An oligosorbent-based aptamer affinity column for selective extraction of aflatoxin B2 prior to HPLC with fluorometric detection

Abstract

The article describes an aptamer affinity column for selective solid-phase extraction of aflatoxin B (AFB). Amino-modified aptamer against AFB was immobilized on CNBr-activated Sepharose through a covalent bond. The effects of oligosorbents based on 3'- or 5'-amino-modified sequences with a C6 or a C7 spacer arm were evaluated by UV spectroscopy at 260 nm. The extraction recovery was evaluated by HPLC with fluorometric detection. The extraction of AFB was optimized. Under the optimum conditions, the aptamer affinity column has a linear response to AFB in the range of 0.5-80 ng, with a capacity of 84.6 ng. Control supports without immobilized aptamers and a nonspecific oligosorbent immobilized with a negative control oligonucleotide were studied in order to demonstrate selectivity. The method was tested with spiked peanut sample (0.5-50 μg·kg AFB) and gave average recoveries of 80.9% and a mean relative standard deviation of 1.9%. The limit of detection is 25 pg·mL. This is much lower than the maximum residue limits suggested by the European Union. The columns can be re-used up to five times without any loss of performance. The oligosorbent was also applied to clean-up of AFB from peanut sample extracts before HPLC analysis. Results were further confirmed by ultra-fast liquid chromatography with tandem mass spectrometry. Conceivably, the method may also be applied to other samples, such as food, agricultural products, and traditional Chinese medicines. Graphical abstract Schematic illustration of the fabrication procedures of aptamer affinity column, AAC (a), its principle of aptamer bound to aflatoxin B (b) and the obtained AAC

Aptamer-based and immunosorbents

“…Values of capacity found in the literature for target molecule on OSs available as SPE cartridges are quite homogeneous and are in the range 0.26 to 69 nmoL/g of OS (4). Such capacity values are associated to binding density values in the range 19 to 37% on Sepharose beads, a binding density of active aptamers of 68% being recently reported (34). Those capacity values for OSs are very close to those obtained with immunosorbents (from 4 to 93.6 nmoL/g of sorbent, e.g., immunosorbents specific to pesticides) (94).…”

“…By plotting the amount of target analyte retained by the sorbent (i.e. recovered in the elution fraction) as a function of its concentration in the percolated sample, a curve is obtained that is characterized by two different parts (17,34,36,37,40). First, for the lowest percolated amounts/concentration levels, a linear part is obtained and corresponds to a range of concentrations for which a constant recovery yield of extraction is obtained, this recovery yield being given by the slope of this linear part.…”

“…mainly hydrophobic and/or electrostatic interactions favored in aqueous media. The comparison of the retention of the target analyte on the IS/OS with the nongrafted solid-support, named control sorbent (17,21,34,36,37,(40)(41)(42)(44)(45)(46)(47)70,71,(73)(74)(75)(76)84), or grafted with another antibody/oligonucleotide sequence, having no affinity for the target (18,21,25,34,36,37,42,47,48,54,55,70,84), is now often reported to evaluate the contribution of these NSIs. The use of these sorbents in parallel to IS/OS is particularly helpful for optimizing the washing step and then obtaining an optimal selectivity (48,57).…”

Immunoaffinity Extraction and Alternative Approaches for the Analysis of Toxins in Environmental, Food or Biological Matrices

“…In another study, the use of a C7 spacer arm gave better results than the C6 spacer arm in terms of extraction efficiency of aflatoxins. Both results were explained by higher aptamer grafting yields with the longer spacer arm [ 173 ].…”

An Automated and High-Throughput Immunoaffinity Magnetic Bead-Based Sample Clean-Up Platform for the Determination of Aflatoxins in Grains and Oils Using UPLC-FLD

“…Sample clean-up is a vital step in AFTs analysis method, which can significantly affect the accuracy and precision of results, but is also the most time-consuming and error-prone step. Widely used clean-up methods in AFT analysis include solid-phase extraction (SPE) [4,5], dispersive liquid–liquid microextraction [6,7], QuEChERS [8,9,10], and diluting crude extract [11,12,13,14,15]. Immunoaffinity columns (IAC) exhibit many advantages against these methods, including high specificity, selectivity, and stability, which is why they are the most popular method for clean-up mycotoxin contaminants in foodstuffs [16].…”