- The utilization of field-effect transistor (FET) devices in biosensing applications have been extensively studied in recent years. Qualitative and quantitative understanding of the contribution of the organic layers constructed on the device gate, and the electrolyte media, on the behavior of the device is thus crucial. In this work we analyze the contribution of different organic layers on the pH sensitivity, threshold voltage, and gain of a silicon-on-insulator based FET device. We further monitor how these properties change as function of the electrolyte screening length. Our results show that in addition to electrostatic effects, changes in the amphoteric nature of the surface also affect the device threshold voltage. These effects were found to be additive for the first (3-aminopropyl)trimethoxysilane linker layer and second biotin receptor layer. For the top streptavidin protein layer, these two effects cancel each other. The number and nature of amphoteric groups on the surface, which changes upon the formation of the layers, was shown also to affect the pH sensitivity of the device. The pH sensitivity reduces with the construction of the first two layers. However, after the formation of the streptavidin protein layer, the protein’s multiple charged side chains induce an increase in the sensitivity at low ionic strengths. Furthermore, the organic layers were found to influence the device gain due to their dielectric properties, reducing the gain with the successive construction of each layer. These results demonstrate the multilevel influence of organic layers on the behavior of the FET devices.