Enzyme sensors, protein stabilization

To overcome the poor stability of ferrocene-mediated enzyme sensors, mediator-modified electrodes have been used. In the case of glucose oxidase, the cofactor FAD is deeply buried within the protein matrix. The depth of the active center is estimated to be 0.87 nm. Therefore, one cannot expect that the mediator covalently attached to the electrode surface via a short spacer retain the possibility of closely approaching the cofactor of the enzyme. 

Second, sensors are often intended for a single use, or for usage over periods of one week or less, and enzymes are capable of excellent performance over these time scales, provided that they are maintained in a nfild environment at moderate temperature and with minimal physical stress. Stabilization of enzymes on conducting surfaces over longer periods of time presents a considerable challenge, since enzymes may be subject to denaturation or inactivation. In addition, the need to feed reactants to the biofuel cell means that convection and therefore viscous shear are often present in working fuel cells. Application of shear to a soft material such as a protein-based film can lead to accelerated degradation due to shear stress [Binyamin and Heller, 1999]. However, enzymes on surfaces have been demonstrated to be stable for several months (see below). 

Despite these improvements, there are other important biosensor limitations related to stability and reproducibility that have to be addressed. In this context, enzyme immobilisation is a critical factor for optimal biosensor design. Typical immobilisation methods are direct adsorption of the catalytic protein on the electrode surface, or covalent binding. The first method leads to unstable sensors, and the second one presents the drawback of reducing enzyme activity to a great extent. A commonly used procedure, due to its simplicity and easy implementation, is the immobilisation of the enzyme on a membrane. The simplest way is to sandwich the enzyme between the membrane and the electrode. Higher activity and greater stability can be achieved if the enzyme is previously cross-linked with a bi-functional reagent. 

Other types of cell membrane interaction have also been examined. For example Wang et al. have used an electrochemical SPR sensor to monitor peroxidase enzyme activity within the plasma membrane [77]. As a means of improving stability and creating a closer approximation of a true cell membrane, interlayer structures between the lower phospholipid leaf and the solid supports have been tested. Cushion layers based on PEG to enable transmembrane protein insertion were tested by Munro et al. [78], while Schuster et al. have reviewed the use of S-layer proteins as a construction element [79, 80]. 

Electrode surfaces modified with a multilayered surface architecture prepared by a layer-by-layer repeated deposition of several enzyme mono-layers show a modulated increase of surface-bound protein with a subsequent increase in output current, which is directly correlated with the number of deposited protein layers. The versatility of this approach allows alternate layers of different proteins for the manufacture of electrode surfaces, which are the basis for multianalyte sensing devices with multiple substrate specificities. The surface chemistry used for the manufacture of multilayered electrode surfaces is similar to that previously described for the preparation of affinity sensors, and is based on the stabilization of self-assembled multilayer assemblies by specific affinity interactions, electrostatic attraction, or covalent binding between adjacent monolayers. 

To demonstrate a way of the use of aptamers in design of biomimetic sensors, two examples will be cited from the recent literature. The piezoelectric sensor for protein IgE has been developed with the use of commercially available anti-IgE aptamer oligonucleotide.167 The obtained sensor shows specificity and sensitivity equivalent to these of immunosensor, but for aptamer-based sensor a less decrease of sensitivity after consecutive cycles of analyte binding and regeneration, as well as relative heat resistance and stability over several weeks was shown. A more complex mechanism of sensing was employed in adenosine aptamer-based sensor.168 Detection was based on enzymatic activity measurements by fluorescence polarization with the use of aptameric enzyme subunit, which was a DNA aptamer composed of enzyme-inhibiting aptamer and adenosine-binding aptamer. 

Enzymes, like most proteins, are unstable molecules. The stability of enzymes is of particular importance to biosensors because the rate of the enzymatically catalysed reaction is especially sensitive to the three-dimensional conformation of the enzyme. Most, if not all, enzymes are fully active only in a hydrated state. However, rarely are enzymes found in a completely aqueous medium in a sensor. Usually, the enzyme is dissolved into a hydrogel, immobilized onto a surface, or entrapped in a polymer matrix, all environments which differ in varying degrees from the natural environment of the enzyme. 

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