**4. Conclusions**

This work presents a comparison on the performances of two aptamers for the colorimetric quantification of FB1. The results indicated that, along with the aptamer sequence, the selected buffer and incubation conditions play an important role in the final sensitivity and specificity of each assay. In this regard, incubation with Tris-HCl and MgCl2 was suitable for the 40 and 96-mer aptamers, respectively. Contrary to previous reports [31], the assay with a short length aptamer (40 nt) was not specific for FB1, as similar results were observed through the incubation with OTA. A different mechanism has been proposed for the long aptamer (96 nt), previously reported for several aptamer-based approaches. In this case, an aptamer–FB1–AuNP conjugate was formulated in the presence of MgCl2 1 mM, showing stability to salt-aggregation at an increasing concentration of FB1(0.001–10 μg/mL). Unlike other aptasensors, the 96 nt aptamer offered a simplified approach as an unmodified ssDNA sequence was applied without the need of end modifications or complementary strands. Analysis of the spectrophotometric signals resulted in LODs similar to other sensitive techniques; however, the exploration of the aggregation profile by AF4 with multidetection (UV/VIS, MALS, DLS) derived in a promising sensing technique with sensitivity in the fg/mL level. The characterization of the complex formation revealed the absence of DNA conformational change upon binding, ye<sup>t</sup> this new mechanism might be suitable for the direct analysis of different food matrices, where there is scope for exploring other targets, such as emerging mycotoxins. To our knowledge, this is the first aptasensing technique for FB1 applying the 96 nt aptamer sequence without any end modification, label or complementary strand. Likewise, there is no evidence for the use of AF4 in the exploration of aptamer–target–AuNPs interactions.

Further validation and standardization steps are still required for the commercial application and possible scaling to paper-based techniques, which might enhance the opportunities for on-site quantifications, while decreasing the total manufacturing cost. Nevertheless, this work established a new mechanism for detecting FB1 with a 96 nt aptamer in bulk, while at the same time presents for the first time the use of a more robust method, as it is AF4, resulting in LODs with strong advantages over more complex designs.

**Supplementary Materials:** The following is available online at https://www.mdpi.com/article/10 .3390/bios11010018/s1.

**Author Contributions:** Conceptualization, V.A.M.-M.; Y.G.; Y.Y.G. and F.M.G.; methodology, V.A.M.- M.; Y.G.-E. and M.C.-G.; software, V.A.M.-M.; validation, V.A.M.-M.; formal analysis, V.A.M.-M.; investigation, V.A.M.-M.; resources, Y.G., Y.Y.G. and F.M.G.; data curation, V.A.M.-M.; writing— original draft preparation, V.A.M.-M.; writing—review and editing, V.A.M.-M., Y.G.-E., M.C.-G. and F.M.G.; visualization, V.A.M.-M.; supervision, Y.Y.G. and F.M.G.; project administration, V.A.M.-M. and F.M.G.; funding acquisition, V.A.M.-M. and F.M.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** V.A.M.-M. was awarded with a scholarship from CONACYT (Mexico) for completing his PhD studies. M.C.G. acknowledges a fellowship for postdoctoral training (20381/PD/17) funded by the Consejería de Empleo, Universidades y Empresa de la CARM, through the Fundación Séneca de la Región de Murcia.

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

**Informed Consent Statement:** Not applicable. **Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to its classification as intellectual property by the University of Leeds.

**Acknowledgments:** Help from G. Nasir Khan for the CD spectroscopy measurements at the Astbury Centre for Structural Molecular Biology, University of Leeds, is gratefully acknowledged. We are also indebted to B. Sabagh from Postnova Analytics UK for his kind advice in the development of the AF4 method.

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