**1. Introduction**

Exposure to FB1 occurs not only in African [1] and Latin-American [2] countries but also in several regions of Asia [3] and Europe [4]. For that reason, monitoring and controlling contamination of food commodities with fumonisins becomes a highly pressing matter for protecting human health worldwide. Fumosinins are a group of toxins generated by diverse fungi including *Fusarium verticillioides* [5], *Alternaria alternata* [6], *Aspergillus niger* [7], *Tolypocladium cylindrosporum*, *Tolypocladium geodes* and *Tolypocladium inflatum* [8]. Their chemical structure commonly consists of alkylamines, whose substitution of up to seven side chains allows the formation of different analogs, group B being the most common in nature [9]. From the latter, Fumonisin B1(C34H59NO15) has been reported to be a latent risk in several food products, such as cereals and beverages.

Classified as group 2B hazard, FB1 (Figure S1) has a possible carcinogenic effect on human health [10], whose toxicity has been related to the disruption of sphingolipid metabolism, oxidative stress and epigenetic changes [11], along with the interruption of barrier functions [12]. Nevertheless, current conventional methods for mycotoxin detection, including enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography-mass spectrometry (LC-MS), are costly, time consuming, are difficult to be applied on site and

**Citation:** Mirón-Mérida, V.A.; González-Espinosa, Y.; Collado-González, M.; Gong, Y.Y.; Guo, Y.; Goycoolea, F.M. Aptamer–Target–Gold Nanoparticle Conjugates for the Quantification of Fumonisin B1. *Biosensors* **2021**, *11*, 18.https://doi.org/10.3390/bios 11010018

Received: 23 December 2020 Accepted: 5 January 2021 Published: 8 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

require experienced users [13]. Therefore, there is a need for developing sensitive, ye<sup>t</sup> quick and affordable methods for quantifying mycotoxins.

Biosensors are a suitable alternative to conventional methods by means of their general mechanism, where any target detection is carried out by a biological receptor and transduced into a signal (optical, electrochemical, mechanical, etc.). From the different biorecognition receptors (enzymes, antibodies, nuclei acids, cells, etc.), the performance of aptamers has been exceptional [14]. Aptamers are single-stranded DNA or RNA molecules with high molecular recognition toward different types of molecules, distinct binding affinities, target selectivity and high capability to discriminate slight chiral differences [15]. Their selection technique called Systematic Evolution of Ligands by Exponential enrichment (SELEX), involves incubating a DNA library with the specific target or other relevant molecules, followed by the amplification of potential binders after several selection and discrimination rounds [16]. In addition to their good performance, compared to antibodies, aptamers are easy to modify, as well as reproducible by solid-phase chemical synthesis, which represents a reduction in cost and time. To date, two aptamers specific for FB1 composed of 96 and 80 nucleotides (nt) have been selected by SELEX and used in up to 31 different biosensing approaches [15,16], through aptamer modifications, hybridization with complementary strands or reduction in the sequence length (Figure S2). Those applications have involved optical, chemiluminescence, electrochemical, surface-enhanced Raman spectroscopy (SERS), mass spectrometry (MS) and bending related signals, where the most sensitive methods were correlated with fluorescent and SERS read outs (Figure S2).

A decisive step during the design of aptamer-based biosensors is the selection of the target-aptamer incubation conditions (buffer, time and temperature). In such approaches, Tris, Tris-HCl, phosphate buffer, NaCl, CaCl2, KCl and MgCl2 are normally added during the binding stage, which normally results in a 3D conformational change of aptamers upon binding [16]. Apart from the sensitive response obtained through aptamer dehybridization from complementary sequences at specific binding sites, mycotoxin detection has been carried out by immobilization of aptamers onto the surface of different platforms such as graphene and gold nanoparticles. In this regard, gold nanoparticles (AuNPs) are suitable for developing colorimetric methods, which are still relevant due to its feasible application for on-site analysis and reduction in engineering costs. The thermodynamic instability of colloidal gold suspensions potentiates their flocculation. This aggregation, however, can be prevented when gold nanoparticles are coated with negatively charged citrate molecules, or at low ionic strength and pH above the isoelectric point of citrate. Particle aggregation in this case occurs when an aqueous dispersant medium containing the nanoparticles has a high enough ionic strength to screen their electrostatic repulsion charges caused by their citrate stabilization (e.g., salt-induced aggregation) [17]. AuNPs are commonly analyzed in terms of their absorbance by using surface plasmon resonance related to their color modification (red to blue) by aggregation with cationic compounds, changes in the suspension pH and ionic strength [18,19]. The SPR spectra of AuNPs are closely related to their particle size, which plays an important role in their absorption, scattering and excitation behavior [20]. Functionalization of AuNPs can be promoted through aptamer (ssDNA) uncoiling, whose bases are exposed to the negative surface of AuNPs, thus enabling their interaction by van der Waals forces [21].

In addition to the spectrophotometric analysis of AuNPs, particle size characterization can be achieved by more robust methods capable of probing the interaction between aptamers and AuNPs in the presence of a target molecule. For instance, asymmetric flow field-flow fractionation (AF4) is a technique that allows the separation of particles and aggregates in a size range from 1 nm to 1 μm, based on their diffusion coefficient in aqueous media. This analytical method offers several advantages, such as minimal sample alteration and efficient quantitative analysis of the physico-chemical parameters of the study materials such as their concentration and particle size by multidetection (UV/VIS, fluorescence, multiangle light scattering (MALS) and differential refractive index (dRI)) [22]. The general AF4 operation principle is based on the generation of a parabolic laminar flow profile within the separation channel transporting the particles. The action of a perpendicular flow, known as cross flow (CF) across the semipermeable membrane, drives separation according to the diffusion coefficient of particles. Some AF4 methods have been developed for studying the properties of AuNPs as either standard samples [22] or additives [23], as well as to study the target-binding relation between aptamers and their specific targeted proteins [24].

In this paper, we developed a bulk colorimetric method to examine the efficiency of two aptamers, namely, the first 96 nt sequence and a shortened version (40 nt) from the 80 nt aptamer, to quantify FB1 in different binding buffers and assessed its potential as a robust biosensing method. The general procedure involved incubating FB1 with aptamers, followed by another incubation stage with AuNPs, addition of NaCl and subsequent analysis by UV/VIS spectroscopy (λ = 400–800 nm). In addition, the characterization of the formation of Aptamer (96 nt)–FB1–AuNP conjugates was carried out by multidetection AF4, thus uncovering a promising application on the sensitive detection of this mycotoxin.

#### **2. Materials and Methods**
