## 3 Results and Discussion
### Detection Performance as a Function of Poly(MMA-co-MAA) Polymer Coating Composition  
In order to compare the performance of uncoated and coated PHBV fiber mats in the actual virus detection, colorimetric sandwich ELISA assay was performed to detect DENV as the target analyte. Figure 6 summarizes the detection results into two plots: detection signal intensities and negative controls (Figure 6a,b respectively). For clear comparison with the clinical standard method, ELISA was performed (in PS as a standard analytical well plate) as control. In order to perform substantial number of trials, the standard deviations in each set of the experiments are the results of 12 successive replicates. The fiber segments of i-ELISA were observed to be highly stable throughout several steps of incubation. As can be observed from Figure 6a, resultant optical density (OD) values are in the range of 0.4-1.2, which is generally in a greater range than previously reported values for DENV detection via conventional ELISA [14; 15; 17]. \begin{table}
\begin{tabular}{c|c|c|c|c|c|c|c|c|c|c|c|c} \hline \multirow{2}{*}{**DENV Status**} & \multicolumn{2}{c|}{**PHBV**} & \multicolumn{2}{c|}{**PMMA**} & \multicolumn{2}{c|}{**Comp.(9:1)**} & \multicolumn{2}{c|}{**Comp.(7:3)**} & \multicolumn{2}{c}{**Comp.(5:5)**} & \multicolumn{2}{c}{**ELISA**} \\ \cline{2-13}  & \(\mathbf{+}\) & \(\mathbf{-}\) & \(\mathbf{+}\) & \(\mathbf{-}\) & \(\mathbf{+}\) & \(\mathbf{-}\) & \(\mathbf{+}\) & \(\mathbf{-}\) & \(\mathbf{+}\) & \(\mathbf{-}\) & \(\mathbf{+}\) & \(\mathbf{-}\) \\ \hline Positive (TP, FP) & 70 & 4 & 71 & 1 & 70 & 3 & 71 & 1 & 69 & 2 & 55 & 5 \\ \hline Negative (FN, TN) & 2 & 20 & 1 & 23 & 2 & 21 & 1 & 23 & 3 & 22 & 17 & 19 \\ \hline Total & 72 & 24 & 72 & 24 & 72 & 24 & 72 & 24 & 72 & 24 & 72 & 24 \\ \hline Sensitivity (\%) & 97.22 & 98.61 & 97.22 & 98.61 & 95.83 & 76.39 \\ \hline Specificity (\%) & 83.33 & 95.83 & 87.50 & 95.83 & 91.67 & 79.17 \\ \hline Accuracy (\%) & 93.75 & 97.91 & 94.79 & 97.91 & 94.79 & 77 \\ \hline LoD (p.f.u/mL) & 413.99 & 9.9 & 14.1 & 16.2 & 10.41 & \(5\times 10^{3}\) \\ \hline \end{tabular}
\end{table} 
Table 1: Calculated sensitivity, specificity, accuracy and limit of detection (LoD) of the assay performed with polymer-coated electrospun PHBV (poly(3-hydroxybutyrate-co-3-hydroxyvalerate)) fiber mats in DENV (dengue virus) detection (the total number of 432 positive and 144 negative replicates was examined to study the assay accuracy). Indicative parameters in the table are as follows: true positive (TP), true negative (TN), false positive (FP), false negative (FN). ELISA: enzyme-linked immunosorbent assay. PMMA: poly methyl methacrylate-co-methacrylic acid. Although pure PHBV fibers have generated slightly higher detection signals than a conventional assay, it was observed that PHBV fibers (without polymer coating) resulted in considerable false positive signals when the assay was performed in the absence of DENV, as presented in Figure 6b. This result can be considered as direct evidence which proves that hydrophobicity of the PHBV fibers does not necessarily protect the surface from the risk of buffer penetration. In a sandwich assay, if primary Abs are entrapped inside the structure of the material, they would not be thoroughly eliminated in the following washing steps of the assay. Entangled primary Abs could couple with the secondary labeled  
Figure 6: Sandwich ELISA performed on different fiber mats (DENV concentration = \(3.5\times 10^{-2}\) p.f.u/mL): (**a**) detection signal intensity resulted from i-ELISA conducted with coated PHBV fiber mats with different compositions of poly(MMA-co-MAA) (data are plotted after subtracting the cut-off values from the actual detection results); and (**b**) negative control signals obtained from the assay conducted in the absence of DENV. Abs in the next step, thus leading to the false positive signal as can be seen in Figure 6b.