Molecular-Charge-Contact-Based Ion-Sensitive Field-Effect Transistor Sensor in Microfluidic System for Protein Sensing

In this paper, we demonstrate the possibility of direct protein sensing beyond the Debye length limit using a molecular-charge-contact (MCC)-based ion-sensitive field-effect transistor (ISFET) sensor combined with a microfluidic device. Different from the MCC method previously reported, biotin-coated magnetic beads are set on the gate insulator of an ISFET using a button magnet before the injection of target molecules such as streptavidin. Then, the streptavidin—a biotin interaction, used as a model of antigen—antibody reaction is expected at the magnetic beads/gate insulator nanogap interface, changing the pH at the solution/dielectric interface owing to the weak acidity of streptavidin. In addition, the effect of the pH or ionic strength of the measurement solutions on the electrical signals of the MCC-based ISFET sensor is investigated. Furthermore, bound/free (B/F) molecule separation with a microfluidic device is very important to obtain an actual electrical signal based on the streptavidin–biotin interaction. Platforms based on the MCC method are suitable for exploiting the advantages of ISFETs as pH sensors, that is, direct monitoring systems for antigen–antibody reactions in the field of in vitro diagnostics.


S1. Previous MCC method
shows the change in interfacial potential (ΔVout) obtained by the previously reported MCC method (Figure 1b). Upon adding the biotin-coated magnetic beads with streptavidin, ΔVout drastically increased owing to the direct attachment of magnetic beads to the gate insulator and subsequently decreased gradually when the measurement solution flowed. That is, the effect of the direct attachment of magnetic beads to the gate insulator on the electrical responses was very large and then it took a long time to determine the baseline of interfacial potential; therefore, the actual signal used for biomolecular recognition may be distorted in the MCC method shown in Figure 1b.

S2. Source-follower circuit for FET real-time measurement
In this study, the change in surface potential was monitored using the source-follower circuit shown in Figure S2 [S1]. Here, VG and VD were set to constant values and VS was controlled at a constant IDS. Figure S2. Electrical circuit. The change in surface potential (ΔVout) at the gate was measured at a constant ID (1 mA) and VD (2.5 V) using the source follower circuit.

S4. Calculation of LOD
Considering the Kaiser method, the lowest concentration of streptavidin that can be detected by the MCC method is calculated from ∆ showing a significant difference from the average ΔV2 at the concentration of streptavidin in the blank (C = 0). C indicates the concentration of streptavidin. In Figure 5, an approximately straight line is drawn in the concentration range from 1.8 μM to 180 μM, within which streptavidin can be detected, and extrapolated to the ΔV2 axis. This relationship is expressed as where the y-intercept (0.93) means the average ΔV2 in the blank. Here, the corrected sample standard deviation ( ) is where ∆ shows the average ∆ of a detected signal and n indicates the number of detected output signals. Moreover, the reliable range of w in the blank is indicated as That is, the LOD ( ) is calculated using equations (1)-(3) on the basis of the output signal ∆ ( ) that shows +3 from the average ΔV2 in the blank (0.93).
Considering the above, the LOD for streptavidin for the MCC method was calculated to be about 2.3 μM.   [S3,S4]. In fact, ΔVT

S5. LOD for streptavidin-biotin interaction
was measured with changes in pH using one of the ISFET sensors in this study ( Figure   S4b). From such electrical characteristics, the average pH sensitivity was found to be about 55 mV/pH at 25 °C for the 6 ISFET sensors used in this study, as shown in Figure   S5c. According to the detection principle, the surface potential at the gate surface was continuously monitored using a source follower circuit, as shown in Figure 3. As a result, the potential change (ΔVout) at the interface between the gate insulator and an aqueous solution, which corresponds to -ΔVT, can be directly output at a constant ID [S1]. ISFET sensor was about 55 mV/pH, which almost showed a Nernstian response at 25 °C.
VG at a pH of 4.01 was offset to 0.