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.


Evaluation of cell growth on aluminium
The cytotoxicity of aluminium was evaluated using trypan blue exclusion assay and Sf21 and HEK293T cells cultured on aluminium-sputtered silicon substrates. Before seeding the cells, the aluminium substrates were immersed in 70% ethanol for 1 h and then in 99.5% ethanol for 30 min for sterilization and drying. 50-µL suspensions of Sf21 cells were seeded on the aluminium substrates in 35-mm plastic cell culture dishes containing 2 mL of insect culture medium, and the cultures were incubated at 27°C. Time-dependent changes in Sf21 and HEK293T cells were measured at 0 (2 h after seeding), 1, 2, and 3 days under a bright-field microscope. At each time point, 1 mL of culture medium was removed from the cell-culture dish and 200 µL, 0.4 w/v % trypan blue solution (Wako Pure Chemical Industries Ltd., Osaka, Japan) was added to each dish.
We measured cell-adhesion areas of Sf21 and HEK293T cells cultured on aluminiumsputtered substrates to evaluate the cytotoxicity of aluminium. Sf21 and HEK293T cells were seeded separately on aluminium-sputtered substrates (test) and plastic cell culture dishes (control). Figures S2 and S3 show the time-dependent changes in the Sf21 and HEK293T cells from 0 to 3 days. Each day, samples were observed by bright-field microscopy after addition of trypan blue. Figure S4 shows the comparison of timedependent changes in Sf21 and HEK293T cell areas on the aluminium substrates and plastic cell culture dishes. The bright-field microscopy images and the graphs indicate that Sf21 cells cultured on aluminium substrates grew and were not stained by trypan blue, similar to the cells cultured in plastic dishes. In contrast, HEK293T cell-adhesion areas on aluminium substrates were not constant, and cell growth was slower than that on plastic dishes. Moreover, some HEK293T cells cultured on the aluminium substrates were stained each day by trypan blue.

All cross-sectional SEM images of attached BmOR3 cells on Al2O3 layers
In this study, we have taken nine cross-sectional SEM images of BmOR3 cells cultured on sputtered Al2O3 layers. One cross-section included multiple cells ( Figure S5 (a)), suggesting that a majority of the cells were tightly attached. BmOR3 cells on sputtered Al2O3 layers on Si substrates ( Figure S5 (b)-(h)) or extended-gate electrodes of the OSFET (Figure S5 (i) and (j)) are shown. These SEM images indicated that a majority of the cells were tightly attached onto Al2O3 layers except the cells shown in Figure S5 (c) and (e). We conducted elaborate analysis of adhesive interface of two cells (cell A and B). -

Brightness value analysis of cell membrane using ImageJ
To confirm that the membrane of the BmOR3 cell was attached to the Al2O3 layer, we measured brightness value of the free membrane and attached membrane of the cell A using ImageJ software ( Figure S6). Results showed that floating parts of the cell had long cleft distances (Line1 to 3 of Figure S6) and attached parts of the cell had short cleft distances (Line4 to 5 of Figure S6). Therefore, we concluded that the cell membrane was certainly attached on the device surface.
Vertical yellow profile lines were drawn at 50 pixel intervals.

Cross-sectional SEM images of BmOR3 cells on aluminium layer
To observe and analyse cleft distances between BmOR3 cells and aluminium layers, we developed cross-sectional specimens using a cross-section polisher similar to that for the cells on Al2O3 layers ( Figure S7 (a)). However, we could not distinguish the plasma membranes of BmOR3 cells ( Figure S7 (b)) and brightness value decreased between the cell-device interfaces.