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Figure 1.4 Schematic of popular enzyme immobilization techniques. (a) Adsorption (b) Polymer entrapment (c) Electrostatic entrapment (d) Covalent bonding and (e) Cross-linking.

Electrostatic entrapment (Figure 1.4c) makes use of the charge in the amino acid residues of the redox enzyme. Depending on the relative values of the protein isoelectric point and environmental pH, enzymes can present a net positive or negative charge. Accordingly, they can be favorably attracted to ionic polymers bearing the opposite charge. Cationic polymers, such as poly(ethyleneimine)s (PEIs) [46], poly(allylamine) [47, 48] and chitosan [42, 49], have therefore been used to immobilize negatively charged enzymes through an anion exchange process. Conveniently, the two most commonly used enzymes, GOx and laccase, are negatively charged at their operating pH (usually 7–7.5 and 4.5–5.5, respectively [7, 50, 51]). This approach has been taken further to form a structure of alternating layers of enzyme and cationic polymer, generally known as layer-by-layer (LbL) deposition. Using this strategy on both anodic and cathodic graphite electrodes, Rengaraj et al. achieved maximum power densities of 103 μW cm⁻² [50]. Although stronger than simple adsorption, the forces involved in electrostatic entrapment are relatively weak. Therefore, enzyme loss is still a problem that prevents this strategy to be reliable at making durable electrodes.

      Chemical bonding of the enzymes to the substrate is an alternative that provides stronger immobilization. The main forms in which it is usually implemented are referred to in literature as covalent bonding and cross-linking. Strictly speaking, however, both methods involve the formation of a covalent bond with the enzyme. In so-called covalent bonding (Figure 1.4d), the functional groups on the enzyme surface react either with the electrode surface directly or with a short molecule that is anchored to the electrode surface. While providing stable enzyme/surface linkage, the main problem of this technique is that the presence of the reactive groups at the electrode surface often causes significant stress on the enzyme’s tertiary structure. Therefore, denaturation is a common problem of these electrodes [52].

Cross-linking is an immobilization technique that attempts to ameliorate the downsides of covalent bonding, while still forming a covalent bond to the enzyme. To this end, cross linking agents are added which react with the surface groups in the enzyme surface (Figure 1.4e). Frequently, the amine groups present in the lysine residues have been targeted. Cross-linkers for this functional group have either aldehyde or epoxy terminated ends. Upon reaction with primary amines, aldehydes form imines (Figure 1.5a), creating a covalent double bond between the enzyme and the cross-linker. One of the most common cross linkers, partly due to its low cost, is glutaraldehyde (GA) (Figure 1.5c). This aliphatic cross linker is used, for example, to create bonds between the lysine residues in GOx, laccase and horseradish peroxidase [13, 53, 54]. While most authors have used the GA in solution [53, 54], some have preferred to expose the enzyme-modified electrode to a vapor of the cross-linker [13, 55, 56]. This method is more economical in that the deposition solution can be used for a longer period. Mixtures of enzymes and cross-linkers are reactive, making these solutions unstable. In fact, some precipitates start appearing within minutes in some solutions containing enzyme and cross-linker.

Figure 1.5 Reactions of (a) primary and (b) secondary amines with aldehydes. (c) Main aldehyde-based cross-linkers used for enzyme immobilization. R₁ and R₂ refer to groups in the enzyme or polymer, while R’ and R” refer to groups in the cross-linker.

Aldehydes can also react with the amine groups present in polymers such as poly(ethyleneimine)s (PEIs) [46]. Branched PEIs (BPEIs)^‡ possess primary, secondary and tertiary amines, while linear PEIs (LPEIs) possess almost exclusively secondary amines [57]. Therefore, when aldehydes react with LPEIs, they produce enamines instead (Figure 1.5b). This cross-linking between enzymes and polymers is expected to strengthen the interaction compared to just using the electrostatic entrapment. Indeed, Chung et al. showed improved stability when cross-linking GOx to BPEI-covered carbon nanotubes using two aldehyde cross-linkers [58].

Kwon’s group has recently introduced the use of terephthalaldehyde (Figure 1.5c) as a cross-linker for glucose oxidase and branched poly(ethyleneimine) [58]. They suggest that the conjugation between the C=N bonds and the phenyl group help improve the electron transfer across the whole composite. Furthermore, they tried different sequence of immobilization steps and found that, if the GOx is cross-linked first, it forms aggregates that can be later cross-linked to PEI-covered nanotubes. Using this methodology, they reported an additional 25% increase in their maximum power density compared to their previous approach СКАЧАТЬ