2.6. Preparation of Lateral Flow Assay Strip

The sample pad (glass fiber, grade 8951, Ahlstrom-Munksjö, Helsinki, Finland) was treated with PBS (1X, pH 7.4) containing 1% BSA and 0.05% Tween 20 and the conjugate pad (glass fiber, grade 8950, Ahlstrom) was treated with sodium phosphate dibasic solution (5 mM, pH 7.4) containing 0.5% BSA and 1% Tween 20. After pre-treatment of the sample and conjugate pad, these pads were dried at 37 °C for 24 h. The dried conjugate pad and nitrocellulose (NC) membrane (CN 110, Sartorius, Göttingen, Germany) were then attached to a plastic backing card (PJEAGO, Seoul, South Korea), and an absorbent pad (cellulose fiber, C083, Merck Millipore, Darmstadt, Germany) was stacked onto the NC membrane. The laminated lateral flow assay strip was cut into 3.8 mm-wide strips using a guillotine cutter (Zeta Corporation, Gunpo, South Korea). In each cut, the conjugate pad was prepared by adding 1 μL of different concentration of AuNP probes and then incubated at 37 °C for 1.5 h. For the test and control line zone, 0.4 μL of anti-cortisol capture antibody (10F10) and anti-mouse IgG was added on the NC membrane and dried at 37 °C for 1.5 h. Then, the LFA test strips were prepared and stored in a 4 °C refrigerator.