ABSTRACT
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.
Subject(s)
Molecular Imprinting , Humans , Matrix Metalloproteinase 1 , Epitopes , Polymers/chemistry , Pepsin A , Peptides , Poly AABSTRACT
A conducting molecularly imprinted polymer (MIP) film was integrated with an extended-gate field-effect transistor (EG-FET) transducer to determine epitopes of matrix metalloproteinase-1 (MMP-1) protein biomarker of idiopathic pulmonary fibrosis (IPF) selectively. Most suitable epitopes for imprinting were selected with Basic Local Alignment Search Tool software. From a pool of MMP-1 epitopes, the two, i.e., MIAHDFPGIGHK and HGYPKDIYSS, the relatively short ones, most promising for MMP-1 determination, were selected, mainly considering their advantageous outermost location in the protein molecule and stability against aggregation. MIPs templated with selected epitopes of the MMP-1 protein were successfully prepared by potentiodynamic electropolymerization and simultaneously deposited as thin films on electrodes. The chemosensors, constructed of MIP films integrated with EG-FET, proved useful in determining these epitopes even in a medium as complex as a control serum. The limit of detection for the MIAHDFPGIGHK and HGYPKDIYSS epitope was â¼60 and 20 nM, respectively. Moreover, the chemosensors selectively recognized whole MMP-1 protein in the 50-500 nM concentration range in buffered control serum samples.
Subject(s)
Biosensing Techniques , Molecular Imprinting , Epitopes , Matrix Metalloproteinase 1 , Molecularly Imprinted PolymersABSTRACT
Molecularly imprinted polymers (MIPs) have been developed to replace antibodies for the recognition of target molecules (such as antigens), and have been integrated into electrochemical sensing approaches by polymerization onto an electrode. Electrochemical sensing is inexpensive and flexible, and has demonstrated utility in point-of-care devices. In this work, several 2D (conductive) materials were employed to improve the performance of MIP sensors. Screen-printed electrodes were coated by the electropolymerization of aniline and metanilic acid, commingled with target molecules and various 2D materials. Tungsten disulfide (WS2) with an average particle size of 2 µm was found to increase the sensitivity of detection of molecularly imprinted conductive polymer-coated electrodes to 17ß-estradiol. As estradiol concentrations are important to eel aquaculture, we screened eel serum samples to determine their 17ß-estradiol concentrations, which were found to be in the range 28.2 ± 3.6 to 73.0 ± 11.6 pg/mL after dilution. These results were in agreement with measurements using commercial immunoanalysis.
