**4. Conclusions**

In conclusion, a WLRS-based biosensing platform for the real-time immunochemical determination of carbendazim in fruit juices was successfully developed. The proposed sensor allowed for the sensitive and fast quantification of carbendazim levels down to 20 ng/mL within less than 30 min. Moreover, the fact that a single chip functionalized with the benzimidazole conjugate could be regenerated and reused for at least 12 times without any effect onto the signal received provides a further advantage. In summary, excellent analytical characteristics and short analysis time combined with the small size of the analytical device render the proposed WLRS biosensor ideal for future on-the-spot determination of carbendazim in food and environmental samples.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/bios11050153/s1, Scheme S1. TMB peroxidase substrate reaction. Figure S1. (a) Absorbance values at 450 nm received for zero carbendazim standard from wells coated with benzimidazoleconjugate concentration 0.5 (black squares), 1 (red circles), 2.5 (blue up triangles), or 5 μg/mL (green down triangles) when assayed with anti-carbendazim antibody concentrations ranging from 0.5 to 8 μg/mL. Each point is the mean value of four wells ± SD. (b) Percent absorbance values obtained for the zero carbendazim standard (orange bars) and a standard containing 200 ng/mL carbendazim (green bars) using different combinations of benzimidazole conjugate for well coating and anticarbendazim antibody. Each point is the mean value of four wells ± SD. Figure S2. Absorbance values at 450 nm received for zero carbendazim standard (orange bars) and a standard containing 200 ng/mL carbendazim (green bars) using the following assay buffers: 10 mM PBS buffer, pH 6.5; 10 mM PBS, pH 7.4; 50 mM Tris-HCl buffer, pH 7.8; 50 mM Tris-HCl buffer, pH 8.25. All buffers contained 0.4% BSA. Each point is the mean value of four wells ± SD. Figure S3. Carbendazim calibration plots obtained without preincubation of carbendazim standards with the anti-carbendazim antibody solution (green down triangles) or with preincubation for 30 min (black squares), 60 min (red circles), and 120 min (blue up triangles). Each point is the mean value of four wells ± SD. Figure S4. Signals received for zero carbendazim standard from WLRS chips coated with benzimidazole conjugate concentrations ranging from 100 to 1000 μg/mL when assayed with anti-carbendazim antibody solutions of 0.5 (black squares), 1 (red circles), 2 (blue up triangles), or 4 μg/mL (green down triangles). Each point is the mean value of measurements obtained by three chips ± SD. Figure S5. Calibration plots obtained from chips coated with 500 μg/mL of benzimidazole conjugate and assayed with anti-carbendazim antibody solutions with concentration 2 (black squares) or 4 μg/mL (red circles). Each point is the mean value of three measurements ± SD.

**Author Contributions:** Conceptualization, G.K., C.-E.K., E.L. and S.K.; methodology, G.K. and C.-E.K.; investigation, G.K. and C.-E.K.; data curation, G.K. and C.-E.K.; resources, I.R., P.P.; funding acquisition, E.L. and S.K.; supervision, E.L. and S.K.; writing—original draft preparation, C.-E.K.; writing—review and editing, G.K., C.-E.K., P.P., E.L., and S.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research is cofinanced by Greece and the European Union (European Social Fund— ESF) through the Operational Program "Human Resources Development, Education and Lifelong Learning 2014–2020" in the context of the project "Development of immunochemical analytical methodology, especially an ELISA and a biosensor assay, for determining benzimidazole-type pesticides" (MIS 5047811).

**Institutional Review Board Statement:** Animal immunization was performed in accordance with the Presidential Decree 56/2013 for the Protection of Animals used for Scientific Purposes (Directive 2010/63/EU) and approved by the local committee of the Animal House (Institute of Biosciences & Applications, NCSR "Demokritos") which is a certified installation (EL 25 BIOexp 039, Prefecture of Attica) and the Prefecture of Attica, Division of Agriculture and Veterinary Medicine (license No 247871/08-04-2020).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy issues.

**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.
