Microcontact surface imprinting of affinity peptide for electrochemical impedimetric detection of neutrophil gelatinase-associated lipocalin

In this study, a specific binding peptide (BP1)-imprinted film comprising crosslinked poly(acrylamide-co-diallyamine-co-N,N′-methylenebisacrylamide) matrix was photochemically fabricated on a gold-coated quartz crystal electrode for detecting neutrophil gelatinase-associated lipocalin (NGAL), a promising biomarker of inflammatory bowel disease. Microcontact surface imprinting was performed using a BP1 template-adsorbed PDMS mold with a concave hemispherical array to form patterned films. The surface morphologies of the molecularly imprinted polymer (MIP) films were characterized using scanning electron microscopy (SEM), and the limit of detection (LOD, 0.07 μg/mL) and limit of quantification (LOQ, 0.24 μg/mL), including sensitivity and reproducibility, were investigated using electrochemical impedance spectroscopy (EIS). Moreover, the sensing properties of (NGAL-BP1)-imprinted MIP (“I-MIP”)-assisted biosensors for detecting NGAL protein were investigated using differential pulse voltammetry (DPV) and EIS. Normalized current peak (I_N) and impedance (R_N) values linearly increased with NGAL ranging from 1 to 300 ng/mL. Moreover, the selectivity coefficient (k* ) of NGAL proteins on the BP1-imprinted film was 3.5–5.8 against bovine serum albumin for the binding process in the entire low-concentration range owing to a high affinity between the imprinted BP1 peptide and target NGAL protein. Remarkable impedance difference was registered with respect to different stages of the Crohn's disease with the biosensors at the adsorption of NGAL proteins using samples from real patients. Moreover, the NGAL concentration measured using the developed (I-MIP)-assisted biosensors was comparable with that estimated using the commercially available enzyme-linked immunosorbent assay (ELISA). Therefore, peptide-imprinted biosensors based on electrochemical impedance sensing platforms are potentially promising for detecting macromolecular proteins.