RESUMO
Heart disease remains a leading cause of global mortality, underscoring the need for advanced technologies to study cardiovascular diseases and develop effective treatments. We introduce an innovative interferometric biosensor for high-sensitivity and label-free recording of human induced pluripotent stem cell (hiPSC) cardiomyocyte contraction in vitro. Using an optical cavity, our device captures interference patterns caused by the contraction-induced displacement of a thin flexible membrane. First, we demonstrate the capability to quantify spontaneous contractions and discriminate between contraction and relaxation phases. We calculate a contraction-induced vertical membrane displacement close to 40 nm, which implies a traction stress of 34 ± 4 mN/mm2. Finally, we investigate the effects of a drug compound on contractility amplitude, revealing a significant reduction in contractile forces. The label-free and high-throughput nature of our biosensor may enhance drug screening processes and drug development for cardiac treatments. Our interferometric biosensor offers a novel approach for noninvasive and real-time assessment of cardiomyocyte contraction.
Assuntos
Técnicas Biossensoriais , Células-Tronco Pluripotentes Induzidas , Interferometria , Contração Miocárdica , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Interferometria/instrumentação , Contração Miocárdica/efeitos dos fármacosRESUMO
Bioelectrical variations trigger different cell responses, including migration, mitosis, and mutation. At the tissue level, these actions result in phenomena such as wound healing, proliferation, and pathogenesis. Monitoring these mechanisms dynamically is highly desirable in diagnostics and drug testing. However, existing technologies are invasive: either they require physical access to the intracellular compartments, or they imply direct contact with the cellular medium. Here, we present a novel approach for the passive recording of electrical signals from non-excitable cells adhering to 3D microelectrodes, based on optical mirroring. Preliminary results yielded a fluorescence intensity output increase of the 5,8% in the presence of a HEK-293 cell on the electrode compared to bare microelectrodes. At present, this technology may be employed to evaluate cell-substrate adhesion and monitor cell proliferation. Further refinements could allow extrapolating quantitative data on surface charges and resting potential to investigate the electrical phenomena involved in cell migration and cancer progression.
Assuntos
Neoplasias , Humanos , Células HEK293 , Neoplasias/patologia , Potenciais da Membrana , Adesão Celular , MicroeletrodosRESUMO
The reliable identification of chronic cardiotoxic effects in in vitro screenings is fundamental for filtering out toxic molecular entities before in vivo animal experimentation and clinical trials. Present techniques such as patch-clamp, voltage indicators, and standard microelectrode arrays do not offer at the same time high sensitivity for measuring transmembrane ion currents and low-invasiveness for monitoring cells over long time. Here, we show that optoporation applied to microelectrode arrays enables measuring action potentials from human-derived cardiac syncytia for more than 1 continuous month and provides reliable data on chronic cardiotoxic effects caused by known compounds such as pentamidine. The technique has high potential for detecting chronic cardiotoxicity in the early phases of drug development.
Assuntos
Cardiotoxicidade , Miócitos Cardíacos , Animais , Humanos , Potenciais de Ação , MicroeletrodosRESUMO
Drug-induced cardiotoxicity is a major barrier to drug development and a main cause of withdrawal of marketed drugs. Drugs can strongly alter the spontaneous functioning of the heart by interacting with the cardiac membrane ion channels. If these effects only surface during in vivo preclinical tests, clinical trials or worse after commercialization, the societal and economic burden will be significant and seriously hinder the efficient drug development process. Hence, cardiac safety pharmacology requires in vitro electrophysiological screening assays of all drug candidates to predict cardiotoxic effects before clinical trials. In the past 10 years, microelectrode array (MEA) technology began to be considered a valuable approach in pharmaceutical applications. However, an effective tool for high-throughput intracellular measurements, compatible with pharmaceutical standards, is not yet available. Here, we propose laser-induced optoacoustic poration combined with CMOS-MEA technology as a reliable and effective platform to detect cardiotoxicity. This approach enables the acquisition of high-quality action potential recordings from large numbers of cardiomyocytes within the same culture well, providing reliable data using single-well MEA devices and single cardiac syncytia per each drug. Thus, this technology could be applied in drug safety screening platforms reducing times and costs of cardiotoxicity assessments, while simultaneously improving the data reliability.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Arritmias Cardíacas/induzido quimicamente , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Lasers , Microeletrodos , Miócitos Cardíacos/efeitos dos fármacos , Técnicas Fotoacústicas/instrumentação , Testes de Toxicidade/instrumentação , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Cardiotoxicidade , Redução de Custos , Análise Custo-Benefício , Frequência Cardíaca/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microeletrodos/economia , Miócitos Cardíacos/metabolismo , Técnicas Fotoacústicas/economia , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Tempo , Testes de Toxicidade/economia , Fluxo de TrabalhoRESUMO
Delivery of molecules into intracellular compartments is one of the fundamental requirements in molecular biology. However, the possibility of delivering a precise number of nano-objects with single-particle resolution is still an open challenge. Here we present an electrophoretic platform based on 3D hollow nanoelectrodes to enable delivery of single nanoparticles into single selected cells and monitoring of the single-particle delivery by surface-enhanced Raman scattering (SERS). The gold-coated hollow nanoelectrode capable of confinement and enhancement of electromagnetic fields upon laser illumination can distinguish the SERS signals of a single nanoparticle flowing through the nanoelectrode. Tight wrapping of cell membranes around the nanoelectrodes allows effective membrane electroporation such that single gold nanorods are delivered on demand into a living cell by electrophoresis. The capability of the 3D hollow nanoelectrodes to porate cells and reveal single emitters from the background in continuous flow is promising for the analysis of both intracellular delivery and sampling.
RESUMO
The dynamic interface between the cellular membrane and 3D nanostructures determines biological processes and guides the design of novel biomedical devices. Despite the fact that recent advancements in the fabrication of artificial biointerfaces have yielded an enhanced understanding of this interface, there remain open questions on how the cellular membrane reacts and behaves in the presence of sharp objects on the nanoscale. Here we provide a multifaceted characterization of the cellular membrane's mechanical stability when closely interacting with high-aspect-ratio 3D vertical nanostructures, providing strong evidence that vertical nanostructures spontaneously penetrate the cellular membrane to form a steady intracellular coupling only in rare cases and under specific conditions. The cell membrane is able to conform tightly over the majority of structures with various shapes while maintaining its integrity.
Assuntos
Adesão Celular , Nanoestruturas/ultraestrutura , Fenômenos Biomecânicos , Linhagem Celular , Membrana Celular/ultraestrutura , Forma Celular , Eletroporação , Células HEK293 , Humanos , Miócitos Cardíacos/citologia , Nanoestruturas/química , Nanotecnologia , Propriedades de SuperfícieRESUMO
Adsorption on nanoporous matrices is characterized by a pronounced hysteresis loop in the adsorption isotherm, when the substrate is loaded and unloaded with adsorbate, the origin of which is a matter of immense debate in the literature. In this work, we report a study of argon adsorption at 85 K on nonconnecting nanopores with one end closed to the surrounding where the effects of different pore cross sections fabricated by electron beam lithography (EBL) are investigated. A polymethylmethacrylate (PMMA) resist is deposited on the electrodes of a sensitive quartz crystal microbalance without degradation of the resonance quality factor or the long-term and short-term stabilities of the device even at cryogenic temperatures. Four different pores' cross sections: circular, square, rectangular, and triangular, are produced from EBL, and the isotherms for these pore shapes exhibit pronounced hysteresis loops whose adsorption and desorption branches are nearly vertical and have almost the same slopes. No difference is observed in the hysteresis loops of the isotherms for the pores with triangular and square cross sections, whereas the hysteresis loop for the pore with circular cross sections is much narrower, suggesting that they are more regular than the other pores. All of these observations suggest that the hysteresis behavior resulted mainly from microscopic geometric irregularities present in these porous matrices.
RESUMO
Three-dimensional vertical micro- and nanostructures can enhance the signal quality of multielectrode arrays and promise to become the prime methodology for the investigation of large networks of electrogenic cells. So far, access to the intracellular environment has been obtained via spontaneous poration, electroporation, or by surface functionalization of the micro/nanostructures; however, these methods still suffer from some limitations due to their intrinsic characteristics that limit their widespread use. Here, we demonstrate the ability to continuously record both extracellular and intracellular-like action potentials at each electrode site in spontaneously active mammalian neurons and HL-1 cardiac-derived cells via the combination of vertical nanoelectrodes with plasmonic optoporation. We demonstrate long-term and stable recordings with a very good signal-to-noise ratio. Additionally, plasmonic optoporation does not perturb the spontaneous electrical activity; it permits continuous recording even during the poration process and can regulate extracellular and intracellular contributions by means of partial cellular poration.
Assuntos
Técnicas Eletroquímicas/métodos , Miócitos Cardíacos/fisiologia , Nanoestruturas/química , Neurônios/fisiologia , Potenciais de Ação , Animais , Citoplasma/metabolismo , Espaço Extracelular/fisiologia , Espaço Intracelular/fisiologia , Microeletrodos , Fenômenos Físicos , Ratos Sprague-Dawley , Razão Sinal-RuídoRESUMO
Janus particles can self-assemble around microfabricated gears in reproducible configurations with a high degree of spatial and orientational order. The final configuration maximizes the torque applied on the rotor leading to a unidirectional and steady rotating motion. The interplay between geometry and dynamical behavior leads to the self-assembly of Janus micromotors starting from randomly distributed particles.
RESUMO
Out-of-plane plasmonic nanoantennas protruding from the substrate are exploited to perform very sensitive surface enhanced Raman scattering analysis of living cells. Cells cultured on three-dimensional surfaces exhibit tight adhesion with nanoantenna tips where the plasmonic hot-spot resides. This fact provides observable cell adhesion sites combined with high plasmonic enhancement, resulting in an ideal system for Raman investigation of cell membranes.
Assuntos
Nanoestruturas , Análise Espectral Raman/métodos , Actinas/química , Animais , Adesão Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Sobrevivência Celular , Campos Eletromagnéticos , Ouro/química , Nanopartículas Metálicas , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Células NIH 3T3 , Nanotecnologia , Tamanho da Partícula , Prata/química , Software , Espectroscopia de Luz Próxima ao Infravermelho , Ressonância de Plasmônio de SuperfícieRESUMO
Every test procedure, scientific and non-scientific, has inherent uncertainties, even when performed according to a standard operating procedure (SOP). In addition, it is prone to errors, defects, and mistakes introduced by operators, laboratory equipment, or materials used. Adherence to an SOP and comprehensive validation of the test method cannot guarantee that each test run produces data within the acceptable range of variability and with the precision and accuracy determined during the method validation. We illustrate here (part I) why controlling the validity of each test run is an important element of experimental design. The definition and application of acceptance criteria (AC) for the validity of test runs is important for the setup and use of test methods, particularly for the use of new approach methods (NAM) in toxicity testing. AC can be used for decision rules on how to handle data, e.g., to accept the data for further use (AC fulfilled) or to reject the data (AC not fulfilled). The adherence to AC has important requirements and consequences that may seem surprising at first sight: (i) AC depend on a test method's objectives, e.g., on the types/concentrations of chemicals tested, the regulatory context, the desired throughput; (ii) AC are applied and documented at each test run, while validation of a method (including the definition of AC) is only performed once; (iii) if AC are altered, then the set of data produced by a method can change. AC, if missing, are the blind spot of quality assurance: Test results may not be reliable and comparable. The establishment and uses of AC will be further detailed in part II of this series.
Assuntos
Disciplinas das Ciências Biológicas , Testes de Toxicidade , Humanos , Projetos de PesquisaRESUMO
Optical stimulation technologies are gaining great consideration in cardiology, neuroscience studies, and drug discovery pathways by providing control over cell activity with high spatio-temporal resolution. However, this high precision requires manipulation of biological processes at genetic level concealing its development from broad scale application. Therefore, translating these technologies into tools for medical or pharmacological applications remains a challenge. Here, an all-optical nongenetic method for the modulation of electrogenic cells is introduced. It is demonstrated that plasmonic metamaterials can be used to elicit action potentials by converting near infrared laser pulses into stimulatory currents. The suggested approach allows for the stimulation of cardiomyocytes and neurons directly on commercial complementary metal-oxide semiconductor microelectrode arrays coupled with ultrafast pulsed laser, providing both stimulation and network-level recordings on the same device.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Raios Infravermelhos , Miócitos Cardíacos/fisiologia , Nanoestruturas/toxicidade , Neurônios/fisiologia , Potenciais de Ação/efeitos da radiação , Animais , Linhagem Celular , Humanos , Metais/química , Camundongos , Microeletrodos , Miócitos Cardíacos/citologia , Nanoestruturas/química , Neurônios/citologia , Porosidade , Ratos , Semicondutores , Dióxido de Silício/químicaRESUMO
The electrophysiological recording of action potentials in human cells is a long-sought objective due to its pivotal importance in many disciplines. Among the developed techniques, invasiveness remains a common issue, causing cytotoxicity or altering unpredictably cell physiological response. In this work, a new approach for recording intracellular signals of outstanding quality and with noninvasiveness is introduced. By taking profit of the concept of mirror charge in classical electrodynamics, the new proposed device transduces cell ionic currents into mirror charges in a microfluidic chamber, thus realizing a virtual mirror cell. By monitoring mirror charge dynamics, it is possible to effectively record the action potentials fired by the cells. Since there is no need for accessing or interacting with the cells, the method is intrinsically noninvasive. In addition, being based on optical recording, it shows high spatial resolution and high parallelization. As shown through a set of experiments, the presented methodology is an ideal candidate for the next generation devices for the reliable assessment of cardiotoxicity on human-derived cardiomyocytes. More generally, it paves the way toward a new family of in vitro biodevices that will lay a new milestone in the field of electrophysiology.
Assuntos
Potenciais de Ação/fisiologia , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Fenômenos Eletrofisiológicos/fisiologia , Miócitos Cardíacos/fisiologia , Materiais Biocompatíveis/química , Linhagem Celular , Equipamentos e Provisões , Humanos , Microeletrodos , Nanoestruturas/química , Compostos de Silício/química , Propriedades de Superfície , Imagens com Corantes Sensíveis à VoltagemRESUMO
Graphene with its unique electrical properties is a promising candidate for carbon-based biosensors such as microelectrodes and field effect transistors. Recently, graphene biosensors were successfully used for extracellular recording of action potentials in electrogenic cells; however, intracellular recordings remain beyond their current capabilities because of the lack of an efficient cell poration method. Here, we present a microelectrode platform consisting of out-of-plane grown three-dimensional fuzzy graphene (3DFG) that enables recording of intracellular cardiac action potentials with high signal-to-noise ratio. We exploit the generation of hot carriers by ultrafast pulsed laser for porating the cell membrane and creating an intimate contact between the 3DFG electrodes and the intracellular domain. This approach enables us to detect the effects of drugs on the action potential shape of human-derived cardiomyocytes. The 3DFG electrodes combined with laser poration may be used for all-carbon intracellular microelectrode arrays to allow monitoring of the cellular electrophysiological state.
RESUMO
High quality attenuated intracellular action potentials from large cell networks can be recorded on multi-electrode arrays by means of 3D vertical nanopillars using electrical pulses. However, most of the techniques require complex 3D nanostructures that prevent the straightforward translation into marketable products and the wide adoption in the scientific community. Moreover, 3D nanostructures are often delicate objects that cannot sustain several harsh use/cleaning cycles. On the contrary, laser optoacoustic poration allows the recording of action potentials on planar nanoporous electrodes made of noble metals. However, these constraints of the electrode material and morphology may also hinder the full exploitation of this methodology. Here, we show that optoacoustic poration is also very effective for porating cells on a large family of MEA electrode configurations, including robust electrodes made of nanoporous titanium nitride or disordered fractal-like gold nanostructures. This enables the recording of high quality cardiac action potentials in combination with optoacoustic poration, providing thus attenuated intracellular recordings on various already commercial devices used by a significant part of the research and industrial communities.
RESUMO
Neuropathological models and neurological disease progression and treatments have always been of great interest in biomedical research because of their impact on society. The application of in vitro microfluidic devices to neuroscience-related disciplines provided several advancements in therapeutics or neuronal modeling thanks to the ability to control the cellular microenvironment at spatiotemporal level. Recently, the introduction of three-dimensional nanostructures has allowed high performance in both in vitro recording of electrogenic cells and drug delivery using minimally invasive devices. Independently, both delivery and recording have let to pioneering solutions in neurobiology. However, their combination on a single chip would provide further fundamental improvements in drug screening systems and would offer comprehensive insights into pathologies and diseases progression. Therefore, it is crucial to develop platforms able to monitor progressive changes in electrophysiological behavior in the electrogenic cellular network, induced by spatially localized injection of biochemical agents. In this work, we show the application of a microfluidic multielectrode array (MEA) platform to record spontaneous and chemically stimulated activity in primary neuronal networks. By means of spatially localized caffeine injection via microfluidic nanochannels, the device demonstrated its capability of combined localized drug delivery and cell signaling recording. The platform could detect activity of the neural network at multiple sites while delivering molecules into just a few selected cells, thereby examining the effect of biochemical agents on the desired portion of cell culture.
RESUMO
Inherited retinal dystrophies and late-stage age-related macular degeneration, for which treatments remain limited, are among the most prevalent causes of legal blindness. Retinal prostheses have been developed to stimulate the inner retinal network; however, lack of sensitivity and resolution, and the need for wiring or external cameras, have limited their application. Here we show that conjugated polymer nanoparticles (P3HT NPs) mediate light-evoked stimulation of retinal neurons and persistently rescue visual functions when subretinally injected in a rat model of retinitis pigmentosa. P3HT NPs spread out over the entire subretinal space and promote light-dependent activation of spared inner retinal neurons, recovering subcortical, cortical and behavioural visual responses in the absence of trophic effects or retinal inflammation. By conferring sustained light sensitivity to degenerate retinas after a single injection, and with the potential for high spatial resolution, P3HT NPs provide a new avenue in retinal prosthetics with potential applications not only in retinitis pigmentosa, but also in age-related macular degeneration.
Assuntos
Pontos Quânticos , Retina/efeitos dos fármacos , Retinose Pigmentar/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Injeções Intraoculares , Masculino , Estimulação Luminosa , Polímeros/administração & dosagem , Polímeros/farmacologia , Pontos Quânticos/administração & dosagem , Pontos Quânticos/uso terapêutico , Ratos , Ratos Sprague-Dawley , Córtex Visual/efeitos dos fármacos , Córtex Visual/metabolismo , Próteses VisuaisRESUMO
In vitro multi-electrode array (MEA) technology is nowadays involved in a wide range of applications beyond neuroscience, such as cardiac electrophysiology and bio-interface studies. However, the cost of commercially available acquisition systems severely limits its adoption outside specialized laboratories with high budget capabilities. Thus, the availability of low-cost methods to acquire signals from MEAs is important to allow research labs worldwide to exploit this technology for an ever-expanding pool of experiments independently from their economic possibilities. Here, we provide a comprehensive toolset to assemble a multifunctional in vitro MEA acquisition system with a total cost 80% lower than standard commercial solutions. We demonstrate the capabilities of this acquisition system by employing it to i) characterize commercial MEA devices by means of electrical impedance measurements ii) record activity from cultures of HL-1 cells extracellularly, and iii) electroporate HL-1 cells through nanostructured MEAs and record intracellular signals.
Assuntos
Técnicas Eletrofisiológicas Cardíacas/instrumentação , Miócitos Cardíacos/fisiologia , Potenciais de Ação/fisiologia , Animais , Linhagem Celular , Análise Custo-Benefício , Técnicas Eletrofisiológicas Cardíacas/economia , Técnicas Eletrofisiológicas Cardíacas/estatística & dados numéricos , Fenômenos Eletrofisiológicos , Eletroporação , Desenho de Equipamento , Camundongos , Microeletrodos , SoftwareRESUMO
Here, we propose an easy method for site-selective deposition of two-dimensional (2D) material flakes onto nanoholes by means of electrophoretic deposition. This method can be applied to both simple flat nanostructures and complex three-dimensional structures incorporating nanoholes. The deposition method is here used for the decoration of large ordered arrays of plasmonic structures with either a single or few layers of MoS2. In principle, the plasmonic field generated by the nanohole can significantly interact with the 2D layer leading to enhanced light-material interaction. This makes our platform an ideal system for hybrid 2D material/plasmonic investigations. The engineered deposition of 2D materials on plasmonic nanostructures is useful for several important applications such as enhanced light emission, strong coupling, hot-electron generation, and 2D material sensors.
RESUMO
3D vertical nanostructures have become one of the most significant methods for interfacing cells and the nanoscale and for accessing significant intracellular functionalities such as membrane potential. As this intracellular access can be induced by means of diverse cellular membrane poration mechanisms, it is important to investigate in detail the cell condition after membrane rupture for assessing the real effects of the poration techniques on the biological environment. Indeed, differences of the membrane dynamics and reshaping have not been observed yet when the membrane-nanostructure system is locally perturbed by, for instance, diverse membrane breakage events. In this work, new insights are provided into the membrane dynamics in case of two different poration approaches, optoacoustic- and electro-poration, both mediated by the same 3D nanostructures. The experimental results offer a detailed overview on the different poration processes in terms of electrical recordings and membrane conformation.