Long-term in vitro recording of cardiac action potentials on microelectrode arrays for chronic cardiotoxicity assessment.

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.

All-Optical and Label-Free Stimulation of Action Potentials in Neurons and Cardiomyocytes by Plasmonic Porous Metamaterials.

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.

Intracellular action potential recordings from cardiomyocytes by ultrafast pulsed laser irradiation of fuzzy graphene microelectrodes.

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.

Improving reliability and reducing costs of cardiotoxicity assessments using laser-induced cell poration on microelectrode arrays.

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.

Mirroring Action Potentials: Label-Free, Accurate, and Noninvasive Electrophysiological Recordings of Human-Derived Cardiomyocytes.

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.

Microfluidic Multielectrode Arrays for Spatially Localized Drug Delivery and Electrical Recordings of Primary Neuronal Cultures.

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.

Intracellular Recording of Human Cardiac Action Potentials on Market-Available Multielectrode Array Platforms.

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.

Cost-effective and multifunctional acquisition system for in vitro electrophysiological investigations with multi-electrode arrays.

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.

On-Demand Intracellular Delivery of Single Particles in Single Cells by 3D Hollow Nanoelectrodes.

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.

Membrane Poration Mechanisms at the Cell-Nanostructure Interface.

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.

Selective intracellular delivery and intracellular recordings combined in MEA biosensors.

Biological studies on in vitro cell cultures are of fundamental importance to investigate cell response to external stimuli, such as new drugs for the treatment of specific pathologies, or to study communication between electrogenic cells. Although three-dimensional (3D) nanostructures brought tremendous improvements on biosensors used for various biological in vitro studies, including drug delivery and electrical recording, there is still a lack of multifunctional capabilities that could help gain deeper insights in several bio-related research fields. In this work, the electrical recording of large cell ensembles and the intracellular delivery of few selected cells are combined on the same device by integrating microfluidic channels on the bottom of a multi-electrode array decorated with 3D hollow nanostructures. The novel platform allows the recording of intracellular-like action potentials from large ensembles of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) and from the HL-1 line, while different molecules are selectively delivered into single/few targeted cells. The proposed approach shows high potential for enabling new comprehensive studies that can relate drug effects to network level cell communication processes.

Author Correction: Live Intracellular Biorthogonal Imaging by Surface Enhanced Raman Spectroscopy using Alkyne-Silver Nanoparticles Clusters.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Plasmonic meta-electrodes allow intracellular recordings at network level on high-density CMOS-multi-electrode arrays.

The ability to monitor electrogenic cells accurately plays a pivotal role in neuroscience, cardiology and cell biology. Despite pioneering research and long-lasting efforts, the existing methods for intracellular recording of action potentials on the large network scale suffer limitations that prevent their widespread use. Here, we introduce the concept of a meta-electrode, a planar porous electrode that mimics the optical and biological behaviour of three-dimensional plasmonic antennas but also preserves the ability to work as an electrode. Its synergistic combination with plasmonic optoacoustic poration allows commercial complementary metal-oxide semiconductor multi-electrode arrays to record intracellular action potentials in large cellular networks. We apply this approach to measure signals from human-induced pluripotent stem cell-derived cardiac cells, rodent primary cardiomyocytes and immortalized cell types and demonstrate the possibility of non-invasively testing a variety of relevant drugs. Due to its robustness and easiness of use, we expect the method will be rapidly adopted by the scientific community and by pharmaceutical companies.

Live Intracellular Biorthogonal Imaging by Surface Enhanced Raman Spectroscopy using Alkyne-Silver Nanoparticles Clusters.

Live intracellular imaging is a valuable tool in modern diagnostics and pharmacology. Surface Enhanced Raman Spectroscopy (SERS) stands out as a non-destructive and multiplexed technique, but intracellular SERS imaging still suffers from interfering background from endogenous components. Here we show the assembly of small colloidal SERS probes with Raman signal in the cell-silent window of 1800-2900 cm-1 for biorthogonal intracellular SERS imaging of dopamine that was undistinguishable from the endogenous cell background. By linking colloidal silver nanoparticles with alkyne-dopamine adducts, clusters are formed by 2-6 nanoparticles spaced by tight interparticle gaps that exhibited high electric field enhancement and strong SERS signals of alkyne and dopamines. Due to the cell-silent signals of the alkyne, intracellular in-vitro Raman imaging shows that the dopamines on the internalized clusters remain distinguishable across the cytoplasm with good spatial resolution. Our method can be a general-purpose method for real-time imaging of biomolecules, such as proteins, peptides, DNA and drugs.

Author Correction: Plasmonic meta-electrodes allow intracellular recordings at network level on high-density CMOS-multi-electrode arrays.

In the version of this Article originally published, the affiliation for the author Francesca Santoro was incorrectly given; it should have been 'Center for Advanced Biomaterials for Healthcare, Istituto Italiano di Tecnologia, Napoli, Italy'. This has now been corrected in all versions of the Article.

Cells Adhering to 3D Vertical Nanostructures: Cell Membrane Reshaping without Stable Internalization.

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.

Soft electroporation for delivering molecules into tightly adherent mammalian cells through 3D hollow nanoelectrodes.

Electroporation of in-vitro cultured cells is widely used in biological and medical areas to deliver molecules of interest inside cells. Since very high electric fields are required to electroporate the plasma membrane, depending on the geometry of the electrodes the required voltages can be very high and often critical to cell viability. Furthermore, in traditional electroporation configuration based on planar electrodes there is no a priori certain feedback about which cell has been targeted and delivered and the addition of fluorophores may be needed to gain this information. In this study we present a nanofabricated platform able to perform intracellular delivery of membrane-impermeable molecules by opening transient nanopores into the lipid membrane of adherent cells with high spatial precision and with the application of low voltages (1.5-2 V). This result is obtained by exploiting the tight seal that the cells present with 3D fluidic hollow gold-coated nanostructures that act as nanochannels and nanoelectrodes at the same time. The final soft-electroporation platform provides an accessible approach for controlled and selective drug delivery on ordered arrangements of cells.