β-Cyclodextrin Polymer-Based Fluorescence Enhancement Strategy via Host-Guest Interaction for Sensitive Assay of SARS-CoV-2

Abstract

Nucleocapsid protein (N protein) is an appropriate target for early determination of viral antigen-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have found that β-cyclodextrin polymer (β-CDP) has shown a significant fluorescence enhancement effect for fluorophore pyrene via host-guest interaction. Herein, we developed a sensitive and selective N protein-sensing method that combined the host-guest interaction fluorescence enhancement strategy with high recognition of aptamer. The DNA aptamer of N protein modified with pyrene at its 3' terminal was designed as the sensing probe. The added exonuclease I (Exo I) could digest the probe, and the obtained free pyrene as a guest could easily enter into the hydrophobic cavity of host β-CDP, thus inducing outstanding luminescent enhancement. While in the presence of N protein, the probe could combine with it to form a complex owing to the high affinity between the aptamer and the target, which prevented the digestion of Exo I. The steric hindrance of the complex prevented pyrene from entering the cavity of β-CDP, resulting in a tiny fluorescence change. N protein has been selectively analyzed with a low detection limit (11.27 nM) through the detection of the fluorescence intensity. Moreover, the sensing of spiked N protein from human serum and throat swabs samples of three volunteers has been achieved. These results indicated that our proposed method has broad application prospects for early diagnosis of coronavirus disease 2019.

Keywords: DNA aptamer; SARS-CoV-2; fluorescence enhancement; host–guest interaction; β-cyclodextrin polymer.

Figure 1

Schematic design of aptamer-based sensitive assay of SARS-CoV-2 N protein using β-cyclodextrin polymer to enhance pyrene fluorescence.

Figure 2

(A) Fluorescence emission spectra in addition to β-CDP or β-CD monomer. (B) Fluorescence intensity of pyrene in addition to β-CDP vs. time. The concentration of β-CDP was 1.5 mg/mL. The concentration of pyrene labeled aptamer was 400 nM. The maximum emission wavelength was set at 376 nm.

Figure 3

Fluorescence emission spectra in addition to pyrene-labeled scrambled DNA sequence and N protein aptamer. The concentration of β-CDP was 1.5 mg/mL. The concentration of N protein was 600 nM. The concentration of pyrene-labeled aptamer or pyrene-labeled scrambled DNA sequence was set at 400 nM. The maximum emission wavelength was set at 376 nm.

Figure 4

(A) CD spectra of aptamer after incubation with N protein at 37 °C for 30 min (black curve), aptamer (red curve), and N protein alone (blue curve). The concentration of N protein and aptamer was equal to 1 μM. (B) Agarose gel electrophoresis. The concentration of N protein, aptamer, and Exo I was 600 nM, 400 nM, and 240 U/mL, respectively.

Figure 5

(A) Optimization of pH value. The activity of Exo I was 240 U/mL. The concentration of N protein, aptamer, and β-CDP was 200 nM, 400 nM, and 1.5 mg/mL, respectively. (B) (F₀–F)/F₀ vs. the centration of β-CDP. The activity of Exo I was 240 U/mL. The concentration of N protein and aptamer was 200 nM and 400 nM, respectively. (C) (F₀–F)/F₀ vs. the activity of Exo I. The concentration of aptamer was 400 nM. The concentration of N protein was 200 nM. The concentration of β-CDP was 1.5 mg/mL. (D) (F₀–F)/F₀ vs. the concentration of aptamer. The concentration of Exo I and N protein were 240 U/mL and 200 nM, respectively. The concentration of β-CDP was 1.5 mg/mL. Error bars indicated the standard deviations of three experiments.

Figure 6

(A) Fluorescence emission spectra of the system under the concentration of N protein from 0 to 600 nM. (B) The linear relationship between the concentration of N protein (the concentration from left to right was 0, 40, 100, 140, 200, and 300 nM, respectively) and (F₀–F)/F₀ (S/N = 3). The concentration of aptamer, Exo I, and β-CDP was 400 nM, 240 U/mL, and 1.5 mg/mL, respectively. The excitation wavelength was set at 345 nm, and the maximum emission wavelength was set at 376 nm.

Figure 7

(F₀–F)/F₀ value of potential interferences in human blood samples (A) and throat swabs (B). The concentration of potential interferences or N protein was all set at 600 nM.

Figure 8

(A) (F₀–F)/F₀ value at various concentrations of N protein spiked to the human serum sample. (B) (F₀–F)/F₀ value at various concentrations of N protein spiked to the throat swabs sample.