[Show abstract][Hide abstract] ABSTRACT: Down scaling of microfluidic cell culture and detection devices for electrochemical monitoring is mostly focused on the miniaturization of the microfluidic chips which are often designed for specific applications and therefore they lack functional flexibility. We present a compact microfluidic cell culture and electrochemical analysis platform with in-built fluid handling and detection, enabling complete cell based assays comprising on-line cleaning, sterilization, surface functionalization, cell seeding, cultivation and electrochemical real-time monitoring of cellular dynamics. To demonstrate the versatility and multifunctionality of the platform, we explored amperometric monitoring of intracellular redox activity in yeast (Saccharomyces cerevisiae) and detection of exocytotically released dopamine from rat pheochromocytoma cells (PC12). Electrochemical impedance spectroscopy was used in both applications for monitoring cell sedimentation and adhesion as well as proliferation in the case of PC12 cells. The influence of flow rate on the signal amplitude in the detection of redox metabolism as well the effect of mechanical stimulation of dopamine release was facilitated by the programmable fluid handling capability. The here presented platform is aimed for application in cell based assays ranging from e.g. monitoring of drug effects in pharmacological studies, characterization of neural stem cell differentiation and screening of genetically modified microorganisms to environmental monitoring.
[Show abstract][Hide abstract] ABSTRACT: We present the application of electrochemical impedance spectroscopy (EIS) as a method for discriminating between different polydimethylsiloxane (PDMS) scaffolds for three-dimensional (3D) cell cultures. The validity of EIS characterisation for scaffolds having different degree of porosity (networks of structured or random channels) is discussed in relation to Archie’s law. Guidelines for EIS analysis are presented and demonstrated to provide porosity information in physiological buffer that agrees well with a more conventional weight-based approach. We also propose frequency ranges that may serve as means of single-frequency measurements for fast scaffold characterization combined with in vitro monitoring of 3D cell cultures.
[Show abstract][Hide abstract] ABSTRACT: Structurally patterned pyrolysed three-dimensional carbon scaffolds (p3D-carbon) are fabricated and applied for differentiation of human neural stem cells (hNSCs) developed for cell replacement therapy and sensing of released dopamine. In the absence of differentiation factors (DF) the pyrolysed carbon material induces spontaneous hNSC differentiation into mature dopamine-producing neurons and the 3D-topography promotes neurite elongation. In the presence and absence of DF, ≈73–82% of the hNSCs obtain dopaminergic properties on pyrolysed carbon, a to-date unseen efficiency in both two-dimensional (2D) and 3D environment. Due to conductive properties and 3D environment, the p3D-carbon serves as a neurotransmitter trap, enabling electrochemical detection of a significantly larger dopamine fraction released by the hNSC derived neurons than on conventional 2D electrodes. This is the first study of its kind, presenting new conductive 3D scaffolds that provide highly efficient hNSC differentiation to dopaminergic phenotype combined with real-time in situ confirmation of the fate of the hNSC-derived neurons.
[Show abstract][Hide abstract] ABSTRACT: A bioimpedance platform is presented as a promising tool for non-invasive real-time monitoring of the entire process of three-dimensional (3D) cell culturing in a hydrogel scaffold. In this study, the dynamics involved in the whole process of 3D cell culturing, starting from polymerisation of a bare 3D gelatin scaffold, to human mesenchymal stem cell (MSC) encapsulation and proliferation, was monitored over time. The platform consists of a large rectangular culture chamber with four embedded vertical gold plate electrodes that were exploited in two- and three terminal (2T and 3T) measurement configurations. By switching between the different combinations of electrode couples, it was possible to generate a multiplexing-like approach, which allowed for collecting spatially distributed information within the 3D space. Computational finite element (FE) analysis and electrochemical impedance spectroscopic (EIS) characterisation were used to determine the configurations' sensitivity field localisation. The 2T setup gives insight into the interfacial phenomena at both electrode surfaces and covers the central part of the 3D cell culture volume, while the four 3T modes provide focus on the dynamics at the corners of the 3D culture chamber. By combining a number of electrode configurations, complementary spatially distributed information on a large 3D cell culture can be obtained with maximised sensitivity in the entire 3D space. The experimental results show that cell proliferation can be monitored within the tested biomimetic environment, paving the way to further developments in bioimpedance tracking of 3D cell cultures and tissue engineering.
[Show abstract][Hide abstract] ABSTRACT: In this paper we demonstrate the fabrication and electrochemical characterization of a microchip with 12 identical but individually addressable electrochemical measuring sites, each consisting of a set of interdigitated electrodes acting as a working electrode as well as two circular electrodes functioning as a counter and reference electrode in close proximity. The electrodes are made of gold on a silicon oxide substrate and are passivated by a silicon nitride membrane. A method for avoiding the creation of high edges at the electrodes (known as lift-off ears) is presented. The microchip design is highly symmetric to accommodate easy electronic integration and provides space for microfluidic inlets and outlets for integrated custom-made microfluidic systems on top.
[Show abstract][Hide abstract] ABSTRACT: This paper introduces the work and diversity of the Council for Frontiers of Knowledge (CFK). In a series of vignettes relating to the intellectual interests of some of the leading academics working with the CFK, both the mission and the trans- disciplinary oversight of the agency are explored.
International Journal of Transdisciplinary Research. 05/2014; 7(1):1 - 26.
[Show abstract][Hide abstract] ABSTRACT: A surface modification of interdigitated gold microelectrodes (IDEs) with a doped polypyrrole (PPy) film for detection of dopamine released from populations of differentiated PC12 cells is presented. A thin PPy layer was potentiostatically electropolymerized from an aqueous pyrrole solution onto electrode surfaces. The conducting polymer film was doped during electropolymerization by introducing counter-ions in the monomer solution. Several counter-ions were tested and the resulting electrode modifications were characterized electrochemically to find the optimal dopant that increases sensitivity in dopamine detection. Overoxidation of the PPy films was shown to contribute to a significant enhancement in sensitivity to dopamine. The changes caused by overoxidation in the electrochemical behavior and electrode morphology were investigated using cyclic voltammetry and SEM as well as AFM, respectively. The optimal dopant for dopamine detection was found to be polystyrene sulfonate anion (PSS(-)). Rat pheochromocytoma (PC12) cells, a suitable model to study exocytotic dopamine release, were differentiated on IDEs functionalized with an overoxidized PSS(-)-doped PPy film. The modified electrodes were used to amperometrically detect dopamine released by populations of cells upon triggering cellular exocytosis with an elevated K(+) concentration. A comparison between the generated current on bare gold electrodes and gold electrodes modified with overoxidized doped PPy illustrates the clear advantage of the modification, yielding 2.6-fold signal amplification. The results also illustrate how to use cell population based dopamine exocytosis measurements to obtain biologically significant information that can be relevant in, for instance, the study of neural stem cell differentiation into dopaminergic neurons.
[Show abstract][Hide abstract] ABSTRACT: Conventionally, microbial bioelectrochemical assays have been conducted using immobilized cells on an electrode that is placed in an electrochemical batch cell. In this paper, we describe a developed microfluidic platform with integrated microelectrode arrays for automated bioelectrochemical assays utilizing a new double mediator system to map redox metabolism and screen for genetic modifications in Saccharomyces cerevisiae cells. The function of this new double mediator system based on menadione and osmium redox polymer (PVI-Os) is demonstrated. "Wiring" of S. cerevisiae cells using PVI-Os shows a significant improvement of bioelectrochemical monitoring in a microfluidic environment and functions as an effective immobilization matrix for cells that are not strongly adherent. The function of the developed microfluidic platform is demonstrated using two strains of S. cerevisiae, ENY.WA and its deletion mutant EBY44, which lacks the enzyme phosphoglucose isomerase. The cellular responses to introduced glucose and fructose were recorded for the two S. cerevisiae strains, and the obtained results are compared with previously published work when using an electrochemical batch cell, indicating that microfluidic bioelectrochemical assays employing the menadione-PVI-Os double mediator system provides an effective means to conduct automated microbial assays.
Analytical and Bioanalytical Chemistry 02/2013; · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Microfluidics systems usually consist of materials like PMMA - poly(methyl methacrylate) and PDMS - poly(dimethylsiloxane) and not polystyrene (PS), which is usually used for cell culture. Cellular and molecular responses in cells grown on PS are well characterized due to decades of accumulated research. In contrast, the experience base is limited for materials used in microfludics chip fabrication.
The effect of different materials (PS, PMMA and perforated PMMA with a piece of PDMS underneath) on the growth and differentiation of PC12 (adrenal phaeochromocytoma) cells into neuronal-like cells was investigated using cell viability, cell cycle distribution, morphology, and gene expression analysis.
After differentiation, the morphology, viability and cell cycle distribution of PC12 cells grown on PS, PMMA with and without PDMS underneath was the same. By contrast, 41 genes showed different expression for PC12 cells differentiating on PMMA as compared to on PS. In contrast, 677 genes showed different expression on PMMA with PDMS underneath as compared with PC12 cells on PS. The differentially expressed genes are involved in neuronal cell development and function. However, there were also many markers for neuronal cell development and functions that were expressed similarly in cells differentiating on PS, PMMA and PMMA with PDMS underneath. In conclusion, it was shown that PMMA has a minor impact and PDMS a major impact on gene expression in PC12 cells.
PLoS ONE 01/2013; 8(1):e53107. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since the use of impedance measurements for label-free monitoring of
cells has become widespread but still the choice of sensing
configuration is not unique though crucial for a quantitative
interpretation of data, we demonstrate the application of a novel custom
multipotentiostat platform to study optimal detection strategies.
Electrochemical Impedance Spectroscopy (EIS) has been used to monitor
and compare adhesion of different cell lines. HeLa cells and 3T3
fibroblasts have been cultured for 12 hours on interdigitated electrode
arrays integrated into a tailor-made cell culture platform. Both
vertical and coplanar interdigitated sensing configuration approaches
have been used and compared on the same cell populations.
Journal of Physics Conference Series 12/2012; 407(1):012029.
[Show abstract][Hide abstract] ABSTRACT: A novel potentiostat containing 54 current amplifiers matched to an array of custom-fabricated 5μm microelectrodes for electrochemical imaging of released neurotransmitters is presented. The board is integrated with a programmable microfluidic cell culture system and the whole assembly is thin and compact enough to be placed under the objective of a standard microscope for simultaneous optical and electrochemical monitoring. Each channel, scanned every 54μs, features 3pA current resolution over a 5kHz bandwidth, suitable for detecting single exocytotic events. The design and electrical characterization of the system are reported together with its functionality, certified by a 54-pixel electrochemical imaging of the diffusion of a 10μl droplet of a target analyte inside the cell culture chamber.
2012 IEEE Biomedical Circuits and Systems Conference (BioCAS 2012); 11/2012
[Show abstract][Hide abstract] ABSTRACT: An electrochemical detection system specifically designed for multi-parameter real-time monitoring of stem cell culturing/differentiation in a microfluidic system is presented. It is composed of a very compact 24-channel electronic board, compatible with arrays of microelectrodes and coupled to a microfluidic cell culture system. A versatile data acquisition software enables performing amperometry, cyclic voltammetry and impedance spectroscopy in each of the 12 independent chambers over a 100 kHz bandwidth with current resolution down to 5 pA for 100 ms measuring time. The design of the platform, its realization and experimental characterization are reported, with emphasis on the analysis of impact of input capacitance (i.e., microelectrode size) and microfluidic pump operation on current noise. Programmable sequences of successive injections of analytes (ferricyanide and dopamine) and rinsing buffer solution as well as the impedimetric continuous tracking for seven days of the proliferation of a colony of PC12 cells are successfully demonstrated.
IEEE Transactions on Biomedical Circuits and Systems 10/2012; 6(5):498-507. · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A microfluidic component library for building systems driving parallel or serial microfluidic-based assays is presented. The components are a miniaturized eight-channel peristaltic pump, an eight-channel valve, sample-to-waste liquid management, and interconnections. The library of components was tested by constructing various systems supporting perfusion cell culture, automated DNA hybridizations, and in situ hybridizations. The results showed that the MainSTREAM components provided (1) a rapid, robust, and simple method to establish numerous fluidic inputs and outputs to various types of reaction chips; (2) highly parallel pumping and routing/valving capability; (3) methods to interface pumps and chip-to-liquid management systems; (4) means to construct a portable system; (5) reconfigurability/flexibility in system design; (6) means to interface to microscopes; and (7) compatibility with tested biological methods. It was found that LEGO Mindstorms motors, controllers, and software were robust, inexpensive, and an accessible choice as compared with corresponding custom-made actuators. MainSTREAM systems could operate continuously for weeks without leaks, contamination, or system failures. In conclusion, the MainSTREAM components described here meet many of the demands on components for constructing and using microfluidics systems.
Journal of the Association for Laboratory Automation 09/2012; · 1.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A modular microfluidic airways model system that can simulate the changes in oxygen tension in different compartments of the cystic fibrosis (CF) airways was designed, developed, and tested. The fully reconfigurable system composed of modules with different functionalities: multichannel peristaltic pumps, bubble traps, gas exchange chip, and cell culture chambers. We have successfully applied this system for studying the antibiotic therapy of Pseudomonas aeruginosa, the bacteria mainly responsible for morbidity and mortality in cystic fibrosis, in different oxygen environments. Furthermore, we have mimicked the bacterial reinoculation of the aerobic compartments (lower respiratory tract) from the anaerobic compartments (cystic fibrosis sinuses) following an antibiotic treatment. This effect is hypothesised as the one on the main reasons for recurrent lung infections in cystic fibrosis patients.
[Show abstract][Hide abstract] ABSTRACT: In this paper we present a series of experiments showing that vertical self-assembled diphenylalanine peptide nanowires (PNWs) are a suitable candidate material for cellular biosensing. We grew HeLa and PC12 cells onto PNW modified gold surfaces and observed no hindrance of cell growth caused by the peptide nanostructures; furthermore we studied the properties of PNWs by investigating their influence on the electrochemical behavior of gold electrodes. The PNWs were functionalized with polypyrrole (PPy) by chemical polymerization, therefore creating conducting peptide/polymer nanowire structures vertically attached to a metal electrode. The electroactivity of such structures was characterized by cyclic voltammetry. The PNW/PPy modified electrodes were finally used as amperometric dopamine sensors, yielding a detection limit of 3,1 microM.
Journal of Nanoscience and Nanotechnology 04/2012; 12(4):3077-83. · 1.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper describes a method to create giant protein vesicles (GPVs) of ≥10 μm by solvent-driven fusion of large vesicles (0.1-0.2 μm) with reconstituted membrane proteins. We found that formation of GPVs proceeded from rotational mixing of protein-reconstituted large unilamellar vesicles (LUVs) with a lipid-containing solvent phase. We made GPVs by using n-decane and squalene as solvents, and applied generalized polarization (GP) imaging to monitor the polarity around the protein transmembrane region of aquaporins labeled with the polarity-sensitive probe Badan. Specifically, we created GPVs of spinach SoPIP2;1 and E. coli AqpZ aquaporins. Our findings show that hydrophobic interactions within the bilayer of formed GPVs are influenced not only by the solvent partitioning propensity, but also by lipid composition and membrane protein isoform.
[Show abstract][Hide abstract] ABSTRACT: This study describes the interaction between sodium dodecyl sulfate (SDS) and membrane proteins reconstituted into large unilamellar lipid vesicles and detergent micelles studied by circular dichroism (CD) and polarity sensitive probe labeling. Specifically, we carried out a comparative study of two aquaporins with high structural homology SoPIP2;1 and AqpZ using identical reconstitution conditions. Our CD results indicate that SDS, when added to membrane-reconstituted aquaporins in concentrations below the SDS critical micelle concentration (CMC, ~8mM), causes helical rearrangements of both aquaporins. However, we do not find compelling evidence for unfolding. In contrast when SDS is added to detergent stabilized aquaporins, SoPIP2;1 partly unfolds, while AqpZ secondary structure is unaffected. Using a fluorescent polarity sensitive probe (Badan) we show that SDS action on membrane reconstituted SoPIP2;1 as well as AqpZ is associated with initial increased hydrophobic interactions in protein transmembrane (TM) spanning regions up to a concentration of 0.1× CMC. At higher SDS concentrations TM hydrophobic interactions, as reported by Badan, decrease and reach a plateau from SDS CMC up to 12.5× CMC. Combined, our results show that SDS does not unfold neither SoPIP2;1 nor AqpZ during transition from a membrane reconstituted form to a detergent stabilized state albeit the native folds are changed.
Biochimica et Biophysica Acta 06/2011; 1808(10):2600-7. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR)=50 more than 10(5) FomA proteins could be incorporated in a bilayer array with a total membrane area of 2mm(2) within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.
Biochemical and Biophysical Research Communications 02/2011; 406(1):96-100. · 2.28 Impact Factor