## 3 Results and Discussion
### Advantages of i-ELISA in Comparison to the Competing Technologies  
Although a variety of different techniques such as printing, patterning, waxing, heating, etc. have been used in fabrication of paper-based analytical devices, such platforms suffer from lack of control over the properties of the paper itself [2]. Paper segments in most of these platforms are chosen from commercially available cellulose products. Known as a hydrophilic material, NC possess WCA of 21\({}^{\circ}\) to 67\({}^{\circ}\), depending on the type of the paper and the specific manufacturer [52]. This level of hydrophilicity is not favorable for protein immobilization as it may induce protein entrapment and subsequently lead to the false positive signal. This phenomenon, to a large extent, is one of the responsible factors for low sensitivity of the paper-based analytical devices. Moreover, NC contains -OH functional groups in its structure. Different treatment techniques have been employed to modify the surface of NC by converting less reactive -OH groups to the more reactive -COOH groups [8; 9; 10; 11]. Nonetheless, as it was discussed before, such surface modification techniques tend to compromise the characteristics of the NC papers while generated -COOH functionalities are not guaranteed to last long. Furthermore, current paper-based platforms mainly rely on the visual readout as opposed to the analytical signal from the optical density or fluorescence measurement that corresponds to the exact concentration of the target analyte in blood serum. Semi-quantitative analysis with such devices is often carried out using tablets, smart phones and/or digital cameras [6]. Therefore, almost none of these paper-based techniques are comparable to the \"gold standard\" method (ELISA) that has been used for more than three decades in laboratories and hospitals. One of the justifications to accept those semi-quantitative methods is that such platforms have shown reliability in remote/rural areas and resource-limited environments that are in major need of such extreme point-of-care (EPOC) devices. Our strategy is to provide more sensitive analytical results even for EPOC applications by replacing the paper strips with more controllable polymer fiber mats. I-ELISA can successfully address a number of drawbacks from the previous paper-based platforms:  
* I-ELISA is the first developed electrospun fiber mat-assisted analytical well plate that strictly follows the principles of the standard ELISA method. * I-ELISA well plates can be fabricated by a slight modification in the current well plate by the injection molding process. Therefore, our platform can be highly cost-effective in comparison to the vying products. * Unlike previously developed paper-based well plates, which are often composed of several different layers that need to be assembled before the application (Figure 7a), i-ELISA consists of only two parts (lid and body of the well plate, Figure 7b), in the same fashion as commercially available analytical well plates. This fact to a considerable extent makes i-ELISA practical as the proper installation of the multilayer platforms is often beyond expertise of the lab technicians. * I-ELISA can be used as a universal approach through which a wide variety of fiber/paper products with their special properties and functionalities can be introduced to the assay. * As a unique platform, i-ELISA can offer the visual detection judgment as the white color of the paper makes the perfect background for colorimetric detection. More importantly, i-ELISA enables routine quantification of the exact signal intensity, as the bottom part of the well plate stays intact for a regular colorimetric readout (Figure 7b). * I-ELISA is privileged with a great stability of the fiber parts as well as the well plate itself. In most of the paper-based well plates, the paper segments are printed or sprayed at the bottom of the well plate [2].