*2.2. Synthesis and Characterization of AuNPs*

All glassware used for the preparation of AuNPs was first immersed thoroughly in aqua regia (1:3 (*v*/*v*) HNO3: HCl), then thoroughly washed with distilled water (dH2O) and dried overnight at 70 ◦C in an oven. AuNPs were prepared via the reduction of HAuCl4·3H2O using sodium citrate as described by Stiolica et al. [41,42]. The spectroscopic characterization of the synthesized AuNPs was carried out using Ultraviolet–visible (UV–vis) spectrophotometry (POLARstar Omega plate reader, BMG Labtech, Offenburg, Germany). The concentration of the AuNPs was evaluated using the UV–vis spectra as described by Haiss et al. [43]. The size distribution and zeta potential (ζ- potential) characterization of the AuNPs were determined using a Malvern NanoZS90 Zetasizer (Malvern Instruments Ltd., Malvern, UK) at a scattering angle of 90◦ at 25 ◦C. For the characterization of the size and morphology of the AuNPs, one drop of the sample solution was loaded onto a carbon-coated copper grid. The grids were dried for a few minutes under a Xenon lamp. High-resolution transmission electron microscope (HR-TEM) images were captured using an FEI Tecnai G<sup>2</sup> 20 field-emission gun (FEG) HRTEM (Hillsboro, OR, USA) operated in bright field mode at an accelerating voltage of 200 kV.

#### *2.3. Development of the RBP-A-Aptasensor*

#### 2.3.1. Optimization of the Aptamer and NaCl Concentration

AuNPs are highly reactive and aggregate easily in the presence of salts; therefore, it is important to monitor the salt-induced AuNPs aggregation percentage for the development of a colorimetric aptasensor [44–46]. To optimize the performance of the developed assay, various conditions such as aptamer and NaCl concentrations were investigated. For this experiment, a total of 600 μL of different concentrations (0, 6.25, 12.5, 25, 50, 100 and 150 nM) of RBP-A was added to 1.08 mL of AuNPs in separate 2 mL Eppendorf tubes, mixed well, and incubated overnight at 25 ◦C. Then, 50 μL of the functionalized RBP-A-AuNPs (aptasensor) was added to a 96-well microtiter plate, containing 50 μL of dH2O. This was followed by the addition of different concentrations of NaCl (0, 20, 40, 60, 80 and 100 mM). The samples were incubated at 25 ◦C for 5 min and observed for colour change. The UV–vis spectra were measured using a plate reader, and the absorbance ratios (A650/A520) were calculated to determine the aggregation percentage of the AuNPs [47]. The experiment was performed in triplicate and the average absorbance ratios were calculated and used to plot bar graphs.

#### 2.3.2. RBP4 Detection Based on the Label-Free AuNPs-Based RBP-A-Aptasensor

For the detection of RBP4, 600 μL of 50 nM RBP-A and 1.08 mL of AuNPs were added to a 2 mL tube, mixed well, and incubated overnight at 25 ◦C. Subsequently, 50 μL of the aptasensor was added into the 96-well microtiter plate containing 25 μL of dH2O. Then, 25 μL of different concentrations of RBP4 (0, 7.8, 15.6, 31.2, 62.5, 125, 250 nM) were added into the aptasensor, mixed thoroughly, and incubated for another 5 min at 25 ◦C, and observed for colour change This was followed by the addition of 6 μL of 1 M NaCl solution (60 mM final concentration) and incubation at 25 ◦C for 5 min. The UV–vis spectra were measured using a plate reader and the absorbance ratios (A650/A520) were calculated to determine the aggregation percentage of the AuNPs. The experiment was performed in triplicate and the average absorbance ratios were calculated and used to plot the linear curve.

#### 2.3.3. Specificity of the Label-Free AuNPs-Based RBP-A-Aptasensor

To evaluate the specificity of the RBP-A-aptasensor, 50 μL of the aptasensor was added into the 96-well microtiter plate, containing 25 μL of dH2O. Then, 25 μL of 250 nM RBP4, A2MG, leptin and BSA were added into the aptasensor, mixed thoroughly, and incubated for another 5 min at 25 ◦C and observed for colour change. This was followed by the addition of 6 μL of 1 M NaCl solution (60 mM final concentration) and incubation at 25 ◦C for 5 min. A water sample was used as a blank. The UV–vis spectra were measured using a plate reader and the absorbance ratios (A650/A520) were calculated to determine the aggregation percentage of the AuNPs. The experiment was performed in triplicate and the average absorbance ratios were calculated and used to plot bar graphs.
