## 3 Results and Discussion
### Enhanced Microfluidic Immunoassay by Applying MET
#### 3.2.1 Transient Immune Response Under MET  
In the simulation of on-chip antigen-antibody binding kinetics, it is assumed that the antibody probes are coated on the sixth electrode from left to right. That is, only the sixth metal strip serves as the functionalized surface where immunoassay is possible to occur, while all other electrodes are bare and in direct contact with the electrolyte. Antigens are freely suspended in the buffer medium and initially released from the left inlet with no external forces. The concentration distribution of incoming antigens within the straight channel at t = 600 s, due to the action of MET, is shown in Figure 6 on application of the hybrid SW/TW signal of different voltage amplitudes at the ideal frequency combination. UnderV\({}_{\text{SW}}\) = 0 V and Vtw = 0 V, as shown in Figure 6A, it can be found that the incoming analyte fails to move to the surface of the sixth electrode under pure thermal diffusion even at t = 600 s, and the concentration of antigen on the functionalized surface is close to 0. Under V\({}_{\text{SW}}\) = 6 V and Vtw = 0 V, as shown in Figure 6B, a series of lateral SWET vortices appear in the solution with no axial pump flow component at t = 600 s, so the concentration of antigen on the surface of the sixth electrode strip approaches zero once again. Under Vsw = 0 V and Vtw = 6 V, as shown in Figure 6C, since the TWET pump motion is induced along the channel length direction, the confined space of the microfluidic channel is filled with antigen at t = 600 s. Although a decrease in antigen concentration occurs near the reaction surface, the thickness of the depletion boundary layer is relatively thin due to a paucity in convective mixing. Under Vsw = 6 V and Vtw = 6 V, both global horizontal pumping and local transverse vortex stirring appeared in the solution, not only the channel volume was filled with antigens that were constantly delivered downstream by TWET pump force, but the local SWET vortex also made the range of the depletion boundary layer of antigen larger on the functionalized surface of the sixth electrode strip, which possesses a semicircular contour and a more homogeneous depleted antigen distribution, indicating the occurrence of an efficient binding reaction. Figure 6: Simulation analysis of improvement of time-dependent immune reaction kinetics by active MET convection. (**A–D**) A surface plot of the concentration field of incoming antigen and a streamline plot of the MET flow field under different Vsw and Vtw at the ideal frequency combination, (**A**) at V\({}_{\text{SW}}\) = 0 V and Vtw = 0 V, (**B**) at Vsw = 6 V and Vtw = 0 V, (**C**) at Vsw = 0 V and Vtw = 6 V, (**D**) at Vsw = 6 V and Vtw = 6 V. (**E**) Transient evolution of the surface averaged BR\({}_{\text{T}}\)\({}^{-1}\) on the sixth metal strip for four different combinations of SW and TW voltage amplitudes. As in Figure 6E, the dynamic evolution of antigen concentration adsorbed on the functionalized surface \(B\) is quantitatively characterized from 0 to 600 s, which can more intuitively reflect the promotion effect of MET convection on microscale binding kinetics. When Vsw = 0 V and Vtw = 0 V, the surface antigen concentration \(B\) does not change with t and is still almost zero at t = 600 s under no electrothermal fluid motion. When Vsw = 6 V and Vtw = 0 V, B appears to rise, but is still very small without TWET transportation. When Vsw = 0 V and VTw = 6 V, B grows significantly with time, and B/Rt attains 0.0112 at t = 600 s due to the action of the TWET pump motion. When Vsw = 6 V and Vtw = 6 V, the enhancement of B with time is the most obvious, and B/Rt rises to 0.0163 at t = 600 s, since the incoming antigen sample is stirred by lateral SWET eddies and transported by horizontal TWET pump streamlines at the same time. As a result, the dynamic binding efficiency between free antigen and fixed antibody probe can only be significantly enhanced by fully combining the respective flow properties of SWET and TWET by using MET under dual-frequency harmonic actuation.