**5.2. Applications**

**5. Lab-on-paper devices**

52 Nanostructured Solar Cells

of urine and blood composition [109].

**5.1. Wax printing technology**

hydrocarbons (e.g., paraffin), and dyes.

Lab-on-paper is a novel technology for fabricating simple, low-cost, portable, and disposable analytical devices holding great promise to aid global health, food quality control, and environmental monitoring [81, 100–105]. Lab-on-paper technology requires minimum fluid samples, compared to common devices, and the porous structure of the paper is solely responsible for fluid transportation. This is a crucial aspect of the diagnostic apparatus, since the substrate must facilitate the diffusion and flow of the solutions within the hydrophilic fiber matrix. The movement of fluids is governed by capillary forces, thus without the need for pumps or power [102, 103]. This technology was introduced in 2007 by Martinez et al. [106] as a method for patterning paper to create well-defined, millimeter-sized channels comprising hydrophilic paper bounded by hydrophobic polymer; but the first evidence of the lab-on-paper technology dates back to 1902 with a patent for paper strips impregnated with hydrophobic materials [107]. Earlier diagnostic applications of lab-on-paper comprise the detection of nickel and copper salt concentrations [108], determination of pH for water testing and biological analysis

To date, researchers have been focused on adapting new developments in nanotechnology, biotechnology, and materials science to paper-based sensors. The production of practical analytical devices, based on those developments and by simple fabrication techniques, can have a positive impact for creating worldwide applications where they are most needed [100, 104, 106, 110, 111]. The fabrication of lab-on-paper devices starts by defining channels and reaction zones onto paper, in order to control the movement of liquids and to assure the confinement of different reagents. A wide range of diverse patterning techniques was already reported and can be divided in three major patterning principles: (i) physical blocking of pores in paper (e.g., photolithography [106], plotting [112], and laser treatment [113]); (ii) physical deposition of reagent on fiber surface (e.g., inkjet etching [114] and wax printing [104, 115]); and (iii) chemical modification of fiber surface (e.g., plasma treatment [116], inkjet printing [117], and silanization [118]). Each technique has its advantages and limitations which have to be taken under consideration according to the type of device, equipment available, material costs, and the intended application, among other factors.

Among the different patterning techniques, wax printing represents one of the most promising technology for paper patterning, given its simple and fast (5–10 min) fabrication process. The printing process uses a solid wax printer, in which the ink is supplied as solid wax and then melted, before being ejected from the print head, and once it rests on the paper surface it immediately solidifies. Since the wax remains only on the paper surface, the printed paper is then processed on a hot plate (120–140°C, 1–5 min; depending on the type of wax and paper) to allow the wax to diffuse vertically through the entire paper thickness, hence creating the hydrophobic barriers to confine the fluids (**Figure 12**). The wax is formulated from a nontoxic resin-based polymer, composed of a mixture of hydrophobic carbamates, The wax printing technology has already been successfully applied in the fabrication of colorimetric paper-based sensors for diagnostic and environmental applications, such as for glucose monitoring, based on enzymatic reactions [104], canine *leishmaniosis* detection through an immunoassay [104], tuberculosis detection by nucleic acid identification [100], and electrochemically active bacteria detection, based on electrochromic detection [81].

#### *5.2.1. Glucose monitoring*

*Diabetes mellitus* is a leading cause of illness and mortality worldwide and a major health problem for most developed societies. This metabolic disorder from insulin deficiency and hyperglycemia results in blood glucose concentrations higher or lower than the normal range (4.4–6.6 mM) [119, 120]. In developed countries where healthcare infrastructure is well established, self-monitoring of blood glucose levels is effective and economically accessible to the public. However, in many resource-limited countries the first priority is generally not given to diabetes care. Even widely used glucose meters and test strips are extremely expensive in those low-income regions; hence, there is an urgent need to develop a simple, low cost, disposable sensor that allows individuals in those countries to self-monitor their blood glucose level [121].

The work of Costa et al. [104] demonstrates how such blood glucose monitor can be fabricated on paper, as a 2D lateral flow device or as a 3D device, using the wax printing technology, for colorimetric analysis. The paper substrate used was *Whatman* filter paper n° 1 (WFP1), which is the most common paper for microfluidic devices. Briefly, the enzyme glucose oxidase (*GOx*) oxidizes glucose to D-glucono-δ-lactone and hydrogen peroxide. The enzyme peroxidase then reacts with the hydrogen peroxide and oxidizes the colorimetric indicators generating a visible color change, which is proportional to the initial amount of glucose in the sample.

**Figure 13** illustrates the layers that comprise the paper-based biosensors and compares their sensibility for two different colorimetric indicators: AB (4-aminoantipyrine + 3,5-dichloro-2-hydroxy-benzenesulfonic acid) and KI (potassium iodide).

The 3D device shows important advantages compared to the 2D lateral flow sensor, given its higher coloration homogeneity. The 2D lateral flow sensor, on the other hand, is more subject to some carryover of the colored products that hinder the coloration intensity. This test runs in less than 5 min and a calibration color gradient scale can be printed directly on the paper device in order to compare with test results, allowing the patient to infer an approximate concentration of glucose in the sample. Eventually, a more complex device able to perform a wide range of analysis could take advantage of a solar cell to power sensors, a CMOS to perform logic calculations and a display to show information.

**Figure 13.** (a) Schematic representation of the 2D lateral flow device—enzyme and colorimetric indicators are deposited together. (b) Schematic representation of the 3D device. (c) Comparison between the 2D and 3D glucose sensors, when tested with increasing glucose concentrations (0.01–100 mM), with the graphical representations of the color intensity of detection zones containing the AB indicator (top) and the KI indicator (bottom). Adapted from Costa et al. [104], with permission from IOP Publishing.

#### *5.2.2. Electrochemically active bacteria detection*

Electrochemically active bacteria (EAB) have the ability to extracellularly transfer their electrons produced during microbial respiration [81, 122, 123]. This ability sparked interest given the multiplicity of applications than can range from bioremediation to electricity production in microbial fuel cells. Although an increasing number of EAB have already been identified, isolated, and characterized, this number is still quite small regarding their ubiquity in the environment. This fact significantly constrains the fundamental knowledge about these bacteria and their role in the environment [81, 124]. To visually detect these bacteria, Marques et al. [81] developed a paper-based sensor, using common office paper (COP) as substrate and tungsten trioxide nanoparticles (WO<sup>3</sup> NPs) as the sensor layer (**Figure 14**), to successfully test the presence of electrochemically active *Geobacter sulfurreducens* DL-1 [125] cells (**Figure 14b**). The change in the WO<sup>3</sup> optical properties (between white and blue) occurs in the presence of EAB which triggers the electron-transfer (redox) process (bioelectrochromic response).

The hexagonal WO<sup>3</sup> NPs, hydrothermally synthesized by microwave, were integrated in a wax-printed office paper platform as an active layer for EAB detection. Common office paper allows a superficial adhesion of the WO<sup>3</sup> NPs, which facilitates the interaction of EAB with the electrochromic nanoparticles, promoting an intense and uniform coloration on the reaction zone, contrarily to chromatography paper (e.g., WFP).

**Figure 14.** (a) Schematic representation of the common office paper device. (b) Digital photograph of a positive result showing a deep blue color, from the tungsten bronze, on the sample well, and nonresponse on the control well. The amplified SEM image shows the interaction between the *Geobacter sulfurreducens* bacteria (in yellow) and the hexagonal WO<sup>3</sup> nanoparticles (in blue). The SEM image is false-colored for better understanding of the different components. Adapted from Marques et al. [81].
