Fluorescence‐Quenching Lateral Flow Immunoassay for “Turn‐On” and Sensitive Detection of Anti‐SARS‐Cov‐2 Neutralizing Antibodies in Human Serum

Abstract The titer of anti‐severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) neutralizing antibodies (NAbs) in the human body is an essential reference for evaluating the acquired protective immunity and resistance to SARS‐CoV‐2 infection. In this study, a fluorescence‐quenching lateral flow immunoassay (FQ‐LFIA) is established for quantitative detection of anti‐SARS‐CoV‐2 NAbs in the sera of individuals who are vaccinated or infected within 10 min. The ultrabright aggregation‐induced emission properties encapsulated in nanoparticles, AIE490NP, are applied in the established FQ‐LFIA with gold nanoparticles to achieve a fluorescence “turn‐on” competitive immunoassay. Under optimized conditions, the FQ‐LFIA quantitatively detected 103 positive and 50 negative human serum samples with a limit of detection (LoD) of 1.29 IU mL−1. A strong correlation is present with the conventional pseudovirus‐based virus neutralization test (R 2 = 0.9796, P < 0.0001). In contrast, the traditional LFIA with a “turn‐off” mode can only achieve a LoD of 11.06 IU mL−1. The FQ‐LFIA showed excellent sensitivity to anti‐SARS‐CoV‐2 NAbs. The intra‐ and inter‐assay precisions of the established method are below 15%. The established FQ‐LFIA has promising potential as a rapid and quantitative method for detecting anti‐SARS‐CoV‐2 NAbs. FQ‐LFIA can also be used to detect various biomarkers.

Table S2.The background information of the serum samples.
Sample NO.First, the absorbance spectra of AIE 490 (in THF/water, v/v = 1/99) with various concentrations by serial dilution were measured (Figure S6a).The absorbance value at 365 nm exhibited a linear relationship with the concentration of AIE 490 .The standard curve was obtained by plotting the absorbance value at 365 nm (Y) against the concentration of AIE 490 (X) as represented by the equation: Y=0.02158*X+0.001525 with a reliable coefficient of determination (R 2 =0.9976) (Figure S6b). 1 mg of AIE 490 NP was freeze-dried and then dissolved in 1 mL of THF to obtain the AIE 490 encapsulated in AIE 490 NP.Subsequently, 10 μL of supernatant was taken in 990 μL of water and the absorbance of the mixture was measured too (Figure S6c).The mass of the AIE 490 molecule contained in 1 mg of PS nanoparticles was calculated to be 89.7 μg, which is equivalent to 7.16×10 16 by the formula of N 1 = (n=6.02×10 23/mol, M=753.97 g/mol).Meanwhile, the number of 1 mg of PS was calculated to be 7.08×10 10 by the formula of N 2 = .The number of AIE 490 in an AIE 490 NP was calculated to be 1.01×10 6 by a formula of N= .curve (e-f) of test strips used to detect sample buffer; the test strips were coated with different concentrations (0.5, 1, 1.5, and 2 mg mL -1 ) of RBD on the test line.Table S6.The anti-SARS-CoV-2 NAbs titers (wild-type strain and Delta-variant strain) of serum samples (pVNT).

Figure S1 .
Figure S1.The absorption and fluorescence spectra of AIE 490 .

Figure S2 .
Figure S2.The AIE properties of AIE 490 .(a) The fluorescence intensity of AIE 490 in THF and THF/water mixture with different water fractions (f w ).(b) The fluorescence integration of AIE 490 in THF and THF/water mixture with different water fractions (f w ).

Figure S3 .
Figure S3.The fluorescence intensity changing trends of AIE 490 under continuous irradiation of white light (100 mW cm -2 ).

Figure
Figure S6.(a) The UV-vis absorption spectra of AIE 490 with various concentrations in THF/water.(B) The linear relationship between the concentration of AIE 490 and the absorbance value at 365 nm.(C) UV-vis absorption spectra of AIE 490 NP dissolved in THF/water.THF/water, v/v = 1/99.

Figure S7 .
Figure S7.The fluorescence intensity of AIE 490 NP and AIE 490 aggregated in water when the concentration of AIE 490 molecule is the same at 0.897 μg mL -1 .

Figure S16 .
Figure S16.The SEM images of the uncoated NC membrane (a) and the coated test (b) or control line (c) position on the NC membrane.

Figure S17 .Figure S18 .
Figure S17.The strips with different concentrations of AIE 490 NP coating on the NC membrane.(a) The picture of the FQ-LFIA strips with test line coated by RBD with a concentration of 1 mg mL -1 and AIE 490 NP-BSA with concentrations of 0.1, 0.2, or 0.3 mg mL -1 .(b) The fluorescence peak area of the test line in (a) obtained by a portable fluorescence reader.

Figure S19 .
Figure S19.The optimization of RBD concentration coated on the test line.The pictures (a-d, left: under white light, right: under UV light with λ ex = 365 nm) and fluorescence readout

Figure S20 .
Figure S20.The fluorescence peak area of the test line (a) and control line 1 (b) of test strips used to detect sample buffer which were coated with different concentrations (0.5, 1, 1.5, and 2 mg mL -1 ) of RBD on the test line.

Figure S23 .FigureFigure S24 .
Figure S23.The dose-response curve (a) and detection results (b) of the commercial ELISA kit (the dotted line was LoD of the method).

Table S3 .
The loss of fluorescence intensity of AIE 490 and AIE 490 NP under different temperatures.

Table S4 .
The loss of fluorescence intensity of AIE 490 and AIE 490 NP under different pH levels.

Table S5 .
The detection results of the 103 positive serum samples and 50 negative serum samples using the pVNT method, the established FQ-LFIA, and the commercial ELISA kit.