Enzyme-based biosensors

Electrocatalysis by Heme Enzymes—Applications in Biosensing

“…Enzyme attachment to the electrode surface is the most critical part of biosensor design. The main types of immobilization account for adsorption, encapsulation, and chemical binding, namely covalent coupling and cross-linking (Figure 2) [7,35,36]. The strategy of choice often results from the combination of several of these methods [37][38][39][40].…”

“…The strategy of choice often results from the combination of several of these methods [37][38][39][40]. In general terms, enzyme immobilization should guarantee (i) biocompatibility, (ii) efficient electrical contact between enzyme and transducer that also ensures exposed substrate binding sites, and (iii) preservation of catalytic activity [7,35,36,41]. For the chemical modification of electrodes, we highlight the use of self-assembled monolayers (SAMs) of alkanethiols, which represent one of the most common electrode coatings [42,43].…”

“…The enzyme solution is typically deposited onto the electrode surface and allowed to incubate for a determined period of time; the weakly adsorbed molecules are afterwards removed by washing. Adsorption is governed by electrostatic and hydrophobic interactions and van der Waals forces, which are considered to be less disruptive for enzyme structure and activity than e.g., covalent binding [35,36]. Nonetheless, the direct contact with bare metal or carbon electrodes can cause enzyme denaturation [28].…”

“…Encapsulation/entrapment into porous matrices (Figure 2b) fixes the protein molecules on the electrode support, while allowing the access of substrate molecules [35,36]. Commonly used materials include carbon pastes, conducting and non-conducting polymers, such as polyaniline (PANI), PPY, Nafion, and silica sol-gels [5].…”

“…In addition, the access of interfering species to the enzyme layer can be selectively blocked by e.g., size exclusion and charge repulsion. The main drawbacks of encapsulation/entrapment are (i) the potentially limited access of substrate to the active site due to the diffusion barrier created by the matrix film, (ii) the reduced enzyme loading capacity, and (iii) the possible alterations to the enzyme structure [7,35,36].…”