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1.
Sci Adv ; 7(16)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33863724

RESUMO

Several important drug targets, e.g., ion channels and G protein-coupled receptors, are extremely difficult to approach with current antibody technologies. To address these targets classes, we explored kinetically controlled proteases as structural dynamics-sensitive druggability probes in native-state and disease-relevant proteins. By using low-Reynolds number flows, such that a single or a few protease incisions are made, we could identify antibody binding sites (epitopes) that were translated into short-sequence antigens for antibody production. We obtained molecular-level information of the epitope-paratope region and could produce high-affinity antibodies with programmed pharmacological function against difficult-to-drug targets. We demonstrate the first stimulus-selective monoclonal antibodies targeting the transient receptor potential vanilloid 1 (TRPV1) channel, a clinically validated pain target widely considered undruggable with antibodies, and apoptosis-inducing antibodies selectively mediating cytotoxicity in KRAS-mutated cells. It is our hope that this platform will widen the scope of antibody therapeutics for the benefit of patients.


Assuntos
Anticorpos Monoclonais , Antígenos , Anticorpos Monoclonais/química , Sítios de Ligação de Anticorpos , Epitopos , Humanos
2.
Sci Rep ; 10(1): 19529, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33173097

RESUMO

Significant strides have been made in the development of in vitro systems for disease modelling. However, the requirement of microenvironment control has placed limitations on the generation of relevant models. Herein, we present a biological tissue printing approach that employs open-volume microfluidics to position individual cells in complex 2D and 3D patterns, as well as in single cell arrays. The variety of bioprinted cell types employed, including skin epithelial (HaCaT), skin cancer (A431), liver cancer (Hep G2), and fibroblast (3T3-J2) cells, all of which exhibited excellent viability and survivability, allowing printed structures to rapidly develop into confluent tissues. To demonstrate a simple 2D oncology model, A431 and HaCaT cells were printed and grown into tissues. Furthermore, a basic skin model was established to probe drug response. 3D tissue formation was demonstrated by co-printing Hep G2 and 3T3-J2 cells onto an established fibroblast layer, the functionality of which was probed by measuring albumin production, and was found to be higher in comparison to both 2D and monoculture approaches. Bioprinting of primary cells was tested using acutely isolated primary rat dorsal root ganglia neurons, which survived and established processes. The presented technique offers a novel open-volume microfluidics approach to bioprint cells for the generation of biological tissues.


Assuntos
Bioimpressão/métodos , Microfluídica/métodos , Impressão Tridimensional , Engenharia Tecidual/métodos , Células 3T3 , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2 , Humanos , Camundongos , Microscopia de Fluorescência , Ratos , Pele/citologia , Pele/efeitos dos fármacos , Tretinoína/farmacologia
3.
Sci Transl Med ; 10(428)2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29444979

RESUMO

Cancer cells adapt to their inherently increased oxidative stress through activation of the glutathione (GSH) and thioredoxin (TXN) systems. Inhibition of both of these systems effectively kills cancer cells, but such broad inhibition of antioxidant activity also kills normal cells, which is highly unwanted in a clinical setting. We therefore evaluated targeting of the TXN pathway alone and, more specifically, selective inhibition of the cytosolic selenocysteine-containing enzyme TXN reductase 1 (TXNRD1). TXNRD1 inhibitors were discovered in a large screening effort and displayed increased specificity compared to pan-TXNRD inhibitors, such as auranofin, that also inhibit the mitochondrial enzyme TXNRD2 and additional targets. For our lead compounds, TXNRD1 inhibition correlated with cancer cell cytotoxicity, and inhibitor-triggered conversion of TXNRD1 from an antioxidant to a pro-oxidant enzyme correlated with corresponding increases in cellular production of H2O2 In mice, the most specific TXNRD1 inhibitor, here described as TXNRD1 inhibitor 1 (TRi-1), impaired growth and viability of human tumor xenografts and syngeneic mouse tumors while having little mitochondrial toxicity and being better tolerated than auranofin. These results display the therapeutic anticancer potential of irreversibly targeting cytosolic TXNRD1 using small molecules and present potent and selective TXNRD1 inhibitors. Given the pronounced up-regulation of TXNRD1 in several metastatic malignancies, it seems worthwhile to further explore the potential benefit of specific irreversible TXNRD1 inhibitors for anticancer therapy.


Assuntos
Citosol/enzimologia , Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Tiorredoxina Redutase 1/antagonistas & inibidores , Animais , Antioxidantes/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Peróxido de Hidrogênio/metabolismo , Masculino , Camundongos SCID , Oxirredução , Relação Estrutura-Atividade , Tiorredoxina Redutase 1/química , Tiorredoxina Redutase 1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Neurosci Methods ; 241: 132-6, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25554414

RESUMO

BACKGROUND: Among the various fluidic control technologies, microfluidic devices are becoming powerful tools for pharmacological studies using brain slices, since these devices overcome traditional limitations of conventional submerged slice chambers, leading to better spatiotemporal control over delivery of drugs to specific regions in the slices. However, microfluidic devices are not yet fully optimized for such studies. NEW METHOD: We have recently developed a multifunctional pipette (MFP), a free standing hydrodynamically confined microfluidic device, which provides improved spatiotemporal control over drug delivery to biological tissues. RESULTS: We demonstrate herein the ability of the MFP to selectively perfuse one dendritic layer in the CA1 region of hippocampus with CNQX, an AMPA receptor antagonist, while not affecting the other layers in this region. Our experiments also illustrate the essential role of hydrodynamic confinement in sharpening the spatial selectivity in brain slice experiments. Concentration-response measurements revealed that the ability of the MFP to control local drug concentration is comparable with that of whole slice perfusion, while in comparison the required amounts of active compounds can be reduced by several orders of magnitude. COMPARISON WITH EXISTING METHOD: The multifunctional pipette is applied with an angle, which, compared to other hydrodynamically confined microfluidic devices, provides more accessible space for other probing and imaging techniques. CONCLUSIONS: Using the MFP it will be possible to study selected regions of brain slices, integrated with various imaging and probing techniques, without affecting the other parts of the slices.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Técnicas Analíticas Microfluídicas/métodos , Animais , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Cultura de Órgãos , Preparações Farmacêuticas/administração & dosagem , Ratos , Ratos Sprague-Dawley
5.
J Am Chem Soc ; 136(42): 14875-82, 2014 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-25254316

RESUMO

Even though gain, loss, or modulation of ion channel function is implicated in many diseases, both rare and common, the development of new pharmaceuticals targeting this class has been disappointing, where it has been a major problem to obtain correlated structural and functional information. Here, we present a microfluidic method in which the ion channel TRPV1, contained in proteoliposomes or in excised patches, was exposed to limited trypsin proteolysis. Cleaved-off peptides were identified by MS, and electrophysiological properties were recorded by patch clamp. Thus, the structure-function relationship was evaluated by correlating changes in function with removal of structural elements. Using this approach, we pinpointed regions of TRPV1 that affect channel properties upon their removal, causing changes in current amplitude, single-channel conductance, and EC50 value toward its agonist, capsaicin. We have provided a fast "shotgun" method for chemical truncation of a membrane protein, which allows for functional assessments of various peptide regions.


Assuntos
Dispositivos Lab-On-A-Chip/métodos , Proteólise , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Ativação do Canal Iônico , Modelos Moleculares , Conformação Proteica , Propriedades de Superfície , Tripsina/metabolismo
6.
Anal Chem ; 85(21): 10126-33, 2013 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-24003961

RESUMO

We report a novel approach for determining the enzymatic activity within a single suspended cell. Using a steady-state microfluidic delivery device and timed exposure to the pore-forming agent digitonin, we controlled the plasma membrane permeation of individual NG108-15 cells. Mildly permeabilized cells (~100 pores) were exposed to a series of concentrations of fluorescein diphosphate (FDP), a fluorogenic alkaline phosphatase substrate, with and without levamisole, an alkaline phosphatase inhibitor. We generated quantitative estimates for intracellular enzyme activity and were able to construct both dose-response and dose-inhibition curves at the single-cell level, resulting in an apparent Michaelis contant Km of 15.3 µM ± 1.02 (mean ± standard error of the mean (SEM), n = 16) and an inhibition constant Ki of 0.59 mM ± 0.07 (mean ± SEM, n = 14). Enzymatic activity could be monitored just 40 s after permeabilization, and five point dose-inhibition curves could be obtained within 150 s. This rapid approach offers a new methodology for characterizing enzyme activity within single cells.


Assuntos
Neuroblastoma/enzimologia , Análise de Célula Única , Animais , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Relação Dose-Resposta a Droga , Fluorescência , Levamisol/farmacologia , Camundongos , Neuroblastoma/patologia , Ratos
7.
J Neurosci Methods ; 219(2): 292-6, 2013 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23969260

RESUMO

We have developed a superfusion method utilizing an open-volume microfluidic device for administration of pharmacologically active substances to selected areas in brain slices with high spatio-temporal resolution. The method consists of a hydrodynamically confined flow of the active chemical compound, which locally stimulates neurons in brain slices, applied in conjunction with electrophysiological recording techniques to analyze the response. The microfluidic device, which is a novel free-standing multifunctional pipette, allows diverse superfusion experiments, such as testing the effects of different concentrations of drugs or drug candidates on neurons in different cell layers with high positional accuracy, affecting only a small number of cells. We demonstrate herein the use of the method with electrophysiological recordings of pyramidal cells in hippocampal and prefrontal cortex brain slices from rats, determine the dependence of electric responses on the distance of the superfusion device from the recording site, document a multifold gain in solution exchange time as compared to whole slice perfusion, and show that the device is able to store and deliver up to four solutions in a series. Localized solution delivery by means of open-volume microfluidic technology also reduces reagent consumption and tissue culture expenses significantly, while allowing more data to be collected from a single tissue slice, thus reducing the number of laboratory animals to be sacrificed for a study.


Assuntos
Encéfalo/citologia , Eletrofisiologia/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Animais , Encéfalo/efeitos dos fármacos , Eletrofisiologia/métodos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Técnicas de Cultura de Órgãos , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
8.
Mol Pain ; 9: 1, 2013 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-23279936

RESUMO

The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.


Assuntos
Colesterol/deficiência , Canais de Cátion TRPV/metabolismo , Animais , Células CHO , Capsaicina/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colesterol/farmacologia , Cricetinae , Cricetulus , Temperatura Alta , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Porosidade , Prótons , beta-Ciclodextrinas/farmacologia
9.
ACS Nano ; 7(1): 308-15, 2013 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-23215045

RESUMO

We use single-molecule fluorescence microscopy to monitor individual hybridization reactions between membrane-anchored DNA strands, occurring in nanofluidic lipid monolayer films deposited on Teflon AF substrates. The DNA molecules are labeled with different fluorescent dyes, which make it possible to simultaneously monitor the movements of two different molecular species, thus enabling tracking of both reactants and products. We employ lattice diffusion simulations to determine reaction probabilities upon interaction. The observed hybridization rate of the 40-mer DNA was more than 2-fold higher than that of the 20-mer DNA. Since the lateral diffusion coefficient of the two different constructs is nearly identical, the effective molecule radius determines the overall kinetics. This implies that when two DNA molecules approach each other, hydrogen bonding takes place distal from the place where the DNA is anchored to the surface. Strand closure then propagates bidirectionally through a zipper-like mechanism, eventually bringing the lipid anchors together. Comparison with hybridization rates for corresponding DNA sequences in solution reveals that hybridization rates are lower for the lipid-anchored strands and that the dependence on strand length is stronger.


Assuntos
DNA/química , DNA/ultraestrutura , Hibridização in Situ Fluorescente/métodos , Bicamadas Lipídicas/química , Membranas Artificiais , Imagem Molecular/métodos , Espectrometria de Fluorescência/métodos , Difusão , Cinética
10.
Anal Chem ; 84(13): 5582-8, 2012 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-22656064

RESUMO

We have developed a microfluidic flow cell where stepwise enzymatic digestion is performed on immobilized proteoliposomes and the resulting cleaved peptides are analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The flow cell channels consist of two parallel gold surfaces mounted face to face with a thin spacer and feature an inlet and an outlet port. Proteoliposomes (50-150 nm in diameter) obtained from red blood cells (RBC), or Chinese hamster ovary (CHO) cells, were immobilized on the inside of the flow cell channel, thus forming a stationary phase of proteoliposomes. The rate of proteoliposome immobilization was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D) which showed that 95% of the proteoliposomes bind within 5 min. The flow cell was found to bind a maximum of 1 µg proteoliposomes/cm(2), and a minimum proteoliposome concentration required for saturation of the flow cell was determined to be 500 µg/mL. Atomic force microscopy (AFM) studies showed an even distribution of immobilized proteoliposomes on the surface. The liquid encapsulated between the surfaces has a large surface-to-volume ratio, providing rapid material transfer rates between the liquid phase and the stationary phase. We characterized the hydrodynamic properties of the flow cell, and the force acting on the proteoliposomes during flow cell operation was estimated to be in the range of 0.1-1 pN, too small to cause any proteoliposome deformation or rupture. A sequential proteolytic protocol, repeatedly exposing proteoliposomes to a digestive enzyme, trypsin, was developed and compared with a single-digest protocol. The sequential protocol was found to detect ~65% more unique membrane-associated protein (p < 0.001, n = 6) based on peptide analysis with LC-MS/MS, compared to a single-digest protocol. Thus, the flow cell described herein is a suitable tool for shotgun proteomics on proteoliposomes, enabling more detailed characterization of complex protein samples.


Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Peptídeos/análise , Proteolipídeos/química , Animais , Células CHO , Cromatografia Líquida , Colagenases/metabolismo , Cricetinae , Desenho de Equipamento , Eritrócitos/química , Humanos , Hidrodinâmica , Proteínas Imobilizadas/química , Proteínas Imobilizadas/isolamento & purificação , Proteínas Imobilizadas/metabolismo , Peptídeo Hidrolases/metabolismo , Proteolipídeos/isolamento & purificação , Proteolipídeos/metabolismo , Espectrometria de Massas em Tandem
11.
Nano Lett ; 12(3): 1372-8, 2012 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-22313341

RESUMO

We report a novel method for the measurement of lipid nanotube radii. Membrane translocation is monitored between two nanotube-connected vesicles, during the expansion of a receiving vesicle, by observing a photobleached region of the nanotube. We elucidate nanotube radii, extracted from SPE vesicles, enabling quantification of membrane composition and lamellarity. Variances of nanotube radii were measured, showing a growth of 40-56 nm, upon increasing cholesterol content from 0 to 20%.


Assuntos
Recuperação de Fluorescência Após Fotodegradação/métodos , Lipídeos/química , Membranas Artificiais , Nanotubos/química , Nanotubos/ultraestrutura , Substâncias Macromoleculares/química , Teste de Materiais , Conformação Molecular , Movimento (Física) , Tamanho da Partícula , Propriedades de Superfície
12.
Lab Chip ; 12(7): 1255-61, 2012 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-22252460

RESUMO

Microfluidics has emerged as a powerful laboratory toolbox for biologists, allowing manipulation and analysis of processes at a cellular and sub-cellular level, through utilization of microfabricated features at size-scales relevant to that of a single cell. In the majority of microfluidic devices, sample processing and analysis occur within closed microchannels, imposing restrictions on sample preparation and use. We present an optimized non-contact open-volume microfluidic tool to overcome these and other restrictions, through the use of a hydrodynamically confined microflow pipette, serving as a multifunctional solution handling and dispensing tool. The geometries of the tool have been optimised for use in optical microscopy, with integrated solution reservoirs to reduce reagent use, contamination risks and cleaning requirements. Device performance was characterised using both epifluorescence and total internal reflection fluorescence (TIRF) microscopy, resulting in ~200 ms and ~130 ms exchange times at ~100 nm and ~30 µm distances to the surface respectively.


Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Animais , Células CHO , Cricetinae , Cricetulus , Soluções/química , Manejo de Espécimes
13.
Langmuir ; 28(6): 3200-5, 2012 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-22204476

RESUMO

We describe a general photolithography-based process for the microfabrication of surface-supported Teflon AF structures. Teflon AF patterns primarily benefit from superior optical properties such as very low autofluorescence and a low refractive index. The process ensures that the Teflon AF patterns remain strongly hydrophobic in order to allow rapid lipid monolayer spreading and generates a characteristic edge morphology which assists directed cell growth along the structured surfaces. We provide application examples, demonstrating the well-controlled mixing of lipid films on Teflon AF structures and showing how the patterned surfaces can be used as biocompatible growth-directing substrates for cell culture. Chinese hamster ovary (CHO) cells develop in a guided fashion along the sides of the microstructures, selectively avoiding to grow over the patterned areas.


Assuntos
Técnicas Analíticas Microfluídicas/métodos , Nanotecnologia/métodos , Politetrafluoretileno/química , Animais , Materiais Biocompatíveis/química , Células CHO , Cricetinae , Cricetulus , Interações Hidrofóbicas e Hidrofílicas , Microscopia Confocal
15.
J Am Chem Soc ; 133(45): 18046-9, 2011 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-21978148

RESUMO

Hydrophobic side groups on a stimuli-responsive polymer, encapsulated within a single giant unilamellar vesicle, enable membrane attachment during compartment formation at elevated temperatures. We thermally modulated the vesicle through implementation of an IR laser via an optical fiber, enabling localized directed heating. Polymer-membrane interactions were monitored using confocal imaging techniques as subsequent membrane protrusions occurred and lipid nanotubes formed in response to the polymer hydrogel contraction. These nanotubes, bridging the vesicle membrane to the contracting hydrogel, were retained on the surface of the polymer compartment, where they were transformed into smaller vesicles in a process reminiscent of cellular endocytosis. This development of a synthetic vesicle system containing a stimuli-responsive polymer could lead to a new platform for studying inter/intramembrane transport through lipid nanotubes.


Assuntos
Membranas Artificiais , Nanotubos/química , Lipossomas Unilamelares/química , Lipídeos/química , Polímeros/química
16.
Nat Protoc ; 6(6): 791-805, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21637199

RESUMO

We describe micromanipulation and microinjection procedures for the fabrication of soft-matter networks consisting of lipid bilayer nanotubes and surface-immobilized vesicles. These biomimetic membrane systems feature unique structural flexibility and expandability and, unlike solid-state microfluidic and nanofluidic devices prepared by top-down fabrication, they allow network designs with dynamic control over individual containers and interconnecting conduits. The fabrication is founded on self-assembly of phospholipid molecules, followed by micromanipulation operations, such as membrane electroporation and microinjection, to effect shape transformations of the membrane and create a series of interconnected compartments. Size and geometry of the network can be chosen according to its desired function. Membrane composition is controlled mainly during the self-assembly step, whereas the interior contents of individual containers is defined through a sequence of microneedle injections. Networks cannot be fabricated with other currently available methods of giant unilamellar vesicle preparation (large unilamellar vesicle fusion or electroformation). Described in detail are also three transport modes, which are suitable for moving water-soluble or membrane-bound small molecules, polymers, DNA, proteins and nanoparticles within the networks. The fabrication protocol requires ∼90 min, provided all necessary preparations are made in advance. The transport studies require an additional 60-120 min, depending on the transport regime.


Assuntos
Biomimética/métodos , Bicamadas Lipídicas/síntese química , Micromanipulação/métodos , Nanotubos/química , Transporte Biológico , Biomimética/instrumentação , Eletroporação/instrumentação , Eletroporação/métodos , Lipídeos/química , Microinjeções/instrumentação , Microinjeções/métodos , Micromanipulação/instrumentação , Nanopartículas/química , Nanotubos/ultraestrutura , Glycine max/química
17.
Nat Mater ; 9(11): 908-12, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20935656

RESUMO

Bilayer membranes envelope cells as well as organelles, and constitute the most ubiquitous biological material found in all branches of the phylogenetic tree. Cell membrane rupture is an important biological process, and substantial rupture rates are found in skeletal and cardiac muscle cells under a mechanical load. Rupture can also be induced by processes such as cell death, and active cell membrane repair mechanisms are essential to preserve cell integrity. Pore formation in cell membranes is also at the heart of many biomedical applications such as in drug, gene and short interfering RNA delivery. Membrane rupture dynamics has been studied in bilayer vesicles under tensile stress, which consistently produce circular pores. We observed very different rupture mechanics in bilayer membranes spreading on solid supports: in one instance fingering instabilities were seen resulting in floral-like pores and in another, the rupture proceeded in a series of rapid avalanches causing fractal membrane fragmentation. The intermittent character of rupture evolution and the broad distribution in avalanche sizes is consistent with crackling-noise dynamics. Such noisy dynamics appear in fracture of solid disordered materials, in dislocation avalanches in plastic deformations and domain wall magnetization avalanches. We also observed similar fractal rupture mechanics in spreading cell membranes.


Assuntos
Membranas/fisiologia , Animais , Fenômenos Biomecânicos , Células CHO , Cricetinae , Cricetulus , Fractais , Bicamadas Lipídicas , Fluidez de Membrana/fisiologia , Membranas/lesões , Membranas/ultraestrutura , Microscopia de Fluorescência
18.
Anal Chem ; 82(11): 4529-36, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20443547

RESUMO

We report on a free-standing microfluidic pipette made in poly(dimethylsiloxane) having a circulating liquid tip that generates a self-confining volume in front of the outlet channels. The method is flexible and scalable as the geometry and the size of the recirculation zone is defined by pressure, channel number, and geometry. The pipette is capable of carrying out a variety of complex fluid processing operations, such as mixing, multiplexing, or gradient generation at selected cells in cell and tissue cultures. Using an uptake assay, we show that it is possible to generate dose-response curves in situ from adherent Chinese hamster ovary cells expressing proton-activated human transient receptor potential vanilloid (hTRPV1) receptors. Using confined superfusion and cell stimulation, we could activate hTRPV1 receptors in single cells, measure the response by a patch-clamp pipette, and induce membrane bleb formation by exposing selected groups of cells to formaldehyde/dithiothreitol-containing solutions, respectively. In short, the microfluidic pipette allows for complex, contamination-free multiple-compound delivery for pharmacological screening of intact adherent cells.


Assuntos
Técnicas Analíticas Microfluídicas , Farmacologia/instrumentação , Animais , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Dimetilpolisiloxanos/química , Relação Dose-Resposta a Droga , Fenômenos Eletrofisiológicos , Humanos , Canais de Cátion TRPV/metabolismo
19.
Anal Bioanal Chem ; 397(8): 3235-48, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20496058

RESUMO

Single-cell electroporation (SCEP) is a relatively new technique that has emerged in the last decade or so for single-cell studies. When a large enough electric field is applied to a single cell, transient nano-pores form in the cell membrane allowing molecules to be transported into and out of the cell. Unlike bulk electroporation, in which a homogenous electric field is applied to a suspension of cells, in SCEP an electric field is created locally near a single cell. Today, single-cell-level studies are at the frontier of biochemical research, and SCEP is a promising tool in such studies. In this review, we discuss pore formation based on theoretical and experimental approaches. Current SCEP techniques using microelectrodes, micropipettes, electrolyte-filled capillaries, and microfabricated devices are all thoroughly discussed for adherent and suspended cells. SCEP has been applied in in-vivo and in-vitro studies for delivery of cell-impermeant molecules such as drugs, DNA, and siRNA, and for morphological observations.


Assuntos
Células/química , Técnicas Citológicas , Eletroporação/métodos , Animais , Células/metabolismo , Eletroporação/instrumentação , Humanos
20.
Langmuir ; 26(1): 1-4, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-19916531

RESUMO

In this letter, a hydrogel compression method for the facile generation of high polymer concentration within single giant unilamelar vesicles (GUV) is presented. A GUV with an internalized poly(N-isopropyl acrylamide) solution is heated above the lower critical solution temperature (LCST) until compartmentalization by collapsed hydrogel microaggregates occurs. Subsequently, the volume of the vesicle is decreased by reducing the area of the enclosing membrane. An increase in concentration by a factor of up to 11 is reached while the membrane remains intact, bringing the model system close to the living cell situation with its high concentration of proteins in the cytoplasm. The polymer-filled vesicles are also in a suitable density range to significantly improve the flow control performance of hydrogel valves embedded in soft matter nanofluidic devices.


Assuntos
Acrilamidas/química , Temperatura Alta , Hidrogéis/química , Polímeros/química , Lipossomas Unilamelares/química , Resinas Acrílicas , Soluções
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