*3.5. Validation of the Aptasensor*

Over the years, there has been a greater focus on the development of colorimetric aptasensors due to their simplicity, which is based on the visual detection of a test result. However, they frequently have sensitivity and reproducibility issues; thus, the reproducibility of the RBP-A-aptasensor was evaluated. The assay was performed in triplicate (at a concentration of 250 nM), on three separate days. As shown in Table 1, the recovery rate of RBP4 was in the range from 90.36 ± 3.60% to 101.49 ± 1.9% and the intra-assay coefficients of variation (CV) calculated ranged from 1,88% to 3,99%. The results for the RBP-A-aptasensor were comparable with the results of a commercially available Human ELISA kit (ab108897) which has a recovery rate of 97% and an intra-assay CV of 4.8%. In contrast, our RPB-A-aptasensor had an intra-assay CV of 3.12%, which is better than that of the ELISA kit (8.5%), further confirming the exceptional reproducibility of the RBP-A-aptasensor.

**Figure 5.** Analysis of the sensitivity of the AuNP-based colorimetric aptasensor. (**a**) Absorption spectra of RBP-A-AuNPs-aptasensor in the presence of increasing concentrations of RBP4. Insert: visual colour changes of the aptasensor incubated with increasing concentrations of RBP4 (final RBP4 concentrations: 0, 7.81, 15.63, 31.25, 62.5, 125 and 250 nM). (**b**) Linear calibration curve for different absorbance ratios (A620/A520) corresponding to different concentrations of RBP4 (0–125 nM).


**Table 1.** Mean Recoveries and Coefficients of Variation for RBP4.

SD: Standard deviation.

#### *3.6. Specificity of the Aptasensor for RBP4*

The specificity of the developed RBP-A-aptasensor for the detection of RBP4 was evaluated against two diabetes-related biomarkers (A2MG and leptin) and one serumabundant protein (BSA). The proteins (at a concentration of 250 nM) were individually incubated with the RBP-A-aptasensor, and the absorbance ratios of A620/A520 were calculated (Figure 6). A water solution was used as the blank. The RBP-A-aptasensor showed a colour change from ruby red to purple/blue in the presence of RBP4 and no colour change was observed in the presence of A2MG, leptin and BSA. This indicated that RBP4 had a very high response to the RBP-A-aptasensor; whereas, A2MG, leptin and BSA displayed no interaction with the RBP-A-aptasensor. Previous studies have also evaluated the specificity of RBP-A towards RBP4 using various proteins such as RBP4, adiponectin, vaspin, nampt, BSA, human serum albumin, human IgG, fibrinogen, insulin and anti-RBP4 antibody and no interference was detected [21,23], further corroborating our results that RBP-A is highly specific to RBP4.
