A microfluidic device to confine a single cardiac myocyte in a sub-nanoliter volume on planar microelectrodes for extracellular potential recordings

A hybrid chip is described which combines a microfluidic network fabricated in a silicone elastomer (PDMS) with planar microelectrodes. It was used to measure extracellular potentials from single adult murine cardiac myocytes in a restricted extracellular space. The recorded variations in the extracellular potentials were caused by transmembrane currents associated with spontaneously initiated intracellular calcium waves. Single cells were trapped inside the 100 pl microchamber by pressure gradients and maintained for several hours by continuous perfusion. In addition, the localized delivery of drugs to a portion of the cell was demonstrated. The impedance of the electrodes was reduced by a factor of 10 to 20 after the electrodeposition of platinum black. Biopotentials recorded from single cells with platinum black electrodes showed a three-fold decrease in the noise, resulting in a maximum signal-to-noise ratio of 15:1. Characteristic variations in the frequency and shape of the extracellular potentials were observed among different cells which are most likely due to differences in the sarcoplasmic reticulum Ca(2+) load. Our device architecture permits the integration of electrochemical and optical sensors for multiparameter recordings.     

High-throughput examination of fluorescence resonance energy transfer-detected metal-ion response in mammalian cells

Fluorescence resonance energy transfer (FRET)-based genetically encoded metal-ion sensors are important tools for studying metal-ion dynamics in live cells. We present a time-resolved microfluidic flow cytometer capable of characterizing the FRET-based dynamic response of metal-ion sensors in mammalian cells at a throughput of 15 cells/s with a time window encompassing a few milliseconds to a few seconds after mixing of cells with exogenous ligands. We have used the instrument to examine the cellular heterogeneity of Zn(2+) and Ca(2+) sensor FRET response amplitudes and demonstrated that the cluster maps of the Zn(2+) sensor FRET changes resolve multiple subpopulations. We have also measured the in vivo sensor response kinetics induced by changes in Zn(2+) and Ca(2+) concentrations. We observed an ～30 fold difference between the extracellular and intracellular sensors.     

Controlled on-chip stimulation of quantal catecholamine release from chromaffin cells using photolysis of caged Ca2+ on transparent indium-tin-oxide microchip electrodes

Photorelease of caged Ca(2+) is a uniquely powerful tool to study the dynamics of Ca(2+)-triggered exocytosis from individual cells. Using photolithography and other microfabrication techniques, we have developed transparent microchip devices to enable photorelease of caged Ca(2+), together with electrochemical detection of quantal catecholamine secretion from individual cells or cell arrays as a step towards developing high-throughput experimental devices. A 100 nm thick transparent indium-tin-oxide (ITO) film was sputter-deposited onto glass coverslips, which were then patterned into 24 cell-sized working electrodes (approximately 20 microm by 20 microm). We loaded bovine chromaffin cells with acetoxymethyl (AM) ester derivatives of the Ca(2+) cage NP-EGTA and Ca(2+) indicator dye fura-4F, then transferred these cells onto the working ITO electrodes for amperometric recordings. Upon flash photorelease of caged Ca(2+), a uniform rise of [Ca(2+)](i) within the target cell leads to quantal release of oxidizable catecholamines measured amperometrically by the underlying ITO electrode. We observed a burst of amperometric spikes upon rapid elevation of [Ca(2+)](i) and a "priming" effect of sub-stimulatory [Ca(2+)](i) on the response of cells to subsequent [Ca(2+)](i) elevation, similar to previous reports using different techniques. We conclude that UV photolysis of caged Ca(2+) is a suitable stimulation technique for higher-throughput studies of Ca(2+)-dependent exocytosis on transparent electrochemical microelectrode arrays.     

Magnetic anisotropy in the excited states of low symmetry lanthanide complexes

Ab initio investigation of multiplet spectrum of lanthanides in archetypal coordination geometries shows an unexpected regular structure consisting of (i) mirror symmetry of anisotropic magnetic properties of doublet states, (ii) high magnetic axiality of low-lying and high-lying doublets, comparable to complexes with ideal axial symmetry, and (iii) the strong rotation of the anisotropy axes of individual doublets. The obtained high axiality of the ground doublet states explains the SMM behaviour of low-symmetry lanthanide complexes.     

Small-angle scattering of polarized light. VI. Sphere doublets at rest and during shear

The polarized or depolarized light scattering by well-defined monodispersed sphere doublets is investigated. Two configurations of doublets are studied. In the first (at rest) the doublets are randomly oriented in a plane, in the second the doublets are oriented in a preferred direction. This is achieved by submitting a suspension of doublets to a shear flow. The scattering patterns are compared to two theoretical predictions based on simplified geometries. In the first approach, the doublet is approximated by two interpenetrating spheres scattering independently, whereas in the second, an ellipsoid geometry is used. A good qualitative comparison is obtained. However, the HV and VH patterns of a randomly dispersed suspension are not similar. The observation of the flow of a doublet suspension in shear shows that the doublets are spiraling around the vorticity axis. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36: 2005–2013, 1998     

Influence of Ca2+ on tetracycline adsorption on montmorillonite

The adsorption of tetracycline (TC) on montmorillonite was studied as a function of pH and Ca(2+) concentration using a batch technique complemented with X-ray diffraction and transmission electron microscopy. In the absence of Ca(2+), TC adsorption was high at low pH and decreased as the pH increased. In the presence of Ca(2+), at least two different adsorption processes took place in the studied systems, i.e., cation exchange and Ca-bridging. Cation exchange was the prevailing process at pH<5, and thus, TC adsorption decreased by increasing total Ca(2+) concentration. On the contrary, Ca-bridging was the prevailing process at pH>5, and thus, TC adsorption increased by increasing Ca(2+) concentration. The pH 5 represents an isoadsorption pH where both adsorption processes compensate each other. TC adsorption became independent of Ca(2+) concentration at this pH. For TC adsorption on Ca(2+)-montmorillonite in 0.01 M NaCl experiments, the ratio adsorbed TC/retained Ca(2+) was close to 1 in the pH range of 5-9, indicating an important participation of Ca(2+) in the binding of TC to montmorillonite. X-ray diffraction and transmission electron microscopy showed that TC adsorption induced intercalation between montmorillonite layers forming a multiphase system with stacking of layers with and without intercalated TC.     

Immobilization of DNAzyme catalytic beacons on PMMA for Pb2+ detection

Due to the numerous toxicological effects of lead, its presence in the environment needs to be effectively monitored. Incorporating a biosensing element within a microfluidic platform enables rapid and reliable determinations of lead at trace levels. A microchip-based lead sensor is described here that employs a lead-specific DNAzyme (also called catalytic DNA or deoxyribozyme) as a recognition element that cleaves its complementary substrate DNA strand only in the presence of cationic lead (Pb(2+)). Fluorescent tags on the DNAzyme translate the cleavage events to measurable, optical signals proportional to Pb(2+) concentration. The DNAzyme responds sensitively and selectively to Pb(2+), and immobilizing DNAzyme in the sensor permits both sensor regeneration and localization of the detection zone. Here, the DNAzyme has been immobilized on a PMMA surface using the highly specific biotin-streptavidin interaction. The strategy includes using streptavidin physisorbed on a PMMA surface to immobilize DNAzyme both on planar PMMA and on the walls of a PMMA microfluidic device. The immobilized DNAzyme retains its Pb(2+) detection activity in the microfluidic device and can be regenerated and reused. The DNAzyme shows no response to other common metal cations and the presence of these contaminants does not interfere with the lead-induced fluorescence signal. While prior work has shown lead-specific catalytic DNA can be used in its solubilized form and while attached to gold substrates to quantitate Pb(2+) in solution, this is the first use of the DNAzyme immobilized within a microfluidic platform for real time Pb(2+) detection.     

Extending optogenetics to a Ca(2+)-selective channel

Many cellular processes are regulated by Ca(2+) signaling. In this issue of Chemistry & Biology, Pham et?al. have developed a photo-activated protein, LOVS1K, which enables the generation of local or global Ca(2+) signals through binding to the Ca(2+)-specific membrane channel Orai.     

Real-time monitoring of intracellular calcium dynamic mobilization of a single cardiomyocyte in a microfluidic chip pertaining to drug discovery

A microfluidic method for real-time quantitative measurement of cellular response pertaining to drug discovery is reported. This method is capable of multiple-step liquid delivery for measuring the drug response of a single cardiomyocyte, due to the improved cell retention by a newly designed chip. The chip, which consists of a cell-retention chamber with a weir structure, was fabricated just by a one-photomask microfabrication procedure followed by on-chip etching. This method differs from the conventional method, which uses two-mask photolithography to fabricate the microchannel (deep etch) and the weir structure (shallow etch). The dimensions of the weir structure have been predicted by a mathematical model, and confirmed by confocal microscopy. Using this microfluidic method, the dynamic [Ca2+]i mobilization in a single cardiomyocyte during its spontaneous contraction was quantified. Furthermore, we measured the cellular response of a cardiomyocyte on (i) a known cardiotonic agent (caffeine), (ii) a cardiotoxic chemotherapeutic drug (daunorubicin), and (iii) an herbal anticancer drug candidate - isoliquiritigenin (IQ) based on the fluorescent calcium measurement. It was found that IQ had produced a less pronounced effect on calcium mobilization( )of the cardiomyocytes whereas caffeine and daunorubicin had much stronger effects on the cells. These three experiments on cardiomyocytes pertaining to drug discovery were only possible after the improved cell retention provided by the new chip design (MV2) required for multiple-step real-time cellular analysis on a microchip, as compared with our old chip design (MV1).     

EPR g factors of trigonal Dy(3+) center in ThO(2) crystal

The perturbation formulas of EPR g factors g(parallel) and g( perpendicular ) for the lowest Kramers doublet of 6H(15/2) of a 4f(9) ion in trigonal symmetry are established in this paper. In these formulas, besides the contribution due to the interaction within the lowest 6H(15/2) manifolds considered in the previous papers, the contributions due to the J-mixing among the 6H(15/2), 6H(13/2) (first excited state) and 6H(11/2) (second excited state) via crystal-field interaction, the admixtures among the states with the same J value via spin-orbit coupling interaction and those between the lowest Kramers doublet Gammagamma and other Kramers doublets Gamma(X) within the states 6H(J) (J=15/2,13/2,11/2) via crystal-field and orbital angular momentum interactions are included. From these formulas, the g factors g(parallel) and g( perpendicular ) for the trigonal Dy(3+) center in ThO(2) crystal are calculated. The results are discussed.     

Electrochemical determination of triple helices: electrocatalytic oxidation of guanine in an intramolecular triplex

Electrocatalytic oxidation of the oligonucleotide 5'- GAA GAG GTT TTT CCT CTT CTT TTT CTT CTC C (TS) by Ru(bpy)(3)(2+) was studied by cyclic voltammetry. This oligonucleotide forms either an intramolecular triplex, hairpin, or single strand, depending on the pH (Plum, G. E.; Breslauer, K. J. J. Mol. Biol. 1995, 248, 679-695). In the triplex form, the guanine doublet in TS is buried inside the folded structure, and as such is less susceptible to oxidation by electrogenerated Ru(bpy)(3)(3+). Digital simulations of the catalytic voltammograms gave a rate constant of 3.5 +/- 0.2 x 10(2) M(-1) s(-1) for oxidation of the triplex form, while oxidation of the duplex and single-stranded forms occurred with much faster rate constants of (3.5-9.1) x 10(4) M(-1) s(-1). Experiments using a truncated form of TS that lacked the third strand of the triplex were consistent with these measurements. The Ru(bpy)(3)(3+) complex was also generated by photolyzing Ru(bpy)(3)(2+) in the presence of Fe(CN)(6)(3-). This reaction produced strand scission following piperidine treatment, which was visualized using high-resolution gel electrophoresis. These experiments showed decreased reactivity for the triplex form, and also gave an unusual reversal of a common selectivity for the 5'-G of GG doublets generally seen in B-form DNA. This reversal was ascribed to strain caused by the location of the GG doublet adjacent to the hairpin loop.     

Morphology of small aggregates of red blood cells

Blood can be considered a two-phase liquid composed of plasma as well as cells and cell aggregates. The degree of cell aggregation is an important determinant of blood rheology: The size and shape of the aggregates affect blood viscosity. The microscopic mechanisms of red blood cell adhesion involve a complex interplay of electrostatic, van der Waals, and a range of specific biochemical inter-membrane interactions. Here we use an effective model of these interactions combined with the membrane elasticity theory to calculate the equilibrium shape of a red blood cell doublet and compare it with the experimentally observed red blood cell aggregates both in vitro and in vivo. Special attention is devoted to the shape of doublets formed by dissimilar cells. A possible effect of doublet shape on pathways of the formation of multicellular aggregates is discussed. Red blood cell rouleau formation is expected to take place at intermediate adhesion strengths where the outer doublet surfaces are either concave or flat, whereas in the strong-adhesion regime where the outer doublet surfaces are convex the cells should form rounded clump-like aggregates.     

A 13C solid-state NMR analysis of steroid compounds

(13)C CP/MAS solid-state NMR spectroscopy has been utilized to analyze six steroid compounds, namely testosterone (Tes), hydrocortisone (Cor), trans-dehydroandrosterone (Adr), prednisolone (Prd), prednisone (Pre) and estradiol (Est). Among them, Tes displays a doublet pattern for all residues, whereas Prd, Pre and Est, exhibit exclusively singlets. For Cor and Adr, the (13)C spectra contain both doublet and singlet patterns. The (13)C doublet signal, with splittings of 0.2-1.5 ppm, are ascribed to local differences in the ring conformations associated with polymorphism. We have assigned all of the (13)C resonances to the different residues in these steroid compounds on the basis of solution NMR data. The C-7, C-8, C-10, C-15 and C-16 residues of Tes, Cor and Adr consistently give rise to singlets or doublets with splittings of less than 0.5 ppm, indicating similar local conformations. Accompanying hydration and dehydration processes, a reversible phase transformation between delta- and alpha-crystal forms has been observed in Tes, corresponding to singlet and doublet (13)C patterns, respectively. To further characterize the ring conformations in the alpha-form, we have successfully extracted chemical shift tensor elements for the (13)C doublets. It is demonstrated that (13)C solid-state NMR spectroscopy provides a reliable and sensitive means of characterizing polymorphism in steroids.     

Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel

Deciphering the signaling pathways that govern stimulation of na?ve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of na?ve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations.     

Design, synthesis, and structure-activity relationships of 3,4-dihydropyridopyrimidin-2(1H)-one derivatives as a novel class of sodium/calcium exchanger inhibitor

Design, synthesis, and structure-activity relationships for 3,4-dihydropyridopyrimidin-2(1H)-one derivatives, which are aza-3,4-dihydro-2(1H)-quinazolinone derivatives, as the sodium/calcium (Na+/Ca2+) exchanger inhibitors are discussed. These studies based on 3,4-dihydro-2(1H)-quinazolinone derivatives led to the discovery of a structurally novel and potent Na+/Ca2+ exchanger inhibitor, 3,4-dihydropyridopyrimidin-2(1H)-one derivative (26), with an IC30 value of 0.02 microM. Compound 26 directly inhibited the Na+-dependent Ca2+ influx via the Na+/Ca2+ exchanger after Na+-free treatment in cardiomyocytes.     

Analysis of intercellular calcium signaling using microfluidic adjustable laminar flow for localized chemical stimulation

The propagation of intercellular calcium signals provides a mechanism to coordinate cell population activity, which is essential for regulating cell behavior and organ development. However, existing analytical methods are difficult to realize localized chemical stimulation of a single cell among a population of cells that are in close contact with one another for studying the propagation of calcium wave. In this work, a microfluidic method is presented for the analysis of contact-dependent propagation of intercellular calcium wave induced by extracellular ATP using multiple laminar flows. Adjacent cells were seeded ∼300 μm downstream the intersection of a Y-shaped microchannel with negative pressure pulses. Consequently, the lateral diffusion distance of the chemical at cell locations was limited to ∼26 μm with a total flow rate of 20 μL min⁻¹, which prevented the interference of diffusion-induced cellular responses. Localized stimulation of the target cell with ATP induced the propagation of intercellular calcium wave among the cell population. In addition, studies on the spread of intercellular calcium wave under octanol inhibition allowed us to characterize the gap junction mediated cell–cell communication. Thus, this novel device will provide a versatile platform for intercellular signal transduction studies and high throughput drug screening.     

Microfluidic wound-healing assay to assess the regenerative effect of HGF on wounded alveolar epithelium

We present a microfluidic epithelial wound-healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow-focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system, a technique that is reported here for the first time. We demonstrate that alveolar epithelial cells cultured in a microfluidic environment preserve their phenotype before and after wounding. In addition, we report a wound-healing benefit induced by addition of HGF to the cell culture medium (19.2 vs. 13.5 μm h(-1) healing rate).     

Role of Ca(2+) in diallyl disulfide-induced apoptotic cell death of HCT-15 cells

Diallyl disulfide (DADS) induced apoptosis through the caspase-3 dependent pathway in leukemia cells was earlier reported from this laboratory. In this study, we investigated the involvement of Ca(2+) in DADS-induced apoptotic cell death of HCT-15, human colon cancer cell line. DADS induced the elevation of cytosolic Ca(2+) by biphasic pattern; rapid Ca(2+) peak at 3 min and following slow and sustained elevation till 3 h after the addition of DADS. Production of H(2)O(2) was also observed with its peak value at 4 h. Apoptotic pathways including the sequence of caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation by DADS were completely blocked by various inhibitors such as specific caspase-3 inhibitor, free radical scavenger, and intracellular Ca(2+) chelator. N-acetylcystein and catalase treatment prevented the accumulation of H2O2 and later caspase-3 dependent apoptotic pathway. However, these radical scavengers did not block the elevation of intracellular Ca(2+). Treatment of cells with 1, 2-bis (2-aminophenoxyethane)-N, N, N-tetraacetic acid tetrakis -acetoxymethyl ester (BAPTA-AM), cellular Ca(2+) chelator, resulted in a complete blockage of the caspase-3 dependent apoptotic pathway of HCT-15 cells. It abolished the elevation of intracellular Ca(2+), and furthermore, completely inhibited the production of H(2)O(2). These results indicate that cytosolic Ca(2+) elevation is an earlier signaling event in apoptosis of HCT-15 cells. Collectively, our data demonstrate that DADS can induce apoptosis in HCT-15 cells through the sequential mechanism of Ca(2+) homeostasis disruption, accumulation of H(2)O(2), and resulting caspase-3 activation.     

Photosensitization reaction-induced acute electrophysiological cell response of rat myocardial cells in short loading periods of talaporfin sodium or porfimer sodium

Electrophysiological responses of rat myocardial cells to exogenous photosensitization reactions for a short period of incubation with two photosensitizers, talaporfin sodium or porfimer sodium, were measured in a subsecond time scale. The loading period of the photosensitizer when the photosensitizer might not be taken up by the cells was selected as 15min, which was determined by the fluorescence microscopic observation. We measured the intracellular Ca(2+) concentration ([Ca(2+) ](in) ) by using a fluorescent Ca(2+) indicator, Fluo-4 AM, under a high-speed confocal laser microscope to evaluate the acute electrophysiological cell response to the photosensitization reaction. The measured temporal change in Fluo-4 fluorescence intensity indicated that the response to the photosensitization reaction might be divided into two phases in both photosensitizers. The first phase is acute response: disappearance of Ca(2+) oscillation when irradiation starts, which might be caused by ion channel dysfunction. The second phase is slow response: [Ca(2+) ](in) elevation indicating influx of Ca(2+) due to the concentration gradient. The continuous Ca(2+) influx followed by changes in cell morphology suggested micropore formation on the surface of the cell membrane, resulting in necrotic cell death.     

Analysis of intercellular communication by flexible hydrodynamic gating on a microfluidic chip

Intercellular Ca²⁺ waves are propagation of Ca²⁺ transients among cells that could be initiated by chemical stimulation. Current methods for analyzing intercellular Ca²⁺ waves are difficult to realize localized chemical stimulations upon the target cell without interfering with adjacent contacting cells. In this paper, a simple and flexible microfluidic method was developed for investigating the intercellular communication of Ca²⁺ signals. A cross-patterned microfluidic chip was designed and fabricated with polydimethylsiloxane as the structural material. Localized chemical stimulation was achieved by a new strategy based on hydrodynamic gating technique. Clusters of target cells were seeded at the location within 300 μm downstream of the intersection of the cross-shaped microchannel. Confined lateral molecular diffusion largely minimized the interference from diffusion-induced stimulation of adjacent cells. Localized stimulation of the target cell with adenosine 5′-triphosphate successfully induced the propagation of intercellular Ca²⁺ waves among a population of adjacent contacting cells. Further inhibition studies verified that the propagation of calcium signals among NIH-3 T3 cells was dependent on direct cytosolic transfer via gap junctions. The developed microfluidic method provides a versatile platform for investigating the dynamics of intercellular communications.