共查询到20条相似文献,搜索用时 31 毫秒
1.
A tool for detection and characterization of intracellular enzyme-substrate and receptor-ligand interactions inside the cytoplasm of single targeted cells or small confined groups of cells is presented. Fluorogenic enzyme substrates and receptor ligands were rapidly delivered by electroosmosis and internalized by electroporation in cells using an electrolyte-filled capillary (EFC) biased at a high voltage. Specifically, alkaline phosphatase and proteases were detected in single NG108-15 cells using fluorescein diphosphate and casein BODIPY FL, respectively. The intracellular 1,4,5-inositol triphosphate (IP3) and ryanodine receptors were detected after EFC introduction of the selective receptor agonists IP3 and cyclic adenosine diphosphate ribose (cADPr), respectively. Receptor activation in both cases resulted in increased cytosolic concentrations of free calcium ions that were measured using the calcium-ion-selective probe, fluo-3. The effect of cADPr could be blocked by coadministration of the ryanodine receptor antagonist ruthenium red. Furthermore, electroporation of a plurality of cells grown in microwell structures (100 x 100 x 45 microm) molded in PDMS is demonstrated. The methods and systems described using an EFC for electroporation and delivery of protein markers, ligands, and substrates might be useful in high-throughput screening of intracellular targets, with applications in proteomics and phenotype profiling, as well as in drug discovery. 相似文献
2.
Biomechanical properties of cells yield important information on the disease state of cells such as transformation and metastasis. Screening of cells based on their biomechanical properties provides rapid tools for label-free diagnosis and staging of cancers. However, existent single-cell techniques for measuring biomechanical properties suffer from low throughput (<1 cell/min). This prevents the application of these assays to a large cell population, which produces information with statistical significance. In this study, we applied microfluidics-based electroporative flow cytometry (EFC) that combined electroporation with flow cytometry to study deformability of cells at the single-cell level with a throughput of approximately 5 cells/s. The cell swelling during flow-through electroporation was recorded in real time. We believe that the degree of such swelling was indicative of the cell deformability and the cytoskeleton mechanics. Three cell types (MCF-10A, MCF-7, and 12- O-tetradecanoylphorbol-13-acetate-treated MCF-7) with different malignancy and metastatic potential were tested using our approach. We found that the more malignant and metastatic cell types exhibited more swelling due to higher cell deformability. Furthermore, the disruption of microtubules by colchicine caused substantial change in the EFC results, which confirmed that EFC data strongly reflected the cytoskeletal mechanics. Finally, the cell type with the highest metastatic potential also suffered the most cell death due to the flow-through electroporation treatment, presumably due to the most substantial cell swelling, which could irreversibly rupture the membrane. EFC provides a new method for examining single-cell biomechanics with high throughput. We believe that this technique will be useful for mechanistic studies of cytoskeleton dynamics and clinical applications such as diagnosis and staging of cancers in general. 相似文献
3.
The ability to measure chemical gradients surrounding single cells provides novel insights into several areas of cell dynamics--particularly metabolism. Detection of metabolic oxygen consumption can be achieved from a single mammalian cell using a modulated amperometric sensor in a self-referencing mode. To date, however, apart from visual cues, we do not have a reliable and cell-compatible method for determining and stabilizing the position of such probes. In this paper, we report on having successfully measured the increase in the uncompensated resistance of an electrochemical cell upon approach to single, living, biological cells, while simultaneously measuring the metabolic oxygen consumption. This was accomplished by applying an ac and a dc excitation signal to the electrode. The applied ac waveform was a 100-kHz sine wave with an amplitude of 10 mV rms, while the dc voltage applied was -600 mV. The two signals were shown not to interfere with one another. Furthermore, it is shown that the sample-probe distance can be measured for approach to single cells on the order of 10-15-microm diameter and 5-microm height, with 100-nm resolution. 相似文献
4.
Jing Liu Juan C. Fraire Stefaan C. De Smedt Ranhua Xiong Kevin Braeckmans 《Small (Weinheim an der Bergstrasse, Germany)》2020,16(22)
Extrinsic probes have outstanding properties for intracellular labeling to visualize dynamic processes in and of living cells, both in vitro and in vivo. Since extrinsic probes are in many cases cell‐impermeable, different biochemical, and physical approaches have been used to break the cell membrane barrier for direct delivery into the cytoplasm. In this Review, these intracellular delivery strategies are discussed, briefly explaining the mechanisms and how they are used for live‐cell labeling applications. Methods that are discussed include three biochemical agents that are used for this purpose—purpose‐different nanocarriers, cell penetrating peptides and the pore‐foraming bacterial toxin streptolysin O. Most successful intracellular label delivery methods are, however, based on physical principles to permeabilize the membrane and include electroporation, laser‐induced photoporation, micro‐ and nanoinjection, nanoneedles or nanostraws, microfluidics, and nanomachines. The strengths and weaknesses of each strategy are discussed with a systematic comparison provided. Finally, the extrinsic probes that are reported for intracellular labeling so‐far are summarized, together with the delivery strategies that are used and their performance. This combined information should provide for a useful guide for choosing the most suitable delivery method for the desired probes. 相似文献
5.
A fritless electroosmotic pump with reduced pH dependence has been fabricated on a glass microchip and its performance characterized. The chip design consists of two 500-microm channels, one packed with anion exchange beads and the other packed with cation exchange beads, which produce convergent electroosmotic flow streams. The electroosmotically pumped solution flows away from the intersection of the two pumping channels through a field-free channel. This simple design allows for the production of a fritless electroosmotic pump and easy replacement of the ion exchange beads whose charged surfaces generate the flow. The pump was found to produce volumetric flow rates of up to 2 microL/min for an applied voltage of 3 kV at a pH of 6.8. Moreover, the electroosmotic pump can generate high flow rates over an extended pH range of at least 2-12, a significant advantage over previously fabricated electroosmotic pumps, which typically have a more limited range in which they can achieve high flow rates. 相似文献
6.
Nanochannel electroporation delivers precise amounts of biomolecules into living cells 总被引:1,自引:0,他引:1
Boukany PE Morss A Liao WC Henslee B Jung H Zhang X Yu B Wang X Wu Y Li L Gao K Hu X Zhao X Hemminger O Lu W Lafyatis GP Lee LJ 《Nature nanotechnology》2011,6(11):747-754
Many transfection techniques can deliver biomolecules into cells, but the dose cannot be controlled precisely. Delivering well-defined amounts of materials into cells is important for various biological studies and therapeutic applications. Here, we show that nanochannel electroporation can deliver precise amounts of a variety of transfection agents into living cells. The device consists of two microchannels connected by a nanochannel. The cell to be transfected is positioned in one microchannel using optical tweezers, and the transfection agent is located in the second microchannel. Delivering a voltage pulse between the microchannels produces an intense electric field over a very small area on the cell membrane, allowing a precise amount of transfection agent to be electrophoretically driven through the nanochannel, the cell membrane and into the cell cytoplasm, without affecting cell viability. Dose control is achieved by adjusting the duration and number of pulses. The nanochannel electroporation device is expected to have high-throughput delivery applications. 相似文献
7.
An on-column mechanism for electrokinetically injecting long sample plugs with simultaneous stacking of neutral analytes in capillary electrokinetic chromatography is presented. On-column stacking methods allow for the direct injection of long sample plugs into the capillary, with narrowing of the analyte peak width to allow for an increase in the detected signal. Low-pressure injections (approximately 50 mbar) are commonly used to introduce sample plugs containing neutral analytes. We demonstrate that injection can be accomplished by applying an electric field from the sample vial directly into the capillary, with neutral analytes injected by electroosmotic flow at up to 1 order of magnitude faster than the corresponding pressure injections. Since stacking occurs simultaneously with electrokinetic injection, stacking is initiated at the capillary inlet, resulting in an increased length of capillary remaining for separation. Reproducibility obtained for peak height and peak area with electroosmotic flow injection is comparable to that obtained with the pressure injection mode, while reproducibility of analysis time is markedly improved. Electrokinetic stacking of neutral analytes utilizing electroosmotic flow is demonstrated with discontinuous (high conductivity, high mobility) as well as continuous (equal conductivity, equal mobility) sample electrolytes. Injecting neutral analytes by electroosmotic flow affords a 10-fold or greater decrease in analysis times when capillaries of 50-microm i.d. or smaller are used. This stacking method should be exportable to dynamic pH junction stacking and electrokinetic chromatography with capillary arrays. Equations describing this electrokinetic injection mode are introduced and stacking of a neutral analyte on a microchip by electrokinetic injection using a simple cross-T channel configuration is demonstrated. 相似文献
8.
Characterization and manipulation of the electroosmotic flow in porous anodic alumina membranes 总被引:1,自引:0,他引:1
Porous anodic alumina membranes (PAAMs) have uniform and high-density nanopores, and the dimension and interval of the pores can be easily controlled by varying the anodization conditions. The application of PAAMs could widely impact the cost and efficiency of the liquid-based nanoscale separations. We report here the property of electroosmotic flow in PAAMs, which plays a significant role in the mass transport across these membranes that have charged pore surfaces. By controlling the solution pH and the magnitude and sign of the applied current, the mass transport through these nanoporous membranes can be spatially and temporally manipulated. The effects of electrosurface properties and electrolyte ionic strength on electroosmotic flow were studied. The anion incorporation and adsorption cause the variation of the electrosurface properties of PAAMs, which in turn influence the rate and direction of the mass transport. As compared to the membrane with fixed surface charge, this diversity makes it possible for the PAAMs to be used in various conditions. 相似文献
9.
Continuous cell introduction for the analysis of individual cells by capillary electrophoresis 总被引:3,自引:0,他引:3
Instrumentation for high-throughput analysis of single cells by capillary electrophoresis is described. A flow-based interface that uses electroosmotic flow (EOF) provides continuous injection of intact cells through an introduction capillary into a cell lysis junction and migration of the resulting cell lysate through a separation capillary for analysis. Specifically, two capillaries were coupled together with 5-mm-long Teflon tubing to create a approximately 5-microm gap, and the junction was immersed in a buffer reservoir. High voltage was applied across both capillaries so that cells were continuously pumped into the first capillary by EOF. Individual cells were lysed on-column at the junction without detergents, presumably owing to mechanical disruption caused by a dramatic change in flow properties at the gap. After each cell was lysed at the junction, the major proteins hemoglobin and carbonic anhydrase were separated by capillary electrophoresis and the resultant analyte zones were detected by laser-induced native fluorescence using 275-nm excitation. The detection limits of hemoglobin and carbonic anhydrase were 37 and 1.6 amol, respectively, which correlate well with the literature. The instrumentation was evaluated with intact red blood cells. The averaged time for complete analysis (i.e., continuous injection, lysis, separation, and detection) of one human erythrocyte was less than 4 min with this capillary-based setup. Moreover, this instrumentation simplifies the introduction of individual, intact cells without the use of a microscope. 相似文献
10.
A method for determining the accurate effective mobility value of an analyte in the presence of a charged interacting agent, such as a charged cyclodextrin, a micellar agent, a protein, or a DNA fragment that binds the traditional electroosmotic flow markers, is presented. Part of the capillary is filled with the charged interacting agent-containing background electrolyte; the other part is filled with the charged interacting agent-free background electrolyte. The analyte band is placed in the charged interacting agent-containing background electrolyte zone, while a neutral marker (electroosmotic flow marker) is placed in the adjacent charged interacting agent-free background electrolyte zone. The initial, preelectrophoresis distance between the analyte band and the neutral marker band is determined by pressure mobilizing the bands past the detector and recording the detector trace. Subsequently, by applying reverse pressure, the bands are moved back into the first portion of the capillary and a brief electrophoretic separation is carried out. Then, the bands are pressure mobilized again past the detector to obtain their final, postelectrophoresis distance. If (i) the neutral marker does not come into contact with the charged interacting agent and (ii) the analyte does not migrate out of the homogeneous portion of the charged interacting agent zone, the accurate effective electrophoretic migration distance of the analyte, corrected for bulk flow transport, can be determined. The actual electric field strengths in the different zones of the heterogeneously filled capillary can be calculated from the integral of the electrophoretic current and the conductivity of the charged interacting agent-containing background electrolyte measured in a separate experiment. Once the effective mobility of an analyte in the charged resolving agent-containing background electrolyte is determined by this method, the analyte becomes a mobility reference probe for that background electrolyte and can be used to calculate the bulk flow mobility in subsequent conventional CE separations utilizing the same charged interacting agent. The new method can also be used to probe the interactions of the charged interacting agents and the wall of the capillary. 相似文献
11.
Wan QH 《Analytical chemistry》1997,69(3):361-363
A plug electroosmotic velocity profile is generally assumed to be characteristic of capillary electrochromatography. However, this ideal plug flow may be illusive in some experiments with packed-capillary columns due to overlap of electrical double layers in flow channels. We report here a theoretical analysis of the double-layer overlap effects in packed-capillary columns, which is based on Rice and Whitehead's theory of electroosmotic flow combined with a capillary tube model for porous packing. The results show that the electroosmotic velocity under the influence of double-layer overlap depends strongly on the operating parameters, which increases with the column porosity, the particle diameter, and the electrolyte concentration. 相似文献
12.
Muro E Fragola A Pons T Lequeux N Ioannou A Skourides P Dubertret B 《Small (Weinheim an der Bergstrasse, Germany)》2012,8(7):1029-1037
The in vivo labeling of intracellular components with quantum dots (QDs) is very limited because of QD aggregation in the cell cytoplasm and/or QD confinement into lysosomal compartments. In order to improve intracellular targeting with QDs, various surface chemistries and delivery methods have been explored, but they have not yet been compared systematically with respect to the QD intracellular stability. In this work, the intracellular aggregation kinetics of QDs for three different surface chemistries based on ligand exchange or encapsulation with amphiphilic polymers are compared. For each surface chemistry, three delivery methods for bringing the nanoparticles into the cells are compared: electroporation, microinjection, and pinocytosis. It is concluded that the QD intracellular aggregation behavior is strongly dependent on the surface chemistry. QDs coated with dihydrolipoic acid-sulfobetaine (DHLA-SB) ligands diffuse freely in cells for longer periods of time than for QDs in the other chemistries tested, and they can access all cytoplasmic compartments. Even when conjugated to streptavidin, these DHLA-SB QDs remain freely diffusing inside the cytoplasm and unaggregated, and they are able to reach a biotinylated target inside HeLa cells. Such labeling was more efficient when compared to commercial streptavidin-conjugated QDs, which may be due to the smaller size of DHLA-SB QDs and/or to their superior intracellular stability. 相似文献
13.
An electroosmotic flow (EOF)-based pump, integrated with a sol-gel stationary phase located in the electric field-free region of a microchip, enabled the separation of six nitroaromatic and nitramine explosives and their degradation products via liquid chromatography (LC). The integrated pump and LC system were fabricated within a single quartz substrate. The pump region consisted of a straight channel (3.0 cm x 230 microm x 100 microm) packed with 5-microm porous silica beads. The sol-gel stationary phase was derived from a precursor mixture of methyltrimethoxy- and phenethyltrimethoxysilanes and was synthesized in the downstream, field-free region of the microchip, resulting in a stationary-phase monolith with dimensions of 2.6 cm x 230 microm x 100 microm. Fluid dynamic design considerations are discussed, especially as they relate to integrating the EOF pump with the LC system. Pump and separation performance, as characterized by flow rate measurements, injection, elution, separation, and detection, point to a viable analytical chemistry platform that encompasses all of the benefits expected of portable, laboratory-on-chip systems, including reduced sample requirements and small packaging. 相似文献
14.
A novel method for speciation analysis of mercury was developed by on-line hyphenating capillary electrophoresis (CE) with atomic fluorescence spectrometry (AFS). The four mercury species of inorganic mercury Hg(II), methymercury MeHg(I), ethylmercury EtHg(I), and phenylmercury PhHg(I) were separated as mercury-cysteine complexes by CE in a 50-cm x 100-microm-i.d. fused-silica capillary at 15 kV and using a mixture of 100 mmol L(-1) of boric acid and 12% v/v methanol (pH 9.1) as electrolyte. A novel technique, hydrostatically modified electroosmotic flow (HSMEOF) in which the electroosmotic flow (EOF) was modified by applying hydrostatical pressure opposite to the direction of EOF was used to improve resolution. A volatile species generation technique was used to convert the mercury species into their respective volatile species. A newly developed CE-AFS interface was employed to provide an electrical connection for stable electrophoretic separations and to allow on-line volatile species formation. The generated volatile species were on-line detected with AFS. The precisions (RSD, n = 5) were in the range of 1.9-2.5% for migration time, 1.8-6.3% for peak area response, and 2.3-6.1% for peak height response for the four mercury species. The detection limits ranged from 6.8 to 16.5 microg L(-1) (as Hg). The recoveries of the four mercury species in the water samples were in the range of 86.6-111%. The developed technique was successfully applied to speciation analysis of mercury in a certified reference material (DORM-2, dogfish muscle). 相似文献
15.
A quantitative investigation of the effect of process parameters such as electrolyte concentration, temperature, anodization duration and anodization potential on the pore pattern (including pore diameter and distribution) in anodic alumina was performed based on aluminum anodization experiments. Using fast Fourier transform (FFT) analysis, we developed a method to quantify the orderedness of pore distribution. We found that at a lower temperature the anodization protocol of a 1 hr first step followed by a 4 hr second step did not cause any change in pore orderedness as opposed to the anodization protocol of a 12 hr first step followed by a 1 hr second step, but at a higher temperature the former improved the pore orderedness. Increasing the electrolyte concentration, improved the pore orderedness. Varying the electrolyte concentration, temperature, and anodization duration did not have any effect on the pore diameter. Increasing the anodization potential, however, not only improved the pore orderedness but also increased the pore diameter. Linear relationships exist between the pore diameter and anodization potential and between the center to center pore spacing and applied anodization potential. 相似文献
16.
Dispersion of a nonelectrolyte solute due to the electroosmotic flow in long straight microchannels was analyzed theoretically. A version of the Aris-Taylor procedure was employed to predict the dispersion coefficient for arbitrary geometry of the microchannel cross section. The analysis was conducted using a thin double-layer approximation, which is valid when the Debye length is much smaller than the characteristic dimensions of the cross section. For thin double layers, the obtained results describe the electroosmotic dispersion for arbitrary surface potential, electrolyte type, and cross-section geometry. Dispersion for several cases of the cross-section geometries was discussed. It was shown that, for given values of the surface potential and the Debye length, both the cross-section geometry and the electrolyte content of the driven solution substantially affect the dispersion of a nonelectrolyte solute. In the relevant particular cases, the obtained results agree with predictions of the previous theories. 相似文献
17.
Choi SO Kim YC Lee JW Park JH Prausnitz MR Allen MG 《Small (Weinheim an der Bergstrasse, Germany)》2012,8(7):1081-1091
The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, intracellular delivery of molecules and transfection with plasmid DNA by electroporation is presented using a novel microneedle electrode array designed for the targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50% of cells and transfection of 12% of cells with a gene for green fluorescent protein is demonstrated at high cell viability. It is concluded that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA, and other biopharmaceuticals. 相似文献
18.
Olofsson J Levin M Strömberg A Weber SG Ryttsén F Orwar O 《Analytical chemistry》2007,79(12):4410-4418
We present a computer-controlled scanning electroporation method. Adherent cells are electroporated using an electrolyte-filled capillary in contact with an electrode. The capillary can be scanned over a cell culture and locally deliver both an electric field and an electroporation agent to the target area without affecting surrounding cells. The instantaneous size of the targeted area is determined by the dimensions of the capillary. The size and shape of the total electroporated area are defined by these dimensions in combination with the scanning pattern. For example, striped and serpentine patterns of electroporated cells in confluent cultures can be formed. As it is easy to switch between different electroporation agents, the method is suitable for design of cell cultures with complex composition. Finite element method simulations were used to study the spatial distributions of the electric field and the concentration of an electroporation agent, as well as the fluid dynamics related to scanning and flow of electroporation agent from the capillary. The method was validated for transfection by introduction of a 9-base-pair-long randomized oligonucleotide into PC12 cells and a pmaxGFP plasmid coding for green fluorescent protein into CHO and WSS cells. 相似文献
19.
Allen PB Rodriguez I Kuyper CL Lorenz RM Spicar-Mihalic P Kuo JS Chiu DT 《Analytical chemistry》2003,75(7):1578-1583
This paper describes a general strategy for the fabrication of a microthermocouple based on the spatially defined electroless deposition of metal, followed by annealing and electroplating. We present scanning electron microscopy and atomic force microscopy characterizations of the deposition and annealing process, as well as the performance of the microfabricated Ni-Ag thermocouple. The temperature-voltage curve for this Ni-Ag microthermocouple is linear over the range 0-50 degrees C with a slope of 61.9 degrees C mV(-1). The sensitivity of our temperature measurement, which is limited by the uncertainty of our calibration curve, is approximately 1 degrees C. The optimum figure of merit (Z(opt)) is 1.0 x 10(-5) for this type of Ag-Ni thermocouple. We have fabricated microthermocouples ranging in size from 50 to 300 microm. The microthermocouple was integrated into microchannels and used to measure the in-channel temperature rise caused by the following: (1) a simple acid-base reaction, HCl + NaOH --> H2O + NaCl, and (2) an enzyme-catalyzed biochemical reaction, H2O2 + catalase --> H2O + 1/2 O2. We have also profiled the temperature increase in the presence of electroosmotic flow for a 100-, 200-, and 300-microm channel. 相似文献
20.
A new method for accurately determining effective mobilities and electroosmotic flow rates for capillary electrophoresis is described. The proposed method can be performed using most commercial capillary electrophoresis instruments. Problems inherent to the conventional mobility determination method such as a variable electroosmotic flow during the run and migration through unthermostated regions of the capillary are eliminated with the use of the proposed method. In addition, very low effective mobilities and electroosmotic flow rates can be measured quickly and reproducibly. Also, cation mobilities and anion mobilities can be measured in a single run regardless of the magnitude or direction of the electroosmotic flow. 相似文献