**3. Results**

The impedance characteristics of biochips PS5 and BS5 are studied under the same experimental conditions. During the experiments, firstly, the ImS on solo biochips are measured without adding anything in the ring top electrode region, secondly, the ImS on biochips are recorded after adding 20 μL Deionized (DI) water. In the third step, for both biochips PS5 and BS5, the additional 1–5 μL DI

water or bacteria suspension are applied within the ring top electrodes. Each measurement is repeated on individual biochip for 3 times, and the corresponding experimental (circular dots) and modeled results (solid lines) with error bars are shown in Figures 4 and 5 for biochip PS5 and BS5, respectively.

**Figure 4.** Experimental and modeled Nyquist plots of the Biochip PS5 with (a, b) no filling and with DI water (20 μL) and (**a**) with additional DI water with volume from 1 μL to 5 μL, and (**b**) with additional bacteria volume of JG-A12 from 1 μL to 5 μL. The error bars are inserted in all Nyquist plots according to 3 repeated Biochip experiments. The experimental results are represented in dots, and modeled results are represented in solid lines.

**Figure 5.** Experimental and modeled Nyquist plots of the Biochip BS5 with (a, b) no filling and with DI water (20 μL) and (**a**) with additional DI water volume from 1 μL to 5 μL and (**b**) with additional bacteria volume of JG-A12 from 1 μL to 5 μL. The error bars are inserted in all Nyquist plots according to 3 repeated Biochip experiments. The experimental results are represented in dots, and modeled results are represented in solid lines.

The equivalent experimental results on two PS5 biochips with the same implantation parameters without filling and with 20 μL DI water are demonstrated in Figure 4a and in Figure 4b as black and red dots, respectively, which reveals the reproducible impedance behavior of the PS5 biochips in the frequency domain. In Figure 4a, additional 1–5 μL DI water is applied after adding the 20 μL DI water, while additional 1–5 μL bacteria is applied in Figure 4b. The additionally added DI water results in an increase of corresponding capacitance and resistance parameters in the equivalent circuits in comparison to the ImS from the case after adding 20 μL DI water, whereas the additional 1–5 μL bacteria cause the decrease of both parameters. Moreover, by adding subsequential bacteria volume from 1 μL to 5 μL as shown in Figure 4b, more significant changes in the third semicircle (CPE3 and R3 as illustrated in Figure 3b) can be recorded in comparison to the impedance characteristics in Figure 4a by adding subsequential 1 μL to 5 μL DI water.

Similarly, based on the equivalent experimental results on two BS5 biochips without filling and with 20 μL DI water in the subfigures in Figure 5, the additional 1–5 μL DI water or bacteria are applied after adding the 20 μL DI water in Figure 5a,b, respectively. Note that, for biochip BS5 after adding the additional 1–5 μL DI water (Figure 5a) or 1–5 μL bacteria (Figure 5b), the decrease of corresponding capacitance and resistance parameters (the thinner curves in Figure 5) in the equivalent circuits in comparison to the ImS from the case after adding 20 μL DI water (thicker red curves in Figure 5a,b) can be recorded, which is di fferent with the biochip PS5. Nevertheless, more significant changes from the impedance characteristics of BS5 after adding additional 1 μL–5 μL bacteria (Figure 5b) can be detected than in the case after adding additional 1–5 μL DI water (Figure 5a), which is consistent to the biochip PS5. These results successfully proved that biochips PS5 and BS5 can be used to detect adhesion of *Lysinibacillus sphaericus* JG-A12 in the ring top electrode region.

Note that the resistance of the boron-implanted biochip BS5 (Figure 5) is typically larger than the resistance of the phosphor-implanted biochip PS5 (Figure 4) due to the lower conductivity of the p-type semiconductor in which the holes are majority carriers in comparison to the n-type semiconductor whereas the electrons are the majority carriers. Conductivity is defined by σ = p.e.μh + n.e.μe, where the mobility of holes and electrons are 505 and 1450 cm<sup>2</sup>/Vs, respectively. By adding analytes, the capacitive impedance is decreased and a third semicircle is formed. Moreover, for the BS5 biochip, the dramatic impedance variation is found after adding additional 1 μL of DI water (violet curve in Figure 5a) and 1 μL of bacterial suspension (violet curve in Figure 5b) than that of biochip PS5 (violet curves in Figure 4). The experimental impedance characteristics from the biochips PS5 can be modeled by the equivalent circuit parameters in the equivalent circuit as shown in Figure 3, which consists of two nonideal capacitances or CPEs (Cp1, Cp2), two parallel resistances (Rp1, Rp2), a contact resistance (Rs) and a contact inductance (Ls). ImS modeling of biochip PS5 is shown in Tables 2 and 3 (first row).

**Table 2.** Modeled equivalent circuit parameters Cp1, Rp1, Cp2, Rp2, Cp3, and Rp3 of the biochip PS5 with 20 μL DI water and di fferent additionally inserted 1–5 μL DI water (Figure 4a).


W20 = 20 μL DI water, W1 = 1 μL DI Water, W2 = 2 μL DI Water, W3 = 3 μL DI Water, W4 = 4 μL DI Water, W5 = 5 μL DI Water.

**Table 3.** Modeled equivalent circuit parameters Cp1, Rp1, Cp2, Rp2, Cp3, and Rp3 of the biochip PS5 and with 20 μL DI water and additionally inserted 1–5 μL bacteria (Figure 4b).


W20 = 20 μL DI water, B1 = 1 μL bacteria, B2 = 2 μL bacteria, B3 = 3 μL bacteria, B4 = 4 μL bacteria, B5 = 5 μL bacteria.

It should be noted that the equivalent circuit of the impedance spectra of the solo biochip PS5 and biochip PS5 with analytes are di fferent due to the additional appeared semicircle. Based on the experimental impedance characteristics from the biochips PS5 after adding analytes, the composition and cell numbers of analytes added to the Au top ring electrode region for biochip PS5 can be determined by modeling the equivalent circuit parameters in the equivalent circuit as shown in Figure 3, which consists of three nonideal capacitors (Cp1, Cp2, Cp3) and three resistors (Rp1, Rp2, Rp3), a contact resistance (Rs), and a contact inductance (Ls). The corresponding ImS modeling results of the biochip PS5 are shown in Table 2 (with DI water) and in Table 3 (with DI water and bacteria).

Similarly, the ImS characteristics of the biochip BS5 can be modeled with two pairs of resistors and nonideal capacitors (Rp1, Rp2, Cp1, Cp2), contact resistance (Rs), and contact inductance (Ls). Furthermore, for the biochip BS5 the electrical equivalent circuit model for biochip with analytes consists of three pairs of resistance-capacitance in addition to the contact resistance and contact inductance. The corresponding ImS modeling results of the biochip BS5 are shown in Table 4 (with DI water) and in Table 5 (with bacteria).

**Table 4.** Modeled equivalent circuit parameters Cp1, Rp1, Cp2, Rp2, Cp3, and Rp3 of the biochip BS5 with 20 μL DI water and additionally inserted 1–5 μL DI water (Figure 5a).


W20 = 20 μL DI water, W1 = 1 μL DI water, W2 = 2 μL DI water, W3 = 3 μL DI water, W4 = 4 μL DI water, W5 = 5 μL DI water.

**Table 5.** Modeled equivalent circuit parameters Cp1, Rp1, Cp2, Rp2, Cp3, and Rp3 of the biochip BS5 and with 20 μL DI water and additionally inserted 1–5 μL bacteria (Figure 5b).


W20 = 20 μL DI water, B1 = 1 μL bacteria, B2 = 2 μL bacteria, B3 = 3 μL bacteria, B4 = 4 μL bacteria, B5 = 5 μL bacteria

With the help of optical microscopy, the calibration between the ImS data and cell concentration observed with the optical density at 600 nm (OD600) has been considered. Calibration of the biochip is achieved in the volume range from 0 μL to 5 μL bacterial suspension in 20 μL DI water. OD600 of 4 corresponds to 2.46E7 cells on the chip if *Lysinibacillus sphaericus* JG-A12 with 1 μL of concentration is applied to 20 μL DI water. For calibration, the dependency of the modeled equivalent circuit elements Rp1, Rp2, Cp1, Cp2 and Rp3 and Cp3 (from impedance modeling) on the nominal number of bacterial cells (from optical microscopy) was evaluated on the basis of the biochip PS5 (Figure 6).

**Figure 6.** Modeled equivalent circuit parameters (dots) and linear fitting curve (red lines) for (**a**) Rp1, and Cp1 and for (**b**) Rp3 and CP3 of the biochip PS5 in dependence on the number of *Lysinibacillus sphaericus* JG-A12.

As demonstrated in Figure 6, 4 equivalent circuit parameters Rp1, Cp1, Rp3, and Cp3 from biochip PS5 have been proved to possess the linear dependence with the number of bacteria. Moreover, 3 equivalent circuit parameters Cp1, Rp3, and Cp3 have the linear relationship with the nominal number of bacterial cells for the biochip BS5 in the range from 2.46E7 to 1.23E8 (Figure 7).

**Figure 7.** Interpolation (red line) of modeled equivalent circuit parameters (**a**) Rp1 and Cp1 and of (**b**) Rp3 and CP3 of the Si Biochip BS5 that depend on number of *Lysinibacillus sphaericus* JG-A12.

Among the modeled electrical elements in the equivalent circuit in Figure 3b, the Rp1 and Cp1 pair represents the Schottky contact at the electrodes/semiconductor interface. If the size of contact area is denoted as A, by adding bacteria suspension to the top electrode region of biochips, the area of the top contact is increased. According to the equation Rp1 = ρ(d/A), where d denotes the thickness of the Schottky barrier, the resistance is reversely related to the area A. Thus, there is reduction in resistance by adding the bacteria suspension. If we consider Cp1 = ε(A/d) with ε as the permittivity of semiconductor, the relationship between Cp1 and A results in the increasing Cp1 with increasing bacteria suspension. The Rp2 and Cp2 pair corresponds to the impedance of semiconductors Si:B in PS5 and Si:P in BS5, and the values are kept the same in Table 2/Table 3 for PS5 and in Table 4/Table 5 for BS5, respectively. The Rp3 and Cp3 pair represents the impedance of bacterial suspension which is added into the Au top electrode region. According to the experimental results from biochip PS5, Rp3 is decreasing linearly with bacteria concentration, while BS5 Rp3 is increasing linearly. Cp3 is increased linearly in both cases.

In detail, the linear impedance change that depends on the bacterial concentration for the Si Biochip PS5 is illustrated in Figure 6 with the 4 model parameters Rp1, Cp1, Rp3, and CP3. Furthermore, the linear impedance change that depends on the bacterial concentration for Si Biochip BS5 is illustrated in Figure 7 with the 3 model parameters Cp1, Rp3, and CP3. Therefore, a multiparameter determination of the bacterial concentration can be performed for both Si Biochips.
