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For the first time the direct electron transfer of an enzyme - cellobiose dehydrogenase, CDH - has been coupled with the hexokinase catalyzed competition for glucose in a sensor for ATP. To enhance the signal output for ATP, pyruvate kinase was coimmobilized to recycle ADP by the phosphoenolpyruvate driven reaction. The new sensor overcomes the limit of 1:1 stoichiometry of the sequential or competitive conversion of ATP by effective enzymatic recycling of the analyte. The anodic oxidation of the glucose converting CDH proceeds at electrode potentials below 0 mV vs. Ag vertical bar AgCl thus potentially interfering substances like ascorbic acid or catecholamines do not influence the measuring signal. The combination of direct electron transfer of CDH with the enzymatic recycling results in an interference-free and oxygen-independent measurement of ATP in the lower mu molar concentration range with a lower limit of detection of 63.3 nM (S/N=3).
Cytochrome P450 enzymes (CYPs) act on more than 90 percent of all drugs currently on the market. The catalytic cycle requires electron supply to the heme iron in the presence of oxygen. Electrochemistry allows to characterise the reaction mechanism of these redox enzymes by observing the electron transfer in real time. According to the number of publications on protein electrochemistry CYP has the third position after glucose oxidase and cytochrome c. CYP based enzyme electrodes for the quantification of drugs, metabolites or pesticides have been developed using different iso-enzymes. A crucial step in the sensor development is the efficiency of coupling the biocatalytic systems with the electrode is. In the 1970s the direct electron transfer of heme and heme peptides called microperoxidases (MPs) was used as model of oxidoreductases. They exhibit a broad substrate spectrum including hydroxylation of selected aromatic substrates, demethylation and epoxidation by means of hydrogen peroxide. It overlaps with that of P450 making heme and MPs to alternate recognition elements in biosensors for the detection of typical CYP substrates. In these enzyme electrodes the signal is generated by the conversion of all substrates thus representing in complex media an overall parameter. By combining the biocatalytic substrate conversion with selective binding to a molecularly imprinted polymer layer the specificity has been improved. Here we discuss different approaches of biosensors based on CYP, microperoxidases and catalytically active MIPs and discuss their potential as recognition elements in biosensors. The performance of these sensors and their further development are discussed. (C) 2013 Elsevier Ltd. All rights reserved.
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.
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.