Tungsten trioxide synthesis

The crystallographic structure of the synthesized WO 3 nanoparticles was determined by X-ray diffraction (XRD) ( Figure 1 ) and corroborated by Fourier transform infrared spectroscopy (FT-IR) ( Fig. S1 ). Tungsten oxides follow a well-known ReO 3 -type structure built up of layers containing distorted corner-shared WO 6 octahedra. The growing process of WO 3 nanostructures can be described in three major steps: (i) formation of the tungstic acid (H 2 WO 4 ), (ii) formation of WO 3 clusters by decomposition of H 2 WO 4 and (iii) growth of WO 3 crystal nucleus29. In the synthesis with sodium tungstate dihydrate (Na 2 WO 4 ·2H 2 O) as precursor and NaCl as structure-directing agent (SDA) (Figure 1A), WO 3 nanoparticles grow in a monoclinic (m-WO 3 ) crystallographic structure (ICDD #00-043-1035) at pH 0.0 and orthorhombic (o-WO 3 ·0.33H 2 O) (ICDD #01-072-0199) at pH 1.8. At pH 0.4 the WO 3 nanoparticles are a mixture of the two phases, monoclinic and orthorhombic, together with the precursor (marked with *) and tungstic acid (marked with ♦). Using Na 2 SO 4 as SDA (Figure 1B), orthorhombic and hexagonal (h-WO 3 ) (ICDD #01-075-2187) phases were obtained at pH 0.4 and pH 1.8, respectively. At pH 0.4 the sample also shows a peak assigned to the acid tungstic (♦) and two unidentified peaks (Δ) that are due to lattice distortions of the crystallographic structure, as previously reported for WO 3 nanoparticles prepared by hydrothermal synthesis30. Finally, using peroxopolytungstic acid (PTA) as precursor (Figure 1C), the crystallographic structure of the synthesized nanopowder is monoclinic for the lowest and higher pH values, which is in agreement with previous reports, although with different crystallographic plane intensities29. For the intermediate pH value, the WO 3 nanoparticles present an orthorhombic phase. The FT-IR analysis is in accordance with the crystallographic structures attributed by XRD. However, the samples prepared with PTA precursor also revealed the presence of a W = O vibration bond that are assigned to some impurities. In general, the formation of nanoparticles is favourable for pH values lower that 2.0, however at pH 0.4 tend to form bundle structures and a mixture of phases and/or impurities31.

Figure 1 XRD diffractograms of the WO 3 nanoparticles. (A) WO 3 nanoparticles synthesized from Na 2 WO 4 ·2H 2 O, NaCl solutions; (B) WO 3 nanoparticles synthesized from Na 2 WO 4 ·2H 2 O, Na 2 SO 4 solutions; (C) WO 3 nanoparticles synthesized from PTA solutions. The peaks marked as * and ♦ are characteristic of Na 2 WO 4 ·2H 2 O and H 2 WO 4 structures. The peaks marked as Δ are non-identified. The crystalline structures were produced with the CrystalMaker software (Centre for Innovation & Enterprise, Oxford). Full size image

Figure 2 shows the morphological analysis performed by scanning electron microscopy (SEM). The WO 3 nanoparticles synthesized with Na 2 WO 4 ·2H 2 O show different morphologies: (i) nanocubes assigned to m-WO 3 , (ii) nanosheets assigned to o-WO 3 ·0.33H 2 O, (iii) nanowires assigned to h-WO 3 and (iv) bundle structures. Regarding the WO 3 nanoparticles synthesized with PTA, the obtained structures present well-defined edges with nanosheet-like morphology at pH 0.0, a mixture of nanowires and nanocubes at pH 0.4 and single nanocubes at pH 1.8.

Figure 2 SEM images of the synthesized WO 3 nanoparticles. The images are false colored (GIMP software) for better understanding. The different colors are related with the XRD diffractograms for each crystalline structure. Full size image

The sulphate ions added to the synthesis process, act as capping agents covering some facets of WO 3 crystal nuclei. At pH 1.8 a faster growth rate along c-axis is observed, yielding to one-dimensional wire/rod-like structures. In the meantime, a certain amount of sodium cations is required as stabilization ions for the hexagonal and triangular tunnels in the formation of h-WO 3 15,31,32,33. When chloride ions are added it is believed that a similar process occurs prompting the growth of the nanoparticles in a specific direction34.

Additionally, electrochemical impedance spectroscopy (EIS) was performed in all the samples, in order to compare the conductivity of the nanostructures, since this affects its electron transfer ability during the electrochromic process. The Bode plots represented in Fig. S2 display, in general, lower impedances values for the orthorhombic and hexagonal crystallographic structures, which is in accordance with the literature35,36,37. The results obtained for hexagonal crystallographic structure ( Table S1 ) are explained by its high surface area and tunnel structure.

Office paper as a platform for EAB identification

The above described WO 3 nanoparticles were used as an active layer in a regular office paper substrate in order to develop a colorimetric electrochemical device for EAB detection. This paper is optimized for printing and therefore has a more uniform surface, lower porosity and higher hydrophobicity (water-contact angle of 106°) when compared to chromatography paper (water-contact angle of 12°)22, the most common type employed in paper-based devices. Office paper allows a superficial adhesion of the WO 3 , which facilitates the interaction of EAB with the electrochromic nanoparticles, promoting an intense and uniform coloration of the test well. SEM-EDS and XRD analysis ( Figure 3A–C ) revealed a high-density structure of intertwined cellulose fibers with a cylindrical and flat shape and the presence of agglomerates, especially calcium carbonate, as confirmed by a FT-IR analysis ( Fig. S3 ).

Figure 3 Office paper characterization. (A) SEM image and EDS map; (B) EDS spectrum; (C) XRD diffractogram; (D) Hydrophobic barriers formation; (E) Photograph of a positive result in the developed paper-based sensor with WO 3 nanoprobes for the colorimetric detection of EAB (Geobacter sulfurreducens cells in yellow and hexagonal WO 3 nanoparticles in blue). The images are false-colored (GIMP software) for better understanding of the different materials. Full size image

The sensor layout was designed to resemble a conventional microplate ( Figure 4A ) for parallel assays and prototyped into single-use sensors containing only a test and a control wells ( Figure 3D–E ). The layout was then patterned by wax printing, a method previously optimized by M. N. Costa et al.22, that increases the surface hydrophobicity (water-contact angle of 119°) ensuring no cross contamination between adjacent samples as well as the confinement of the WO 3 nanoparticles dispersion to one particular area. A thermal analysis of the office paper ( Fig. S4 ) was also performed in order to guarantee that the material could withstand the heating process. The patterned office paper was then impregnated with the synthetized WO 3 nanoparticles by a drop casting process. A drop was spotted on each well delimited by the hydrophobic pattern, in order to create the electrochromic layer.

Figure 4 EAB detection. (A) Paper-based sensor photograph of the Colorimetric assays of all synthesized WO 3 nanoparticles at 5 g/L; (B) RGB analyses for all the samples in contact with Geobacter sulfurreducens (Gs) cells. (Results recorded after 4 hours) The horizontal line represents the threshold of 1 considered for discrimination between positive and negative and the results represent the average of three independent measurements with the respective error bars indicative of the standard deviation. Full size image

Colorimetric assays

A screening colorimetric assay of all the synthesized WO 3 nanoparticles was performed in the developed paper-based sensor ( Figure 4A ) and in the conventional 96-well plate ( Fig. S5 ). For the paper platform, an RGB analysis of the results was carried out using ImageJ software and the ratio of the average intensities in blue and red channels was recorded ( Figure 4B ). An electrochromic response translated by the deep blue color of the tungsten bronze (Equation 1) was achieved with sample 6, which corresponds to the synthesized hexagonal WO 3 nanowires. The h-WO 3 nanoparticles has attracted much attention due to its well-known tunnel structure where openness degree is higher when compared to the layered structure of orthorhombic or monoclinic geometries. This feature results in an easier intercalation of cations to form tungsten bronzes and concomitant enhancement of the electrochromic properties14,38. Moreover, the one-dimensional nanowire shape originates a structure with a high surface area and increased surface atom density that can easily interact with the EAB. Additionally, the electrochromic response was also observed in the conventional assay with other crystallographic WO 3 structures, due to the higher concentration of the nanoparticles and enhanced interaction with EAB (Fig. S5).

For the fabricated paper-based sensor, the bioelectrochromic response is conditioned by the time that the cell suspension drops (V = 50 µL) take to dry (approximately 4 hours). Therefore, an equal scale down on the well's diameter (d = 3.38 mm) and in the sample volume (V = 20 µL) was carried out, with a reduction (2.5 times smaller) from the first paper-based sensor (Figure 4A). Additionally, an optimization assay was carried out ( Figure 5A ) to evaluate the influence of the h-WO 3 nanoparticles concentration in the detection of EAB. Figure 5B represents the RGB analysis of the resulting color of the Geobacter sulfurreducens cells in contact with h-WO 3 nanoparticles. From this analysis it is possible to conclude that 15 g/L and 20 g/L h-WO 3 nanoparticles dispersion renders higher RGB ratios when compared to the other studied concentrations. Moreover, 15 g/L nanoparticles dispersion presents a linear response to the increasing G. sulfurreducens cells concentration. Therefore, henceforward the sensors were produced with h-WO 3 nanoparticles dispersion at 15 g/L, with the same ratio nanoparticles per area than the first sensor (0.014 g/mm2). With the described scale-down the response time decreased to half the time (approximately 2 hours).

Figure 5 EAB detection. (A) Paper-based sensor photograph of the Colorimetric assays of h-WO 3 nanoparticles at different concentrations; (B) RGB analyses of h-WO 3 nanoparticles at 15 g/L in contact with G. sulfurreducens cells, with the negative control E. coli and a blank test. (Results recorded after 2 hours) The horizontal line represents the threshold of 1 considered for discrimination between positive and negative and the results represent the average of three independent measurements with the respective error bars indicative of the standard deviation. Full size image