## 3 Results and Discussion
In the present case, polymer-coated PHBV fibers create the platform for the mentioned physical forces to play their role in protein immobilization (Figure 4a,c). Figure 4d outlines the possible interactions that might occur between an Ab and a PHBV fiber mat coated with poly(MMA-co-MAA). While \(-\)COO\({}^{-}\) groups can electrostatically bind \(-\)NH\({}_{3}\)\({}^{+}\) groups (within appropriate pH range), the hydrophobic \(-\)COOCH\({}_{3}\) groups of the surface promote the eminent hydrophobic force that bind Ab molecules to the fibrous surface [36]. At the same time, though in a completely different manner, available \(-\)COOH groups of the platforms can bind with \(-\)NH\({}_{2}\) groups of the Ab molecules via H-bonding and provide an additional mean of interaction [36]. Abundant oxygen from methacrylate \(-\)COOCH\({}_{3}\) groups might also contribute interfacial binding with amine groups of the Ab's (Figure 4d). Looking into the chemical structures depicted in Figure 1, it can be considered that there is a major competition  
Figure 3: Water-in-air contact angle (WCA) analysis of the coated and uncoated PHBV fibers: (**a**) results with excluded (negligible) standard deviations; (**b**) droplet images of the contact angles measured for a commercial 96-well plate made from PS (polystyrene); and (**c**) representative droplet image on PMMA-coated PHBV electrospun fiber mat. between hydrophobic interaction and H-bonding in attracting Abs to the surface. PMMA-coated PHBV platform has shown the highest WCA among all the developed platforms that, in turn, facilitates biomolecule immobilization via hydrophobic interaction. On the other hand, PHBV coated with PMMA is privileged with the abundance of oxygen atoms in its structure. This gives an overall electronegativity to the surface that highly promotes electrostatic interaction even though this force contributes in protein immobilization in a weaker manner [36]. Figure 4: Application of i-ELISA platform in sandwich assay: (**a,b**) i-ELISA principle and magnified view of the pillar sections and the electrospun fibers; (**c**) sandwich ELISA at the interface with coated PHBV fiber mat through direct (physical) attachment of the biomolecules to the surface; and (**d**) major forces that contribute in the biomolecular interaction via surface functional groups. In addition to hydrophobic character of all the coated samples (WCA-109\({}^{\circ}\)-115\({}^{\circ}\) for Comp. (5:5)-(9:1); Figure 4a) poly(MMA-co-MAA) macromolecules also contain pendant -COOH groups that can trap Ab molecules through H-bonding, which could strongly affect the sensitivity of the detection platform. The complexity of heterogeneous (or interfacial) macromolecular binding is extended further by taking under consideration the surface concentration of -COOH groups. For instance, overly functionalized surfaces (such as the case of Comp. (5:5) composition with the highest concentration of -COOH groups) could often cause relatively inefficient protein immobilization due to the steric repulsion or multiple surface binding [24; 38]. In opposite cases when the concentration of surface functional groups is insufficient, the immobilized proteins could lose their activity due to close proximity to the solid platform. Overall, it is difficult to predict such phenomena and therefore any proposed method for biorecognition through heterogenic macromolecular interactions must be experimentally investigated and optimized in detail [23; 37; 39; 40]. The application of cross-linkers for covalent immobilization of the proteins to the surface is often recommended; however, driven by our previous findings, we have selected the method of physical immobilization for this study [37]. Covalent protein immobilization is often performed by carbodiimide chemistry as the covalent attachment of the proteins to the surface is expected to be stronger than physical immobilization [41; 42].