3D-Printed Bubble-Free Perfusion Cartridge System for Live-Cell Imaging.

The advent of 3D-printing technologies has had a significant effect on the development of medical and biological devices. Perfusion chambers are widely used for live-cell imaging in cell biology research; however, air-bubble invasion is a pervasive problem in perfusion systems. Although 3D printing allows the rapid fabrication of millifluidic and microfluidic devices with high resolution, little has been reported on 3D-printed fluidic devices with bubble trapping systems. Herein, we present a 3D-printed millifluidic cartridge system with bent and flat tapered flow channels for preventing air-bubble invasion, irrespective of bubble volume and without the need for additional bubble-removing devices. This system realizes bubble-free perfusion with a user-friendly interface and no-time-penalty manufacturing processes. We demonstrated the bubble removal capability of the cartridge by continually introducing air bubbles with different volumes during the calcium imaging of Sf21 cells expressing insect odorant receptors. Calcium imaging was conducted using a low-magnification objective lens to show the versatility of the cartridge for wide-area observation. We verified that the cartridge could be used as a chemical reaction chamber by conducting protein staining experiments. Our cartridge system is advantageous for a wide range of cell-based bioassays and bioanalytical studies, and can be easily integrated into portable biosensors.

Frustoconical porous microneedle for electroosmotic transdermal drug delivery.

A truncated cone-shaped porous microneedle (PMN) made of poly-glycidyl methacrylate was studied as a minimally invasive tool for transdermal drug delivery. The transdermal electrical resistance of a pig skin was evaluated during the indentation of the PMNs, revealing that the frustoconical PMN (300 µm height) significantly reduced the resistance of the skin by expanding the stratum corneum without penetrating into the skin. A thin film of poly (2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) was grafted onto the inner wall of the microchannels of the frustoconical PMN to generate electroosmotic flow (EOF) upon current application in the direction of injection of the drug into the skin. Owing to the synergy of the expansion of the stratum corneum and the EOF-promotion, the PAMPS-modified frustoconical PMN effectively enhances the penetration of larger (over 500 Da) molecules, such as dextran (∼10 kDa).

Totally Organic Hydrogel-Based Self-Closing Cuff Electrode for Vagus Nerve Stimulation.

An intrinsically soft organic electrode consisting of poly(3,4-ethylenedioxythiophene)-modified polyurethane (PEDOT-PU) is embedded into a bilayer film of polyvinyl alcohol (PVA) hydrogels for developing a self-closing cuff electrode for neuromodulation. The curled form of the PVA hydrogel is prepared by releasing internal stress in the bilayer structure. The inner diameter of the cuff electrode is set to less than 2 mm for immobilization to the vagus nerve (VN) of humans and pigs. The stability of the immobilization is examined, while the pressure applied to a nerve bundle is at a harmless level (≈200 Pa). Since the electrode is totally organic, MRI measurements can be conducted without image artifacts. The large electric capacitance of the PEDOT-PU (≈27 mF cm-2 ) ensures a safe stimulation of living tissues without Faradaic reactions. The practical performance of the cuff electrode for VN stimulation is demonstrated by observation of bradycardia induction in a pig.

Low Surface Potential with Glycoconjugates Determines Insect Cell Adhesion at Room Temperature.

Cell-coupled field-effect transistor (FET) biosensors have attracted considerable attention because of their high sensitivity to biomolecules. The use of insect cells (Sf21) as a core sensor element is advantageous due to their stable adhesion to sensors at room temperature. Although visualization of the insect cell-substrate interface leads to logical amplification of signals, the spatiotemporal processes at the interfaces have not yet been elucidated. We quantitatively monitored the adhesion dynamics of Sf21 using interference reflection microscopy (IRM). Specific adhesion signatures with ring-like patches along the cellular periphery were detected. A combination of zeta potential measurements and lectin staining identified specific glycoconjugates with low electrostatic potentials. The ring-like structures were disrupted after cholesterol depletion, suggesting a raft domain along the cell periphery. Our results indicate dynamic and asymmetric cell adhesion is due to low electrostatic repulsion with fluidic sugar rafts. We envision the logical design of cell-sensor interfaces with an electrical model that accounts for actual adhesion interfaces.

Electroantennography-based Bio-hybrid Odor-detecting Drone using Silkmoth Antennae for Odor Source Localization.

Small drones with chemical or biosensor devices that can detect airborne odorant molecules have attracted considerable attention owing to their applicability in environmental and security monitoring and search-and-rescue operations. Small drones with commercial metal-oxide-semiconductor (MOX) gas sensors have been developed for odor source localization; however, their real-time-odor-detection performance has proven inadequate. However, biosensing technologies based on insect olfactory systems exhibit relatively high sensitivity, selectivity, and real-time response with respect to odorant molecules compared to commercial MOX gas sensors. In such devices, excised insect antennae function as portable odorant biosensor elements and have been found to deliver excellent sensing performance. This study presents experimental protocols for odorant-molecule detection in the air using a small autonomous bio-hybrid drone based on a mountable electroantennography (EAG) device incorporating silkmoth antennae. We developed a mountable EAG device including sensing/processing parts with a Wi-Fi module. The device was equipped with a simple sensor enclosure to enhance the sensor directivity. Thus, odor source localization was conducted using the spiral-surge algorithm, which does not assume an upwind direction. The experimental bio-hybrid odor-detecting drone identified real-time odorant-concentration differences in a pseudo-open environment (outside a wind tunnel) and localized the source. The developed drone and associated system can serve as an efficient odorant molecule-detection tool and a suitable flight platform for developing odor source localization algorithms owing to its high programmability.

Highly effective volatile organic compound dissolving strategy based on mist atomization for odorant biosensors.

The detection of volatile organic compound (VOC) mixtures is crucial in the medical and security fields. Receptor-based odorant biosensors sensitively and selectively detect odorant molecules in a solution; however, odorant molecules generally exist as VOCs in the air and exhibit poor water solubility. Therefore, techniques that enable the dissolution of poorly water-soluble VOCs using portable systems are essential for practical biosensors' applications. We previously proposed a VOC dissolution method based on water atomization to increase the surface area via the generation of fine bubbles, as a proof-of-concept; however, the system was lab-based (non-mobile) and the dissolution was limited to one VOC. In this study, we established a highly effective VOC dissolution method based on mist atomization that can be used in the field. This new method demonstrated a rapid dissolution potential of a sparsely-soluble VOC mixture with various functional groups in distilled water (DW) within 1 min, without the use of any organic solvents. Calcium imaging revealed that odorant receptor 13a-expressing Sf21 cells (Or13a cells) responded to 1-octen-3-ol in the mixture. Further, we successfully developed a field-deployable prototype vacuum and dissolution system with a simple configuration that efficiently captured and rapidly dissolved airborne 1-octen-3-ol in DW. This study proposes a field-deployable system that is appropriate for solubilizing various airborne odorant molecules and therefore is a practical strategy to use in the context of odorant biosensors.

Increasing cell-device adherence using cultured insect cells for receptor-based biosensors.

Field-effect transistor (FET)-based biosensors have a wide range of applications, and a bio-FET odorant sensor, based on insect (Sf21) cells expressing insect odorant receptors (ORs) with sensitivity and selectivity, has emerged. To fully realize the practical application of bio-FET odorant sensors, knowledge of the cell-device interface for efficient signal transfer, and a reliable and low-cost measurement system using the commercial complementary metal-oxide semiconductor (CMOS) foundry process, will be indispensable. However, the interfaces between Sf21 cells and sensor devices are largely unknown, and electrode materials used in the commercial CMOS foundry process are generally limited to aluminium, which is reportedly toxic to cells. In this study, we investigated Sf21 cell-device interfaces by developing cross-sectional specimens. Calcium imaging of Sf21 cells expressing insect ORs was used to verify the functions of Sf21 cells as odorant sensor elements on the electrode materials. We found that the cell-device interface was approximately 10 nm wide on average, suggesting that the adhesion mechanism of Sf21 cells may differ from that of other cells. These results will help to construct accurate signal detection from expressed insect ORs using FETs.