**4. Conclusions**

O-PADs provide a more convenient, reliable, and easy-to-use approach for target sample detection. In this study, we successfully used an O-PAD to detect rabbit IgG level with a low LOD. Due to the limited information regarding the IgG LOD for ophthalmic viral infection or inflammation, we could not compare our results to the references. Overall, this study demonstrated two potential areas for clinical application of OPADs that could provide significant scientific and health benefits. Our device provides two advantages: (1) to reduce test complexity and the number of required reagents through pre-loaded reagents; and, (2) to provide a wet environment for moisture-sensitive analysis, e.g., paraquat detection. However, there are still several limitations associated with our device. First, while providing advantages over paper-based ELISA, our device still could not reach the sensitivity of conventional ELISA; this limitation may be improved by optimizing the assay to suppress the background signal. Second, the long-term storage stability of preloaded reagents such as ascorbic acid, which oxidizes if exposed to oxygen, may limit usage. We believe that additional developments, especially those aimed at improving long-term dry-state storage, would improve our device and broaden its applicability.

**Author Contributions:** Conceptualization, Z.-K.K. and C.-M.C.; methodology, C.-M.C.; software, T.-H.C.; validation, Y.-S.C.; formal analysis, T.-H.C.; investigation, Z.-K.K.; resources, C.-M.C.; data curation, Y.-S.C.; writing—original draft preparation, Z.-K.K., C.-Y.T.; writing—review and editing, C.-Y.T., C.-M.C.; visualization, Z.-K.K.; supervision, C.-M.C.; project administration, C.-M.C.; funding acquisition, C.-M.C., C.-Y.T.

**Funding:** This project is funded by the Ministry and Science Technology, Taiwan, no.: MOST 108-2622-E-007-020 -CC3 and the Fu Jen Catholic University, no: PL201908002B.

**Acknowledgments:** This study was supported by grants (107-2628-E-007-001-MY3 and 108-2622-E-007-020-CC3) from the Ministry of Science and Technology, Taiwan, and a gran<sup>t</sup> (PL-201809004-V) from the Fu Jen Catholic University Hospital, Taiwan.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
