D. Gosselin, M. Gougis, M. Baque, V. Maffini-Alvaro, P. Mailley, A.G. Bourdat, F. Revol-Cavalier, S. Vignoud, F. Navarro, M.N. Belgacem, D. Chaussy, J. Berthier
Keywords: LAMP, screen printing, polyaniline, potentiometry, embossing
Summary:Introduction: Diagnostic is probably the most important step in the fight against infectious diseases and epidemics. While it is necessary to give a treatment to diseased people, it is just as important to give the appropriate one to avoid any overtreatment which may result in an increased antimicrobial resistance [1,2]. Thus reliable diagnostic tests are needed. Because laboratories are not always available in developing countries, development of Point-of-Care (PoC) devices is of utmost importance. The World Health Organization (WHO) recommends that these diagnostic tests fulfill the following criteria: Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free and Delivered . In order to comply with these criteria, a low-cost screen printed and embossed LAMP (Loop-mediated isothermal amplification) reactor has been developed. Materials and Methods: To provide highly portability, the developed LAMP reactor integrates a micro-heater and an electrochemical sensor, both screen printed. The micro-heaters are made of resistive carbon ink and are able to provide a stable temperature of 65°C over one hour  with low energy consumption. The electrochemical detection of the LAMP reaction is made by potentiometric measurement of pH. In fact, during the LAMP, when the DNA polymerase adds a dNTP (desoxyribonucleotides tri-phosphate) while synthetizing a DNA strand, a hydronium ion is released, decreasing the pH of the solution. In this work, a silver/silver chloride reference electrode is used with a PolyAniline (PAni) working electrode. While the electrical potential of the reference electrode does not depend on pH, the potential of the PAni electrode does . The degree of oxidation of the PAni depends on its protonation and therefore on the pH of the solution in contact with it. Thus the measurement of the voltage variation between these electrodes reflects the pH variation of the solution. Results: These two functional elements have been successfully screen printed on PolyCarbonate (PC) sheets (Figure 1a) which can be hot embossed afterwards to produce a microfluidic chip (Figure 1b). The electrochemical sensor shows a linear response to the pH level (Figure 2) with a slope of -80mV/pH unit. Such a sensor has been used to monitor in real-time a LAMP reaction (Figure 3). At 6 minutes a change in voltage is detected. This corresponds to the starting of the heating. After 10 to 15 minutes, an increase of voltage is observed. This increase corresonds to the acidification of the solution due to the DNA amplification. In a near future, we plan to adapt the system to waterproof, hydrophilic embossed paper substrates  (Figure 4). It has been recently shown that such devices were compatible with spontaneous capillary flows and could be interesting alternate solutions to plastic. Conclusion: It is demonstrated that the combination of screen printing and embossing technologies is a promising way to design low-cost Point-of-Care devices. By stacking a screen printed microheater and a screen printed potentiometric sensor, it is shown that a LAMP reaction can be performed and monitored. Besides this stacking can be embossed to design directly a microfluidic chip.