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Molecularly imprinted polymers (MIPs) mimic the binding sites of antibodies by substituting the amino acid-scaffold of proteins by synthetic polymers. In this work, the first MIP for the recognition of the diagnostically relevant enzyme butyrylcholinesterase (BuChE) is presented. The MIP was prepared using electropolymerization of the functional monomer o-phenylenediamine and was deposited as a thin film on a glassy carbon electrode by oxidative potentiodynamic polymerization. Rebinding and removal of the template were detected by cyclic voltammetry using ferricyanide as a redox marker. Furthermore, the enzymatic activity of BuChE rebound to the MIP was measured via the anodic oxidation of thiocholine, the reaction product of butyrylthiocholine. The response was linear between 50 pM and 2 nM concentrations of BuChE with a detection limit of 14.7 pM. In addition to the high sensitivity for BuChE, the sensor responded towards pseudo-irreversible inhibitors in the lower mM range.
Electrochemical methods offer the simple characterization of the synthesis of molecularly imprinted polymers (MIPs) and the readouts of target binding. The binding of electroinactive analytes can be detected indirectly by their modulating effect on the diffusional permeability of a redox marker through thin MIP films. However, this process generates an overall signal, which may include nonspecific interactions with the nonimprinted surface and adsorption at the electrode surface in addition to (specific) binding to the cavities. Redox-active low-molecular-weight targets and metalloproteins enable a more specific direct quantification of their binding to MIPs by measuring the faradaic current. The in situ characterization of enzymes, MIP-based mimics of redox enzymes or enzyme-labeled targets, is based on the indication of an electroactive product. This approach allows the determination of both the activity of the bio(mimetic) catalyst and of the substrate concentration.
An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V ( vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 mu M sulfite with a sensitivity of 2.19 mA M-1 sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.
Electrosynthesized molecularly imprinted polyscopoletin nanofilms for human serum albumin detection
(2017)
Molecularly imprinted polymers (MIPs) rendered selective solely by the imprinting with protein templates lacking of distinctive properties to facilitate strong target-MIP interaction are likely to exhibit medium to low template binding affinities. While this prohibits the use of such MIPs for applications requiring the assessment of very low template concentrations, their implementation for the quantification of high-abundance proteins seems to have a clear niche in the analytical practice. We investigated this opportunity by developing a polyscopoletin-based MIP nanofilm for the electrochemical determination of elevated human serum albumin (HSA) in urine. As reference for a low abundance protein ferritin-MIPs were also prepared by the same procedure. Under optimal conditions, the imprinted sensors gave a linear response to HSA in the concentration range of 20-100 mg/dm(3), and to ferritin in the range of 120-360 mg/dm(3). While as expected the obtained limit of detection was not sufficient to determine endogenous ferritin in plasma, the HSA-sensor was successfully employed to analyse urine samples of patients with albuminuria. The results suggest that MIP-based sensors may be applicable for quantifying high abundance proteins in a clinical setting. (c) 2017 Elsevier B.V. All rights reserved.
A tailor-made horseradish peroxidase (HRP) bulk composite electrode was developed on the basis of pyrolyzed cobalt tetramethoxyphenylporphyrin (CoTMPP) by modifying pore size and surface area of the porous carbon material through varying amounts of iron oxalate and sulfur prior to pyrolyzation. The materials were used to immobilize horseradish peroxidase (HRP). These electrodes were characterized in terms of their efficiency to reduce hydrogen peroxide. The heterogeneous electron transfer rate constants of different materials were determined with the rotating disk electrode method and a k(S) (401 +/- 61 s(-1)) exceeding previously reported values for native HRP was found.
For the first time a molecularly imprinted polymer (MIP) with direct electron transfer (DET) and bioelectrocatalytic activity of the target protein is presented. Thin films of MIPs for the recognition of a hexameric tyrosine-coordinated heme protein (HTHP) have been prepared by electropolymerization of scopoletin after oriented assembly of HTHP on a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold electrodes. Cavities which should resemble the shape and size of HTHP were formed by template removal. Rebinding of the target protein sums up the recognition by non-covalent interactions between the protein and the MIP with the electrostatic attraction of the protein by the SAM. HTHP bound to the MIP exhibits quasi-reversible DET which is reflected by a pair of well pronounced redox peaks in the cyclic voltammograms (CVs) with a formal potential of -184.4 +/- 13.7 mV vs. Ag/AgCl (1 M KCl) at pH 8.0 and it was able to catalyze the cathodic reduction of peroxide. At saturation the MIP films show a 12-fold higher electroactive surface concentration of HTHP than the non-imprinted polymer (NIP).
Molecularly imprinted polymers (MIPs) for the recognition of proteins are expected to possess high affinity through the establishment of multiple interactions between the polymer matrix and the large number of functional groups of the target. However, while highly affine recognition sites need building blocks rich in complementary functionalities to their target, such units are likely to generate high levels of nonspecific binding. This paradox, that nature solved by evolution for biological receptors, needs to be addressed by the implementation of new concepts in molecular imprinting of proteins. Additionally, the structural variability, large size and incompatibility with a range of monomers made the development of protein MIPs to take a slow start. While the majority of MIP preparation methods are variants of chemical polymerization, the polymerization of electroactive functional monomers emerged as a particularly advantageous approach for chemical sensing application. Electropolymerization can be performed from aqueous solutions to preserve the natural conformation of the protein templates, with high spatial resolution and electrochemical control of the polymerization process. This review compiles the latest results, identifying major trends and providing an outlook on the perspectives of electrosynthesised protein-imprinted MIPs for chemical sensing. (C) 2016 Elsevier B.V. All rights reserved.
Biomimetic binders and catalysts have been generated in order to substitute the biological pendants in separation techniques and bioanalysis. The two major approaches use either "evolution in the test tube" of nucleotides for the preparation of aptamers or total chemical synthesis for molecularly imprinted polymers (MIPs). The reproducible production of aptamers is a clear advantage, whilst the preparation of MIPs typically leads to a population of polymers with different binding sites. The realization of binding sites in the total bulk of the MIPs results in a higher binding capacity, however, on the expense of the accessibility and exchange rate. Furthermore, the readout of the bound analyte is easier for aptamers since the integration of signal generating labels is well established. On the other hand, the overall negative charge of the nucleotides makes aptamers prone to non-specific adsorption of positively charged constituents of the sample and the "biological" degradation of non-modified aptamers and ionic strength-dependent changes of conformation may be challenging in some application.
A new electrochemical MIP sensor for the most frequently used drug paracetamol (PAR) was prepared by electropolymerization of mixtures containing the template molecule and the functional monomers ophenylenediamine, resorcinol and aniline. The imprinting factor of 12 reflects the effective target binding to the MIP as compared with the non-imprinted electropolymer. Combination of the MIP with a nonspecific esterase allows the measurement of phenacetin - another analgesic drug. In the second approach the PAR containing sample solution was pretreated with tyrosinase in order to prevent electrochemical interferences by ascorbic acid and uric acid. Interference-free indication at a very low electrode potential without fouling of the electrode surface was achieved with the o-phenylenediamine: resorcinol-based MIP.