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Peptide Selection of MMP-1 for Electrochemical Sensing with Epitope-Imprinted Poly(TPARA-<i>co</i>-EDOT)s
oleh: Mei-Hwa Lee, Cheng-Chih Lin, Piyush Sindhu Sharma, James L. Thomas, Chu-Yun Lin, Zofia Iskierko, Paweł Borowicz, Chien-Yu Lin, Wlodzimierz Kutner, Chien-Hsin Yang, Hung-Yin Lin
Format: | Article |
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Diterbitkan: | MDPI AG 2022-11-01 |
Deskripsi
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-<i>co</i>-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)<sub>6</sub><sup>3−</sup>/Fe(CN)<sub>6</sub><sup>4−</sup> 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.