**1. Introduction**

Biochips [1], as one of the most advancing technologies in the biomedical field, have attracted lots of attention in the past decades due to their promising functionalities, e.g., for the detection and recognition of biomaterial in a considerable wide range [2]. In the application field of microbiology, in comparison to optical microscopy, the biochips can prevent human errors and offer faster and easier functional operation as lab measurement tools for biosensing purposes. Thus, the biochips can be helpful for the disease diagnosis with high reliability and time efficiency. Biochips possess many advantages such as mass production, simple immobilization, high density, and high throughput [3].

In this work, the miniaturized p-n junction-based Si biochips are proposed with well-defined gold ring top electrodes and unstructured platinum bottom electrodes, which offer the advantages for sensing the biomaterial such as cost-effectiveness and high portability. The impedance spectroscopy (ImS) [4] has been used to characterize the novel designed biochips. After applying the biomaterial in the Au ring top electrode region, the two-phase electrode structure has been successfully developed and investigated

for establishing the functioning electrical equivalent circuit of biochips, which can be utilized for interpreting the impedance properties that recorded between the top and bottom electrodes [5]. Based on the two-phase electrode structure, the straight-forward linear relationship between the specific equivalent circuit parameters and the cell numbers has been discovered, which o ffers the opportunity for determining biomaterial concentration with low cost and high e fficiency [6].

Furthermore, the novel p-n junction-based Si biochips possess the advantages from di fferent perspectives. First, the analysis cost required by p-n junction-based Si biochips for determining the cell density of the biomaterials is considerably lower than that required by other methods [7]. For example, in comparison to analytical techniques such as mass spectrophotometry, gas chromatography, or liquid chromatography [8], the proposed biochips need no special treatments to the biomaterials and they can be kept alive during the detection process. In perspective applications, the number of biomaterials will be determined in a large concentration range by using p-n junction-based Si biochips in conduction with their impedance characterization. Second, the newly developed two-phase electrode structure enhanced the sensitivity of the corresponding equivalent circuit and improved the analytical accuracy of the recorded impedance properties of biochips, which enabled the possibility for sensing biomaterial with considerable low cost [9]. In this paper, the bacteria *Lysinibacillus sphaericus* JG-A12 [10] has been studied due to its potential industrial applications in metal remediation or selective recovery of metals in recycling processes. The S-layer protein in *Lysinibacillus sphaericus* JG-A12 is mainly responsible for such outstanding metal-binding capabilities. The impedance spectroscopy of p-n junction-based Si biochips may o ffer a new possibility for online monitoring the biomass during the cultivation process.

The paper is structured as follows: In Materials and Methods section, we describe the structure of proposed p-n junction-based Si biochips and introduce electrical equivalent circuit. In Results section, the systematical experimental study of impedance properties of biochips is demonstrated, and the equivalent circuit parameters are extracted. In Discussions section, the origin of two-phase electrode structure is studied and validated by the experimentally recorded impedance data. The paper is summarized and an outlook is given in Conclusion section.

#### **2. Materials and Methods**

The bacteria used for the investigation in the work is *Lysinibacillus sphaericus* JG-A12, i.e., a Gram-positive, rod-shaped soil bacterium isolated from the uranium mining waste pile "Haberland" near Johanngeorgenstadt in Saxony, Germany. They were cultivated in nutrient broth (8 g/L, Mast Group) overnight in Erlenmeyer flasks at 30 ◦C with shaking at 100 rpm. Cell density was determined by measuring the optical density at 600 nm (OD600) using a UV–Vis spectrometer. Correlation between OD600 and cell number was achieved by cell counting under a microscope using a Neubauer counting chamber.

Detection and culture purity of the *Lysinibacillus sphaericus* JG-A12 is usually done with help of microscopy, including the morphology of colonies on agar plates, the growth behavior, even the smell of the culture. To make sure that a microorganism is *Lysinibacillus sphaericus* JG-A12, one has to use genetics means for detecting the 16S rDNA sequence. All these tests are time-consuming. The cell number could be estimated by optical density measurements (OD 600 nm) by putting culture samples into UV–VIS spectrophotometer and using established correlation between cell count by microscopy and OD values. The proposed p-n junction-based Si biochip could be used in a bypass of a culture vessel to measure cell density.

## *2.1. Structural Description*

As illustrated in Figure 1, phosphor or boron ions have been implanted into p- or n-type silicon wafers with a thickness of 525 μm, which results in an n-p junction or a p-n junction, respectively. The 150 nm thick gold (Au) ring top electrodes have been deposited by dc-magnetron sputtering with inner and outer diameters of 6.7 mm and 8.0 mm (Figure 1b). A ring electrode has been chosen because of the homogenous field distribution between top and bottom electrodes. In the work, the biochips

PS5 and BS5 have been manufactured, measured, and modeled to analyze influence of bacteria on the biochips. Table 1 lists the overview of the implantation parameters for the manufacturing of the biochips PS5 and BS5 with ring top electrodes.

**Figure 1.** Schematic sketch of the p-n junction-based Si biochip with a ring top electrode with (**a**) boron ions implanted into Si:P or with (**c**) phosphorous ions implanted into Si:B. (**b**) Photograph of a socketed p-n junction-based Si biochip with Au ring electrode. Top and bottom electrodes have been wire-bonded to a diode socket and connected to an Agilent 4294A impedance bridge.

**Table 1.** Implantation parameters of biochips PS5 (phosphor into Si:B) and BS5 (boron into Si:P). The Au ring top electrodes and unstructured Pt bottom contacts have been prepared after ion implantation.


The impedance characteristics of biochips PS5 and BS5 have been recorded within the frequency range from 40 Hz to 1 MHz under normal daylight at room temperature. These measurements were taken using the Agilent 4294A precision impedance analyzer. In the impedance experiments, the solvent (Deionized water) and the bacteria (*Lysinibacillus sphaericus* JG-A12) are added into the Au ring top electrode region.

In order to visualize the different concentrations of *Lysinibacillus sphaericus* JG-A12, the optical microscopic images have been taken before adding (Figure 2a) and after adding bacteria with corresponding optical density at 600 nm (OD600) (Figure 2b–d). The Au ring top electrodes are deposited on a glass slide for utilizing the phase contrast mode of microscope. The OD600 is a common measure for microbial cell density, which can be correlated to the cell number per volume depending on the chosen bacteria. In this work, the OD600 of 4 up to 16 are applied in the Au ring top electrode region for further impedance characterization, which corresponds to bacteria concentration of 1.23E9 up to 6.15E9 cfu/mL under the assumption that all of the cells are alive.

**Figure 2.** Top view optical microscopic image of a section of ring electrode on glass with (**a**) Deionized water, (**b**) bacteria in water at OD600 0.25, (**c**) bacteria in water at OD600 1, (**d**) bacteria in water at OD600 2 in the ring top electrode region. Here a transparent glass substrate has been used to illuminate the sample with light from the backside. The thickness of the ring top electrodes was 150 nm and was large enough to keep the inserted liquid in the ring top electrode.
