This protocol outlines the synthesis and use of engineered hyaluronan-based hydrogels for 3D cell culture and bioprinting of human induced pluripotent stem cell (hiPSC)-derived neuroepithelial stem cells (lt-NES). Key steps include hydrogel formation using bioorthogonal chemistries, cell encapsulation, and 3D bioprinting with a Cellink BioX printer, enabling the creation of complex tissue models. The protocol ensures high cell viability and supports differentiation, essential for neuroscience research and drug development.

Controlled breakdown has emerged as an effective method for fabricating solid-state nanopores in thin suspended dielectric membranes for various biomolecular sensing applications. On an unpatterned membrane, the site of nanopore formation by controlled breakdown is random. Nanopore formation on a specific site on the membrane has previously been realized using local thinning of the membrane by lithographic processes or laser-assisted photothermal etching under immersion in an aqueous salt solution. However, these approaches require elaborate and expensive cleanroom-based lithography processes or involve intricate procedures using custom-made equipment. Here, we present a rapid cleanroom-free approach using single pulse femtosecond laser exposures of 50 nm thick silicon nitride membranes in air to localize the site of nanopore formation by subsequent controlled breakdown to an area less than 500 nm in diameter on the membrane. The precise positioning of the nanopores on the membrane could be produced both using laser exposure powers which caused significant thinning of the silicon nitride membrane (up to 60% of the original thickness locally), as well as at laser powers which caused no visible modification of the membrane at all. We show that nanopores made using our approach can work as single-molecule sensors by performing dsDNA translocation experiments. Due to the applicability of femtosecond laser processing to a wide range of membrane materials, we expect our approach to simplify the fabrication of localized nanopores by controlled breakdown in a variety of thin film material stacks, thereby enabling more sophisticated nanopore sensors.

Food waste is a global challenge that needs to be mitigated in the development of more sustainable societies. From manufacturers to customers, food biosensors could effectively reduce the amount of discarded food and provide more precise predictions of freshness with respect to pre-decided expiration dates. In this study, we developed a novel organic electrochemical transistor (OECT)-based xanthine biosensor. The OECT-based biosensor is based on the p-type conjugated polymer, p(g42T-TT) as the channel, and incorporated xanthine oxidase (XOD) as the biorecognition element. The OECT thus acts as a transducer and amplifier of the enzymatic oxidation of xanthine. Real-time monitoring of xanthine using the OECT-based biosensor led to a linear range between 5 and 98 μM (R2=0.989), 3.28 μM limit of detection, and high sensitivity up to 21.8 mA/mM. Real sample tests showed that the biosensor can detect the accumulation of xanthine in fish meat from 0 to 6 days of degradation. Interference tests with ascorbic acid and uric acid and spike-and-recovery tests with fish samples indicated that as-designed biosensors have good selectivity and accuracy. The developed biosensors show great potential for point-of-care testing applied to food monitoring.

Solid-state nanopores offer unique possibilities for biomolecule sensing; however, scalable production of sub-5 nm pores with precise diameter control remains a manufacturing challenge. In this work, we developed a scalable method to fabricate sub-5 nm nanopores in silicon (Si) nanomembranes through metal-assisted chemical etching (MACE) using gold nanoparticles. Notably, we present a previously unreported self-limiting effect that enables sub-5 nm nanopore formation from both 10 and 40 nm nanoparticles in the 12 nm thick monocrystalline device layer of a silicon-on-insulator substrate. This effect reveals distinctive etching dynamics in ultrathin Si nanomembranes, enabling precise control over nanopore dimensions. The resulting nanopore sensor, suspended over self-aligned spheroidal oxide undercuts with diameters of just a few hundred nanometers, exhibited low electrical noise and high stability due to encapsulation within dielectric layers. In DNA translocation experiments, our nanopore platform could distinguish folded and unfolded DNA conformations and maintained stable baseline conductance for up to 6 h, demonstrating both sensitivity and robustness. Our scalable nanopore fabrication method is compatible with wafer-level and batch processing and holds promise for advancing biomolecular sensing and analysis.

In recent years, considerable advancements have been made in developing in vitro models to better understand the complex dynamics of the blood-brain barrier (BBB) and its critical role in neurological health and disease. Incorporating astrocytes into these models introduces an essential layer of complexity, allowing for a more comprehensive investigation of the cellular interactions and regulatory mechanisms that maintain BBB integrity and functionality. Despite these advances, the specific influence of astrocytes on endothelial cells in in vitro systems remains inadequately explored. This study addresses this gap by examining the transcriptional changes in primary human brain microvascular endothelial cells (HBMECs) cocultured with human astrocytes (HAs). Our findings demonstrate that astrocytes profoundly modulate endothelial pathways involved in cell cycle regulation and division while upregulating genes associated with BBB integrity, protective mechanisms, and transporter activity. Furthermore, astrocytes significantly enhanced transendothelial electrical resistance (TEER) and reduced permeability to tracer Cascade Blue dye, confirming their functional impact on BBB models. By providing a comprehensive human primary cell dataset, this research underscores the pivotal role astrocytes play in shaping endothelial cell gene expression and function in contact coculture systems. These results emphasize the necessity of incorporating astrocytes into in vitro BBB models to accurately replicate neurovascular interactions. Ultimately, this study advances our understanding of BBB physiology and highlights the importance of refining in vitro models to better reflect the complexity of the human neurovascular environment, with potential implications for studying neurological disorders and drug delivery strategies.

Hybridizing biological cells with man-made sensors enable the detection of a wide range of weak physiological responses with high specificity. The anterior chamber of the eye (ACE) is an ideal transplantation site due to its ocular immune privilege and optical transparency, which enable superior non-invasive longitudinal analyses of cells and microtissues. Engraftment of biohybrid microstructures in the ACE might, however, be affected by the pupillary response and dynamics. Here, sutureless transplantation of biohybrid microstructures, 3D printed in IP-Visio photoresin, containing a precisely localized pancreatic islet to the ACE of mice is presented. The biohybrid microstructures allow mechanical fixation in the ACE, independent of iris dynamics. After transplantation, islets in the microstructures successfully sustain their functionality for over 20 weeks and become vascularized despite physical separation from the vessel source (iris) and immersion in a low-viscous liquid (aqueous humor) with continuous circulation and clearance. This approach opens new perspectives in biohybrid microtissue transplantation in the ACE, advancing monitoring of microtissue-host interactions, disease modeling, treatment outcomes, and vascularization in engineered tissues.

Organic electrochemical transistors (OECTs) are promising devices for bioelectronics, such as biosensors. However, current cleanroom-based microfabrication of OECTs hinders fast prototyping and widespread adoption of this technology for low-volume, low-cost applications. To address this limitation, a versatile and scalable approach for ultrafast laser microfabrication of OECTs is herein reported, where a femtosecond laser to pattern insulating polymers (such as parylene C or polyimide) is first used, exposing the underlying metal electrodes serving as transistor terminals (source, drain, or gate). After the first patterning step, conducting polymers, such as poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS), or semiconducting polymers, are spin-coated on the device surface. Another femtosecond laser patterning step subsequently defines the active polymer area contributing to the OECT performance by disconnecting the channel and gate from the surrounding spin-coated film. The effective OECT width can be defined with high resolution (down to 2 µm) in less than a second of exposure. Micropatterning the OECT channel area significantly improved the transistor switching performance in the case of PEDOT:PSS-based transistors, speeding up the devices by two orders of magnitude. The utility of this OECT manufacturing approach is demonstrated by fabricating complementary logic (inverters) and glucose biosensors, thereby showing its potential to accelerate OECT research.

Tunnel junctions have been suggested as high-throughput electronic single molecule sensors in liquids with several seminal experiments conducted using break junctions with reconfigurable gaps. For practical single molecule sensing applications, arrays of on-chip integrated fixed-gap tunnel junctions that can be built into compact systems are preferable. Fabricating nanogaps by electromigration is one of the most promising approaches to realize on-chip integrated tunnel junction sensors. However, the electrical behavior of fixed-gap tunnel junctions immersed in liquid media has not been systematically studied to date, and the formation of electromigrated nanogap tunnel junctions in liquid media has not yet been demonstrated. In this work, we perform a comparative study of the formation and electrical behavior of arrays of gold nanogap tunnel junctions made by feedback-controlled electromigration immersed in various liquid and gaseous media (deionized water, mesitylene, ethanol, nitrogen, and air). We demonstrate that tunnel junctions can be obtained from microfabricated gold nanoconstrictions inside liquid media. Electromigration of junctions in air produces the highest yield (61–67%), electromigration in deionized water and mesitylene results in a lower yield than in air (44–48%), whereas electromigration in ethanol fails to produce viable tunnel junctions due to interfering electrochemical processes. We map out the stability of the conductance characteristics of the resulting tunnel junctions and identify medium-specific operational conditions that have an impact on the yield of forming stable junctions. Furthermore, we highlight the unique challenges associated with working with arrays of large numbers of tunnel junctions in batches. Our findings will inform future efforts to build single molecule sensors using on-chip integrated tunnel junctions.

Limitations with cell cultures and experimental animal-based studies have had the scientific and industrial communities searching for new approaches that can provide reliable human models for applications such as drug development, toxicological assessment, and in vitro pre-clinical evaluation. This has resulted in the development of microfluidic-based cultures that may better represent organs and organ systems in vivo than conventional monolayer cell cultures. Although there is considerable interest from industry and regulatory bodies in this technology, several challenges need to be addressed for it to reach its full potential. Among those is a lack of guidelines and standards. Therefore, a multidisciplinary team of stakeholders was formed, with members from the US Food and Drug Administration (FDA), the National Institute of Standards and Technology (NIST), European Union, academia, and industry, to provide a framework for future development of guidelines/standards governing engineering concepts of organ-on-a-chip models. The result of this work is presented here for interested parties, stakeholders, and other standards development organizations (SDOs) to foster further discussion and enhance the impact and benefits of these efforts.

Tunnel junctions have long been used to immobilize and study the electronic transport properties of single molecules. The sensitivity of tunneling currents to entities in the tunneling gap has generated interest in developing electronic biosensors with single molecule resolution. Tunnel junctions can, for example, be used for sensing bound or unbound DNA, RNA, amino acids, and proteins in liquids. However, manufacturing technologies for on-chip integrated arrays of tunnel junction sensors are still in their infancy, and scalable measurement strategies that allow the measurement of large numbers of tunneling junctions are required to facilitate progress. Here, we describe an experimental setup to perform scalable, high-bandwidth (>10 kHz) measurements of low currents (pA–nA) in arrays of on-chip integrated tunnel junctions immersed in various liquid media. Leveraging a commercially available compact 100 kHz bandwidth low-current measurement instrument, we developed a custom two-terminal probe on which the amplifier is directly mounted to decrease parasitic probe capacitances to sub-pF levels. We also integrated a motorized three-axis stage, which could be powered down using software control, inside the Faraday cage of the setup. This enabled automated data acquisition on arrays of tunnel junctions without worsening the noise floor despite being inside the Faraday cage. A deliberately positioned air gap in the fluidic path ensured liquid perfusion to the chip from outside the Faraday cage without coupling in additional noise. We demonstrate the performance of our setup using rapid current switching observed in electromigrated gold tunnel junctions immersed in deionized water.