RESUMO
Enumeration of circulating tumor cells (CTCs) has proved valuable for early detection and prognosis in cancer treatment. This paper describes an automated high-throughput counting method for CTCs based on microfluidics and line-confocal microscopy. Peripheral blood was directly labeled with multiple antibodies, each conjugated with a different fluorophore, pneumatically pumped through a microfluidic channel, and interrogated by a line-confocal microscope. On the basis of the fluorescence signals and labeling schemes, the count of CTCs was automatically reported. Due to the high flow rate, 1 mL of whole blood can be analyzed in less than 30 min. We applied this method in analyzing CTCs from 90 stage IV breast cancer patient samples and performed a side-by-side comparison with the results of the CellSearch assay, which is the only method approved by the U.S. Food and Drug Administration at present for enumeration of CTCs. This method has a recovery rate for cultured breast cancer cells of 94% (n = 9), with an average of 1.2 counts/mL of background level of detected CTCs from healthy donors. It detected CTCs from breast cancer patients ranging from 15 to 3375 counts/7.5 mL. Using this method, we also demonstrate the ability to enumerate CTCs from breast cancer patients that were positive for Her2 or CD44(+)/CD24(-), which is a putative cancer stem cell marker. This automated method can enumerate CTCs from peripheral blood with high throughput and sensitivity. It could potentially benefit the clinical diagnosis and prognosis of cancer.
Assuntos
Contagem de Células/métodos , Células Neoplásicas Circulantes , Anticorpos/química , Anticorpos/imunologia , Antígenos de Neoplasias/imunologia , Automação , Moléculas de Adesão Celular/imunologia , Contagem de Células/instrumentação , Molécula de Adesão da Célula Epitelial , Corantes Fluorescentes/química , Humanos , Células MCF-7 , Técnicas Analíticas Microfluídicas , Microscopia ConfocalRESUMO
Protein sorting represents a potential point of regulation in neurotransmission because it dictates the protein composition of synaptic vesicles, the organelle that mediates transmitter release. Although the average number of most vesicle proteins has been estimated using bulk biochemical approaches (Takamori et al., 2006), no information exists on the intervesicle variability of protein number, and thus on the precision with which proteins are sorted to vesicles. To address this, we adapted a single molecule quantification approach (Mutch et al., 2007) and used it to quantify both the average number and variance of seven integral membrane proteins in brain synaptic vesicles. We report that four vesicle proteins, SV2, the proton ATPase, Vglut1, and synaptotagmin 1, showed little intervesicle variation in number, indicating they are sorted to vesicles with high precision. In contrast, the apparent number of VAMP2/synaptobrevin 2, synaptophysin, and synaptogyrin demonstrated significant intervesicle variability. These findings place constraints on models of protein function at the synapse and raise the possibility that changes in vesicle protein expression affect vesicle composition and functioning.
Assuntos
Proteínas de Membrana/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Encéfalo/metabolismo , Técnicas In Vitro , Transporte Proteico , Ratos , Ratos Sprague-DawleyRESUMO
Biological cells are highly sensitive to variation in local pressure because cellular membranes are not rigid. Unlike microbeads, cells deform under pressure or even lyse. In isolating or enriching cells by mechanical filtration, pressure-induced lysis is exacerbated when high local fluidic velocity is present or when a filter reaches its intended capacity. Microfabrication offers new possibilities to design fluidic environments to reduce cellular stress during the filtration process. We describe the underlying biophysics of cellular stress and general solutions to scale up filtration processes for biological cells.
Assuntos
Fenômenos Fisiológicos Celulares , Microfluídica , Animais , Linhagem Celular Tumoral , Humanos , Microfluídica/instrumentação , Microfluídica/métodos , ReologiaRESUMO
Microfluidic systems can conveniently be used for rapid analysis of biological samples. Here we describe a single re-circulating flow, or microvortex, that can generate a maximum fluid rotational velocity of up to 12 m s(-1) and a corresponding radial acceleration in excess of 10(6)g. Such microvortices may be exploited in centrifugal microdevices to investigate the effects of high radial acceleration on biological and chemical processes.
RESUMO
We recently reported a new UV-curable polyurethane-methacrylate (PUMA) resin that has excellent qualities as a disposable microfluidic substrate for clinical diagnostic applications. This article discusses strategies to improve the production yield of PUMA chips that contain dense and high-aspect-ratio features, which presents unique challenges in demolding and bonding steps. These fabrication improvements were deployed to produce a microfiltration device that contained closely spaced and high-aspect-ratio columns, suitable for retaining and concentrating cells or beads from a highly diluted suspension.
Assuntos
Metacrilatos/química , Técnicas Analíticas Microfluídicas/instrumentação , Filtros Microporos , Poliuretanos/química , Neoplasias da Mama/diagnóstico , Fracionamento Celular/instrumentação , Linhagem Celular Tumoral , Desenho de Equipamento , Feminino , HumanosRESUMO
As microfluidic systems transition from research tools to disposable clinical-diagnostic devices, new substrate materials are needed to meet both the regulatory requirement as well as the economics of disposable devices. This paper introduces a UV-curable polyurethane-methacrylate (PUMA) substrate that has been qualified for medical use and meets all of the challenges of manufacturing microfluidic devices. PUMA is optically transparent, biocompatible, and exhibits high electroosmotic mobility without surface modification. We report two production processes that are compatible with the existing methods of rapid prototyping and present characterizations of the resultant PUMA microfluidic devices.
Assuntos
Comércio/instrumentação , Desenho de Equipamento/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica/instrumentação , Farmacopeias como Assunto/normas , Materiais Biocompatíveis/química , Metacrilatos/química , Microscopia Eletrônica de Varredura , Poliuretanos/química , Espectrofotometria Ultravioleta , Especificidade por Substrato , Propriedades de Superfície , Estados UnidosRESUMO
Preparation of calibration standards for cell enumeration is critical in characterizing the performance of any method or apparatus intended for recovering rare cells. Diluting a cell suspension serially is prone to statistical sampling errors as the cell suspension becomes more dilute, whereas transferring and injecting cells individually into a diluent with a micromanipulator is time-consuming. We developed a simple and robust method using a surface-modified glass capillary to siphon and eject cells. One-dimensional confinement of cells offered by the capillary made cell enumeration by visual counting simple and rapid, and cell ejection from the capillary was near 100% when the appropriate surface coating and cell solution was used. The residence time of cells in the capillary, however, could affect the percentage of cells that was ejected from the capillary. To characterize the performance of this method, we enumerated the ejected cell using both visual counting under a microscope and automated detection using a chip-based flow cytometer.
Assuntos
Contagem de Células/normas , Citometria de Fluxo/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Vidro/química , Humanos , Padrões de Referência , Silanos/química , Coloração e Rotulagem , Propriedades de SuperfícieRESUMO
This paper describes the shrinkage and re-expansion of individual femtoliter-volume aqueous droplets that were suspended in an organic medium and held in an optical vortex trap. To elucidate the mechanism behind this phenomenon, we constructed a heat- and mass-transfer model and carried out experimental verifications of our model. From these studies, we conclude that an evaporation mechanism sufficiently describes the shrinkage of aqueous droplets held in a vortex trap, whereas a mechanism based on the supersaturation of the organic phase by water that surrounds the droplet adequately explains the re-expansion of the shrunk droplet. The proposed mechanisms correlated well with experimental observations using different organic media, when H2O was replaced with D2O and when an optical tweezer was used to induce droplet shrinkage rather than an optical vortex trap. For H2O droplets, the temperature rise within the droplet during shrinkage was on the order of 1 K or less, owing to the rapid thermal conduction of heat away from the droplet at the microscale and the sharp increase in solubility for water by the organic phase with slight elevations in temperature. Because most chemical species confined to droplets can be made impenetrable to the aqueous/organic interface, a change in the volume of aqueous droplets translates into a change in concentration of the dissolved species within the droplets. Therefore, this phenomenon should find use in the study of fundamental chemical processes that are sensitive to concentration, such as macromolecular crowding and protein nucleation and crystallization.
Assuntos
Modelos Químicos , Acetofenonas/química , Água/químicaAssuntos
Células Sanguíneas/citologia , Células Neoplásicas Circulantes , Anticorpos/imunologia , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Molécula de Adesão da Célula Epitelial , Citometria de Fluxo , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Humanos , Microscopia Confocal , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismoRESUMO
This paper presents a systematic study that illustrates the importance of the topologies of microchannels on electrokinetically based separation. Using theoretical and numerical analyses, we designed and showed that topologies that significantly increased the surface-to-volume ratio of the channel can provide dramatic improvement in the ability of the channel both to dissipate the heat generated by Joule heating and to reduce the axial dispersion associated with the siphoning effect. The incremental benefit and tradeoff of geometric complexity was also evaluated. The improvement offered by topographically patterned channels, such as finned structures, is especially pertinent in the development of preparative or semi-preparative scale electrokinetically driven separations, such as capillary electrophoresis and capillary electrochromatography, in which large cross sections of channels are required to achieve the needed volumetric throughput.
Assuntos
Eletroforese/métodos , Microfluídica , Eletroforese/instrumentaçãoRESUMO
As more microfluidic applications emerge for clinical diagnostics, the choice of substrate and production method must be considered for eventual regulatory approval. In this review, we survey recent developments in disposable microfluidic substrates and their fabrication methods. We note regulatory approval for disposable microfluidic substrates will be more forthcoming if the substrates are developed with the United States Pharmacopeia's biocompatibility compliance guidelines in mind. We also review the recent trend in microfluidic devices constructed from a hybrid of substrates that takes advantage of each material's attributes.
Assuntos
Técnicas de Laboratório Clínico/instrumentação , Equipamentos Descartáveis , Técnicas Analíticas Microfluídicas/instrumentação , Animais , Equipamentos Descartáveis/estatística & dados numéricos , Humanos , Polímeros/químicaRESUMO
Microfluidic platforms offer exquisite capabilities in controlling mass transport for biological studies. In this review, we focus on recent developments in manipulating chemical concentrations at the microscale. Some techniques prevent or accelerate mixing, whereas others shape the concentration gradients of chemical and biological molecules. We also highlight several in vitro biological studies in the areas of organ engineering, cancer, and blood coagulation that have benefited from accurate control of mass transfer.
Assuntos
Microfluídica/instrumentação , Coagulação Sanguínea , Difusão , Neoplasias , Engenharia TecidualRESUMO
Maturation of Plasmodium falciparum decreases the deformability of infected red blood cells (RBCs), increasing their clearance as they attempt to pass through endothelial slits of the splenic sinus. Previous studies of Plasmodium vivax-infected RBCs led to opposite conclusions with respect to cellular deformability. To resolve this controversy, P. vivax-infected RBCs were passed through a 2-microm microfluidic channel. In contrast to P. falciparum-infected RBCs, mature P. vivax-infected RBCs readily became deformed through 2-microm constrictions. After this extreme deformation, 67% of P. vivax-infected RBCs recovered a normal appearance; however, 15% of uninfected RBCs were destroyed. Results suggest mechanisms for both avoidance of splenic clearance and anemia in vivax malaria.
Assuntos
Deformação Eritrocítica , Eritrócitos/citologia , Eritrócitos/parasitologia , Microfluídica , Plasmodium vivax/fisiologia , Animais , Humanos , Malária Falciparum/sangue , Malária Vivax/sangue , Plasmodium falciparum/fisiologiaRESUMO
In fluorescence microscopy, images often contain puncta in which the fluorescent molecules are spatially clustered. This article describes a method that uses single-molecule intensity distributions to deconvolve the number of fluorophores present in fluorescent puncta as a way to "count" protein number. This method requires a determination of the correct statistical relationship between the single-molecule and single-puncta intensity distributions. Once the correct relationship has been determined, basis histograms can be generated from the single-molecule intensity distribution to fit the puncta distribution. Simulated data were used to demonstrate procedures to determine this relationship, and to test the methodology. This method has the advantages of single-molecule measurements, providing both the mean and variation in molecules per puncta. This methodology has been tested with the avidin-biocytin binding system for which the best-fit distribution of biocytins in the sample puncta was in good agreement with a bulk determination of the avidin-biocytin binding ratio.
Assuntos
Avidina/metabolismo , Lisina/análogos & derivados , Modelos Biológicos , Vesículas Sinápticas/metabolismo , Algoritmos , Animais , Encéfalo , Células Cultivadas , Simulação por Computador , Interpretação de Imagem Assistida por Computador/métodos , Lisina/metabolismo , Modelos Estatísticos , Ratos , Distribuições Estatísticas , Vesículas Sinápticas/ultraestruturaRESUMO
The ability to generate individual picoliter- and femtoliter-volume aqueous droplets on demand is useful for encapsulating and chemically manipulating discrete chemical and biological samples. This paper characterizes the effects of orifice dimensions and material choices on generating such droplets in an immiscible oil phase by using single high-voltage pulses with various amplitudes and durations. We have examined microfluidic orifices as small as 1.7 microm in equivalent radii and found that the electrohydrodynamic jet lengths and the subsequent formation of droplets are affected by the axial aspect ratios of the orifices (length of an orifice divided by its equivalent radius). As higher voltages were used to compensate for the increased capillary pressure and hydrodynamic resistance in ultrasmall orifices, we observed secondary jet protrusions and droplet formations that were not of classical electrohydrodynamic origin. The droplets generated from secondary jets traveled at relatively lower velocities as compared to those of electrohydrodynamic origin, and these slow individual droplets are potentially more useful for applications in microscale chemical reactions.
Assuntos
Técnicas Analíticas Microfluídicas , Água , EletroquímicaRESUMO
This article describes the first single-vesicle study of proton permeability across the lipid membrane of small (approximately 100 nm) uni- and multilamellar vesicles, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To follow proton permeation into the internal volume of each vesicle, we encapsulated carboxyfluorescein, a pH-sensitive dye whose fluorescence was quenched in the presence of excess protons. A microfluidic platform was used for easy exchange of high- and low-pH solutions, and fluorescence quenching of single vesicles was detected with single-molecule total internal reflection fluorescence (TIRF) microscopy. Upon solution exchange and acidification of the extravesicular solution (from pH 9 to 3.5), we observed for each vesicle a biphasic decay in fluorescence. Through single-vesicle analysis, we found that rate constants for the first decay followed a Poisson distribution, whereas rate constants for the second decay followed a normal distribution. We propose that proton permeation into each vesicle first arose from formation of transient pores and then transitioned into the second decay phase, which occurred by the solubility-diffusion mechanism. Furthermore, for the bulk population of vesicles, the decay rate constant and vesicle intensity (dependent on size) correlated to give an average permeability coefficient; however, for individual vesicles, we found little correlation, which suggested that proton permeability among single vesicles was heterogeneous in our experiments.
Assuntos
Fluoresceínas/química , Corantes Fluorescentes/química , Lipossomos/química , Fosfatidilcolinas/química , Permeabilidade da Membrana Celular , Cinética , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Fluorescência , PrótonsRESUMO
This paper presents a simple procedure for the fabrication of thermoset polyester (TPE) microfluidic systems and discusses the properties of the final devices. TPE chips are fabricated in less than 3 h by casting TPE resin directly on a lithographically patterned (SU-8) silicon master. Thorough curing of the devices is obtained through the combined use of ultraviolet light and heat, as both an ultraviolet and a thermal initiator are employed in the resin mixture. Features on the order of micrometers and greater are routinely reproduced using the presented procedure, including complex designs and multilayer features. The surface of TPE was characterized using contact angle measurements and X-ray photoelectron spectroscopy (XPS). Following oxygen plasma treatment, the hydrophilicity of the surface of TPE increases (determined by contact angle measurements) and the proportion of oxygen-containing functional groups also increases (determined by XPS), which indicates a correlated increase in the charge density on the surface. Native TPE microchannels support electroosmotic flow (EOF) toward the cathode, with an average electroosmotic mobility of 1.3 x 10(-4) cm(2) V(-1) s(-1) for a 50-microm square channel (20 mM borate at pH 9); following plasma treatment (5 min at 30 W and 0.3 mbar), EOF is enhanced by a factor of 2. This enhancement of the EOF from plasma treatment is stable for days, with no significant decrease noted during the 5-day period that we monitored. Using plasma-treated TPE microchannels, we demonstrate the separation of a mixture of fluorescein-tagged amino acids (glycine, glutamic acid, aspartic acid). TPE devices are up to 90% transparent (for approximately 2-mm-thick sample) to visible light (400-800 nm). The compatibility of TPE with a wide range of solvents was tested over a 24-h period, and the material performed well with acids, bases, alcohols, cyclohexane, n-heptane, and toluene but not with chlorinated solvents (dichloromethane, chloroform).
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Silício , Microscopia Eletrônica de Varredura/métodos , Osmose , Poliésteres , Propriedades de SuperfícieRESUMO
This letter describes a new strategy for initiating a chemical reaction that is based on the laser-induced breakdown of a nanoscopic barrier, which physically separates the reactants in space. Because the breakdown of the barrier is fast ( approximately 0.3 micros) and owing to the nanometer dimension of the barrier, the reactants can be brought together and the reaction can be initiated rapidly. The time scale most suited for this method (from microseconds to tens of milliseconds) bridges nicely between the faster time scales that are accessible mostly with laser-based triggering experiments and the slower time scales that are studied most frequently with flow-based devices.
Assuntos
Cinética , Lasers , Membranas Artificiais , Nanotecnologia , Espectrometria de FluorescênciaRESUMO
With advances in III-V nitride manufacturing processes, high-power light-emitting diode (LED) chips in the blue and UV wavelengths are now commercially available at reasonable cost and can be used as excitation sources in optical sensing. We describe the use of these high-power blue and UV LEDs for sensitive fluorescence detection, including chip-based flow cytometry, capillary electrophoresis (CE), and single-molecule imaging. By using a blue LED with a focusable power of approximately 40 mW as the excitation source for fluorescent beads, we demonstrate a simple chip-based bead sorter capable of enriching the concentration of green fluorescent beads from 63% to 95%. In CE experiments, we show that a mixture of analyte solution containing 30 nM 6-carboxyrhodamine 6G and 10 nM fluorescein can be separated and detected with excellent signal-to-noise ratio (approximately 17 for 10 nM fluorescein) using the collimated emission from a blue LED; the estimated mass detection limit was approximately 200 zmol for fluorescein. We also demonstrated ultrasensitive fluorescence imaging of single rhodamine 123 molecules and individual lambda-DNA molecules. At a small fraction of the cost of an Ar+ laser, high-power blue and UV LEDs are effective alternatives for lasers and arc lamps in fluorescence applications that demand portability, low cost, and convenience.