**5. Conclusions**

In this paper, an innovative method for the electrical characterization of cellulosebased membranes was proposed towards the development of low-cost, quantitative paperbased biosensors. It consists in the use of a simple parallel-plate setup as sensor to perform impedance measurements on the membrane inserted within the electrodes. The sensing principle was studied and validated by detecting saline solutions of different molar concentrations spread within nitrocellulose membranes. We then demonstrated the proofof-concept of the detection of bacteria: impedance-based detection of 10<sup>8</sup> CFU mL−<sup>1</sup> of *B. thuringiensis* cells was presented without labeling nor signal enhancement strategies. The bacteria were detected through an overall increase in ions in the membrane caused by their presence. Impedance measurements were also performed with interdigital electrodes integrated on the membrane and confirmed the parallel-plates results. Finally, newly discovered endolysin CBD were introduced as specific bioreceptors and deposited inside the nitrocellulose membrane, enabling successful bacterial cell capture over the whole membrane volume.

In conclusion, by combining the benefits of a cellulose-based membrane, novel protein bioreceptors and precise impedance measurements with a reusable plug-and-play setup, we obtained promising results towards the development of an affordable and sensitive

biosensor with a speed of response under 5 min. To overcome the limitations presented by our system and reach the very low limit of detection required for example for drinking water assessment, it is necessary to integrate signal enhancement strategies. Future research works will explore the use of nanoparticles as labels to increase the analytical performances of the device. The development of such a versatile tool creates novel opportunities in situations that require rapid and frequent pathogen detection, such as the detection of *E. coli* in drinking water. Sensing applications could be extended to the detection of various pathogens and viruses as well, through appropriate and direct modification of the biointerface. This may prove particularly useful in emergency situations in light of the recent coronavirus disease 2019 (COVID-19) pandemic.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/bios11020057/s1, Table S1: Results from the fitting of impedance data (Figure 5) to the simple electrical equivalent model used for the parallel-plate setup. Table S2: Results from the fitting of impedance data (Figure 6) to the simple electrical equivalent model used for the parallel-plate setup.

**Author Contributions:** Conceptualization, G.L.B., M.H. and J.-P.R.; methodology, G.L.B., M.H. and A.L.; software, G.L.B. and M.H.; validation, G.L.B. and M.H.; formal analysis, G.L.B. and M.H.; investigation, G.L.B. and M.H.; resources, J.-P.R., K.G. and J.M.; data curation, G.L.B. and M.H.; writing—original draft preparation, G.L.B. and M.H.; writing—review and editing, J.-P.R., K.G., A.L., J.M.; visualization, G.L.B.; supervision, J.-P.R., K.G., J.M.; project administration, J.-P.R., K.G., J.M.; funding acquisition, G.L.B., J.-P.R., K.G., J.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Fund for Scientific Research (FRIA gran<sup>t</sup> to G.L.B. and FNRS gran<sup>t</sup> to J.M.) and the Research Department of the Communauté française de Belgique (Concerted Research Action, ARC no.17/22-084, gran<sup>t</sup> to A.L.).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We thank all the engineers and technicians of the UCL-WINFAB cleanrooms and WELCOME platform for IDE depositions and help in electrical measurements, respectively; W. Xu (IMCN, UCLouvain) for her help in biofunctionalization protocol development; M.-C. Eloy (LIBST, UCLouvain) for confocal experiments; and researchers from the MIAE lab for protein and bacteria productions. K.G. is a Senior Research Associate of F.R.S.-FNRS.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
