Fully Integrated 3D Microelectrode Arrays with Polydopamine-Mediated Silicon Dioxide Insulation for Electrophysiological Interrogation of a Novel 3D Human, Neural Microphysiological Construct.

Advances within in vitro biological system complexity have enabled new possibilities for the "Organs-on-a-Chip" field. Microphysiological systems (MPS) as such incorporate sophisticated biological constructs with custom biological sensors. For microelectromechanical systems (MEMS) sensors, the dielectric layer is critical for device performance, where silicon dioxide (SiO2) represents an excellent candidate due to its biocompatibility and wide utility in MEMS devices. Yet, high temperatures traditionally preclude SiO2 from incorporation in polymer-based BioMEMS. Electron-beam deposition of SiO2 may provide a low-temperature, dielectric serving as a nanoporous MPS growth substrate. Herein, we enable improved adherence of nanoporous SiO2 to polycarbonate (PC) and 316L stainless steel (SS) via polydopamine (PDA)-mediated chemistry. The resulting stability of the combinatorial PDA-SiO2 film was interrogated, along with the nature of the intrafilm interactions. A custom polymer-metal three-dimensional (3D) microelectrode array (3D MEA) is then reported utilizing PDA-SiO2 insulation, for definition of novel dorsal root ganglion (DRG)/nociceptor and dorsal horn (DH) 3D neural constructs in excess of 6 months for the first time. Spontaneous/evoked compound action potentials (CAPs) are successfully reported. Finally, inhibitory drugs treatments showcase pharmacological responsiveness of the reported multipart biological activity. These results represent the initiation of a novel 3D MEA-integrated, 3D neural MPS for the long-term electrophysiological study.

Microfabricated polymer-metal biosensors for multifarious data collection from electrogenic cellular models.

Benchtop tissue cultures have become increasingly complex in recent years, as more on-a-chip biological technologies, such as microphysiological systems (MPS), are developed to incorporate cellular constructs that more accurately represent their respective biological systems. Such MPS have begun facilitating major breakthroughs in biological research and are poised to shape the field in the coming decades. These biological systems require integrated sensing modalities to procure complex, multiplexed datasets with unprecedented combinatorial biological detail. In this work, we expanded upon our polymer-metal biosensor approach by demonstrating a facile technology for compound biosensing that was characterized through custom modeling approaches. As reported herein, we developed a compound chip with 3D microelectrodes, 3D microfluidics, interdigitated electrodes (IDEs) and a microheater. The chip was subsequently tested using the electrical/electrochemical characterization of 3D microelectrodes with 1 kHz impedance and phase recordings and IDE-based high-frequency (~1 MHz frequencies) impedimetric analysis of differential localized temperature recordings, both of which were modeled through equivalent electrical circuits for process parameter extraction. Additionally, a simplified antibody-conjugation strategy was employed for a similar IDE-based analysis of the implications of a key analyte (l-glutamine) binding to the equivalent electrical circuit. Finally, acute microfluidic perfusion modeling was performed to demonstrate the ease of microfluidics integration into such a polymer-metal biosensor platform for potential complimentary localized chemical stimulation. Overall, our work demonstrates the design, development, and characterization of an accessibly designed polymer-metal compound biosensor for electrogenic cellular constructs to facilitate comprehensive MPS data collection.

Plotter Cut Stencil Masks for the Deposition of Organic and Inorganic Materials and a New Rapid, Cost Effective Technique for Antimicrobial Evaluations.

Plotter cutters in stencil mask prototyping are underutilized but have several advantages over traditional MEMS techniques. In this paper we investigate the use of a conventional plotter cutter as a highly effective benchtop tool for the rapid prototyping of stencil masks in the sub-250 µm range and characterize patterned layers of organic/inorganic materials. Furthermore, we show a new diagnostic monitoring application for use in healthcare, and a potential replacement of the Standard Kirby-Bauer Diffusion Antibiotic Resistance tests was developed and tested on both Escherichia coli and Xanthomonas alfalfae as pathogens with Oxytetracycline, Streptomycin and Kanamycin. We show that the reduction in area required for the minimum inhibitory concentration tests; allow for three times the number of tests to be performed within the same nutrient agar Petri dish, demonstrated both theoretically and experimentally resulting in correlations of R ≈ 0.96 and 0.985, respectively for both pathogens.

Rapid Makerspace Microfabrication and Characterization of 3D Microelectrode Arrays (3D MEAs) for Organ-on-a-Chip Models.

Integrated sensors in "on-a-chip" in vitro cellular models are a necessity for granularity in data collection required for advanced biosensors. As these models become more complex, the requirement for the integration of electrogenic cells is apparent. Interrogation of such cells, whether alone or within a connected cellular framework, are best achieved with microelectrodes, in the form of a microelectrode array (MEA). Makerspace microfabrication has thus far enabled novel and accessible approaches to meet these demands. Here, resin-based 3D printing, selective multimodal laser micromachining, and simple insulation strategies, define an approach to highly customizable and "on-demand" in vitro 3D MEA-based biosensor platforms. The scalability of this approach is aided by a novel makerspace microfabrication assisted technique denoted using the term Hypo-Rig. The MEA utilizes custom-defined metal microfabricated microelectrodes transitioned from planar (2D) to 3D using the Hypo-Rig. To simulate this transition process, COMSOL modeling is utilized to estimate transitionary forces and angles (with respect to normal). Practically, the Hypo-Rig demonstrated a force of ~40N, as well as a consistent 70° average angular transitionary performance which matched well with the COMSOL model. To illustrate the scalability potential, 3 × 3, 6 × 6, and 8 × 8 versions of the device were fabricated and characterized. The 3D MEAs, demonstrated impedance and phase measurements in the biologically relevant 1 kHz range of 45.4 kΩ, and -34.6° respectively, for polystyrene insulated, ~70µm sized microelectrodes.

Fabrication and Characterization of 3D Printed, 3D Microelectrode Arrays for Interfacing with a Peripheral Nerve-on-a-Chip.

We present a nontraditional fabrication technique for the realization of three-dimensional (3D) microelectrode arrays (MEAs) capable of interfacing with 3D cellular networks in vitro. The technology uses cost-effective makerspace microfabrication techniques to fabricate the 3D MEAs with 3D printed base structures with the metallization of the microtowers and conductive traces being performed by stencil mask evaporation techniques. A biocompatible lamination layer insulates the traces for realization of 3D microtower MEAs (250 µm base diameter, 400 µm height). The process has additionally been extended to realize smaller electrodes (30 µm × 30 µm) at a height of 400 µm atop the 3D microtower using laser micromachining of an additional silicon dioxide (SiO2) insulation layer. A 3D microengineered, nerve-on-a-chip in vitro model for recording and stimulating electrical activity of dorsal root ganglion (DRG) cells has further been integrated with the 3D MEA. We have characterized the 3D electrodes for electrical, chemical, electrochemical, biological, and chip hydration stability performance metrics. A decrease in impedance from 1.8 kΩ to 670 Ω for the microtower electrodes and 55 to 39 kΩ for the 30 µm × 30 µm microelectrodes can be observed for an electrophysiologically relevant frequency of 1 kHz upon platinum electroless plating. Biocompatibility assays on the components of the system resulted in a large range (∼3%-70% live cells), depending on the components. Fourier-transform infrared (FTIR) spectra of the resin material start to reveal possible compositional clues for the resin, and the hydration stability is demonstrated in in-vitro-like conditions for 30 days. The fabricated 3D MEAs are rapidly produced with minimal usage of a cleanroom and are fully functional for electrical interrogation of the 3D organ-on-a-chip models for high-throughput of pharmaceutical screening and toxicity testing of compounds in vitro.

Biocompatibility of Blank, Post-Processed and Coated 3D Printed Resin Structures with Electrogenic Cells.

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.

Makerspace microfabrication of a stainless steel 3D microneedle electrode array (3D MEA) on a glass substrate for simultaneous optical and electrical probing of electrogenic cells.

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.

SeedEZ™ Interdigitated Electrodes and Multifunctional Layered Biosensor Composites (MLBCs): A Paradigm Shift in the Development of In Vitro BioMicrosystems.

We have developed a new technology for the realization of composite biosensor systems, capable of measuring electrical and electrophysiological signals from electrogenic cells, using SeedEZ™ 3D cell culture-scaffold material. This represents a paradigm-shift for BioMEMS processing; 'Biology-Microfabrication' versus the standard 'Microfabrication-Biology' approach. An Interdigitated Electrode (IDE) developed on the 3D cell-scaffold was used to successfully monitor acute cardiomyocyte growth and controlled population decline. We have further characterized processability of the 3D scaffold, demonstrated long-term biocompatibility of the scaffold with various cell lines and developed a multifunctional layered biosensor composites (MLBCs) using SeedEZ™ and other biocompatible substrates for future multilayer sensor integration.