Magnetic SERS Strip for Sensitive and Simultaneous Detection of

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Magnetic SERS-strip for sensitive and simultaneous detection of respiratory viruses Chongwen Wang, Chaoguang Wang, Xiaolong Wang, Keli Wang, Yanhui Zhu, Zhen Rong, Weiyun Wang, Rui Xiao, and Shengqi Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03920 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 6, 2019

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ACS Applied Materials & Interfaces

Magnetic SERS-strip for sensitive and simultaneous detection of respiratory viruses Chongwen Wanga,b,e, Chaoguang Wangd, Xiaolong Wangc, Keli Wangb, Yanhui Zhub, Zhen Rongb, Weiyun Wanga*, Rui Xiaoa,b,*, Shengqi Wanga,b,e,* a.

College of Life Sciences, Anhui Agricultural University, Hefei 230036, PR China. Beijing Key Laboratory of New Molecular Diagnosis Technologies for Infectious Disease, Beijing Institute of Radiation Medicine, Beijing 100850, PR China. c Shandong Provincial Key Laboratory of Traditional Chinese Medicine for Basic research, Shandong University of Traditional Chinese Medicine, Jinan 250355, PR China. d College of Mechatronics Engineering and Automation, National University of Defense Technology, Changsha 410073, PR China. e Anhui Provincial Key Laboratory of Veterinary Pathobiology and Disease Control, Anhui Agricultural University, Hefei 230036, PR China. b

ABSTRACT Rapid and early diagnosis of respiratory viruses is key to preventing infections from spreading and guiding treatments. Here, we developed a sensitive and quantitative surface-enhanced Raman scattering-based lateral flow immunoassay (SERS-based LFIA) strip for simultaneous detection of influenza A H1N1 virus and human adenovirus (HAdV) by using Fe3O4@Ag nanoparticles as magnetic SERS nanotags. The new type of Fe3O4@Ag magnetic tags, which were conjugated with dual-layer Raman dye molecules and target virus-capture antibodies, perform the following functions: specific recognition and magnetic enrichment of target viruses in the solution, and SERS detection of the viruses on the strip. Based on this strategy, the magnetic SERS-strip can directly be used for real biological samples without any sample pretreatment steps. The limits of detection (LODs) for H1N1 and HAdV measured 50 and 10 pfu/mL, respectively, which were 2000 times more sensitive than those from standard colloidal gold strip method. Moreover, the proposed magnetic-strip is easy to operate, rapid, stable and can achieve high throughput and is thus a potential tool for early detection of virus infection. Key words: Lateral flow assay, magnetic-SERS-strip, Fe3O4@Ag, respiratory virus, SERS

1. INTRODUCTION Common respiratory viruses mainly include influenza A virus (FluA), influenza B

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virus (FluB), human adenovirus (HAdV), parainfluenza virus (PIV) and respiratory syncytial virus (RSV), are still serious public health concerns associated with considerable morbidity and mortality, as well as high economic losses.1-3 Children and infants, elderly, and individuals with compromised immune systems are easily infected with respiratory viruses.4-5 Moreover, the caused illnesses can mimic other viral and bacterial infections and could lead to unnecessary treatments when misdiagnosed.6 According to the World Health Organization, acute respiratory viral infections cause 3.9 million deaths in humans annually.7 Many diagnostic tools, such as cell culture,8 polymerase chain reaction (PCR),9 sequencing-based tests,10 and enzyme-linked immunosorbent assays (ELISA),11 have been invented and progressively applied to accurately detect respiratory viruses at the early stages and guide appropriate treatments. However, all these classic methods have one or more disadvantages. For example, cell culture is complex and requires 1–2 weeks to replicate and additional days for confirmation by immunological or molecular biological methods. PCR, sequencing, and ELISA can achieve rapid microbiological identification with assay times of several hours, but these methods involve tedious procedures, expensive equipment, or skilled personnel, thereby limiting their application in field detection. Lateral flow immunoassay (LFIA) strip has become a popular point-of-care testing (POCT) technology because of its simple operation, easy production, low cost, short assay time, and robustness in various applications.12-14 However, conventional LFIA, which mainly uses colloidal gold as reporters and is based on color visualization, can only provide poor detection sensitivity and limited qualitative or semiquantitative detection capability. Several novel signal-enhancement strategies, including fluorescence signal-based strategy,15-16 magnetic signal-based strategy,17-18 enzyme-amplified signal system,19 double-targeted nanogold method,20 and surface-enhanced Raman scattering (SERS) signal-based system,21-23 have been recently reported to improve the sensitivity and quantitative detection capability of LFIA strips. Among these methods, SERS-based LFIA strips have recently attracted attention owing to their integration of high sensitivity and quantitative analysis by using functional SERS-encoded nanoparticles (SERS tags) instead of colloidal gold as signal reporters. A typical SERS nanotag consists of three basic components: a Au/Ag nanoparticle as Raman enhanced substrate, adsorbed Raman reporter dyes to produce characteristic SERS signals, and specific antibodies to bind targets. Studies have shown that the combination of SERS tags and LFIA strips enables the quantitative evaluation of specific analytes (e.g., staphylococcal enterotoxin B, pneumolysin, cardiac biomarkers, and DNA markers) with high sensitivity.24-28 All these


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applications are based on colloidal Au/Ag SERS tags but have not been used for live virus detection yet. Additionally, in complex samples, colloidal Au/Ag NPs are easily affected by environmental factors, such as interference, pH value, and salt ions strength.29 However, the compositions of clinical respiratory specimens are complex and can adversely affect the practical application of SERS-based LFIA strips. In addition, specific areas could be improved to increase the performance of SERS-based LFIA: (i) optimization of particle size and shape of SERS tags to achieve lower non-specific signal on the strip; (ii) improvement of SERS activity by controlling stable hot spots in the nanostructure, (iii) good stability of SERS tags for biological sample detection, and (iv) SERS tags with analyte enrichment ability. In recent years, Au/Ag-coated Fe3O4 magnetic NPs (Fe3O4@Ag or Fe3O4@Au MNPs) have attracted wide attention for their excellent versatility and stability, especially for complex sample detection.30-31 Given the combination of SERS capability of Au/Ag NPs and sample enrichment capability of Fe3O4 MNPs, these magnetic SERS substrates can be used to separate the target analyte from complex solution by an external magnetic field and serve as stable SERS substrates to enhance the Raman signal of target.32-34 However, all applications of these Au/Ag-coated MNPs in SERS detection are limited to act as magnetic separation tool and SERS substrates. No study has reported the use of Au/Ag-coated Fe3O4 MNPs as magnetic SERS tags in LFIA strips. In this study, we designed and synthesized new dual dye-loaded Ag-coated Fe3O4 MNPs (Fe3O4@Ag tags) as magnetic SERS tags. Fe3O4@Ag SERS tags possess good dispersity, stability, enrichment capability, and excellent SERS activity and potentially act as high-performance SERS tags and fully meet the actual requirements of clinical sample SERS-LFIA detection. Based on the high-performance Fe3O4@Ag SERS tags, a new type of SERS-LFIA strip was further proposed for simultaneous and highly sensitive detection of two common respiratory viruses (FluA H1N1 and HAdV) in biological samples. For our assay, magnetic SERS tags were used instead of Au/Ag colloidal SERS tags, and both served as the concentration and SERS tool in the SERS-LFIA system. The

designed

Fe3O4@Ag

SERS

tags

comprised

three

parts:

150

nm

superparamagnetic Fe3O4@Ag cores to provide sufficient magnetic response property and SERS activity, two layer of dye molecule 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) to produce strong Raman signal, and surface-modified virus-capture antibodies to specifically recognize target respiratory viruses. Based on the stability and enrichment ability of MNPs, the proposed magnetic SERS-strip can be directly used for real biological samples without sample pretreatment steps. The characteristic



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SERS signal intensities of Fe3O4@Ag tags trapped on two test lines were utilized for quantitative detection of H1N1 and HAdV. The sensitivity values of the magnetic SERS-strip for H1N1 and HAdV detection were as low as 50 and 10 pfu/mL, respectively, which were 2000-fold lower than those of the standard colloidal gold strip. As far as we know, this work is the first to introduce magnetic SERS tags into lateral flow immunoassay strip. The magnetic SERS-strip offers new perspectives for sensitive detection of virus with POCT use in biological samples.

2. EXPERIMENTAL SECTION 2.1. Materials and chemicals Sodium borohydride (NaBH4), chloroauric acid tetrahydrate (HAuCl4·4H2O), silver nitrate (AgNO3), and ferric chloride (FeCl3·6H2O) were obtained from Shanghai Chemical Reagent Co. (China). Polyethyleneimine (PEI) with a MW of 25 kDa, MES monohydrate, polyvinylpyrrolidone (PVP) with a MW of 40 kDa, DTNB, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

hydrochloride

(EDC),

and

N-hydroxysuccinimide (NHS) were obtained from Sigma-Aldrich (USA). The binding buffer solution was prepared by 10 mM phosphate-buffered saline (PBS, pH 7.4). 10 mM PBS solution containing 1% Tween 20, 10% fetal bovine serum (FBS) and 1% bovine serum albumin (BSA) was used as running buffer. All the aqueous solutions were prepared by deionized water which was purified with a Millipore Milli-Q system (18.2 MΩ·cm−1). Nitrocellulose (NC) membrane (UniSart CN95) with 15 μm pore size was obtained from Sartorius (Spain). The conjugate pad, absorbent pad, and Millipore glass fiber sample pad were provided by GE Healthcare (UK). Mouse monoclonal anti-adenovirus antibodies (ADEN-001, ADEN-002) were produced by ACTHTEAM, LLC (USA). Mouse monoclonal anti-Flu A antibodies (10-1241, 10-1243) were provided by Fitzgerald Industries International, Inc. (USA). HAdV (serotype 5) and influenza A/FM/1/86 (H1N1) virus were obtained from the Institute of Disease Control and Prevention, PLA, Beijing. The viruses were cultured in A549 cells, quantified by a plaque assay method, and then frozen at −70 °C for future use. The mean diameters of HAdV and H1N1 particle were approximately 60 and 100 nm, respectively, as shown in Fig. S1. Other common respiratory viruses, influenza B (FluB), respiratory syncytial virus (RSV), and parainfluenza (PIV) were also obtained from the Institute for Disease Control and Prevention, PLA, Beijing. Human biological samples were obtained from Affiliated Hospital of Xuzhou Medical University. All experimental operations were performed following the guidelines approved by the Ethics Committee of the Beijing Institute of Radiation Medicine.


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2.2. Instrumentation Transmission electron microscopy (TEM) images were obtained with a Hitachi H-7650 TEM instrument operated at an accelerating voltage of 80 kV. High-resolution TEM (HRTEM) images were obtained with a FEI Tecnai G2 F20 electron microscopy at an accelerating voltage of 200 kV. Scanning electron microscope (SEM) images were taken on a JEOL JSM-7001F instrument operated at 10 kV. The magnetic properties of the products were studied via a superconducting quantum interference device magnetometer (SQUID, MPMSXL-7) at 300 K. UV-vis absorption spectra of MNPs were measured via a Shimadzu UV-2600 spectrometer. Superconducting quantum interference device magnetometer (MPMSXL-7) was used to investigate the magnetic properties of the as-prepared MNPs at 300 K. SERS spectra were acquired on a Renishaw inVia plus Raman system with a 785 nm laser and maximum laser power of 300 mW. Olympus BX51 optical microscope was used to couple incident radiation. The laser was focused to a 50 μm diameter spot by using a 20× objective. The illumination area was fixed at 5 × 50 μm. For SERS measurement, the laser power was set to 3 mW, and the acquisition time was 5 s. The obtained spectra were baseline-subtracted and smoothed by using Renishaw Wire 4.2. Ten random tests were measured on the test line for each sample. For each spectrum, ten independent spectra were collected and averaged before use. The mapping images of the T line of magnetic SERS-strip with SERS signal at 1332 cm−1 were obtained by using a 20× objective lens. For each T line, a 44 µm (x axis) and 100 µm (y axis) range (total 275 pixels) area was scanned in 4 µm × 4 µm steps by using a computer-controlled x-y translational stage. The laser power was set to 1% of the maximum power, and the acquisition time at each pixel was set to 1 s. 2.3. Preparation of dual-DTNB Fe3O4@Ag SERS tags for magnetic SERS-strip Dual DTNB-modified magnetic SERS tags were fabricated by attaching dye molecules (DTNB) in and on the Ag nanoshells of Fe3O4@Ag MNPs. The monodispersed DTNB-inserted Fe3O4@Ag MNPs were designed and synthesized through our proposed PEI-interlayered strategy with specific modifications.35-36 The detailed synthesis process is provided in Supporting Information S1. Then, 0.1 mL freshly prepared DTNB ethanol solution (10 mM) was added to 20 mL Fe3O4@Ag ethanol solution, and the mixed solution was vigorously sonicated for more than 2 h. Then, the formed DTNB-modified Fe3O4@Ag MNPs were activated through immersion in a MES buffer (100 mM) containing 1 mM EDC and 2 mM NHS for 15 min. After being magnetic separation and resuspension in PBS buffer, the capture antibody (10 µg) was added into the tube. After 2 h incubation, the Fe3O4@Ag SERS tags were then blocked with 10% BSA for an additional 1 h. Finally, the Fe3O4@Ag



SERS tags were magnetically collected, washed twice with PBST, and resuspended with 0.5 mL stock solution (10 mM PBS containing 0.1% BSA) for future use. 2.4. Preparation of LFIA strip for detecting two respiratory viruses The control line of the NC membrane was coated by spraying 0.8 mg/mL polyclonal goat anti-mouse IgG, and the two test lines were separately coated by spraying HAdV detection antibody (1 mg/mL) and H1N1 detection antibody (1 mg/mL) to detect two viruses. These antibodies were sprayed onto the line area of NC membrane with a constant dispense rate (0.1 μL/mm) via a line dispensing instrument (BioDot XYZ3050). The control line and two test lines were positioned at a fixed interval (4 mm). The prepared NC membrane was dried at 37 °C for more than 2 h. Finally, the NC membrane, a sample pad, and a absorbent pad were assembled onto a plastic backing plate and finally cut into individual 3.5 mm strips for future use. 2.5. Viral DNA extraction and PCR amplification of HAdV DNA Three HAdV samples were harvested. Viral DNA was extracted using a commercial kits, TIANamp Virus DNA/RNA Kit (Tiangen Biotech) following the instructions. In brief, 3 μL of DNA extracted from HAdV was used as a template, and highly conserved genomic sequences of the adenovirus hexon gene were amplified using the extracted viral DNA via PCR.37-38 The PCR products were determined using classical gel electrophoresis to obtain the bands. The primer sequences for the adenovirus hexon gene were as follows: Forward: 5ʹ-GCCACGGTGGGGTTTCTAAACTT-3ʹ and Reverse: 5ʹ-GCCCCAGTGGTCTTACATGCACATC-3ʹ. 2.6. Viral plaque assay The experimental section for viral plaque assay is provided in Supporting Information S2.


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Scheme 1. (a) Synthetic route for antibody-modified Fe3O4@Ag magnetic tags and (b) schematic diagram of magnetic SERS-strip for detecting two respiratory viruses.

3. RESULTS AND DISCUSSION 3.1. Operating principle of magnetic SERS-strip The principle of the magnetic SERS-strip for two respiratory viruses detection is illustrated in Scheme 1. In this detection system, we used Fe3O4@Ag MNPs as SERS nanotags instead of colloidal gold, which is commonly used in the standard strip method, to improve the sensitivity and quantitative capability of LFIA. The Fe3O4@Ag SERS tag is a critical factor for the performance of the magnetic SERS-strip, which consisted of the following three parts: Fe3O4@Ag MNP as a magnetic capturer and enhancing substrate, dual-labeled DTNB molecules to generate strong and stable SERS signals, and virus-capture antibodies modified with magnetic tag surface to specifically recognize HAdV and H1N1 (Scheme 1a). It should be noted that the 150 nm Fe3O4@Ag cores of the Fe3O4@Ag SERS tags are fabricated based on our previously reported PEI-mediated seed growth method,35-36 which possess strong SERS activity, good dispersity, and enough magnetic responsiveness, and are stable magnetic carrier materials for SERS based immunoassay in complex solution. Herein, the dual dye-modified Fe3O4/DTNB@Ag/DTNB nanostructure was elaborately designed as a high-performance magnetic SERS tags by modifying DTNB on the surface and embedded in the Fe3O4@Ag MNPs to create strong SERS signal.



Owing to the abundant intraparticle hot spots and good chemical stability of Fe3O4@Ag MNPs, the dual-DTNB modified magnetic tags could act as high-performance SERS nanotags for LFIA strip use. Scheme 1b shows the operating procedure of magnetic SERS-strip for the sensitive and simultaneous detection of two respiratory viruses (HAdV and H1N1) by using Fe3O4@Ag SERS tags. The magnetic SERS-LFIA system is composed of a NC membrane, a sample application pad, an absorbing pad, test line 1 for H1N1, test line 2 for HAdV, and a control line for quality control. The magnetic SERS-strip based on antibody–antigen interaction and was performed in a microtube and a strip in sequence. First, the Fe3O4@Ag SERS tags were added in virus samples and shaken at room temperature. The HAdV or H1N1 were captured by the antibody-conjugated magnetic tags during incubation. After the target viruses were captured and separated by Fe3O4@Ag SERS tags from the sample solution, the Fe3O4@Ag SERS tag–viruses complexes were resuspended in 70 μL of running buffer (containing 10 mM PBS, 1% Tween 20, and 1% BSA), and then vigorously whirled for 10 s. The complexes were dropped to the sample pad, continued to migrate along the NC membrane through capillary action, and were caught by the immobilized virus detection antibodies on the test lines. Thus, the magnetic SERS tag–viruses–detection antibody sandwich-like immunocomplexes were formed and accumulated at the test lines (test line 1 for H1N1 and test line 2 for HAdV). The excess Fe3O4@Ag SERS tags continually migrated along the NC membrane of strip and were captured by goat anti-mouse IgG immobilized on the control line of strip. Consequently, two dark bands appeared in the test lines in the presence of the two target viruses, whereas only test line 1 or test line 2 turned to one dark band in the presence of one target virus H1N1 or HAdV. When no target virus existed, one dark band of the control line could be observed. This phenomenon verified that the magnetic SERS-strip was effective, and the modified antibodies were active. Finally, ultrasensitive and quantitative detection was conducted by testing the SERS intensity of the test lines using a portable Raman spectrometer. Our SERS-strip facilitates an indirect test, and the specific SERS signal is produced by the Fe3O4@Ag magnetic tags. Thus, the sensitivity and reliability of the method highly depends on the performance of Fe3O4@Ag MNPs. Too small Fe3O4@Ag MNPs usually caused for the speed and loss issues of magnetic enrichment, whereas huge Fe3O4@Ag MNPs easily blocked the pores of NC membrane and consequently resulted in a high non-specific signal. Herein, Fe3O4@Ag SERS tag of proper size (150 nm) was designed and fabricated to achieve fast separation, highly sensitive, and specific detection of two target viruses on the


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LFIA strip. 3.2 Characterization of the properties of Fe3O4@Ag SERS tags The synthetic route of Fe3O4@Ag magnetic tags is elucidated in Scheme 1a. Fe3O4 MNPs with a diameter of about 120 nm were first prepared through a modified solvothermal reaction (Fig. 1a). The Fe3O4 MNPs were ultrasonically treated with PEI aqueous solution to form positively charged Fe3O4@PEI MNPs. After the Fe3O4@PEI MNPs were coated with 3 nm Au NPs, tiny Au seeds adhered to the all over Fe3O4 surface (Fig. 1b). The as-obtained Fe3O4-Au seed MNPs were modified with Raman dye molecules under ultrasonic irradiation for 1 h. During this process, DTNB molecules easily coupled with Au seeds by forming the Au–S chemical bond. Finally, a continuous and rough Ag shell outside the Fe3O4 core was formed through the reduction of AgNO3 onto the absorbed Au seeds under sonication. The Fe3O4@Ag MNPs are uniform with a size of approximately 150 nm, and the Ag shell is 15 nm thick (Fig. 1c). Shown in Fig. 1d-1f are magnified TEM images for single Fe3O4, Fe3O4-Au seed, and Fe3O4@Ag MNP, respectively. Moreover, the SEM image (Fig. 2a) confirmed that the Fe3O4@Ag tags were successfully synthesized with a rough Ag shell and relatively uniform size.

Figure 1. TEM images of the prepared (a) Fe3O4, (b) Fe3O4-Au seed, and (c) Fe3O4@Ag MNPs, with their corresponding magnified TEM images of single MNP in (d), (e), and (f), respectively. Elemental composition of Fe3O4@Ag MNPs was confirmed through X-ray diffraction (XRD) and X-ray mapping. Fig. 2b displays the relative elemental mapping of an individual Fe3O4@Ag MNP. Fe element was mainly situated in the Fe3O4 core, whereas Au signal was found distributed in the middle of the MNP owing to the absorbed Au seeds. Ag element was mainly distributed on the shell, thus



confirming that a uniform Ag shell was successfully coated on the surface of Fe3O4 MNP. Powder XRD also confirmed the crystal structure and phase purity of the Fe3O4@Ag MNPs as shown in Fig. 2c. Additionally, the X-ray photoelectron spectroscopy (XPS) spectrum of DTNB-modified Fe3O4@Ag MNPs (Fig. S2) indicated the presence of strong Ag 3d5/2 (~367.6 eV) and Ag 3d3/2 (~373.6 eV) peaks, indicative of the Ag shell formation.39 The appearance of C 1s (~285.0 eV) and N 1s (~398.4 eV) was attributed to DTNB molecules. No obvious Fe peaks could be observed, which confirmed that a complete and thick Ag nanoshell was coated on the Fe3O4 MNPs. Fig. S3 shows the UV–visible spectra of Fe3O4@Ag MNPs in Milli-Q water and reflectance spectrum of the synthesized Fe3O4@Ag SERS tags on the strip. This red shift phenomenon can be ascribed to localized surface plasmon resonance wavelength of Fe3O4@Ag aggregates; this wavelength is much longer than that of the isolated single Fe3O4@Ag MNP. The magnetic properties of the magnetic SERS tags were showed in Fig. 2d. The saturation magnetization (MS) values of the obtained Fe3O4 and Fe3O4@Ag tags are found to be 75.9 and 42.3 emu/g, respectively. The MS value is weakened after the formation of Ag nanoshell. However, the relatively high magnetite content (ca. 55.7 wt%) enabled the Fe3O4@Ag tags to rapidly separate from the sample solution within only 30 s upon application of an external magnetic field (Fig. 2e). These results demonstrate that such Fe3O4@Ag MNP could be used as a strong magnetic core of SERS tags for the rapid separation and enrichment of targets.


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Figure 2. (a) SEM image of Fe3O4@Ag MNPs. (b) Elemental mapping images of a typical Fe3O4@Ag MNP. The red, blue, and yellow colors show the element distributions of Fe, Au, and Ag in one MNP, respectively. (c) Typical XRD pattern of the Fe3O4@Ag MNPs. (d) Magnetic hysteresis curves of Fe3O4@Ag MNPs, and (e) magnetic separation behavior of Fe3O4@Ag MNPs in the solution. DTNB molecules were used to build dual-dye magnetic SERS tags due to their high Raman scattering cross-section, unambiguous vibrational modes, and free carboxyl groups as antibody conjugation sites.40-42 When DTNB molecules were mixed with Fe3O4@Ag MNPs, the double sulfur bond of DTNB broke down, thus leaving the thiol groups anchored onto the Ag shell and forming an Ag–S bond.43 Two other kinds of single-DTNB-modified Fe3O4@Ag tags (Fe3O4/DTNB@Ag and Fe3O4@Ag/DTNB) were prepared, and their SERS activity was compared with that of the dual DTNB-loaded Fe3O4@Ag SERS tags. All these Fe3O4@Ag SERS tags were modified with the same concentration DTNB under sonication for 2 h. The SERS signals of the three kinds of Fe3O4@Ag nanotags and Fe3O4-Au seed/DTNB are shown in Fig. S4. All the feature peaks at 738, 848, 1063, 1152, 1332, and 1556 cm−1 of DTNB were observed and consistent with the previously published data,44 and the main peak at 1332 cm−1 was utilized as standard for comparison. The intensity of SERS spectra of the dual-DTNB modified Fe3O4@Ag SERS tags was 13.5 times higher than that of the Fe3O4-Au seed/DTNB, and 1.8 and 2.1 times higher than those of Fe3O4@Ag/DTNB and Fe3O4/DTNB@Ag tags, respectively. The advantages of the dual DTNB-modified Fe3O4@Ag SERS tags are the density hot spots built in the nanostructure and the flexibility for bioconjugation of the detection antibody. We considered the suitable capillary speed rate of NC membrane for running Fe3O4@Ag MNPs. Two kinds of NC membrane (CN95 and CN140) with different pore sizes were tested. As shown in Fig. S5, the CN95 membrane with a larger pore size provided good transport of the Fe3O4@Ag MNPs, whereas obvious Fe3O4@Ag agglomerates were observed in the CN140 membrane. Moreover, no characteristic Raman signal of the Fe3O4@Ag tags was detected in the CN95 membrane. Thus, the CN95 NC membrane was chosen. The running buffer is another important factor in ensuring the good performance of the SERS-LFIA strip as it strongly affects the flow rate of SERS tags and antibody binding capacity of the test line. Various kinds of commonly used running buffer solutions were tested, and 10 mM PBS (pH 7.4) was selected as it causes no effect on stability of antibody-conjugated SERS tags. Tween 20 was added in the PBS buffer as surfactant to prevent the pore of NC membrane from being blocked by magnetic SERS tags. With the increasing concentration of



Tween 20 in the PBS buffer (0%–1%), the fluidity of SERS tags on the NC membrane gradually improved (Fig. S6a). The PBST (1% Tween 20) can ensure that 150 nm Fe3O4@Ag MNPs are unimpeded on the strip. Fig. S6b indicate that addition of 10% FBS and 1% BSA to the above PBS running buffer (10 mM, 1% Tween 20) can effectively reduce nonspecific adsorption and improve the signal-to-noise ratio of the test lines. Therefore, we selected 10 mM PBS solution containing 1% Tween 20, 10% FBS, and 1% BSA as the final running buffer. Notably, the fluorescence backgrounds of the NC membrane differed under different laser excitations, which may disturb SERS detection. As shown in Fig. S7, the fluorescence background of CN95 membrane was higher under the laser source of 532 and 633 nm than at 785 nm. The high fluorescence background will affect the quantification of SERS signal and detection sensitivity. Additionally, the 785 nm excitation wavelength was more suitable to reduce the damage to biological samples.45-46 As a consequence, the 785 nm laser device was employed for SERS detection of respiratory viruses in the magnetic SERS-strip. 3.3. Enrichment performance of the antibody-conjugated Fe3O4@Ag SERS tags The absorption band of protein at 280 nm was investigated for confirmation of antibody conjugation on the surface of magnetic SERS tags. Fig. S8 shows that the absorbance intensity at 280 nm of the liquid supernatant of the magnetic SERS tags remarkably decreased after the antibody modification. Furthermore, Dylight 488-conjugated goat anti-mouse antibody was used to treat the mouse monoclonal HAdV antibody-modified Fe3O4@Ag SERS tags. As shown in Figs. 3a–c, many green fluorescent second antibodies were specifically bonded to the Fe3O4@Ag SERS tags. These results indicate that the virus-capture antibody was effectively modified on the surface of Fe3O4@Ag SERS tags. In this study, Fe3O4@Ag MNPs were modified with capture antibodies to realize the capture and separation of two respiratory viruses. Moreover, the capture ability of magnetic SERS tags was investigated. HAdV (serotype 5) was used as the model virus, and the viral titer was adjusted to 103 pfu/mL. Anti-HAdV antibody-modified Fe3O4@Ag MNPs were incubated with HAdV sample at room temperature for 20 min with shaking. The formed complexes were magnetically separated. Then, 20 μL of the supernatant from each group was tested in viral plaque assay. Photos of culture plates revealed that most of HAdV was captured by the Fe3O4@Ag SERS tags, and only a few viruses remained in the supernatant (Figs. 3d–3e). The capturing of HAdV on Fe3O4@Ag SERS tags was further investigated through PCR. The Fe3O4@Ag SERS tags–HAdV complexes were extracted by using a viral nucleic acid extraction kit, and the obtained product was amplified by using PCR. The bright bands of Fe3O4@Ag


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SERS tags–HAdV complexes were consistent with those of the positive control, thus indicating that a large number of HAdV was captured by Fe3O4@Ag SERS tags (Fig. 3f).

Figure 3. (a) Brightfield, (b) fluorescence, and (b) merged images of mouse monoclonal HAdV antibody modified-Fe3O4@Ag SERS tags conjugated with Dylight 488-labelled goat anti-mouse IgG. Plaque assay showing the amounts of adenovirus in the supernatant (d) before and (e) after Fe3O4@Ag SERS tag capture. (f) PCR amplification of adenoviral genomic DNAs of the magnetically enriched Fe3O4@Ag SERS tags/adenovirus complexes. Samples 1–3 under the same condition, sample 4 is blank control, and sample 5 using pure adenovirus is positive control. 3.4. Detection capability of magnetic-SERS strip As expected from our design, the Fe3O4@Ag SERS tags–viruses were captured on the test lines of strip by the “capture antibody-HAdV-detection antibody” sandwich-like immunoassay mechanism, and the presence of HAdV and H1N1 can be easily measured by a color change on the test zones. Fig. 4a displays the photographs of magnetic-SERS strip for the simultaneous detection of two target viruses. The corresponding color changes of the test lines of strips can be observed for different viral titers of HAdV and H1N1. Figs. 4b-4e show the SERS signals of two test lines for the four test magnetic-SERS strips. The independent qualitative analysis was performed by watching the color changes of two individually test lines: test line 1 for H1N1 and test line 2 for HAdV. No cross reaction of two respiratory viruses were present in the magnetic-SERS strip. Notably, the C lines turned black regardless of the existence of target viruses or not, which can prove that the SERS-strips were working normally. Moreover, a strong SERS signal was observed on the test lines of groups containing the target viruses, whereas no SERS activity was detected on the same zone of the negative group. SEM images showed the corresponding “positive–



negative” conditions of target viruses in test lines. Many Fe3O4@Ag SERS tags were observed in the paper fiber pores of the positive group (Fig. S9a), whereas no immunocomplexes were attached on the negative group (Fig. S9b). These results strongly prove that Fe3O4@Ag SERS tag-based immunocomplexes were formed and accumulated on the test lines when the target viruses appeared, and they caused the color change to black and acted as source of SERS signals. Thus, target viruses can be quantitatively detected through the SERS signal of the Fe3O4@Ag tags on the test lines. The SERS signal strength was proportional to the number of Fe3O4@Ag tags accumulated on the test zones.24, 47

Figure 4. (a) Photographs and (b) corresponding SERS spectra of magnetic-SERS strip in the presence of (1) HAdV, 105 pfu/mL; H1N1, 105 pfu/mL; (2) HAdV, 0 pfu/mL; H1N1, 105 pfu/mL; (3) HAdV, 105 pfu/mL; H1N1, 0 pfu/mL; (4) HAdV, 0 pfu/mL; H1N1, 0 pfu/mL. HAdV and H1N1 samples with different concentration were prepared and tested to assess the sensitivity and linearity range of magnetic SERS-strip for detecting the two target viruses. The concentrations of two respiratory viruses were determined by using plaque assay. The detection process is described in Section 3.1 and Scheme 1b. Fig. 5b shows the photographs and SERS mapping images of two independent test lines of Raman peak at 1332 cm−1 for different concentrations (107 pfu/mL to 10 pfu/mL) of two target viruses. The color of Fe3O4@Ag SERS tags immobilized onto the test lines was clearly observed at high viral titer and gradually decreased with decreasing virus concentration. The black band of test lines can be observed with the naked eye at HAdV and H1N1 concentrations at 104 and 105 pfu/mL, respectively. For


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the magnetic SERS-strip, two target viruses were quantitatively detected through the main Raman peak (1332 cm−1) of Fe3O4@Ag tags on the test lines. The corresponding SERS intensity maps across two independent test lines for different concentrations of viruses are also shown in Fig. 5b. The test lines of high viral-concentration groups (107-104 pfu/mL) showed uniform SERS intensity over the whole line area, whereas the SERS signal intensity of low viral concentration groups (103-10 pfu/mL) was mainly concentrated at the front of the test lines. This phenomenon occurred as the front of test line was the first site encountered by the fluid as it migrated along the NC membrane. Previous studies have shown that sampling errors could be reduced by increasing the sample size of the test.28, 48 In this study, ten spots on the test line were randomly measured for each sample, and the experiments for low-concentration groups were performed twice. Each spectrum was the average of all spectra individually collected in each sample. Figs. 5c and 5d show the SERS spectra of different concentrations of H1N1 and HAdV, respectively. The SERS signal generated from DTNB increased significantly with increasing virus concentration as well. The corresponding calibration curves were constructed by the logarithmic relationship of virus concentrations and the SERS signal strength at 1332 cm−1 (Figs. 5e and 5f). Error bars represent the standard deviations from three repetitive experiments. The limit of detection (LOD) values of the magnetic-SERS biosensor based on Fe3O4@Ag tags were calculated by using the IUPAC guidelines (LOD = yblank + 3 × SDblank, where yblank is the average signal intensity at zero and SDblank is the standard deviation of the blank measurements).15, 49 According to this method, the LODs of H1N1 and HAdV were calculated to be 50 and 10 pfu/mL, respectively. Notably, the biosensor sensitivity depended on the effect of target virus antibodies used for detection. For this reason, the lower LOD of magnetic SERS strip for HAdV than for H1N1 can be acceptable. The magnetic SERS-strip for HAdV and H1N1 simultaneous detection also revealed a good dynamic range of six orders.



Figure 5. (a) Photographs of commercially-available colloidal gold strips for H1N1 and HAdV detection. (b) Photographs and SERS mapping images of T lines of magnetic SERS-strips at different concentrations of H1N1 and HAdV. (c-d) Averaged SERS spectrum of two test lines at different concentrations of H1N1 and HAdV. (e-f) Corresponding calibration lines of H1N1 and HAdV. Error bars represent standard deviation (n = 3). To directly compare the sensitivity of the magnetic SERS-strip with that of the standard colorimetric analysis, commercially-available colloidal gold strips for two target viruses were used. The results reveal that the LOD for standard colloidal gold strips with the naked-eye is 105 pfu/mL (Fig. 5a). For comparison, the visualization limit and LOD of the Fe3O4@Ag SERS tag-based strip are 10 and more than 2000 times lower than those of the standard colloidal gold strip, respectively. The two virus concentrations in test samples were also evaluated by using conventional plaque assays (Figs. S10 and S11). The plaque unit was formed by single live virus-infected cells, and the number of H1N1 or HAdV is consistent with our SERS results. These results indicate that the accuracy and sensitivity of the magnetic SERS-strip are comparable with those of plaque assay, the gold standard for live H1N1 or HAdV detection. ELISA analysis for two target respiratory viruses was also conducted, and the testing results are shown in Fig. S12. The proposed magnetic SERS-strip exhibited higher detection sensitivity and wider dynamic range than ELISA by using the same immunoreagents. In addition, our magnetic SERS-strip is 100 times more sensitive than the conventional ELISA assays for H1N1 or HAdV detection in literatures.50-51


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The repeatability of magnetic SERS strip was measured at the same concentration of target viruses on five different batches of SERS-LFIA strips using the same protocol, and the results are shown in Fig. S13. The SERS peak intensity of DTNB at 1332 cm−1 on the T lines exhibited good signal reproducibility for both target viruses. The relative standard deviation (RSD) values of SERS signals are calculated to be 4.1% and 2.5% for HAdV and H1N1, respectively. 3.5. Selectivity and specificity of magnetic-SERS strip for respiratory virus detection The specificities of the proposed magnetic SERS-strip for target H1N1 and HAdV was tested by comparing the visual and SERS signal with other common respiratory viruses in the serum. FluB, PIV, and RSV, another three of the most common respiratory viruses, were selected as unspecific viruses, and 107 pfu/mL of these viruses was prepared for the SERS strip. All the three interfering viruses can cause respiratory tract infections and exhibit similar symptoms to H1N1 or HAdV. The visual detection results (Fig. 6) display two black bands for H1N1 and HAdV groups (105 pfu/mL), whereas only the control line appeared for the other three viruses (107 pfu/mL). The corresponding SERS spectra showed that SERS intensity of the positive groups (H1N1 or HAdV) was much higher than that of the negative virus groups. These results reveal that the magnetic SERS-strip has a good selectivity toward H1N1 and HAdV detection. Thereafter, stability tests were carried out in 0.1 M PBS buffer at pH 5.0 to 9.0 to study the inﬂuence of pH and salt concentration on the magnetic SERS-strip. HAdV was used as model virus, and standard virus solution was prepared at a concentration of 106 pfu/mL in different PBS buffers (0.1 M, pH 5-9). Then, Fe3O4@Ag SERS tags were added to the above HAdV solution, and the detection process was conducted according to optimized conditions. The results are shown in Fig. S14. No notable effect of pH (5-9) and high salt concentration (0.1 M) was observed on the performance of SERS-LFIA strip. It is been well reported that immunomagnetic beads have good stability and biocompatibility, and can be applied to complex solutions, including real biological samples.52-56 Thus, target viruses should be easily captured and enriched by the capture-antibody-modified Fe3O4@Ag SERS tags in clinical samples. Controlled experiments were conducted to detect H1N1 and HAdV spike in real biological samples, such as human whole blood, serum and sputum (containing viral titer of 105 pfu/mL), and to evaluate the accuracy and practical applications of our immunomagnetic SERS tag-based strip. For real samples, Fe3O4@Ag tags were first added to the test specimens. After incubation for 20 min, the Fe3O4@Ag SERS tags



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were magnetically separated and resuspended with 70 µL running buffer. Detection tests were then carried out according to the developed conditions. Blank PBS solution was used as negative group. Fig. S15 shows that all the positive complex samples produced strong SERS signals similar to those of the PBS groups, whereas almost no SERS intensity was detected in the negative groups. The good coincidence in spiked test confirmed that the magnetic SERS-strip could be competent for practical virus detection in complex samples with sufficient accuracy. Furthermore, the magnetic SERS-strips showed no obvious signal intensity changes in test line after storage for two months at 4 °C (Fig. S16); this finding indicates the good stability of our proposed magnetic SERS-strip.

Figure 6. Specificity of magnetic SERS-strip. The inset shows the photographs of magnetic SERS-strip in the presence of 107 pfu/mL of FluB, RSA, PIV, and 105 pfu/mL of H1N1 and HAdV. Error bars represent standard deviation (n = 3). Table 1. Overall performance of the magnetic SERS-strip compared with other respiratory virus detection techniques. Detection method

Virus

Detection limit

Total time

Reference

Immunosensor

HAdV

1000 pfu/mL

2h

Caygill et al. 201257

Electrochemical sensor

HAdV

30 pfu/mL

2h

Lin et al. 201558

Electrochemical Assay

H1N1

102 pfu/mL

1h

Zhang et al. 20152

Paper immunoassay

H1N1, H3N2

2×103, 2×104 pfu/mL

1h

Lei et al. 201551

SERS biosensor

H3N2

102 TCID50/mL

25 min

Sun et al. 201759

Optoelectronic sensor

HAdV

10 pfu/mL

2h

Ahmed et al. 201850

Electrochemical sensor

Inactive H1N1

0.9 pg/μL

1h

Bai et al. 201860

SERS biosensor

HAdV, H1N1

10, 50 pfu/mL

30 min

This work

Table 1 displays the typical features of the proposed magnetic SERS-strip and its comparison with other recently developed techniques for H1N1 or HAdV detection.2, 50-51, 57-60

As shown in this table, the key indicators of our method are superior or


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equivalent to those of other studies, such as sensitivity, detection time and throughput. Importantly, the proposed magnetic SERS-strip can be directly used for real biological samples without any sample pretreatment steps. On the contrary, common nanoparticle-based biosensors are easily affected by circumstantial factors, such as interference, pH value, and salt ion strength, in complex samples.

4. CONCLUSIONS In this work, we proposed a new SERS-based strip based on the Fe3O4@Ag SERS tags for the rapid, highly sensitive, and quantitative detection of two respiratory viruses. The Fe3O4@Ag SERS tag acted as a multifunctional tool for the specific recognition and magnetic enrichment of target viruses in the solution, and SERS detection of such viruses on the strip. Based on this strategy, sensitivity values of the magnetic SERS-strip for H1N1 and HAdV detection were as low as 50 and 10 pfu/mL, respectively. Our results indicate that the visualization limit and SERS detection limit of the magnetic SERS-strip were 10 and 2000 times lower, respectively, than those of the standard colloidal gold strip. In addition, the proposed magnetic SERS-strip has many other notable merits, including rapid, simple operation, stability, high specificity, and reproducibility. As far as we know, this work is the first to introduce Fe3O4@Ag SERS tags into LFIA strip. By changing specific antibodies, the SERS strip can be used for rapid detection of other targets, such as biomarkers, toxins, and bacterial pathogens, in complex specimens. In the future, the accuracy and specificity of our proposed magnetic SERS strip will be verified by a large number of clinical real-world samples. We expect that the magnetic SERS strip can exert a positive effect on clinical diagnosis.

ASSOCIATED CONTENT Appendix A. Supporting information Additional

experimental

description

(S1.

Preparation

of

monodisperse

DTNB-embedded Fe3O4@Ag MNPs; S2. Viral plaque assay; S3. Enzyme-linked immunosorbent assay), and supporting figures mentioned in the main text.

AUTHOR INFORMATION The authors Chongwen Wang, Chaoguang Wang and Xiaolong Wang contributed equally to this work.

*Corresponding author Phone: +86-551-65786703; e-mail: [email protected] (W. Wang), Phone: +86-10-66931422-5; e-mail: [email protected]

(R. Xiao),



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Phone: +86-10-66932211; e-mail: [email protected] (S. Wang).

ORCID Chongwen Wang: 0000-0002-7096-6527 Shengqi Wang: 0000-0003-0380-5659

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS W.C.W., K.L.W., Z.R., R.X., and S.Q.W. acknowledge support from the National Natural Science Foundation of China (Grant no 81830101). C.G.W. acknowledges support from the National Natural Science Foundation of China (Grant no 51605486). X.L.W., Y.H.Z., and S.Q.W. acknowledge support from the

Beijing

Municipal

Science

&

Technology

Commission

(No.

Z161100000116040).

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A magnetic SERS tag-based lateral flow biosensor was reported for the first time to enable rapid, sensitive and quantitative detection of two respiratory viruses in biological samples.



Scheme 1


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Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



Figure 6


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Magnetic SERS Strip for Sensitive and Simultaneous Detection of

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