**2. Paper-based analytical devices**

## **2.1 Substrate material**

Paper is a complex material and a promising support for the development of biosensor analytical devices. Its main features, such as versatility, low-cost, and biocompatibility, generate simple and disposable bioanalytical tools using low reagent consumption (in the order of microliters) [8, 11, 12]. Paper is mainly constituted of cellulose fibers. The cellulose is a hydrophilic polymer, which makes paper substrate permeable to aqueous liquids [15].

There are different types of paper that are used for fabrication of paper-based devices (**Table 1**). Filter papers have been widely used as substrate material to paper-based devices for biosensor application [16–19]. There are a vast range of commercially available high-quality filter papers, mainly constituted of alpha cellulose, a highly stable form of cellulose. The filter papers can be classified according to different properties, such as particle retention, pore size, thickness, and flow rate.

The filter paper grade 1, considered as a medium retention and flow filter paper, has been functionalized to obtain paper-based immunosensors [16, 17]. Irvine et al. adsorbed metallothioneins in grade 1 filter paper for the detection of heavy metals [18]. In the same way, other filter paper grades have been used, such as the slow filter paper grade 42 (pore size of 2.5 μm) for the incorporation of an in vitro transcription/translation system [19].

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*Paper-Based Biosensors for Analysis of Water DOI: http://dx.doi.org/10.5772/intechopen.84131*

Cellulose

Nitrocellulose membrane

**Table 1.**

chromatography paper

**Type of paper Examples Features**

Whatman® chromatographic paper grades 1, and 2

Boise®Aspen® 30 multiuse recycled copy paper

Cellulose filter paper Whatman® filter paper grades 1 and 2

Printing paper Fabriano 5 HP paper,

*Types and features of papers used in biosensors described recently.*

immunoagglutination assay.

three layers of printing.

**2.2 Fabrication procedures**

or as a low-cost alternative to microplates [28].

Cellulose chromatography paper is also an alternative as high-quality substrate in paper-based biosensors. These papers can be differentiated by their flow rate and thickness. Vijitvarasan et al. [20] developed a paper-based device taking in advantage of the separative properties of chromatographic paper in order to enhance the concentration of gold nanoparticles (AuNPs) on the surface of the paper. Thus, a lower level of reduced silver particles was detected on the surface of AuNPs. Chromatography papers have also been applied in the development of microfluidic devices based on capillary flow measurement [21, 22]. McCraken et al. [22] tested two different chromatography papers (grade 1 Chr and grade 2 Chr) to optimize the separation of immunoagglutinated particles. The chromatography paper providing the lower flow rate (grade 2, 115 mm/30 min) was selected for the

Hydrophilic polymer, permeable to aqueous liquids; available with different

immobilization of complex biological structures by electrostatic interactions

3D structures can be easily printed in its surface, providing microchannels or

screen-printed electrodes

pore sizes and thickness

Millipore Hi-Flow Plus HF240 Hydrophobic polymer; adequate for

Allows the concentration of nanostructures in its surface and the separation of nanoparticles in agglutination-based assays

Derivatives of cellulose, such as nitrocellulose membranes, have been applied as substrates of paper-based devices. These membranes are naturally hydrophobic and demonstrate to be adequate for the immobilization of enzymes and proteins by electrostatic interactions [11]. For instance, Lopez-Marzo et al. [23] developed a lateral flow immunodevice with nitrocellulose membrane for the detection of Cd2+ in water, taking advantage of the immobilization of antibodies (2A81G5 mouse antibody and antibovine serum albumin (BSA) mouse antibody), to create zones where the probe conjugate and the positive control containing BSA would interact. A similar approach was reported for immobilization of BSA conjugate and control

Concerning electrochemical paper-based devices, printing paper can be applied

In addition, both chromatographic paper and multiuse recycled copy paper were used for printing wax wells in paper-based devices as a confinement strategy [20],

There are different techniques that can be applied in order to obtain paper-based biosensor devices with variable properties, such as functionalized platforms with

in the development of screen-printed electrodes. Hence, carbon-based conductive ink is printed onto paper surface [25, 26]. In this context, Rengaraj et al. [27] fabricated a paper-based electrode with the high-quality printing paper using only

goat antimouse immunoglobulin for the detection of U(VI) [24].


#### **Table 1.**

*Biosensors for Environmental Monitoring*

MCL for lead is 15 [5] and 10 μg L<sup>−</sup><sup>1</sup>

according to United States Environmental Protection

[6]. Uranium presents the higher MCL, which

Agency (EPA) and World Health Organization (WHO), respectively. Moreover, the

which provided microfluidic features to the paper-based devices [12].

contaminants in water, focusing on work developed in the last 3 years.

**2. Paper-based analytical devices**

permeable to aqueous liquids [15].

transcription/translation system [19].

**2.1 Substrate material**

Reviews concerning the application of paper-based devices in different fields such as food, water analysis, environmental monitoring, and health diagnostics are available [8, 11, 13]. Furthermore, the application of biosensors has been extensively discussed regarding both their usefulness on assessing environmental and urban pollutants [1], and also their role as part of portable biochemical detection systems [14]. However, gathering information about the implementation of paperbased techniques coupled with biosensor devices to water analysis is still lacking. Hence, the aim of this work is to provide a description of the state of the art about the development and application of paper-based analytical biosensors to detect

Paper is a complex material and a promising support for the development of biosensor analytical devices. Its main features, such as versatility, low-cost, and biocompatibility, generate simple and disposable bioanalytical tools using low reagent consumption (in the order of microliters) [8, 11, 12]. Paper is mainly constituted of cellulose fibers. The cellulose is a hydrophilic polymer, which makes paper substrate

There are different types of paper that are used for fabrication of paper-based devices (**Table 1**). Filter papers have been widely used as substrate material to paper-based devices for biosensor application [16–19]. There are a vast range of commercially available high-quality filter papers, mainly constituted of alpha cellulose, a highly stable form of cellulose. The filter papers can be classified according to different properties, such as particle retention, pore size, thickness, and flow rate. The filter paper grade 1, considered as a medium retention and flow filter paper,

has been functionalized to obtain paper-based immunosensors [16, 17]. Irvine et al. adsorbed metallothioneins in grade 1 filter paper for the detection of heavy metals [18]. In the same way, other filter paper grades have been used, such as the slow filter paper grade 42 (pore size of 2.5 μm) for the incorporation of an in vitro

[5, 6]. Cadmium's maximum contaminant level corresponds to 3 and

 according to WHO and EPA, respectively. With respect to the pathogens targeted in the paper-based biosensors, the EPA [7] recommends that *Escherichia coli* cannot exceed 126 CFU per 100 mL in fresh recreational water, while *Enterococcus* should present a maximum of 35 CFU per 100 mL in marine and freshwater. In this context, paper-based biosensor devices combine the main features of paper substrates (cost-effectiveness, easy manipulation, and compatibility with proteins and biomolecules), with the high specificity and selectivity of the biorecognition systems of biosensors [1, 8, 9]. Furthermore, paper-based assays can be a solution in resource-limited contexts, as both sample and reagents can be introduced without any flow device, through imbibition and filtration via capillary action [10]. The first types of paper-based devices were related to semiquantitative analysis of glucose in urine and immunoassays on chromatographic paper test strips (or lateral flow) [11]. In the last decade, a new fabrication method based on wax patterning was introduced, allowing the design of well-defined channels on paper surface,

for mercury is 2 or 6 μg L<sup>−</sup><sup>1</sup>

is 30 μg L<sup>−</sup><sup>1</sup>

5 μg L<sup>−</sup><sup>1</sup>

**82**

*Types and features of papers used in biosensors described recently.*

Cellulose chromatography paper is also an alternative as high-quality substrate in paper-based biosensors. These papers can be differentiated by their flow rate and thickness. Vijitvarasan et al. [20] developed a paper-based device taking in advantage of the separative properties of chromatographic paper in order to enhance the concentration of gold nanoparticles (AuNPs) on the surface of the paper. Thus, a lower level of reduced silver particles was detected on the surface of AuNPs. Chromatography papers have also been applied in the development of microfluidic devices based on capillary flow measurement [21, 22]. McCraken et al. [22] tested two different chromatography papers (grade 1 Chr and grade 2 Chr) to optimize the separation of immunoagglutinated particles. The chromatography paper providing the lower flow rate (grade 2, 115 mm/30 min) was selected for the immunoagglutination assay.

Derivatives of cellulose, such as nitrocellulose membranes, have been applied as substrates of paper-based devices. These membranes are naturally hydrophobic and demonstrate to be adequate for the immobilization of enzymes and proteins by electrostatic interactions [11]. For instance, Lopez-Marzo et al. [23] developed a lateral flow immunodevice with nitrocellulose membrane for the detection of Cd2+ in water, taking advantage of the immobilization of antibodies (2A81G5 mouse antibody and antibovine serum albumin (BSA) mouse antibody), to create zones where the probe conjugate and the positive control containing BSA would interact. A similar approach was reported for immobilization of BSA conjugate and control goat antimouse immunoglobulin for the detection of U(VI) [24].

Concerning electrochemical paper-based devices, printing paper can be applied in the development of screen-printed electrodes. Hence, carbon-based conductive ink is printed onto paper surface [25, 26]. In this context, Rengaraj et al. [27] fabricated a paper-based electrode with the high-quality printing paper using only three layers of printing.

In addition, both chromatographic paper and multiuse recycled copy paper were used for printing wax wells in paper-based devices as a confinement strategy [20], or as a low-cost alternative to microplates [28].

#### **2.2 Fabrication procedures**

There are different techniques that can be applied in order to obtain paper-based biosensor devices with variable properties, such as functionalized platforms with

#### **Figure 1.**

*Examples of fabrication schemes for production of (A) wax printed paper-based well devices and (B) stencil-printed transparency film-based carbon electrodes. Adapted and reprinted with permission from [28]. Copyright 2017 American Chemical Society.*

biomolecules or cell suspensions, conductive characteristics for electrochemical analysis, and create barriers to define the reaction zones.

Wax printing is a process used to create hydrophobic barriers that define reaction microzones or fluid reservoirs [29], as exemplified in **Figure 1**. Different works [16, 17, 20, 22, 28] applied this technique using graphic design software [16, 17, 22] or stencils [28] to define the microchannel areas. The incorporation of the wax onto the microfluidic channel is performed by printing the wax onto the paper surface with subsequent heating to allow wax penetration in the paper.

Screen printing is another technique used to fabricate paper-based devices, particularly for electrochemical analysis (**Figure 1B**). For example, Rengaraj et al. [27] fabricated a paper-based electrode by printing three layers of a carbon-based conductive ink onto hydrophobic printer paper. Other fabrication techniques include a simple procedure of cutting by punching [18, 19], obtaining discs with millimetric dimensions that can be functionalized and/or introduced into devices, such as commercial screen-printed electrodes [18].

Furthermore, lateral flow immune-based devices can be fabricated by assembling different layers, which include the conjugation of pad strip (signal producer), the nitrocellulose membrane, as well as the sample and absorption pad [23, 24]. Stocker et al. [30] applied a simple technique based on premarking the spots with a pencil, with subsequent physical deposition of a cell suspension and drying of the paper strips.

### **3. Integrated biosensors methods**

#### **3.1 Transducers**

Biosensors can be defined as analytical devices, which integrate or associate a biorecognition element and a transducer. The bioelement recognizes the target analyte and the transducer converts the biochemical interaction to a measurable signal [1]. The most frequently applied transducers are based on optical, electrochemical, thermal, and piezoelectric properties. This work focused on the

**85**

every five frames.

**3.2 Biorecognition elements**

inhibiting bacterial protein synthesis [19].

*Paper-Based Biosensors for Analysis of Water DOI: http://dx.doi.org/10.5772/intechopen.84131*

(electrochemical, optical, and piezoelectric).

transducer types most used in the paper-based biosensors for analysis of water

The electrochemical paper-based biosensors are based on the modification of paperbased platforms placed in commercial screen-printed electrodes [17, 18], or rely on the fabrication of lab-made functionalized paper-based screen-printed electrodes [27]. Concerning the optical approaches used in the paper-based devices, the most applied strategies were based on colorimetric measurement [19, 20, 28] using image processing algorithms as an analytical system. In this context, a colorimetric based approach for the detection of Pb2+ and for U(VI) resorted to the acquisition of images of the paper spots with a digital camera, and images processed using the ImageJ software [20, 24]. On the other hand, Adkins et al. [28] developed a paperbased colorimetric method for the detection of *Escherichia coli*, which was based on a smartphone for image acquisition and ImageJ software for image processing.

Other example of mobile-based strategy involves the quantification of different pathogens (*E. coli* and Zika virus) as a function of capillary flow rate using a smartphone as a photometric detector [21]. This photometric approach was based on video recording of an immunoassay followed by comparison of the capillary flows between different analyte concentrations. Other colorimetric method was based on the detection of Cd2+ [23] in drinking water with a lateral flow immunosensor device. The measurement of color intensity was performed with COZART™ RapidScan color intensity portable reader. Colorimetry in paper-based biosensor devices was developed as a semiquantitative approach for the detection of arsenite [30]. This method was based on a bacterial biosensor deposited onto a paper strip. The developed color measurement was performed by comparison with spots containing known arsenite concentrations. Furthermore, a method based on fluorescence was applied as transducer for the detection of ethinylestradiol in a paper-based immunoassay [16]. For this, a LED-based system was constructed and used as excitation source. The fluorescence emission was measured with a scientific-grade spectrometer.

A piezoelectric strategy was also implemented for measurement of immunoagglutinated samples for the detection of *E. coli* and Zika virus [22]. This approach was based on particle rheology of the immunoagglutinated samples. In order to monitor the movement of the suspension of particles in a microfluidic paper-based platform, videos were taken with a smartphone and flow distance was measured

The biorecognition element has a strong and selective affinity to the target. There are several types of biorecognition elements, such as natural biomolecules (nucleic acids, antibodies, enzymes, and other proteins), synthetic bioelements (molecularly imprinted polymers, aptamers), or whole cells (**Figure 2**) [1].

Different types of biorecognition elements have been applied for the development of paper-based biosensors for the detection of target analytes in water. An in vitro transcription/translation system reconstituted from purified recombinant components necessary for *E. coli* translation of β-galactosidase enzyme was immobilized on paper as a turn on/turn off switcher for the presence of antibiotics

Concerning the application of antibodies as biorecognition elements, specific antibodies (polyclonal rabbit anti-EE2) have been applied for the detection of the estrogen ethinylestradiol in river water samples [16, 17]. In addition, suspensions of antibody-conjugated particles were used for the detection of two target pathogens (*E. coli* K12 and Zika virus) [21]. For the detection of U(VI), immobilized U(VI)- 2,9-dicarboxyl-1,10-phenanthroline-BSA conjugate worked as a competitive probe

#### *Paper-Based Biosensors for Analysis of Water DOI: http://dx.doi.org/10.5772/intechopen.84131*

*Biosensors for Environmental Monitoring*

biomolecules or cell suspensions, conductive characteristics for electrochemical

*Examples of fabrication schemes for production of (A) wax printed paper-based well devices and (B) stencil-printed transparency film-based carbon electrodes. Adapted and reprinted with permission from [28].* 

Wax printing is a process used to create hydrophobic barriers that define reaction microzones or fluid reservoirs [29], as exemplified in **Figure 1**. Different works [16, 17, 20, 22, 28] applied this technique using graphic design software [16, 17, 22] or stencils [28] to define the microchannel areas. The incorporation of the wax onto the microfluidic channel is performed by printing the wax onto the paper surface

Screen printing is another technique used to fabricate paper-based devices, particularly for electrochemical analysis (**Figure 1B**). For example, Rengaraj et al. [27] fabricated a paper-based electrode by printing three layers of a carbon-based conductive ink onto hydrophobic printer paper. Other fabrication techniques include a simple procedure of cutting by punching [18, 19], obtaining discs with millimetric dimensions that can be functionalized and/or introduced into devices,

Furthermore, lateral flow immune-based devices can be fabricated by assembling different layers, which include the conjugation of pad strip (signal producer), the nitrocellulose membrane, as well as the sample and absorption pad [23, 24]. Stocker et al. [30] applied a simple technique based on premarking the spots with a pencil, with subsequent physical deposition of a cell suspension and drying of the paper strips.

Biosensors can be defined as analytical devices, which integrate or associate a biorecognition element and a transducer. The bioelement recognizes the target analyte and the transducer converts the biochemical interaction to a measurable signal [1]. The most frequently applied transducers are based on optical, electrochemical, thermal, and piezoelectric properties. This work focused on the

analysis, and create barriers to define the reaction zones.

*Copyright 2017 American Chemical Society.*

with subsequent heating to allow wax penetration in the paper.

such as commercial screen-printed electrodes [18].

**3. Integrated biosensors methods**

**84**

**3.1 Transducers**

**Figure 1.**

transducer types most used in the paper-based biosensors for analysis of water (electrochemical, optical, and piezoelectric).

The electrochemical paper-based biosensors are based on the modification of paperbased platforms placed in commercial screen-printed electrodes [17, 18], or rely on the fabrication of lab-made functionalized paper-based screen-printed electrodes [27].

Concerning the optical approaches used in the paper-based devices, the most applied strategies were based on colorimetric measurement [19, 20, 28] using image processing algorithms as an analytical system. In this context, a colorimetric based approach for the detection of Pb2+ and for U(VI) resorted to the acquisition of images of the paper spots with a digital camera, and images processed using the ImageJ software [20, 24]. On the other hand, Adkins et al. [28] developed a paperbased colorimetric method for the detection of *Escherichia coli*, which was based on a smartphone for image acquisition and ImageJ software for image processing.

Other example of mobile-based strategy involves the quantification of different pathogens (*E. coli* and Zika virus) as a function of capillary flow rate using a smartphone as a photometric detector [21]. This photometric approach was based on video recording of an immunoassay followed by comparison of the capillary flows between different analyte concentrations. Other colorimetric method was based on the detection of Cd2+ [23] in drinking water with a lateral flow immunosensor device. The measurement of color intensity was performed with COZART™ RapidScan color intensity portable reader. Colorimetry in paper-based biosensor devices was developed as a semiquantitative approach for the detection of arsenite [30]. This method was based on a bacterial biosensor deposited onto a paper strip. The developed color measurement was performed by comparison with spots containing known arsenite concentrations. Furthermore, a method based on fluorescence was applied as transducer for the detection of ethinylestradiol in a paper-based immunoassay [16]. For this, a LED-based system was constructed and used as excitation source. The fluorescence emission was measured with a scientific-grade spectrometer.

A piezoelectric strategy was also implemented for measurement of immunoagglutinated samples for the detection of *E. coli* and Zika virus [22]. This approach was based on particle rheology of the immunoagglutinated samples. In order to monitor the movement of the suspension of particles in a microfluidic paper-based platform, videos were taken with a smartphone and flow distance was measured every five frames.

#### **3.2 Biorecognition elements**

The biorecognition element has a strong and selective affinity to the target. There are several types of biorecognition elements, such as natural biomolecules (nucleic acids, antibodies, enzymes, and other proteins), synthetic bioelements (molecularly imprinted polymers, aptamers), or whole cells (**Figure 2**) [1].

Different types of biorecognition elements have been applied for the development of paper-based biosensors for the detection of target analytes in water. An in vitro transcription/translation system reconstituted from purified recombinant components necessary for *E. coli* translation of β-galactosidase enzyme was immobilized on paper as a turn on/turn off switcher for the presence of antibiotics inhibiting bacterial protein synthesis [19].

Concerning the application of antibodies as biorecognition elements, specific antibodies (polyclonal rabbit anti-EE2) have been applied for the detection of the estrogen ethinylestradiol in river water samples [16, 17]. In addition, suspensions of antibody-conjugated particles were used for the detection of two target pathogens (*E. coli* K12 and Zika virus) [21]. For the detection of U(VI), immobilized U(VI)- 2,9-dicarboxyl-1,10-phenanthroline-BSA conjugate worked as a competitive probe

#### **Figure 2.**

*Examples of biorecognition elements found in paper-based biosensors.*

for the antibody 12F6-AuNP conjugate, as the antibody 12F6 has an increased affinity to U(VI)-2,9-dicarboxyl-1,10-phenanthroline complex [24].

Biomolecules aiming the detection of toxic metals can also be referred. A complex comprising magnetic beads, gold nanoparticles (AuNPs), and the functional nucleotide GR5-DNAzyme was applied as a biorecognition element of lead ion [20]. In another work, the recombinant human metallothionein 1a, a metal-binding protein [18], was used for the recognition of As3+ and Hg2+ in water. The tetrameric protein lectin concanavalin A (obtained from *Canavalia ensiformis*), selective to carbohydrates on bacterial cells, was selected as a biorecognition element of bacterial cultures from sewage sludge [27].

Finally, whole-cell living bacterial biosensors for arsenite detection were based on genetically engineered *E. coli*, where the *ars* operon (set of structural and regulatory genes whose expression is controlled through arsenite binding) was modified with a sequence for expression of β-galactosidase as a reporter protein in the presence of the target analyte [30]. Whole cells (biofilm formed from anaerobic sludge) were also employed in the biosensor proposed by Chouler et al. [25] for the assessment

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*Paper-Based Biosensors for Analysis of Water DOI: http://dx.doi.org/10.5772/intechopen.84131*

**4. Applications**

0.1 ng L<sup>−</sup><sup>1</sup>

nition element are summarized.

(0.5, 2.1, 0.8, and 6.1 μg mL<sup>−</sup><sup>1</sup>

screening methodology.

of toxic compounds in water in a microbial fuel cell device. The detection principle was based on the conversion of the chemical energy contained in organic matter into electricity via the metabolic processes of microorganisms. Hence, a microbial biofilm is placed on the anode surface, where the electroactive bacteria mediate the transference of electrons to the electrode upon their metabolic activity. Any factor disrupting this (water pollution for instance) will disrupt this signal. A similar approach was

In this section, the application of paper-based biosensors for the detection of different types of target analytes in water samples is discussed. In **Table 2**, the main features of the target analyte, the sample type, the paper substrate, and the fabrication method of the paper-based device, the method of detection, and the biorecog-

Pharmaceuticals are among the targets. Indeed, they are considered emerging environmental contaminants, as they can be harmful to human health and to aquatic life. In this context, the synthetic hormone—ethinylestradiol, one of the main compounds of oral contraceptives, is considered an emerging pollutant due to its potential high estrogenic effect on the biota. Scala-Benuzzi et al. developed two different methods for the detection of ethinylestradiol in river water samples using an antiethinylestradiol specific antibody [16, 17]. In both approaches, the water samples were filtered, and pH was adjusted to 7.0 with phosphate buffer before the analysis. In one work, a fluorescent paper-based biosensor was implemented [16].

, which is

This methodology presented a limit of detection (LOD) of 0.05 ng L<sup>−</sup><sup>1</sup>

, suitable for environmental analysis.

mainly related to the high sensitivity of fluorescence methods. In another work, ethinylestradiol was detected in river water with a paper-based immunosensor based on electrochemical analysis [17], which also reached a low LOD value of

Antibiotics are another group of pollutants of great concern due to the global threat of antimicrobial resistance and the excessive, and sometimes abusive, use of these compounds. A colorimetric biosensor for screening of several antibiotics (paromomycin, tetracycline, chloramphenicol, and erythromycin) inhibiting bacterial protein synthesis was applied for the detection of antibiotics in surface water [19]. The method was based on the ability of these antimicrobials to inhibit β-galactosidase synthesis. When a water sample without the target antibiotics was placed in the paper-based device, the enzyme β-galactosidase was synthetized and its activity induced a color change on the paper disc surface. However, when antibiotics were present, the inhibition of β-galactosidase synthesis prevented the change of color. Despite the limit of detection was on the microgram per milliliter level

erythromycin, respectively), this biosensor can be applied as a simple and portable

Heavy metals are naturally present in the environment. However, these elements can be toxic to human and aquatic organisms even at low concentrations. Moreover, their presence can be increased by industrial and agriculture activities. Vijitvarasan et al. implemented a paper-based scanometric biosensor for the detection of lead in water [20]. The biosensor was applied to river water samples. These samples were filtered, diluted 10 times with 10 mM tris-acetate buffer and spiked with different Pb2+ concentrations before analysis. An LOD value of 0.9 nM was determined. Furthermore, a method using a sensitive gold nanoparticle-based lateral flow immunodevice [23] was applied for the quantification of cadmium. Drinking water

for paromomycin, tetracycline, chloramphenicol, and

proposed by Xu et al. [26] using a wastewater bacteria consortium.

*Paper-Based Biosensors for Analysis of Water DOI: http://dx.doi.org/10.5772/intechopen.84131*

of toxic compounds in water in a microbial fuel cell device. The detection principle was based on the conversion of the chemical energy contained in organic matter into electricity via the metabolic processes of microorganisms. Hence, a microbial biofilm is placed on the anode surface, where the electroactive bacteria mediate the transference of electrons to the electrode upon their metabolic activity. Any factor disrupting this (water pollution for instance) will disrupt this signal. A similar approach was proposed by Xu et al. [26] using a wastewater bacteria consortium.
