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This paper reported a micro flow cytometer capable of high-throughput characterization of single-cell electrical and structural features based on constrictional microchannels and deep neural networks. When single cells traveled through microchannels with constricted cross-sectional areas, they effectively blocked concentrated electric field lines, producing large impedance variations. Meanwhile, the traveling cells were confined within the cross-sectional areas of the constrictional microchannels, enabling the capture of high-quality images without losing focuses. Then single-cell features from impedance profiles and optical images were extracted from customized recurrent and convolution networks (RNN and CNN), which were further fused for cell-type classification based on support vector machines (SVM). As a demonstration, two leukemia cell lines (e.g., HL60 vs. Jurkat) were analyzed, producing high-classification accuracies of 99.3% based on electrical features extracted from Long Short-Term Memory (LSTM) of RNN, 96.7% based on structural features extracted from Resnet18 of CNN and 100.0% based on combined features enabled by SVM. The microfluidic flow cytometry developed in this study may provide a new perspective for the field of single-cell analysis.
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Microfluídica , Redes Neurales de la Computación , Microfluídica/métodos , Citometría de Flujo/métodos , Impedancia Eléctrica , Línea CelularRESUMEN
Fast and quantitative estimation of single-cell proteins with various distribution patterns remains a technical challenge. Here, a microfluidic flow cytometer with a uniform optical field (Uni-µFCM) was developed, which enabled the translation of multicolor fluorescence signals of bound antibodies into targeted protein numbers with arbitrary distributions of biological cells. As the core of Uni-µFCM, a uniform optical field for optical excitation and fluorescence detection was realized by adopting a microfabricated metal window to shape the optical beam for excitation, which was modeled and validated by both numerical simulation and experimental characterization. After the validation of Uni-µFCM in single-cell protein quantification by measuring single-cell expressions of three transcriptional factors from four cell lines of variable sizes and origins, Uni-µFCM was applied to (1) quantify membrane and cytoplasmic markers of myeloid and lymphocytic leukocytes to classify cell lines and normal and patient blood samples; (2) measure single-cell expressions of key cytokines affiliated with gene stabilities, differentiating paired oral and colon tumor cell lines with varied malignancies, and (3) quantify single-cell stemming markers of liver tumor cell lines, cell subtypes, and liver patient samples to determine a variety of lineage hierarchy. By quantitatively assessing complex cellular phenotypes, Uni-µFCM substantially expanded the phenotypic space accessible to single-cell applications in leukemia gating, tumor classification, and hierarchy determination of cancer stem cells.
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Leucemia , Microfluídica , Humanos , Línea Celular Tumoral , Anticuerpos , Células Madre NeoplásicasRESUMEN
TiO2-based photocatalytic building materials can keep the building surface clean, and have decontamination, antibacterial effects and so on, which greatly reduces the maintenance cost and the risk of cleaning work, and these materials have great application potential in pollution and carbon reduction in the future. However, due to the wide band gap of TiO2, the low utilization of solar energy and the instability of super hydrophilicity have always been the difficulties in the field of photocatalysis. Based on the relevant research of TiO2-based photocatalytic materials in recent years, this review summarizes the modification strategies that can effectively improve the photocatalytic activity of TiO2-based photocatalytic materials. At the same time, the influence of complex environmental factors and substrate properties on the self-cleaning behavior of TiO2-based building materials was analyzed. This paper aims to provide effective guidance for the future application of TiO2-based photocatalysts in the construction field, improve people's understanding of photocatalytic building materials (PBM) and photocatalytic self-cleaning characteristics, and provide more possibilities for the extensive application of photocatalysis technology in the construction field, as well as to promote the realization of global carbon neutrality and other strategic goals of pollution and carbon reduction.
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This article presents an approach of microfluidic flow cytometry capable of continuously characterizing cytoplasmic viscosities of single cells. The microfluidic system consists of a major constriction channel and a side constriction channel perpendicularly crossing each other. Cells are forced to rapidly travel through the major channel and are partially aspirated into the side channel when passing the channel junction. Numerical simulations were conducted to model the time dependence of the aspiration length into the side channel, which enables the measurement of cytoplasmic viscosity by fitting the model results to experimental data. As a demonstration for high-throughput measurement, the cytoplasmic viscosities of HL-60 cells that were native or treated by N-Formylmethionine-leucyl-phenylalanine (fMLP) were quantified with sample sizes as large as thousands of cells. Both the average and median cytoplasmic viscosities of native HL-60 cells were found to be about 10% smaller than those of fMLP-treated HL-60 cells, consistent with previous observations that fMLP treatment can increase the rigidity of white blood cells. Furthermore, the microfluidic system was used to process granulocytes from three donors (sample size >1,000 cells for each donor). The results revealed that the cytoplasmic viscosity of granulocytes from one donor was significantly higher than the other two, which may result from the fact that this donor just recovered from an inflammation. In summary, the developed microfluidic system can collect cytoplasmic viscosities from thousands of cells and may function as an enabling tool in the field of single-cell analysis. © 2019 International Society for Advancement of Cytometry.
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Técnicas Analíticas Microfluídicas , Microfluídica , Constricción , Citoplasma , Humanos , ViscosidadRESUMEN
Surface plasmon resonance (SPR) biosensors are an extremely sensitive optical technique used to detect the changes in refractive index occurring at the sensor interface. Fluorescence involves the emission of light by a substance that has absorbed light or other electromagnetic radiation, and the parameters of the absorbed and emitted radiation are used to identify the presence and the amount of specific molecules in a specimen. SPR biosensors and fluorescence analysis are both effective methods for real-time detection. The combination of these technologies would improve the quantitative detection sensitivity of fluorescence analysis and the specificity of SPR detection. We designed and developed an SPR and fluorescence synchronous detection system. The SPR module was based on two kinds of modulation methods, and the fluorescence module was capable of switching between four wavelengths. The fluorescence microspheres and A549 cells of different concentration were both detected by the SPR and fluorescence method synchronously in real time. The fluorescent signal and the optical signal of the SPR were shown to correlate. The correlation coefficient for fluorescent microspheres detection reached up to 0.9866. The system could be used in cell analysis and molecule diagnosis in the future.
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This paper presents a crossing constriction channel-based microfluidic system for high-throughput characterization of specific membrane capacitance (Csm) and cytoplasm conductivity (σcy) of single cells. In operations, cells in suspension were forced through the major constriction channel and instead of invading the side constriction channel, they effectively sealed the side constriction channel, which led to variations in impedance data. Based on an equivalent circuit model, these raw impedance data were translated into Csm and σcy. As a demonstration, the developed microfluidic system quantified Csm (3.01 ± 0.92 µF cm-2) and σcy (0.36 ± 0.08 S m-1) of 100 000 A549 cells, which could generate reliable results by properly controlling cell positions during their traveling in the crossing constriction channels. Furthermore, the developed microfluidic impedance cytometry was used to distinguish paired low- and high-metastatic carcinoma cell types of SACC-83 (ncell = â¼100 000) and SACC-LM cells (ncell = â¼100 000), distinguishing significant differences in both Csm (3.16 ± 0.90 vs. 2.79 ± 0.67 µF cm-2) and σcy (0.36 ± 0.06 vs.0.41 ± 0.08 S m-1). As high-throughput microfluidic impedance cytometry, this technique may add a new marker-free dimension to flow cytometry in single-cell analysis.
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Membrana Celular/metabolismo , Capacidad Eléctrica , Citometría de Flujo/métodos , Microfluídica/instrumentación , Microfluídica/métodos , Análisis de la Célula Individual/métodos , Células A549 , HumanosRESUMEN
This article presents a microfabricated 96-well wound-healing assay enabling high-throughput measurement of cellular migration capabilities. Within each well, the middle area is the wound region, made of microfabricated gold surface with self-assembled PEG repellent for cell seeding. After the formation of a cellular confluent monolayer around the wound region, collagen solution was applied to form three-dimensional matrix to cover the PEG surface, initiating the wound-healing process. By interpreting the numbers of migrated cells into the wound regions as a function of specific stimuli with different concentrations, EC50 (half-maximal effective concentration) was obtained. Using H1299 as a model, values of EC50 were quantified as 8% and 160 ng/ml for fetal bovine serum and CXCL12, respectively. In addition, the values of EC50 were demonstrated not to be affected by variations in compositions of extracellular matrix and geometries of wounds, which can thus be regarded as an intrinsic marker. Furthermore, the migration capabilities of a second cell type (HeLa) were characterized by the developed wound-healing assay, producing EC50 of 2% when fetal bovine serum was used as the stimuli. These results validated the proposed high-throughput wound-healing assay, which may function as an enabling tool in studying cellular capabilities of migration and invasion. © 2017 International Society for Advancement of Cytometry.
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Movimiento Celular/fisiología , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Cicatrización de Heridas/fisiología , Línea Celular Tumoral , HumanosRESUMEN
This paper presents a 96-well microfabricated assay to study three-dimensional (3D) invasion of tumor cells. A 3D cluster of tumor cells was first generated within each well by seeding cells onto a micro-patterned surface consisting of a central fibronectin-coated area that promotes cellular attachment, surrounded by a poly ethylene glycol (PEG) coated area that is resistant to cellular attachment. Following the formation of the 3D cell clusters, a 3D collagen extracellular matrix was formed in each well by thermal-triggered gelation. Invasion of the tumor cells into the extracellular matrix was subsequently initiated and monitored. Two modes of cellular infiltration were observed: A549 cells invaded into the extracellular matrix following the surfaces previously coated with PEG molecules in a pseudo-2D manner, while H1299 cells invaded into the extracellular matrix in a truly 3D manner including multiple directions. Based on the processing of 2D microscopic images, a key parameter, namely, equivalent invasion distance (the area of invaded cells divided by the circumference of the initial cell cluster) was obtained to quantify migration capabilities of these two cell types. These results validate the feasibility of the proposed platform, which may function as a high-throughput 3D cellular invasion assay.
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Bioensayo , Técnicas de Cultivo de Célula/métodos , Movimiento Celular , Microtecnología/métodos , Células A549 , Técnicas de Cultivo de Célula/instrumentación , Línea Celular Tumoral , Colágeno/química , Cámaras de Difusión de Cultivos , Dimetilpolisiloxanos/química , Matriz Extracelular/química , Fibronectinas/química , Humanos , Microtecnología/instrumentación , Especificidad de Órganos , Poliésteres/química , Polietilenglicoles/químicaRESUMEN
We herein introduce a novel multi-well stretching device that is made of three polydimethylsiloxane layers, consisting of a top hole-punched layer, middle thin membrane, and bottom patterned layer. It is the first time that such a simple device has been used to supply axisymmetric and nonuniform strains to cells cultured on well bottoms that are stretchable. These mechanical stimuli can somewhat mimic the stretching at the bending sites of blood vessels, where the strains are complicated. In this device, nonuniform strain is given to cells through the deformation of a membrane from a flat surface to a spherical cap during the injection of a certain volume of water into the chamber between the middle membrane and bottom layer. EA.hy926 cells (a human umbilical vein endothelial cell line) were seeded on the well bottoms and exposed to axisymmetric strain under a 5, 10, 15, and 20% degree of deformation of the membrane. The cellular responses were characterized in terms of cell morphology, cell viability, and expression of inflammatory mRNAs and proteins. With increasing the degree of deformation, the cells exhibited an inclination toward detachment and apoptosis; meanwhile the expression of inflammatory mRNAs and proteins, such as MCP-1, IL-8, IL-6 and ICAM-1, showed a significant increment. The obtained results demonstrate that the inflammatory responses of EA.hy926 cells can be induced by increasing the magnitude of the strain. This simple device provides a useful tool for in vitro investigation of the inflammatory mechanisms related to vascular diseases.
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Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Inflamación/metabolismo , Técnicas Analíticas Microfluídicas/instrumentación , Línea Celular , Supervivencia Celular , Dimetilpolisiloxanos/química , HumanosRESUMEN
This paper presents a tubing-free microfluidic wound healing assay to quantify the migration of vascular smooth muscle cells (VSMCs), where gravity was used to generate a laminar flow within microfluidic channels, enabling cell seeding, culture, and wound generation. As the first systemic study to quantify the migration of VSMCs within microfluidic environments, the effects of channel geometries, surface modifications and chemokines on cellular migration were investigated, revealing that 1) height of the micro channels had a significant impact on cell migration; 2) the surface coating of collagen induced more migration of VSMCs than fibronectin coated surfaces and 3) platelet derived growth factor resulted in maximal cell migration compared to tumor necrosis factor alpha and fetal bovine serum. Furthermore, migrations of five types of VSMCs (e.g., the human vascular smooth muscle cell line, two types of primary vascular smooth cells, and VSMCs isolated from two human samples) were quantified, finding that VSMCs from the cell line and human samples demonstrated comparable migration distances, which were significantly lower than the migration distances of two primary cell types. As a platform technology, this wound healing assay may function as a new model to study migration of VSMCs within microfluidic environments.
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Técnicas Analíticas Microfluídicas/métodos , Músculo Liso Vascular/citología , Cicatrización de Heridas , Animales , Aorta/citología , Becaplermina , Bovinos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Colágeno/química , Fibronectinas/química , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Proteínas Proto-Oncogénicas c-sis/farmacología , Ratas , Suero/química , Factor de Necrosis Tumoral alfa/farmacología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
This paper presents a microfluidic device (poly-dimethylsiloxane micro channels bonded with glass slides) enabling culture of MLO-Y4 osteocyte like cells. In this study, on-chip collagen coating, cell seeding and culture, as well as staining were demonstrated in a tubing-free manner where gravity was used as the driving force for liquid transportation. MLO-Y4 cells were cultured in microfluidic channels with and without collagen coating where cellular images in a time sequence were taken and analyzed, confirming the positive effect of collagen coating on phenotype maintaining of MLO-Y4 cells. The proliferating cell nuclear antigen based proliferation assay was used to study cellular proliferation, revealing a higher proliferation rate of MLO-Y4 cells seeded in microfluidic channels without collagen coating compared to the substrates coated with collagen. Furthermore, the effects of channel dimensions (variations in width and height) on the viability of MLO-Y4 cells were explored based on the Calcein-AM and propidium iodide based live/dead assay and the Hoechst 33258 based apoptosis assay, locating the correlation between the decrease in channel width or height and the decrease in cell viability. As a platform technology, this microfluidic device may function as a new cell culture model enabling studies of osteocytes.