# Advancements in Glucose Monitoring: From Traditional Methods to Wearable Sensors
## 2 Invasive Methods
### Electrochemical Glucose Sensors  
Electrochemical glucose biosensors, which dominate the commercial glucometer market, typically consist of a three-electrode system: working, reference, and counter electrodes [21]. Each of these electrodes serves a distinct function. The working electrode acts as the sensor or transducer that responds to the electrochemical reaction occurring during the measurement process. The reference electrode provides a stable, known electrode potential and is often based on a saturated calomel electrode (SCE) or a silver-silver chloride (Ag/AgCl) electrode. The counter electrode completes the electrical circuit by facilitating current flow between the electrocatalytic solutions and the working electrode within the electrochemical cell. Counter electrodes are typically made from inert materials, such as platinum (Pt), gold (Au), graphite, or glassy carbon. Among the three electrodes, the working electrode plays the crucial role in determining the sensitivity and specificity of glucose detection [21]. Electrochemical glucose biosensors can be broadly categorized into two types: enzymatic and non-enzymatic. Enzymatic glucose biosensors are widely used due to the high specificity and sensitivity provided by the immobilized glucose oxidase (GOD) enzyme [22]. The process of immobilizing the GOD enzyme on the surface of the working electrode is a key consideration in the development of these biosensors. Glucose oxidase (GOD) is a flavoprotein that facilitates the oxidation of \(\beta\)-d-glucose to d-glucono-\(\delta\)-lactone and hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)), with molecular oxygen acting as the electron acceptor [22; 23]. This reaction occurs in two steps: a reductive and an oxidative reaction (Figure 1a) [23]. In the reductive reaction, GOD catalyzes the conversion of \(\beta\)-d-glucose into d-glucono-\(\delta\)-lactone, which is then hydrolyzed non-enzymatically to form gluconic acid. This process reduces the flavin adenine dinucleotide (FAD) in GOD to FADH\({}_{2}\). During the oxidative reaction, FADH\({}_{2}\) is reoxidized by oxygen, producing hydrogen peroxide. Catalase (CAT) then breaks down the hydrogen peroxide into water and oxygen. Additionally, [PERSON] et al. (1992) identified the enzyme lactonase (EC 3.1.1.17) from _Aspergillus niger_ as the catalyst responsible for hydrolyzing glucono-\(\delta\)-lactone to gluconic acid [24]. However, enzyme activity is highly sensitive to factors like temperature, humidity, and interference from other substances. Furthermore, the electron transfer process can be hindered by the thick enzyme layer, limiting the efficiency of these sensors [21]. As a result, there has been increasing interest in non-enzymatic glucose detection methods, which offer advantages such as improved stability and reduced interference. For instance, [PERSON] et al. demonstrated the general reaction pathway of glucose on metal surfaces (Figure 1b) [25]. For both \(\alpha\)- and \(\beta\)-glucose, the hydrogen atom attached to the C1 carbon becomes activated because the acidity of the hemiacetal OH group (pKa = 12.3) is stronger than that of the alcoholic OH group (pKa = 16). As a result, the electrochemical oxidation of \(\alpha\)- and \(\beta\)-glucose primarily produces glucono-\(\delta\)-lactone, which is then hydrolyzed to gluconic acid. This hydrolysis occurs with a half-life of about 10 min at pH 7.5. On the other hand, the electrochemical oxidation of \(\gamma\)-glucose directly yields gluconic acid. Whether glucono-\(\delta\)-lactone is involved as an intermediate or not, gluconic acid is the ultimate stable product of glucose oxidation through a two-electron process [25]. The conventional blood glucose-monitoring methods involves collecting blood through a fingertip trick, followed by measurement with an enzyme-based glucome  
Figure 1: (**a**) Schematic representation of glucose oxidation in presence of GOD. Reproduced with permission from [23]. (**b**) Redox mechanism of glucose demonstrating pathways of electrochemical electron transfer.