In this project, UPV/EHU will use the stress-associated sRNAs found and characterized in the other two projects (CSIC and UPV) as specific biomarkers for the development of point-of-need sensors based on microfluidic paper-based analytical device (microPAD) technology. The final devices will be used for the early, easy, and low-cost detection of the damage induced by stress conditions in crops, in particular, tomato plants. In order to do that, we will first investigate the design and fabrication of the microPADs with the specific characteristics that fulfill the ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid, Equipment free and Delivered (small and portable)) and that will permit the generation of point-of-need sensor devices for agriculture. The microPADs will be fabricated by wax printing methods using different substrates. Then, a cartridge will be generated to hold the device. Finally, the manipulation of the sample in the device will be investigated through the integration of self-powered microfluidic pumps to overtake the fluidic limitations offered by conventional paper devices.

Then, we will investigated the integration of the assay to detect syn-sRNAs, as a proof of concept. A holistic approach for integration will be followed, considering first the abundancy of the sRNAs proposed by the other members in the real processed samples and second, the most adequate optical detection method, e.g. colorimetric. The assay will be encapsulated in a

polymeric matrix (alginate, ionogels) and latter integrated in the microPADs. An RNAzyme based colorimetric assay will be developed for the interrogation of the samples. In addition, different approaches will be followed for the transducer such as: (1) naked eye interrogation to obtain a qualitative response; (2) color analysis of images took by a mobile phone in a controlled environment and (3) Integrated optical device based on a LED/photodiode system, both providing with quantitative values.

Subsequently, we will investigate the performance of the microfluidic devices. In order to do that, a systematic evaluation of their accuracy, precision and trueness, when possible, will be done assuring the success of the device and thus its further development towards a

marketable product. Moreover, their limits of detection and quantification, and their reproducibility will be evaluated. It needs to be considered that this project is not a monolithic block of work thus an iterative protocol will be followed by testing the devices, all over the

first and a half-year of the project, to reach the desired final prototype. Finally, the device will be validated in field using real samples and in the lab by the CSIC group using conventional, well stablished techniques, for comparison.