570 Biowissenschaften; Biologie
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- Electropolymerization (2)
- Redox marker (2)
- immobilization (2)
- molecularly imprinted polymers (2)
- Biomarker (1)
- Cytochrome P450 (1)
- Daptomycin (1)
- Direct electron transfer (1)
- Epitope imprinting (1)
- Hangman porphyrin (1)
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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.
The combination of the biocatalytic features of enzymes with the unique physical properties of nanoparticles in a biohybrid system provides a promising approach for the development of advanced bioelectrocatalytic devices. This study describes the construction of photoelectrochemical signal chains based on CdSe/ZnS quantum dot (QD) modified gold electrodes as light switchable elements, and low molecular weight redox molecules for the combination with different biocatalysts. Photoelectrochemical and photoluminescence experiments verify that electron transfer can be achieved between the redox molecules hexacyanoferrate and ferrocene, and the QDs under illumination. Since for both redox mediators a concentration dependent photocurrent change has been found, light switchable enzymatic signal chains are built up with fructose dehydrogenase (FDH) and pyrroloquinoline quinone-dependent glucose dehydrogenase ((PQQ) GDH) for the detection of sugars. After immobilization of the enzymes at the QD electrode the biocatalytic oxidation of the substrates can be followed by conversion of the redox mediator in solution and subsequent detection at the QD electrode. Furthermore, (PQQ) GDH has been assembled together with ferrocenecarboxylic acid on top of the QD electrode for the construction of a funtional biohybrid architecture, showing that electron transfer can be realized from the enzyme over the redox mediator to the QDs and subsequently to the electrode in a completely immobilized fashion. The results obtained here do not only provide the basis for light-switchable biosensing and bioelectrocatalytic applications, but may also open the way for self-driven point-of-care systems by combination with solar cell approaches (power generation at the QD electrode by enzymatic substrate consumption).
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 polymer (MP) nanofilrns for transferrin (Trf) have been synthesized on gold surfaces by electro-polymerizing the functional monomer scopoletin in the presence of the protein target or around pre-adsorbed Trf. As determined by atomic force microscopy (AFM) the film thickness was comparable with the molecular dimension of the target. The target (re)binding properties of the electro-synthesized MIP films was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) through the target-binding induced permeability changes of the MIP nanofilms to the ferricyanide redox marker, as well as by surface plasmon resonance (SPR) and surface enhanced infrared absorption spectroscopy (SEIRAS) of the immobilized protein molecules. For Trf a linear concentration dependence in the lower micromolar range and an imprinting factor of similar to 5 was obtained by SWV and SPR. Furthermore, non-target proteins including the iron-free apo-Trf were discriminated by pronounced size and shape specificity. Whilst it is generally assumed that the rebinding of the target or of cross-reacting proteins exclusively takes place at the polymer here we considered also the interaction of the protein molecules with the underlying gold transducers. We demonstrate by SWV that adsorption of proteins suppresses the signal of the redox marker even at the bare gold surface and by SEIRAS that the treatment of the MIP with proteinase K or NaOH only partially removes the target protein. Therefore, we conclude that when interpreting binding of proteins to directly MIP-covered gold electrodes the interactions between the protein and the gold surface should also be considered.
The thiophene-modified iron porphyrin FeT3ThP and the respective iron Hangman porphyrin FeH3ThP, incorporating a carboxylic acid hanging group in the second coordination sphere of the iron center, were electropolymerized on glassy carbon electrodes using 3,4-ethylenedioxythiophene (EDOT) as co-monomer. Scanning electron microscopy images and Resonance Raman spectra demonstrated incorporation of the porphyrin monomers into a fibrous polymer network. Porphyrin/polyEDOT films catalyzed the reduction of molecular oxygen in a four-electron reaction to water with onset potentials as high as +0.14V vs. Ag/AgCl in an aqueous solution of pH7. Further, FeT3ThP/polyEDOT films showed electrocatalytic activity towards reduction of hydrogen peroxide at highly positive potentials, which was significantly enhanced by introduction of the carboxylic acid hanging group in FeH3ThP. The second coordination sphere residue promotes formation of a highly oxidizing reaction intermediate, presumably via advantageous proton supply, as observed for peroxidases and catalases making FeH3ThP/polyEDOT films efficient mimics of heme enzymes.
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.
Widely used diagnostic tools make use of antibodies recognizing targeted molecules, but additional techniques are required in order to alleviate the disadvantages of antibodies. Herein, molecular dynamic calculations are performed for the design of high affinity artificial protein binding surfaces for the recognition of neuron specific enolase (NSE), a known cancer biomarker. Computational simulations are employed to identify particularly stabile secondary structure elements. These epitopes are used for the subsequent molecular imprinting, where surface imprinting approach is applied. The molecular imprints generated with the calculated epitopes of greater stability (Cys-Ep1) show better binding properties than those of lower stability (Cys-Ep5). The average binding strength of imprints created with stabile epitopes is found to be around twofold and fourfold higher for the NSE derived peptide and NSE protein, respectively. The recognition of NSE is investigated in a wide concentration range, where high sensitivity (limit of detection (LOD) = 0.5 ng mL(-1)) and affinity (dissociation constant (K-d) = 5.3 x 10(-11)m) are achieved using Cys-Ep1 imprints reflecting the stable structure of the template molecules. This integrated approach employing stability calculations for the identification of stabile epitopes is expected to have a major impact on the future development of high affinity protein capturing binders.
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.
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.