*3.7. Comparison of the Aptasensor with Other Aptamer-Based Approaches for RBP4 Detection*

Since the identification of RBP-A in 2008 by Lee et al. [36], there have been only three studies published detailing aptamer-based assays for the detection of RBP4. A comparison of the results of these assays is provided in Table 2. The LODs for ELAAS, SPR and chemiluminescence were 3.39 nM, 75 nM, and 0.04 pM, respectively [36,53,54]. In contrast, the aptasensor developed in this study had a limit of detection of 90.76 nM. This indicated that the developed aptasensor was less sensitive when compared to the aptamer-based SPR, ELAAS and chemiluminescence sensors. Nevertheless, the results in this study were still within the normal detection range. More importantly, the results for this assay were obtained within 5 min, whereas the results for the other assays were obtained after approximately 2 h, indicating that the developed aptasensor was more rapid. Additionally, confirmation of the test results is based on visual detection of colour changes without the need for any advanced instruments, a laboratory or a power source. This further indicates that the aptasensor is suitable for PoCT.

**Figure 6.** Specificity of the RBA-A-aptasensor for RBP4 detection. The specificity experiment was performed at a concentration of 250 nM for all proteins. The blank sample contained water.


**Table 2.** Comparison of the RBP-A-aptasensor with other methods used for RBP4 detection.

The incubation time of the aptasensor with RBP4 was also evaluated. This was carried out by incubating RBP4 (at a concentration of 250 nM) with the aptasensor for different time periods (0–30 min), followed by the UV–vis measurements and calculation of the absorbance ratios (A620/A520) to determine the degree of aggregation of the AuNPs (Figure 7). The results indicated that the aptasensor had lower aggregation in the presence of NaCl, as indicated by the lower absorbance ratio. When the aptasensor was incubated with RBP4 for 5 to 30 min, followed by the addition of NaCl, the aggregation increased and reached saturation levels between 5 and 30 min, indicating that 5 min incubation of the aptasensor and RBP4 was sufficient for binding. The interaction of RBP-A with RBP4 was further analyzed using the MicroScale Thermophoresis (MST) technique (Monolith NT.115, Nanotemper). As shown in Table 3, MST revealed that RBP-A bound with a dissociation constant (Kd) of 3.523 nM within 1.5 s and continued until it reached a Kd of 13.438 nM within 20 s. This is attributed to the high affinity of the aptamer for the target thus allowing rapid recognition. One of the major advantages of PoCT is that it provides much faster access to test results, allowing for more rapid clinical decision-making and more appropriate treatments and interventions. This means that standard RBP4 detection assays such as ELISAs, which require longer incubation periods, may be replaced by the aptasensor at the PoC, allowing the process from sample collection to data analysis to happen within 10 min.

**Figure 7.** Aggregation of the RBP-A-aptasensor in the presence of 60 mM NaCl at various incubation periods.

**Table 3.** MST analysis of the interaction of RBP-A with RBP4.


#### **4. Conclusions**

In this study, a label-free colorimetric aptasensor for the detection of RBP4 using ssDNA aptamer and unmodified AuNPs was successfully developed. The assay is based on colour change from ruby red to blue/purple due to the binding of RBP-A to RBP4, which leaves AuNPs exposed to the phenomenon of salt-induced AuNP aggregation. The assay was sensitive, with an LOD of 90.76 ± 2.81 nM. Further development using clinical samples on non-diabetic and diabetic patients is required to assess the utility of the aptasensor for the early detection of T2DM.

**Author Contributions:** K.L.M., T.M.L. and M.J.-R.—data collection, data analysis, and validation; K.L.M. and N.R.S.S.—writing of manuscript; C.K.O., M.M. and A.M.M.—conceptualisation and student supervision; M.M. and A.M.M.—funding acquisition, resources and project administration. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors acknowledge the NRF Equipment Related Travel Grant (Ref. ERT180801351351) for funding K.L.M.'s travelling expenses to Spain (Universität Rovira I Virgili, Tarragona).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data generated in this study has been represented as Tables and Figures in the manuscript and available from the corresponding author upon request.

**Acknowledgments:** The authors acknowledge DSI/Mintek NIC and the NRF Masters Scholarship for the financial support. The authors also acknowledge the Electron Microscope Unit at the University of Cape Town for assistance with HR-TEM analysis, and the Drug Delivery Research Proto-Unit (Department of Pharmaceutical Sciences, University of KwaZulu-Natal) and Riziki Martin (Biolabels Research Node) for assistance with MST analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.
