Magnetic Separation-Based Multiple SELEX for Effectively Selecting Aptamers against Saxitoxin, Domoic Acid, and Tetrodotoxin

Abstract

In this study, a novel magnetic separation-based multiple systematic evolution of ligands by exponential enrichment (SELEX) was applied to select aptamers simultaneously against three kinds of marine biotoxins, including domoic acid (DA), saxitoxin (STX), and tetrodotoxin (TTX). Magnetic reduced graphene oxide (MRGO) was prepared to adsorb unbound ssDNAs and simplify the separation step. In the multiple SELEX, after the initial twelve rounds of selection against mixed targets and the subsequent four respective rounds of selection against each single target, the three resulting ssDNA pools were cloned, sequenced, and analyzed. Several aptamer candidates were selected and subjected to the binding affinity and specificity test. Finally, DA-06 ( K = 62.07 ± 19.97 nM), TTX-07 ( K = 44.12 ± 15.38 nM), and STX-41 ( K = 61.44 ± 23.18 nM) showed high affinity and good specificity for DA, TTX, and STX, respectively. They were also applied to detect and quantify DA, TTX, and STX successfully. The other two multitarget aptamers, DA-01 and TTX-27, were also obtained, which can bind with either DA or TTX. These aptamers provide alternative recognition molecules to antibodies for biosensor applications.

Generation of ssDNA aptamers as diagnostic tool for Newcastle avian virus

“…One of the described protocols, based on high salt concentration for elution of the binders, and limited number of amplifications of the aptamers that bind to the target on the dot blot was used in this study. This protocol that was first reported by Arnold et al [ 17 ] and later was successfully used in our previous work with some modification [ 15 ], allowed generation of aptamers using very few selection rounds in comparison to other protocols, which often require up to 10 rounds of selection [ 26 , 27 ].…”

Recent Advances in Micro/Nanomaterial-Based Aptamer Selection Strategies

“…MBs are among the representative beads used in SELEX that enable simple and rapid partitioning of target-bound and unbound aptamers through the use of a magnet [51,58,62,81,82]. The quenching of MBs against fluorescence [83,84] can also be used for aptamers.…”

“…The surface of GOx also strongly absorbs free ssDNA through hydrophobic and π–π stacking interactions between the nucleobases of the ssDNA and the GOx [ 46 , 47 , 48 , 49 ]; therefore, upon the exposure to target molecules, the unbound aptamers bind to GOx, while the target-bound aptamers remain in the solution. After centrifugation, ssDNA-bound GOx can be removed, and target-bound aptamers can be recovered [ 50 , 51 ].…”

Magnetic Beads in Marine Toxin Detection: A Review

“…Changes in the optical thickness and mass density of biosensor layer were measured, resulting in an LOD of 0.04 ng/L. Gu and collaborators [40] developed a magnetic separation-based multiple SELEX to simultaneously select aptamers against three different marine biotoxins: DA, STX and TTX. The first 12 rounds entailed mixed screening against the three toxins, and the subsequent four rounds of single screening were against each individual toxin.…”