# Gating Hysteresis as an Indicator for Silicon Nanowire FET Biosensors
## 3 Results and Discussion
### Hysteresis of the Transfer Curve Versus Subthreshold Shift \(dV_{T}\) for thrombin Detection  
Figure 3a summarizes the thrombin sensing experiments upon the step by step addition of the thrombin molecules into the solution. It demonstrates the concurrent measurements of threshold voltage shift \(dV_{T}\) (blue curve) and hysteresis evolution \(V_{Inst}\) (black curve) for the color-coded concentrations of thrombin (200 pM-200 nM) in a gate sweep measurement as a function of \(V_{G}\) and time. The analyzed data allow us to make a number of conclusions discussed in detail below: (i) the fluid flow rate (on/off) influences the measurements dramatically; (ii) an irreversible \(dV_{T}\) upon increase of thrombin concentration was observed; (iii) the (reversible) increase of hysteresis \(V_{Inst}\) was found to be dependent on thrombin concentration (Figure 3b,c). This is especially the case in the range of high analyte concentrations (nM range). The first aspect, related to the dependence of the sensor signal on the pumping rate, is eventually caused by the perturbation of the formed Debye layer; i.e., the removal of the diffusive outer layer in the electric double layer induced by the on/off operation of the fluidic pump injecting the analyte solutions (100 uL/min during buffer or thrombin injection followed by incubation in static state). Such effects were previously described for ion-sensitive field effect transistors (ISFETs) [51]. _Subthreshold shift \(dV_{T}\) for thrombin detection_: Furthermore we analyze \(dV_{T}\) upon addition of the thrombin into the solution. Magnified details of the corresponding \(I_{SD}\)-\(V_{G}\) curves are displayed in Figure 3d. It demonstrates the accumulation of the thrombin-induced voltage shifts upon increasing concentrations from 0 (lowest concentration tested: 200 pM) to 200 nM, from the right (buffer curve) to the left (maximal concentration 200 nM). Note the progressively smaller voltage shifts at higher concentrations of the injected thrombin, which is potentially attributed to a gradual saturation of binding sites on the sensor surface. To analyze \(dV_{\text{Ti}}\) induced by thrombin attachment, the corresponding \(V_{G}\) at a fixed \(I_{SD}\) at different times \(t_{i}\) (with \(t=t_{i}\) after thrombin attachment and rinsing for each concentration \(C_{i}\)) are determined at a level where the subthreshold slope is steepest in the semilogarithmic plot. Although this point is in the subthreshold regime and therefore below the conventional threshold of field effect transistors, we deliberately define this point of measurement as our sensor threshold voltage \(V_{T}\) since there is only a constant offset to the point where the transistor actually turns on. The \(V_{T}\) value is calculated as the mean value of both branches of the FET gating hysteresis at each time step. Apart from the instantaneous change of \(V_{T}\), we plot the fragments of the transfer characteristics, depicting the negative shift of the curve upon increase of the thrombin  
Figure 3: Thrombin detection experiment with the bio-functionalized FET. (**a**) Real-time results upon step by step addition of thrombin, resulting in threshold voltage hysteresis (black line) and threshold voltage shift (blue line). (**b**) Example hysteretic transfer curves before thrombin addition, after thrombin injection and after rinsing. (**c**) Calibration of the hysteresis in the logarithmic scale of thrombin concentration. (**d**) Magnification of the transfer curves after different thrombin concentrations demonstrating an accumulative negative shift. (**e**) Calibration curve of the threshold voltage shift for thrombin detection using the TBA (red line) and the RDM (black line). concentration from 0 (200 pM lowest concentration) to 200 nM (Figure 3d). For each \(dV_{T}\) value at different thrombin concentrations, the running mean of 15 neighboring \(V_{T}\) values is taken after the signal saturation and is averaged to flatten the curve, resulting in a small error bar of \(dV_{T}\) of less than 1 mV.