Molecularly Imprinted Polyscopoletin for the Electrochemical Detection of the Chronic Disease Marker Lysozyme

Abstract

Herein we report the electropolymerization of a scopoletin based molecularly imprinted polymer (MIP) for the detection of lysozyme (Lyz), an enzymatic marker of several diseases in mammalian species. Two different approaches have been used for the imprinting of lysozyme based, respectively, on the use of a monomer-template mixture and on the covalent immobilization of the enzyme prior to polymer synthesis. In the latter case, a multi-step protocol has been exploited with preliminary functionalization of gold electrode with amino groups, via 4-aminothiophenol, followed by reaction with glutaraldehyde, to provide a suitable linker for lysozyme. Each step of surface electrode modification has been followed by cyclic voltammetry and electrochemical impedance spectroscopy, which has been also employed to test the electrochemical responses of the developed MIP. The sensors show good selectivity to Lyz and detect the enzyme at concentrations up to 292 mg/L (20 μM), but with different performances, depending on the used imprinting approach. An imprinting factor equal to 7.1 and 2.5 and a limit of detection of 0.9 mg/L (62 nM) and 2.1 mg/L (141 nM) have been estimated for MIPs prepared with and without enzyme immobilization, respectively. Competitive rebinding experiment results show that this sensing material is selective for Lyz determination. Tests were performed using synthetic saliva to evaluate the potential application of the sensors in real matrices for clinical purposes.

Artificial Biomimetic Electrochemical Assemblies

“…Scopoletin provides functional groups that can participate in hydrogen bonds, van der Waals forces, and hydrophobic interactions with the template [ 161 ]. Therefore, scopoletin as a functional monomer has proven to be very efficient for MIP electrosynthesis, especially for protein imprinting [ 162 ]. Although satisfactory results have been obtained with polyscopoletin-based MIPs using a variety of protein templates, such as cyt c [ 117 ], heme protein [ 163 ], and tyrosine [ 146 ], this polymer remains an underestimated material in the field of imprinting [ 162 ].…”

“…Recently, an imprinted polyscopoletin was used for the impedimetric detection of lysozyme [ 162 ]. Lysozyme is a potent antimicrobial enzyme that is abundant in nature.…”

“Out of Pocket” Protein Binding—A Dilemma of Epitope Imprinted Polymers Revealed for Human Hemoglobin

“…They introduced an intermediate step by generating the template for MIP-synthesis via hemoglobin immobilization to silica nanoparticles, through the N-terminal valines, followed by digestion of the protein using trypsin. This MIP-synthetic approach resulted only in a two-fold higher HbA binding as compared with the non-imprinted polymer (NIP) [9].…”

“…The concentration of HbA and HbA1c in blood are routinely measured for the determination of the degree of glycated HbA-the long-term diabetic parameter [6]. Recently MIP based sensors which apply the whole spectrum of electrochemical and optical transducers made inroads in the determination of protein biomarkers [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] including SARS-COV-2 antigens [8]. For many years, Bülow's group made great efforts to create HbA-specific polymer beads for chromatographic separation of HbA variants from protein mixtures and crude cell 2 of 13 extracts [27][28][29].…”

Temperature-Responsive Magnetic Nanoparticles for Bioanalysis of Lysozyme in Urine Samples

“…Emerging techniques for lysozyme measurement in biological samples include antibody [31][32][33], aptamer [34], MIP [35] or nanoparticle-based [36] biosensors and the use of core-shell polymer NPs as selective extraction sorbents in the sample cleanup process. Most of the newly developed lysozyme nanoadsorbents use a molecular imprinting strategy [37][38][39][40][41][42][43][44][45][46] and only one of them relies on a polymeric material [18] with optimized composition of special functional monomers to achieve selectivity.…”