RESUMO
A major process of iron homeostasis in whole-body iron metabolism is the release of iron from the macrophages of the reticuloendothelial system. Macrophages recognize and phagocytose senescent or damaged erythrocytes. Then, they process the heme iron, which is returned to the circulation for reutilization by red blood cell precursors during erythropoiesis. The amount of iron released, compared to the amount shunted for storage as ferritin, is greater during iron deficiency. A currently accepted model of iron release assumes a passive-gradient with free diffusion of intracellular labile iron (Fe2+) through ferroportin (FPN), the transporter on the plasma membrane. Outside the cell, a multi-copper ferroxidase, ceruloplasmin (Cp), oxidizes ferrous to ferric ion. Apo-transferrin (Tf), the primary carrier of soluble iron in the plasma, binds ferric ion to form mono-ferric and di-ferric transferrin. According to the passive-gradient model, the removal of ferrous ion from the site of release sustains the gradient that maintains the iron release. Subcellular localization of FPN, however, indicates that the role of FPN may be more complex. By experiments and mathematical modeling, we have investigated the detailed mechanism of iron release from macrophages focusing on the roles of the Cp, FPN and apo-Tf. The passive-gradient model is quantitatively analyzed using a mathematical model for the first time. A comparison of experimental data with model simulations shows that the passive-gradient model cannot explain macrophage iron release. However, a facilitated-transport model associated with FPN can explain the iron release mechanism. According to the facilitated-transport model, intracellular FPN carries labile iron to the macrophage membrane. Extracellular Cp accelerates the oxidation of ferrous ion bound to FPN. Apo-Tf in the extracellular environment binds to the oxidized ferrous ion, completing the release process. Facilitated-transport model can correctly predict cellular iron efflux and is essential for physiologically relevant whole-body model of iron metabolism.
Assuntos
Simulação por Computador , Homeostase/fisiologia , Ferro/metabolismo , Macrófagos/metabolismo , Modelos Biológicos , Biologia Computacional , Humanos , Espaço Intracelular/metabolismo , Macrófagos/citologiaRESUMO
We report here on differential linear scan voltammetry, DLSV, that combines the working principles of linear scan voltammetry, LSV, and the numerous existing pulsed voltammetry techniques. DLSV preserves the information from continuous interrogation in voltage and high accuracy that LSV provides about electrochemical processes, and the much better sensitivity of differential pulsed techniques. DLSV also minimizes the background current compared to both LSV and pulsed voltammetry. An early version of DLSV, derivative stationary electrode polarography, DSEP, had been proposed in the 1960s but soon abandoned in favor of the emerging differential pulsed techniques. Relative to DSEP, DLSV takes advantage of the flexibility of discrete smoothing differentiation that was not available to early investigators. Also, DSEP had been explored in pure solutions and with reversible electrochemical reactions. DLSV is tested in this work in more challenging experimental contexts: the measurement of oxygen with a carbon fiber microelectrode in buffer, and with a gold microdisc electrode exposed to a live biological preparation. This work compares the analytical performance of DLSV and square wave voltammetry, the most popular pulsed voltammetry technique.
Assuntos
Técnicas Eletroquímicas/métodos , Oxigênio/análise , Carbono , Fibra de Carbono , Técnicas Eletroquímicas/instrumentação , Desenho de Equipamento , Ouro , Microeletrodos , Sensibilidade e EspecificidadeRESUMO
Scanning electrochemical microscopy visualizes concentration profiles. To determine the location of the probe relative to topographical features of the substrate, knowledge of the probe-to-sample distance at each probe position is required. The use of electrochemical impedance spectroscopy for obtaining information on the substrate-to-probe distance and on the concentration of interest using the electrochemical probe alone is suggested. By tuning the frequencies of interrogation, the probe-to-substrate distance can be derived followed by interrogation of processes that carry information on concentration at lower frequencies. These processes may include charge-transfer relaxation, diffusional relaxation at the electrode, and open-circuit potential at zero frequency. A potentiometric chloride sensing microprobe is used herein to reconstruct both topography and the concentration field at a microscopic diffusional source of chloride.
Assuntos
Técnicas Biossensoriais/métodos , Microscopia de Varredura por Sonda/métodos , Técnicas Biossensoriais/instrumentação , Cloretos/análise , Impedância Elétrica , Eletroquímica , Eletrodos Seletivos de Íons , Microeletrodos , Microscopia de Varredura por Sonda/instrumentação , Modelos Químicos , Potenciometria , Soluções , Propriedades de SuperfícieRESUMO
A significant amount of Ca²+ is contained in secretory mucin granules. Exchange of Ca²+ for monovalent cations drives the process of mucin decondensation and hydration after fusion of granules with the plasma membrane. Here we report direct observation of calcium secretion with a Ca²+ ion-selective electrode (ISE) in response to apical stimulation with ATP from HT29-Cl.16E cells, a subclone of the human colonic cancer cell line HT29. No increase in Ca²+ level was seen for the sister cell line Cl.19A, which lacks mucin granules, or for Cl.16E cells after inhibition of granule fusion with wortmannin. Further, the measured concentration was used to estimate the time-resolved rate of release of Ca²+ from the cell monolayer, by use of a deconvolution-based method developed previously (Nair and Gratzl in Anal Chem 77:2875-2881, 2005). The results argue that Ca²+ release by Cl.16E cells is associated specifically with mucin secretion, i.e., that the measured Ca²+ increase in the apical solution is derived from granules after fusion and mucin exocytosis. The Ca²+ ISE in conjunction with deconvolution provides a minimally disturbing method for assessment of Ca²+ secretion rates. The release rates provide estimates of exocytosis rates and, when combined with earlier capacitance measurements, estimates of post-stimulation endocytosis rates also.
Assuntos
Cálcio/metabolismo , Células Epiteliais/metabolismo , Mucinas/metabolismo , Técnicas de Patch-Clamp/métodos , Trifosfato de Adenosina/metabolismo , Cátions Bivalentes/metabolismo , Endocitose , Exocitose , Células HT29 , Humanos , Eletrodos Seletivos de ÍonsRESUMO
Both potentiometric and voltammetric measurements report on the concentration of the analyte in the closest layer of the sample solution that is unperturbed by the measurement. Besides this local concentration, the solution composition within the thin boundary layer adjacent to the electrode|solution interface is influenced also by local mass transport and other electrochemical processes necessary for signal transduction. This local perturbation of concentrations is typically corrected for by calibration so that the ultimate output of the measurement is the local concentration, at a distance of a few micrometers to about 100 microm from the electrode surface. In contrast to many optical techniques, the electrochemical approach is therefore only capable of measuring local concentrations but cannot be used to assess three-dimensional (3D)-averaged bulk concentrations of inhomogeneous samples. This may pose a problem in very small samples where homogenization by stirring is difficult. We present here the concept of spatially averaging electrodes that can, due to their special design, report 3D spatially averaged bulk concentration of inhomogeneous samples that have some type of symmetry. Within a given type of symmetry an infinite variation of concentration distribution may exist. We illustrate the concept of spatially averaging electrodes with results obtained in microliter-sized hemispherical samples with a source in the center of the drop.
Assuntos
Técnicas Eletroquímicas/métodos , Algoritmos , Técnicas Eletroquímicas/instrumentação , Eletrodos , Ferricianetos/química , Platina/químicaRESUMO
Enzyme activities in body fluids are often used as diagnostic markers for physiological conditions and diseases. Common enzyme assays use optical methods that often require the use of pseudosubstrates and associated dyes. Here we introduce a reagent-free micro pH-stat that can determine absolute enzyme activity without the need for exogenic reagents. This approach employs electrolysis for precise dosing of the requisite acid or base titrant to stat the pH of the sample. The micro pH-stat is based on the rotating sample system (RSS), a convection platform for microliter drops. Activities of serum cholinesterase in fetal bovine serum and human serum were analyzed with this approach. The performance of this system is comparable to that of standard techniques (r(2)=0.99), yet it offers a broader range of detection. The reagent-free micro pH-stat has potential to be developed as a miniaturized device for point-of-care testing.
Assuntos
Colinesterases/sangue , Técnicas e Procedimentos Diagnósticos , Técnicas e Procedimentos Diagnósticos/instrumentação , Eletroquímica , Humanos , Concentração de Íons de Hidrogênio , Indicadores e Reagentes/química , MicroquímicaRESUMO
The introduction of specific molecules into live cells is a widely used approach to probe cellular mechanisms. Recently, we have reported on the sustained dosing of molecules into single cells via a microscopic diffusion port. Here we describe temporal ratiometry, a method to reconstruct intracellular concentration distribution of the delivered molecules as it varies in time during dosing. To characterize this method, we analyzed fluorescence intensity maps obtained during delivery of Lucifer Yellow CH, LY, a polar fluorophore into A7r5 vascular smooth muscle cells, normal rat kidney epithelial cells (NRKE), and MCF-7 human breast cancer cells. Temporal ratiometry indicates a linear increase in concentration of LY in these cells with a nearly uniform distribution during 20 min of delivery. The method cancels the effects of varying cell height and variable accessible volume on the measured intensities at different locations within the cell. Temporal ratiometry will be useful to estimate dynamic changes in intracellular concentration distributions and thus, facilitate the understanding of transport, binding, sequestration, and efflux of molecules introduced into cells.
Assuntos
Citoplasma/metabolismo , Células Epiteliais/citologia , Isoquinolinas/análise , Microscopia de Fluorescência/métodos , Miócitos de Músculo Liso/citologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Difusão , Humanos , Processamento de Imagem Assistida por Computador , Rim/citologia , RatosRESUMO
Electrochemical sensing techniques are increasingly used to study biological processes by monitoring concentration changes of the molecule of interest close to cells. The measured concentration is the result of cellular transport across the cell membrane and diffusion of the released molecules from the cells to the sensing electrode. The objective of such experiments is to understand the cellular processes underlying the observed changes in concentration. Thus, the influence of mass transport on the measured concentration trace has to be removed. This is done by deconvolution of the impulse response function of diffusion from the concentration data. We have recently observed that measuring concentration at a sampling rate that satisfies the Nyquist criterion for the observed concentration dynamics may not be sufficient to correctly reconstruct cellular flux. This is because the impulse response function of diffusion also has to be represented with sufficient temporal resolution. We discuss this problem here using the example of monitoring drug efflux from a monolayer of cancer cells with microvoltammetry, and chloride secretion from an epithelial cell monolayer monitored with an ion-selective electrode.
Assuntos
Células/metabolismo , Eletroquímica/métodos , Animais , Antineoplásicos/metabolismo , Células CHO , Cricetinae , Cricetulus , Difusão , Doxorrubicina/metabolismo , Eletrodos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
pH-stating is a common technique for monitoring kinetics of various biochemical reactions that involve the generation of hydrogen or hydroxyl ions. In this work, we describe a reagentless electrochemical micro-pH-stat where the titrant of acid or base is produced by water electrolysis on the rotating sample system (RSS) platform. RSS originated from the authors' laboratory as a convective platform to support different analytical techniques in microliter-sized samples. As water electrolysis induces no volume change and the current that generates the reagent can be precisely measured even at low levels, very small samples in the microliter range become accessible for pH-stating: a reduction of more than an order of magnitude in specimen size relative to the most sensitive conventional methods. Nearly 100% current efficiency has been achieved with this system using a 250 microm Pt minidisc working electrode for electrolysis. The developed micro-pH-stat has been validated by the determination of the activity of erythrocyte acetylcholinesterase as a function of substrate concentration and pH. The optimal pH and activity profile obtained are in good agreement with those determined with standard techniques. The micro-pH-stat has the potential for applications for enzyme assays, reagentless pH control, acidity/alkalinity, and buffer capacity measurements in very small samples of biomedical and environmental origin.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Animais , Soluções Tampão , Bovinos , Condutividade Elétrica , Concentração de Íons de Hidrogênio , Indicadores e Reagentes/química , Nitratos/química , Compostos de Potássio/química , Reprodutibilidade dos Testes , Rotação , Especificidade por SubstratoRESUMO
We report here on a method for preparation of precise laboratory solutions using controlled diffusion for dosing. The desired chemical is delivered into the target solution from a stock solution through a mass-transport-limiting delivery port that blocks convection but allows reproducible diffusive transport. Solution making with this approach involves a single step irrespective of how low the desired concentration is. Diffusional delivery of chemicals involves no appreciable movement of water and, thus, no addition of volume. The approach is therefore particularly suitable for standard addition. Precise solutions of usual laboratory volumes can be made within a short time period with proper design of the delivery port or ports. Comparison with the performance of conventional methods of routine solution preparation shows that better precision can be achieved with less labor using this approach.
Assuntos
Soluções/análise , Soluções/química , Calibragem , DifusãoRESUMO
A novel experimental approach using the rotating sample system (RSS) is proposed here for the determination of the critical micelle concentration (CMC) of surfactants. The RSS has been conceived in our laboratory as a convection platform for physicochemical studies and analyses in microliter-sized sample drops. The scheme allows for vigorous rotation of the drop despite its small size through efficient air-liquid mechanical coupling. Thus, changes in surface properties of aqueous samples result in corresponding modulation of the hydrodynamic performance of the RSS, which can be utilized to investigate interfacial phenomena. In this work, we demonstrate that the RSS can be used to study the effects of surfactants on the surface and in the bulk of very small samples with hydrodynamic electrochemistry. Potassium ferrocyanide is employed here with cyclic voltammetry to probe the air-water interface of solutions containing Triton X-100. The CMC of this surfactant determined using this approach is 140 ppm, which agrees well with reported values obtained with conventional methods in much larger samples. The results also demonstrate that besides the CMC, variations in bulk rheological properties can also be investigated in very small specimens using the RSS with a simple method.
Assuntos
Micelas , Reologia/métodos , Tensoativos/química , Eletroquímica/métodos , Octoxinol , RotaçãoRESUMO
A micro-miniature array of sensing capsules for optical monitoring of pH, potassium and glucose is described. Optode technology translates the respective ionic levels into variable colors of ionophore/dye/polymeric liquid micro-beads stuffed into individual capsules. Glucose is monitored indirectly, by coupling through glucose oxidase (GOX) immobilized in cellulose acetate phthalate (CAP) based microscopic beads that are stuffed into another microcapsule together with pH sensitive optical microscopic beads. The electrolyte and glucose sensing capsules are embedded in a transparent cellulose acetate bar 300-500 microm wide and 2-2.5 mm long called the sliver sensor that includes also a white capsule made of micro-beads without dye for optical reference. By adding further capsules custom combinations of analytes can be monitored in biomedical and non-biological contexts. In this work, as an example, design, fabrication and testing of a sliver sensor that could be developed for in vivo use are described.
Assuntos
Técnicas Biossensoriais/métodos , Glicemia/análise , Eletrólitos/análise , Análise em Microsséries/métodos , Técnicas Biossensoriais/instrumentação , Boratos/química , Celulose/análogos & derivados , Celulose/química , Éteres de Coroa/química , Ácidos Decanoicos/química , Glucose/química , Glucose Oxidase/química , Concentração de Íons de Hidrogênio , Ionóforos/química , Análise em Microsséries/instrumentação , Cloreto de Polivinila/química , Potássio/análise , Prata/químicaRESUMO
In Part 1 of this series (Anal. Sci., 2006, 22, 383), design, fabrication, and optical data acquisition of an array of tiny color changing capsules embedded in a cellulose acetate bar, called the "sliver sensor", have been described. Capsule colors are read by a CCD camera and translated into blue, red and green Kubelka-Munk variables for quantitative analysis. The respective concentrations are determined using prior calibration. The approach may be adapted to different non-biological analytical problems, as well as in vitro and in vivo applications. To demonstrate this adaptability to potential in vivo use as an example, sensitivity for each target ion was tuned to cover the respective interstitial levels by varying the relative amount of ionophore used in the corresponding microscopic beads. After optimizing the ratio of glucose oxidase (GOX)-containing beads relative to the coupled pH sensing beads and their composition, reversible color response to glucose was obtained in the entire clinically relevant glucose concentration range (10 to 600 mg/dL, 0.55 to 33 mM). Decoupling of pH and glucose sensing from possible variations in interstitial sodium level and buffer capacity is currently being optimized for future in vivo use. In vitro and non-biological applications are also being explored.
Assuntos
Técnicas Biossensoriais/métodos , Celulose/análogos & derivados , Glucose/química , Potássio/química , Celulose/química , Celulose/metabolismo , Glucose Oxidase/química , Concentração de Íons de Hidrogênio , Íons/química , Óptica e Fotônica , Sensibilidade e EspecificidadeRESUMO
The Rotating Sample System (RSS) has been conceived in the authors' laboratory as a convection platform for microliter-sized solution volumes. Convection is achieved by rotating a small drop of sample on a stationary substrate by humidified gas jets directed tangentially at the drop base with the working electrode and a liquid junction embedded in it. Simplicity and portability of the device, and substrates complete with microfabricated electrode and junction made potentially disposable, are further competitive advantages with respect to competing, conventional analytical systems. In this work the RSS' performance with variation of system parameters such as the position and size of gas jets used for sample rotation, and position of the working electrode in the substrate are studied. Trace levels of Pb could be detected with this system and is reported here.
Assuntos
Chumbo/análise , Técnicas Analíticas Microfluídicas/instrumentação , Água/química , Eletroquímica , Técnicas Analíticas Microfluídicas/métodos , Rotação , Oligoelementos/sangueRESUMO
We have developed a novel approach to allow for continuous imaging of concentration fields that evolve at surfaces due to release, uptake, and mass transport of molecules, without significant interference of the concentration fields by the chemical imaging itself. The technique utilizes optical "reporter" microbeads immobilized in a thin layer of transparent and inert hydrogel on top of the surface. The hydrogel has minimal density and therefore diffusion in and across it is like in water. Imaging the immobilized microbeads over time provides quantitative concentration measurements at each location where an optical reporter resides. Using image analysis in post-processing these spatially discrete measurements can be transformed into contiguous maps of the dynamic concentration field across the entire surface. If the microbeads are small enough relative to the dimensions of the region of interest and sparsely applied then chemical imaging will not noticeably affect the evolution of concentration fields. In this work colorimetric optode microbeads a few micrometers in diameter were used to image surface concentration distributions on the millimeter scale.
Assuntos
Ágar/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Microscopia/instrumentação , Cor , Difusão , Desenho de Equipamento , Concentração de Íons de Hidrogênio , Membranas Artificiais , Microesferas , Porosidade , Propriedades de SuperfícieRESUMO
Nanomaterial-based carrier systems hold great promise to deliver therapies with increased efficacy and reduced side effects. While the state-of-the-art carrier system is a sphere, recent data indicate that elongated rods and filaments have advantageous flow and margination properties, resulting in enhanced vascular targeting and tumor homing. Here, we report on the distinct diffusion rates of two bio-inspired carrier systems: 30 nm-sized spherical cowpea mosaic virus (CPMV) and 300×18 nm-sized tobacco mosaic virus (TMV) with a tubular structure, using a spheroid model of the tumor microenvironment and fluorescent imaging.
RESUMO
Direct delivery of molecules into the cytosol of live cells is required in many areas of biology and clinical research. Molecules of interest include indicator dyes, biomolecules, and pharmacological agents. In this work we describe continuous delivery of molecules into single cells using a diffusional microburet, DMB. The DMB is a pulled glass micropipette with a fine tip that contains a microscopic plug made of a hydrogel such as agar or polyacrylamide. This plug prevents flow but allows diffusive delivery of the molecule of interest from the DMB body into the cytosol, driven by its concentration gradient. This leads to a scheme of sustained intracellular dosing that is highly reproducible and quantifiable yet does not require the addition of solution volume to the cell. Potential loss of biomolecules from the cytosol through the plug of the DMB can be greatly reduced by proper choice of the pore size and tortuosity of the hydrogel in the DMB tip. The intracellular concentration of fluorescent molecules during delivery can be obtained calibration free. In this work we demonstrate dosing of Lucifer Yellow CH, LY, a charged fluorescent dye, into individual a7r5 vascular smooth muscle cells with a DMB. New types of quantitative analytical experiments on single live cells that the DMB technology enables are titration of intracellular ions and ligands, binding sites, and efflux pathways such as those that are involved in drug resistance.
Assuntos
Permeabilidade da Membrana Celular , Corantes Fluorescentes/farmacocinética , Isoquinolinas/farmacocinética , Miócitos de Músculo Liso/metabolismo , Animais , Aorta/citologia , Linhagem Celular , Difusão , RatosRESUMO
Short-circuit current (Isc) measurement is used to quantify transepithelial ion flux. This technique provides a direct measure of net charge transport across a cell monolayer. Isc however, lacks chemical selectivity. Chemically resolved ion fluxes may be much greater than Isc, and differ in different biological processes. This work describes a novel experimental approach and deconvolution method to obtain temporally resolved ion fluxes at epithelial cell monolayers. HT29-Cl.16E cells, a sub clone of the human colonic cancer cell line HT29 was used as a model cell line to validate this approach in the context of epithelial transport studies. This cell line is known to secrete chloride in response to purinergic stimulation. Changes in chloride concentration after stimulation with 1 mM ATP plus 50 nM phorbol-myristate acetate (PMA) are recorded with a chloride ion-selective electrode (ISE) at a short distance (approximately 50 microm) from the monolayer. The recorded concentrations are transformed to corresponding chloride flux across the monolayer using a deconvolution algorithm for extracellular mass transport based on minimization of the shape error function (Nair and Gratzl in Anal Chem 77:2875-2888, 2005). Simultaneous voltage clamp yields the associated net electrical charge flux (Isc). The dynamics of Cl(-) flux did correlate with that of the electrical flux, but was found to be greater in amplitude. This suggests that Cl(-) may not be the only ion secreted. The method of simultaneously assessing ionic and electrical fluxes with a temporal resolution of seconds provides unique information about the dynamics of solute fluxes across the apical membrane.
Assuntos
Cloretos/química , Células Epiteliais/metabolismo , Mucinas/química , Transporte Biológico , Transporte Biológico Ativo , Linhagem Celular Tumoral , Canais de Cloreto , Eletrodos , Eletrofisiologia/métodos , Epitélio/patologia , Desenho de Equipamento , Humanos , Transporte de Íons , Íons , Fatores de TempoRESUMO
The need for precise delivery of minute quantities of substances for solution preparation and other applications is well-known in research, clinical, industrial, and environmental settings. Currently available techniques for solution preparation in the laboratory include traditional transfer pipettes, micropipettes based on air displacement, and motorized devices using some form of a piston system. These techniques control the amount delivered by controlling the delivered volume. In this work we test the practicality of the concept of using a constant rate-limiting hydrodynamic resistance to achieve controlled reagent flow for solution preparation. The delivered amount is determined in this approach by time, pressure, flow resistance, or a combination of these. Good results are achieved comparable to conventional techniques without the use of fine mechanical instrumentation. This approach holds promise as an alternative to current methods of solution preparation and reagent delivery for routine laboratory use.
Assuntos
Água/química , Calibragem , SoluçõesRESUMO
In many fields of science and engineering including several areas in analytical chemistry, deconvolution needs to be performed on measured data to extract meaningful information. This situation arises when a variable of interest has to be indirectly estimated from a measurable quantity that depends on this variable in some known manner. This dependence, called the "forward problem", has to be computationally undone to obtain the information sought from the experimental results. Solving this "inverse problem" requires deconvolution whenever the forward problem involves convolution. Despite its ubiquitous importance, however, performance of the methodologies used for deconvolution remains often unsatisfactory. An example is in bioanalytical applications where microsensing at live preparations is performed to obtain information on biological transport. It is in this context that a novel approach to solve inverse problems, shape error optimization, is proposed and tested in this work. The experimental paradigm addressed is in the area of multidrug resistance (MDR) in cancer that gives rise to passive and active drug efflux from cells. Doxorubicin (DOX) concentration is monitored with a carbon fiber microelectrode in vitro at close proximity to a monolayer of cells expressing MDR. The measured local concentration is the result of convolution of cellular efflux with the impulse response of diffusion in the extracellular medium. Hence, estimating DOX efflux, which is the biologically meaningful information, leads to a deconvolution problem. Performance of deconvolution via shape error optimization is compared with that of two conventional techniques: discrete Fourier transform and square error optimization. The results obtained are also applicable to other areas of science and engineering where deconvolution is commonly used for processing experimental data.