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We introduce a practically generic approach for the generation of epitope-imprinted polymer-based microarrays for protein recognition on surface plasmon resonance imaging (SPRi) chips. The SPRi platform allows the subsequent rapid screening of target binding kinetics in a multiplexed and label-free manner. The versatility of such microarrays, both as synthetic and screening platform, is demonstrated through developing highly affine molecularly imprinted polymers (MIPs) for the recognition of the receptor binding domain (RBD) of SARS-CoV-2 spike protein. A characteristic nonapeptide GFNCYFPLQ from the RBD and other control peptides were microspotted onto gold SPRi chips followed by the electrosynthesis of a polyscopoletin nanofilm to generate in one step MIP arrays. A single chip screening of essential synthesis parameters, including the surface density of the template peptide and its sequence led to MIPs with dissociation constants (K-D) in the lower nanomolar range for RBD, which exceeds the affinity of RBD for its natural target, angiotensin-convertase 2 enzyme. Remarkably, the same MIPs bound SARS-CoV-2 virus like particles with even higher affinity along with excellent discrimination of influenza A (H3N2) virus. While MIPs prepared with a truncated heptapeptide template GFNCYFP showed only a slightly decreased affinity for RBD, a single mismatch in the amino acid sequence of the template, i.e. the substitution of the central cysteine with a serine, fully suppressed the RBD binding.
Insights in electrosynthesis, target binding, and stability of peptide-imprinted polymer nanofilms
(2021)
Molecularly imprinted polymer (MIP) nanofilms have been successfully implemented for the recognition of different target molecules: however, the underlying mechanistic details remained vague.
This paper provides new insights in the preparation and binding mechanism of electrosynthesized peptide-imprinted polymer nanofilms for selective recognition of the terminal pentapeptides of the beta-chains of human adult hemoglobin, HbA, and its glycated form HbA1c.
To differentiate between peptides differing solely in a glucose adduct MIP nanofilms were prepared by a two-step hierarchical electrosynthesis that involves first the chemisorption of a cysteinyl derivative of the pentapeptide followed by electropolymerization of scopoletin.
This approach was compared with a random single-step electrosynthesis using scopo-letin/pentapeptide mixtures. Electrochemical monitoring of the peptide binding to the MIP nanofilms by means of redox probe gating revealed a superior affinity of the hierarchical approach with a Kd value of 64.6 nM towards the related target.
Changes in the electrosynthesized non-imprinted polymer and MIP nanofilms during chemical, electrochemical template removal and rebinding were substantiated in situ by monitoring the characteristic bands of both target peptides and polymer with surface enhanced infrared absorption spectroscopy.
This rational approach led to MIPs with excellent selectivity and provided key mechanistic insights with respect to electrosynthesis, rebinding and stability of the formed MIPs.
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.
Effective recognition of enzymatically active tetrameric acetylcholinesterase (AChE) is accomplished by a hybrid nanofilm composed of a propidium-terminated self-assembled monolayer (Prop-SAM) which binds AChE via its peripheral anionic site (PAS) and an ultrathin electrosynthesized molecularly imprinted polymer (MIP) cover layer of a novel carboxylate-modified derivative of 3,4-propylenedioxythiophene. The rebinding of the AChE to the MIP/Prop-SAM nanofilm covered electrode is detected by measuring in situ the enzymatic activity. The oxidative current of the released thiocholine is dependent on the AChE concentration from approximate to 0.04 x 10(-6) to 0.4 x 10(-6)m. An imprinting factor of 9.9 is obtained for the hybrid MIP, which is among the best values reported for protein imprinting. The dissociation constant characterizing the strength of the MIP-AChE binding is 4.2 x 10(-7)m indicating the dominant role of the PAS-Prop-SAM interaction, while the benefit of the MIP nanofilm covering the Prop-SAM layer is the effective suppression of the cross-reactivity toward competing proteins as compared with the Prop-SAM. The threefold selectivity gain provided by i) the shape-specific MIP filter, ii) the propidium-SAM, iii) signal generation only by the AChE bound to the nanofilm shows promise for assessing AChE activity levels in cerebrospinal fluid.
Hypothesis: Electrosynthesis of the MIP nano-film after binding of the separated domains or holocytochrome BM3 via an engineered anchor should result in domain-specific cavities in the polymer layer. Experiments: Both the two domains and the holo P450 BM3 have been bound prior polymer deposition via a N-terminal engineered his6-anchor to the electrode surface. Each step of MIP preparation was characterized by cyclic voltammetry of the redox-marker ferricyanide. Rebinding after template removal was evaluated by quantifying the suppression of the diffusive permeability of the signal for ferricyanide and by the NADH-dependent reduction of cytochrome c by the reductase domain (BMR). Findings: The working hypothesis is verified by the discrimination of the two domains by the respective MIPs: The holoenzyme P450 BM3 was ca. 5.5 times more effectively recognized by the film imprinted with the oxidase domain (BMO) as compared to the BMR-MIP or the non-imprinted polymer (NIP). Obviously, a cavity is formed during the imprinting process around the hiss-tag-anchored BMR which cannot accommodate the broader BMO or the P450 BM3. The affinity of the MIP towards P450 BM3 is comparable with that to the monomer in solution. The hiss-tagged P450 BM3 binds (30 percent) stronger which shows the additive effect of the interaction with the MIP and the binding to the electrode.
Objectives: In this work, a simple and rapid liquid chromatographic method for the simultaneous determination of irbesartan (IRBE) and hydrochlorothiazide (HCT) was developed and validated by reverse phase high performance liquid chromatography (RP-HPLC). <br /> Materials and Methods: Experimental conditions such as different buffer solutions, various pH values, temperature, composition of the mobile phase, and the effect of flow rate were optimized. <br /> Results: The developed RP-HPLC method for these antihypertensive agents was wholly validated and IRBE was detected in the linear range of 0.1-25 mu g mL(-1) and HCT was detected in the linear range of 0.25-25 mu g mL(-1). Moreover, the suggested chromatographic technique was successfully applied for the determination of the drugs in human serum and pharmaceutical dosage forms with limit of detection values of 0.008 mu g mL(-1) for IRBE and 0.012 mu g mL(-1) for HCT. <br /> Conclusion: The proposed rapid analysis method of these antihypertensive drugs can be easily used and applied by pharmaceutical companies for which the analysis time is important.
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.
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.
Binding of heme to the amyloid peptides A beta 40/42 is thought to be an initial step in the development of symptoms in the early stages of Alzheimer's disease by enhancing the intrinsic peroxidatic activity of heme. We found considerably higher acceleration of the reaction for the physiologically relevant neurotransmitters dopamine and serotonin than reported earlier for the artificial substrate 3,3',5,5'-tetramethylbenzidine (TMB). Thus, the binding of hemin to A beta peptides might play an even more crucial role in the early stages of Alzheimer's disease than deduced from these earlier results. To mimic complex formation, a new surface architecture has been developed: The interaction between the truncated amyloid peptide A beta 1-16 and hemin immobilized on an aminohexanethiol spacer on a gold electrode has been analyzed by cyclic voltammetry. The resulting complex has a redox pair with a 25 mV more cathodic formal potential than hemin alone.
In this work, a novel electrochemical molecularly imprinted polymer (MIP) sensor for the detection of the lipopeptide antibiotic Daptomycin (DAP) is presented which integrates gold decorated platinum nanoparticles (Au-Pt NPs) into the nanocomposite film. The sensor was prepared by electropolymerization of o-phenylenediamine (o-PD) in the presence of DAP using cyclic voltammetry. Cyclic voltammetry and differential pulse voltammetry were applied to follow the changes in the MIP-layer related to rebinding and removal of the target DAP by using the redox marker [Fe(CN)(6)](3-/4-). Under optimized operational conditions, the MIP/Au-Pt NPs/ GCE nanosensor exhibits a linear response in the range of 1-20 pM towards DAP. The limit of detection and limit of quantification were determined to be 0.161pM +/- 0.012 and 0.489pM +/- 0.012, respectively. The sensitivity towards the antibiotics Vancomycin and Erythromycin and the amino acids glycine and tryptophan was below 7 percent as compared with DAP. Moreover, the nanosensor was also successfully used for the detection of DAP in deproteinated human serum samples.
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.
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.
Surface-Tuned Electron Transfer and Electrocatalysis of Hexameric Tyrosine-Coordinated Heme Protein
(2015)
Molecular modeling, electrochemical methods, and quartz crystal microbalance were used to characterize immobilized hexameric tyrosine-coordinated heme protein (HTHP) on bare carbon or on gold electrodes modified with positively and negatively charged self-assembled monolayers (SAMs), respectively. HTHP binds to the positively charged surface but no direct electron transfer (DET) is found due to the long distance of the active sites from the electrode surfaces. At carboxyl-terminated surfaces, the neutrally charged bottom of HTHP can bind to the SAM. For this "disc" orientation all six hemes are close to the electrode and their direct electron transfer should be efficient. HTHP on all negatively charged SAMs showed a quasi-reversible redox behavior with rate constant k(s) values between 0.93 and 2.86 s(-1) and apparent formal potentials E-app(0)' between -131.1 and -249.1 mV. On the MUA/MU-modified electrode, the maximum surface concentration corresponds to a complete monolayer of the hexameric HTHP in the disc orientation. HTHP electrostatically immobilized on negatively charged SAMs shows electrocatalysis of peroxide reduction and enzymatic oxidation of NADH.
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).
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).
Natürliche Evolution hat geschaffenBiopolymereauf der Basis von Aminosäuren undNukleotidezeigt hohe chemische Selektivität und katalytische Kraft. Die molekulare Erkennung durch Antikörper und die katalytische Umwandlung der Substratmoleküle durch Enzyme findet in sogenannten Paratopen oder katalytischen Zentren des Makromoleküls statt, die typischerweise 10-15 Aminosäuren umfassen. Die konzertierte Wechselwirkung zwischen den Reaktionspartnern führt zu Affinitäten bis zu nanomolaren Konzentrationen für die Antigenbindung und nähert sich einer Million Umsätze pro Sekunde anEnzym-katalysierte Reaktionen.
Biosensors representing the technological counterpart of living senses have found routine application in amperometric enzyme electrodes for decentralized blood glucose measurement, interaction analysis by surface plasmon resonance in drug development, and to some extent DNA chips for expression analysis and enzyme polymorphisms. These technologies have already reached a highly advanced level and need minor improvement at most. The dream of the "100-dollar' personal genome may come true in the next few years provided that the technological hurdles of nanopore technology or of polymerase-based single molecule sequencing can be overcome. Tailor-made recognition elements for biosensors including membrane-bound enzymes and receptors will be prepared by cell-free protein synthesis. As alternatives for biological recognition elements, molecularly imprinted polymers (MIPs) have been created. They have the potential to substitute antibodies in biosensors and biochips for the measurement of low-molecular-weight substances, proteins, viruses, and living cells. They are more stable than proteins and can be produced in large amounts by chemical synthesis. Integration of nanomaterials, especially of graphene, could lead to new miniaturized biosensors with high sensitivity and ultrafast response. In the future individual therapy will include genetic profiling of isoenzymes and polymorphic forms of drug-metabolizing enzymes especially of the cytochrome P450 family. For defining the pharmacokinetics including the clearance of a given genotype enzyme electrodes will be a useful tool. For decentralized online patient control or the integration into everyday "consumables' such as drinking water, foods, hygienic articles, clothing, or for control of air conditioners in buildings and cars and swimming pools, a new generation of "autonomous' biosensors will emerge.
Electrochemical synthesis and signal generation dominate among the almost 1200 articles published annually on protein-imprinted polymers. Such polymers can be easily prepared directly on the electrode surface, and the polymer thickness can be precisely adjusted to the size of the target to enable its free exchange. In this architecture, the molecularly imprinted polymer (MIP) layer represents only one ‘separation plate’; thus, the selectivity does not reach the values of ‘bulk’ measurements. The binding of target proteins can be detected straightforwardly by their modulating effect on the diffusional permeability of a redox marker through the thin MIP films. However, this generates an ‘overall apparent’ signal, which may include nonspecific interactions in the polymer layer and at the electrode surface. Certain targets, such as enzymes or redox active proteins, enables a more specific direct quantification of their binding to MIPs by in situ determination of the enzyme activity or direct electron transfer, respectively.
A nanohybrid consisting of poly(3-aminobenzenesulfonic acid-co-aniline) and multiwalled carbon nanotubes [MWCNT-P(ABS-A)]) on a gold electrode was used to immobilize the hexameric tyrosine-coordinated heme protein (HTHP). The enzyme showed direct electron transfer between the heme group of the protein and the nanostructured surface. Desorption of the noncovalently bound heme from the protein could be excluded by control measurements with adsorbed hemin on aminohexanthiol-modified electrodes. The nanostructuring and the optimised charge characteristics resulted in a higher protein coverage as compared with MUA/MU modified electrodes. The adsorbed enzyme shows catalytic activity for the cathodic H2O2 reduction and oxidation of NADH.
The first molecularly imprinted polymer (MIP) for the recognition of the copper-enzyme laccase was successfully prepared by electropolymerizing scopoletin in the presence of alkaline-inactivated enzyme. Laccase-MIP and the control polymer without laccase (nonimprinted polymer, NIP) were characterized by voltammetry using the redox marker ferricyanide. After electropolymerization, the signals for ferricyanide for both the MIP and the NIP were almost completely suppressed and increased after removal of the target from the polymer layer. Rebinding of both inactivated and active laccase decreased the ferricyanide peak currents to almost equal extent. The relative decrease of signal suppression approached saturation above 10 nM. Furthermore, the surface activity of rebound laccase toward the oxidation of catechol was investigated. The surface activity approached saturation above 10 nM, a value close to the value of the measurements with ferricyanide. Interaction of NIP with laccase brought about a six times smaller signal of catechol oxidation.