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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.
Human sulfite oxidase (hSO) is a homodimeric two-domain enzyme central in the biological sulfur cycle. A pyranopterin molybdenum cofactor (Moco) is the catalytic site and a heme b(5) group located in the N-terminal domain. The two domains are connected by a flexible linker region. Electrons produced at the Moco in sulfite oxidation, are relayed via heme b(5) to electron acceptors or an electrode surface. Inter-domain conformational changes between an open and a closed enzyme conformation, allowing "gated" electron transfer has been suggested. We first recorded cyclic voltammetry (CV) of hSO on single-crystal Au(111)-electrode surfaces modified by self-assembled monolayers (SAMs) both of a short rigid thiol, cysteamine and of a longer structurally flexible thiol, omega-amino-octanethiol (AOT). hSO on cysteamine SAMs displays a well-defined pair of voltammetric peaks around -0.207 V vs. SCE in the absence of sulfite substrate, but no electrocatalysis. hSO on AOT SAMs displays well-defined electrocatalysis, but only "fair" quality voltammetry in the absence of sulfite. We recorded next in situ scanning tunnelling spectroscopy (STS) of hSO on AOT modified Au(111)-electrodes, disclosing, a 2-5 % surface coverage of strong molecular scale contrasts, assigned to single hSO molecules, notably with no contrast difference in the absence and presence of sulfite. In situ STS corroborated this observation with a sigmoidal tunnelling current/overpotential correlation.
An amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O-2 reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O-2 scavenging membrane. The TMAO sensor operates at a potential of -0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 mu M and 15 mM, with a sensitivity of 2.75 +/- 1.7 mu A/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10% human serum, where the lowest detectable concentration is of 10 mu M TMAO.
The epitope imprinting approach applies exposed peptides as templates to synthesize Molecularly Imprinted Polymers (MIPs) for the recognition of the parent protein. While generally the template protein binding to such MIPs is considered to occur via the epitope-shaped cavities, unspecific interactions of the analyte with non-imprinted polymer as well as the detection method used may add to the complexity and interpretation of the target rebinding. To get new insights on the effects governing the rebinding of analytes, we electrosynthesized two epitope-imprinted polymers using the N-terminal pentapeptide VHLTP-amide of human hemoglobin (HbA) as the template. MIPs were prepared either by single-step electrosynthesis of scopoletin/pentapeptide mixtures or electropolymerization was performed after chemisorption of the cysteine extended VHLTP peptide. Rebinding of the target peptide and the parent HbA protein to the MIP nanofilms was quantified by square wave voltammetry using a redox probe gating, surface enhanced infrared absorption spectroscopy, and atomic force microscopy. While binding of the pentapeptide shows large influence of the amino acid sequence, all three methods revealed strong non-specific binding of HbA to both polyscopoletin-based MIPs with even higher affinities than the target peptides.
A biosensor for phenolic compounds based on a chemically modified laccase from Coriolus hirsula immobilized on functionalized screen-printed carbon electrodes (SPCEs) was achieved. Different enzyme modifications and immobilization strategies were analyzed. The electrochemical response of the immobilized laccase on SPCEs modified with carboxyl functionalized multi-walled carbon nanotubes (COOH-MWCNT) was the highest when laccase was aminated prior to the adsorption onto the working electrode. The developed lactase biosensor sensitivity toward different phenolic compounds was assessed to determine the biosensor response with several phenolic compounds. The highest response was obtained for ABTS with a saturation value of I-max = 27.94 mu A. The electrocatalytic efficiency (I-max/K-m(app)) was the highest for ABTS (5588 mu A mu M-1) followed by syringaldazine (3014 mu A.mu M-1). The sensors were considerably stable, whereby 99.5, 82 and 77% of the catalytic response using catechol as substrate was retained after 4, 8 and 10 successive cycles of reuse respectively, with response time average of 5 s for 12 cycles. No loss of activity was observed after 20 days of storage.
Electrochemical biosensors employing natural and artificial heme peroxidases on semiconductors
(2020)
Heme peroxidases are widely used as biological recognition elements in electrochemical biosensors for hydrogen peroxide and phenolic compounds. Various nature-derived and fully synthetic heme peroxidase mimics have been designed and their potential for replacing the natural enzymes in biosensors has been investigated. The use of semiconducting materials as transducers can thereby offer new opportunities with respect to catalyst immobilization, reaction stimulation, or read-out. This review focuses on approaches for the construction of electrochemical biosensors employing natural heme peroxidases as well as various mimics immobilized on semiconducting electrode surfaces. It will outline important advances made so far as well as the novel applications resulting thereof.
The heme-undecapeptide microperoxidase-11 (MP-11) was immobilized on mesoporous antimony-doped tin oxide (ATO) thin-film electrodes modified with the positively charged binding promotor polydiallyldimethylammonium chloride. Surface concentrations of MP-11 of 1.5 nmol cm(-2) were sufficiently high to enable spectroelectrochemical analyses. UV/Vis spectroscopy and resonance Raman spectroscopy revealed that immobilized MP-11 adopts a six-coordinated low-spin conformation, as in solution in the presence of a polycation. Cathodic reduction of hydrogen peroxide at potentials close to +500mV versus Ag/AgCl indicates that the reaction proceeds via a Compound I-type like intermediate, analogous to natural peroxidases, and confirms mesoporous ATO as a suitable host material for adsorbing the heme-peptide in its native state. A hydrogen peroxide sensor is proposed by using the bioelectrocatalytic properties of the MP-11-modified ATO.
Molecularly imprinted polymers (MIPs) have the potential to complement antibodies in bioanalysis, are more stable under harsh conditions, and are potentially cheaper to produce. However, the affinity and especially the selectivity of MIPs are in general lower than those of their biological pendants. Enzymes are useful tools for the preparation of MIPs for both low and high-molecular weight targets: As a green alternative to the well-established methods of chemical polymerization, enzyme-initiated polymerization has been introduced and the removal of protein templates by proteases has been successfully applied. Furthermore, MIPs have been coupled with enzymes in order to enhance the analytical performance of biomimetic sensors: Enzymes have been used in MIP-sensors as tracers for the generation and amplification of the measuring signal. In addition, enzymatic pretreatment of an analyte can extend the analyte spectrum and eliminate interferences.
Modulating the Molybdenum Coordination Sphere of Escherichia coli Trimethylamie N-Oxide Reductase
(2018)
The well-studied enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. We report on a system for the in vitro reconstitution of TorA with molybdenum cofactors (Moco) from different sources. Higher TMAO reductase activities for TorA were obtained when using Moco sources containing a sulfido ligand at the molybdenum atom. For the first time, we were able to isolate functional bis-MGD from Rhodobacter capsulatus formate dehydrogenase (FDH), which remained intact in its isolated state and after insertion into apo-TorA yielded a highly active enzyme. Combined characterizations of the reconstituted TorA enzymes by electron paramagnetic resonance spectroscopy and direct electrochemistry emphasize that TorA activity can be modified by changes in the Mo coordination sphere. The combination of these results together with studies of amino acid exchanges at the active site led us to propose a novel model for binding of the substrate to the molybdenum atom of TorA.
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.