ σ ...
... In addition, molecules can be physically adsorbed or be covalently bound to the CP surface by including functional anchors in the polymer. Reproduced with permission from [36], C WILEY-VCH Verlag GmbH & Co. KGaA (2017). Reproduced with permission from [38], C Elsevier (2014). ...
... It should be noted that reduced mechanical integrity and electrochemical performance could be associated with the relatively large molecular weight (>10 kDa) of biological dopants as well as their poorly conducting nature [35]. Covalent modification of the CP surface, although more synthetically complex, has provided an alternative route to improve biological integration of CP microelectrodes ( Figure 3A) [36]. One example involves functionalization of the conventional EDOT monomer to produce EDOT acid, which, upon polymerization exposes a carboxylic acid functional group (COOH) on the polymer surface. ...
Article
The widespread use of conducting polymers, especially poly(3,4-ethylene dioxythiophene) (PEDOT), within the space of bioelectronics has enabled improvements, both in terms of electrochemistry and functional versatility, of conventional metallic electrodes. This short review aims to provide an overview of how PEDOT coatings have contributed to functionalizing existing bioelectronics, the challenges which meet conducting polymer coatings from a regulatory and stability point of view and the possibilities to bring PEDOT-based coatings into large-scale clinical applications. Finally, their potential use for enabling new technologies for the field of bioelectronics as biodegradable, stretchable and slow-stimulation materials will be discussed.
... However, systematic 3D nano-and micro-structuring and patterning of organic semiconductors can involve complex and time-consuming processes due to the sensitivity of conjugated polymers to traditional cleanroom-based patterning methods. 14,15 Furthermore, the characterization of the interface between those 3D electroactive materials and cells requires investigations at the nanoscale of soft interfaces, which presents significant challenges. ...
... Laser ablation of polymer materials has been studied widely 17 and various reports on laser patterning for cell control demonstrate a range of applications, 18 such as directing cell migration 19 and preferential cell adhesion by laser ablation of self-assembled monolayers. 15 However, in this work, we demonstrate for the first time a rapid out-of-cleanroom direct-write laser patterning method to increase the conducting polymer/cell attachment, allowing for a direct measurement of interface impedance. ...
Article
Interfacing soft materials with biological systems holds considerable promise for both biosensors and recording live cells. However, the interface between cells and organic substrates is not well studied, despite its crucial role in the effectiveness of the device. Furthermore, well-known cell adhesion enhancers, such as micro-grooves, have not been implemented on these surfaces Here, we present a nanoscale characterization of the cell-substrate interface for 3D laser-patterned organic electrodes by combining electrochemical impedance spectroscopy (EIS) and scanning electron microscopy/focused ion beam (SEM/FIB). We demonstrate that introducing 3D micro-patterned grooves on organic surfaces enhances the cell adhesion of electrogenic cells.
... In particular, by patterning cell-repelling (perfluorodecyltrichlorosilane, FDTS) SAMs on top of PEDOT:PSS films, we were able to spatially confine cells in laser-ablated regions of various shapes and sizes, with remarkable precision. 224 Greco et al. demonstrated a novel method for the preparation of micro-or nanowrinkles on CP surfaces. 215 As shown in Figure 11d, wrinkle-like structures were produced via a heat-shrinking process with a thermo-retractable polystyrene substrate. ...
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Bioelectronics have made strides in improving clinical diagnostics and precision medicine. The potential of bioelectronics for bidirectional interfacing with biology through continuous, label-free monitoring on one side and precise control of biological activity on the other has extended their application scope to in vitro systems. The advent of microfluidics and the considerable advances in reliability and complexity of in vitro models promise to eventually significantly reduce or replace animal studies, currently the gold standard in drug discovery and toxicology testing. Bioelectronics are anticipated to play a major role in this transition offering a much needed technology to push forward the drug discovery paradigm. Organic electronic materials, notably conjugated polymers, having demonstrated technological maturity in fields such as solar cells and light emitting diodes given their outstanding characteristics and versatility in processing, are the obvious route forward for bioelectronics due to their biomimetic nature, among other merits. This review highlights the advances in conjugated polymers for interfacing with biological tissue in vitro, aiming ultimately to develop next generation in vitro systems. We showcase in vitro interfacing across multiple length scales, involving biological models of varying complexity, from cell components to complex 3D cell cultures. The state of the art, the possibilities, and the challenges of conjugated polymers toward clinical translation of in vitro systems are also discussed throughout.
... 170,171 It is also possible to link biological species through functional groups on the CP film surface 172 or via the use of selfassembled monolayers (SAMs). 173,174 CP films can be designed to bear side-chains, such as polymer brushes, comprising biocomponents. 175−177 Some CPs possess a charged surface that can be exploited in a layer-by-layer (LbL) assembly type strategy to link the biorecognition units. ...
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Electrochemical detection of metabolites is essential for early diagnosis and continuous monitoring of a variety of health conditions. This review focuses on organic electronic material-based metabolite sensors and highlights their potential to tackle critical challenges associated with metabolite detection. We provide an overview of the distinct classes of organic electronic materials and biorecognition units used in metabolite sensors, explain the different detection strategies developed to date, and identify the advantages and drawbacks of each technology. We then benchmark state-of-the-art organic electronic metabolite sensors by categorizing them based on their application area (in vitro, body-interfaced, in vivo, and cell-interfaced). Finally, we share our perspective on using organic bioelectronic materials for metabolite sensing and address the current challenges for the devices and progress to come.
... (7) On/off patterning is achieved by interrupting the jet with a mechanical shutter. Wang et al., 2016;Ohayon et al., 2017). In addition, PEDOT:PSSbased substrates have been shown to allow the direct electrical stimulation of electrogenic cells (e.g., neurons and muscle cells), regulating or inducing several biological functions (Gomez et al., 2007;Lundin et al., 2011). ...
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Digitally driven manufacturing technologies such as aerosol jet printing (AJP) can make a significant contribution to enabling new capabilities in the field of tissue engineering disease modeling and drug screening. AJP is an emerging non-contact and mask-less printing process which has distinct advantages over other patterning technologies as it offers versatile, high-resolution, direct-write deposition of a variety of materials on planar and non-planar surfaces. This research demonstrates the ability of AJP to print digitally controlled patterns that influence neuronal guidance. These consist of patterned poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) tracks on both glass and poly(potassium 3-sulfopropyl methacrylate) (PKSPMA) coated glass surfaces, promoting selective adhesion of SH-SY5Y neuroblastoma cells. The cell attractive patterns had a maximum height ≥0.2 μm, width and half height ≥15 μm, Ra = 3.5 nm, and RMS = 4.1. The developed biocompatible PEDOT:PSS ink was shown to promote adhesion, growth and differentiation of SH-SY5Y neuronal cells. SH-SY5Y cells cultured directly onto these features exhibited increased nuclei and neuronal alignment on both substrates. In addition, the cell adhesion to the substrate was selective when cultured onto the PKSPMA surfaces resulting in a highly organized neural pattern. This demonstrated the ability to rapidly and flexibly realize intricate and accurate cell patterns by a computer controlled process.
... What governs the process of vesicle adsorption and rupture is not fully understood, but the first requirement is a hydrophilic substrate. To promote vesicle fusion on PEDOT:PSS an oxygen plasma treatment can be done to introduce OH-groups at the surface, which renders the surface more hydrophilic (33,34). There are two techniques for monitoring the formation of an SLB through vesicle fusion applied in Paper II. ...
... Another approach to patterning PEDOT:PSS as an ordered thin-film structure for application in organic electronic devices suggests the use of self-assembled monolayers (SAMs) for the modification of the surface free energy and morphology of the polymer to effectively adjust its W f for better energy level matching between the polymer electrodes and the active layers of the devices [117,118]. ...
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An organic conductive polymer, poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS), is an attractive candidate for a low-cost, low-temperature, and solution-processed electrode material for achieving high-performance flexible and stretchable thin-film devices. Unlike most organic materials, this water-soluble conjugated polymer is highly stable against chemical and physical exposure. It exhibits the most superior mechanical flexibility and the highest optical transparency and electrical conductivity among all organic conductors. Therefore, this conductive polymer is among the most promising alternatives to the expensive, rigid, and brittle metal oxide- and even metal-based electrode materials, such as indium tin oxide (ITO) and gold, in the future solution-processed electronic devices. Nevertheless, the intrinsic conductivity of PEDOT:PSS is typically below 1 S cm⁻¹, which is too low for such devices. Fortunately, the material properties of PEDOT:PSS, including its conductivity, are easily tuned by employing a large number of simple approaches. In this paper, the reports on the successful application of PEDOT:PSS to a wide range of solution-processed organic devices, such as organic light-emitting diodes (OLEDs), organic thin-film transistors (OTFTs), and organic photovoltaics (OPVs), are reviewed. The recent progress in the development of highly conductive PEDOT:PSS-based films for electrode applications in the field of organic electronics is the main focus of the discussion herein.
... The compatibility of PEDOT:PSS with cell cultures is wellestablished (Ramuz et al., 2015;Ohayon et al., 2017). Several organic electronic schemes, developed by us and others, utilize this material with biological models (e.g., barrier forming cells) for biosensing and monitoring applications (Pappa et al., 2018;Pitsalidis et al., 2018b). ...
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... better compatibilities with cellular networks) [43,44]. A recent work that incorporated nanostructured PEDOT:PSS layer into epithelial sensing platform has marked the start of using this technology for the improvement on cell-based applications [45]. ...
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... While the first reported OECTs were based on polypyrrole (12), the workhorse material used typically as the channel is the conducting polymer poly (3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT: PSS) due to its notable stability in its oxidized and reduced forms. Especially important for cell-based applications, its optical transparency and its amenability to surface functionalization allow parallel optical and electrical assessment of cells (13) as well as controlled cell attachment and growth on its surface (14). ...
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... [10][11][12] In addition, these materials offer more micro-patterning options than the traditional metallic conductors and can be used for instance to engineer surfaces with selectively cell-adhesive or cell-repulsive areas. 13 Center for Advanced Biomaterials for Healthcare, Istituto Italiano di Tecnologia, 80125, Naples, Italy. E-mail: francesca.santoro@iit.it ...
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Thesis
A plethora of biologically relevant processes depends directly on the effective secretion of biomolecules, from regulatory molecules to structural components. Thus, the analysis of complex biological processes requires the development of novel biosensing tools. Therefore, the aim of this thesis is to provide versatile strategies for the generation of innovative biosensors and biochips based on Surface Plasmon Resonance (SPR). As a result from this research, an indirect photofunctionalization method was developed. This procedure allowed the generation of protein microarrays in fully aqueous conditions while preserving the functionality of the grafted proteins. Furthermore, we created and evaluated a novel microstructured SPR biochip for real-time monitoring of cellular secretions. This microstructured biochip presents two different optical phenomena which could be used for cell detection and the monitoring of their secretions. Finally, multiple functionalization strategies were evaluated for the conception of a nanostructured fiber-bundle SPR biochip. Among the approaches, the generation of photoreactive self-assembled monolayers was the most adapted to this system and currently is being optimized. Once achieved, this nanostructured biochip could pave the way for further development of promising in vivo biosensing systems.
Thesis
Rapid and early diagnosis of disease plays a major role in preventative healthcare. Undoubtedly, technological evolutions, particularly in microelectronics and materials science, have made the hitherto utopian scenario of portable, point-of-care personalized diagnostics a reality. Organic electronic materials, having already demonstrated a significant technological maturity with the development of high tech products such as displays for smartphones or portable solar cells, have emerged as especially promising candidates for biomedical applications. Their soft and fuzzy nature allows for an almost seamless interface with the biological milieu rendering these materials ideally capable of bridging the gap between electronics and biology. The aim of this thesis is to explore and validate the capabilities of organic electronic materials and devices in real-world biological sensing applications focusing on metabolite sensing, by combining both the right materials and device engineering. We show proof-of-concept studies including microfluidic integrated organic electronic platforms for multiple metabolite detection in bodily fluids, as well as more complex organic transistor circuits for detection in tumor cell cultures. We finally show the versatility of organic electronic materials and devices by demonstrating other sensing strategies such as nucleic acid detection using a simple biofunctionalization approach. Although the focus is on in vitro metabolite monitoring, the findings generated throughout this work can be extended to a variety of other sensing strategies as well as to applications including on body (wearable) or even in vivo sensing.
Article
Organic semiconductors are being increasingly used for a variety of biological applications, such as biochemical sensors, drug delivery, and neural interfaces. However, the poor adhesion of cells to the typically hydrophobic, neutrally charged and low surface energy of semiconducting thin films limit their use in in vitro, cell integrated bioelectronic devices. In this work, we investigate the influence of lysine side chain units incorporated in a diketopyrrolopyrrole (DPP) semiconducting polymer on neural cell adhesion and growth, as well as evaluate their function in electrical devices. Synthesis of such biofunctionalized polymers obviates the need of biological coating steps while changing the surface physiochemistry, promising for applications in bioelectronics.
Article
The lipid bilayer is the elemental structure of cell membrane, forming a stable barrier between the interior and exterior of the cell while hosting membrane proteins that enable selective transport of biologically important compounds and cellular recognition. Monitoring the quality and function of lipid bilayers is thus essential and can be performed using electrically active substrates that allow for transduction of signals. Such a promising electronic transducer material is the conducting polymer poly(3,4-ethylenedioxythiophene) doped with poly(styrene sulfonate) (PEDOT:PSS) which has provided a plethora of novel bio transducing architectures. The challenge is however in assembling a bilayer on the conducting polymer surface, which is defect-free and has high mobility. Herein, we investigate the fusion of zwitterionic vesicles on a variety of PEDOT:PSS films, but also on an electron transporting, negatively charged organic semiconductor, in order to understand the surface properties that trigger vesicle fusion. The PEDOT:PSS films are prepared from dispersions containing different concentrations of ethylene glycol included as a formulation additive, which gives a handle to modulate surface physicochemical properties without a compromise on the chemical composition. The strong correlation between the polarity of the surface, the fusion of vesicles and the mobility of the resulting bilayer aides extracting design principles for the development of future conducting polymers that will enable the formation of lipid bilayers.
Article
Mesoangioblasts are outstanding candidates for stem cell therapy and are already being explored in clinical trials. However, a crucial challenge in regenerative medicine is the limited availability of undifferentiated myogenic progenitor cells, since growth is typically accompanied by differentiation. Here reversible myogenic-differentiation-switching during proliferation is achieved by functionalizing the glass substrate with high-density ZnO nanowires. Specifically, mesoangioblasts grown on ZnO nanowires present a spherical viable undifferentiated cell state without lamellopodia formation during all the observation time (8 days). Consistently, the Myosin Heavy Chain, typically expressed in skeletal muscle tissue and differentiated myogenic progenitors, is completely absent. Remarkably, nanowires do not induce any damage while reversibly block differentiation, so that the differentiation capabilities are completely recovered upon cells removal from the nanowires-functionalized substrate and re-plating on standard culture glass. This is the first evidence of a reversible myogenic-differentiation switch which does not affect viability. These results can be the first step toward the in vitro growth of a large number of undifferentiated stem/progenitor cells and therefore can represent a breakthrough for cell based therapy and tissue engineering.
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Article
The polydioxythiophenes PEDOT and more recently ProDOT have emerged as champion materials in the field of organic bioelectronics, both in the domain of biosensing and also for integration with living cells (both in vitro and in vivo). Although polydioxythiophenes in their pristine forms have shown great promise for bioelectronics, in order to broaden the spectrum of applications, a biofunctionalization step is essential. In this review we summarise the methods that have been used thus far to biofunctionalize polydioxythiophenes in an effort to improve the biotic/abiotic interface. We provide an introduction to this class of materials, focusing particularly on the different methods of synthesis (chemical oxidative polymerization, vapor phase polymerization or direct electrochemical polymerization) and discuss the implications of synthesis on biofunctionalization. Rather than provide an exhaustive review, we chose to highlight key examples of biofunctionalization techniques for polydioxythiophenes for specific biomedical applications. Finally, we conclude with a brief discussion of the importance of biofunctionalization methods in future bioelectronics applications, and some ideas for future directions in this field.
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Article
Although adhesive interactions between cells and nanostructured interfaces have been studied extensively, there is a paucity of data on how nanostructured interfaces repel cells by directing cell migration and cell-colony organization. Here, by using multiphoton ablation lithography to pattern surfaces with nanoscale craters of various aspect ratios and pitches, we show that the surfaces altered the cells' focal-adhesion size and distribution, thus affecting cell morphology, migration and ultimately localization. We also show that nanocrater pitch can disrupt the formation of mature focal adhesions to favour the migration of cells towards higher-pitched regions, which present increased planar area for the formation of stable focal adhesions. Moreover, by designing surfaces with variable pitch but constant nanocrater dimensions, we were able to create circular and striped cellular patterns. Our surface-patterning approach, which does not involve chemical treatments and can be applied to various materials, represents a simple method to control cell behaviour on surfaces.
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Article
We report the fabrication of three dimensional (3D) macroporous scaffolds made from poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) via an ice-templating method. The scaffolds offer tunable pore size and morphology, and are electrochemically active. When a potential is applied to the scaffolds, reversible changes take place in their electrical doping state, which in turn enables precise control over the conformation of adsorbed proteins (e.g., fibronectin). Additionally, the scaffolds support the growth of mouse fibroblasts (3T3-L1) for 7 days, and are able to electrically control cell adhesion and pro-angiogenic capability. These 3D matrix-mimicking platforms offer precise control of protein conformation and major cell functions, over large volumes and long cell culture times. As such, they represent a new tool for biological research with many potential applications in bioelectronics, tissue engineering, and regenerative medicine.
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Article
We hereby report a method to fabricate addressable micropatterns of e-surfaces based on the conducting polymer poly(3,4-ethylenedioxythiophene) doped with the anion tosylate (PEDOT:Tos) to gain dynamic control over the spatial distribution of platelets in vitro. With thin film processing and microfabrication techniques, patterns down to 10 μm were produced to enable active regulation of platelet adhesion at high spatial resolution. Upon electronic addressing, both reduced and oxidized surfaces were created within the same device. This surface modulation dictates the conformation and/or orientation, rather than the concentration, of surface proteins, thus indirectly regulating the adhesion of platelets. The reduced electrode supported platelet adhesion, whereas the oxidized counterpart inhibited adhesion. PEDOT:Tos electrode fabrication is compatible with most of the classical patterning techniques used in printing as well as in the electronics industry. The first types of tools promise ultra-low-cost production of low-resolution (>30 μm) electrode patterns that may combine with traditional substrates and dishes used in a classical analysis setup. Platelets play a pronounced role in cardiovascular diseases and have become an important drug target in order to prevent thrombosis. This clinical path has in turn generated a need for platelet function tests to monitor and assess platelet drug efficacy. The spatial control of platelet adherence presented here could prove valuable for blood cell separation or biosensor microarrays, e.g. in diagnostic applications where platelet function is evaluated.
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Article
This review describes different strategies of surface elaboration for a better control of biomolecule adsorption. After a brief description of the fundamental interactions between surfaces and biomolecules, various routes of surface elaboration are presented dealing with the attachment of functional groups mostly thanks to plasma techniques, with the grafting to and from methods, and with the adsorption of surfactants. The grafting of stimuli-responsive polymers is also pointed out. Then, the discussion is focused on the protein adsorption phenomena showing how their interactions with solid surfaces are complex. The adsorption mechanism is proved to be dependent on the solid surface physicochemical properties as well as on the surface and conformation properties of the proteins. Different behaviors are also reported for complex multiple protein solutions.
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Article
Laser selective micro patterning of thin film of Poly (3,4-ethylenedioxythiophene):Poly (styrene sulfonate) (PEDOT:PSS) blend is investigated. Picosecond pulsed laser with 355 nm and 1064 nm wavelengths are used to study the ablation behavior of PEDOT:PSS thin film coated on glass. We present and discuss ablation thresholds for different film thicknesses as well as ablation lines with different overlapping rates. The results observed by SEM and white-light interference microscopy reveal that PEDOT:PSS film on glass substrates can be selectively patterned by optimized laser parameters.
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Article
The purpose of the present study is to explore topographical patterns produced with femtosecond laser pulses as a means of controlling the behaviour of living human cells (U2OS) on stainless steel surfaces and on negative plastic imprints (polycarbonate). The results show that the patterns on both types of material strongly affect cell behaviour and are particularly powerful in controlling cell spreading/elongation, localization and orientation. Analysis by fluorescence and scanning electron microscopy shows that on periodic 1D grating structures, cells and cell nuclei are highly elongated and aligned, whereas on periodic 2D grid structures, cell spreading and shape is affected. The results also show that the density and morphology of the cells can be affected. This was observed particularly on pseudo-periodic, coral-like structures which clearly inhibited cell growth. The results suggest that these patterns could be used in a variety of applications among the fields of clinical research and implant design, as well as in diagnosis and in cell and drug research. Furthermore, this article highlights the noteworthy aspects and the unique strengths of the technique and proposes directions for further research. Electronic supplementary material The online version of this article (doi:10.1007/s10544-012-9726-8) contains supplementary material, which is available to authorized users.
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The emergence of organic electronics represents one of the most dramatic technological developments of the past two decades. Perhaps the most important frontier of this field involves the interface with biology. The “soft” nature of organics offers better mechanical compatibility with tissue than traditional electronic materials, while their natural compatibility with mechanically flexible substrates suits the nonplanar form factors often required for implants. More importantly, the ability of organics to conduct ions in addition to electrons and holes opens up a new communication channel with biology. In this article, we consider a few examples that illustrate the coupling between organic electronics and biology and highlight new directions of research.
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Article
Stem cell function is regulated by intrinsic as well as microenvironmental factors, including chemical and mechanical signals. Conducting polymer-based cell culture substrates provide a powerful tool to control both chemical and physical stimuli sensed by stem cells. Here we show that polypyrrole (PPy), a commonly used conducting polymer, can be tailored to modulate survival and maintenance of rat fetal neural stem cells (NSCs). NSCs cultured on PPy substrates containing different counter ions, dodecylbenzenesulfonate (DBS), tosylate (TsO), perchlorate (ClO(4)) and chloride (Cl), showed a distinct correlation between PPy counter ion and cell viability. Specifically, NSC viability was high on PPy(DBS) but low on PPy containing TsO, ClO(4) and Cl. On PPy(DBS), NSC proliferation and differentiation was comparable to standard NSC culture on tissue culture polystyrene. Electrical reduction of PPy(DBS) created a switch for neural stem cell viability, with widespread cell death upon polymer reduction. Coating the PPy(DBS) films with a gel layer composed of a basement membrane matrix efficiently prevented loss of cell viability upon polymer reduction. Here we have defined conditions for the biocompatibility of PPy substrates with NSC culture, critical for the development of devices based on conducting polymers interfacing with NSCs.
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Article
In the body, cells encounter a complex milieu of signals, including topographical cues, in the form of the physical features of their surrounding environment. Imposed topography can affect cells on surfaces by promoting adhesion, spreading, alignment, morphological changes, and changes in gene expression. Neural response to topography is complex, and it depends on the dimensions and shapes of physical features. Looking toward repair of nerve injuries, strategies are being explored to engineer guidance conduits with precise surface topographies. How neurons and other cell types sense and interpret topography remains to be fully elucidated. Studies reviewed here include those of topography on cellular organization and function as well as potential cellular mechanisms of response.
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In vivo, cardiomyocytes are exposed to multiple biochemical and physical cues including topographical and electrical cues. During prolonged in vitro cultivation in standard tissue culture set-ups, cardiomyocytes are known to de-differentiate due to the lack of appropriate micro-environmental cues. Most currently available cell culture systems provide only a single biophysical cue, thus development of advanced cell cultivation systems incorporating multiple cues is urgently needed. We report here the development of a microfabricated system, incorporating topographical and electrical cues on a single chip, which enables cultivation of differentiated cardiomyocytes. The cell culture chips were created by hot embossing of polystyrene, to create microgrooves and microridges of precisely defined depth, width and periodicity. Substrates consisting of 0.5 microm-wide grooves and 0.5 microm-wide ridges (1 microm period) and those consisting of 3 microm-wide grooves and 1 microm-wide ridges (4 microm period) were investigated, with smooth surfaces used as controls. The depth of the microgrooves was 400 nm. The two gold electrodes were electrodeposited 1 cm apart such that the microgrooves in-between were oriented either parallel or perpendicular to the electrodes, enabling studies of interaction between topographical and electrical cues. Neonatal rat cardiomyocytes cultivated on microgrooved substrates for 7 days were elongated and aligned along the microgrooves forming a well developed contractile apparatus, as evidenced by sarcomeric alpha-actinin staining, with a more pronounced effect on substrates with 1 microm compared to 4 microm periodicity. Importantly, simultaneous application of biphasic electrical pulses and topographical cues resulted in gap junctions confined to the cell-cell end junctions rather than the punctate distribution found in neonatal cells. Electrical field stimulation further enhanced cardiomyocyte elongation when microgrooves were oriented parallel to the electric field. Due to the compatibility of the described cell culture chips with fluorescence and optical microscopy as well as the ability to independently control field stimulation parameters, biochemical and topographical cues on each chip, this system may in the future become a useful tool in drug development and maturation of cardiomyocytes derived from stem cells.
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Article
The cytoarchitecture of nervous tissue is lost during the dissociation procedures used to form primary cell cultures. As a first step toward reestablishing an ordered arrangement of these cells in vitro, we developed a set of procedures for patterning the outgrowth of cells cultured on 2-dimensional substrates. These procedures used a combination of surface chemistry and photolithographic techniques. The adhesive properties of either silicon or silicon dioxide (quartz) surfaces were controlled by covalently binding small organic molecules to the surface with silane coupling agents. The attachment and growth of either embryonic mouse spinal cells or perinatal rat cerebellar cells were found to be promoted by binding certain amine derivatives to the surface. In particular, cells grown on surfaces bound with diamines and triamines, but not with monoamines, formed cultures whose morphology was similar to that of cells cultured on conventional substrates, i.e., glass coated with poly(D-lysine). The attachment of cells to a substrate was inhibited by binding alkane chains (e.g., n-tetradecane) to the surface and plating the cells in media containing 5-10% (vol/vol) serum. Patterns of selected adhesivity were formed using photochemical resist materials and lithographic masking techniques compatible with the silane chemistry. Cultures of either spinal cord cells or cerebellar cells could be confined to square regions on the scale of 50 micron. Cerebellar cells could be confined to grow on lines with widths less than 10 micron. This width is comparable to the diameter of granule cell somata. The patterned growth of cerebellar cells was maintained up to 12 d in vitro. Over this time period the granule cells were observed to develop electrical excitability and immunoreactivity for neuron-specific enolase. Purkinje neurons also developed electrical excitability when grown on the chemically modified surfaces. Immunochemical reactivity of the patterned cultures for glial fibrillary acid protein (GFAP) showed that glia are patterned along with the associated granule cells. Interestingly, the GFAP-positive glia that proliferated on surfaces bound with amine derivatives attained primarily a tile-shaped, fibroblast-like morphology, while those proliferating on glass coated with poly(D-lysine) developed primarily a spindle-shaped, process-bearing morphology. Granule cells preferentially associated with the spindle-shaped glia.
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Article
Damage to peripheral nerves often cannot be repaired by the juxtaposition of the severed nerve ends. Surgeons have typically used autologous nerve grafts, which have several drawbacks including the need for multiple surgical procedures and loss of function at the donor site. As an alternative, the use of nerve guidance channels to bridge the gap between severed nerve ends is being explored. In this paper, the electrically conductive polymer--oxidized polypyrrole (PP)--has been evaluated for use as a substrate to enhance nerve cell interactions in culture as a first step toward potentially using such polymers to stimulate in vivo nerve regeneration. Image analysis demonstrates that PC-12 cells and primary chicken sciatic nerve explants attached and extended neurites equally well on both PP films and tissue culture polystyrene in the absence of electrical stimulation. In contrast, PC-12 cells interacted poorly with indium tin oxide (ITO), poly(L-lactic acid) (PLA), and poly(lactic acid-co-glycolic acid) surfaces. However, PC-12 cells cultured on PP films and subjected to an electrical stimulus through the film showed a significant increase in neurite lengths compared with ones that were not subjected to electrical stimulation through the film and tissue culture polystyrene controls. The median neurite length for PC-12 cells grown on PP and subjected to an electrical stimulus was 18.14 micron (n = 5643) compared with 9.5 micron (n = 4440) for controls. Furthermore, animal implantation studies reveal that PP invokes little adverse tissue response compared with poly(lactic acid-co-glycolic acid).
Article
Self-assembly is possibly the most effective and versatile strategy for surface functionalization. Self-assembled monolayers (SAMs) can be formed on (semi-)conductor and dielectric surfaces, and have been used in a variety of technological applications. This work aims to review the strategy behind the design and use of self-assembled monolayers in organic electronics, discuss the mechanism of interaction of SAMs in a microscopic device, and highlight the applications emerging from the integration of SAMs in an organic device. The possibility of performing surface chemistry tailoring with SAMs constitutes a versatile approach towards the tuning of the electronic and morphological properties of the interfaces relevant to the response of an organic electronic device. Functionalisation with SAMs is important not only for imparting stability to the device or enhancing its performance, as sought at the early stages of development of this field. SAM-functionalised organic devices give rise to completely new types of behavior that open unprecedented applications, such as ultra-sensitive label-free biosensors and SAM/organic transistors that can be used as robust experimental gauges for studying charge tunneling across SAMs.
Article
Photo-induced processes have high potential in in situ patterning and controlling living cells, whose developments are introduced and recent progresses by utilizing femtosecond laser are described. Photochemical and photothermal surface modification performed by conventional light and nanosecond laser irradiation is summarized and their applicability is considered. Femtosecond laser ablation has superior features due to its photomechanical mechanism, which is confirmed by ultrafast spectroscopy and imaging of a model film under laser ablation. Femtosecond laser ablation of physiological solutions generates shockwave and cavitation bubbles, which is employed for patterning and manipulating living cells. Femtosecond laser ablation fabricating cytophobic and cytophilic domains enable us to form living cell patterns and to study cell migration and cell–cell interaction. Finally summary and perspective are presented.
Book
Surface modification (also known as treatment, pre-treatment and activation) of different materials (metals, ceramics, polymers, composites,) is sine quanon to impart surface characteristics for their applications for a legion of purposes. The beauty of surface modification is that it provides the requisite surface properties without tempering with the bulk, thus retaining the desirable attributes of bulk materials.
Article
Unlabelled: We propose a facile and reproducible method, based on ultra thin porous alumina membranes, to produce cm(2) ordered arrays of nano-pores and nano-pillars on any kind of substrates. In particular our method enables the fabrication of conducting polymers nano-structures, such as poly[3,4-ethylenedioxythiophene]:poly[styrene sulfonate] ( Pedot: PSS). Here, we demonstrate the potential interest of those templates with controlled cell adhesion studies. The triggering of the eventual fate of the cell (proliferation, death, differentiation or migration) is mediated through chemical cues from the adsorbed proteins and physical cues such as surface energy, stiffness and topography. Interestingly, as well as through material properties, stiffness modifications can be induced by nano-topography, the ability of nano-pillars to bend defining an effective stiffness. By controlling the diameter, length, depth and material of the nano-structures, one can possibly tune the effective stiffness of a (nano) structured substrate. First results indicate a possible change in the fate of living cells on such nano-patterned devices, whether they are made of conducting polymer (soft material) or silicon (hard material).
Article
This book book is an attempt to review the recent progress in both electronics and computational tools developed to analyze the functional operations of large ensembles of neurons and to provide the readers with a sense of the applications made possible by these technological tools. While considerable progress has been made over the last decades in our understanding of electrophysiological processes at the single channel, single synapse, and single neuron levels, our understanding of electrophysiological processes at the neuronal network level is still in its infancy. This book includes: Technological overviews of multi-electrode arrays (MEAs) and related electronics and software, Applications of MEAs to study ensemble activity of dissociated cell cultures including cardiac myocytes, Applications of the technology for drug discovery using acute and cultured slices. Examples of studies directed at understanding the origins and functions of various rhythmic activities in neuronal ensembles. © 2006 Springer Science+Business Media, Inc. All rights reserved.
Article
Electrical, label-free monitoring of cells is a non-invasive method for dynamically assessing the integrity of cells for diagnostic purposes. The organic electrochemical transistor (OECT) is a device that has been demonstrated to be advantageous for interfacing with biological systems and had previously been shown to capable of monitoring electrically tight, resistant, barrier type tissue. Herein, the OECT is demonstrated not only for monitoring of barrier tissue such as MDCK I cells, but also for other, non-barrier tissue adherent cells including HeLa cells and HEK epithelial cells. Transistor performance, expressed as transconductance (gm) is measured as a function of frequency; barrier tissue type cells are shown to have a more abrupt drop in transconductance compared to non-barrier tissue cells, however both tissue types are clearly distinguishable. Simple modelling of the cell layers on the transistor allows extraction of a resistance term (Rc). OECT monitoring shows that barrier tissue cells lose their barrier function in a standard calcium switch assay, but remain adhered to the surface. Re-addition of calcium results in recovery of barrier tissue function. The entire process is continuously followed both electronically and optically. Finally, high resolution fluorescence imaging of live cells labelled with a red fluorescent actin marker demonstrates the versatility of this method for tracking molecular events optically, with direct correlation to electronic readouts.
Article
Micropatterning strategies, which enable control over cell and tissue architecture in vitro, have emerged as powerful platforms for modelling tissue microenvironments at different scales and complexities. Here, we provide an overview of popular micropatterning techniques, along with detailed descriptions, to guide new users through the decision making process of which micropatterning procedure to use, and how to best obtain desired tissue patterns. Example techniques and the types of biological observations that can be made are provided from the literature. A focus is placed on microcontact printing to obtain co-cultures of patterned, confluent sheets, and the challenges associated with optimizing this protocol. Many issues associated with microcontact printing, however, are relevant to all micropatterning methodologies. Finally, we briefly discuss challenges in addressing key limitations associated with current micropatterning technologies.
Article
In this review, we provide insight into protein interactions with organic conducting polymers, a class of “intelligent” and dynamic materials that offer unique strategies for controlling protein interactions as a prelude to developing a wide range of bioapplications. Following a general introduction on the importance of protein interactions, this review initially focuses on the areas of bioseparation and biosensor applications. These applications amount to an extensive body of work; however, we provide only a brief overview for the purpose of introducing palpable examples of translating the ability to control organic conducting polymer–protein interactions into practical and useful applications. Because organic conducting polymers are breaking new ground for implantable electrodes and tissue regeneration/engineering, we duly turn to the importance of protein interactions and role organic conducting polymers can play in advancing these applications. Lastly, for those not familiar with organic conducting polymers, we take a step back and examine the unique properties underlying their innate ability to control protein interactions, particularly the use of external electrical control to reversibly switch the physical surface properties. Several characterization techniques identified as being critical to our understanding at the macroscopic down to the single molecule level are also highlighted.
Article
Chemical, mechanical, and topographic extracellular matrix (ECM) cues have been extensively studied for their influence on cell behavior. These ECM cues alter cell adhesion, cell shape, and cell migration, and activate signal transduction pathways to influence gene expression, proliferation, and differentiation. ECM elasticity and topography, in particular, have emerged as material properties of intense focus based on strong evidence these physical cue can partially dictate stem cell differentiation. Cells generate forces to pull on their adhesive contacts, and these tractional forces appear to be a common element of cells' responses to both elasticity and topography. This review focuses on recently published work that links ECM topography and mechanics and their influence on differentiation and other cell behaviors, We also highlight signaling pathways typically implicated in mechanotransduction that are (or may be) shared by cells subjected to topographic cues. Finally, we conclude with a brief discussion of the potential implications of these commonalities for cell based therapies and biomaterial design.
Article
Direct interfacing of neurons with electronic devices has been investigated for both prosthetic and neuro-computing applications. In vitro neuronal networks provide great tools not only for improving neuroprostheses but also to take advantage of their computing abilities. However, it is often difficult to organize neuronal networks according to specific cell distributions. Our aim was to develop a cell-type specific immobilization of neurons on individual electrodes to produce organized in vitro neuronal networks on multi-electrode arrays (MEAs). We demonstrate the selective capture of retinal neurons on antibody functionalized surfaces following the formation of self-assembled monolayers from protein-thiol conjugates by simple contact and protein-polypyrrole deposits by electrochemical functionalization. This neuronal selection was achieved on gold for either cone photoreceptors or retinal ganglion neurons using a PNA lectin or a Thy1 antibody, respectively. Anti-fouling of un-functionalized gold surfaces was optimized to increase the capture efficiencies. The technique was extended to electrode arrays by addressing electropolymerization of pyrrole monomers and pyrrole-protein conjugates to active electrodes. Retinal ganglion cell recording on the array further demonstrated the integrity of these neurons following their selection on polypyrrole-coated electrodes. Therefore, this protein-polypyrrole electrodeposition could provide a new approach to generate organized in vitro neuronal networks.
Article
In this paper we present a potentially fast method for high resolution micro structuring of organic electronics via laser patterning. An investigation of the absorption spectrum in the UV/VIS regime of poly (3,4-ethylene dioxythiophene) poly (styrene-sulfonate) (PEDOT/PSS) has shown that UV-laser radiation should be used for optimal laser ablation of the material. Hence, the ablation characteristics of PEDOT/PSS with two different excimer lasers are compared with each other. The optimal fluence for the ablation of the material has been determined. The lasers used in this study are ArF (λ=193nm) and KrF (λ=248nm) excimer lasers.
Article
A simple and versatile fabrication process is used to define conducting polymer microelectrode arrays (MEAs), patterning at the same time the recording electrodes as well as the insulating layer. Thanks to the low impedance of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) electrodes, these MEAs allow in vitro recording of action potentials from rat hippocampus slices.
Article
Periodic grating structures self-formed on a metal surface under the irradiation of a femtosecond laser pulse are characterized by grating spaces which are shorter than the laser wavelength, as well as by dependence on the laser fluence. This Brief Report presents a different interpretation of these features in terms of the process of parametric decay of laser light to surface plasma waves. Depending on the electron density, grating spaces with lengths of 680 nm to as short as 400 nm can be produced for 800 nm laser wavelength as a result of the interaction of laser pulses with laser-produced surface plasma.
Article
At present, the prime methodology for studying neuronal circuit-connectivity, physiology and pathology under in vitro or in vivo conditions is by using substrate-integrated microelectrode arrays. Although this methodology permits simultaneous, cell-non-invasive, long-term recordings of extracellular field potentials generated by action potentials, it is 'blind' to subthreshold synaptic potentials generated by single cells. On the other hand, intracellular recordings of the full electrophysiological repertoire (subthreshold synaptic potentials, membrane oscillations and action potentials) are, at present, obtained only by sharp or patch microelectrodes. These, however, are limited to single cells at a time and for short durations. Recently a number of laboratories began to merge the advantages of extracellular microelectrode arrays and intracellular microelectrodes. This Review describes the novel approaches, identifying their strengths and limitations from the point of view of the end users - with the intention to help steer the bioengineering efforts towards the needs of brain-circuit research.
Article
We report on the selective cell detachment from nanoengineered gold nanoparticle (AuNP) surfaces triggered by laser irradiation, which occurs in a nonthermal manner. The gold nanoparticle-based surfaces reveal good adhesion of NIH3T3 fibroblast cells. Patterning is achieved by lithographic microcontact printing, selective gold nanoparticle deposition, and by laser beam profiling. It is shown that the effectiveness of fibroblast cell detachment depends on the cell age, laser power, and AuNP patterning profile. Heat distribution and temperature rise around gold nanoparticle functionalized surfaces is modeled, revealing low heating of nanoparticles by laser illumination. The nonthermal photochemical mechanism of cell detachment due to production of reactive oxygen species under illumination of gold nanoparticles by green laser light is studied. We also demonstrate that cells migrate from unirradiated areas leading to their reattachment and surface recovery which is important for controlled spatial organization of cells in wound healing and tissue engineering. Research presented in this work is targeted at designing biointerfaces for cell cultures.
Article
We have investigated the effects of thickness variation and thermal treatment of the electrode polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonic acid) (PEDOT:PSS) in photovoltaic and photodetector devices using conjugated polymer blends as the photoactive material. By variation of the PEDOT:PSS layer thickness between 25 and 150 nm, we found optimum device performance, in particular low dark current and high external quantum efficiency (EQE) and open-circuit voltage (Voc), at around 70 nm. This has been observed for two different active layers. Annealing studies on the PEDOT:PSS films, with temperatures varied between 120 and 400 °C, showed an optimum device performance, in particular EQE and Voc at 250 °C. This optimum performance was found to be associated with loss of water from the PSS shell of the PEDOT:PSS grains. For annealing temperatures above 260 °C, device performance was dramatically reduced. This was associated with chemical decomposition leading to loss of sulfonic acid, although this did not significantly affect the in-plane conductivity.
Article
This report demonstrates proof-of-concept application of two established electrochemical techniques affecting self-assembled monolayersthe thermodynamic control of monolayer assembly at gold and the electrochemical desorption of self-assembled monolayers from goldto pattern monolayers onto gold microelectrodes. When combined with a simple laser-based microfabrication technique, using commercially available microscope adaptations, to form arrays of individually-addressable gold microelectrodes from continuous thin gold films on glass, two new patterning methodologies result that enable fabrication of monolayer-based affinity arrays for potential use in chemical and biosensors. The described microfabrication technique is central to the success of these patterning methodologies because it allows SAM patterning of metal arrays immediately after their fabrication, so as to avoid problems associated with forming SAMs on commercially-supplied arrays. In this paper, each patterning methodology is used to fabricate an interdigitated microelectrode array in which one set of electrodes is modified with an active antibody and the other set of electrodes is resistant to nonspecific protein adsorption.
Article
This paper describes the use of patterned self-assembled monolayers to fabricate small, two-dimensional patterns of electrically functional polypyrrole on a gold electrode. These patterns can be transferred to an insulating polydimethylsiloxane substrate. Thin (10-100 mu m) polypyrrole wires retain their electrical conductivity over millimeter length scales. Interdigitated fingers of polypyrrole spaced 50 mu m apart are electrically insulated from one another. Polypyrrole can be electrochemically polymerized on patterns with size features of 2 mu m into films that are 200-400 nm thick.
Article
A laser-based transfer technique, termed matrix assisted pulsed laser evaporation direct write (MAPLE DW), has been modified to rapidly and accurately deposit mammalian cells in a non-contact manner. While this technology was originally developed for deposition of inorganic materials, it has shown the ability to transfer of a wide range of biological materials. Two types of mammalian cells, human osteosarcoma and rat cardiac cells were deposited into a biopolymer matrix via MAPLE DW. Current results show that it is possible to deposit cells in a stepwise manner and build cellular ‘stacks’ 50–100 μm tall. Furthermore, the technique is now capable of depositing cells with near single cell resolution. Post-transfer results of live/dead viability/cytotoxicity assays show that the cells are unaffected by the process with near 100% viability. The ability to build cellular structures in three dimensions and deposit small numbers of cells accurately has potential application to tissue engineering.
Article
Patterned arrangement of living cells to form tissues on a given surface is the prerequisite of successful bioen-gineering, tissue building and biosensor technology. The aim of the present study was to deposit various biomaterials onto a given substrate to establish a patterned cellular matrix using laser-based technologies. We used the Pulsed Laser Deposi-tion and the Absorbing Film-Assisted Laser-Induced Forward Transfer methods to deposit various biomaterials, such as fibronectin, collagen and endothelial cell growth supplement. Subpicosecond KrF excimer laser was used for irradiation of the target materials. When cultured neuroectodermal stem cells, astrocytes, endothelial and neuroblastoma cells were layered on the deposited biomaterial patterns a guided growth of these cells was induced along the patterned thin film. Some cell types showed various interactions when approached each other. The above methods are suitable to build an ar-chitecture of substrates which supports and guides the growth of cells and may enable the cells to induce directed and rapid repair of injured tissues.
Article
In many fields and applications, a good knowledge of the wetting behaviour of solvents on a surface is crucial. Self-assembled monolayers (SAMs) have enabled improved control over surface properties, while more recent fields such as organic electronics gave rise to new applications and requirements regarding solvent–substrate interactions. However, most reported wettability studies are limited to practically less relevant solvents such as water, diiodomethane or hexadecane. Herein we report static contact angle measurements of various, typical SAM-modified surfaces, characterizing these surfaces' wettabilities over a wide range of practically relevant solvents. Surface energies, both the polar and the disperse component, of these SAM-modified surfaces are extracted with various methods from the contact angle data. Reliable methods for surface energy extraction, such as the Owens–Wendt–Rabel–Kaelble method and the method after Wu, yield values which could be expected from the chemical structure and nature of the self-assembled molecules and which correspond well to the few reported literature values. We also determined wetting envelopes for the various surfaces which allow easy prediction of the surfaces' wettability for a certain solvent and which ensure relevance for current and future solvents.
Article
Femtosecond laser patterning of octadecylsiloxane monolayers on quartz glass at λ=800 nm , τ≪30 fs , and ambient conditions has been investigated. Selective decomposition of the coating with single laser pulses at subwavelength resolution can be carried out over a wide range of fluences from 4.2 down to 3.1 J / cm <sup>2</sup> . In particular, at a 1/e laser spot diameter of 1.8 μ m , structures with a width down to 250 nm and below were fabricated. This opens up a facile route towards laser fabrication of transparent templates with chemical structures down into the sub- 100- nm -regime.
Article
The use of biologically active dopants in conductive polymers allows the polymers to be tailored for specific application. The incorporation of nerve growth factor (NGF) as a co-dopant in the electrochemical properties of the NGF-modified conducting polymers are studied by impedance spectroscopy and cyclic voltammetry. Impedance measurements at the neurobiologically important frequency of 1 kHz reveal that the minimum impedance of the NGF-modified polypyrrole (PPy) film, 15 k Omega, is lower than the minimum impedance of peptide-modified PPy film (360 k Omega).Similar results are found with NGF-modified poly(3,4-ethylene dioxythiophene) (PEDOT). The microstructure of the conductive polymerfilms is characterized by optical microscopy and electron microscopy and indicates that the NGF-functionalized polymer surface topology is similar to that of the unmodified polymer film. Optical and fluorescence microscopy reveal that (rat pheochromacytoma) cells adhered to the NGF-modified substrate and extended neurites on both PPy and PEDOT, indicating that the NGF in the polymer film is biologically active. Taken together these data indicate that the incorporation of NGF can modify the biological interactions of the electrode without compromising the conductive properties or the morphology of the polymeric film.
Article
A series of mixed self-assembled monolayers of functionalized (Br(CH2)11SiCl3) and unfunctionalized (CH3(CH2)10SiCl3) alkyltrichlorosilanes of different compositions have been prepared on bulk silicon substrates. By in situ modification of these monolayers the bromo end groups were transformed to amino end groups as shown by X-ray photoelectron spectroscopy measurements. The change of hydrophilicity was monitored by water contact angle measurements, showing the expected decrease of contact angles with increase amino group content on the surface. These substrates were used to initiate N-carboxyanhydrides in dioxane to yield α-helical polypeptides grafted from the surface. The thicknesses of the obtained polymer layers were measured with ellipsometry and X-ray reflectometry. Fourier transform infrared measurements confirm that the grafted polymers are in the α-helical conformation.
Article
A simple technique for in situ measurements of pulsed Gaussian-beam spot sizes is reported. This technique is particularly useful for measurements on highly focused beam spots. It can also be used for absolute calibration of the threshold-energy fluences for pulsed-laser-induced effects. The thresholds for several effects in picosecondlaser-induced phase transformation on silicon-crystal surfaces are calibrated with this technique.
Article
Conducting polymers are soft, flexible materials, exhibiting material properties that can be reversibly changed by electrochemically altering the redox state. Surface chemistry is an important determinant for the molecular events of cell adhesion. Therefore, we analyzed whether the redox state of the conducting polymer PEDOT:Tosylate can be used to control epithelial cell adhesion and proliferation. A functionalized cell culture dish comprising two adjacent electrode surfaces was developed. Upon electronic addressing, reduced and oxidized surfaces are created within the same device. Simultaneous analysis of how a homogenous epithelial MDCK cell population responded to the electrodes revealed distinct surface-specific differences. Presentation of functional fibronectin on the reduced electrode promoted focal adhesion formation, involving alpha(v)beta(3) integrin, cell proliferation, and ensuing formation of polarized monolayers. In contrast, the oxidized surface harbored only few cells with deranged morphology showing no indication of proliferation. This stems from the altered fibronectin conformation, induced by the different surface chemistry of the PEDOT:Tosylate electrode in the oxidized state. Our results demonstrate a novel use of PEDOT:Tosylate as a cell-hosting material in multiple-electrode systems, where cell adhesion and proliferation can be controlled by electrochemical modulation of surface properties.
Article
Electrically conducting polymers such as polypyrrole (PPy) are important biomaterials in neural engineering applications, including neural probes, nerve conduits, and scaffolds for tissue and nerve regeneration. Surface modification of these polymers can introduce other valuable characteristics for neural interfacing in addition to electrical conductivity, such as topographical features and chemical bioactivity. Here, the patterning of PPy to create topographical cues for cells is reported. In particular, 1 and 2 µm wide PPy microchannels are fabricated using electron-beam (e-beam) lithography and electropolymerization. A systematic analysis of parameters controlling PPy micropatterning is performed, and finds that microchannel depth, roughness, and morphology are highly dependent on the e-beam writing current, polymerization current, PPy/dopant concentrations, and the polymerization time. Embryonic hippocampal neurons cultured on patterned PPy polarize (i.e., defined an axon) faster on this modified material, with a twofold increase in the number of cells with axons compared to cells cultured on unmodified PPy. These topographical features also have an effect on axon orientation but do not have a significant effect on overall axon length. This is the first investigation that studies controlled PPy patterning with small dimensions (i.e., less than 5 µm) for biological applications, which demonstrates the relevance of expanding microelectronic materials and techniques to the biomedical field.
Article
The interaction of mammalian cells with nanoscale topography has proven to be an important signaling modality in controlling cell function. Naturally occurring nanotopographic structures within the extracellular matrix present surrounding cells with mechanotransductive cues that influence local migration, cell polarization, and other functions. Synthetically nanofabricated topography can also influence cell morphology, alignment, adhesion, migration, proliferation, and cytoskeleton organization. We review the use of in vitro synthetic cell-nanotopography interactions to control cell behavior and influence complex cellular processes, including stem-cell differentiation and tissue organization. Future challenges and opportunities in cell-nanotopography engineering are also discussed, including the elucidation of mechanisms and applications in tissue engineering.
Article
A prototype photothermal procedure: Alkylsiloxane monolayers on surface-oxidized silicon substrates are processed in gaseous bromine using a focused Ar+-laser operated at λ=514 nm. Monolayer bromination remains confined to a micrometer-sized reaction zone, where laser irradiation results in a local temperature rise (see figure).
Article
Bioactive coatings for neural electrodes that are tailored for cell interactions have the potential to produce superior implants with improved charge transfer capabilities. In this study synthetically produced anionically modified laminin peptides DEDEDYFQRYLI and DCDPGYIGSR were used to dope poly(3,4-ethylenedioxythiophene) (PEDOT) electrodeposited on platinum (Pt) electrodes. Performance of peptide doped films was compared to conventional polymer PEDOT/paratoluene sulfonate (pTS) films using SEM, XPS, cyclic voltammetry, impedance spectroscopy, mechanical hardness and adherence. Bioactivity of incorporated peptides and their affect on cell growth was assessed using a PC12 neurite outgrowth assay. It was demonstrated that large peptide dopants produced softer PEDOT films with a minimal decrease in electrochemical stability, compared to the conventional dopant, pTS. Cell studies revealed that the YFQRYLI ligand retained neurite outgrowth bioactivity when DEDEDYFQRYLI was used as a dopant, but the effect was strongly dependant on initial cell attachment. Alternate peptide dopant, DCDPGYIGSR was found to impart superior cell attachment properties when compared to DEDEDYFQRYLI, but attachment on both peptide doped polymers could be enhanced by coating with whole native laminin.
Article
This study investigated a novel multi-electrode-array (MEA) design capable of long-term and highly selective recordings of axonal signals using PDMS microtunnels. We successfully grew neurons in culture so that only axons extended through narrow (10 microm wide by 3 microm high) and long (750 microm) microtunnels under which multiple electrodes were integrated. This permitted the recording of relatively large (up to 200 microV) electrical signals, including the propagation speed and direction of these travelling action potentials. To further demonstrate the operation of the device as a diagnostic tool for drug screening assays, the drug mepivacaine was applied in washout experiments. Here, we identified significant changes in mean spiking rate and conduction velocity.
Article
An identified neuron of the leech, a Retzius cell, has been attached to the open gate of a p-channel field-effect transistor. Action potentials, spontaneous or stimulated, modulate directly the source-drain current in silicon. The electronic signals match the shape of the action potential. The average voltage on the gate was up to 25 percent of the intracellular voltage change. Occasionally weak signals that resemble the first derivative of the action potential were observed. The junctions can be described by a model that includes capacitive coupling of the plasma membrane and the gate oxide and that accounts for variable resistance of the seal.
Article
Electrically conducting polymers are novel in that their surface properties, including charge density and wettability, can be reversibly changed with an applied electrical potential. Such properties might render conducting polymers unique for biological applications. However, the majority of research on conducting polymers has been carried out under nonbiological conditions. We synthesized optically transparent polypyrrole thin films and studied them in environments suitable for protein adsorption and mammalian cell culture. In vitro studies demonstrated that extracellular matrix molecules, such as fibronectin, adsorb efficiently onto polypyrrole thin films and support cell attachment under serum-free conditions. When aortic endothelial cells were cultured on fibronectin-coated polypyrrole (oxidized) in either chemically defined medium or the presence of serum, cells spread normally and synthesized DNA. In contrast, when the polymer was switched to its neutral state by applying an electrical potential, both cell extension and DNA synthesis were inhibited without affecting cell viability. Application of a similar electrical potential to cells cultured on indium tin oxide surfaces had no effect on cell shape or function. These data suggest that electrically conducting polymers may represent a type of culture substrate which could provide a noninvasive means to control the shape and function of adherent cells, independent of any medium alteration.
Article
Toward the goal of creating patterns of primary hippocampal neurons in low density culture, we investigated techniques to fabricate microminiature grids of organofunctional silanes on glassy surfaces. A new photoresist (PR) process, Selective Silane Removal (SSR), was developed and compared to two previously developed techniques which use PR and laser patterning. The grid patterns consisted of 27 combinations of path width, length, and intersection (node diameter). The background consisted of squares bounded by the paths. The best neuron patterning was observed on substrates produced by the SSR process where cytophilic aminosilane is uniformly deposited and selectively removed from the background. Controlling water during aminosilane deposition was critical to good neuronal growth and patterning. Oxygen plasma etching of background regions prior to cytophobic phenylsilane binding significantly reduced off-pattern cell growth. Up to 90% of somata grown on these substrates complied to the pattern, and an average of 77% of background regions were free of neurites or cells connected to the pattern. The highest laser energy density, 120 mJ/cm2, produced the best compliance on lased substrates, with an average of 35% of background regions free of connected cells and neurites, but considerable variation across the surface. On substrates with excellent patterning, compliance to nodes was found to be dependent on pattern dimensions, with 20-micron node diameters and 80-micron internodal path lengths increasing compliance.
Article
Cell shapes induced by cell-substratum interactions are linked with proliferation, differentiation or apoptosis of cells. To clarify the relevance of specific surface characteristics, we applied self-assembled monolayers (SAM) of alkyl silanes exhibiting a variety of terminating functional groups. We first characterised the SAMs on glass or silicon wafers by measuring wettability, layer thickness and roughness. Water contact angle data revealed that methyl (CH(3)), bromine (Br), and vinyl (CH=CH(2)) groups lead to hydrophobic surfaces, while amine (NH(2)) and carboxyl (COOH) functions lead to moderately wettable surfaces, and polyethylene glycol (PEG) and hydroxyl (OH) groups created wettable substrata. The surfaces were found to be molecular smooth except for one type of NH(2) surface. The SDS-PAGE analysis of proteins adsorbed from bovine serum to the SAMs showed less protein adsorption to PEG and OH than to CH(3), NH(2) and COOH. Immunoblotting revealed that a key component of adsorbed proteins is vitronectin while fibronectin was not detectable. The interaction of human fibroblasts with CH(3), PEG and OH terminated SAMs was similarly weak while strong attachment, spreading, fibronectin matrix formation and growth were observed on COOH and NH(2). The strong interaction of fibroblasts with the latter SAMs was linked to an enhanced activity of integrins as observed after antibody-tagging of living cells.
Article
The development of a methodology to manipulate surface properties of a self-assembled monolayer (SAM) of alkanethiol on a gold film using direct laser patterning is the objective of this paper. The present study demonstrates proof of the concept for the feasibility of laser patterning monolayers and outlines theoretical modeling of the process to predict the resulting feature size. This approach is unique in that it eliminates the need for photolithography, is noncontact, and can be extended to other systems such as SAMs on silicon wafers or potentially polymeric substrates. A homogeneous SAM made of 1-hexadecanethiol is formed on a 300-A sputtered film of gold (supported by a soda lime glass substrate). Localized regions are then desorbed by scanning the focal spot of a 488-nm continuous-wave argon ion laser beam under a nitrogen atmosphere. The desorption occurs as a result of a high substrate temperature produced by the moving laser beam with a Gaussian spatial profile at a constant speed of 200 microm/s. After completing the scans, the sample is dipped into a dilute solution of 16-mercaptohexadecanoic acid and a hydrophilic monolayer self-assembles along the previously irradiated regions. The resultant lines are viewed, and line widths are measured using both wetting with tridecane under a light microscope and scanning electron microscopy. Using the direct laser patterning method, we have produced straight line patterns with widths of 28-170 microm. A thermal model was constructed to predict the line width of the desorbed monolayer. The effect of the laser power, beam waist, and temperature dependence of the substrate conductivity on the theoretical predictions is considered. It is shown that the theoretical predictions are in good agreement with the experimental results, and, thus, the model can effectively be used to predict experimental results.
Article
Porous silicon is a promising biomaterial that is non-toxic and biodegradable. Surface modification can offer control over the degradation rate and can also impart properties that promote cell adhesion. In this study, we modified the surface of porous silicon surface by ozone oxidation, silanisation or coating with collagen or serum. For each surface, topography was characterised using atomic force microscopy, wettability by water contact angle measurements, degradation in aqueous buffer by interferometric reflectance spectroscopy and surface chemistry by Fourier-transform infrared spectroscopy. The adhesion of rat pheochromocytoma (PC12) and human lens epithelial cells to these surfaces was investigated. Cells were incubated on the surfaces for 4 and 24 h, and adhesion characteristics were determined by using a fluorescent vital stain and cell counts. Collagen coated and amino silanised porous silicon promoted cell attachment for both cell lines whereas cells attached poorly to ozone oxidised and polyethylene glycol silanised surfaces. We showed that the two cell lines had different adhesion characteristics on the various surfaces at different time points. The use of the vitality assays Alamar Blue (redox based assay) and neutral red (active cellular uptake assay) with porous silicon was also investigated. We reveal incompatibilities between certain resazurin (Alamar Blue), lysosomal incorporation assays (neutral red) and porous silicon.