key: cord-0759118-yeize40i authors: Wang, Chongwen; Yang, Xingsheng; Zheng, Shuai; Cheng, Xiaodan; Xiao, Rui; Li, Qingjun; Wang, Wenqi; Liu, Xiaoxian; Wang, Shengqi title: Development of an ultrasensitive fluorescent immunochromatographic assay based on multilayer quantum dot nanobead for simultaneous detection of SARS-CoV-2 antigen and influenza A virus date: 2021-06-29 journal: Sens Actuators B Chem DOI: 10.1016/j.snb.2021.130372 sha: 42fdd851c64d44a1aaadf32b2e88f7b7f273a2d9 doc_id: 759118 cord_uid: yeize40i Rapid and accurate diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (FluA) antigens in the early stages of virus infection is the key to control the epidemic spread. Here, we developed a two-channel fluorescent immunochromatographic assay (ICA) for ultrasensitive and simultaneous qualification of the two viruses in biological samples. A high-performance quantum dot nanobead (QB) was fabricated by adsorption of multilayers of dense quantum dots (QDs) onto the SiO(2) surface and used as the highly luminescent label of the ICA system to ensure the high-sensitivity and stability of the assay. The combination of monodispersed SiO(2) core (∼180 nm) and numerous carboxylated QDs formed a hierarchical shell, which ensured that the QBs possessed excellent stability, superior fluorescence signal, and convenient surface functionalization. The developed ICA biosensor achieved simultaneous detection of SARS-CoV-2 and FluA in one test within 15 min, with detection limits reaching 5 pg/mL for SARS-CoV-2 antigen and 50 pfu/mL for FluA H1N1. Moreover, our method showed high accuracy and specificity in throat swab samples with two orders of magnitude improvement in sensitivity compared with traditional AuNP-based ICA method. Hence, the proposed method is a promising and convenient tool for detection of respiratory viruses. The ongoing coronavirus disease 2019 (COVID- 19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread to over 214 countries since December 2019 and resulted in more than 104 million J o u r n a l P r e -p r o o f infected individuals and over 2.27 million deaths [1] [2] [3] . SARS-CoV-2 is spread through close person-to-person contact and has high infectiousness rate. The early symptoms (mainly cough and fever) of SARS-CoV-2 infection mimic those of common respiratory viruses, such as influenza A virus (FluA), influenza B virus (FluB), and respiratory syncytial virus (RSV) [4] [5] [6] ; as such, controlling the epidemic spread and guiding clinical treatment are difficult. On another hand, FluA has received extensive attention for a long time because its spread causes seasonal epidemics and raging pandemic [7] . For example, the pandemic of 2009 (swine flu) was caused by FluA H1N1 strain (A/2009/H1N1) and resulted in more than 201,200 deaths [8] . Notably, recent report indicated patients with coinfecting FluA and SARS-CoV-2 have appeared [9, 10] . Thus, methods for accurate and rapid diagnosis of FluA/SARS-CoV-2 are urgently needed. To date, serological testing of virus specific immunoglobulins (e.g., IgM, IgG) in blood samples and direct detection of viral components (e.g., RNA or protein) are the two main strategies for diagnosis of respiratory virus infection [11] [12] [13] . Considering that the initial antibody responses to viral antigens are usually detected in the late stage of infection (7-14 days after virus exposure), serological antibody tests cannot achieve accurate screening of asymptomatic populations or early cases [14, 15] . Current recommended methods for SARS-CoV-2 and FluA detection is real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunoassays for antigens, such as enzyme-linked immunosorbent assay (ELISA) [16] [17] [18] [19] . Although PCR and ELISA could provide accurate results with high specificity for respiratory J o u r n a l P r e -p r o o f tract specimens (nasal or throat swab) in the early stage of infection, the disadvantages include long processing time (> 2h) and tedious procedures, which severely limit their application in point-of-care testing (POCT) area. Immunochromatographic assay (ICA) is a mature POCT technology with the merits of rapidity, low cost, portability, and simple operation and has been widely used in the field of food safety control, human health monitoring, and clinical diagnosis [20] [21] [22] [23] [24] . Developing an ICA-based method for respiratory virus is an ideal approach to improve the detection capability of SARS-CoV-2/FluA infections owing to the following advantages. First, this assay can be used directly to respiratory tract specimens without sample pretreatment steps and provide results quickly (generally 10-20 min). Second, the ICA strip is ready-to-use by ordinary people and can be applied to any public places, such as hospitals, communities, and schools, and is thus suitable for rapid screening virus of infected persons. Third, simultaneous detection of SARS-CoV-2/FluA in the ICA platform can be easily realized by setting two test (T) lines on the strip, thereby improving the detection efficiency. However, traditional ICA method has two inherent defects including limited sensitivity and poor quantitative ability due to the use of colloidal gold (AuNP)-based colorimetric analysis [25, 26] . These defects hinder the large-scale implementation of ICA for respiratory virus diagnosis. In recent years, a new class of luminescent materials named quantum dot (QD) has been introduced into ICA systems to replace colorimetric labels [27] [28] [29] [30] . These materials have advantages of high photostability, narrow fluorescence emission spectra, and strong luminescence. As such, QD J o u r n a l P r e -p r o o f nanoprobes can provide intense and quantifiable fluorescence signal for ICA. Moreover, QD beads (QBs) can be fabricated by encapsulating many QDs into a polymer or silica micelles, which can provide high luminescence and stability [31] [32] [33] [34] [35] . Thus, integrating high-performance QBs into an ICA system is likely to overcome the deficiencies of traditional ICA method and achieve sensitive detection of respiratory viruses. Herein, we developed a two-channel fluorescent ICA method for ultrasensitive and simultaneous detection of SARS-CoV-2/FluA in real biological samples by using a novel silica-QD nanocomposite with triple-QD shell (SiTQD) as the advance signal probe. The innovations of the proposed SiTQD-ICA could be summarized in three points: (i) a novel QB label was fabricated for the first time by adsorption of three layers (thousands) of carboxylated QDs onto monodispersed 180 nm SiO2 surface, which greatly enhanced the fluorescence signal of ICA; (ii) high-performance SiTQD QBs confer the ICA system with high stability and sensitivity for clinical sample testing; and (iii) a two-channel platform was established to simultaneously detect SARS-CoV-2 and FluA. The fluorescence signals of SiTQD labels on the two T lines could be easily observed using a UV light source for qualitative detection and quickly measured by a commercial fluorescent reader for quantitative analysis of SARS-CoV-2 and FluA. Under the optimal conditions, the sensitivity of SiTQD-ICA for detection of SARS-CoV-2 and FluA H1N1 reached 5 pg/mL and 50 pfu/mL, respectively. Moreover, the practical detection capability of the method was evaluated by testing inactivated SARS-CoV-2 samples. The specificity, accuracy, and stability of the J o u r n a l P r e -p r o o f proposed method were fully demonstrated, indicating the strong potential of SiTQD-ICA for POCT use. SiO2 NPs (~180 nm) were synthesized by a typical Stöber method with slight modification [40] . In brief, 1.6 mL of ammonia aqueous solution (28 wt%) and 2.4 mL of deionized water were mixed together in 40 mL of ethanol. The mixture was stirred with a magnetic stirrer at 600 r/min. After 2 min of stirring, 1.6 mL of TEOS was rapidly injected, and the mixture was continuously reacted for 3 h at 25 °C. The resulting SiO2 NPs were collected by centrifugation (6000 rpm, 6 min), rinsed with ethanol twice, and dried at 70 °C in a vacuum oven. The SiTQD nanocomposite with a triple QD-shell was prepared via PEI-mediated The SARS-CoV-2 NP antigen detecting antibody and FluA detecting antibody were The two-channel ICA strip was consisted of a sample pad, a conjugate pad containing two specific immuno-SiTQD labels, a NC membrane with two test lines (T lines) and All the ICA components were placed into a drying oven at 37 °C for 4 h and then attached onto the plastic backing card in sequence. The assembled ICA card was cut into 3 mm-wide strips and preserved in desiccator until use. The testing processes for inactivated SARS-CoV-2 virion and active FluA H1N1 viruses were carried out inside a Class II biological safety cabinet. First, the concentration of FluA H1N1 sample was determined via the classical plaque assays, and the results were displayed in Fig. S1 . Then, the concentration-determined H1N1 virion (1 × 10 8 pfu/mL) were prepared according to the results of plaque assays. After Scheme 1a illustrates the preparation of multilayer SiTQD QBs based on a layer-bylayer (LbL) assembly strategy via PEI-mediated electrostatic adsorption of three layers of QDs onto the surface of SiO2 sphere. The high-performance SiTQD QBs were introduced into the two-channel ICA instead of the common QD with the following characteristics: (i) a ~180 nm SiO2 core as the monodispersed supporter to provide hydrophilicity and colloidal stability; (ii) a triple-layer QD shell containing thousands of QDs to generate high luminescence; and (iii) abundant surface carboxyl groups for convenient surface functionalization. The fabricated SiTQD QBs were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Figs. 1a-1d display the high-resolution TEM images of SiO2, SiQD, SiDQD with dual QD shell, and SiTQD with triple QD shell, respectively. The prepared SiO2 sphere (~180 nm) exhibited a spherical morphology with a smooth surface (Fig. 1a) . Our previous works indicated the cationic PEI could directly coat onto the SiO2 surface through ultrasonic driving [41] . As observed in With the successive adsorption of dual and triple layers of QD, the resulting SiDQD with dual QD shell (Fig. 1c) and SiTQD with triple QD shell (Fig. 1d) were successfully fabricated. The TEM images indicated that the number of QDs loaded onto the SiO2 surface increased obviously with the number of PEI-driven assembly, and numerous QDs densely covered the SiTQD surface to form a hierarchical structure. The average diameter of SiTQD QB increased to approximately 240 nm after triple-QD shell formation (Fig. S3) . The maximum loading amount of QDs for SiQD, SiDQD, and SiTQD were calculated to be approximately 923, 2050, and 3400, respectively (Supporting information S1/ Fig. S4) . Importantly, the SiO2, PEI, and carboxylated QDs were hydrophilic materials, thereby ensuring the excellent dispersity of SiTQD nanocomposites. Figs. 1e-1f reveal that the SiTQD NPs possess high monodispersity as well as SiO2 spheres. The SEM images in Fig. 1g and 1h showed the surface morphology of SiTQD before and after QDs adsorption. The SiTQD surface became rough, which provided large surface site and benefitted antibody conjugation. We employed energy-dispersive X-ray spectroscopy (EDS) mapping for analysis of the elemental distributions in SiTQD. As revealed in Fig. 1i , dense Cd, Se, and Zn were well distributed outside of the Si core, which clearly exhibited the layered shell respectively. Such regular changes in zeta potential confirmed that the fabrication of SiTQD was driven by PEI-mediated electrostatic adsorption [42] . The practical sensing capability of SiTQD NCs was assessed by systematically studying their optical property and colloidal stability. The photographs and fluorescence spectra of the SiO2-based NCs are displayed in Fig. 1k . The SiO2 and SiO2@PEI exhibited no obvious fluorescence signal, whereas NCs with one to triple QD shells all showed bright red fluorescence. By measuring signal intensity of the major emission peak at ~618 nm, the luminescence of SiTQD was 3.58 and 1.76 times higher than those of SiQD and SiDQD, respectively, due to the larger amount of loaded QDs of the triple QD shell. The SiTQD exhibited excellent stability due to the highly stable SiO2 core. As revealed in Fig. S5 , the fluorescence intensity of SiTQD was unaffected by the high-salt environment and long-term preservation. Moreover, the SiTQD showed stable fluorescence intensity in aqueous samples over a wide range of pH values of 4-12 (Fig. 1l) . The superior fluorescence properties and stability of SiTQD ensured its wide applications in complex samples. Two kinds of immuno-SiTQD labels for two target respiratory viruses and an ICA strip with two test lines were prepared for simultaneous detection of SARS-CoV-2 and FluA, as illustrated in Scheme 1b and 1c, respectively. The triple layers of carboxylated QDs formed shell of SiTQD, providing not only sufficient carboxyl J o u r n a l P r e -p r o o f groups for antibody conjugation but also larger external surface area than the common QD with smooth shell, thereby increasing the efficiency of antibody coupling. Using the carbodiimide cross-linking method [43] , the SARS-CoV-2 NP monoclonal antibody and FluA monoclonal antibody were directly immobilized on the surface of SiTQD, respectively. As indicated in Fig. S6 , the zeta potential values of immuno-SiTQD significantly declined after the antibody coupling and remained stable at -13. Therefore, suitable antibodies with affinity and specificity are the key to construct the two-channel ICA. Our previous work screened a pair of efficient mAbs for FluA detection [44] . The SARS-CoV-2 NP mAbs used in this study were selected as shown in Fig. S7 . In addition, some important parameters of paper chromatography strip with J o u r n a l P r e -p r o o f big nanotags (~200 nm) were well studied in our previous works [45] [46] [47] . From these experiences, the NC membrane type and running buffer ingredient needed to be first optimized because they are closely related to the transport of SiTQD/virus complexes on the strip. As revealed in Fig. S8a , using the CN140 membrane with 8 μm pore size achieved the highest signal-to-noise ratio (SNR) for detection of both target viruses. NP-40 was added into the running buffer to effectively lyse the SARS-CoV-2 virion and fully release the NP antigen [48] . Under the optimal conditions, the detection performance of SiTQD-ICA for clinical specimens was evaluated by detecting a series of throat swab samples spiked with different conditions of SARS-CoV-2 NP and H1N1. As shown in the fluorescence image of ICA strips in Fig. 3a(i) , the red fluorescence bands on the test lines became brighter with increasing concentrations of the SARS-CoV-2 NP/H1N1 in the wide J o u r n a l P r e -p r o o f detection ranges of 0.01-100 ng/mL and 100-10 5 pfu/mL, respectively. The fluorescence test lines for 0.01 ng/mL of SARS-CoV-2 and 100 pfu/mL of H1N1 were clearly distinguished from those of negative control by the naked eye. Notably, an obvious "hook effect" [49, 50] was observed at the T2 line for 10 6 where yblank and SDblank are the average fluorescence intensity and standard deviation of the blank groups, respectively [51] [52] [53] . Thus, the LOD values for SARS-CoV-2 NP and H1N1 were estimated as 5 pg/mL and 50 pfu/mL in this assay. As a sensitive POCT tool for respiratory viruses, the performance of SiTQD-ICA should be compared with the traditional AuNP-based ICA strips, which used the same pairs of mAbs. The preparation of AuNP-based ICA was provided in Supporting information S2. As displayed in Fig. 3d , the visual sensitivity levels based on the naked eye of AuNP-based ICA strips for H1N1 and SARS-CoV-2 NP were 5000 pfu/mL and 1 ng/mL. By comparison, the sensitivity of SiTQD-ICA for target virus detection was at least 100 times higher than that of the AuNP-ICA based method. We further compared the testing ability of our method and commercially available ELISA kits. The ELISA analysis for SARS-CoV-2 NP and H1N1 was carried out with the The tested strips of SiTQD-ICA showed higher distinguishable SARS-CoV-2 levels by eye observation. Moreover, the fluorescence intensity values of both T/C lines of SiTQD-ICA strips were much higher than those of SiQD-ICA and SiDQD-ICA (Fig. 4b) . The results confirmed that using SiTQD QBs with superior performance can improve the detection sensitivity of ICA biosensor. In addition, the SiTQD with a huge SiO2 core ensured its monodispersity and excellent stability in sample solution, which benefit the reproducibility of the ICA. As revealed in Fig. 4c , the two-channel SiTQD-ICA exhibited good fluorescence signal reproducibility on the T lines for one virus testing or simultaneous detection of two target viruses. The coefficients of variation (CV) for the fluorescence signals of T1 and T2 lines are below 4.3% and 2.76%, respectively, suggesting the high reliability of our method. Different concentrations of H1N1 virions (2000-100 pfu/mL) and SARS-CoV-2 NP (1-0.05 ng/mL) were spiked into the throat swab samples and tested by SiTQD-ICA to further assess the precision of our method. As demonstrated in Table 1 , the average recoveries of our ICA biosensor ranged from 91.6% to 114.4% for H1N1 samples and ranged from 97.7% to 110.8% for SARS-CoV-2 NP samples, respectively, with a relative low CV ranging from 3.64% to 9.39%. These results indicated the acceptable accuracy of SiTQD-ICA for quantification of two target viruses. The specificity of the SiTQD-ICA was assessed against other respiratory viruses considering the advantages of low cost, highly stable, easy operation, multiplex detection capacity, and short testing time, the proposed method is a promising POCT tool for the rapid and accurate diagnosis of respiratory viruses in real specimens. In this work, we proposed a novel fluorescent SiTQD-ICA for sensitive and simultaneous diagnosis of SARS-CoV-2 and FluA antigens and demonstrated its high performance (sensitivity, stability, specificity, and reproducibility) in biological samples. Highly luminescent and monodispersed SiTQD QB was fabricated by J o u r n a l P r e -p r o o f coating a three-layer QDs formed shell onto the 180 nm SiO2 core via our proposed PEI-mediated LbL assembly method and introduced into ICA system as the advanced optical nanoprobe. Under the optimal conditions, the SiTQD-ICA simultaneously detected SARS-CoV-2 NP and FluA H1N1 in throat swab samples with LOD values of 5 pg/mL and 50 pfu/mL, respectively. The compared test results verified that the sensitivity of SiTQD-ICA was improved by about 100 times than that of traditional AuNP-based ICA method and over 20 times than that of ELISA kits. To the best of our knowledge, this work is the first to develop a two-channel ICA for simultaneous detection of SARS-CoV-2 and FluA. We believe that the proposed method could be an efficient POCT tool for direct, rapid, and accurate detection of pathogenic respiratory viruses. The authors declare no conflict of interest. Her work focuses on the development of fluorescent biosensors. Chongwen Wang is currently a professor at Anhui Agricultural University in China. His work focuses on the preparation and application of novel metal and magnetic nanomaterials. Shengqi Wang is currently a professor at the Beijing Institute of Radiation Medicine. His research interests include DNA/protein biosensing, virology, and pharmacology. 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its immune cross-reactivity with SARS-CoV Unraveling the Hook Effect: A Comprehensive Study of High Antigen Concentration Effects in Sandwich Lateral Flow Immunoassays Sensitive and hook effect-free lateral flow assay integrated with cascade signal transduction system Simultaneous Detection of Dual Nucleic Acids Using a SERS-Based Lateral Flow Assay Biosensor Quantitative and ultrasensitive detection of multiplex cardiac biomarkers in lateral flow assay with core-shell SERS nanotags Dual-Signal Readout Nanospheres for Rapid Point-of-Care Detection of Ebola Virus Glycoprotein Biographies Xingsheng Yang is a master student under the guidance of Prof. Chongwen Wang at Anhui Agricultural University in China. His work focuses on the development of ICAbased biosensors Shuai Zheng is a currently a doctoral candidate under the guidance of Prof. Rui Xiao at Anhui Agricultural University in China. His work focuses on the development of POCT methods Xiaodan Cheng is a master student under the guidance of Prof. Chongwen Wang at Anhui Agricultural University in China. Her work focuses on the development of ICAbased biosensors Rui Xiao is currently a professor at the Beijing Institute of Radiation Medicine. Her research interests include SERS-based biosensor and nanomaterials-based sensors Shengqi Wang at Shandong University of Traditional Chinese Medicine in China. His work focuses on the development of detection method for viruses Wenqi Wang is a master student under the guidance of Prof. Chongwen Wang at Anhui Agricultural University in China. His work focuses on the development of ICA-based biosensors Supporting Information is available from the author. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.