Dual-Signal Readout Nanospheres for Rapid Point-of-Care Detection

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A Dual-Signal Readout Nanosphere for Rapid Pointof-Care Detection of Ebola Virus Glycoprotein Jiao Hu, Yong-Zhong Jiang, Ling-Ling Wu, Zhen Wu, Yuhai Bi, Gary Wong, Xiang-Guo Qiu, Jian-Jun Chen, Dai-Wen Pang, and Zhi-Ling Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02222 • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 18, 2017

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Analytical Chemistry

A Dual-Signal Readout Nanosphere for Rapid Point-of-Care Detection of Ebola Virus Glycoprotein Jiao Hu,† Yong-Zhong Jiang,† Ling-Ling Wu,† Zhen Wu,† Yuhai Bi,‡,ǁ,⊥ Gary Wong,ǁ,⊥ Xiangguo Qiu,# Jianjun Chen*,§,⊥ Dai-Wen Pang,† Zhi-Ling Zhang,*,† †

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, People’s Republic of China ‡ Shenzhen Key Laboratory of Pathogen and Immunity, State Key Discipline of Infectious Disease, Shenzhen Third People’s Hospital, Shenzhen 518112, People’s Republic of China § CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Hubei 430071, People’s Republic of China ǁ CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Beijing 100101, People’s Republic of China ⊥

Center for Influenza Research and Early warning (CASCIRE), Chinese Academy of Sciences, Beijing 100101, People’s Republic of China # Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba R3E 3R2, Canada

ABSTRACT: Rapid detection of highly contagious pathogens is the key to increasing the probability of survival and reducing infection rates. We developed a sensitive and quantitative lateral flow assay for detection of EBOV glycoprotein with a novel multifunctional nanosphere (RNs@Au) as a reporter. Each RNs@Au contains hundreds of quantum dots and dozens of Au nanoparticles and can achieve enhanced dual-signal readout (fluorescence signal for quantitative detection and colorimetric signal for visual detection). Antibody (Ab) and streptavidin (SA) were simultaneously modified onto the RNs@Au to label the target as well as to act as the signal enhancer. After the target was labeled by the Ab-RNs@Au-SA and captured on the test line, biotin modified RNs@Au was used to amplify the dualsignal by the reaction of SA with biotin. The assay enables naked-eye detection of 2 ng/mL glycoprotein within 20 min, and the quantitative detection limit is 0.18 ng/mL. Additionally, the assay has been successfully tested in field work for detecting EBOV in spiked urine, plasma and tap water samples, and is thus a promising candidate for early diagnosis of suspect infections in EBOV-stricken areas.

The 2014–16 Ebola virus (EBOV) infections have resulted in over 28,500 cases and 11,300 deaths.1 This virus is highly infectious, with a case fatality rate of 40–90%.2,3 The high number of deaths was at least partly due to the lack of rapid diagnostic methods,4 which can provide effective diagnosis for medical professionals and contribute to efforts to contain virus spread. It has been reported that if 60% of patients are diagnosed rapidly and isolated within one day of illness, the attack rate drops from 80% 1 Environment ACS Paragon Plus


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to nearly 0%.5 This finding indicates the tremendous effect of a rapid diagnosis of Ebola virus disease. Rapid diagnostic tests are also crucial for infected patients during early in the course of disease, at a time when they do not yet transmit widespread infection to others, and with a greater chance of survival with clinical intervention. Currently, the most widely-used diagnostic techniques for EBOV infections are enzyme linked immunosorbent assay (ELISA)6,7 and reverse transcription-polymerase chain reaction (RT-PCR).8–10 Since these methods are laboratory-based and difficult to perform at a point-of-care (POC) facility in the field, 3–10 days are typically needed to conclusively diagnose the infected patients after the onset of symptoms, resulting in a delay in therapy.11 Recently, many sensitive assays were developed for EBOV detection, such as optofluidic biosensors based on plasmonic nanoholes,12 interferometric measurement technique,13 the luminescence resonance energy transfer strategy,14 and the electroluminescence method.15 However, these methods are tedious to operate in the field and are not readily compatible with POC use without extensive, existing infrastructure. Hence, a POC assay allowing the rapid, sensitive, and quantitative detection of EBOV is vitally important for diagnosis in resource-poor areas. Lateral flow-based test strip assays are currently the most promising format for POC diagnosis, since its advantages include portability, speed, cost-effectiveness, and ease-of-use.16,17 Besides, the result can be determined visually without any specialist facilities or healthcare professionals due to the colorimetric readout of Au nanoparticles (Au NPs).18,19 Over the years, lateral flow test strips have been used in the detection of metal ions, small molecules, nucleic acids, and proteins.20 Recently, a few lateral flow assays for diagnosis of EBOV infections have been developed, 21–23 in which, the OraQuick Ebola Rapid Antigen test was approved by WHO. Still, a drawback of these assays is low sensitivity, which is problematic as POC assay need to be highly sensitive in order to be useful in the clinic. In addition, the 15% false positive rate of the OraQuick Ebola Rapid Antigen test limits its application. 21 Fluorescent materials can increase the assay sensitivity due to their inherent higher signal-to-noise ratios than Au NPs.24–26 Our previous work has suggested that fluorescent nanospheres have excellent fluorescence signal and strong stability.27–29 Using them as a readout in the lateral flow assay has many advantages, including quantitative determination and enhanced sensitivity.30 However, the fluorescent materials require an extra excitation light source to observe the emission light, which may impede application for POC diagnosis, especially for areas with poor medical conditions. Herein, we designed a novel reporter (RNs@Au) as the reporter of lateral flow assay. It was fabricated by adhering Au NPs onto the surface of the red fluorescent nanospheres (RNs). Each RNs@Au contains approximately hundreds of quantum dots30 and dozens of Au NPs, which enhances both the fluorescence and the colorimetric signals compared to a single quantum dot or Au nanoparticle. EBOV infects the host cells via glycoproteins, which are presented on the outside of the virus.31 Hence, 2 Environment ACS Paragon Plus

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it is significance to detect EBOV glycoprotein for EBOV diagnosis. To develop the enhanced EBOV glycoprotein-based POC assay, specific antibody (Ab) to the EBOV glycoprotein and streptavidin (SA) were simultaneously modified onto the surface of RNs@Au to prepare the Ab-RNs@Au-SA conjugates, which act as a dual-signal probe (visual and fluorescence readout), as well as the signal enhancer based on the multivalent interactions of SA with biotin modified RNs@Au (biotin-RNs@Au). As illustrated in Scheme 1, the test was started by loading the mixture of sample, Ab-RNs@Au-SA and 1% BSA–1% Tween 20–PBS onto the sample pad. A few minutes later, the biotin-RNs@Au was applied to the sample pad. The signal can be enhanced by one order of magnitude sensitive due to the formation of biotin-SA multivalent network. With the enhanced lateral flow assay, as low as 2 ng/mL EBOV glycoprotein can be visualized with the naked eye, and a quantitative detection limit of 0.18 ng/mL were achieved within 20 min by measuring the fluorescence intensity with an EMCCD. The detection limit was lowered 555fold compared to Au-based lateral flow assay. In addition, human-origin proteins (ie. CRP, HSA, CEA, AFP), H1N1, H3N2, H9N2 influenza virus antigens and three kinds of malaria were negative using this method, indicating excellent specificity. The assay was successfully applied for quantitative detection of the EBOV glycoprotein, as well as inactivated whole virion in spiked samples (urine, plasma and tap water) in the field. Only a smartphone is required as an apparatus and data processing is simple. Our results demonstrate the feasibility of the RNs@Au-based lateral flow assay to visually and quantitatively detect EBOV-spiked samples, and potentially a broader range of diseases, with high sensitivity and specificity.

Scheme 1. Two-step detection of Ebola with RNs@Au based lateral flow immunoassay.

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EXPERIMENTAL SECTION Reagents and Instrument. EBOV glycoprotein, inactivated EBOV whole virion and horse antibody against EBOV glycoprotein were supplied by Prof. Xiangguo Qiu. N-hydroxysuccinimide (NHS) was bought from Thermo. Human alpha fetoprotein (AFP) was ordered from Linc-Bio Science Co., Ltd. (Shanghai). C-reaction protein (CRP) was from Prospecbio. Carcinoembryonic antigen (CEA) was bought from Fitzgerald Industries International, Inc. Streptavidin, biotin hydrazide, FITC-biotin, N-(3dimethylaminopropyl-N′-ethylcarbodiimide hydrochloride (EDC) and albumin from human serum (HSA) were purchased from Sigma-Aldrich. Alexa Fluor 488-conjugated AffiniPure goat anti-horse IgG and goat anti-horse IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. Fetal bovine serum (FBS) was obtained from Gibco. Hydrophobic CdSe/ZnS QDs were purchased from JiaYuan Quantum Dots Co., Ltd. (Wuhan). All other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai). Nitrocellulose membrane, sample pad, absorbent pad, and plastic adhesive card were purchased from JieYi BioTech Co., Ltd. (Shanghai). Transmission electron microscopy (TEM) images were acquired on a FEI Tecnai G2 20 TWIN electron microscope. Scanning electron microscopy (SEM) images were recorded with a Zeiss Sigma FE-SEM. Fluorescence emission spectra were collected on a Fluorolog-3 fluorescence spectrometer (HORIBA JOBIN YVON). The absorbance spectra were obtained with a UV-vis spectrophotometer (Shimadzu). Hydrodynamic size and zeta potential of nanospheres were measured by Nano ZS-ZEN 3600 from Malvern. Preparation of Gold Nanoparticles (Au NPs). Au NPs were synthesized using the citrate-reduction method developed by Frens.32 Briefly, 100 mL of 0.01% HAuCl4 solution was heated until boiling, then 5 mL of 1% trisodium citrate solution was injected rapidly under vigorous stirring. The solution was heated for another 20 min, during which the color changed from pale yellow to burgundy. After cooling to room temperature while stirring, the colloids were purified by ultrafiltration. Synthesis of RNs. The red fluorescent nanospheres (RNs) were prepared according to the embedding method previously developed by our group.33,34 Hundreds of hydrophobic red quantum dots (QDs) were embedded into one hydrophilic carboxyl-terminated poly(styrene/acrylamide) copolymer nanosphere (Pst-AAm-COOH) to obtain red fluorescent nanosphere. Preparation of RNs@Au. The RNs@Au was obtained by adhering Au NPs onto the surface of the RNs as illustrated in Figure 1A. The citrated-capped Au NPs were added to the solution of RNs. After reaction for approximately 1 h at room temperature with gentle agitation in 0.01 M phosphate-buffered saline (PBS) (pH = 7.2), the Au NPs were tethered onto the surface of RNs via Au-COOH bonds to form RNs@Au. The RNs@Au were then separated by centrifugation and washed once with ultrapure water. The precipitation were then suspended in ultrapure water and stored at 4°C for use. 4 Environment ACS Paragon Plus

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Fabrication of the Antibody and Streptavidin Dual-Labeled RNs@Au and Biotin Labeled RNs@Au. To fabricate the dual-labeled RNs@Au conjugates, 2 mg RNs@Au were first activated in 10 mM EDC and 10 mM NHS in 300 µL of PBS buffer (0.01 M, pH = 6.8) at room temperature with continuous shaking for 20 min. The activated RNs@Au were then centrifuged and washed twice with PBS (0.01 M, pH = 7.2) at 12,000 rpm for 5 min, and then resuspended in 300 µL of PBS buffer (0.01 M, pH = 7.2) to react with the EBOV glycoprotein-specific antibody (Ab) and streptavidin (SA). After incubation with a vigorous shaking for 4 h, the dual-labeled RNs@Au (Ab-RNs@Au-SA) were washed five times with PBS (0.01 M, pH = 7.2), resuspended in 100 µL PBS (0.01 M, pH = 7.2) and stored at 4°C for use. Similarly, we prepared biotin modified RNs@Au (biotin-RNs@Au). The conjugation procedure was the same as above, except only biotin hydrazide was added to the activated RNs@Au. The biotin-RNs@Au were washed five times to remove redundancy biotin and resuspended in PBS (0.01 M, pH = 7.2). The Ab-RNs@Au-SA and biotin-RNs@Au were blocked with 10% BSA in 0.01 M PBS (0.01 M, pH = 7.2) for 10 h and washed five times with PBS (0.01 M, pH = 7.2). Then AbRNs@Au-SA and biotin-RNs@Au were resuspended in 100 µL PBS (0.01 M, pH = 7.2) respectively and stored at 4°C for use. Detection of EBOV Glycoprotein and Whole Virion. 50 µL aliquots of the target (either EBOV glycoprotein or whole virion) solutions in buffer (0.01 M, pH = 7.4 PBS containing 1% BSA and 1% Tween 20) with 4 µL Ab-RNs@Au-SA were added onto the sample pad, with the solution migrating towards the absorption pad by capillary action. Five minutes later, 30 µL 1% BSA-1% Tween 20-1×PBS (containing 12 µL biotin-RNs@Au) was used to enhance the signals. For The intensity of the red band on the test line was visualized and estimated by the naked eye. The band also can be observed with a portable ultraviolet lamp, and the fluorescence photographs of the test strips were acquired with an Alpha Innotech. The fluorescence intensity of red band was collected and analyzed by the Inverted fluorescence Microscope mounted with an EMCCD or a smartphone as the apparatus with simple data processing. The concentration of EBOV whole virion and other virions were quantified by Bradford method. Detection of EBOV Glycoprotein and Whole Virion in Complex Matrices. Tap water, fetal bovine serum, urine and plasma were mixed with the EBOV glycoprotein or whole virion suspension to get the spiked samples. The samples were tested using the enhanced RNs@Au based lateral flow assay following the procedure. Control experiments were done the same except neither EBOV glycoprotein nor whole virion was added. For cloudy urine sample, it is necessary to centrifuge before detection. Whole blood samples need to remove blood cells before detection. The whole blood samples of three patients who effected with three kinds of malaria (Plasmodium falciparum, Plasmodium ovale and Plasmodium vivax) were lysed the red blood cells before use. 5 Environment ACS Paragon Plus


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RESULTS AND DISCUSSION Characterization of RNs@Au. RNs@Au was obtained based on the RNs and Au NPs (Figure 1A). Transmission electron microscopy (TEM) shows that there were some 20 nm Au NPs (red arrows) on the nanosphere besides numerous QDs (blue arrows) (Figure 1B). We calculated that dozens of Au NPs were adhered onto the surface of one hydrophilic carboxyl-terminated poly (styrene/acrylamide) (PStAAm) (for 100 RNs@Au). Additionally, there were approximately hundreds of QDs in one PStAAm.30,33,34 From the scanning electron microscopy (SEM) image, we can also see the Au NPs (red arrows) on the surface of the nanosphere clearly (Figure 1C). The TEM and SEM results indicate that the RNs@Au was successfully fabricated. This point was also proved by dynamic light scattering (DLS) results. The hydrodynamic size of RNs@Au was 286.6 nm, which is 27 nm larger than the size of RNs (260.9 nm). Besides, the polydispersity index (PDI) of RNs@Au was 0.049, indicating the RNs@Au were monodispersed in solution (Figure 1D). Compared to RNs, the zeta potential of RNs@Au also changed, from –37.2 mV to –46.1 mV (Figure 1E). To investigate whether the RNs@Au possesses the surface plasmon resonance signal, the UV-vis spectrum was used to further explore the absorption of the RNs@Au. In contrast to RNs, the RNs@Au clearly showed an absorption peak at 527 nm (Figure 1F), showing a considerable shift of the absorbance to red (from 520 to 527 nm) versus Au NPs. From the inset picture (Figure 1F), RNs@Au showed bright red color (inset picture 3) compared to RNs (inset picture 2). In addition, it also exhibited strong fluorescence (Figure 1F inset picture 4, Figure 1G), indicating unique fluorescence properties of the RNs@Au.


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Figure 1. (A) Schematic illustration of the preparation of RNs@Au. (B) TEM image of the RNs@Au. Blue arrows and red arrows indicate QD and Au, respectively. (C) SEM image of the RNs@Au. Red arrows indicate Au NPs bounded onto the surface of the RNs. (D) Hydrodynamic size of RNs and RNs@Au. (E) Zeta potential of RNs and RNs@Au. (F) UV-vis absorption spectra of Au, RNs and RNs@Au. Corresponding bright field photographs of Au (1), RNs (2) and RNs@Au (3), respectively, and fluorescence photograph of RNs@Au (4) are shown in the inset. (G) Fluorescence microscope image of RNs@Au.

These results suggest that the RNs@Au were successfully fabricated and it possesses both colorimetric and fluorescence signals. The RNs@Au was then modified with specific antibody (Ab) to the EBOV glycoprotein and streptavidin (SA) to obtain Ab-RNs@Au-SA. Biotin modified RNs@Au was also fabricated. Alexa Fluor 488-conjugated AffiniPure goat anti-horse IgG and FITC-biotin were used to react with AbRNs@Au-SA, respectively. The results demonstrate that the RNs@Au was successfully modified with antibody and SA (Figure S3, Supporting Information). The hydrodynamic size and zeta potential of RNs@Au were significantly changed after modification with antibody and SA (Figure S4 A,B, Supporting Information) and biotin (Figure S4 C,D, Supporting Information), respectively, further indicating the successful fabrication of the conjugates. Principle of EBOV Glycoprotein and EBOV Whole Virion Detection Using Enhanced Strip Biosensor. As illustrated in Scheme 2, the Ab-RNs@Au-SA was used to detect EBOV glycoprotein or EBOV whole virion via two steps. In the first step, the target was labeled with Ab-RNs@Au-SA and captured by the test line of antibody to glycoprotein via antigen-antibody reaction. In the second step, the biotin-RNs@Au solution was applied to the sample pad. The biotin-RNs@Au were adhered to the test line by Ab-RNs@Au-SA via biotin-SA affinity, and then a network of RNs@Au can form on the 7 Environment ACS Paragon Plus


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test line through multivalent interactions of biotin-SA, in which a darker and brighter signal was obtained. The signal can be read with the naked eye due to the localized surface plasmon resonance effect of the Au NPs. The signal can also be seen with a portable ultraviolet lamp due to the super-strong fluorescence of RNs. Additionally, quantitative analysis of glycoprotein can be achieved by monitoring the fluorescence intensity of the RNs@Au/glycoprotein complexes in the test line with an EMCCD or a smart phone for different conditions. The dual-signal readout of RNs@Au enables the assay to detect target visually and quantitatively, especially for poor-resource areas.

Scheme 2. Schematic illustration of EBOV detection using enhanced RNs@Au based test strip.

The Proof-of-Concept Assay for the Detection of EBOV Glycoprotein. In the present work, the proof-of-concept of the assay for the Ebola glycoprotein detection is introduced. In the absence of glycoprotein, no color change was observed (“negative”, Figure 2A) because no RNs@Au/glycoprotein complexes can be captured on the test line. In this case, neither colorimetric signal nor fluorescence signal was observed on the test line. From Figure 2B, the red signal can be read from the positive sample with the naked eye due to the localized surface plasmon resonance effect of Au NPs. Simultaneously, a bright fluorescence line can also be seen with a portable ultraviolet lamp due to the super-strong fluorescence of RNs. In addition, according to the SEM image of positive sample, the RNs@Au can be seen clearly (Figure 2D) while no RNs@Au exists in the test line of the negative sample (Figure 2C). Consequently, EBOV glycoprotein can be detected by the assay visually. Additionally, quantitative analysis of glycoprotein can be achieved by monitoring the fluorescence intensity of the RNs@Au/glycoprotein complexes.


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Figure 2. Representative photographic images (A, B) and SEM images (C, D) of the lateral flow test in the absence (negative, A, C) and presence (positive, B, D) of glycoprotein, respectively. Blue arrows and red arrows indicate Au NPs and RNs, respectively.

Detection of EBOV Glycoprotein. To evaluate the sensitivity and dynamic range of the enhanced lateral flow test biosensor for EBOV glycoprotein detection, under optimized detection conditions (Figure S5), different concentrations of glycoprotein were loaded onto the strip biosensor. As shown in Figure 3A, in the absence of glycoprotein, there was no red band observed on the test line. For the positive samples, the intensity of red lines on the test line increased with increasing concentrations of glycoprotein. The red band on the test zone is visible even at 2 ng/mL of glycoprotein, which can be used as the threshold for the visual determination of glycoprotein without any need for instrumentation. In addition, the fluorescence intensity on the test line also increased with the concentration of glycoprotein (Figure 3B). Direct comparison of signals from Au and RNs, demonstrates that the latter results in brighter and clearer signal, the fluorescence signal is easily discerned by the naked eye at target concentrations of 2 ng/mL (Figure 3A, B). Their corresponding optical responses were further confirmed by recording the fluorescence intensities of the red bands on the test line with an EMCCD. The resulting calibration curve shows that the fluorescence intensities were proportional to the concentration of glycoprotein in the range between 2–1000 ng/mL (Figure 3C). The detection limit (LOD) was calculated to be 0.18 ng/mL. The LOD was estimated by the IUPAC standard method (LOD=yblank+3×SDblank, where yblank is the average signal intensity at zero and SDblank is the standard deviation of the blank measurements).35



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Figure 3. Photographs (A) and fluorescence pictures (B) of the test strips for 0, 2, 10, 40, 100, 400, 1000 ng/mL glycoprotein in buffer, respectively. (C) Enhanced RNs@Au based lateral flow assay linear response for the detection of glycoprotein at a concentration range of 2 to 1000 ng/mL in buffer. Error bars indicate the standard errors of three independent experiments of three strips.

Compared with other assays for EBOV, the assay developed in this work showed better performance (Table 1).7,21–23 It realized visual detection without any instrument, which is suitable for application in areas with poor medical conditions. The detection limit of our method is estimated to be 294-fold lower than the commercial product for EBOV detection, which was developed by the OraQuick,22 providing a large window for the early detection of viral glycoprotein at lower concentrations. In addition, perhaps the observed detection limit can be used to screen for EBOV infections. Moreover, intra-assay and interassay precision and accuracy analysis indicated that the assay owns good reproducibility (Table S1). Table 1. Summary of the Analytical Performances of EBOV Detection with Different Methods method Enzyme-linked immunosorbent assay; Nanozyme-strip; OraQuick Ebola Rapid Antigen test;

target recombinant nucleoprotein; glycoprotein; recombinant nucleoprotein;

detection limit and assay time

linearity range

ref

300 ng/mL; unknown;

unknown;

7

1 ng/mL; 30 min;

qualitative;

21

53 ng/mL; 30 min;

qualitative;

22

Ag-based strip;

glycoprotein;

75 ng/mL; unknown;

qualitative;

23

Enhanced RNs@Au based strip;

glycoprotein;

0.18 ng/mL; 20 min;

2–1000 ng/mL;

current method

Superiority of the Enhanced Strategy over the Conventional Test Strip and the Unenhanced Strategy. Since each RNs@Au contains dozens of Au NPs, and biotin-RNs@Au further amplify the 10 Environment ACS Paragon Plus

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signal of Au NPs, more Au NPs can be captured on the test line. Therefore, the assay has advantages in sensitivity over the conventional test strip, which is based on Au NPs. As shown in Figure 4A, the visualization limit of the Au-based lateral flow assay is 100 ng/mL glycoprotein, which is 50-fold higher than the enhanced assay in this work. Moreover, the signal-to-noise ratio of fluorescent materials is much higher than Au NPs used in colorimetry-based detection. Therefore, the detection limit of the enhanced assay is 555-fold lower than the Au-based lateral flow assay. Compared with the unenhanced RNs@Au based lateral flow assay, the method is also superiority by introducing biotin-SA multivalent reaction to form a network for signal amplification. The visualization limit of the unenhanced RNs@Au-based lateral flow assay is 40 ng/mL glycoprotein (Figure 4B), while at the same concentration the color of the enhanced assay is darker. In addition, the detection limit of the enhanced assay is 0.18 ng/mL, which is one order of magnitude lower than a value of 6.09 ng/mL of the unenhanced assay. This dual labeling of RNs@Au treatment and the introduction of biotin-RNs@Au leads to the dual signal enhancement on test line of the strip and improvement of the sensitivity.

Figure 4. (A) Photographs of the Au-based test strips for 0, 40, 100, 200, 400, 800, 1000 ng/mL glycoprotein in buffer. (B) Photographs and fluorescence pictures of the unenhanced RNs@Au-based test strips for 0, 20, 40, 100, 200, 400, 1000 ng/mL glycoprotein in buffer. (C) Unenhanced RNs@Au-based lateral flow test strip linear response for glycoprotein detection; concentration range is 20 to 1000 ng/mL in buffer. Error bars indicate the standard errors of three independent experiments of three strips.

Robustness of the Enhanced Lateral Flow Assay for Clinical Application. After demonstrating that the enhanced RNs@Au based lateral flow assay has better performance than the unenhanced RNs@Au 11 Environment ACS Paragon Plus


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based lateral flow assay and conventional Au-based lateral flow assays, the specificity of this strategy was evaluated. After infected with Ebola virus, the patient presents fever symptoms. Given the lack of Marburg virus, Lassa virus and yellow fever virus, which are relevant species to distinguish Ebola infections by the fever symptoms36, other proteins (CEA, AFP, HSA, CRP) and other viruses (H1N1, H3N2, H9N2) and three malaria patients samples (Plasmodium falciparum, Plasmodium ovale and Plasmodium vivax, respectively) were chose as negative controls to test the specificity of the assay. Malaria and influenza virus can also cause the human body exhibit symptoms of fever. Meanwhile, the CEA, AFP, HSA and CRP were human-origin proteins, the existence of these proteins may interfere the assay. Especially, the CRP has robust association with viral and bacterial infections. As shown in Figure 5, the results can be determined with the naked eye and the fluorescence intensity can be collected with a smartphone camera and measured with Image-Pro Plus. From Figure 5A, it can be seen that the fluorescence intensity of glycoprotein was much higher than negative controls. The results suggested that this strategy had strong specificity, and could be applied to the detection in complex samples. The assay was then used to detect inactivated EBOV whole virion spiked in buffer, tap water, urine and plasma (the total protein concentration of EBOV is 400 ng/mL) to investigate the accuracy and practical applications of this method. As shown in Figure 5B, all positive samples presented fluorescence signals, while fluorescence was not detected in negative controls. In addition, the assay showed excellent performance in the detection of FBS samples (Figure S6). These results suggest that the dual-readout enhanced lateral flow assay maybe meet the requirement of assays that need rapid and sensitive detection, such as the diagnosis of EBOV infections.


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Figure 5. (A) Histogram for the specificity of the enhanced lateral flow assay method for glycoprotein detection. (B) Detection results of the spiked samples for inactivated EBOV whole virion. Error bars indicate the standard errors of three independent experiments of three strips.

CONCLUSIONS In summary, we designed a novel dual-signal readout nanosphere (RNs@Au) by the integration of many Au NPs and QDs with one nanosphere. The RNs@Au can provide both colorimetric signal and fluorescence signal to meet requirements from different clinical conditions facility. Based on the RNs@Au and the introduction of biotin-streptavidin system to amplify the dual signals, we present a dual-readout, enhanced lateral flow assay for EBOV detection with a high sensitivity. Compared with other reported lateral flow assay for EBOV glycoprotein, the dual-readout enhanced lateral flow assay is competitive as a rapid assay and also can increase the sensitivity by two orders of magnitude, and realize quantitative detection of EBOV glycoprotein. Moreover, this method is easy to operate, timesaving, and its success in detecting EBOV-spiked samples validates its use in virus detection in real clinical samples. The merits of the novel reporter, dual-signal readout, ability for quantitation, high sensitivity, and anti-interference ability may substantially broaden the applications of lateral flow assays for early detection of disease biomarkers, and holds great potential in fields such as clinical diagnosis, food safety, environmental monitoring and so forth.



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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Sections S.1−S.5, and Figures S1−S6, Table S1. Figure S1, characterization of Au Nanoparticles. Figure S2, characterization of the red fluorescent nanospheres. Figure S3 and Figure S4, verifies the fabrication of Ab-RNs@Au-SA and biotin-RNs@Au. Figure S5, optimization of the detection conditions. Figure S6, application of the enhanced detection assay in FBS samples. Table S1, reproducibility analysis of the enhanced RNs@Au-Based test strip. (Table S1) (PDF). AUTHOR INFORMATION Corresponding Author *Zhi-Ling Zhang, Email: [email protected]; Phone: 0086-27-68756759, Fax: 0086-27-68754067. *Jianjun Chen, Email: [email protected]; Phone: 0086-27-87198739, Fax: 0086-027-87198167. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21475099, 21535005), National Science and Technology Major Project (2016ZX10004222), and the intramural special grants for influenza virus research from the Chinese Academy of Sciences (KJZD-EW-L15). Y. B. is supported by the Youth Innovation Promotion Association of Chinese Academy of Sciences (CAS) (2017122). G. W. is the recipient of a Banting Postdoctoral Fellowship from the Canadian Institutes of Health Research (CIHR) and the President’s International Fellowship Initiative from the CAS. REFERENCES (1) WHO Ebola situation report. 2016. http://apps.who.int/ebola/current-situation/ebola-situation-report-30-march2016. (2) Hoenen, T.; Groseth, A.; Falzarano, D.; Feldmann, H. Trends Mol. Med. 2006, 12, 206–215. (3) Roberts, L. Science 2015, 347, 1189–1189. (4) Aylward, B.; Barboza, P.; Bawo, L.; Bertherat, E.; Bilivogui, P.; Blake, I.; Brennan, R.; Briand, S.; Chakauya, J. M.; Chitala, K.; et al. N. Engl. J. Med. 2014, 371, 1481–1495. 14

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Dual-Signal Readout Nanospheres for Rapid Point-of-Care Detection

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