Preparation of a Specific ssDNA Aptamer for Brevetoxin-2 Using SELEX

Abstract

The existing assays for detecting brevetoxin (BTX) depend on expensive equipment with a professional operator or on an antibody with limited stability, which requires complex processes, a high cost, and a considerable amount of time. The development of an alternative detection probe is another promising research direction. This paper reports the use of aptamers binding to BTX-2 in an analytical assay using the systematic evolution of ligands by exponential enrichment (SELEX). After 12 rounds of selection, the secondary structures of 25 sequences were predicted. Compared to other aptamers, Bap5 has relatively high affinity with the lowest dissociation constant of 4.83 μM, and IC50 is 73.81 ng mL−1. A good linear regression formula of y = 30.688x − 7.329 with a coefficient correlation of R 2 = 0.9798 was obtained using a biotin-avidin ELISA. Moreover, there is no cross-reaction with the detected marine toxins, except for BTX-2. Thus, Bap5 has potential to detect BTX-2 in shellfish in the future as a substitute for the recognition probe.

Aptamer-Based Biosensors to Detect Aquatic Phycotoxins and Cyanotoxins

“…Consumption of these organisms by humans causes Neurotoxic Shellfish Poisoning (NSP). BTX-2 is the most common toxin responsible for NSP and only BTX has aptamers developed against it [ 37 , 66 ]. One aptasensor was developed upon one of these aptamers [ 37 ].…”

“…Spiked shellfish extracts were analyzed using this aptasensor, showing non-significant interference from the matrix on the aptasensor response. In this study, the secondary structures of most of the obtained aptamers were typical stem-loop and hairpin-loop structures [ 66 ]. The aptamer selected with the highest affinity has a K d of 4.83 μM, a LOD of 3.125 μg/L, and a LRD over the range of 3.125–200 μg/L.…”

Aptamers and Aptasensors for Highly Specific Recognition and Sensitive Detection of Marine Biotoxins: Recent Advances and Perspectives

“… Thirdly , some new selection methods promote efficient aptamer selection for marine biotoxins. In 2016, Tian et al [ 64 ] completed a selection of aptamers binding to BTX-2 based on microwell-SELEX. The positive selection process is illustrated in Figure 3 .…”

Recent Microdevice-Based Aptamer Sensors

“…The binding affinity of an aptamer to detect brevetoxins and gonyautoxin-1/4 has been tested. The lowest dissociation constants for brevetoxin were 4.83 µM [ 104 ] and for gonyautoxin ¼ 17.7 nM [ 105 ].…”