## 3 Results and Discussion  
The optimization of the conditions of surface treatment for fabricated membranes is especially important in improving the efficiency of adhesion between solid phases and biomolecules. The adhesion ability of a solid surface depends on many factors such as the pH and concentration of the modified buffer. To find out the optimal conditions for measurement, we performed a calibration of IgG antigen with varying incubation times, concentration of chemicals, and on different days. Figure 2 shows the changes of slope and linearity of the calibration according to the reaction incubation time with 3-APTES. The optimized incubation time with 3-APTES treatment is measured to be 120 min as can be seen in Figure 2a. The results show that the optical density with an 3-APTES reaction time less than 90 min was decreased by 1.5 times at 50 ng/mL IgG antigen as compared with more than 90 min. In terms of the low concentration range, i.e., below 40 ng/mL, which is the main range concerning IgG, the calibration results showed better linearity than results with other incubation times. The linearity changes against concentrations with varying pH of GA solution in 0.1 M phosphate buffer were measured by optical density (Figure 2b,c). The GA solution of 0.5%, 1%, and 2% GA containing each concentration in phosphate buffer and 2% GA solution showed the maximum difference between 0 ng/mL and 100 ng/mL of antigen concentration (Figure 2b). As can be seen in Figure 2c, the calibration linearity based on optical density was measured to be similar in pH 6.2 and 7.2 of phosphate buffer solution. Figure 2d shows the changes in calibration linearity during a four-week period. Five separate plastic membranes underwent surface pretreatment and were then stored in EtOH solution. Each of the five membranes was tested independently using the ELISA process with a period of one week. Interestingly, the membrane stored for only a day showed a higher intensity as compared to those stored for a longer period of time which showed no significant difference in optical densities. The aforementioned surface treatments were applied to a plastic membrane and the optical density results of the IgG interaction were obtained before they were compared with those obtained using a typical ELISA. The optical density level of the ELISA system using a hybrid interface shows a 1.67-fold increased binding ability at 100 ng/mL in the presence of IgG antigen as compared with the conventional ELISA. In addition, the developed membrane could detect as low as 0.15625 ng/mL of IgG antigen resulting in increases of 1.75-fold over the traditional ELISA. As shown in Figure 3a, the meshed polystyrene (PS) membrane increases linearly as the concentration of IgG increases, especially in the case of over 50 ng/mL of antigen, by more than traditional ELISA. The PS membrane ELISA method increased the reaction surface area due to its structural advantage and thus increased the optical density accordingly. Figure 3b shows the effect of such materials as polypropylene (PP) and polymethyl methacrylate (PMMA) on the optical density of the ELISA system. In addition, the structure is made with machining in order to have a bar-shape and size of 0.35 \(\times\) 1.2 cm (r \(\times\) l). The bar-shape membrane was also treated with optimal chemicals as KOH, APTES, and GA prior to conjugation with antibodies. They were measured using spectrophotometer to find out optical densities against different concentrations of antigens. As can be seen in Figure 3b, although the bar-shaped membrane showed lower optical density levels than those of the meshed shaped membrane, both of their slopesagainst antigen concentration showed good linearity. Overall, the structure shape played the main role in increasing the area of reaction which determined the amount of overall reactions. Finally, for the clinical feasibility test, we have applied our membrane to measure the optical density changes against TSH concentrations.