## 2 Application of BRBS in the Detection of Diabetic Nephropathy Biomarkers
### Cystatin C (Cys-C)  
Cys-C is a 13 kDa non-glycoslated polypeptide chain consisting of 120 amino acid residues. It is a small protein that can be freely filtered by the renal glomerulus. The average serum Cys-C level in patients with diabetic nephropathy (1.87 \(\pm\) 0.51 mg/L) is higher than that in patients with non-diabetic nephropathy (1.025 \(\pm\) 0.30 mg/L). In addition, serum Cys-C levels are higher in patients with elevated serum Crn levels, suggesting a correlation between Cys-C and Crn. Studies have shown that serum Cys-C detection can help to identify early renal impairment in diabetic patients, with better efficacy than serum Crn [34]. Another study showed that the average serum cystatin C value was 1.73 in type 2 diabetic patients with normal urinary albumin levels, while the average serum cystatin C value was 2.07 in type 2 diabetic patients with microalbuminuria. These results suggest that serum cystatin C, as a renal glomerular filtration biomarker, is an earlier indicator of diabetic nephropathy than urinary microalbumin and serum Crn [35]. Multifunctional monomer imprinting can enhance non-covalent binding between functional monomers and TM via multiple synergistic interaction sites [36]. This is particularly effective in improving the selectivity and adsorption of MIPs for large molecules. Therefore, it is an effective technique to use two or more functional monomers simultaneously, \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Category of Technology** & **Functional Monomer** & **Detection Limit** & **Ref.** \\ \hline Traditional-MIT & methacrylic acid & 0.1 ppm & [25] \\ N-MIT & nickel nanoclusters & 0.5 nmol/L & [31] \\ Surface MIT & dopamine hydrochloride & 2 \(\times\) 10\({}^{-2}\) pg/mL & [33] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The developed BRBS for the detection of Crn. Figure 2: Schematic representation for the preparation of GNP/PDA-MIP. (**a**) The fabrication and application process of GNP/PDA-MIP and the EIS/DPV response of the GNP/PDA-MIP modified electrode towards different concentration of creatinine in electrolyte. (**b**) The formation mechanism of DA spontaneous oxidative polymerization [33]. forming multiple complementary interaction sites with TM in different areas. However, the selection, combination, design, synthesis, and full utilization of their synergistic effect to prepare ideal MIPs still require continuous exploration. In addition, polypyrrole (PPy) is a conductive polymer that is well-known for its use in MIP materials [37]. Carbon nanotubes (CNTs) can improve electron transfer and surface area in electrochemical systems, thereby enhancing the electrical properties of electrochemical biosensors. Therefore, after adding CNTs to PPy, a 3D structure with increased electrical conductivity is formed due to the strong \(\pi\)-\(\pi\) bonding between the Ppy-conjugated structure and CNTs [38]. The PPy/CNT nanocomposite exhibits good charge transfer and energy/electron storage performance, making it suitable for supercapacitors [39]. Based on this strategy, [PERSON] [40] et al. first formed a mixed system of Cys-C, multi-walled carbon nanotubes (MWCNTs), pyrrole (Py), and carboxylated pyrrole (Py-COOH), and electrochemically polymerized it on the surface of a carbon fiber net screen-printed electrode (SPE) to form an MPPy nanocomposite. Subsequently, the Cys-C template was removed by the urea method, constructing a disposable biosensor based on molecular imprinting capable of accurately detecting Cys-C. Because Cys-C has a higher affinity with binding sites when surrounded by bifunctional monomers, the recognition ability of the sensor is enhanced. Additionally, introducing MWCNTs into the PPy matrix resulted in a more porous structure, larger surface area, and better sensitivity.