key: cord-0762910-v2jma4qv
authors: Hu, Ruibin; Liao, Tao; Ren, Yan; Liu, Wenming; Ma, Rui; Wang, Xinyuan; Lin, Qihui; Wang, Guoxin; Liang, Yongye
title: Sensitively detecting antigen of SARS-CoV-2 by NIR-II fluorescent nanoparticles
date: 2022-05-10
journal: Nano Res
DOI: 10.1007/s12274-022-4351-1
sha: 9f1add26e05cac45937a6b0126cf69d384bfca7f
doc_id: 762910
cord_uid: v2jma4qv

Early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is an efficient way to prevent the spread of coronavirus disease 2019 (COVID-19). Detecting SARS-CoV-2 antigen can be rapid and convenient, but it is still challenging to develop highly sensitive methods for effective diagnosis. Herein, a lateral flow assay (LFA) based on fluorescent nanoparticles emitting in the second near-infrared (NIR-II) window is developed for sensitive detection of SARS-CoV-2 antigen. Benefiting from the NIR-II fluorescence with high penetration and low autofluorescence, such NIR-II based LFA allows enhanced signal-to-background ratio, and the limit of detection is down to 0.01 ng·mL(−1) of SARS-CoV-2 antigen. In the clinical swab sample tests, the NIR-II LFA outperforms the colloidal gold LFA with higher overall percent agreement with the polymerase chain reaction test. The clinical samples with low antigen concentrations (∼ 0.015−∼ 0.068 ng·mL(−1)) can be successfully detected by the NIR-II LFA, but fail for the colloidal gold LFA. The NIR-II LFA can provide a promising platform for highly sensitive, rapid, and cost-effective method for early diagnosis and mass screening of SARS-CoV-2 infection. [Image: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material (the operation procedure and cost of the materials needed of NIR-II lateral flow assays, the dynamic light scattering spectrum of the NIR-II nanoparticles, the components and testing principle, optimization of main parameters pertaining to the LFA performance, the colloidal gold LFA strip, the fluorescence intensity distribution curves and the T/C values of the strips for clinical samples by NIR-II LFA, and results of clinical swab samples detected by colloidal gold LFA) is available in the online version of this article at 10.1007/s12274-022-4351-1.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), identified as the causative agent of the coronavirus disease 2019 [1, 2] , has caused a worldwide pandemic and posed a huge challenge for global public health. By February 2022, the number of patients with SARS-CoV-2 pneumonia reaches 380 million and the death toll exceeds 5 million [3] . The data still increase every day. The direct human to human transmission, the indefinite incubation period of carriers, and the nonspecific symptoms of carriers are the main reasons for such high infectivity of SARS-CoV-2 [4, 5] . To prevent transmission and control pandemic of COVID-19, it is necessary to exploit advanced methods to detect it in the early stage of infection.

Up to now, researchers have developed numerous early diagnostic kits, which can be divided into two categories: genetic material-based molecular diagnosis or antigen/antibody-based immunoassays. For nucleic acid quantification analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR) is regarded as the gold standard for COVID-19 detection because of its mature technique and high sensitivity [6, 7] . However, it requires specific equipment and laboratory, which is apparently not suitable for rapidly detecting the virus in point of care (POC) setting.

Immune-based assays are based on the high affinity between antigen and antibody, affording high specificity and rapid response (about a few minutes) [8] , and they are very suitable for rapid diagnosis of COVID-19. These methods mainly include enzyme-linked immunosorbent assays (ELISA) [9] [10] [11] , chemiluminescent immunoassays (CLIA) [12] [13] [14] , and lateral flow assays (LFA) [15] [16] [17] . LFA technique is advantageous for its simplicity, convenience, rapidity, and cost-efficiency. As early as February 2020, SARS-CoV-2 antibody detection kit was developed [18] . So far, a variety of SARS-CoV-2 antibody assay kits based on colloidal gold LFA have been widely used [19] [20] [21] . However, the antibody test cannot be used alone as a diagnostic indicator for screening of SARS-CoV-2 infection as it generally takes more than 7 days for the body to produce antibodies after infection [22] [23] [24] . In this window period, the viral load increased rapidly along with the acceleration of reproduction and the infection risk increases further [25, 26] . In contrast, antigen can be detected at the earlier stage of infection. Rapid antigen detection by LFA has been developed for case finding and contract tracing of COVID-19 [27] . However, the concentration of virus contained in the sampling could be low as it is generally acquired from the upper respiratory tract, but COVID-19 mainly infringes on the lower respiratory tract areas. In this situation, highly sensitive viral antigen detection technique is urgently required.

In the past two years, improved antigen detection methods of SARS-CoV-2 have been reported [28] [29] [30] [31] . For instance, Guo et al. developed an up-conversion nanoparticles based LFA for quantitative detection of spike (S) protein and nucleocapsid (N) protein of SARS-CoV-2. In this platform, the sensor could detect the S protein and N protein with limit of detection (LoD) of 1.6 and 2.2 ng·mL −1 , respectively [32] . Grant et al. demonstrated a half-strip LFA for detection of N protein, achieving a LoD of 0.65 ng·mL −1 with an optical reader [33] . Liu et al., reported a nanozyme-based test with the lateral flow strip. Co-Fe@heminperoxidase nanozymes were constructed for catalyzing chemiluminescence similar with the natural horseradish peroxidase (HRP), leading to the amplification of the detection signals. And the LoD is improved to 0.1 ng·mL −1 [34] . However, the determination of clinical samples with lower concentration viruses is still challenging. Thus, a robust analytical technology with higher sensitivity is urgently needed.

Recently, fluorophores emitting in the second near-infrared (NIR-II) window with wavelengths ranging from 1,000 to 1,700 nm have achieved visualization of deep anatomical features in in vivo biological imaging [35] [36] [37] [38] [39] . Compared with the visible and the first near infrared window (NIR-I, ~ 700-1,000 nm), the imaging in the NIR-II window can reduce scattering and absorption of photons and lower background auto-fluorescence, affording improved signal to background ratio (SBR) [40, 41] . The coupling of NIR-II fluorescent dyes with biomarkers of SARS-CoV-2 may offer improved visualization modality to understand virus function in vivo. In another side, these features are also favored for detection of biological in vitro assays. However, NIR-II fluorescence has been rarely explored for biological in vitro assay application.

In this study, we applied the NIR-II fluorescent nanoparticles for LFA, and demonstrated sensitive detection of SARS-CoV-2 antigen. The sample concentration was analyzed by the fluorescent signal of over 1,000 nm from the NIR-II fluorescent nanoparticles, affording low background interference and high sensitivity. LoD of 0.01 ng·mL −1 and a wide analytical range of 0.02-120 ng·mL −1 were achieved for N protein of SARS-CoV-2. In a preliminary measurement of 30 clinical pharyngeal swabs (18 COVID-19 infected and 12 uninfected), the NIR-II LFA could successfully detect all the positive samples due to the enhanced SBR. In contrast, the colloidal gold LFA failed to detect 10 positive samples with viral load below ~ 0.068 ng·mL −1 .

The NIR-II fluorescent nanoparticles were prepared by encapsulating organic dyes in polystyrene (PS) nanoparticles (obtained from WWHS Biotech, Inc.). The nanoparticle surface was modified with carboxyl groups, allowing conjugation with the amine functionalized monoclonal antibodies (mAbs117). Scanning electron microscopy (SEM) images showed the nanoparticle size increased and the surface became rough after conjugating with mAbs117 ( Fig. 1(a) ). The dynamic light scattering (DLS) measurement indicated that the mean diameter of the NIR-II nanoparticles was 295.3 ± 5 nm. The size of the labeled NIR-II nanoparticles increased by 47 nm, reaching 342.0 ± 1.3 nm (Fig. S1 in the Electronic Supplementary Material (ESM)), suggesting that the antibodies were successfully conjugated to the surface of NIR-II nanoparticles. The aqueous dispersion of the nanoparticles was light green, and showed bright NIR-II fluorescence ( Fig. 1(b) ). The ultraviolet-visible-NIR (UV-Vis-NIR) absorption spectrum of the NIR-II nanoparticles displayed a peak at 806 nm, whereas the fluorescence emission was peaked at 1,046 nm ( Fig. 1(c) ).

NIR-II fluorescent nanoparticles were introduced to design a rapid LFA for SARS-CoV-2 diagnosis (Fig. 2) . The LFA strip consisted of five parts, sample pad, conjugation pad, nitrocellulose (NC) membrane, absorbent pad, and plastic backing. The as-prepared antibody labeled nanoparticles (NIR-II nanoparticles@mAbs117) were immobilized on conjugation pad. And the N protein capture antibodies (mAbs30) and streptavidin were dispensed on the test line (T line) and control line (C line) on the NC membrane respectively, and the gap between the T and C lines was ~ 3 mm. After being loaded onto the sample pad, sample liquid flowed along the strip to the absorbent pad by capillary forces. During the flowing, the mAbs117 labeled nanoparticles@SARS-CoV-2 N protein complexes formed on the conjugation pad were captured by the mAbs30 antibodies at the T line via the antigen-antibody interaction between SARS-CoV-2 N protein and mAbs30, forming a sandwich immune-complexes structure. The more N proteins in the sample, the more sandwich immune-complexes were aggregated at test line. As the liquid continued to flow, the NIR-II nanoparticles labeled with biotin were trapped by the streptavidin at C line. An 808 nm light-emitting diode was employed to excite the NIR-II nanoparticles and the fluorescent signals were recorded by a low-cost InGaAs photodetector operated at room temperature after filtering with a 1,000 nm longpass filter ( Fig. S2 (a) in the ESM). And the fluorescent signal was positively correlated with the number of the fluorescent nanoparticles. Quantitative sample concentration could be obtained by establishing the corresponding relationship between antigen concentration and fluorescent intensity through integrating the fluorescent signal intensity at the T and C lines ( Fig. S2(b) in the ESM). Based on this principle, a highly sensitive SARS-CoV-2 antigen lateral flow assay method was developed, which could be applied to the SARS-CoV-2 early diagnosis by quantifying its antigen.

The optimized antibody loading on NIR-II nanoparticles and immunoassay time were first screened. It showed that the T/C signal intensity gradually increased with the antibody concentration up to 240 ng·mL −1 . Then the T/C signal intensity did not change significantly when the antibodies concentration ranged from 240 to 360 ng·mL −1 (Fig. S3(a) in the ESM). The immunoassay time was also investigated, and the result indicated that the T/C signal intensity increased rapidly in the first 15 min, then tended to be saturated after 15 min ( Fig. S3(b) in the ESM). Thus, the antibodies concentration of 240 ng·mL −1 and immunoassay time of 15 min were chosen for later studies.

Subsequently, the sensitivity of the NIR-II based LFA was evaluated by detecting SARS-CoV-2 recombinant antigen with varying concentration. The SARS-CoV-2 N protein solutions (100 μL) with the concentration range of 0 to 120 ng·mL −1 were loaded onto the sample pad, respectively. With the increase of N protein concentration, the T/C signal intensity increased accordingly ( Fig. 3(a) ). There was a good linearity relationship between the T/C intensity and the concentration of N protein in the range of 0.02-120 ng·mL −1 , and the correlation coefficient (R 2 ) was over 0.99. Further, a series of low concentration experiments were carried out near the detection threshold. And the LoD was down to 0.01 ng·mL −1 , which was calculated by the mean signal intensity of blank plus triples of standard deviation ( Fig. 3(b) ). Other SARS-CoV-2 antigen detection methods based on LFA were then introduced to compare, and the NIR-II LFA is advantageous for its high sensitivity (Table 1 ). The reproducibility is a key factor in evaluating the effectiveness of a method. Herein, it was evaluated by intraday, intreday variations, and recovery rates. Coefficient of variation (CV) was below 11.6% for intraday and below 12.8% for interday. Recovery rates at N protein concentrations of 0.5, 10, and 100 ng·mL -1 were estimated to 102.6%, 98.9%, and 109.1%, respectively (Fig. 3(c) ). We also verified the specificity of our strips by measuring the influenza A, influenza B, S protein (SARS-CoV-2 recombinant spike protein), and N protein, respectively. The results showed that our strips could specifically recognize SARS-CoV-2, and there were no cross reactions with other proteins (Fig. 3(d) ). It demonstrated that our NIR-II LFA showed high specificity for SARS-CoV-2.

From the test of low antigen concentrations, it could be found that the T line was clearly distinguished from the background signal and the SBR was up to 3.46 when the antigen concentration was down to 0.1 ng·mL −1 (Fig. 4(a) ). When the antigen concentration was as low as 0.01 ng·mL −1 , the SBR could still reach 1.21, though the T line looks blurred ( Fig. 4(a) ).

Subsequently, commercial colloidal gold-based assay kits were accessed for comparison. In the colloidal gold-based assay kits prepared with the same antibody, the T line was indistinct when the antigen concentration was diluted to 0.1 ng·mL −1 and SBR at 0.5 ng·mL −1 was only 1.23 ( Fig. 4(b) ). In another commercial colloidal gold-based SARS-CoV-2 antigen kits (Wandfo Biotech Co., Ltd., cat. W19610617), the T line was indistinct when the antigen concentration was diluted to 0.5 ng·mL −1 (Fig. S4 in the ESM). These results suggested that the NIR-II LFA was more sensitive than colloidal gold LFAs for SARS-CoV-2 antigen detection.

To further validate the NIR-II LFA, we measured 30 clinical swab samples including 18 COVID-19 positive samples and 12 COVID-19 negative samples (PCR-confirmed, from Shenzhen Center for Disease Control and Prevention, China). In NIR-II LFAs, the positive samples and negative samples could be clearly distinguished by comparing the fluorescence intensity distribution curves (Figs. S5(a) and S5(b) in the ESM) and T/C values (Fig.  S5(c) in the ESM). For the tested negative samples, the T/C values were around 0.035-0.039. In the positive samples, the detected N protein concentrations derived from the T/C values were between 0.015 and 13.906 ng·mL −1 . Of which, 10 cases had low antigen concentrations ranging from 0.015 to 0.068 ng·mL −1 (Fig. 5(a) ). Afterwards, we tested the performance of colloidal gold strips (WWHS Biotech, Inc.). Only 8 cases with high antigen concentrations were detected from the 18 positive samples (Fig.  5 (b) and the red boxes in Fig. S6 in the ESM). The remaining 10 cases with low antigen concentrations (< 0.068 ng·mL −1 ) could not be distinguished from the negative samples based on the established cutoff values (the purple ones showed in Fig. 5(b) and Fig. S6 in the ESM). As summarized in Fig. 5(c) , the NIR-II LFA outperformed the colloidal gold LFA as it could reduce the number of false negative samples. It should be pointed that the sample preservation solution and preservation time may affect the accuracy of antigen quantification in clinical samples. Current results imply that the NIR-II method can achieve more sensitive COVID-19 determination in case of suspected cases or earlier stage of infection with lower viral loads, but more detailed study of clinical COVID-19 samples is needed for future applications.

In summary, we have reported a sensitive LFA for SARS-CoV-2 antigen detection based on NIR-II fluorescent nanoparticles loaded with the capture antibody of N protein. A remarkable feature of the platform is that the NIR-II fluorescence detection afford higher SBR due to its advance in reducing photo scattering, light absorption, and auto-fluorescence, affording a low LoD of 0.01 ng·mL −1 for detecting SARS-CoV-2 antigen. Impressively, the clinical swab samples with the antigen content range from 0.015 to 0.068 ng·mL −1 could be successfully detected by using the NIR-II LFA, while the colloidal gold LFA failed within this concentration range. This NIR-II LFA assay enables not only rapidness but also high sensitivity, meeting the demand of early SARS-CoV-2 diagnosis with lower viral load. It should be pointed out that a lowcost InGaAs photodetector is adopted in present method, and we believe the detection sensitivity can be further increased by improving the performance of the reader. By estimating the consumables including lysis buffer and test card, the cost for each test was ca. U.S.$1.0 (Table S1 in the ESM), which was be equivalent to commercial colloidal gold LFA kits. Hence, the NIR-II LFA with cost-effectiveness, high sensitivity, and high specificity, is suitable for rapid/mass screening of COVID-19. It is also promising for point-of-care diagnosis with enhanced performances.

The NIR-II nanoparticles were provided by WWHS Biotech. Inc., (Shenzhen, China). Bovine serum albumin (BSA), biotin, streptavidin, boric acid, sodium tetraborate, 2morpholinoethanesulfonic acid (MES), ethylenediaminetetraacetic acid disodium salt (EDTA·2Na), Tris-HCl, Tween-20, ethanolamine, 1-ethyl-3-(3-dimethytaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and phosphate buffered saline (PBS) were obtained from Aladdin reagent (Shanghai, China). NaN 3 was purchased from Sigma Aldrich. N protein (SARS-CoV-2 Nucleocapsid-His recombinant antigen protein), S protein (SARS-CoV-2 spike recombinant antigen protein), mAbs30 (SARS-CoV-2 N protein capture antibody), and mAbs117 (SARS-CoV-2 N protein binding antibody) were purchased from FRPON Biotech Co., Ltd. (Dong guan, China). Influenza A virus antigen, influenza B virus antigen were purchased from Biocom Biotech Co., Ltd. (Nanjing, China). Fiberglass pads, absorbent pads, and PVC plastic boards were purchased from Kinbio Tech. Co., Ltd. (Shanghai, China). Nitrocellulose membrane was purchased from Nirmidas Inc., (Palo Alto, USA). Colloidal gold LFA kits were purchased from WWHS Biotech. Inc. and Wandfo Biotech Co., Ltd. Other chemicals used in this study were of analytic grade. Deionized water (≥ 18.2 MΩ·cm) purified using a Milli-Q purification system was used in all experiments.

The morphology of NIR-II nanoparticles, NIR-II nanoparticles labeled with antibody was analyzed by SEM (TESCAN MIRA3 LM microscope) images, and the size distribution of the NIR-II nanoparticles and NIR-II nanoparticles@mAbs117 was also characterized by DLS (Zetasizer Pro, Malvern Panalytical) measurements. All NIR-II images were taken by the DeepVision™ Imaging System (Nirmidas Biotech Inc.). All data were analyzed by origin 2017.

200 μL 1% NIR-II nanoparticles were dispersed into 1 mL 0.1 M MES buffer (pH 6.0) by sonication. 50 μL EDC (10 mg·mL −1 in 0.1 M MES buffer) and 150 μL NHS (10 mg·mL −1 in 0.1 M MES buffer) were added to activate the carboxylic acid groups on the NIR-II nanoparticles' surface for 12 min at room temperature. After that, the activated NIR-II nanoparticles were washed with the conjugation buffer (140 mM boric acid and 15 mM sodium tetraborate, pH 6.5) three times and finally dispersed into 400 μL conjugation buffer. 48 μg mAbs117 was then added to conjugation buffer with gentle stirring for 3 h at room temperature, and the nanoparticles were coupled with antibodies by EDC/NHS agents. Then, 1 mL blocking solution (0.5% BSA, 0.1% casein in 50 mM Tris-HCl, and pH 8.0) and 3 μL ethanolamine were added to quench and block the reaction for 2 h. Further, the nanoparticles were washed with the washing buffer (1 × PBS containing 0.5% BSA, 0.1% NaN 3 , 0.4% Tween-20, and pH 7.4) for three times. And the nanoparticles were finally re-dispersed into 1 mL washing buffer. Finally, the antibody-labeled NIR-II nanoparticles were stored at 4 °C for later studies. Accordingly, the biotin-labeled NIR-II nanoparticles (NIR-II nanoparticles@biotin) were obtained by a similar procedure.

The strip was composed of a plastic backing, a sample pad, a conjugation pad, a NC membrane and an absorption pad. The sample pad was fiberglass treated with the buffer (0.4% BSA, 1.2% Tris-HCl, 0.12% EDTA, 0.1% NaN 3 , 0.4% Tween-20, and pH 8.0), and the conjugation pad was fiberglass treated with the buffer (10% trehalose, 0.5% BSA, 0.1% casein, and pH 8.0). The treated sample pad and conjugation pad were dried at 37 °C and 25% relative humidity overnight. 25 μL, mAbs30 (1.0 mg·mL −1 ) and 25 μL streptavidin (1.0 mg·mL −1 ) were dispensed at test and control region on the NC membrane, respectively, using a HM3030XYZ dispenser (KinbioTech Co., Ltd., Shanghai, China) at a rate of 2.25 μL·cm −1 and a speed of 10 cm·s −1 and then dried at 37 °C and 25% relative humidity overnight. 20 μL antibodylabeled NIR-II nanoparticles, 5 μL biotin-labeled NIR-II nanoparticles mixed with 2 mL conjugation pad buffer (10% trehelose, 0.5% BSA, 0.1% casein, and pH 8.0) were used to treat the conjugation pads, and dried at 37 °C and 25% relative humidity overnight. As a result, the labeled NIR-II nanoparticles were immobilized on the conjugation pads. The absorption pad, NC membrane, pretreated conjugation pad, and sample pad were attached to the plastic backing and assembled as a trip with a 1-2 mm overlap sequentially. The assembled plate was cut into pieces with a width of 3 mm, using an automatic strip cutter ZQ2000 (KinbioTech Co., Ltd., Shanghai, China). The strips were stored at room temperature and 25% relative humidity.

The antigen of N protein was diluted to 120, 80, 40, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.04, 0.03, 0.02, 0.01, and 0 ng·mL −1 successively with the dilution buffer (1 × PBS including 0.5% BSA, 0.5% Tween-20, 0.1% NaN 3 , and pH 7.4), and 100 μL diluted antigen was loaded onto the sample pad of the strips. No less than three parallel measurements were carried out for each concentration. The strips were scanned after 15 min post-loading. The results were denoted as the fluorescence peak area of the T line and C line. Subsequently, the relationship between T/C value and N protein concentration was established. In addition, the stability and specificity were investigated by using the pure antigen of N protein.

Nasopharyngeal swabs from COVID-19 clinical samples (18 positive cases and 12 negative cases, confirmed by qRT-PCR) were stored in a universal transport medium. In the process, the viruses were inactivated in the preservation solution and then stored at −80 °C for later use. The experiment was carried out as follows: 100 μL diluted swabs were loaded onto the sample pad of the strip. And the strip was scanned after 15 min post-loading. The signal was collected by the optical reader. In addition, the performance of colloidal gold strips (WWHS Biotech, Inc.) was evaluated by testing these clinical samples. All tests were conducted in a standard P2 laboratory by trained personnel, avoiding potential safety risks. Innovation Commission general project (No. JCYJ20180504165657443). Also, we are grateful to the Shenzhen Center for Disease Control and Prevention for providing and detecting the clinical samples.

Supplementary material (the operation procedure and cost of the materials needed of NIR-II lateral flow assays, the dynamic light scattering spectrum of the NIR-II nanoparticles, the components and testing principle, optimization of main parameters pertaining to the LFA performance, the colloidal gold LFA strip, the fluorescence intensity distribution curves and the T/C values of the strips for clinical samples by NIR-II LFA, and results of clinical swab samples detected by colloidal gold LFA) is available in the online version of this article at https://doi.org/10.1007/s12274-022-4351-1.

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