## 2 Application of BRBS in the Detection of Diabetic Nephropathy Biomarkers
### Vascular Endothelial Growth Factor (VEGF)  
VEGF is a 45 kDa homodimeric glycoprotein encoded by a single gene. Currently, five human VEGF microRNAs (mRNAs) have been found by splicing of VEGF mRNA, encoding VEGF with 121, 145, 165, 189, and 206 amino acids, respectively [62; 63]. It has been found that the glomerular basement membrane has a high affinity for VEGF, which can cause an increase in the permeability of the glomerular basement membrane, thereby promoting the production of NO, endothelin, etc., further changing the renal hemodynamics of diabetic patients, leading to proteinuria and inducing diabetic nephropathy [64]. Therefore, the development of highly sensitive and selective VEGF detection methods is of great significance for early diagnosis and patient recovery assessment.  
[PERSON] [65] and others proposed a highly sensitive label-free impedance sensor based on molecularly imprinted polymers (MIP) and graphite screen-printed electrodes (GSPE) as a detection platform for vascular endothelial growth factor (VEGF) (Figure 5). To improve the reproducibility of electrode detection and remove any impurities generated during the electrode manufacturing process, the GSPE was electrochemically pretreated before use. Then, MIP was formed on the pretreated GSPE surface by electropolymerization of ortho-phenylenediamine (o-PD) in the presence of the VGFR template. Under optimized conditions, the sensor used [Fe(CN)]\({}^{3-/4-}\) as a redox probe and employed a label-free method based on EIS measurement to detect VGFR, with good analytical performance in the range of 20-200 pg\(\cdot\)mL\({}^{-1}\) and a detection limit of 0.08 pg mL\({}^{-1}\) (Table 3). Due to the disposable nature of the sensor, it is more suitable for clinical needs.  
\begin{table}
\begin{tabular}{c c c c} \hline
**Category of Technology** & **Functional Monomer/APT** & **Detection Limit** & **Ref.** \\ \hline N-MIT & dopamine hydrochloride & 11.81 fmol L\({}^{-1}\) & [54] \\ MAMT & APT & 1 uM & [56] \\ SMAT & APT & 0.01 uM & [61] \\ \hline \end{tabular}
\end{table}
Table 2: The developed BRBS for the detection of Hcy.  
Figure 4: Schematic illustration of stepwise homocysteine aptasensor fabrication process [61].  
[PERSON] [66] and others constructed a convenient and ultra-sensitive ECL APT biosensor for detecting VEGF165. Firstly, the synthesized graphite-like carbon nitride (g-C\({}_{3}\)N\({}_{4}\)) nanosheets were adhered to CdSe using polydiallyldimethylammonium chloride (PDDA) as an adhesive, followed by assembling the obtained g-C\({}_{3}\)N\({}_{4}\)/PDDA/CdSe composite material onto the GCE surface. Finally, DNA\({}_{1}\) and AuNP-labeled DNA\({}_{2}\) were sequentially incubated on the modified GCE surface. In this strategy, AuDNA\({}_{2}\) showed a significant spectral overlap with the g-C\({}_{3}\)N\({}_{4}\)/PDDA/CdSe composite, thus allowing for effective energy transfer. In the presence of VEGF165, the sensor impedance decreased significantly after the Au-DNA\({}_{2}\) binds to VEGF165, and the ECL signal increased significantly. The ECL signal of the constructed biosensor increased with the increasing concentration of VEGF165, showing a prominent linear relationship from 2 pg mL\({}^{-1}\) to 2 ng mL\({}^{-1}\), and the detection limit of VEGF165 was as low as 0.68 pg mL\({}^{-1}\) (Table 3). The excellent sensitivity, stability, repeatability, and selectivity of the ECL biosensor indicate further potential applications in clinical diagnosis.