Comparison of the Performances of Two Aptamers on a Colorimetric Assay for the Quantification of Fumonisin B1

Abstract

Fumonisin B1 (FB1), a mycotoxin commonly produced by Fusarium verticillioides and classified as a group 2B hazard, has been identified in various food products; hence, sensitive and rapid analytical detection methods are needed. Since the first reported aptamer (96 nt ssDNA) for the highly specific molecular recognition of FB1, only 30 aptamer-based biosensors have been published. A critical point, yet commonly overlooked during the design of aptasensors, is the selection of the binding buffer. In this work, a colorimetric assay was designed by incubating a folded aptamer with FB1 and the subsequent addition of gold nanoparticles (AuNPs). The changes in the aggregation profile of AuNPs by a 40 nt aptamer and a 96 nt aptamer were tested after the addition of FB1 under different buffer conditions, where the incubation with Tris-HCl and MgCl2 exhibited the most favorable performances. The assay with the longest aptamer was specific to FB1 and comparable to other aptasensors with a limit of detection (LOD) of 3 ng/mL (A650/520 ratio). Additionally, the application of asymmetric-flow field-flow fractionation (AF4) with multidetection allowed for the analysis of the peak area (λ) and multi-angle light scattering (MALS) with LODs of up to the fg/mL level.

Aptamer-Based Detection of Fumonisin B1: A Systematic Comparison with Conventional and Other Novel Methods

“…Hence, as indicated in Figure 5, LC-MS [56,75], immunoassays with optical [99,107,135], Raman (due to its quick procedure) [140], fluorescent readouts [147] and electrochemical MIPs [191] were correlated to the combination of low LODs with short assay times. However, such statistical analysis did not show the advantages of aptamer-based methods, which was also observed on the correlation of short assay preparation times with LC-MS, immunologic and only three aptasensors [159,167,200]. This was shown by PCA, where the main drawbacks from aptamer-based sensors for FB1 was their long assay and assay preparation times denoted by is absence of correlation in both components when compared to other methods.…”

“…Nevertheless, some sensors qualified as fast required overnight steps and long incubation times for the whole system arrangement, especially when the synthesis of nanoparticles and drying phases were required. Assay times below ten minutes were achieved through chromatographic, immunoassays, and some innovative methods, nonetheless the more sensitive assays were secured with aptamer-based biosensors [170,177,200], immunosensors with carbon nanotubes [101,109], and molecularly imprinted polymer nanoparticles (MIPs) [191], as indicated in Figures 4a and 4c.…”

“…This indirect electrochemical analysis was translated into a colorimetric signal by means of the smartphone detection of deposited PB at the ITO electrode, submerged in the reporting solution along with the Pt wire [199]. In contrast to previous reports, the sole application of the unmodified sequence (96 nt) was reported by our research group in an aptamer-FB1-AuNPs conjugate, stable to salt-induced aggregation at an increasing target concentration, under the presence of MgCl2 [200].…”

Aptamer-Based Detection of Fumonisin B1: A Systematic Comparison with Conventional and Other Novel Methods

“…Nevertheless, some sensors qualified as fast required overnight steps and long incubation times for the whole system arrangement, especially when the synthesis of nanoparticles and drying phases were required. Assay times below ten minutes were achieved through chromatographic, immunoassays, and some innovative methods, nonetheless the more sensitive assays were secured with aptamer-based biosensors [170,177,200], immunosensors with carbon nanotubes [101,109], and molecularly imprinted polymer nanoparticles (MIPs) [191], as indicated in Figures 4a and 4c.…”

“…On the other hand, the shortest assay times were correlated to applications with the 96 nt aptamer in its end-modified [157,160,163,168] and hybridized forms [158,165,173,174], as well as electrochemical designs with some shorter sequences including a thiol modified 80 nt aptamer [156] and an unmodified 40 nt [164] sequence. Likewise, the assay preparation time showed high correlation to 60 nt fluorescent [159], 40 nt electrochemical [167], and 96 nt colorimetric [200] aptasensors. Nevertheless, as already stated, the high correlation of the 96 nt aptamer with a high sensitivity (low LODs) in combination with its convenient specificity, were relevant for the existence of more biosensors based on this long length sequence.…”