## 4 Conclusions  
A new label-free analytical method for the simultaneous determination of VEGF-A and FGF-2 in serum has been developed, based on SPRi biosensors. The formation of successive biosensor layers was confirmed by plotting SPR curves fitted to the experimental data. Shifts towards higher angle values confirmed that successive layers were formed on the surface of the biosensor. The method presented can also be applied as two separate and independent analytical methods, due to the validation and verification of each of them separately. As \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{4}{c}{**Biomarker**} \\ \cline{2-5}
**Sample** & \multicolumn{2}{c}{**VEGF-A**} & \multicolumn{2}{c}{**FGF-2**} \\ \cline{2-5}  & \multicolumn{2}{c}{**SPRi Biosensor**} & \multicolumn{2}{c}{**ELISA**} & \multicolumn{2}{c}{**SPRi Biosensor**} & \multicolumn{2}{c}{**ELISA**} \\  & [pg/mL] & [pg/mL] & [pg/mL] & [pg/mL] \\ \hline L1 & 673.95 & 695.20 & 9.02 & 8.77 \\ L2 & 387.54 & 394.12 & 6.15 & 6.09 \\ L3 & 376.12 & 366.50 & 8.62 & 7.73 \\ L4 & 445.29 & 434.31 & 10.54 & 9.72 \\ C1 & 234.59 & 227.35 & 3.45 & 3.23 \\ C2 & 45.73 & 46.51 & 5.29 & 5.67 \\ C3 & 135.71 & 134.40 & 4.89 & 4.30 \\ C4 & 192.23 & 174.52 & 3.96 & 3.43 \\ C5 & 165.93 & 145.78 & 3.57 & 3.11 \\ C6 & 224.21 & 227.35 & 2.73 & 2.64 \\ \hline \hline \end{tabular}
\end{table} 
Table 6: Comparison of concentration values obtained with the developed biosensor and with the ELISA test. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline  & \multicolumn{6}{c}{**Biomarker**} \\ \cline{2-8}
**Parameter** & \multicolumn{6}{c}{**VEGF-A**} & \multicolumn{6}{c}{**FGF-2**} \\ \cline{2-8}  & \multicolumn{2}{c}{**CRM [pg/mL]**} & \multicolumn{2}{c}{**Real**} & \multicolumn{2}{c}{**CRM [pg/mL]**} & \multicolumn{2}{c}{**Real**} \\ \cline{2-8}  & **20** & **30** & **50** & **Sample** & **5** & **20** & **50** & **Sample** \\ \hline X\({}_{\text{C}}\) [pg/mL] & 21.47 & 30.62 & 51.19 & 45.73 & 4.93 & 19.88 & 51.46 & 5.62 \\ SD [pg/mL] & 0.51 & 0.39 & 0.42 & 0.81 & 0.16 & 0.62 & 0.55 & 0.43 \\ Recovery [\%] & 107.35 & 102.07 & 102.98 & - & 98.60 & 99.40 & 102.92 & - \\ CV [\%] & 2.38 & 1.27 & 0.82 & 1.77 & 3.25 & 3.12 & 1.07 & 7.65 \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Repeatability data. a result of the validation of the methods, LOD and LOQ values were determined for each of them. These values were: for VEGF-A, LOD = 1.07 pg/mL, LOQ = 3.21 pg/mL; and for FGF-2, LOD = 1.64 pg/mL, LOQ = 4.92 pg/mL. The proposed method is also precise, as evidenced by the small standard deviations of concentrations determined in samples of standard solutions of precisely known concentration and the CVs not exceeding 12%. It has been shown that significant changes in the analytical procedure affect the stability of the results. Changes in the temperature and pH of the solutions had the greatest impact on the concentration values obtained. In the sample stored correctly, no changes in the concentration values were observed compared with a sample prepared just before the analysis. The optimal interaction time of the analyte with the ligand for the VEGF-A-sensitive biosensor was 6 min, and for the FGF-2-sensitive biosensor, it was 8 min. Above these values, no further changes in analyte concentrations were observed. To control the accuracy and reliability of the results, recovery studies were performed. For this purpose, a known amount of standard solution of VEGF-A and FGF-2 was added to a real biological sample, namely a sample with a very complex matrix. Then, based on the difference in analyte concentrations between the samples with and without the standard addition, the added amounts of VEGF-A and FGF-2 were determined. The recovery results did not exceed 103%, which proves the high accuracy and reliability of the results obtained.