Optimization and characterization of electrochemical protein Imprinting on hemispherical porous gold patterns for the detection of trypsin

In this study, protein-imprinted sensors with thin bulk films were electrochemically fabricated on gold-coated quartz crystal electrodes with hemispherical porous gold patterns for detecting trypsin (Trp). The gold patterns were electrodeposited on a polystyrene colloidal monolayer and then rinsed using toluene. For Trp imprinting on the gold patterned electrodes, a thin layer with a poly(o-phenylenediamine) and Trp protein was formed using a cyclic voltammetry method under optimized conditions. In addition, a two-dimensional molecularly imprinted polymer (2D-MIP) film was prepared on a planar gold electrode under the same conditions to compare to the dependence of Trp selective recognition on three-dimensional (3D) thin MIP structure, and each corresponding nonimprinted polymer film were constructed by electropolymerization, in the absence of Trp template, to compare molecular imprinting effects. The sensing properties of Trp imprinted sensors were investigated using electrochemical, such as cyclic voltammetry and electrochemical impedance spectroscopy, and microgravimetric methods to confirm the sensitivity and selectivity of MIP films. The 3D-MIP films demonstrated a higher imprinting factor (3.51) in 48-μg/mL of Trp concentration than the 2D-MIP film, and the limit of detection was calculated to be 70.9-ng/mL. In addition, the films exhibited higher electrochemical sensing responses due to increased Trp recognition by the effective molecular imprinting over a larger surface area. Thus, the construction of 3D-MIP films for the protein imprinting could provide excellent specificity, faster kinetics, and higher sensitivity for detecting macromolecular proteins than 2D-MIP films.