2.2. Microfluidic device and experimental procedures
The polydimethylsiloxane (PDMS) microfluidic device used for zebrafish electrical response studies, under exposure to chemicals in Table 1, is depicted with key features labelled in Fig. 1A,1B and 1C. It consisted of three layers shown in Fig. 1C with various microchannels to enable loading and screening of four larvae simultaneously. The top section of the three-layer device included the main inlet and outlets from which the fish entered and exited the device, the indirect flow inlet and outlet to help direct the fish into the trapping regions (TRs) and the reverse flow to aid in positioning and orienting the fish. The screening pools were designed to provide enough space allowing complete tail strokes without any collision with the device walls. This, in combination with limitations associated with our microscope field of view (FOV), restricted the number of larvae that could be tested at the same time. The bottom layer of the device included valve channels containing air. The middle layer consisted of a thin membrane, approximately 200 μm thick, separating the top and bottom layers. When the bottom valve was pressurized, the membrane deflected into the upper layer channel to prevent the fish from swimming out of the TR. Two anodic copper wire electrodes were attached to the outlets and a copper wire was run through the indirect flow channel (Fig. 1A), at the middle of the device, to function as the cathode.