Surfactant-Assisted Label-Free Fluorescent Aptamer Biosensors and Binding Assays

Abstract

Using DNA staining dyes such as SYBR Green I (SGI) and thioflavin T (ThT) to perform label-free detection of aptamer binding has been performed for a long time for both binding assays and biosensor development. Since these dyes are cationic, they can also adsorb to the wall of reaction vessels leading to unstable signals and even false interpretations of the results. In this work, the stability of the signal was first evaluated using ThT and the classic adenosine aptamer. In a polystyrene microplate, a drop in fluorescence was observed even when non-binding targets or water were added, whereas a more stable signal was achieved in a quartz cuvette. Equilibrating the system can also improve signal stability. In addition, a few polymers and surfactants were also screened, and 0.01% Triton X-100 was found to have the best protection effect against fluorescence signal decrease due to dye adsorption. Three aptamers for Hg²⁺, adenosine, and cortisol were tested for their sensitivity and signal stability in the absence and presence of Triton X-100. In each case, the sensitivity was similar, whereas the signal stability was better for the surfactant. This study indicates that careful control experiments need to be designed to ensure reliable results and that the reliability can be improved by using Triton X-100 and a long equilibration time.

Keywords: aptamers; biosensors; fluorescence; surfactant.

Figure 1

(A) Scheme of adenosine binding to the adenosine aptamer detected by ThT. Initially, ThT binds to the aptamer, producing a strong fluorescence signal. Adenosine binding displaces ThT, resulting in a decrease in fluorescence. Binding assay in a polystyrene microplate by (B) titrating adenosine and guanosine, and (C) titrating water. Binding assay in a quartz cuvette by (D) titrating adenosine and guanosine, and (E) titrating water. WT: wild-type adenosine aptamer; MUT: Ade Apt M2 mutant. Fluorescence intensities were normalized to their respective baseline value (absent of target) before titration.

Figure 2

(A) Time-dependent fluorescence intensity of the adenosine aptamer and ThT mixture in a polystyrene microplate and a quartz cuvette. (B) A scheme showing the adsorption of dyes such as SGI and ThT to the wall of a reaction vessel.

Figure 3

(A) Effect of surfactant on signal stability in Corning 96-well microplate. The samples contained 200 nM DNA1, 0.02× SGI, and 0.01% different surfactants in Buffer 1. (B) Effect of surfactant on signal stability in a quartz cuvette. The solutions contained 1 µM DNA1, 0.1× SGI, and 0.01% different surfactants in Buffer 1. Effect of Triton X-100 concentration on the stability of fluorescence in (C) a Corning microplate, (D) a Greiner microplate, and (E) a quartz cuvette. (F) Structure of Triton X-100. (G) Effect of Triton X-100 on SGI pre-stained microplate well containers. Initially, buffer and 1 µM SGI were incubated for 15 min, then solution was removed. To the same wells, buffer with and without 0.01% Triton X-100 was added alongside 70-mer double-stranded DNA. (H) Titration curve of 70-mer double-stranded DNA titrated by SGI in the presence of 0.01% Triton X-100.

Figure 4

(A) A scheme of Hg²⁺ binding to a thymine-rich DNA detected by SGI. Hg²⁺ binds to the DNA and folds into a structure with a long duplex region, resulting in a strong SGI fluorescence. (B) Titration curve of 200 nM T₃₀ using mercury acetate with and without 0.01% Triton X-100. (C) Effect of Triton X-100 on signal stability of T₃₀/SGI in the presence of 2 µM Hg²⁺.

Figure 5

(A) Titration curve of 1 µM adenosine aptamer by adenosine without and with 0.01% Triton X-100. (B) Effect of Triton X-100 on signal stability of the adenosine aptamer/ThT system in the presence of 100 µM adenosine.

Figure 6

(A) The scheme of cortisol binding to its aptamer detected by SYBR Green I. Initially, the combination of SGI and aptamer produces weak fluorescence, and after the addition of cortisol, aptamer–cortisol binding results in enhanced fluorescence. (B) Titration curve of 1 µM CSS.1-42 with cortisol with and without Triton X-100. (C) Effect of Triton X-100 on signal stability of CSS.1-42 Apt-SGI-cortisol titration.