## 3 Printing Technologies to Deliver Disposable and Miniaturised Devices
### Paper-Based Printed Devices  
Paper-based electrochemical (bio)sensors have recently acquired huge interest by the scientific community due to their sustainable features in terms of plastic-free measure and low cost. In addition, the measurements using paper-based biosensors can be carried out by unskilled personnel and these devices overcome the major drawbacks of microfluidic devices, including the requirement for micropumps, the bubble issues, and high costs for microfluidic pattern fabrication. A further added value in the defence contest relies on the capability of being incinerated after the measure, reducing the management of the waste contaminated with chemical warfare agents. As is well known, when a drop of water wets the paper, the water flows in the cellulosic network, and it also wets the electrical contacts and hinders the correct electrochemical measure. To overcome this drawback, we exploited the wax printing technique to easily create the hydrophilic region surrounded by the hydrophobic one to contain the working solution within the electrochemical cell. As an example, I report a paper-based bioassay for the detection of mustard agents using a portable instrument (Figure 3C(a)) [33]. For the manufacturing of paper-based screen-printed electrodes, the hydrophobic zone was printed onto office paper sheets, using a ColorQube 8580 Xerox printer, and treated at 100 \({}^{\circ}\)C for 2 min to let the wax homogeneously permeate the paper, producing the hydrophobic pattern designed. Then, the three electrodes were printed by using the screen-printing technique (Figure 3C(b)). For mustard agent detection, choline oxidase enzyme was used as a biocomponent, measuring the degree of inhibition with an office paper-based electrochemical sensor bulk modified with Prussian Blue nanoparticles and carbon black with the overriding goal of measuring the enzymatic by-product H\({}_{2}\)O\({}_{2}\) at a low applied potential by the chronoamperometricmode. This bioassay was conceived to work in a drop in the presence of choline oxidase and chemical warfare agent. It was successfully applied for the measurement of sulphur mustard Yprite with a detection limit of 0.9 mM. Figure 3: (**A**) Amperograms using the screen-printed electrodes modified with Prussian blue and butyrylcholinesterase before and after the exposure to Sarin gas [27]. (**B**) Screen-printed based butyrylcholinesteraseerase biosensor integrated into a lab on a chip [28]. (**C**) Paper-based screen-printed electrodes as a platform to detect mustard agents using choline oxidase as a biocomponent. (**a**–**e**) procedural steps for the realisation of wax-printed office paper sergiraphic printing of conductive inks [33]. (**D**) Paper-based screen-printed electrode loaded with butyrylcholinesteraseerase to detect paraoxon as a nerve agent simulant using the following steps (A–F) [34]. The first paper-based biosensor for reagent-free analysis of nerve agents was reported by our group, thanks to the combination of a strip of nitrocellulose membrane loaded with the substrate and a paper-based test screen-printed electrode functionalized with butyrylcholinesteraseterase [34]. As the paper-based device loaded with the reagents was able to carry out a single measure, the measurement of nerve agent simulant was carried out by using two platforms that compared the non-inhibited chronoamperometric signal to the inhibited one. In detail, the blank signal was obtained by adding 10 \(\upmu\)L of distilled water on the edge of the Predator(r) membrane where butyrylthiocholine was adsorbed (Figure 3D(B)). Then, the water flow pushed butyrylthiocholine toward the test area (Figure 3D(C)), wetting the electrochemical cell where the enzyme with the phosphate buffer salts were loaded (Figure 3D(D)). When the water reached the testing area (ca. 30 s), we waited for 2 min for the enzymatic reaction, producing the enzymatic by-product thiocholine (Figure 3D(E)). To measure paraoxon concentration, the testing area was firstly loaded with 5 \(\upmu\)L of the nerve agent simulant.