**1. Introduction**

Access to safe and sufficient water is a prerequisite for to the development of human communities and the enhancement of economic activities [1]. Identification, control and prevention of ground and surface water pollution require both frequent water quality testing and diligent water management. The detection of pathogenic bacteria such as *Escherichia coli*—used as an indicator for fecal water contamination—usually deploys techniques that include colony count, DNA analysis after polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA) [2,3]. Despite the favorable performance

Leprince, A.; Glinel, K.; Mahillon, J.; Raskin, J.-P. Electrical Characterization of Cellulose-Based Membranes towards Pathogen Detection in Water . *Biosensors* **2021**, *11*, 57. https://doi.org/10.3390/ bios11020057

**Citation:** Le Brun, G.; Hauwaert, M.;

Received: 31 January 2021 Accepted: 19 February 2021 Published: 21 February 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

(detection limits of 1 CFU mL−1) of these techniques, their high cost, their requirement of well-equipped laboratory facilities, and the time constraint (at least several hours per test) have prompted the development of portable, simple and low-cost tools suitable for a rapid (less than 1 h) and precise detection methods of pathogens. Furthermore, the sanitary pandemic caused by the infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlighted the field of action of point-of-care (PoC) devices because it can meet the need of mass screening, in particular to screen the virus in water environments. Wastewaterbased epidemiology (WBE) is a promising approach to predict the potential spread of the infection by testing for infectious agents in wastewater, and has been approved as an effective way to obtain information on diseases and pathogens [4,5].

Microfluidic paper analytical devices (μPADs), on the one hand, are the dominant PoC biosensors for the rapid detection of pathogens in both healthcare and environmental monitoring, especially in situations with scarce resources [6,7]. Given the accessibility of this technology, it is appropriate for either water scientists or citizen groups, without requiring specific training [8]. Paper is a valuable platform for biodetection as it presents several assets. First, it has beneficial spontaneous microfluidic properties through capillarity. Second, it facilitates the attachment of bioreceptors which are often proteins such as antibodies, that contribute to specificity towards pathogens. Third, it is low-priced and it allows for straightforward manufacturing and disposability. These benefits are already used in lateral flow assays (LFA) such as pregnancy test strips, in which analytes of interest are passively drained through a nitrocellulose (NC) membrane, a paper derivative, towards the detection zone where they are immobilized by specific bioreceptors. Current paperbased sensors mostly require the use of labels (such as gold nanoparticles, which have an intense red color), conjugated with antibodies to achieve specific optical detection of the immobilized analytes [9]. However, they have two main disadvantages that inhibit their use in the field of water potability, which has demanding limits of detection for pathogens and pollutants. Optical LFA have a reduced sensitivity since only the top 10 μm depth of the paper contributes to the colorimetric signal due to the opaqueness of the membrane [10]. Additionally, the measurement result is mostly qualitative or semi-quantitative.

Electrical biosensors, on the other hand, rely on the monitoring of changes in material electrical properties when bacteria bind in close proximity from the surface of, e.g., interdigital electrodes (IDE) designed on a solid substrate [11,12]. The signal response, often proportional to the number of bacteria, is used as an electrical fingerprint of the sample to provide fast, precise and quantified information about the bacteria presence in water. However, the particular mechanisms of electron transfer between electrodes and specific bacterial cells, as well as within the cells, are still under fundamental studies in bioelectrochemistry [13]. Furthermore, the production and usage of electrical biosensors face significant obstacles. Grafting a biorecognition layer, e.g., bacteriophages (phages) or antibodies, on the surface of conventional surface-based electrical biosensors faces problems such as reproducibility, uniformity and stability over time [14]. The functionalization protocol needs to be adapted to every surface material and grafting molecule. In addition, the capture percentage of bacteria by the biorecognition layer is relatively low since only bacteria in close vicinity to the surface bind to the specific receptors. Many of the target pathogens thus flow over the electrode without binding, decreasing the sensor sensitivity. Finally, conventional surface-based electrical biosensors utilize gold electrodes functionalized with bioreceptors/antibodies using classical thiol chemistry [15,16]. However, insufficient chemical stability of thiolates is one of the most serious problems for their applications in ambient and aqueous environments [17].

Despite the aforementioned drawbacks of both individual sensing technologies, studies have shown that the integration of highly sensitive electronic detection methods with LFA is an attractive approach to circumvent these and capitalize on the advantages of paper substrates and electrical biosensors [18]. Given the favorable electrostatic properties of nitrocellulose, bioreceptors are readily immobilized through the whole pore volume of the paper membrane, thus drastically increasing the number of interactions with targeted

pathogens. Unlike surface-based methods, electrical measurements taking advantage of paper porosity thus allow to quantify the number of bioreceptor-bacteria conjugates in the whole tested sample volume. However, the development of such sensors remains very challenging for three main reasons.

First, one of the key factors affecting the analytical performance of μPADs is the bioreceptor used to capture the bacteria in the test zone. Antibodies, commonly used as a biointerface in μPADs, are rather expensive. As a result, there is a growing interest in developing proteins as alternative receptors for LFA. Particularly promising are bacteriophages, viruses that specifically infect bacteria and produce lytic enzymes called endolysins that show strong affinity and high specificity towards target bacteria [19,20].

Second, due to significantly different properties of paper-based and more conventional substrates for electronic circuits, innovative design methods are needed. Alternative manufacturing techniques, e.g., printing sensors such as IDE, are being explored as integrated sensing devices [21–23]. However, integrating electronics on paper substrates is difficult to implement because of the inhomogeneous nature of the paper, resulting in low electrode resolutions (~hundreds of μm) and high electrical resistances [24] with respect to the classical design of microelectrodes suitable for bacteria sensing (a few μm finger gaps) [25].

Third, prior works that attempted to accommodate electrical bacteria detection on common μPADs were mostly based on direct charge transfer measurements [26,27]. However, these require cumbersome redox probes. Furthermore, electrical measurements are influenced by the ionic strength of the analyzed solution. Direct current measurements are particularly affected by this ionic background noise, since they only measure the solution resistance, inversely proportional to the number of ions. When dealing with aqueous samples presenting varying electrical conductivities, therefore, it is challenging to calibrate the sensor and to differentiate the electrical response of target compounds from the electrical background signal.

In this paper we address the aforementioned challenges by emphasizing three contributions towards paper-based electrical biosensing for simple, rapid and affordable bacteria detection in water.

First, we capitalize on the natural capillarity of the NC membrane to wick bacterial suspensions to the testing zone, where the membrane is functionalized with the recently characterized cell-wall binding domain (CBD) derived from the PlyB221 endolysin encoded by phage Deep-Blue targeting *Bacillus thuringiensis* [28], used as model microorganism in this work. This ensures high binding and immobilizing capacity towards this specific bacteria.

Second, we demonstrate the relevance of the simple parallel-plate setup presented in [29]. This common material dielectric measurement system can be judiciously used as sensor to perform electrical measurements on NC membranes inserted in between the electrodes. This plug-and-play setup eliminates the unpractical need to integrate electronics components directly onto the paper matrix.

Third, impedance sensing is proposed to monitor the porous NC membrane permittivity changes caused by various electrolyte solutions. Since no electron transfer occurs at the electrode surface, changes in electrical properties are mainly observed through volumic impedance properties. By measuring the later with an AC signal, both resistive and capacitive properties are estimated at high frequencies, which enables the label-free and non-intrusive detection of bacteria immobilized by specific bioreceptors. Indeed, their presence in the membrane affects both the global conductivity and permittivity [25,30].

The remainder of this paper is organized as follows. We began by validating the CBD biointerface, both electrically and optically. In order to characterize the system behavior and ionic noise for aqueous solutions with different ionic strengths, we then analyzed the sensor response and resolution to saline solutions at different salt concentrations, used as models of real aqueous samples. An equivalent electrical model of the sensing system was developed to quantify the impact of ionic concentration on the total measured impedance. Then, the sensing principle was validated in the presence of bacterial cells. A proof of concept of the simple and rapid (<5 min) parallel-plate biosensor was demonstrated by detecting *B. thuringiensis* cells in low-conductive buffers. Finally, the bacterial detection results with the plug-and-play parallel-plate setup were compared with a planar fringing field electrodes system, composed of IDE directly applied on a single side of the NC membrane. The sensing principles of both parallel-plate and IDE devices were modeled and analyzed using small-signal electrical equivalent circuits, highlighting the contribution of ions in both bacterial detection mechanism. Their potentials, advantages and limitations are also discussed.

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