(5)
Considering an average loading time of 20.4±4.1 s, we achieved a testing per fish time of 44.4±4.8 s for the proposed quadruple-fish device which was 60% faster than the time spent for testing in the single-fish device[16]. Our quadruple-fish device also offered loading and orientation efficiencies of 87.5±5.6% and 90±7.1%, respectively. These data clearly demonstrate the advantage of our quadruple-fish device when aiming to facilitate larger sample sizes in a shorter period of time.
Electric current and flow field analyses were conducted using COMSOL to ensure their uniformity throughout the device. According to the electric simulation, applying a total electric current of 12 μA between the anode and cathode electrodes (shown in Fig. 1A) resulted in a uniform voltage drop of 1.1 V (as seen in trap B in Fig. 2) and electric current of 3 μA across each trap, consistent with the current used in our previous single-fish device[6,16].
The flow in the indirect flow channel ran opposite to that in the main channel to generate comparable pressure drops across each TR, ensuring uniform loading conditions. The analysis of the flow dynamics within the chip showed a pressure drop and therefore a hydrodynamic force pointing from the main channel towards the TRs enabling loading and immobilization within the TRs. The pressure contour plot across each trap closely resembled the contour shown in Fig. 2 for trap C. The shear stress in the TRs was also obtained by multiplying the water viscosity by the velocity gradient at the wall, as shown in trap D in Fig. 2. The maximum shear stress experienced by zebrafish during the loading process was 10 Pa which was less than the 45 Pa threshold to avoid injury according to the studies done by Ulanowicz and Morgan[35,36].