Tyrosinase Immobilization Strategies for the Development of Electrochemical Biosensors-A Review

Abstract

The development of enzyme biosensors has successfully overcome various challenges such as enzyme instability, loss of enzyme activity or long response time. In the electroanalytical field, tyrosinase is used to develop biosensors that exploit its ability to catalyze the oxidation of numerous types of phenolic compounds with antioxidant and neurotransmitter roles. This review critically examines the main tyrosinase immobilization techniques for the development of sensitive electrochemical biosensors. Immobilization strategies are mainly classified according to the degree of reversibility/irreversibility of enzyme binding to the support material. Each tyrosinase immobilization method has advantages and limitations, and its selection depends mainly on the type of support electrode, electrode-modifying nanomaterials, cross-linking agent or surfactants used. Tyrosinase immobilization by cross-linking is characterized by very frequent use with outstanding performance of the developed biosensors. Additionally, research in recent years has focused on new immobilization strategies involving cross-linking, such as cross-linked enzyme aggregates (CLEAs) and magnetic cross-linked enzyme aggregates (mCLEAs). Therefore, it can be considered that cross-linking immobilization is the most feasible and economical approach, also providing the possibility of selecting the reagents used and the order of the immobilization steps, which favor the enhancement of biosensor performance characteristics.

Keywords: biosensor; cross-linking; entrapment; enzymatic activity; selectivity.

Figure 1

Components of a typical biosensor.

Figure 2

Geometry of the binuclear copper-binding site for isolated and purified tyrosinase from Agaricus bisporus. Reprinted with permission from [35]. Copyright 2023, copyright Elsevier.

Figure 3

Reactions catalyzed by tyrosinase.

Figure 4

Classification and representation of different enzyme immobilization techniques. Reprinted with permission from [105]. Copyright 2023, copyright Elsevier.

Figure 5

Construction of the electrochemical biosensor [113].

Figure 6

Reactions occurring between sublayer, capture agent and enzyme.

Figure 7

Schematic of ND-PS/GCE and of Tyr-ND-PS/GCE preparation. Step 1: ND was dispersed in PS solution. Step 2: ND-PS dispersion was ultrasonicated (30 min). Step 3: A volume of 5.0 µL ND-PS were dropped on the GCE surface. Step 4: ND-PS/GCE was immersed in a solution containing 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) and N-hydroxysuccinimide (NHS) for 2 h. Step 5: ND-PS/GCE was immersed in a solution containing 25 units of Tyr. Step 6: Detection of catechol in samples of tap and river water with the new biosensor. Reprinted with permission from [130]. Copyright 2023, copyright Elsevier.

Figure 8

Schematic illustration of the TYR/GA/GO/GCE biosensor for the electrochemical determination of phenol derivatives: (a), GO casting; (b), covalent bonding of GO and GA; (c), covalent bonding of GA and TYR; and (d), electrochemical detection of phenol derivatives. Reprinted with permission from [80]. Copyright 2023, copyright Elsevier.

Figure 9

Formation of cross-linked enzyme aggregates (CLEAs) [145].

Figure 10

Schematic of the Gold-Electrode/Cysteamine/Carbon Dots/Tyrosinase biosensor [184].

Figure 11

EAPC protocol, consisting of enzyme adsorption, precipitation and crosslinking. Reprinted with permission from [78]. Copyright 2023, copyright Elsevier.

Figure 12

Schematic of the dual enzymatic immobilization process.