**4. Discussion**

The main objective of this work was to accommodate electrical sensing on paper substrates towards simple, rapid, quantitative bacteria detection in aqueous solutions.

To this end, we first developed a biointerface by functionalizing the NC membrane with endolysin cell-wall binding domain (CBD) aimed at capturing bacterial cells through the whole membrane pore surface. The efficiency of CBD specific binding to *B. thuringiensis* was demonstrated in [28]. In this paper, we validated their potential as immobilized bioreceptors on porous NC membranes, taking advantage of their highly selective binding capacity to capture *B. thuringiensis* bacterial cells present in low-salinity buffers. As the activity of the endolysins was shown to decrease at salt concentrations higher than 200 mM [22], it is necessary to assess the binding capacity of the CBD biointerface towards bacteria under different NaCl concentrations. This assessment is required to determine the range of aqueous solutions that can be considered for bacteria detection without loss of the sensor specificity. Also, the specificity of the detection systems towards *B. thuringiensis* is usually assessed with the absence of electrical signals in the absence of bioreceptors or in the presence of only non-target pathogens. Therefore, the detection specificity remains to be studied, and will be the topic of future works. When considering specificity of the detection, it will be of upmost importance to use a complete lateral flow assay with controlled flow as support to perform the electrical detection and a related robust detection protocol integrating the necessary washing steps.

Second, we took advantage of the NC support to develop an innovative volumebased electrical detection setup by applying the membrane between the electrodes of a parallel-plate prober for dielectric measurements. This setup forms a simple plug-andplay sensing device which is responsive to the electrical properties of the NC membrane. A nitrocellulose membrane functionalized with a specific biointerface is indeed an adequate substrate to support the volume-based electrical detection of bacterial suspensions. Such a detection scheme is attractive as the whole sample volume contributes to the sensing, increasing the contribution from targeted bacteria with respect to surface-based methods. The parallel-plate detection scheme based on impedance spectroscopy proposed in this work was much more straightforward both in fabrication and handling than most of the paper-based electrical biosensors encountered in the literature. They often rely on direct current monitoring requiring to apply more complex electrode systems to the paper (usually three electrodes, for reference, working and counter electrode) and involving electrochemical reactions which are relatively complex to interpret in ionic medium [40,41]. These paper-based sensors usually rely either on the application of conducting pastes showing high electrode and contact resistance (generally requiring custom, hand-made electrical connections) [41,42], or more complex and expansive fabrication processes from the microelectronics industry.

Third, we quantified the presence of 10<sup>7</sup> to 10<sup>8</sup> CFU mL−<sup>1</sup> *B. thuringiensis* in diluted physiological buffer (PBS diluted 1000×). An important feature is the rapidity of the sensing mechanism: the whole detection protocol lasts less than 5 min. Using a simple electrical model including the electrical properties of the NC membrane and the capacitive contribution of the polyester backing, the interplay of both capacitive and resistive properties of the electrolyte are observed. The detection mechanism, based on ion concentration, was determined by the parallel-plate characterization of NC membrane filled with different saline solutions, and helped us understanding the sensor differential response for various electrolyte solutions. Similarities can be drawn in the complex impedance difference between the saline solutions and for biological buffer with and without bacterial cells. In particular, close correspondence is observed between electrical properties of PBS:1000 and ~2 × 10−<sup>4</sup> M NaCl solution, while signals from the 10<sup>8</sup> CFU mL−<sup>1</sup> bacterial suspensions are representative of those of the ~10−<sup>4</sup> M condition. These results, supported by dielectric measurements, sugges<sup>t</sup> that the electrical model proposed for the sensing of NC membranes soaked with ionic solutions can be extended to bacterial solutions. Furthermore, it also endorses the concept of bacteria detection through its surroundings ions, which

has already been discussed in [25]: the differences in sensor response are attributed to the slight difference in ionic content, i.e., electrical conductivity, between sterile PBS:1000 and bacterial resuspension in PBS:1000. Indeed, centrifugation steps lead bacterial cells to release ions due to osmotic pressure and damaged cell walls [43].

Both for saline solutions and bacterial suspensions, an interesting phase shift occurs in the phase peaks between 10 and 1000 kHz, driven by changes in *f3* due to both *CNC* and *RNC* variations. Although the resistive character of the sensor is predominant (changes in RNC affect the total module more than *CNC*), the possibility to monitor NC membrane property changes through variation of *CNC* renders this setup versatile in use and potentially more robust against ionic noise, strongly affecting *RNC*. To assess the relative advantage of the parallel-plate setup over traditional paper-based sensors, we also applied gold microelectrodes (IDE) to the NC membrane using a standard microfabrication process. The IDE sensor is only reactive towards *RNC*, and shows a high sensitivity to bacteria over a larger frequency range than the parallel-plate. Indeed, the electrode disposition does not prevent a direct conduction path between the two electrodes.

Despite its dependence on bacterial concentration, two reasons make the detection of bacteria through higher ionic content unsuitable for bacterial sensing. First, the resulting signal is strongly affected by experimental procedures such as manipulation, contamination and experimental conditions, all affecting the baseline sample conductivity. Second, such a sensing principle is useless for real applications dealing with detection in highly saline solutions, whose high electrical conductivity is hardly impacted by bacterial ion release. Thus, the detection of bacteria through ionic contribution has the main disadvantage that it lacks robustness in a complex environment with various living organisms that are potential ion-sources, resulting in a low signal-to-noise ratio (SNR). The specificity and sensitivity of the electrical detection can be improved by nanoparticles (NP) to specifically label whole bacterial cells. In [44–46], *E. coli* O157:H7 were detected through amplified conductance and permittivity changes by means of the conjugation of specific graphene or gold NPs to the bacteria. In other works, the highly intensive response of Si-NPs and Au-NPs conjugated to bioanalytes at radio frequencies (RF) was used to amplify the dielectric contrast of bioanalytes in solutions [47,48] or to act as microantenna in NC membranes [49].

The ability of the parallel-plate setup to monitor changes of both conductive and dielectric properties in the NC membrane makes it possible to select the type of NP (conductive or dielectric) that increases the SNR the most. Conjugating diverse types of NP with bacterial cells offers promising perspectives for highly specific electrical bacterial detection on lateral flow assays, and will be the focus of upcoming works.
