*3.2. Principles of AuNPs-Based RBP-A-Aptasensor*

The principles of the AuNPs-based RBP-A-aptasensor for the detection of RBP4 is illustrated in Figure 2. Colloidal AuNPs produce a ruby-red colour in solution that is highly dependent on the interparticle distance [32,34,35]. In the presence of NaCl, the AuNPs undergo aggregation, leading to a visible colour change from ruby red to purple/blue. The presence of salt electrolytes decreased the interparticle distance of the AuNPs, thus reducing the ζ- potential and enabling electromagnetic interaction among the negatively charged AuNPs consequently leading to visible colour change [46]. In contrast, binding of RBP-A on the surface of AuNPs stabilized the AuNPs and prevented the NaCl-induced aggregation of the AuNPs [54,55]. The solution retained the ruby-red colour even at high NaCl concentrations (up to 60 mM). However, in the presence of RBP4, owing to the high affinity between RBP4 and RBP-A, RBP-A detached from the AuNPs and left the AuNPs

unprotected. The bare AuNPs aggregated in the presence of NaCl, which led to a visible colour change from ruby red to purple/blue.

**Figure 1.** Characterization of AuNPs. (**a**) UV–vis spectrum of AuNPs; (**b**) a representative HR-TEM photomicrograph and (**c**) size distribution.

**Figure 2.** Schematic illustration of the colorimetric aptasensor for the detection of RBP4.

The compatibility of the AuNPs-based RBP-A-aptasensor for the detection of RBP4 was validated using four techniques: UV–vis spectroscopy, TEM, ζ- potential, and FTIR. As shown in Figure 3a, the AuNPs had an SPR peak at 519 nm which shifted to 560 nm upon the addition of NaCl, indicating aggregation. In the presence of RBP-A, the AuNPs were strongly protected against NaCl-induced aggregation and the SPR peak remained inert at 519 nm. Upon the addition of NaCl, the SPR peak shifted to 600 nm as a result of aggregation. This could be because, in the presence of RBP4, RBP-A detached from the AuNPs' surface and bound to RBP4, leaving the AuNPs' surface unprotected against saltinduced aggregation. HR-TEM was used to confirm these observations (Figure 3b). Bare AuNPs were monodispersed in the solution; however, in the presence of NaCl, the AuNPs aggregated and clumped together. The aptasensor without RBP4 showed no aggregation in the presence of NaCl, but in the presence of RBP4 and NaCl, the AuNPs aggregated.

**Figure 3.** (**a**) UV–vis spectra, (**b**) HR-TEM of (i) AuNPs, (ii) AuNPs + NaCl, (iii) AuNPs + RBP-A + NaCl and (iv) AuNPs + RBP-A + RBP4 + NaCl, (**c**) ζ- potential and (**d**) FTIR analysis of the interaction between the RBP-A-aptasensor and RBP4. RBP4 was used at a concentration of 250 nM.

The ζ- potential was also measured to monitor the changes on the AuNPs' surface during the development of the aptasensor and when the aptasensor was incubated with RBP4 (Figure 3c). AuNPs exhibited a ζ- potential of −27.7 mV, which is in agreement with previously published data [56]. In contrast, the ζ- potential of −20.5 mV was obtained when RBP-A was incubated with the AuNPs solution, indicating that negative charges are partially neutralized by the aptamer covering the surface of the particles [47]. Finally, when RBP4 was added, the ζ- potential was restored to −29.6 mV, indicating that the RBP-A bound to RBP4 and left the AuNPs naked, thus returning them to their original ζpotential. Using the same technique, Lerga et al. developed a AuNPs-based aptasensor for the detection of histamine. In their study, AuNPs exhibited a ζ- potential of − 47.6 mV which decreased to − 28.6 mV upon incubation of the aptamer with the AuNPs. The ζpotential was restored to approximately − 50 mV after the addition of histamine [47].

Finally, FTIR (PerkinElmer Spectrum One FTIR spectrophotometer) analysis was carried out to monitor elemental changes to the AuNPs during the development of the aptasensor and when the aptasensor was incubated with RBP4 (Figure 3d). The FTIR spectrum of AuNPs features characteristic peaks at 3305.81, 2082.74, 1638.55, 1400.83, 1277.32 and 686.42 cm−1. The peak at 3305.81 cm−<sup>1</sup> is attributed to the stretching vibration of OH and NH2. The peaks at 1400.82 cm−<sup>1</sup> and 1638.55 cm−<sup>1</sup> are attributed to the carboxylate symmetric and asymmetric stretching bonds of the carboxylate group (COO−) in citrate ions [57], respectively. The peak at 2083.74 cm−<sup>1</sup> is attributed to the S-H group on the AuNPs [58], further validating successful synthesis of the AuNPs. Upon adsorption of the RBP-A, two weak peaks (1064.23 and 723.88 cm−1) were observed in RBP-A-AuNPs conjugate spectra which were absent in the spectrum for the AuNPs. The peak at 1064.23 cm−<sup>1</sup> is attributed to the symmetric C=O stretching band of the phosphate backbone and the peak at 723.88 cm−<sup>1</sup> is attributed to the C–H out of plane base vibration of the RBP-A [59], indicating successful adsorption of RBP-A on the surface of the AuNPs. Finally, when RBP4 is added, the peak attributed to the carboxylate asymmetric stretching band appeared at 1460.05 cm<sup>−</sup>1. This peak was also visible in the AuNPs, further supporting the assertion that RBP-A was bound to RBP4 and detached from the AuNPs.

#### *3.3. Determination of the Optimum Concentration of NaCl and Aptamer*

The flocculation assay was used to determine the stability of the AuNPs-based RBP-A-aptasensor in the presence of different concentrations of NaCl. From Figure 4, it can be deduced that AuNPs and the RBP-A-AuNPs conjugates at all RBP-A concentrations showed no aggregation in the absence of NaCl. The AuNPs and the RBP-A-AuNPs conjugates at all RBP-A concentrations showed minimal aggregation below 20 mM NaCl; whereas, some aggregation was observed at 40 mM NaCl. The minimal changes of the absorbance at low NaCl concentrations were due to the low ionic strength of the electrolyte. At these NaCl concentrations, the aggregation of AuNPs is slow and the mean size of the formed aggregate is close to the size of the original AuNPs, rendering the changes insignificant. However, the AuNPs and the RBP-A-AuNPs conjugate solutions at lower RBP-A concentrations (6.25–12.5 nM) changed colour from ruby red to purple/blue with increasing concentrations of NaCl (60–100 mM), with the AuNPs reaching saturation levels between 60 and 100 mM NaCl. This observation suggested that the Na+ and Cl<sup>−</sup> ions destroyed the ionic environment and led to the aggregation of AuNPs in the absence of aptamers [60]. In contrast, higher concentrations of RBP-A (25–150 nM) showed excellent protection efficiency against NaCl-induced aggregation of AuNPs, which was indicated by the retention of a ruby-red colour and a lower aggregation percentage. Overall, the results indicated that the addition of RBP-A caused a dose-dependent protection against NaCl-induced aggregation for all concentrations of NaCl. To ensure the assay is more cost effective, the optimal concentrations of RBP-A and NaCl for the development of the aptasensor were selected as 50 nM and 60 mM, respectively.

**Figure 4.** Stability of the AuNPs and RBP-A-aptasensor in the presence of increasing concentrations of NaCl. Final aptamer concentrations: 0, 6.25, 12.5, 25, 50, 100 and 150 nM; final NaCl concentrations: 0, 20, 40, 60, 80 and 100 mM.

#### *3.4. Sensitivity of the Aptasensor for RBP4 Detection*

The sensitivity of the AuNPs-based RBP-A-aptasensor for the detection of RBP4, under the optimized experimental conditions (50 nM RBP-A and 60 mM NaCl), was assessed by incubation with different concentrations of RBP4 and by calculating the absorbance ratios (A650/A520) to evaluate the aggregation of the AuNPs. Figure 5a and the insert indicate that the addition of NaCl caused a dose-dependent aggregation of the AuNPs, which is indicated by the gradual colour change of the AuNPs from ruby red to purple/blue and the shift in SPR from 519 to 600 nm. The results demonstrate that RBP-A bound to RBP4; thus, detaching from the AuNPs and leaving the AuNPs unprotected. In the presence of 60 mM NaCl, the AuNPs aggregated in a dose-dependent manner, with a more visible colour change between 62.5 and 250 nM. The assay was sensitive with a limit of detection (LOD) of 90.76 ± 2.81 nM. Moreover, the assay was rapid, and the test results were visually detectable within 5 min. The LOD was calculated as described by the International Union of Pure and Applied Chemistry (IUPAC) [44] as follows:

$$\text{LOD} = 3 \times \text{SD/S}$$

where SD is the standard deviation of the response (y-intercept of the regression line) and S is the slope of the calibration curve.
