*2.2. Biological Procedures*

#### 2.2.1. Bacterial Strains and Growth Conditions

*B. thuringiensis GBJ002 expressing the cyan fluorescent protein (CFP)* was used as proof-ofconcept for this study. Bacteria were plated on lysogeny broth (LB)-agar plate containing kanamycin (50 μg/mL) and acid nalidixic (25 μg/mL) and grown overnight (O/N) at 30 ◦C. Next, an individual colony was used to inoculate 5 mL of LB supplemented with antibiotics and the suspension was incubated O/N at 30 ◦C with agitation (120 rpm). Then, the culture was washed twice with an appropriate buffer (water or diluted PBS) to remove residual LB (centrifugation at 10,000× *g* for 5 min at room temperature (RT)). The pellet was finally re-suspended in the appropriate buffer yielding a bacterial suspension of ca. 10<sup>8</sup> CFU mL−1.

#### 2.2.2. Phage Endolysins Expression and Purification

A detailed description of the expression and purification of the CBD of PlyB221 endolysin, encoded by phage Deep-Blue can be found in [28]. The CBD was fused to a green fluorescent protein (GFP) for fluorescence assays. The protein concentration was adjusted to 1 mg/mL.

#### 2.2.3. Preparation and Characterization of the Cell-Wall Binding Domain (CBD) Biointerface for Specific Bacteria Capture

A protocol for optimal biofunctionalization of the NC membranes with endolysin CBD based on simple physisorption was developed (Figure 1). Membranes were prepared by individually depositing and shaking 50 μL of 1 mg/mL solution of purified CBD on the NC. Membranes were then dried in an oven for 60 min at 37 ◦C followed by desiccation for 30 min at RT to fix the proteins to the membrane. Following desiccation, membranes were washed twice during 2 min with deionized (DI) water to remove proteins in excess. Membranes were wiped, dried, and finally stored in a desiccation chamber at RT. Effective binding of specific bacteria to the biointerface was assessed by depositing a droplet of *B. thuringiensis* suspension on the biofunctionalized membranes followed by a washing step (Figure 1). Confocal laser scanning microscopy (Zeiss LSM 710) experiments were performed to observe bacteria immobilization in the membrane depth.

**Figure 1.** Protocol of the nitrocellulose (NC) membrane biofunctionalization with endolysin cell-wall binding domain (CBD) and validation of the biointerface through capture of *B. thuringiensis* cells. (1) The deposition of the phage endolysin CBD in the NC membrane is followed by drying, washing and desiccator steps. (2) The bacteria are then deposited on the membrane and specifically captured by the CBD.

### *2.3. Parallel-Plate Setup*

### 2.3.1. Impedance Sensing

Figure 2A shows a schematic of the experimental system for the simple parallel-plate measurements. The nitrocellulose membranes were held between the two parallel-plates of the dielectric test fixture (16451B, Agilent, Santa Clara, CA, USA), connected to an impedance analyzer (LCR 4284A, Agilent, Santa Clara, CA, USA). The impedance spectroscopy measurements were carried out with the LCR, remotely controlled by a computer through the Labview software (Labview National Instrument, Austin, TX, USA) to perform an automatic sweep from 1 kHz to 1 MHz, at voltage amplitude of 200 mV. Before impedance measurement, an open-circuit calibration was performed. The impedance data were extracted in a magnitude-phase data-structure. The ZVIEW software (Scribner Associates Inc., Southern Pines, NC, USA) is used to fit the electrical equivalent model of the parallel-plate sensing system, as presented in Figure 2A, and extract the model parameter values from the measurements.

The material real and imaginary dielectric constant are derived for a given membrane thickness by measuring its capacitance and dissipation factor. An electrical model is used to differentiate the actual dielectric properties of the NC from those of the polyester backing (Figure 2A).
