**5. Conclusions**

Intracellular ROS are increasingly recognized as critical determinants of cellular signaling and can be used as markers of 5-FU-mediated anti-tumor e ffects. Biosensors are widely used for the assessment of physiological and pathological applications since they are non-invasive methods for the detection of crucial biomarkers of cancer cell cytotoxicity without the need for extensive sample pretreatment. In this study, we present a cell-based biosensor tool suitable for cell culture superoxide monitoring without the need for cell transfer and staining, which could a ffect cellular responses due to extrinsic chemical and mechanical stress. This biosensor is applied for the estimation of total superoxide levels released from the cancer cells into the culture medium after treatment with the chemotherapeutic agent. Our results are in agreemen<sup>t</sup> with the findings of the biochemical assays for the assessment of mitochondrial superoxide and caspase-3 activities. Further validation is required of the feasibility of our biosensor-based approach for monitoring alterations in the in vitro redox status of cancer cells treated with anticancer agents, e.g., by testing more and di fferent cancer cell lines crossed with chemotherapeutical drug combinations. In a longer perspective, the availability of a reliable, high throughput and rapid system for superoxide determination in cancer cells will allow for the accurate assessment of chemoresistance in cervical and other cancer cells, at least as far as its association with redox balance is concerned [38,64–66].

**Author Contributions:** Methodology, S.M., V.T., M.-A.K., G.M.; investigation, S.M., V.T., M.-A.K., G.M.; data curation, S.M., V.T.; writing—original draft preparation, S.M., V.T., S.K., G.M.; writing—review and editing, S.K.; supervision, S.K.; project administration, G.M.

**Funding:** This work is supported by the Operational Program "Human Resources Development, Education and Lifelong Learning", Priority Axes 6, 8, 9 co-financed by European Social Fund entitled "Support of Researchers with Emphasis on Senior Researchers" (NSRF 2014-2020).

**Conflicts of Interest:** The authors declare no conflict of interest.
