key: cord-1050486-1x6m6qgo
authors: Zhang, Guo-Qiang; Gao, Zhiyuan; Zhang, Jingtian; Ou, Hanlin; Gao, Heqi; Kwok, Ryan T.K.; Ding, Dan; Tang, Ben Zhong
title: Wearable AIEgen-Based Lateral Flow Test Strip for Rapid Detection of SARS-CoV-2 RBD Protein and N Protein
date: 2022-01-17
journal: Cell Rep Phys Sci
DOI: 10.1016/j.xcrp.2022.100740
sha: 21189c4d4bffb1d9f2594df47a06d5073c4de463
doc_id: 1050486
cord_uid: 1x6m6qgo

Accurate and rapid detection of SARS-CoV-2 is significant for early tracing, isolating and treating infected patients, which will efficiently prevent large-scale transmission of COVID-19. Herein, two kinds of test strips for RBD and N antigens of SARS-CoV-2 are established with high sensitivity and specificity, in which AIE luminogens are utilized as reporters. Because of the high brightness and resistance of quenching properties in aqueous solution, the limit of detection can be as low as 6.9 ng/mL for RBD protein and 7.2 ng/mL for N protein. As an antigen collector, the N95 mask equipped with the test strip with excellent enrichment effect would efficiently simplify the sampling procedures. Compared with the test strip based on Au nanoparticles or FITC, the AIEgen-based test strip shows high anti-interference capacity in complex biosamples. Therefore, the AIEgen-based test strip assay could be built as a promising platform for emergency usage during the pandemic.

that the N protein, as the predominantly expressed structural protein, has been identified as one of the best early diagnostic targets in SARS-CoV, broke out in 2003, and could be detected before the antibody appears in serum [22] [23] [24] . RBD protein is located on the surface of the virus 25 , so it would be a suitable diagnosis epitope for simplifying the pretreatment procedures because the whole virions could be directly tested without virus lysis.

Typical AIE luminogens (AIEgens) are often equipped with peripheral phenyl rings leading to 3D twisting of the molecular structure, which extremely reduces intermolecular interactions such as π-π stacking when aggregated and greatly decreases the nonradiative decay of excited states [26] [27] [28] . According to the Jablonski diagram, it is competitive between the radiation and nonradiation energy dissipation pathways 29 . Therefore, the emergence of AIEgens offers an opportunity to maximize the absorbed energy flowing to the fluorescence emission pathway 30 . Therefore, AIEgens are desirable organic fluorophores with high brightness and resistance to quenching properties in aqueous solution.

Herein, based on the AIEgen-based lateral flow test strip, we developed a rapid and sensitive assay strategy for the detection of RBD protein and N protein from SARS-CoV-2. As a proof of concept, the AIEgen-based lateral flow test strip assay was first carried out for C-reactive protein (CRP) , and the results demonstrated that the strategy could accurately detect CRP with a linear relationship from 0.001 μg/mL to 20 μg/mL and an ultralow limit of detection (LOD) of 5.5 ng/mL. Subsequently, two kinds of AIEgen-based lateral flow test strips were fabricated to detect the RBD protein and N protein of SARS-CoV-2. Both of them presented excellent testing performance with ultralow LODs of 6.9 ng/mL for RBD protein and 7.2 ng/mL for N protein. Within 20 min, the testing results could be obtained with high sensitivity and specificity, which would offer an alternative detection tool for SARS-CoV-2 screening, similar to RT-qPCR. We also assembled the test strip on the remodeled N95 mask, which was utilized as a collector to enrich the droplet containing virions or nanoparticles. These results demonstrated the efficient enrichment effect for AIE NPs 2. With the collection strategy, the AIEgen-based test strip was able to detect low concentrations of antigens due to its ultrahigh sensitivity and strong anti-interference ability in complex biosamples.

Five rationally designed AIE fluorescence molecules equipped with carboxyl groups for linking J o u r n a l P r e -p r o o f to the antibodies were synthesized via Knoevenagel condensation between the aldehyde group and the methylene at rhodanine-3-acetic acid 31, 32 (Figure 1a , Schemes S1-S4), whose structures were characterized by 1 H-NMR, 13 C-NMR and HRMS ( Figures S15-S23) . The ultraviolet-visible (UV) absorption spectra of five AIEgens were investigated, as shown in Figure 1b , and the absorption peak values ranged from 438 nm to 504 nm. The fluorescence (FL) emission spectra of five AIEgens were subsequently measured. There was almost no FL emission or weak emission in DMSO solution, which indicated that the active dynamics of the phenyl ring rotors dissipate energy through nonradiative pathways for the reduced fluorescence emissions. To demonstrate their AIE characteristics, fluorescence spectra were also recorded in DMSO/toluene mixtures with different toluene fractions. When the toluene fraction was less than 70%, there was merely weak emission.

However, by further increasing the toluene fraction, the fluorescence intensity of five AIEgens 

With the brightest AIE 2 in hand, the detection antibodies were labeled through a condensation reaction between the carboxyl group of AIE-COOH and the amino groups of antibodies via the NHS active ester method. Herein, the selected AIE 2 was activated by NHS and EDC to form the active ester in dry DMSO at room temperature, which was directly employed to label antibodies without any further purification ( Figure 2a 

The designed test strip comprises a PVC backing card, sample pad, nitrocellulose membrane (NC membrane) and absorption pad (Figure 3a 

CRP is identified as an acute inflammatory response biomarker in the clinic, and after effective treatment, the CRP concentration decreases 34-36 , whose detection methods have been well Figure   4b ), which led to a limit of detection of 5.5 ng/mL. The specificity of the AIEgen-based lateral flow test strip assay for CRP detection was also evaluated. Samples containing AFP, HCG, CEA, CA125, HSA, N protein, RBD protein, S2 protein and FBS were used to detect nonspecific interference. Two FL signals were clearly present on the test and control lines only when testing the CRP antigen, and a nonspecific FL signal was hardly detected for other samples (Figure 4c) . The quantitative data of the FL intensity of the test line for CRP were much higher than those of other samples (Figure 4d ), which indicated that the testing strategy dominated the excellent specificity toward the target antigen. Based on the testing results, the strategy holds tremendous advantages, such as rapid, portability, cost-effectiveness, ease of use, ultrahigh sensitivity and specificity for CRP testing.

Due to its superior properties, the AIEgen-based lateral flow test strip assay could be expanded to detect other antigens as a reliable and general platform. SARS-CoV-2 infects host cells via the RBD of the spike protein, which mediates binding with angiotensin-converting enzyme J o u r n a l P r e -p r o o f (ACE2) to attack respiratory cells 13 . The RBD is present on the outside of the virus. Hence, it would offer a delicate testing strategy for SARS-CoV-2 RBD, in which all virions could be detected directly without a virus lysis procedure.

Before testing the antigens, the incubation time and incubation temperature of the detection antibody and RBD protein were optimized through orthogonal experimental design, as shown in Figure   5b ). This test can be quantified by measuring the brightness of the test line. A linear relationship was obtained in the range of 10 ng/mL to 20 μg/mL with R 2 of 0.99 (Figure 5b ). The limit of detection was determined to be 6.9 ng/mL. In addition, the negative results of AFP, CEA, N protein, S2 protein, FBS and CRP detection indicated the excellent specificity of the test strip ( Figure S14 ).

The N protein not only plays a vital role in the transcription and replication of SARS-CoV-2 but is also abundantly expressed, which can be detectable even after just one day of infection 38 . It would provide rapid, higher sensitivity when it is used as a target protein 39 .

The detection sensitivity of the AIEgen-based lateral flow test strip assay for the N protein (Figure 5c ). This test could be quantified by measuring the FL intensity of the test line. A linear relationship was obtained between 1 ng/mL and 20 μg/mL of N protein with a linear correlation coefficient (R 2 ) of 0.98 (Figure 5d ), whose LOD was as low as 7.2 ng/mL. Figure 5e , the specificity was also evaluated via the samples containing AFP, HCG, CEA, CA125, HSA, S2 protein, RBD protein, FBS and CRP, which clearly showed that only the N protein was specifically bound on the test line, and almost no nonspecific signal was detected for the others. Consequently, the FL intensity of the N protein on the test line was much higher than that of other samples (Figure 5f ), which indicated the ultrahigh specificity of the AIEgen-based lateral flow test strip toward the N protein.

The strategy was successfully applied to the quantitative detection of CRP, N protein and RBD protein. Our results demonstrated the feasibility of the AIEgen-based lateral flow test strip assay to detect the target antigens in PBS with high sensitivity and specificity.

Masks, as personal protective equipment (PPE), have presented huge potential for blocking the transmission of pathogenic pathogens from humans to humans. An N95 face mask, whose name was obtained from blocking 95% of very small (0.3 μm) particles during testing 40 , was designed to minimize facial seal leakage because of tight fit and prevent inhalation of small airborne particles 41 . The WHO and CDC have recommended that physicians on the front lines wear the N95 face mask 42 . For patients, the virus could be restricted by the N95 face mask, which was also identified as a collector to enrich the particles, such as AIE nanoparticles, virus or antigens, for our design.

As a proof of concept, we designed a respiratory tract model, and three holes were drilled on the location of the nose and mouse on the front side as the air outlet ( Figure 6a) and one on the bottom as the air inlet (Figure 6b ), which could simulate respiration. The test strip was equipped on the mask. The sample pad, which could adsorb the analytes, was just located on the breather valve pore to seal, as shown in Figure 6c and d. It is worth mentioning that the weight of the N95 mask with the test strip is only approximate 10 g, which is easy to accept without any wearing burden. To test the enrichment effect, AIE 2 was coprecipitated with DSPE-PEG2000 as a matrix to formulate AIE 2 nanoparticles (AIE NPs 2). Dynamic light scattering (DLS) measurements J o u r n a l P r e -p r o o f indicated that AIE NPs 2 had an average hydrodynamic diameter of ∼100 nm (Figure 6f) , which was close to the results by transmission electron microscopy (TEM) (Figure 6f, inset) , and TEM revealed that AIE NPs 2 had a uniform spherical morphology. Furthermore, the stability of AIE NPs 2 in PBS buffer was also measured by monitoring the size variation using DLS. The average diameter remained nearly the same, and no precipitation was observed after storage at room temperature for seven days (Figure 6g ).

The NPs solution was sprayed into the respiratory tract model through an atomizer at 0.5 mL/min inspiratory flow for 5 min (Figure 6e ). After that, mask images were obtained through the IVIS imaging system, as shown in Figure 7a , which indicated that AIE NPs 2 efficiently accumulated around the breather valve pore compared with the mask without nanoparticle enrichment.

Furthermore, the test strips were also imaged under IVIS, and it was obvious that AIE NPs 2 was attached to the sample pad ( Figure 7b) . Conclusively, the design of a respiratory tract model could efficiently cause droplets to accumulate on the sample pad.

Based on the exciting results, another two kinds of N protein detection antibodies, which were labeled with colloidal gold and FITC, were prepared. All three detection antibodies were incubated with N protein to form binary complexes, followed by spraying into the respiratory tract model. 

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AIEgen-based test strip is developed as a general platform for emergency usage

The antigens of SARS-CoV-2 can be detected by the test strip with high sensitivity

eTOC: Zhang et al. report a general test strip platform, which is based on AIE luminogens as reporter, for SARS-CoV-2 antigen detection. The assay offers a rapid, scale-up detection tool for SARS-CoV-2 screening with high sensitivity, specificity, and good reproducibility