2.5. Quantifications of Cu-DOTA and AMP bound to the PADA coating

The PADA film was deposited onto the AT-cut 5 MHz Au-coated quartz crystal and set in the quartz crystal microbalance with dissipation (QCM-D) equipment chamber. To make the QCM-D baseline stabilized, we injected the PBS (pH 7.4) solution with the rate of 50 μL/min continuously. Then, the WSC solution contain DOTA (0.1 mg/mL) was injected until the curve equilibrated. After that, PBS (pH 7.4) was perfused to rinse the unbound DOTA. Subsequently, the CuCl₂·2H₂O (0.1 mg/mL) were injected to realize the chelation of Cu²⁺ with DOTA for obtained Cu-DOTA-coated surface. After that, PBS was injected to remove the unchelated Cu²⁺ until obtaining the stable baseline. Before grafting of AMP, the PBS (pH 7.4) solution containing Azido-dPEG4-NHS (2 mg/mL) was supplemented to the chamber for obtaining azide-functionalized surface based carbodiimide chemistry between NHS with –NH₂ retained on the Cu-DOTA-coated surface. After complete reaction and rinsing by PBS, 2 mg/mL of DBCO-AMP dissolved by PBS (pH 7.4) was injected into the chamber persistently. Based on the click chemistry between DBCO and Azide, the AMP was finally immobilized on the surface. In the end, the mass of the immobilized molecular species was calculated according to the Sauerbrey equation.