Selection, Characterization, and Optimization of DNA Aptamers against Challenging Marine Biotoxin Gymnodimine-A for Biosensing Application

Abstract

Gymnodimines (GYMs), belonging to cyclic imines (CIs), are characterized as fast-acting toxins, and may pose potential risks to human health and the aquaculture industry through the contamination of sea food. The existing detection methods of GYMs have certain defects in practice, such as ethical problems or the requirement of complicated equipment. As novel molecular recognition elements, aptamers have been applied in many areas, including the detection of marine biotoxins. However, GYMs are liposoluble molecules with low molecular weight and limited numbers of chemical groups, which are considered as "challenging" targets for aptamers selection. In this study, Capture-SELEX was used as the main strategy in screening aptamers targeting gymnodimine-A (GYM-A), and an aptamer named G48nop, with the highest K_D value of 95.30 nM, was successfully obtained by screening and optimization. G48nop showed high specificity towards GYM-A. Based on this, a novel aptasensor based on biolayer interferometry (BLI) technology was established in detecting GYM-A. This aptasensor showed a detection range from 55 to 1400 nM (linear range from 55 to 875 nM) and a limit of detection (LOD) of 6.21 nM. Spiking experiments in real samples indicated the recovery rate of this aptasensor, ranging from 96.65% to 109.67%. This is the first study to report an aptamer with high affinity and specificity for the challenging marine biotoxin GYM-A, and the new established aptasensor may be used as a reliable and efficient tool for the detection and monitoring of GYMs in the future.

Keywords: aptamer; aptasensor; biolayer interferometry; gymnodimine-A.

Figure 1

Chemical structures of GYMs and their congeners [6].

Figure 2

A general workflow of Capture-SELEX. Each round of Capture-SELEX mainly consists of four steps: (1) hybridization of the library and capture oligo by base pairing; (2) immobilization of the hybrid on beads (gray) via the strong interaction between streptavidin (on beads) and biotin (on capture oligos); (3) incubation with the positive target and elution of ssDNA (before being incubated with the positive target, the beads are washed several times with the selection buffer to remove ssDNA that are less strongly bound to the beads and incubated with the negative target to remove those ssDNA, which can bind to the negative target, thereby improving specificity); (4) PCR amplification of the eluted ssDNA and preparation ssDNA from these PCR products for the selection of next round.

Figure 3

Secondary structure prediction of aptamer G48 (A) and aptamer G48nop (B) with their respective lowest Gibbs free energy value by mfold program. The folding temperature is 25 °C, and the concentrations of Na⁺ and Mg²⁺ were 100 mM and 2 mM, respectively.

Figure 4

CD assays of aptamer G48nop and GYM-A samples. The cyan line stands for the CD spectra of GYM-A, the blue line stands for the CD spectra of aptamer G48nop, and the red line stands for the CD spectra of GYM-A and aptamer G48nop. The sample buffer is 20 mM Tris-HCl (pH 7.6), 100 mm NaCl, 2 mM MgCl₂, 5 mM KCl, and 1 mM CaCl₂.

Figure 5

Identification of affinity and specificity of G48nop for GYM-A. (A) Identification of affinity of G48nop for GYM-A. The blue lines stand for the spectral shift of aptamer G48nop with GYM-A (7 μM (top), 3.5 μM (middle), and 1.75 μM (bottom)). (B) Identification of specificity of G48nop for GYM-A. The specificity of G48nop measured by BLI for eleven kinds of marine biotoxins (GYM-A, OA, BTX, DTX, PLTX, GTX, STX, MC-LR, NOD-R, SPX, and PnTX) and mixture is shown as response values caused by spectral shift. A random sequence fixed on the biosensor was used as an aptamer control. The final concentration of each marine biotoxin is 0.5 µM and the final concentration of each marine biotoxin in the mixture is also 0.5 µM. Every sample was tested three times. **** p < 0.0001 vs. GYM-A.

Figure 6

MST assays for aptamer G48nop (blue) and control sequence (red). The K_D value of G48nop is 34.50 ± 1.72 nM. The control sequence shows no binding affinity for GYM-A.

Figure 7

Evaluation of the performance of the BLI-based aptasensor. (A) The principle of BLI assay for detection [85,86]. (B) Response characterization of BLI-based biosensor measuring various concentrations (55–14,000 nM) of GYM-A. (C) Calibration curve of response values with the changing of various concentrations (55–14,000 nM) of GYM-A. The error bar stands for standard deviation. (D) Linear range of the calibration curve of GYM-A. A plot of response values with the changing of various concentrations (55–875 nM) of GYM-A. (E) Specificity of the BLI-based biosensor with eleven different kinds of toxins (GYM-A, OA, BTX, DTX, PLTX, GTX, STX, MC-LR, NOD-R, SPX, and PnTX, each at 1.5 µM) and the mixture of those toxins (each toxin in mixture was of a concentration of 1.5 µM). Every sample was tested three times. **** p < 0.0001 vs. GYM-A.