RESUMO
We report on a compact two-electrode voltage clamping system composed of microfabricated electrodes and a fluidic device for Xenopus laevis oocytes. The device was fabricated by assembling Si-based electrode chips and acrylic frames to form fluidic channels. After the installation of Xenopus oocytes into the fluidic channels, the device can be separated in order to measure changes in oocyte plasma membrane potential in each channel using an external amplifier. Using fluid simulations and experiments, we investigated the success rates of Xenopus oocyte arrays and electrode insertion with respect to the flow rate. We successfully located each oocyte in the array and detected oocyte responses to chemical stimuli using our device.
Assuntos
Oócitos , Animais , Microeletrodos , Xenopus laevis , Constrição , Oócitos/metabolismo , Eletrodos ImplantadosRESUMO
Hydrogels have various promising prospects as a successful platform for detecting biomarkers, and human serum albumin (HSA) is an important biomarker in the diagnosis of kidney diseases. However, the difficult-to-control passive diffusion kinetics of hydrogels is a major factor affecting detection performance. This study focuses on using hydrogels embedded with aggregation-induced emission (AIE) fluorescent probe TC426 to detect HSA in real time. The vortex fluidic device (VFD) technology is used as a rotation strategy to control the reaction kinetics and micromixing during measurement. The results show that the introduction of VFD could significantly accelerate its fluorescence response and effectively improve the diffusion coefficient, while VFD processing could regulate passive diffusion into active diffusion, offering a new method for future sensing research.
Assuntos
Corantes Fluorescentes , Albumina Sérica Humana , Humanos , Espectrometria de Fluorescência/métodos , HidrogéisRESUMO
The current study describes a new technology, effective for readily preparing a fluorescent (FL) nanoprobe-based on hyperbranched polymer (HB) and aggregation-induced emission (AIE) fluorogen with high brightness to ultimately develop FL hydrogels. We prepared the AIE nanoprobe using a microfluidic platform to mix hyperbranched polymers (HB, generations 2, 3, and 4) with AIE (TPE-2BA) under shear stress and different rotation speeds (0-5 K RPM) and explored the FL properties of the AIE nanoprobe. Our results reveal that the use of HB generation 4 exhibits 30-times higher FL intensity compared to the AIE alone and is significantly brighter and more stable compared to those that are prepared using HB generations 3 and 2. In contrast to traditional methods, which are expensive and time-consuming and involve polymerization and post-functionalization to develop FL hyperbranched molecules, our proposed method offers a one-step method to prepare an AIE-HB nanoprobe with excellent FL characteristics. We employed the nanoprobe to fabricate fluorescent injectable bioadhesive gel and a hydrogel microchip based on polyvinyl alcohol (PVA). The addition of borax (50 mM) to the PVA + AIE nanoprobe results in the development of an injectable bioadhesive fluorescent gel with the ability to control AIEgen release for 300 min. When borax concentration increases two times (100 mM), the adhesion stress is more than two times bigger (7.1 mN/mm2) compared to that of gel alone (3.4 mN/mm2). Excellent dimensional stability and cell viability of the fluorescent microchip, along with its enhanced mechanical properties, proposes its potential applications in mechanobiology and understanding the impact of microstructure in cell studies.
Assuntos
Corantes Fluorescentes/química , Hidrogéis/química , Álcool de Polivinil/química , Nanopartículas/química , Espectrometria de FluorescênciaRESUMO
Protein folding is important for protein homeostasis/proteostasis in the human body. We have established the ability to manipulate protein unfolding/refolding for ß-lactoglobulin using the induced mechanical energy in the thin film microfluidic vortex fluidic device (VFD) with monitoring as such using an aggregation-induced emission luminogen (AIEgen), TPE-MI. When denaturant (guanidine hydrochloride) is present with ß-lactoglobulin, the VFD accelerates the denaturation reaction in a controlled way. Conversely, rapid renaturation of the unfolded protein occurs in the VFD in the absence of the denaturant. The novel TPE-MI reacts with exposed cysteine thiol when the protein unfolds, as established with an increase in fluorescence intensity. TPE-MI provides an easy and accurate way to monitor the protein folding, with comparable results established using conventional circular dichroism. The controlled VFD-mediated protein folding coupled with in situ bioprobe AIEgen monitoring is a viable methodology for studying the denaturing of proteins.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas/química , Dicroísmo Circular/métodos , Cisteína/química , Guanidina/química , Humanos , Cinética , Lactoglobulinas/química , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Redobramento de Proteína , Desdobramento de Proteína , Proteostase/fisiologia , Espectrometria de Fluorescência/métodosRESUMO
In recent decades, microfluidic techniques have been extensively used to advance hydrogel design and control the architectural features on the micro- and nanoscale. The major challenges with the microfluidic approach are clogging and limited architectural features: notably, the creation of the sphere, core-shell, and fibers. Implementation of batch production is almost impossible with the relatively lengthy time of production, which is another disadvantage. This minireview aims to introduce a new microfluidic platform, a vortex fluidic device (VFD), for one-step fabrication of hydrogels with different architectural features and properties. The application of a VFD in the fabrication of physically crosslinked hydrogels with different surface morphologies, the creation of fluorescent hydrogels with excellent photostability and fluorescence properties, and tuning of the structure-property relationship in hydrogels are discussed. We conceive, on the basis of this minireview, that future studies will provide new opportunities to develop hydrogel nanocomposites with superior properties for different biomedical and engineering applications.
Assuntos
Hidrogéis , Microfluídica/instrumentação , Microfluídica/métodos , Engenharia Tecidual , Materiais Biocompatíveis , Análise EspectralRESUMO
This paper describes a method for fabricating protein-based capsules with semipermeable and enzyme-degradable surface barriers. It involves the use of a simple fluidic device to generate water-in-oil emulsion droplets, followed by cross-linking of proteins at the water-oil interface to generate a semipermeable surface barrier. The capsules can be readily fabricated with uniform and controllable sizes and, more importantly, show selective permeability toward molecules with different molecular weights: small molecules like fluorescein sodium salt can freely diffuse through the surface barrier while macromolecules such as proteins can not. The proteins, however, can be released by digesting the surface barrier with an enzyme such as pepsin. Taken together, the capsules hold great potential for applications in controlled release, in particular, for the delivery of protein drugs.
Assuntos
Cápsulas/química , Pepsina A/metabolismo , Proteínas/metabolismo , Animais , Cápsulas/toxicidade , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada , Emulsões/química , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Camundongos , Células NIH 3T3 , Óleos/química , Permeabilidade , Polímeros/química , Proteínas/química , Albumina Sérica/química , Albumina Sérica/metabolismo , Propriedades de Superfície , Água/químicaRESUMO
Healthcare facilities produce millions of tons of waste annually, with a significant portion consisting of diagnostic plasticware. Here, we introduce a new detection platform that completely replaces traditional assay plates with a piece of membrane, offering a much greener and more sustainable alternative. The membrane, integrated within the portable vortex fluidic device (P-VFD), enables rapid detection of a clinically relevant protein biomarker, urinary p75ECD. This biomarker is utilized to evaluate the prognosis, disease severity, and progression of amyotrophic lateral sclerosis (ALS). This assay has a limit-of-detection (LOD) of 4.03â pg, which is comparable to the plate-based assay (2.24â pg) and has been optimised through a full factorial design of experiments (DOE) and response surface methodology (RSM). P-VFD has great potential in quantifying p75ECD in human biofluids and can significantly reduce the assay time to 5â min compared to the current plate-based p75ECD ELISA assay (3â days), with at least a 4.4-fold reduction in the usage of the detection antibody.
RESUMO
The design of new and more sustainable synthetic protocols to access new materials or valuable compounds will have a high impact on the broader chemistry community. In this sense, continuous-flow photochemistry has emerged as a powerful technique which has been employed successfully in various areas such as biopharma, organic chemistry, as well as materials science. However, it is important to note that chemical processes must not only advance towards new or improved chemical transformations, but also implement new technologies that enable new process opportunities. For this reason, the design of novel photoreactors is key to advancing photochemical strategies. In this sense, the use of equipment and techniques embracing processes intensification is important in developing more sustainable protocols. Among the most recent applications, spinning continuous flow reactors, such as rotor reactors or vortex reactors, have shown promising performance as new synthetic tools. Nevertheless, there is currently no review in the literature that effectively summarizes and showcases the most recent applications of such type of photoreactors. Herein, we highlight fundamental aspects and applications of two categories of spinning reactors, the Spinning Disc Reactors (SDRs) and Thin Film Vortex reactors, critiquing the scope and limitations of these advanced processing technologies. Further, we take a view on the future of spinning reactors in flow as a synthetic toolbox to explore new photochemical transformations.
RESUMO
Based on a water-in-oil-in-water emulsion system, porous and hollow polydimethylsiloxane (PDMS) beads containing cells using a simple fluidic device with three flow channels are fabricated. Poly(ethylene glycol) (PEG) in the PDMS oil phase is served as a porogen for pore development. The feasibility of the porous PDMS beads prepared with different PEG concentrations (10, 20, and 30 wt%) for cell encapsulation in terms of pore size, protein diffusion, and cell proliferation inside the PDMS beads is evaluated. The PDMS beads prepared with PEG 30 wt% are exhibited a highly porous structure and facilitated fast diffusion of protein from the core domain to the outer phase, eventually leading to enhanced cell proliferation. The results clearly indicate that hollow PDMS beads with a porous structure could provide a favorable microenvironment for cell survival due to the large porous structure.
Assuntos
Materiais Biocompatíveis/química , Dimetilpolisiloxanos/química , Fibroblastos/citologia , Animais , Materiais Biocompatíveis/síntese química , Bovinos , Proliferação de Células , Sobrevivência Celular , Difusão , Dimetilpolisiloxanos/síntese química , Camundongos , Estrutura Molecular , Células NIH 3T3 , Tamanho da Partícula , Polietilenoglicóis/química , Porosidade , Soroalbumina Bovina/química , Propriedades de SuperfícieRESUMO
Introduction: Zebrafish is a suitable animal model for molecular genetic tests and drug discovery due to its characteristics including optical transparency, genetic manipulability, genetic similarity to humans, and cost-effectiveness. Mobility of the zebrafish reflects pathological conditions leading to brain disorders, disrupted motor functions, and sensitivity to environmental challenges. However, it remains technologically challenging to quantitively assess zebrafish's mobility in a flowing environment and simultaneously monitor cellular behavior in vivo. Methods: We herein developed a facile fluidic device using mechanical vibration to controllably generate various flow patterns in a droplet housing single zebrafish, which mimics its dynamically flowing habitats. Results: We observe that in the four recirculating flow patterns, there are two equilibrium stagnation positions for zebrafish constrained in the droplet, i.e., the "source" with the outward flow and the "sink" with the inward flow. Wild-type zebrafish, whose mobility remains intact, tend to swim against the flow and fight to stay at the source point. A slight deviation from streamline leads to an increased torque pushing the zebrafish further away, whereas zebrafish with motor neuron dysfunction caused by lipin-1 deficiency are forced to stay in the "sink," where both their head and tail align with the flow direction. Deviation angle from the source point can, therefore, be used to quantify the mobility of zebrafish under flowing environmental conditions. Moreover, in a droplet of comparable size, single zebrafish can be effectively restrained for high-resolution imaging. Conclusion: Using the proposed methodology, zebrafish mobility reflecting pathological symptoms can be quantitively investigated and directly linked to cellular behavior in vivo.
RESUMO
Biodegradable, biomass derived kombucha cellulose films with increased mechanical strength from 9.98 MPa to 18.18 MPa were prepared by vortex fluidic device (VFD) processing. VFD processing not only reduced the particle size of kombucha cellulose from approximate 2 µm to 1 µm, but also reshaped its structure from irregular to round. The increased mechanical strength of these polysaccharide-derived films is the result of intensive micromixing and high shear stress of a liquid thin film in a VFD. This arises from the incorporation at the micro-structural level of uniform, unidirectional strings of kombucha cellulose hydrolysates, which resulted from the topological fluid flow in the VFD. The biodegradability of the VFD generated polymer films was not compromised relative to traditionally generated films. Both films were biodegraded within 5 days.
Assuntos
Alginatos , Celulose , Ágar/química , Celulose/química , Biomassa , Fenômenos FísicosRESUMO
BACKGROUND: Articular cartilage (AC)'s main function is to resist to a stressful mechanical environment, and chondrocytes are responding to mechanical stress for the development and homeostasis of this tissue. However, current knowledge on processes involved in response to mechanical stimulation is still limited. These mechanisms are commonly investigated in engineered cartilage models where the chondrocytes are included in an exogeneous biomaterial different from their natural extracellular matrix. The aim of the present study is to better understand the impact of mechanical stimulation on mesenchymal stromal cells (MSCs)-derived chondrocytes generated in their own extracellular matrix. METHODS: A fluidic custom-made device was used for the mechanical stimulation of cartilage micropellets obtained from human MSCs by culture in a chondrogenic medium for 21 days. Six micropellets were positioned into the conical wells of the device chamber and stimulated with different signals of positive pressure (amplitude, frequency and duration). A camera was used to record the sinking of each micropellet into their cone, and micropellet deformation was analyzed using a finite element model. Micropellets were harvested at different time points after stimulation for RT-qPCR and histology analysis. RESULTS: Moderate micropellet deformation was observed during stimulation with square pressure signals as mean von Mises strains between 6.39 and 14.35% were estimated for amplitudes of 1.75-14 kPa superimposed on a base pressure of 50% of the amplitude. The compression, tension and shear observed during deformation did not alter micropellet microstructure as shown by histological staining. A rapid and transient increase in the expression of chondrocyte markers (SOX9, AGG and COL2B) was measured after a single 30-min stimulation with a square pressure signal of 3.5 kPa amplitude superimposed on a minimum pressure of 1.75 kPa, at 1 Hz. A small change of 1% of cyclical deformations when using a square pressure signal instead of a constant pressure signal induced a fold change of 2 to 3 of chondrogenic gene expression. Moreover, the expression of fibrocartilage (COL I) or hypertrophic cartilage (COL X, MMP13 and ADAMTS5) was not significantly regulated, except for COL X. CONCLUSIONS: Our data demonstrate that the dynamic deformation of cartilage micropellets by fluidic-based compression modulates the expression of chondrocyte genes responsible for the production of a cartilage-like extracellular matrix. This lays the foundations for further investigating the chondrocyte mechanobiology and the cartilage growth under mechanical stimulation.
Assuntos
Cartilagem , Condrócitos , Humanos , Materiais Biocompatíveis , Condrogênese/genética , Expressão GênicaRESUMO
Neuroblastoma is an extracranial solid tumor which develops in early childhood and still has a poor prognosis. One strategy to increase cure rates is the identification of patient-specific drug responses in tissue models that mimic the interaction between patient cancer cells and tumor environment. We therefore developed a perfused and micro-vascularized tumor-environment model that is directly bioprinted into custom-manufactured fluidic chips. A gelatin-methacrylate/fibrin-based matrix containing multiple cell types mimics the tumor-microenvironment that promotes spontaneous micro-vessel formation by embedded endothelial cells. We demonstrate that both, adipocyte- and iPSC-derived mesenchymal stem cells can guide this process. Bioprinted channels are coated with endothelial cells post printing to form a dense vessel-tissue barrier. The tissue model thereby mimics structure and function of human soft tissue with endothelial cell-coated larger vessels for perfusion and micro-vessel networks within the hydrogel-matrix. Patient-derived neuroblastoma spheroids are added to the matrix during the printing process and grown for more than two weeks. We demonstrate that micro-vessels are attracted by and grow into tumor spheroids and that neuroblastoma cells invade the tumor-environment as soon as the spheroids disrupt. In summary, we describe the first bioprinted, micro-vascularized neuroblastoma-tumor-environment model directly printed into fluidic chips and a novel medium-throughput biofabrication platform suitable for studying tumor angiogenesis and metastasis in precision medicine approaches in future.
Assuntos
Células-Tronco Mesenquimais , Neuroblastoma , Pré-Escolar , Células Endoteliais , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Neuroblastoma/tratamento farmacológico , Medicina de Precisão , Impressão Tridimensional , Engenharia Tecidual , Microambiente TumoralRESUMO
Alginate scaffold has been used widely for controlled release applications because of its ability to provide three-dimensional supports for formation of a gel matrix. Alginate gel scaffolds for drug delivery matrices were prepared using a fluidic device. N2 gas was used in the fluidic device to generate bubbles in the gel layer. The hydrogel matrices with induced voids were compared with hydrogel matrices without voids. This study attempted to identify the release mechanism of vitamin B12 from the two types of prepared scaffolds, and the data were fitted with different release kinetic models. The results revealed that the alginate scaffold exhibited a controlled release profile and that the corresponding release mechanism followed a first-order kinetic model. Hydrogel scaffolds fabricated with biocompatible polymers using fluidic methods could be promising for controlled drug delivery systems.
Assuntos
Alginatos/química , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Hidrogéis/química , Cinética , Resistência ao Cisalhamento , Vitamina B 12/química , Vitamina B 12/metabolismoRESUMO
In single cell analysis, transport of foreign substances into a cell is an important technique. In particular, for accurate analysis, a method to transport a small amount (pico-liter order) of substance into the cell without leakage while retaining the cell shape is essential. Because the fusion of the cell and the giant liposome is a closed system to the outside, it may be possible to transport a precise, small amount of substances into the cell. Additionally, there is no possibility that a leaked substance would affect other systems. To develop the liposome-cell transportation system, knowledge about the behavior of substances in the liposome and the cell is important. However, only a few studies have observed the substance transport between a liposome and a cell. Here, we report observation of small amount of substance transport into a single C2C12 cell by using a giant liposome. Substance transport occurred by electrofusion between the cell and the giant liposome containing the substance, which is a closed system. First, to observe the electrofusion and substance transport from the moment of voltage application, we fabricated a microfluidic device equipped with electrodes. We introduced suspensions of cells and liposomes into the microfluidic device and applied alternating current (AC) and direct current (DC) voltages for electrofusion. We observed a small amount (22.4 ± 0.1%, 10.3 ± 0.4% and 9.1 ± 0.1%) of fluorescent substance (Calcein) contained in the liposomes was transported into the cell without leakage outside the cell, and we obtained the diffusion coefficient of Calcein in the cell as 137 ± 18 µm²/s. We anticipate that this system and the knowledge acquired will contribute to future realization of more accurate single cell analysis in a wide range of fields.
RESUMO
The cancer community continues to search for an efficient and cost-effective technique to isolate and characterize circulating cells (CTCs) as a 'real-time liquid biopsy'. Existing methods to isolate and analyze CTCs require various transfer, wash, and staining steps that can be time consuming, expensive, and led to the loss of rare cells. To overcome the limitations of existing CTC isolation strategies, we have developed an inexpensive 'lab on a chip' device for the enrichment, staining, and analysis of rare cell populations. This device utilizes immunomagnetic positive selection of antibody-bound cells, isolation of cells through an immiscible interface, and filtration. The isolated cells can then be stained utilizing immunofluorescence or used for other downstream detection methods. We describe the construction and initial preclinical testing of the device. Initial tests suggest that the device may be well suited for the isolation of CTCs and could allow the monitoring of cancer progression and the response to therapy over time.
Assuntos
Separação Imunomagnética/instrumentação , Separação Imunomagnética/métodos , Células Neoplásicas Circulantes/patologia , Linhagem Celular Tumoral , Humanos , Dispositivos Lab-On-A-Chip , Masculino , Neoplasias da Próstata/patologia , Propriedades de SuperfícieRESUMO
This paper describes the design and fabrication of doxorubicin (Dox)-conjugated nanodiamond (ND) clusters with controlled sizes and cellular uptake behaviors of free Dox and Dox-conjugated ND clusters. The ND clusters with an average size of 45.84 nm exhibited a higher amount of cellular uptake as compared to the ND clusters with larger sizes. The amount of Dox taken up as free Dox increased initially and then decreased over time. In contrast, the amount of Dox taken up as Dox-ND clusters continuously increased and reached a plateau, resulting in high ablation efficiency. At the same Dox concentration, the cell viabilities after treatment with free Dox and Dox-ND clusters were 26.38 and 5.31%, respectively. The Dox-ND clusters potentially could be employed as efficient drug carriers for efficient cancer therapy.
Assuntos
Doxorrubicina/uso terapêutico , Endocitose/efeitos dos fármacos , Nanodiamantes/química , Neoplasias/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Humanos , Microfluídica , Nanodiamantes/ultraestrutura , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade EstáticaRESUMO
The purpose of this study was to fabricate BMP-2-immobilized porous poly(lactide-co-glycolide) (PLGA) microspheres (PMS) modified with heparin for bone regeneration. A fluidic device was used to fabricate PMS and the fabricated PMS was modified with heparin-dopamine (Hep-DOPA). Bone morphogenic protein-2 (BMP-2) was immobilized on the heparinized PMS (Hep-PMS) via electrostatic interactions. Both PMS and modified PMS were characterized using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). MG-63 cell activity on PMS and modified PMS were assessed via alkaline phosphatase (ALP) activity, calcium deposition, and osteocalcin and osteopontin mRNA expression. Immobilized Hep-DOPA and BMP-2 on PMS were demonstrated by XPS analysis. BMP-2-immobilized Hep-PMS provided significantly higher ALP activity, calcium deposition, and osteocalcin and osteopontin mRNA expression compared to PMS alone. These results suggest that BMP-2-immobilized Hep-PMS effectively improves MG-63 cell activity. In conclusion, BMP-2-immobilized Hep-PMS can be used to effectively regenerate bone defects.
Assuntos
Desenvolvimento Ósseo , Microesferas , Engenharia Tecidual , Linhagem Celular , Perfilação da Expressão Gênica , HumanosRESUMO
The aim of this study was to develop lactoferrin (LF)-impregnated porous poly(lactide-co-glycolide) (PLGA) microspheres (PMs) to induce osteogenic differentiation of rabbit adipose-derived stem cells (rADSCs). Porous PLGA PMs were fabricated by a fluidic device and their surfaces were modified with heparin-dopamine (Hep-DOPA). Then, LF (100µg, 500µg, and 1000µg) was impregnated on the surface of heparinized PMs (Hep-PMs) via electrostatic interactions to yield LF-impregnated PMs. PMs and modified PMs were characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Osteogenic differentiation of rADSCs on PMs and modified PMs was demonstrated by alkaline phosphatase (ALP) activity, calcium deposition, and mRNA expression of osteocalcin and osteopontin. Successful immobilization of Hep-DOPA and LF on the surface of PMs was confirmed by XPS analysis. LF-impregnated PMs generated significantly greater ALP activity, calcium deposition, and mRNA expression of osteocalcin and osteopontin compared with PMs. These results suggested that LF-impregnated PMs effectively induced osteogenic differentiation of rADSCs.