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1.
Biomed Microdevices ; 25(3): 21, 2023 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-37284878

RESUMO

In recent years biomedical scientific community has been working towards the development of high-throughput devices that allow a reliable, rapid and parallel detection of several strains of virus or microparticles simultaneously. One of the complexities of this problem lies on the rapid prototyping of new devices and wireless rapid detection of small particles and virus alike. By reducing the complexity of microfluidics microfabrication and using economic materials along with makerspace tools (Kundu et al. 2018) it is possible to provide an affordable solution to both the problems of high-throughput devices and detection technologies. We present the development of a wireless, standalone device and disposable microfluidics chips that rapidly generate parallel readouts for selected, possible virus variants from a nasal or saliva sample, based on motorized and non-motorized microbeads detection, and imaging processing of the motion tracks of these beads in micrometers. Microbeads and SARS-CoV-2 COVID-19 Delta variant were tested as proof-of-concept for testing the microfluidic cartridges and wireless imaging module. The Microbead Assay (MA) system kit consists of a Wi-Fi readout module, a microfluidic chip, and a sample collection/processing sub-system. Here, we focus on the fabrication and characterization of the microfluidic chip to multiplex various micrometer-sized beads for economic, disposable, and simultaneous detection of up to six different viruses, microparticles or variants in a single test, and data collection using a commercially available, Wi-Fi-capable, and camera integrated device (Fig. 1).


Assuntos
COVID-19 , Técnicas Analíticas Microfluídicas , Humanos , Microfluídica , Microesferas , Análise Custo-Benefício , SARS-CoV-2 , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos
2.
Adv Funct Mater ; 29(8)2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-35586798

RESUMO

The goal of human-on-a-chip systems is to capture multi-organ complexity and predict the human response to compounds within physiologically relevant platforms. The generation and characterization of such systems is currently a focal point of research given the long-standing inadequacies of conventional techniques for predicting human outcome. Functional systems can measure and quantify key cellular mechanisms that correlate with the physiological status of a tissue, and can be used to evaluate therapeutic challenges utilizing many of the same endpoints used in animal experiments or clinical trials. Culturing multiple organ compartments in a platform creates a more physiologic environment (organ-organ communication). Here is reported a human 4-organ system composed of heart, liver, skeletal muscle and nervous system modules that maintains cellular viability and function over 28 days in serum-free conditions using a pumpless system. The integration of non-invasive electrical evaluation of neurons and cardiac cells and mechanical determination of cardiac and skeletal muscle contraction allows the monitoring of cellular function especially for chronic toxicity studies in vitro. The 28 day period is the minimum timeframe for animal studies to evaluate repeat dose toxicity. This technology could be a relevant alternative to animal testing by monitoring multi-organ function upon long term chemical exposure.

3.
Res Sq ; 2022 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-36597542

RESUMO

In recent years biomedical scientific community has been working towards the development of high-throughput devices that allow a reliable, rapid and parallel detection of several strains of virus or microparticles simultaneously. One of the complexities of this problem lies on the rapid prototyping of new devices and wireless rapid detection of small particles and virus alike. By reducing the complexity of microfluidics microfabrication and using economic materials along with makerspace tools (Avra Kundu, Ausaf, and Rajaraman 2018) it is possible to provide an affordable solution to both the problems of high-throughput devices and detection technologies. We present the development of a wireless, standalone device and disposable microfluidics chips that rapidly generate parallel readouts for selected, possible virus variants from a nasal or saliva sample, based on motorized and non-motorized microbeads detection, and imaging processing of the motion tracks of these beads in micrometers. Microbeads and SARS-CoV-2 COVID-19 Delta variant were tested as proof-of-concept for testing the microfluidic cartridges and wireless imaging module. The Microbead Assay (MA) system kit consists of a WiFi readout module, a microfluidic chip, and a sample collection/processing sub-system. Here, we focus on the fabrication and characterization of the microfluidic chip to multiplex various micrometer-sized beads for economic, disposable, and simultaneous detection of up to six different viruses, microparticles or variants in a single test, and data collection using a commercially available, WiFi-capable, and camera integrated device (Fig. 1).

4.
Biosensors (Basel) ; 10(11)2020 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-33105886

RESUMO

The widespread adaptation of 3D printing in the microfluidic, bioelectronic, and Bio-MEMS communities has been stifled by the lack of investigation into the biocompatibility of commercially available printer resins. By introducing an in-depth post-printing treatment of these resins, their biocompatibility can be dramatically improved up to that of a standard cell culture vessel (99.99%). Additionally, encapsulating resins that are less biocompatible with materials that are common constituents in biosensors further enhances the biocompatibility of the material. This investigation provides a clear pathway toward developing fully functional and biocompatible 3D printed biosensor devices, especially for interfacing with electrogenic cells, utilizing benchtop-based microfabrication, and post-processing techniques.


Assuntos
Materiais Biocompatíveis , Impressão Tridimensional , Técnicas de Cultura de Células , Microfluídica , Polímeros
5.
RSC Adv ; 10(68): 41577-41587, 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-35516576

RESUMO

Microfabrication and assembly of a Three-Dimensional Microneedle Electrode Array (3D MEA) based on a glass-stainless steel platform is demonstrated involving the utilization of non-traditional "Makerspace Microfabrication" techniques featuring cost-effective, rapid fabrication and an assorted biocompatible material palette. The stainless steel microneedle electrode array was realized by planar laser micromachining and out-of-plane transitioning to have a 3D configuration with perpendicular transition angles. The 3D MEA chip is bonded onto a glass die with metal traces routed to the periphery of the chip for electrical interfacing. Confined precision drop casting (CPDC) of PDMS is used to define an insulation layer and realize the 3D microelectrodes. The use of glass as a substrate offers optical clarity allowing for simultaneous optical and electrical probing of electrogenic cells. Additionally, an interconnect using 3D printing and conductive ink casting has been developed which allows metal traces on the glass chip to be transitioned to the bottomside of the device for interfacing with commercial data acquisition/analysis equipment. The 3D MEAs demonstrate an average impedance/phase of ∼13.3 kΩ/-12.1° at 1 kHz respectively, and an average 4.2 µV noise. Lastly, electrophysiological activity from an immortal cardiomyocyte cell line was recorded using the 3D MEA demonstrating end to end device development.

6.
Alzheimers Dement (N Y) ; 6(1): e12029, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32490141

RESUMO

INTRODUCTION: The quest to identify an effective therapeutic strategy for neurodegenerative diseases, such as mild congitive impairment (MCI) and Alzheimer's disease (AD), suffers from the lack of good human-based models. Animals represent the most common models used in basic research and drug discovery studies. However, safe and effective compounds identified in animal studies often translate poorly to humans, yielding unsuccessful clinical trials. METHODS: A functional in vitro assay based on long-term potentiation (LTP) was used to demonstrate that exposure to amyloid beta (Aß42) and tau oligomers, or brain extracts from AD transgenic mice led to prominent changes in human induced pluripotent stem cells (hiPSC)-derived cortical neurons, notably, without cell death. RESULTS: Impaired information processing was demonstrated by treatment of neuron-MEA (microelectrode array) systems with the oligomers and brain extracts by reducing the effects of LTP induction. These data confirm the neurotoxicity of molecules linked to AD pathology and indicate the utility of this human-based system to model aspects of AD in vitro and study LTP deficits without loss of viability; a phenotype that more closely models the preclinical or early stage of AD. DISCUSSION: In this study, by combining multiple relevant and important molecular and technical aspects of neuroscience research, we generated a new, fully human in vitro system to model and study AD at the preclinical stage. This system can serve as a novel drug discovery platform to identify compounds that rescue or alleviate the initial neuronal deficits caused by Aß42 and/or tau oligomers, a main focus of clinical trials.

7.
J Tissue Eng Regen Med ; 13(3): 385-395, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30636354

RESUMO

Microelectrode arrays (MEAs) have become important tools in high throughput assessment of neuronal activity. However, geometric and electrical constraints largely limit their ability to detect action potentials to the neuronal soma. Enhancing the resolution of these systems to detect axonal action potentials has proved both challenging and complex. In this study, we have bundled sensory axons from dorsal root ganglia through a capillary alginate gel (Capgel™) interfaced with an MEA and observed an enhanced ability to detect spontaneous axonal activity compared with two-dimensional cultures. Moreover, this arrangement facilitated the long-term monitoring of spontaneous activity from the same bundle of axons at a single electrode. Finally, using waveform analysis for cultures treated with the nociceptor agonist capsaicin, we were able to dissect action potentials from multiple axons on an individual electrode, suggesting that this model can reproduce the functional complexity associated with sensory fascicles in vivo. This novel three-dimensional functional model of the peripheral nerve can be used to study the functional complexities of peripheral neuropathies and nerve regeneration as well as being utilized in the development of novel therapeutics.


Assuntos
Potenciais de Ação/fisiologia , Alginatos/farmacologia , Axônios/fisiologia , Géis/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Capsaicina/farmacologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/fisiologia , Microeletrodos , Ratos
8.
Biomaterials ; 29(29): 3927-35, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18621415

RESUMO

Three-dimensional surface reconstructions from high resolution image stacks of biological specimens, observed by confocal microscopy, have changed the perspective of morphological understanding. In the field of cell-cell or cell-substrate interfaces, combining these two techniques leads to new insights yet also creates a tremendous amount of data. In this article, we present a technique to reduce large, multidimensional data sets from confocal microscopy into one single curve: a membrane allocation profile. Reconstructed cells are represented in a three-dimensional surface from image sections of individual cells. We virtually cut segments of the reconstructed cell membrane parallel to the substrate and calculate the surface areas of each segment. The obtained membrane allocation profiles lead to morphological insights and yield an in vivo ratio of attached and free membrane areas without cell fixation. As an example, glass substrates were modified with different proteins (fibronectin, laminin, concavalin A, extracellular matrix gel, and both isomers of poly-lysine) and presented to HEK293 cells to examine differences in cell morphology and adhesion. We proved that proteins on a substrate could increase the attached portion of a cell membrane, facing the modified substrate, from an average of 32% (glass) to 45% (poly-lysine) of the total membrane surface area.


Assuntos
Adesão Celular , Membrana Celular/metabolismo , Forma Celular , Matriz Extracelular/química , Imageamento Tridimensional , Linhagem Celular , Membrana Celular/química , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Propriedades de Superfície
9.
J R Soc Interface ; 5(19): 213-22, 2008 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-17609177

RESUMO

An emerging number of micro- and nanoelectronics-based biosensors have been developed for non-invasive recordings of physiological cellular activity. The interface between the biological system and the electronic devices strongly influences the signal transfer between these systems. Little is known about the nanoscopic structure of the cell-sensor interface that is essential for a detailed interpretation of the recordings. Therefore, we analysed the interface between the sensor surface and attached cells using transmission electron microscopy (TEM). The maximum possible resolution of our TEM study, however, was restricted by the quality of the interface preparation. Therefore, we complemented our studies with imaging ellipsometry. We cultured HEK293 cells on substrates, which had been precoated with different types of proteins. We found that contact geometry between attached cell membrane and substrate was dependent on the type of protein coating used. In the presence of polylysine, the average distance of the membrane-substrate interface was in the range of 35-40 nm. However, the cell membrane was highly protruded in the presence of other proteins like fibronectin, laminin or concanavalin-A. The presented method allows the nanoscopic characterization of the cell-sensor interface.


Assuntos
Membrana Celular/ultraestrutura , Células Epiteliais/citologia , Adesão Celular/fisiologia , Linhagem Celular , Membrana Celular/química , Humanos , Microscopia Eletrônica de Transmissão , Proteínas/metabolismo , Silício , Propriedades de Superfície
10.
Biomaterials ; 166: 64-78, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29547745

RESUMO

There are currently no functional neuromuscular junction (hNMJ) systems composed of human cells that could be used for drug evaluations or toxicity testing in vitro. These systems are needed to evaluate NMJs for diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy or other neurodegenerative diseases or injury states. There are certainly no model systems, animal or human, that allows for isolated treatment of motoneurons or muscle capable of generating dose response curves to evaluate pharmacological activity of these highly specialized functional units. A system was developed in which human myotubes and motoneurons derived from stem cells were cultured in a serum-free medium in a BioMEMS construct. The system is composed of two chambers linked by microtunnels to enable axonal outgrowth to the muscle chamber that allows separate stimulation of each component and physiological NMJ function and MN stimulated tetanus. The muscle's contractions, induced by motoneuron activation or direct electrical stimulation, were monitored by image subtraction video recording for both frequency and amplitude. Bungarotoxin, BOTOX® and curare dose response curves were generated to demonstrate pharmacological relevance of the phenotypic screening device. This quantifiable functional hNMJ system establishes a platform for generating patient-specific NMJ models by including patient-derived iPSCs.


Assuntos
Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Junção Neuromuscular , Engenharia Tecidual , Técnicas de Cocultura , Avaliação Pré-Clínica de Medicamentos/métodos , Estimulação Elétrica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios Motores/citologia , Contração Muscular , Fibras Musculares Esqueléticas/citologia
11.
ACS Biomater Sci Eng ; 3(12): 3525-3533, 2017 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-29250595

RESUMO

Microelectrode arrays (MEAs) are innovative tools used to perform electrophysiological experiments for the study of electrical activity and connectivity in populations of neurons from dissociated cultures. Reliance upon neurons derived from embryonic tissue is a common limitation of neuronal/MEA hybrid systems and perhaps of neuroscience research in general, and the use of adult neurons could model fully functional in vivo parameters more closely. Spontaneous network activity was concurrently recorded from both embryonic and adult rat neurons cultured on MEAs for up to 10 weeks in vitro to characterize the synaptic connections between cell types. The cultures were exposed to synaptic transmission antagonists against NMDA and AMPA channels, which revealed significantly different receptor profiles of adult and embryonic networks in vitro. In addition, both embryonic and adult neurons were evaluated for NMDA and AMPA channel subunit expression over five weeks in vitro. The results established that neurons derived from embryonic tissue did not express mature synaptic channels for several weeks in vitro under defined conditions. Consequently, the embryonic response to synaptic antagonists was significantly different than that of neurons derived from adult tissue sources. These results are especially significant because most studies reported with embryonic hippocampal neurons do not begin at two to four weeks in culture. In addition, the utilization of MEAs in lieu of patch-clamp electrophysiology avoided a large-scale, labor-intensive study. These results establish the utility of this unique hybrid system derived from adult hippocampal tissue in combination with MEAs and offer a more appropriate representation of in vivo function for drug discovery. It has application for neuronal development and regeneration as well as for investigations into neurodegenerative disease, traumatic brain injury, and stroke.

12.
Biomaterials ; 32(18): 4267-74, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21453966

RESUMO

Cardiac side effects are one of the major causes of drug candidate failures in preclinical drug development or in clinical trials and are responsible for the retraction of several already marketed therapeutics. Thus, the development of a relatively high-throughput, high information content tool to screen drugs and toxins would be important in the field of cardiac research and drug development. In this study, recordings from commercial multielectrode arrays were combined with surface patterning of cardiac myocyte monolayers to enhance the information content of the method; specifically, to enable the measurement of conduction velocity, refractory period after action potentials and to create a functional re-entry model. Two drugs, 1-Heptanol, a gap junction blocker, and Sparfloxacin, a fluoroquinone antibiotic, were tested in this system. 1-Heptanol administration resulted in a marked reduction in conduction velocity, whereas Sparfloxacin caused rapid, irregular and unsynchronized activity, indicating fibrillation. As shown in these experiments, patterning of cardiac myocyte monolayers solved several inherent problems of multielectrode recordings, increased the temporal resolution of conduction velocity measurements, and made the synchronization of external stimulation with action potential propagation possible for refractory period measurements. This method could be further developed as a cardiac side effect screening platform after combination with human cardiomyocytes.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Microeletrodos , Miócitos Cardíacos/citologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Antituberculosos/farmacologia , Células Cultivadas , Ensaios Clínicos como Assunto , Meios de Cultura Livres de Soro , Fluoroquinolonas/farmacologia , Heptanol/farmacologia , Humanos
13.
J Pharmacol Toxicol Methods ; 59(3): 146-52, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19268710

RESUMO

INTRODUCTION: Simultaneous recording of electrical potentials from multiple cells may be useful for physiological and pharmacological research. The present study aimed to establish an in vitro cardiac hypoxia experimental platform on the microelectrode array (MEA). METHODS: Embryonic rat cardiac myocytes were cultured on the MEAs. Following >or=90 min of hypoxia, changes in lactate production (mM), pH, beat frequency (beats per min, bpm), extracellular action potential (exAP) amplitude, and propagation velocity between the normoxic and hypoxic cells were compared. RESULTS: Under hypoxia, the beat frequency of cells increased and peaked at around 42.5 min (08.1+/-3.2 bpm). The exAP amplitude reduced as soon as the cells were exposed to the hypoxic medium, and this reduction increased significantly after approximately 20 min of hypoxia. The propagation velocity of the hypoxic cells was significantly lower than that of the control throughout the entire 90+ min of hypoxia. The rate of depolarisation and Na(+) signal gradually reduced over time, and these changes had a direct effect on the exAP duration. DISCUSSION: The extracellular electrophysiological measurements allow a partial reconstruction of the signal shape and time course of the underlying hypoxia-associated physiological changes. The present study showed that the cardiac myocyte-integrated MEA may be used as an experimental platform for the pharmacological studies of cardiovascular diseases in the future.


Assuntos
Miócitos Cardíacos/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Hipóxia Celular , Células Cultivadas , Eletrofisiologia , Coração/efeitos dos fármacos , Coração/fisiologia , Concentração de Íons de Hidrogênio , Lactatos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Microeletrodos , Miócitos Cardíacos/efeitos dos fármacos , Concentração Osmolar , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley
14.
Nano Lett ; 6(3): 453-7, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16522041

RESUMO

A biohybrid system composed of neuronal cells and silicon-supported nanoporous membranes has been designed to facilitate control of the biochemical environment of neuronal networks with cellular resolution. The membranes may exhibit variable pore sizes and interpore distances and are interfaced to a microfluidic device. Different porosity parameters give rise to changes in the transconductance of the nanopores and can therefore be used to control diffusion of molecules through the membranes. It was shown that the porous membranes are biocompatible with primary vertebrate as well as insect neurons. Our results indicate that nanoporous membranes may be used to interface with biological materials in a biohybrid system, for example as an artificial chemical synapse interface.


Assuntos
Modelos Biológicos , Nanoestruturas/química , Neurônios/metabolismo , Sinapses/metabolismo , Animais , Linhagem Celular , Humanos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Nanoestruturas/ultraestrutura , Porosidade , Ratos , Silício/química
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