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1.
Nat Commun ; 12(1): 1823, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758197

RESUMO

The body naturally and continuously secretes sweat for thermoregulation during sedentary and routine activities at rates that can reflect underlying health conditions, including nerve damage, autonomic and metabolic disorders, and chronic stress. However, low secretion rates and evaporation pose challenges for collecting resting thermoregulatory sweat for non-invasive analysis of body physiology. Here we present wearable patches for continuous sweat monitoring at rest, using microfluidics to combat evaporation and enable selective monitoring of secretion rate. We integrate hydrophilic fillers for rapid sweat uptake into the sensing channel, reducing required sweat accumulation time towards real-time measurement. Along with sweat rate sensors, we integrate electrochemical sensors for pH, Cl-, and levodopa monitoring. We demonstrate patch functionality for dynamic sweat analysis related to routine activities, stress events, hypoglycemia-induced sweating, and Parkinson's disease. By enabling sweat analysis compatible with sedentary, routine, and daily activities, these patches enable continuous, autonomous monitoring of body physiology at rest.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Regulação da Temperatura Corporal/fisiologia , Microfluídica/métodos , Suor/metabolismo , Sudorese/fisiologia , Dispositivos Eletrônicos Vestíveis , Corpo Humano , Humanos , Concentração de Íons de Hidrogênio , Hipoglicemia/metabolismo , Levodopa/metabolismo , Microfluídica/instrumentação , Doença de Parkinson/metabolismo , Descanso/fisiologia , Estresse Fisiológico/fisiologia , Suor/fisiologia , Caminhada/fisiologia
2.
Methods Mol Biol ; 2265: 265-276, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704721

RESUMO

Liquid biopsy has emerged as the next generation target for diagnostics and therapeutic monitoring of many diseases including cancer. Liquid biopsy offers noninvasive analysis of aberrant biomolecular changes (e.g., aberrant protein expression, DNA mutation) which can provide crucial information on disease stages and therapy responses. As a diagnostically important biomarker for melanoma, the detection of the BRAFV600E aberration at the DNA and protein level in liquid biopsies confers an attractive option. This method describes the preparation and operation of an integrated multimolecular sensor (IMMS) for simultaneous detection of the BRAFV600E aberration in both molecular forms from circulating melanoma cells in liquid biopsy. IMMS integrates specific melanoma cell capture, cell release, cell lysis, and electrochemical BRAFV600E detection on a single device. IMMS is demonstrated for a sample-to-answer workflow of plasma spiked with melanoma cells.


Assuntos
Técnicas Biossensoriais/métodos , Imunoensaio/métodos , Dispositivos Lab-On-A-Chip , Melanoma/metabolismo , Microfluídica/instrumentação , Microfluídica/métodos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Cutâneas/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Técnicas Biossensoriais/instrumentação , Técnicas de Cultura de Células/métodos , Humanos , Imunoensaio/instrumentação , Biópsia Líquida/métodos , Melanoma/genética , Melanoma/patologia , Mutação , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia
3.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670545

RESUMO

Microfluidics is a relatively newly emerged field based on the combined principles of physics, chemistry, biology, fluid dynamics, microelectronics, and material science. Various materials can be processed into miniaturized chips containing channels and chambers in the microscale range. A diverse repertoire of methods can be chosen to manufacture such platforms of desired size, shape, and geometry. Whether they are used alone or in combination with other devices, microfluidic chips can be employed in nanoparticle preparation, drug encapsulation, delivery, and targeting, cell analysis, diagnosis, and cell culture. This paper presents microfluidic technology in terms of the available platform materials and fabrication techniques, also focusing on the biomedical applications of these remarkable devices.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Nanopartículas/administração & dosagem , Preparações Farmacêuticas/administração & dosagem , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica/instrumentação , Biologia Molecular/instrumentação , Biologia Molecular/métodos , Nanopartículas/química
4.
Lab Chip ; 21(5): 962-975, 2021 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-33511381

RESUMO

The latest developments in thin-film-transistor digital-microfluidics (TFT-DMF, also known by the commercial name aQdrop™) are reported, and proof of concept application to molecular diagnostics (e.g. for coronavirus disease, COVID-19) at the point-of-need demonstrated. The TFT-DMF array has 41 thousand independently addressable electrodes that are capable of manipulating large numbers of droplets of any size and shape, along any pathway to perform multiple parallel reactions. Droplets are continually tracked and adjusted through closed-loop feedback enabled by TFT based sensors at each array element. The sample-to-answer molecular in vitro diagnostic (IVD) test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) includes nucleic acid extractions from saliva, removal of dsDNA and quantitative reverse transcription polymerase chain reaction (RT-PCR). This proof of concept illustrates how the highly configurable TFT-DMF technology can perform many reactions in parallel and thus support the processing of a range of sample types followed by multiple complex multi-step assays.


Assuntos
/diagnóstico , Microfluídica/métodos , Transistores Eletrônicos , /virologia , Humanos , Microfluídica/instrumentação , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , /isolamento & purificação , Saliva/virologia
5.
J Agric Food Chem ; 69(5): 1466-1477, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33507744

RESUMO

The purpose of this study was to construct a delivery system using a microfluidic chip to protect procyanidins (PCs) and to achieve their pH-controlled release in simulated gastrointestinal fluid. The microfluidic chip was designed and fabricated to generate water-in-water-in-oil (W/W/O) templates for the preparation of sodium alginate/chitosan microparticles with a uniform size and core-shell structure, using an internal-external gelation method. Compared with free PCs, the stability of PCs embedded in microparticles was improved and a pH stimulus-responsive release of PCs from microparticles was observed under neutral pH conditions. The delivery system of microparticles was nontoxic and showed an inhibitory effect on the decrease of mitochondrial membrane potential in Caco-2 cells caused by H2O2 and acrylamide. This work provided a method for fabricating compact microfluidic chips to prepare a pH stimulus-responsive PCs delivery system with improved stability, which may have potential applications in the delivery of other nutrients.


Assuntos
Biflavonoides/química , Catequina/química , Preparações de Ação Retardada/química , Composição de Medicamentos/métodos , Microfluídica/métodos , Proantocianidinas/química , Biflavonoides/farmacologia , Células CACO-2 , Catequina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada/farmacologia , Composição de Medicamentos/instrumentação , Humanos , Concentração de Íons de Hidrogênio , Microfluídica/instrumentação , Microesferas , Tamanho da Partícula , Proantocianidinas/farmacologia
6.
Nat Protoc ; 16(3): 1452-1475, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33514945

RESUMO

Norovirus is a widespread public health threat and has a very low infectious dose. This protocol presents the extremely sensitive mobile detection of norovirus from water samples using a custom-built smartphone-based fluorescence microscope and a paper microfluidic chip. Antibody-conjugated fluorescent particles are immunoagglutinated and spread over the paper microfluidic chip by capillary action for individual counting using a smartphone-based fluorescence microscope. Smartphone images are analyzed using intensity- and size-based thresholding for the elimination of background noise and autofluorescence as well as for the isolation of immunoagglutinated particles. The resulting pixel counts of particles are correlated with the norovirus concentration of the tested sample. This protocol provides detailed guidelines for the construction and optimization of the smartphone- and paper-based assay. In addition, a 3D-printed enclosure is presented to incorporate all components in a dark environment. On-chip concentration and the assay of higher concentrations are presented to further broaden the assay range. This method is the first to be presented as a highly sensitive mobile platform for norovirus detection using low-cost materials. With all materials and reagents prepared, a single standard assay takes under 20 min. Although the method described is used for detection of norovirus, the same protocol could be adapted for detection of other pathogens by using different antibodies.


Assuntos
Microfluídica/instrumentação , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Fluorescência , Dispositivos Lab-On-A-Chip , Microfluídica/métodos , Norovirus/isolamento & purificação , Norovirus/patogenicidade , Smartphone , Água/análise , Microbiologia da Água
7.
Methods Mol Biol ; 2237: 69-82, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33237409

RESUMO

Electrochemistry is a multidisciplinary field encompassing the study of analytes in solution for detection and quantification. For the medical field, this brings opportunities to the clinical practice of disease detection through measurements of disease biomarkers. Specifically, panels of biomarkers offer an important future option that can enable physicians' access to blood, saliva, or urine bioassays for screening diseases, as well as monitoring the progression and response to therapy. Here, we describe the simultaneous detection of eight protein cancer biomarkers in a 30-min assay by a microfluidic electrochemical immunoarray.


Assuntos
Biomarcadores Tumorais/análise , Técnicas Eletroquímicas/métodos , Testes Imunológicos/métodos , Microfluídica/métodos , Análise Serial de Proteínas/métodos , Técnicas Eletroquímicas/instrumentação , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Testes Imunológicos/instrumentação , Microfluídica/instrumentação , Análise Serial de Proteínas/instrumentação
8.
Methods Mol Biol ; 2186: 197-211, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32918739

RESUMO

Because of the high sensitivity of lipid bilayers to external pressure fluctuations, a major challenge in functional studies of biological pores or ion channels is the difficulty in exchanging solutions rapidly while maintaining the stability of the lipid bilayer in a model membrane. Here we describe a droplet-interface bilayer-based perfusion system that has been routinely used in our research and is currently the most robust and stable perfusion system that provides prompt solution exchange surrounding a lipid bilayer. In this model membrane system, solutions can be completely exchanged within 1-2 s to obtain prompt responses of a lipid bilayer or membrane pores to the membrane environments. Also, our system is stable enough to sustain continuous perfusions up to at least dozens of minutes. To demonstrate, we show that acidification-induced protein channel insertion, substrate binding to protein channels, and pH gradient-driven protein translocation of anthrax toxin can be sequentially initiated by continuous perfusions in our system. Moreover, by rapidly switching the solutions, the protein translocation based on ratchet mechanisms can be paused and reinitiated iteratively in our system. Overall, this perfusion system provides a controllable and reliable solution exchange platform for investigations of pores and translocations on lipid bilayers.


Assuntos
Canais Iônicos/química , Bicamadas Lipídicas/química , Microfluídica/métodos , Técnicas de Patch-Clamp/métodos , Transporte de Íons , Potenciais da Membrana , Microfluídica/instrumentação , Técnicas de Patch-Clamp/instrumentação , Prótons
9.
Methods Mol Biol ; 2197: 253-269, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32827142

RESUMO

This chapter describes the synthesis of stealth and cationic liposomes and their complexation with plasmid DNA to generate lipoplexes for gene delivery applications. Two techniques are presented: a top-down approach which requires a second step of processing for downsizing the liposomes (i.e., ethanol injection method) and a microfluidic technique that explores the diffusion of ethanol in water to allow the proper lipid self-assembly. The synthesis of stealth liposomes is also a challenge since the use of poly(ethylene glycol) favors the formation of oblate micelles. In this protocol, the stealth cationic liposome synthesis by exploring the high ionic strength to overcome the formation of secondary structures like micelles is described. Finally, the electrostatic complexation between cationic liposomes and DNA is described, indicating important aspects that guarantee the formation of uniform lipoplexes.


Assuntos
Cátions/química , Técnicas de Química Sintética , Técnicas de Transferência de Genes , Lipossomos/síntese química , Microfluídica , DNA/administração & dosagem , DNA/química , Desenho de Equipamento , Lipídeos/química , Lipossomos/química , Microfluídica/instrumentação , Microfluídica/métodos , Plasmídeos/administração & dosagem , Plasmídeos/química , Plasmídeos/genética , Soluções
10.
Nat Protoc ; 16(2): 937-964, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33318693

RESUMO

Microtissues with specific structures and integrated vessels play a key role in maintaining organ functions. To recapitulate the in vivo environment for tissue engineering and organ-on-a-chip purposes, it is essential to develop perfusable biomimetic microscaffolds. We developed facile all-aqueous microfluidic approaches for producing perfusable hydrogel microtubes with diverse biomimetic sizes and shapes. Here, we provide a detailed protocol describing the construction of the microtube spinning platforms, the assembly of microfluidic devices, and the fabrication and characterization of various perfusable hydrogel microtubes. The hydrogel microtubes can be continuously generated from microfluidic devices due to the crosslinking of alginate by calcium in the coaxial flows and collecting bath. Owing to the mild all-aqueous spinning process, cells can be loaded into the alginate prepolymer for microtube spinning, which enables the direct production of cell-laden hydrogel microtubes. By manipulating the fluid dynamics at the microscale, the composable microfluidic devices and platforms can be used for the facile generation of six types of biomimetic perfusable microtubes. The microfluidic platforms and devices can be set up within 3 h from commonly available and inexpensive materials. After 10-20 min required to adjust the platform and fluids, perfusable hydrogel microtubes can be generated continuously. We describe how to characterize the microtubes using scanning electron or confocal microscopy. As an example application, we describe how the microtubes can be used for the preparation of a vascular lumen and how to perform barrier permeability tests of the vascular lumen.


Assuntos
Materiais Biomiméticos/química , Hidrogéis/síntese química , Técnicas Analíticas Microfluídicas/métodos , Alginatos/química , Biomimética/métodos , Humanos , Hidrogéis/química , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica/instrumentação , Microfluídica/métodos , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos
11.
Nat Commun ; 11(1): 5271, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077832

RESUMO

Three-dimensional (3D) cell culture technologies, such as organoids, are physiologically relevant models for basic and clinical applications. Automated microfluidics offers advantages in high-throughput and precision analysis of cells but is not yet compatible with organoids. Here, we present an automated, high-throughput, microfluidic 3D organoid culture and analysis system to facilitate preclinical research and personalized therapies. Our system provides combinatorial and dynamic drug treatments to hundreds of cultures and enables real-time analysis of organoids. We validate our system by performing individual, combinatorial, and sequential drug screens on human-derived pancreatic tumor organoids. We observe significant differences in the response of individual patient-based organoids to drug treatments and find that temporally-modified drug treatments can be more effective than constant-dose monotherapy or combination therapy in vitro. This integrated platform advances organoids models to screen and mirror real patient treatment courses with potential to facilitate treatment decisions for personalized therapy.


Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Microfluídica/métodos , Organoides/efeitos dos fármacos , Automação , Técnicas de Cultura de Células , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Humanos , Microfluídica/instrumentação , Neoplasias Pancreáticas/tratamento farmacológico
12.
Proc Natl Acad Sci U S A ; 117(29): 16839-16847, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641515

RESUMO

Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.


Assuntos
Separação Celular/métodos , Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos , Células Neoplásicas Circulantes/classificação , Linhagem Celular Tumoral , Separação Celular/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Leucaférese/métodos , Campos Magnéticos , Microfluídica/instrumentação
13.
J Vis Exp ; (160)2020 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-32628171

RESUMO

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.


Assuntos
DNA/química , Microfluídica/métodos , Microtecnologia/instrumentação , Biossíntese de Proteínas , Proteínas/metabolismo , DNA/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microfluídica/instrumentação , Microscopia de Fluorescência , Água/química
14.
Nucleic Acids Res ; 48(14): 8165-8177, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32609820

RESUMO

In synthetic circuits, CRISPR-Cas systems have been used effectively for endpoint changes from an initial state to a final state, such as in logic gates. Here, we use deactivated Cas9 (dCas9) and deactivated Cas12a (dCas12a) to construct dynamic RNA ring oscillators that cycle continuously between states over time in bacterial cells. While our dCas9 circuits using 103-nt guide RNAs showed irregular fluctuations with a wide distribution of peak-to-peak period lengths averaging approximately nine generations, a dCas12a oscillator design with 40-nt CRISPR RNAs performed much better, having a strongly repressed off-state, distinct autocorrelation function peaks, and an average peak-to-peak period length of ∼7.5 generations. Along with free-running oscillator circuits, we measure repression response times in open-loop systems with inducible RNA steps to compare with oscillator period times. We track thousands of cells for 24+ h at the single-cell level using a microfluidic device. In creating a circuit with nearly translationally independent behavior, as the RNAs control each others' transcription, we present the possibility for a synthetic oscillator generalizable across many organisms and readily linkable for transcriptional control.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Microfluídica/métodos , Periodicidade , RNA Guia/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli , Microfluídica/instrumentação , RNA Guia/genética , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
15.
Proc Natl Acad Sci U S A ; 117(29): 16743-16748, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32611809

RESUMO

Mammalian nervous systems, as natural ionic circuitries, stand out in environmental perception and sophisticated information transmission, relying on protein ionic channels and additional necessary structures. Prosperously emerged ionic regulated biomimetic nanochannels exhibit great potentialities in various application scenarios, especially signal transduction. Most reported direct current systems possess deficiencies in informational density and variability, which are superiorities of alternating current (AC) systems and necessities in bioinspired nervous signal transmission. Here, inspired by myelinated saltatory conduction, alternating electrostatic potential controlled nanofluidics are constructed with a noncontact application pattern and MXene nanosheets. Under time-variant external stimuli, ions confined in the interlaminar space obtain the capability of carriers for the AC ionic circuit. The transmitted information is accessible from typical sine to a frequency-modulated binary signal. This work demonstrates the potentiality of the bioinspired nervous signal transmission between electronics and ionic nanofluidics, which might push one step forward to the avenue of AC ionics.


Assuntos
Potenciais de Ação , Materiais Biomiméticos/química , Condutividade Elétrica , Microfluídica/métodos , Modelos Neurológicos , Nanoestruturas/química , Dimetilpolisiloxanos/química , Equipamentos e Provisões Elétricas , Transporte de Íons , Microfluídica/instrumentação
16.
J Chromatogr A ; 1625: 461270, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709322

RESUMO

In sample pre-treatment, millifluidic electromembrane platforms have been developed to extract and pre-concentrate target molecules with good clean-up that minimize matrix effects. Optimal operation conditions are normally determined experimentally, repeating the extractions at different conditions and determining the efficiencies by an analytical technique. To shorten and simplify the optimization protocol, millifluidic platforms have been electrically characterized by impedance spectroscopy. The magnitude of the resistance of the electromembrane has been found very predictive of the migration capacity and extraction efficiency of three different parabens on real time. The optimal conditions (4 V of applied potential) (Electromembrane extraction low voltage) have been successfully applied in the extraction of parabens from urine samples, that not only improves the extraction efficiency (100% for all compounds) but also provides a very low current intensity (7 µA), which is very important in electromembrane to minimize electrolysis phenomena. The possibility to optimize one of the most critical and important parameters such as the voltage with a simple electrical model may accelerate the production of application-specific millifluidic electromembrane platforms in a short development time. The results showed that millifluidic electromembrane extraction based low voltage has a future potential as a simple, selective, and time-efficient sample preparation technique allowing a simple battery as power supply.


Assuntos
Impedância Elétrica , Membranas Artificiais , Microfluídica/instrumentação , Modelos Teóricos , Parabenos/isolamento & purificação , Adulto , Eletrólise , Feminino , Humanos , Limite de Detecção , Reprodutibilidade dos Testes , Reologia , Soluções
17.
J Vis Exp ; (159)2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32510519

RESUMO

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network. By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules. The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.


Assuntos
Coagulação Sanguínea , Plaquetas/metabolismo , Células Endoteliais/metabolismo , Microfluídica/métodos , Adesividade Plaquetária , Agregação Plaquetária , Trombose/fisiopatologia , Células Endoteliais/citologia , Fibrinólise , Hemostasia , Humanos , Microfluídica/instrumentação
18.
Nat Commun ; 11(1): 2982, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532969

RESUMO

Non-invasive and label-free calorimetry could become a disruptive technique to study single cell metabolic heat production without altering the cell behavior, but it is currently limited by insufficient sensitivity. Here, we demonstrate microfluidic single-cell calorimetry with 0.2-nW sensitivity, representing more than ten-fold enhancement over previous record, which is enabled by (i) a low-noise thermometry platform with ultralow long-term (10-h) temperature noise (80 µK) and (ii) a microfluidic channel-in-vacuum design allowing cell flow and nutrient delivery while maintaining a low thermal conductance of 2.5 µW K-1. Using Tetrahymena thermophila as an example, we demonstrate on-chip single-cell calorimetry measurement with metabolic heat rates ranging from 1 to 4 nW, which are found to correlate well with the cell size. Finally, we perform real-time monitoring of metabolic rate stimulation by introducing a mitochondrial uncoupling agent to the microchannel, enabling determination of the spare respiratory capacity of the cells.


Assuntos
Calorimetria/métodos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Análise de Célula Única/métodos , Temperatura , Tetrahymena thermophila/metabolismo , Metabolismo Basal , Calorimetria/instrumentação , Microfluídica/instrumentação , Mitocôndrias/metabolismo , Consumo de Oxigênio , Análise de Célula Única/instrumentação , Tetrahymena thermophila/citologia , Condutividade Térmica
19.
Proc Natl Acad Sci U S A ; 117(20): 10639-10644, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32350139

RESUMO

The lack of rapid antibiotic susceptibility tests adversely affects the treatment of bacterial infections and contributes to increased prevalence of multidrug-resistant bacteria. Here, we describe an all-electrical approach that allows for ultrasensitive measurement of growth signals from only tens of bacteria in a microfluidic device. Our device is essentially a set of microfluidic channels, each with a nanoconstriction at one end and cross-sectional dimensions close to that of a single bacterium. Flowing a liquid bacteria sample (e.g., urine) through the microchannels rapidly traps the bacteria in the device, allowing for subsequent incubation in drugs. We measure the electrical resistance of the microchannels, which increases (or decreases) in proportion to the number of bacteria in the microchannels. The method and device allow for rapid antibiotic susceptibility tests in about 2 h. Further, the short-time fluctuations in the electrical resistance during an antibiotic susceptibility test are correlated with the morphological changes of bacteria caused by the antibiotic. In contrast to other electrical approaches, the underlying geometric blockage effect provides a robust and sensitive signal, which is straightforward to interpret without electrical models. The approach also obviates the need for a high-resolution microscope and other complex equipment, making it potentially usable in resource-limited settings.


Assuntos
Antibacterianos/toxicidade , Microfluídica/métodos , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Impedância Elétrica , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/fisiologia , Testes de Sensibilidade Microbiana/instrumentação , Testes de Sensibilidade Microbiana/métodos , Microfluídica/instrumentação
20.
Sci Rep ; 10(1): 8493, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444830

RESUMO

Acoustic standing wave devices offer excellent potential applications in biological sciences for drug delivery, cell manipulation and tissue engineering. However, concerns have been raised about possible destructive effects on cells due to the applied acoustic field, in addition to other produced secondary factors. Here, we report a systematic study employing a 1D resonant acoustic trapping device to evaluate the cell viability and cell metabolism for a healthy cell line (Human Dermal Fibroblasts, HDF) and a cervical cancer cell line (HeLa), as a function of time and voltages applied (4-10 Vpp) under temperature-controlled conditions. We demonstrate that high cell viability can be achieved reliably when the device is operated at its minimum trapping voltage and tuned carefully to maximise the acoustic standing wave field at the cavity resonance. We found that cell viability and reductive metabolism for both cell lines are kept close to control levels at room temperature and at 34 °C after 15 minutes of acoustic exposure, while shorter acoustic exposures and small changes on temperature and voltages, had detrimental effects on cells. Our study highlights the importance of developing robust acoustic protocols where the operating mode of the acoustic device is well defined, characterized and its temperature carefully controlled, for the application of acoustic standing waves when using live cells and for potential clinical applications.


Assuntos
Acústica/instrumentação , Fibroblastos/metabolismo , Microfluídica/instrumentação , Neoplasias do Colo do Útero/metabolismo , Sobrevivência Celular , Feminino , Fibroblastos/citologia , Humanos , Neoplasias do Colo do Útero/patologia , Vibração
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