## 3 Results and Discussion  
Morphology Analysis of the Coated and Uncoated Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) Electrospun Fiber Mats  
Surface morphologies of the uncoated and polymer-coated PHBV electrospun fibers, recorded by SEM, are presented in Figure 1. A very clear morphology alteration by PMMA-co-MAA coating of the fibers can be observed by SEM image comparison between pristine fibers and polymer-coated PHBV mats (Figure 1a,d). In particular, Figure 1d depicts the highly porous structure of the polymer-coated fibers with the average pore size of \(\sim\)3 \(\upmu\)m (measurements performed for 100 random pores in five different coated samples). General pore size reduction of the coated samples is essential as it prevents the entrapment of the biomolecules in the network of the fibers (this phenomenon is further discussed in Section 3.4). Note that the images in Figure 1a,d were chosen as representative from SEM analysis (complete SEM analysis can be found in supplementary section; Figure A1). Representative cross-section images of the uncoated and polymer-coated PHBV fibers are also shown in Figure 1e,f, respectively. Coverage of the interstitial spaces between the electrospun fibers by coated segments can be recognized by comparing these images as well (Figure 1e,d). Overall, our method enables full cover of 3D fibrous structures used in this experiment. Similar fibrous structure was observed for almost all the polymer-coated PHBV fibers. This result is consistent with our previous findings where the similar coatings were fabricated on different type of polymer fibers [24]. In this study, however, a different approach was utilized by integrating the fiber onto the specially designed ELISA plate lids (Figure 2). With this fabrication strategy of well plates, i-ELISA introduces paper segments or polymer fiber mats from the top of the well plate into the assay. Contact Angle Analysis and the Fundamental Aspect of Physical Interaction between Protein Molecules and Poly Methyl Methacrylate-Co-Methacrylic (poly(MMA-co-MAA))-Coated Fibers  
Surface wettability of the developed platforms was analyzed by WCA measurement and results are presented in Figure 3. The uncoated PHBV fiber mat has shown the average contact angle of \(\sim\)110\({}^{\circ}\), which is in agreement with the previously reported data [33]. PMMA coating has increased the hydrophobicity of electrospun PHBV fibers up to the contact angle of \(\sim\)115\({}^{\circ}\). As expected, WCA for the coated fibers has shown a gradual decrease in WCA as the ratio of MAA monomer in polymer compositions increased from Comp. (9:1) to Comp.(5:5). The deliberate alteration of MMA/MAA ratio in polymer backbone (Figure 1) was used to control surface concentration of -COOH groups, which in turn resulted in WCA presented in Figure 3a. It is important to note that we measured the WCA of PS(commercial ELISA well plate) to be \(\sim\)60\({}^{\circ}\), which is in agreement with the reported results [34]. This drastic difference between two platforms (i-ELISA and PS conventional well plate used as control) is expected to have a strong influence on detection sensitivity. General hydrophobicity of the developed fiber mats, in this study, has drawn a great deal of importance when analyte-surface interaction is concerned. However, the complexity of such heterogenic interactions involves both surface chemistry and morphology that must be taken under consideration [35]. Among several different forces that have an impact on biomolecular interfaces, three main interactions are known as the most influential driving forces in analyte-surface binding. The forces that have frequently been reported in the literature for their considerable impact on protein immobilization are as follows: electrostatic (ionic) attraction, hydrophobic interaction and hydrogen (H) bonding [24; 36; 37]. The ionic attraction is sensitive to pH changes and is relatively weak in comparison to other two forces while H-bonding facilitates the most substantial protein attachment [36].