Platforms for enzyme immobilization and biosensors

Biosensors and devices may also include fiber components. Biosensors essentially contain two main components. These are the sensing element, which senses the molecule of interest, and the transducer, which generates a signal. Amyloid fibrils and other protein nanofibers could be used in biosensors in two ways. The first is as a scaffold to immobilize the sensing element on a nanoscale as described above. The second use is as a coating on the transducer where the presence of the protein fiber could enhance device performance. This enhancement could be carried out either with or without the aid of a further conductive coating (see Section 4.1 above). 

The amyloidogenic Sup35 protein has been successfully used to immobilize three enzymes bamase, carbonic anhydrase, and glutathione S-transferase (Baxa et al., 2002). Each of these enzymes was linked to the Sup35 sequence which drives assembly creating three different fusion proteins which successfully formed fibrils displaying functional enzymes on the fibril surface. 

Artificial catalysts have also been incorporated into amphiphilic structures (Guler and Stupp, 2007). These catalysts were imidazolyl-functiona-lized peptides, which demonstrate a greater rate of 2,4-dinitrophenyl acetate hydrolysis when immobilized on the peptide amphiphile than the rate observed when the same enzyme is present in solution. Although the density of the enzymes on the fiber surface has not been established, the authors attribute the increase in enzymatic activity to the likely concentration of enzyme along the fiber surface, and this study illustrates one of the advantages of enzyme immobilization. 

The hydrophobias are a case where protein nanofibers can play a dual role in creating a biosensor. They can aid in the immobilization of bioactive components within a biosensor and also add further functionality to the transducing element of a biosensor device. Hydrophobins are self-assembling [3-sheet structures observed on the hyphae of filamentous fungi. They are surface active and aid the adhesion of hyphae to hydrophobic surfaces (Corvis et al., 2005). These properties can be used to create hydrophobia layers on glass electrodes. These layers can then facilitate the adsorption of two model enzymes glucose oxidase (GOX) and hydrogen peroxidase (HRP) to the electrode surface. The hydrophobin layer also enhances the electrochemical properties of the electrodes. 

Enzyme immobilization on a hydrophobin layer does not appear to significantly decrease enzyme properties, and these enzymes display similar substrate affinity to free enzymes. The specific activity of these enzymes may be lower than enzymes that are free in solution, but the immobilized enzymes also have the advantage of increased stability from 1 to 3 months (HRP and GOX, respectively). 

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