## 2 Application of BRBS in the Detection of Diabetic Nephropathy Biomarkers
### Epidermal Growth Factor Receptor (EGFR)  
In recent years, EGFR has been proven to be one of the potential biomarkers for diabetic nephropathy [67]. Generally, the preparation of MIPs typically involves a single kind of template molecule/ion. However, MIPs based on a single template cannot recognize and remove multiple targets simultaneously. Multi-template MIT uses two or more target substances simultaneously as templates. [PERSON] et al. achieved simultaneous detection of dopamine and uric acid using dual-template technology, allowing the formation of MIPs containing multiple specific recognition sites. This provides a feasible strategy for simultaneously enriching, recognizing, detecting, and removing multiple targets [68]. [PERSON] et al. [69] constructed a MIP biosensor based on gold nanoparticle-modified screen-printed electrodes (Au-SPE) and used antibody-coupled nanoliposome amplification for simultaneous detection of EGFR and vascular VEGF (Figure 6). First, DSP was assembled onto the Au-SPE surface by self-assembly, and then the target proteins (EGFR and VEGF) were attached covalently through amide bonds to the modified SPE using multi-template molecular imprinting. Unbound sites were then blocked with acrylamide (AAM). Under the presence of persulfate, the AAM and \(N\)\(N^{\prime}\)-methylenebis(acrylamide) functional monomers were  
Figure 5: Scheme representation of molecular imprinting and the recognition principle: (**a**) electropolymerization with o-PD and VEGF; (**b**) removal of VEGF template; (**c**) incubation with VEGF sample solution; (**d**) washing and EIS measurements [65].  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Category of Technology** & **Functional Monomer/APT** & **Detection Limit** & **Ref.** \\ \hline Surface MIT & ortho-phenylenediamine & 0.08 pg mL\({}^{-1}\) & [65] \\ MAMT & APT & 0.68 pg mL\({}^{-1}\) & [66] \\ \hline \hline \end{tabular}
\end{table}
Table 3: The developed BRBS for the detection of VEGF.  
polymerized around the EGFR and VEGF templates, and the polymerization termination time was controlled by mixing the phenol solution with ethanol (w/t, 1%). Finally, by utilizing the ability of oxalic acid (OXA) to destroy peptide bonds, the template molecule is successfully removed from the imprinting layer. This strategy aims to produce reliable electrochemical signals by using nanoliposomes loaded with Cd(II) and Cu(II) ions, and modified with EGFR and VEGF-specific antibodies as the detection targets. In the analysis step, the potentiostatic adsorption analysis electrochemical technique is employed to indirectly determine the trace amounts of EGFR and VEGF in serum based on the content of these ions. Under optimal conditions, the detection limits for EGFR and VEGF analysis are 0.01 and 0.005 pg mL\({}^{-1}\), respectively. The sensor has good sensitivity, repeatability, and specificity. In clinical practice, it is often necessary to analyze a series of biomarkers to make a final diagnostic result, and this SPE-based biosensor can be successfully integrated with lab-on-a-chip, microfluidics, or micro-total analytical systems, opening new avenues for the development of multiplexed sensing of biomarkers. However, it is worth noting that due to the dilution of each template's binding sites, the selectivity of multi-template MIPs is lower than MIPs synthesized with a single template.