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1.
Food Chem ; 306: 125300, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31562927

RESUMO

Chlorophyll is a valuable bioactive compound, which is used as a natural food coloring agent and a photosensitizer for photodynamic therapy because of its antioxidant properties, antimutagenic ability, and near-infrared fluorescence. However, chlorophyll is unstable when it comes to retaining its antioxidant activity, when exposed to oxygen, high temperature, or light environments. To enhance the stability of chlorophyll, a polymer encapsulation method was proposed. Polycaprolactone (PCL) was employed to encapsulate the chlorophyll, and the particles size of the composites was controlled through droplet microfluidics. The composites (chlorophyll-encapsulated PCL particles) were characterized through UV-VIS spectrometry, SEM, optical microscopy, and light exposure. The particles were spherical, with diameters adjustable from 68 to 247 µm. Additionally, the chlorophyll-encapsulated PCL particles exhibited considerably prolonged chlorophyll stability. The solid microparticle is more convenient for storage and transportation, and have great potential for application in the food industry.


Assuntos
Clorofila/química , Poliésteres/química , Microfluídica/métodos , Tamanho da Partícula
2.
Nat Protoc ; 14(12): 3366-3394, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31666743

RESUMO

Epigenetic mechanisms such as histone modifications play critical roles in adaptive tuning of chromatin structures. Profiling of various histone modifications at the genome scale using tissues from animal and human samples is an important step for functional studies of epigenomes and epigenomics-based precision medicine. Because the profile of a histone mark is highly specific to a cell type, cell isolation from tissues is often necessary to generate a homogeneous cell population, and such operations tend to yield a low number of cells. In addition, high-throughput processing is often desirable because of the multiplexity of histone marks of interest and the large quantity of samples in a hospital setting. In this protocol, we provide detailed instructions for device fabrication, setup, and operation of microfluidic oscillatory washing-based chromatin immunoprecipitation followed by sequencing (MOWChIP-seq) for profiling of histone modifications using as few as 100 cells per assay with a throughput as high as eight assays in one run. MOWChIP-seq operation involves flowing of chromatin fragments through a packed bed of antibody-coated beads, followed by vigorous microfluidic oscillatory washing. Our process is semi-automated to reduce labor and improve reproducibility. Using one eight-unit device, it takes 2 d to produce eight sequencing libraries from chromatin samples. The technology is scalable. We used the protocol to study a number of histone modifications in various types of mouse and human tissues. The protocol can be conducted by a user who is familiar with molecular biology procedures and has basic engineering skills.


Assuntos
/instrumentação , Microfluídica/instrumentação , Animais , Cromatina/genética , Imunoprecipitação da Cromatina/métodos , Epigênese Genética/genética , Epigenômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Código das Histonas/genética , Código das Histonas/fisiologia , Histonas/metabolismo , Humanos , Microfluídica/métodos , Processamento de Proteína Pós-Traducional , Análise de Sequência de DNA/métodos
3.
Soft Matter ; 15(46): 9565-9578, 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31724682

RESUMO

The performance of orally administered lipid-based drug formulations is crucially dependent on digestion, and understanding the colloidal structures formed during digestion is necessary for rational formulation design. Previous studies using the established bulk pH-stat approach (Hong et al. 2015), coupled to synchrotron small angle X-ray scattering (SAXS), have begun to shed light on this subject. Such studies of digestion using in situ SAXS measurements are complex and have limitations regarding the resolution of intermediate structures. Using a microfluidic device, the digestion of lipid systems may be monitored with far better control over the mixing of the components and the application of enzyme, thereby elucidating a finer understanding of the structural progression of these lipid systems. This work compares a simple T-junction microcapillary device and a custom-built microfluidic chip featuring hydrodynamic flow focusing, with an equivalent experiment with the full scale pH-stat approach. Both microfluidic devices were found to be suitable for in situ SAXS measurements in tracking the kinetics with improved time and signal sensitivity compared to other microfluidic devices studying similar lipid-based systems, and producing more consistent and controllable structural transformations. Particle sizing of the nanoparticles produced in the microfluidic devices were more consistent than the pH-stat approach.


Assuntos
Lipase/metabolismo , Lipídeos/química , Lipossomos/química , Microfluídica/métodos , Nanopartículas/química , Difração de Raios X/métodos , Composição de Medicamentos/métodos , Microfluídica/instrumentação , Espalhamento a Baixo Ângulo , Difração de Raios X/instrumentação
4.
Pharm Res ; 36(12): 183, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31741058

RESUMO

Research conducted in microgravity conditions has the potential to yield new therapeutics, as advances can be achieved in the absence of phenomena such as sedimentation, hydrostatic pressure and thermally-induced convection. The outcomes of such studies can significantly contribute to many scientific and technological fields, including drug discovery. This article reviews the existing traditional microgravity platforms as well as emerging ideas for enabling microgravity research focusing on SpacePharma's innovative autonomous remote-controlled microgravity labs that can be launched to space aboard nanosatellites to perform drug research in orbit. The scientific literature is reviewed and examples of life science fields that have benefited from studies in microgravity conditions are given. These include the use of microgravity environment for chemical applications (protein crystallization, drug polymorphism, self-assembly of biomolecules), pharmaceutical studies (microencapsulation, drug delivery systems, behavior and stability of colloidal formulations, antibiotic drug resistance), and biological research, including accelerated models for aging, investigation of bacterial virulence , tissue engineering using organ-on-chips in space, enhanced stem cells proliferation and differentiation.


Assuntos
Simulação de Ausência de Peso/instrumentação , Simulação de Ausência de Peso/métodos , Ausência de Peso , Fatores Etários , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Cristalização/instrumentação , Cristalização/métodos , Dimerização , Composição de Medicamentos/instrumentação , Composição de Medicamentos/métodos , Sistemas de Liberação de Medicamentos/instrumentação , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/instrumentação , Descoberta de Drogas/métodos , Resistência Microbiana a Medicamentos , Humanos , Microfluídica/instrumentação , Microfluídica/métodos , Pesquisa Farmacêutica/instrumentação , Pesquisa Farmacêutica/métodos , Fenômenos Físicos , Proteínas/química , Voo Espacial , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos
5.
Nature ; 574(7777): 228-232, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31597972

RESUMO

Microfluidic systems can deliver portable point-of-care diagnostics without the need for external equipment or specialist operators, by integrating all reagents and manipulations required for a particular assay in one device1. A key approach is to deposit picogram quantities of dried reagents in microchannels with micrometre precision using specialized inkjet plotters2-5. This means that reagents can be stored for long periods of time and reconstituted spontaneously when adding a liquid sample. But it is challenging to carry out complex operations using multiple reagents, because shear flow enhances their dispersion and they tend to accumulate at moving liquid fronts, resulting in poor spatiotemporal control over the concentration profile of the reconstituted reagents6. One solution is to limit the rate of release of reagents into the liquid7-10. However, this requires the fine-tuning of different reagents, conditions and targeted operations, and cannot readily produce the complex, time-dependent multireagent concentration pulses required for sophisticated on-chip assays. Here we report and characterize a capillary flow phenomenon that we term self-coalescence, which is seen when a confined liquid with a stretched air-liquid interface is forced to 'zip' back onto itself in a microfluidic channel, thereby allowing reagent reconstitution with minimal dispersion. We provide a comprehensive framework that captures the physical underpinning of this effect. We also fabricate scalable, compact and passive microfluidic structures-'self-coalescence modules', or SCMs-that exploit and control this phenomenon in order to dissolve dried reagent deposits in aqueous solutions with precise spatiotemporal control. We show that SCMs can reconstitute multiple reagents so that they either undergo local reactions or are sequentially delivered in a flow of liquid. SCMs are easily fabricated in different materials, readily configured to enable different reagent manipulations, and readily combined with other microfluidic technologies, so should prove useful for assays, diagnostics, high-throughput screening and other technologies requiring efficient preparation and manipulation of small volumes of complex solutions.


Assuntos
Indicadores e Reagentes/análise , Microfluídica/métodos , Técnicas de Química Analítica/instrumentação , Técnicas de Química Analítica/métodos , Testes Diagnósticos de Rotina , Ensaios Enzimáticos/instrumentação , Ensaios Enzimáticos/métodos , Fluorometria , Glucosefosfato Desidrogenase/análise , Glucosefosfato Desidrogenase/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/isolamento & purificação , Humanos , Microfluídica/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos
6.
Anal Chim Acta ; 1089: 108-114, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31627807

RESUMO

Droplet microfluidics has the ability to greatly increase the throughput of screening and sorting of enzymes by carrying reagents in picoliter droplets flowing in inert oils. It was found with the use of a specific surfactant, the interfacial tension of droplets can be very sensitive to droplet pH. This enables the sorting of droplets of different pH when confined droplets encounter a microfabricated trench. The device can be extended to sort enzymes, as a large number of enzymatic reactions lead to the production of an acidic or basic product and a concurrent change in solution pH. The progress of an enzymatic reaction is tracked from the position of a flowing train of droplets. We demonstrate the sorting of esterase isoenzymes based on their enzymatic activity. This label-free technology, that we dub droplet sorting by interfacial tension (SIFT), requires no active components and would have applications for enzyme sorting in high-throughput applications that include enzyme screening and directed evolution of enzymes.


Assuntos
Hidrolases de Éster Carboxílico/isolamento & purificação , Ensaios Enzimáticos/métodos , Acetatos/química , Animais , Hidrolases de Éster Carboxílico/química , Ensaios Enzimáticos/instrumentação , Fluorcarbonetos/química , Isoenzimas/química , Isoenzimas/isolamento & purificação , Dispositivos Lab-On-A-Chip , Fígado/enzimologia , Microfluídica/instrumentação , Microfluídica/métodos , Óleos/química , Fenóis/química , Reprodutibilidade dos Testes , Tensão Superficial , Suínos , Água/química
7.
Int J Mol Sci ; 20(18)2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31533368

RESUMO

Diseases caused by multi-drug resistant pathogens have become a global concern. Therefore, new approaches suitable for treating these bacteria are urgently needed. In this study, we analyzed genetically encoded photosensitizers (PS) related to the green fluorescent protein (GFP) or light-oxygen-voltage (LOV) photoreceptors for their exogenous applicability as light-triggered antimicrobial agents. Depending on their specific photophysical properties and photochemistry, these PSs can produce different toxic ROS (reactive oxygen species) such as O2•- and H2O2 via type-I, as well as 1O2 via type-II reaction in response to light. By using cell viability assays and microfluidics, we could demonstrate differences in the intracellular and extracellular phototoxicity of the applied PS. While intracellular expression and exogenous supply of GFP-related PSs resulted in a slow inactivation of E. coli and pathogenic Gram-negative and Gram-positive bacteria, illumination of LOV-based PSs such as the singlet oxygen photosensitizing protein SOPP3 resulted in a fast and homogeneous killing of these microbes. Furthermore, our data indicate that the ROS type and yield as well as the localization of the applied PS protein can strongly influence the antibacterial spectrum and efficacy. These findings open up new opportunities for photodynamic inactivation of pathogenic bacteria.


Assuntos
Anti-Infecciosos/farmacologia , Luz , Fármacos Fotossensibilizantes/farmacologia , Proteínas Recombinantes/farmacologia , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Biomarcadores , Relação Dose-Resposta a Droga , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Reporter , Microfluídica/instrumentação , Microfluídica/métodos
8.
Nat Protoc ; 14(11): 3144-3161, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31554957

RESUMO

The analysis of bacteria at the single-cell level is essential to characterization of processes in which cellular heterogeneity plays an important role. BACMMAN (bacteria mother machine analysis) is a software allowing fast and reliable automated image analysis of high-throughput 2D or 3D time-series images from experiments using the 'mother machine', a very popular microfluidic device allowing biological processes in bacteria to be investigated at the single-cell level. Here, we describe how to use some of the BACMMAN features, including (i) segmentation and tracking of bacteria and intracellular fluorescent spots, (ii) visualization and editing of the results, (iii) configuration of the image-processing pipeline for different datasets and (iv) BACMMAN coupling to data analysis software for visualization and analysis of data subsets with specific properties. Among software specifically dedicated to the analysis of mother machine data, only BACMMAN allows segmentation and tracking of both bacteria and intracellular spots. For a single position, single channel with 1,000 frames (2-GB dataset), image processing takes ~6 min on a regular computer. Numerous implemented algorithms, easy configuration and high modularity ensure wide applicability of the BACMMAN software.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Processamento de Imagem Assistida por Computador/métodos , Microfluídica/métodos , Análise de Célula Única/métodos , Software , Imagem com Lapso de Tempo/métodos , Evolução Biológica , Escherichia coli/genética , Microfluídica/instrumentação , Microscopia de Fluorescência/métodos , Mutação
9.
Int J Mol Sci ; 20(19)2019 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-31546717

RESUMO

Niosomes are non-ionic surfactant-based vesicles with high promise for drug delivery applications. They can be rapidly prepared via microfluidics, allowing their reproducible production without the need of a subsequent size reduction step, by controlled mixing of two miscible phases of an organic (lipids dissolved in alcohol) and an aqueous solution in a microchannel. The control of niosome properties and the implementation of more complex functions, however, thus far are largely unknown for this method. Here we investigate microfluidics-based manufacturing of topotecan (TPT)-loaded polyethylene glycolated niosomes (PEGNIO). The flow rate ratio of the organic and aqueous phases was varied and optimized. Furthermore, the surface of TPT-loaded PEGNIO was modified with a tumor homing and penetrating peptide (tLyp-1). The designed nanoparticular drug delivery system composed of PEGNIO-TPT-tLyp-1 was fabricated for the first time via microfluidics in this study. The physicochemical properties were determined through dynamic light scattering (DLS) and zeta potential analysis. In vitro studies of the obtained formulations were performed on human glioblastoma (U87) cells. The results clearly indicated that tLyp-1-functionalized TPT-loaded niosomes could significantly improve anti-glioma treatment.


Assuntos
Sistemas de Liberação de Medicamentos , Lipossomos , Microfluídica , Linhagem Celular Tumoral , Portadores de Fármacos/química , Composição de Medicamentos , Liberação Controlada de Fármacos , Humanos , Lipossomos/química , Microfluídica/métodos , Tamanho da Partícula
10.
Mater Sci Eng C Mater Biol Appl ; 104: 109705, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31499950

RESUMO

Microfluidics-based microfibers have been widely used as bottom-up scaffolds for tissue engineering applications. Different forms of microfibers with certain thickness of shell have been developed during the past decade. Ultra-thin microfiber, as a special and promising carrier of cells, was less explored. In this work, by using the interfacial ionic interaction between sodium alginate (NaA) and chitosan (CS), a novel ultra-thin polyelectrolyte hollow microfiber with the diameter of ~200 µm and the shell thickness of 1.3 ±â€¯0.3 µm was fabricated via a microfluidic device for liver tissue engineering. The fluorescence of FITC labeled CS confirmed the inner CS layer of the fabricated microfiber and the SEM results illustrated its ultra-thin characteristic. Although there are only two layers in the ultra-thin polyelectrolyte hollow microfiber, the following cells encapsulation experiments indicated that it could bear cells loading and the hollow space of the microfibers could encapsulate sufficient number of cells for tissue engineering applications. The presence of inner CS layer in the microfiber promoted cell adhesion and ultra-thin shell characteristic facilitated the exchange of nutrient substance and O2 and thus promoted cell proliferation. HepG2 cells encapsulated in the microfibers maintained favorable viability, proliferation ability and hepatic specific functions during 10 days' culture. These results suggest that the established polyelectrolyte microfibers hold great potential applications in the field of liver tissue engineering. We believe this work will lead to the development of innovative methodologies and materials for both cell culture and biomedical application.


Assuntos
Microfluídica/métodos , Polieletrólitos/química , Alginatos/química , Células Imobilizadas/citologia , Quitosana/química , Fluorescência , Células Hep G2 , Humanos , Reologia , Soluções
11.
Nat Commun ; 10(1): 4049, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492867

RESUMO

Food production in green crops is severely limited by low activity and poor specificity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in natural photosynthesis (NPS). This work presents a scientific solution to overcome this problem by immobilizing RuBisCO into a microfluidic reactor, which demonstrates a continuous production of glucose precursor at 13.8 µmol g-1 RuBisCO min-1 from CO2 and ribulose-1,5-bisphosphate. Experiments show that the RuBisCO immobilization significantly enhances enzyme stabilities (7.2 folds in storage stability, 6.7 folds in thermal stability), and also improves the reusability (90.4% activity retained after 5 cycles of reuse and 78.5% after 10 cycles). This work mimics the NPS pathway with scalable microreactors for continuous synthesis of glucose precursor using very small amount of RuBisCO. Although still far from industrial production, this work demonstrates artificial synthesis of basic food materials by replicating the light-independent reactions of NPS, which may hold the key to food crisis relief and future space colonization.


Assuntos
Enzimas Imobilizadas/metabolismo , Glucose/biossíntese , Microfluídica/métodos , Fotossíntese , Ribulose-Bifosfato Carboxilase/metabolismo , Dióxido de Carbono/metabolismo , Produtos Agrícolas/metabolismo , Estabilidade Enzimática , Glucose/química , Folhas de Planta/metabolismo , Reprodutibilidade dos Testes , Ribulosefosfatos/metabolismo , Temperatura Ambiente
12.
Nat Commun ; 10(1): 3544, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391463

RESUMO

Simultaneous measurement of proteins and mRNA in single cells enables quantitative understanding and modeling of cellular functions. Here, we present an automated microfluidic system for multi-parameter and ultra-sensitive protein/mRNA measurements in single cells. Our technology improves the sensitivity of digital proximity ligation assay by up to 55-fold, with a detection limit of 2277 proteins per cell and with detection efficiency of as few as 29 protein molecules. Our measurements using this system reveal higher mRNA/protein correlation in single mammalian cells than previous estimates. Furthermore, time-lapse imaging of herpes simplex virus 1 infected epithelial cells enabled by our device shows that expression of ICP4 -a major transcription factor regulating hundreds of viral genes- is only partially correlated with viral protein counts, suggesting that many cells go through abortive infection. These results highlight the importance of high-sensitivity protein/mRNA quantification for understanding fundamental molecular mechanisms in individual cells.


Assuntos
Proteínas/isolamento & purificação , RNA Mensageiro/isolamento & purificação , Análise de Célula Única/métodos , Animais , Dosagem de Genes , Humanos , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Dispositivos Lab-On-A-Chip , Limite de Detecção , Microfluídica/instrumentação , Microfluídica/métodos , Análise de Célula Única/instrumentação , Imagem com Lapso de Tempo/instrumentação , Imagem com Lapso de Tempo/métodos , Células Vero
13.
Biosensors (Basel) ; 9(3)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394810

RESUMO

This paper presents focusing of microparticles in multiple paths within the direction of the flow using dielectrophoresis. The focusing of microparticles is realized through partially perforated electrodes within the microchannel. A continuous electrode on the top surface of the microchannel is considered, while the bottom side is made of a circular meshed perforated electrode. For the mathematical model of this microfluidic channel, inertia, buoyancy, drag and dielectrophoretic forces are brought up in the motion equation of the microparticles. The dielectrophoretic force is accounted for through a finite element discretization taking into account the perforated 3D geometry within the microchannel. An ordinary differential equation is solved to track the trajectories of the microparticles. For the case of continuous electrodes using the same mathematical model, the numerical simulation shows a very good agreement with the experiments, and this confirms the validation of focusing of microparticles within the proposed perforated electrode microchannel. Microparticles of silicon dioxide and polystyrene are used for this analysis. Their initial positions and radius, the Reynolds number, and the radius of the pore in perforated electrodes mainly conduct microparticles trajectories. Moreover, the radius of the pore of perforated electrode is the dominant factor in the steady state levitation height.


Assuntos
Microfluídica/métodos , Modelos Teóricos , Técnicas Biossensoriais , Eletrodos , Eletroforese/métodos , Microfluídica/instrumentação , Tamanho da Partícula , Poliestirenos/análise , Dióxido de Silício/análise
14.
Molecules ; 24(16)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394856

RESUMO

Paper-based microfluidic devices have advanced significantly in recent years as they are affordable, automated with capillary action, portable, and biodegradable diagnostic platforms for a variety of health, environmental, and food quality applications. In terms of commercialization, however, paper-based microfluidics still have to overcome significant challenges to become an authentic point-of-care testing format with the advanced capabilities of analyte purification, multiplex analysis, quantification, and detection with high sensitivity and selectivity. Moreover, fluid flow manipulation for multistep integration, which involves valving and flow velocity control, is also a critical parameter to achieve high-performance devices. Considering these limitations, the aim of this review is to (i) comprehensively analyze the fabrication techniques of microfluidic paper-based analytical devices, (ii) provide a theoretical background and various methods for fluid flow manipulation, and iii) highlight the recent detection techniques developed for various applications, including their advantages and disadvantages.


Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Microfluídica/instrumentação , Microfluídica/métodos , Algoritmos , Desenho de Equipamento , Humanos , Modelos Teóricos , Papel
15.
Anal Bioanal Chem ; 411(24): 6399-6407, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31372700

RESUMO

As microfluidic cell culture progresses, the need for robust and reproducible intracellular analyses grows. In particular, intracellular metabolites are subject to perturbation and degradation during the lysing process. The reliability of intracellular metabolomic analysis in microfluidic devices depends on the preservation of metabolite integrity during sample preparation and storage. Described here is a novel automated microfluidic system exhibiting the necessary rapid cellular lysis and quenching of enzymatic activity. Quenching efficiency was assessed using a novel ratiometric MALDI-MS-based assay of exogenous isotopic adenosine triphosphate (ATP) hydrolysis to isotopic adenosine diphosphate (ADP) as a marker of metabolite degradation. The lysis system of the microfluidic device was enhanced using a Peltier cooler to chill the lysate and quench aberrant enzymatic activity. Parameter optimization (flow rate, collection time, and temperature control) improved the endogenous and exogenous ADP/ATP ratios by 44.9% and 39.8% respectively consistent with traditional quenching techniques. The effects of chilling/quenching on metabolism were evaluated resulting in over 500 significant features compared to non-chilled from untargeted capillary LC-MS metabolomic analyses. These include increased levels of tryptophan, histidine, and pyruvate as well as decreased levels in UDP-N-acetylglucosamine. The results illustrate the need for both rapid lysis and quenching in microfluidic cell culture platforms. Graphical abstract.


Assuntos
Trifosfato de Adenosina/metabolismo , Metabolômica , Microfluídica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Difosfato de Adenosina/metabolismo , Automação
16.
Eur J Pharm Biopharm ; 143: 51-60, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31445156

RESUMO

Extensive research has been undertaken to investigate the effect of liposome size in vitro and in vivo. However, it is often difficult to generate liposomes in different size ranges that offer similar low polydispersity and lamellarity. Conventional methods used in the preparation of liposomes, such as lipid film hydration or reverse phase evaporation, generally give rise to liposomal suspensions displaying broad, multimodal size distribution combined with uncontrolled degree of lamellarity. In contrast, microfluidics allows highly homogeneous liposome dispersions to be produced and adjustment of microfluidic operating parameters (flow rate ratio (FRR) and total flow rate (TFR)) can offer size-tuning of liposomes (up to 300 nm, depending on the formulation). Herein, we demonstrate a novel method which allows the production of highly monodisperse, cationic liposomes over a wide particle size range (up to 750 nm in size). This is achieved through controlling the concentration of the aqueous buffer during production. Using this method, liposomes composed of 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium (DDA) - DOPE:DOTAP and DOPE:DDA liposomes - of up to 750 nm were prepared and investigated. These investigations demonstrate that the in vitro cellular uptake of small (40 nm) and large (>500 nm) liposomes in bone marrow-derived macrophages (BMDM) is similar terms of percentage of liposome+ cells and mean fluorescence intensity (MFI). However, significant differences are observed in BMDM uptake when represented in terms of number of liposomes, liposome surface area or liposome internal volume. In vivo biodistribution studies in mice show that by creating small (<50 nm) liposomes we can modify the clearance rates of these liposomes from the injection site and increase accumulation to the draining lymphatics.


Assuntos
Cátions/química , Cátions/metabolismo , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Animais , Transporte Biológico/fisiologia , Química Farmacêutica/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microfluídica/métodos , Tamanho da Partícula , Compostos de Amônio Quaternário/química , Distribuição Tecidual/fisiologia
17.
Sensors (Basel) ; 19(15)2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31362399

RESUMO

A novel microcantilever sensor was batch fabricated for Yersinia detection. The microcantilever surface modification method was optimized by introducing a secondary antibody to increase the number of binding sites. A novel microfluidic platform was designed and fabricated successfully. A 30 µL solution could fully react with the microcantilever surface. Those routines enhanced the binding efficiency between the target and receptor on the microcantilever. With this novel designed microfluidic platform, the specific adsorption of 107 Yersinia on the beam surface with modified F1 antibody was significantly enhanced.


Assuntos
Anticorpos/química , Técnicas Biossensoriais , Yersiniose/diagnóstico , Yersinia/isolamento & purificação , Anticorpos/imunologia , Sítios de Ligação , Humanos , Microfluídica/métodos , Propriedades de Superfície , Yersinia/química , Yersinia/imunologia , Yersiniose/imunologia , Yersiniose/microbiologia
18.
Sensors (Basel) ; 19(15)2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31382441

RESUMO

In this paper, we demonstrate the possibility of direct protein sensing beyond the Debye length limit using a molecular-charge-contact (MCC)-based ion-sensitive field-effect transistor (ISFET) sensor combined with a microfluidic device. Different from the MCC method previously reported, biotin-coated magnetic beads are set on the gate insulator of an ISFET using a button magnet before the injection of target molecules such as streptavidin. Then, the streptavidin-a biotin interaction, used as a model of antigen-antibody reaction is expected at the magnetic beads/gate insulator nanogap interface, changing the pH at the solution/dielectric interface owing to the weak acidity of streptavidin. In addition, the effect of the pH or ionic strength of the measurement solutions on the electrical signals of the MCC-based ISFET sensor is investigated. Furthermore, bound/free (B/F) molecule separation with a microfluidic device is very important to obtain an actual electrical signal based on the streptavidin-biotin interaction. Platforms based on the MCC method are suitable for exploiting the advantages of ISFETs as pH sensors, that is, direct monitoring systems for antigen-antibody reactions in the field of in vitro diagnostics.


Assuntos
Microfluídica/métodos , Proteínas/análise , Transistores Eletrônicos , Reações Antígeno-Anticorpo , Biotina/química , Biotina/metabolismo , Concentração de Íons de Hidrogênio , Íons/química , Limite de Detecção , Microfluídica/instrumentação , Concentração Osmolar , Estreptavidina/química , Estreptavidina/metabolismo
19.
Virchows Arch ; 475(3): 313-323, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31267199

RESUMO

Breast cancer is a highly heterogeneous disease. The efficacy of tailored therapeutic strategies relies on the precise detection of diagnostic biomarkers by immunohistochemistry (IHC). Therefore, considering the increasing incidence of breast cancer cases, a concomitantly time-efficient and accurate diagnosis is clinically highly relevant. Microfluidics is a promising innovative technology in the field of tissue diagnostic, enabling for rapid, reliable, and automated immunostaining. We previously reported the microfluidic-based HER2 (human epidermal growth factor receptor 2) detection in breast carcinomas to greatly correlate with the HER2 gene amplification level. Here, we aimed to develop a panel of microfluidic-based IHC protocols for prognostic and therapeutic markers routinely assessed for breast cancer diagnosis, namely HER2, estrogen/progesterone receptor (ER/PR), and Ki67 proliferation factor. The microfluidic IHC protocol for each marker was optimized to reach high staining quality comparable to the standard procedure, while concomitantly shortening the staining time to 16 min-excluding deparaffinization and antigen retrieval step-with a turnaround time reduction up to 7 folds. Comparison of the diagnostic score on 50 formaldehyde-fixed paraffin-embedded breast tumor resections by microfluidic versus standard staining showed high concordance (overall agreement: HER2 94%, ER 95.9%, PR 93.6%, Ki67 93.7%) and strong correlation (ρ coefficient: ER 0.89, PR 0.88, Ki67 0.87; p < 0.0001) for all the analyzed markers. Importantly, HER2 genetic reflex test for all discordant cases confirmed the scores obtained by the microfluidic technique. Overall, the microfluidic-based IHC represents a clinically validated equivalent approach to the standard chromogenic staining for rapid, accurate, and automated breast cancer diagnosis.


Assuntos
Neoplasias da Mama/diagnóstico , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Biomarcadores Tumorais/metabolismo , Mama/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente , Antígeno Ki-67/metabolismo , Prognóstico , Receptor ErbB-2/metabolismo , Receptores Estrogênicos/metabolismo , Receptores de Progesterona/metabolismo
20.
Int J Nanomedicine ; 14: 4187-4209, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31289440

RESUMO

Circulating tumor cells (CTCs) are disseminated cancer cells. The occurrence and circulation of CTCs seem key for metastasis, still the major cause of cancer-associated deaths. As such, CTCs are investigated as predictive biomarkers. However, due to their rarity and heterogeneous biology, CTCs' practical use has not made it into the clinical routine. Clearly, methods for the effective isolation and reliable detection of CTCs are urgently needed. With the development of nanotechnology, various nanosystems for CTC isolation and enrichment and CTC-targeted cancer therapy have been designed. Here, we summarize the relationship between CTCs and tumor metastasis, and describe CTCs' unique properties hampering their effective enrichment. We comment on nanotechnology-based systems for CTC isolation and recent achievements in microfluidics and lab-on-a-chip technologies. We discuss recent advances in CTC-targeted cancer therapy exploiting the unique properties of nanomaterials. We conclude by introducing developments in CTC-directed nanosystems and other advanced technologies currently in (pre)clinical research.


Assuntos
Biomarcadores Tumorais/análise , Separação Celular/métodos , Nanomedicina/métodos , Células Neoplásicas Circulantes/patologia , Biomarcadores Tumorais/isolamento & purificação , Materiais Biomiméticos , Grafite , Humanos , Dispositivos Lab-On-A-Chip , Microfluídica/instrumentação , Microfluídica/métodos , Nanoestruturas/química , Nanotecnologia/métodos , Nanotubos de Carbono
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