Aptamer-Conjugated Multi-Quantum Dot-Embedded Silica Nanoparticles for Lateral Flow Immunoassay

Abstract

Lateral flow immunoassays (LFIAs) are widely used for their low cost, simplicity, and rapid results; however, enhancing their reliability requires the meticulous selection of ligands and nanoparticles (NPs). SiO₂@QD@SiO₂ (QD²) nanoparticles, which consist of quantum dots (QDs) embedded in a silica (SiO₂) core and surrounded by an outer SiO₂ shell, exhibit significantly higher fluorescence intensity (FI) compared to single QDs. In this study, we prepared QD²@PEG@Aptamer, an aptamer conjugated with QD² using succinimidyl-[(N-maleimidopropionamido)-hexaethyleneglycol]ester, which is 130 times brighter than single QDs, for detecting carbohydrate antigen (CA) 19-9 through LFIA. For LFIA optimization, we determined the optimal conditions as a 1.0:2.0 × 10^-2 ratio of polyethylene glycol (PEG) to aptamer by adjusting the amounts of PEG and aptamer, phosphate-buffered saline containing 0.5% Tween^® 20 as a developing solution, and 0.15 μg NPs by setting the NP weight during development. Under these conditions, QD²@PEG@Aptamer selectively detected CA19-9, achieving a detection limit of 1.74 × 10^-2 mg·mL^-1. Moreover, FI remained stable for 10 days after detection. These results highlight the potential of QD² and aptamer conjugation technology as a reliable and versatile sensing platform for various diagnostic applications.

Keywords: aptamer; carbohydrate antigen 19-9; lateral flow immunoassay; quantum dot.

Figure 1

(a) Schematic of QD²@PEG@Aptamer fabrication. (b) Schematic of reaction process for conjugation of succinimidyl-[(N-maleimidopropionamido)-hexaethyleneglycol]ester and aptamer to QD².

Figure 2

(a) Transmission electron microscopy–energy-dispersive X-ray spectroscopic images of (i) SiO₂ and (ii) QD² NPs, and (iii) energy-dispersive X-ray spectroscopic mapping images of QD² (Si, Cd, and Zn) (scale bar, 50 nm). (b) Image showing hydrophilicity or hydrophobicity of quantum dots (QDs) and QD² in vials containing equal volumes of toluene and distilled water. (c) Comparison of fluorescence intensities of QDs and QD² under same particle concentrations (3.22 × 10¹² particle·mL⁻¹). (d) Ultraviolet–visible absorbance of SiO₂, QD², and QD²–PEG–Aptamer (50 µg·mL⁻¹). (e) Zeta potential of NPs at each synthesis step.

Figure 3

(a) A fluorescence image of NPs synthesized by controlling the amount of polyethylene glycol (PEG) and aptamer applied to a strip. (b) The fluorescence of strips with antigen at a concentration of 3 × 10⁻¹ mg·mL⁻¹ (PEG–aptamer). (c) A fluorescence image of NPs developed on a strip by adjusting the concentration of Tween^® 20 in phosphate-buffered saline (PBS) [0.5, 1.0, 1.5, and 2.0% Tween^® 20 in PBS (PBST)]. (d) The fluorescence intensity (FI) of strips obtained using different concentrations of Tween^® 20 in PBS solution (0.5, 1.0, 1.5, and 2.0% PBST). (e) A fluorescence image representing the optimal amount of NPs in strip development (antigen, 3 × 10⁻¹ mg·mL⁻¹). (f) The FIs of strips developed with different amounts of NPs.

Figure 4

(a) Fluorescence image of strip with developed QD²–PEG–Aptamer containing different concentrations of antigen (0, 1.5 × 10⁻², 3.0 × 10⁻², 1.5 × 10⁻¹, and 3.0 × 10⁻¹ mg·mL⁻¹). (b) FIs measured on strip detecting CA19-9 using QD²–PEG–Aptamer. (c) Fluorescence images of NPs developed on different strips after drying CA19-9, PSA, and amyloid beta 1-40 (Aβ40) to determine selectivity of aptamer. (d) FIs measured on strips with CA19-9, PSA, and Aβ40. (e) Strip image showing antigen detected at concentration of 3 × 10⁻¹ mg·mL⁻¹ on test paper over 10-day period (days 0–10). (f) FIs measured from strips photographed over 10-day period (days 0–10).