Peptide Selection of MMP-1 for Electrochemical Sensing with Epitope-Imprinted Poly(TPARA-co-EDOT)s

• Published in: Biosensors (Basel) Vol. 12; no. 11; p. 1018
• Main Authors: Lee, Mei-Hwa, Lin, Cheng-Chih, Sharma, Piyush Sindhu, Thomas, James L., Lin, Chu-Yun, Iskierko, Zofia, Borowicz, Paweł, Lin, Chien-Yu, Kutner, Wlodzimierz, Yang, Chien-Hsin, Lin, Hung-Yin
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 15.11.2022; MDPI
• Subjects: Acetic acid; Amino acids; Biomarkers; Cancer; Cell culture; Cell migration; Cell proliferation; Collagen; Collagenase; Communication; conductive polymer; Copolymerization; Crystal structure; Electrical conductivity; Electrical resistivity; electrochemical sensing; Electrochemistry; Electrodes; Enzyme-linked immunosorbent assay; epitope imprinting; Epitopes; Humans; Indium tin oxides; Inflammation; Interstitial collagenase; Lung cancer; Lung carcinoma; Matrix metalloproteinase; Matrix Metalloproteinase 1; Matrix metalloproteinases; Metalloproteinase; Molecular Imprinting; Monomers; Pepsin; Pepsin A; Peptides; pH effects; Poly A; Polymer films; Polymerization; Polymers; Polymers - chemistry; Proteins; Ratios; Sensors; Trypsin; Voltammetry; electrochemical sensing; epitope imprinting; conductive polymer; matrix metalloproteinase-1
• ISSN: 2079-6374; 2079-6374
• DOI: 10.3390/bios12111018

Instead of molecularly imprinting a whole protein molecule, imprinting protein epitopes is gaining popularity due to cost and solubility issues. Belonging to the matrix metalloproteinase protein family, MMP-1 is an interstitial collagenase that degrades collagen and may be involved in cell migration, cell proliferation, the pro-inflammatory effect, and cancer progression. Hence, it can serve as a disease protein biomarker and thus be useful in early diagnosis. Herein, epitopes of MMP-1 were identified by screening its crystal structure. To identify possible epitopes for imprinting, MMP-1 was cleaved in silico with trypsin, pepsin at pH = 1.3, and pepsin at pH > 2.0 using Peptide Cutter, generating peptide fragments containing 8 to 12 amino acids. Five criteria were applied to select the peptides most suitable as potential epitopes for MMP-1. The triphenylamine rhodanine-3-acetic acid (TPARA) functional monomer was synthesized to form a stable pre-polymerization complex with a selected template epitope. The complexed functional monomer was then copolymerized with 3,4-ethoxylenedioxythiophene (EDOT) using potentiodynamic electropolymerization onto indium–tin–oxide (ITO) electrodes. The composition of the molecularly imprinted poly(TPARA-co-EDOT) (MIP) was optimized by maximizing the film’s electrical conductivity. Cyclic voltammetry was used to determine MMP-1 concentration in the presence of the Fe(CN)63−/Fe(CN)64− redox probe actuating the “gate effect.” A calibration curve was constructed and used to determine the usable concentration range and the limit of detection as ca. 0.001 to 10.0 pg/mL and 0.2 fg/mL MMP-1, respectively. Finally, the MMP-1 concentration in the A549 human lung (carcinoma) culture medium was measured, and this determination accuracy was confirmed using an ELISA assay.