Insulator-based dielectrophoresis (iDEP) has attracted considerable attention due to its ability to precisely capture and manipulate nanoparticles and biomolecules. A distinctive approach for effective manipulation of nanometer-sized proteins employing iDEP technique by generating higher electric field (E) and gradient (??2) in the iDEP microfluidic devices is delineated. Strategies to generate higher ??2 in the iDEP devices were outlined using numerical simulations. Intriguingly, the numerical simulation results demonstrated that by decreasing the post-to-post gap in the iDEP microfluidic devices, the ??2 was increased by ⁓12 fold. Furthermore, the inclusion of channel constrictions, such as rectangular constriction or curved constriction into the straight channel iDEP microfluidic device led to a significant increase in ??2. In addition, the inclusion of rectangular constrictions in the straight channel iDEP microfluidic device resulted in a greater increase in ??2 compared to the incorporation of curved constrictions in the same device. Moreover, the straight channel device with horizontal post-to-post gap of 20 μm and vertical post-to-post gap of 10 μm generated the lowest ??2 and the ??2 was uniform across the device. The rectangular constriction device with horizontal and vertical post-to-post gap of 5 μm generated the highest ??2 and the ??2 was non-uniform across the device. Subsequently, suitable candidate devices were fabricated using soft lithography as well as high resolution 3D printing and the DEP behavior of ferritin examined under various experimental conditions. Positive streaming DEP could be observed for ferritin at low frequency in the device generating the lowest ??2, whereas at higher frequency of 10 kHz no DEP trapping characteristics were apparent in the same device. Importantly, in the device geometry resulting in the highest ??2 at 10 kHz, labeled ferritin exhibited pDEPtrapping characteristics. This is an indication that the DEP force superseded diffusion and became the dominant force.