Organisms perceive odorants in the environment through the use of a large number of olfactory receptors. Various odor biosensors have been researched and developed in order to mimic this olfactory mechanism. This study examines the quantification of odorant concentrations through the use of a sensor array comprised of several types of cell-based odor sensors expressing insect olfactory receptors with nonlinear characteristics. The sensor system utilized an active sensing method in order to compare the responses of a target odorant and a prepared odorant in determining the relative concentration of the target odorant. By combining an active sensing method with a real-time reference method in which the target odorant was measured every time the prepared odorant was measured, the relative concentrations were successfully determined even when the response fluctuation was large or odorant sensor cell responses varied as measurement time increased. For proof of concept purposes, the study primarily focused on quantifying odorant concentrations composed of one or two odorant components. It was confirmed that an algorithm to find the optimal relative odorant concentration among a limited number of odorant concentrations is achievable. Though this study is still in the initial stage of the developing odor sensors and has many challenges, it can provide insight into paving the way towards a new type of odor biosensor with active sensing.

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.

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.

The extensive diversity of volatile organic compounds, along with their minor structural variations, presents significant challenges in the development of chemosensory-based biosensors. Previously, we generated sensor cells expressing insect odorant receptors (ORs) in Sf21 cells, demonstrating their potential as cell-based odorant sensor elements. However, it remains unclear whether the selectivity of cells expressing ORs in vitro for diverse compounds aligns with the receptor’s in vivo performance, aside from the response to target compounds. To address this, we assessed the ligand responses of sensor cells expressing ORs from Drosophila melanogaster using a high-throughput calcium imaging system. Our results demonstrate that in vitro receptor responses exhibit ligand selectivity comparable to in vivo conditions across different chemical categories. Broadly tuned OR-expressing sensor cells (Or13a, Or47a, and Or98a) displayed differential affinities, whereas the narrowly tuned Or56a-expressing sensor cells selectively responded to geosmin. Moreover, cell responses varied with subtle differences in chemical structure, including carbon chain length and functional group positioning. These findings provide valuable insights into insect OR–ligand interactions in vitro, demonstrating that receptor selectivity in sensor cells closely mirrors in vivo conditions. In addition to this consistency, our results highlight the subtle ligand differentiation capabilities of sensor cells enabling fluorescence-based visualization of receptor–ligand interactions.

A technology that enables highly sensitive, selective on-site detection of odorants without sophisticated equipment could be valuable for a range of applications, from water quality testing to disease diagnosis. In this study, we introduce a portable technology using Sf21 cells engineered to express Drosophila melanogaster odorant receptors, co-receptors, and calcium-sensitive fluorescent proteins for geosmin detection—a natural compound known for its distinct musty odor. Sensor cartridges were fabricated by immobilizing sensor cells on the glass surface inside tubes. When coupled with a portable fluorometer, the system successfully detected geosmin at concentrations as low as 100 pM (18.2 ppt) without requiring any concentration steps. Moreover, the system was deployed at a lakeside dam and detected geosmin in real field samples. This is the first successful detection of target odorants using biosensors comprising transfected sensor cells bearing specific odorant receptor genes. The results highlight the practicality of insect odorant receptor-expressing sensor cells and reveal the potential of this technology in various applications, such as food, beverage, and water quality testing, where on-site odorant detection is required.