*2.2. Synthesis of Antigens*

The synthesis method of the coating antigen and immunogen was appropriately improved by Meng et al. (2015) [15]. First, pyrene butyric acid (PBA) (110 mg), EDC (130 mg) and NHS (210 mg) were dissolved in 3 mL DMF. The solution was gently mixed at 4 ◦C overnight, which was called activated solution. BSA (70 mg) was dissolved in 9 mL of PBS (0.01 mol/L, pH = 7.4). Preparation of immunogen (PBA-BSA) with different coupling ratios: three glass bottles were used, and 3 mL BSA solution was added, respectively. Then, 400 μL, 600 μL, and 800 μL of activation solution was slowly added to the bottle successively and stirred at 4 ◦C for 10 h away from light. Three immunogens were represented by A1, A2 and A3.

The coating antigen (PBA-OVA) was prepared using the same method as the immunogen. Briefly, OVA (90 mg) was dissolved in 12 mL PBS, the above activated solution (200 μL, 300 μL, 400 μL) was added to three glass bottles containing 4 mL OVA protein solution. These solutions were gently stirred at 4 ◦C for 12 h. Three coating antigens were represented by B1, B2 and B3. They were exhaustively dialyzed with 0.1M PBS at 4 ◦C for 5 days and the dialysate was changed every 12 hours. The antigens were collected and centrifuged at 10,000× *g* for 5 min. The supernatant was obtained and stored in a −20 ◦C refrigerator for later use. Antigen synthesis was verified by 8453 UV–Visible spectrophotometer and the ratios of hapten/protein were estimated.
