## 3 Results and Discussion
### Analytical Method Optimization: Calibration and Evaluation of the Assay  
A sandwich ELISA was carried out for a wide range of DENV concentration (3.5 \(\times\) 10\({}^{-5}\) p.f.u/mL to 3.5 \(\times\) 10\({}^{5}\) p.f.u/mL) in order to assess the performance of the developed platforms as a function of DENV concentration. Figure 5a, shows an initial increase of OD (optical density) values as a function of increasing concentrations of DENV. This increase in detection signal was limited to the certain threshold (3.5 \(\times\) 10\({}^{-2}\) p.f.u/mL) from which the signal intensity decreased in the higher range of DENV concentrations. This phenomenon is most likely the result of the large size of biomolecules used in the assay as such macromolecules might lose their activity by denaturation caused by the steric repulsion [46]. Therefore, lower concentrations of biomolecules facilitate biomolecular interaction in a more efficient manner. Nonetheless, developed platforms have continuously generated the positive detection signals that are considerably greater than that of conventional ELISA assay. Sandwich ELISA in different concentrations of DENV (3.5 \(\times\) 10\({}^{-5}\) p.f.u/mL to 3.5 \(\times\) 10\({}^{-2}\) p.f.u/mL) was performed in order to carefully calibrate the assay. In a general observation from the plotted calibration curves in Figure 5b, it is evident that the higher level of confidence was achieved for the generated detection signals by coated electrospun fibers in comparison to the uncoated fibers or the conventional ELISA. Among coated PHBV fibers with different compositions of the polymer, the highest correlation coefficient value (\(R^{2}\) = 0.9994) was obtained for the coated fibers with Comp.(9:1). While coated fibers with PMMA and Comp. (5:5) have also shown comparative linearity, both compositions refer to the higher slopes for their respective calibrations plots than the rest. Coated fibers, in general, have proven to have a higher level of performance compared to the pure PHBV fibers and the actual ELISA, regardless of the polymer composition used for the coating. Even uncoated PHBV fibers have shown greater \(R^{2}\) value (0.9612) than ELISA, which confirms that the conventional analytical assay requires considerable improvements when it operates in lower concentrations of the target analyte. Figure 5: Detection range ELISA results: (**a**) coated and uncoated electrospun PHBV fiber mats in comparison to the conventional ELISA (depicted results are plotted after excluding the cut-off values); (**b**) calibration curves obtained for the assay conducted at DENV (dengue virus) concentration range of 3.5 \(\times\) 10\({}^{-2}\) p.f.u/mL to 3.5 \(\times\) 10\({}^{-5}\) p.f.u/mL. When the assay is conducted in the absence of target analyte, accurate analytical systems are likely to generate minor or no positive detection signal, as the biomolecular chain that leads to generation of the final signal is intentionally broken. As one of the evaluation methods, non-specific bindings at deliberate negative and positive replicates can be quantitatively measured and compared to the true negative and positive results thus establishing sensitivity and specificity of the assay [22]. In our investigation, a total number of 72 positive and 24 negative controls from an overall of 96 replicates were examined to assess the performance of each individual uncoated/coated fiber mats in the sandwich assay. Table 1 represents the evaluation parameters for the assays conducted with coated and uncoated PHBV fibers. Proposed fiber-based platforms in this study exhibited an average sensitivity of 97.49%, which was greatly improved in contrast to the clinical method, ELISA (76.39%, Table 1). Specificity of the proposed platforms was also found to be in a higher range than the clinical assay (Table 1, average 90.83%). Additional data analysis demonstrated significantly higher level of accuracy (average 95.83%) for the developed platforms in comparison to the conventional ELISA (77%).