In this study, anti-CRP nanobodies were anchored to the WE through the formation of covalent bonds. First, anodic pretreatment was performed on the SPGE, wherein a constant potential of 1.5 V vs Ag/AgCl was maintained for 120 s. This process generated hydroxyl groups (-OH) on the WE surface, as described in detail previously . Then the electrode was rinsed with DI water and treated with a mixed solution of 2.1 M LiCl and 40 mM NaIO4 (5 μL) to convert the surface functional groups from hydroxyl (-OH) to aldehyde (-CHO) groups. The modified electrode was allowed to incubate in the dark for 15 min before being washed with DI water. Successively, the modified electrode was functionalized with either 1 μg mL -1 or 10 μg mL -1 of anti-CRP nanobodies for 1 h at room temperature (RT) and further washed using phosphate buffered saline (PBS, pH 7.4). The nanobody covalent binding on the modified electrode was obtained by means of a Schiff base reaction, resulting in the formation of an imine bond (C=N). To ensure the stability of the covalent bond, 1 mg mL -1 solution of NaBH3CN was applied to the activated electrode for 15 min, followed by PBS washing. Subsequently, 3 mg mL -1 of casein were added (30 min at RT) to block unsaturated residues and avoid non-specific interactions. After a final PBS washing step, the ready-to-use SPGEs were stored in a freezer at -20°C. An overview of the overall immobilization procedure is presented in Fig. .