Digital Single Virus Immunoassay for Ultrasensitive Multiplex Avian

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Biological and Medical Applications of Materials and Interfaces

Digital Single Virus Immunoassay for Ultrasensitive Multiplex Avian Influenza Virus Detection Based on Fluorescent Magnetic Multifunctional Nanospheres Zhen Wu, Tao Zeng, Wen-Jing Guo, Yi-Yan Bai, Dai-Wen Pang, and Zhi-Ling Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18898 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 30, 2019

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ACS Applied Materials & Interfaces

Digital Single Virus Immunoassay for Ultrasensitive Multiplex Avian Influenza Virus Detection Based on Fluorescent Magnetic Multifunctional Nanospheres Zhen Wu, Tao Zeng, Wen-Jing Guo, Yi-Yan Bai, 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, Wuhan University, Wuhan, 430072, P. R. China. *Corresponding author: Zhi-Ling Zhang, Email: [email protected].

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ABSTRACT: Fluorescence method has made great progress in the construction of sensitive sensors, but the background fluorescence of matrix and photobleaching limit its broad application in clinical diagnosis. Here, we propose a digital single virus immunoassay for multiplex virus detection by using fluorescent magnetic multifunctional nanospheres as both capture carriers and signal labels. The superparamagnetism and strong magnetic response ability of nanospheres can realize efficient capture and separation of targets without sample pretreatment. Due to its distinguishable fluorescence imaging and photostability, the nanospheres enable single-particle counting for ultrasensitive multiplexed detection. Furthermore, the integration of digital analysis provided a reliable quantitative strategy for rare targets detection. Based on multifunctional nanospheres and digital analysis, a digital single virus immunoassay was proposed for simultaneous detection of H9N2, H1N1 and H7N9 avian influenza virus (AIV) without complex signal amplification, whose detection limits were 0.02 pg/mL. Owing to its good specificity and anti-interference ability, the method showed great potential in single biomolecules, multiplexed detection and early diagnosis of diseases.

KEYWORDS: Multifunctional nanospheres, Digital analysis, Avian influenza virus, Single virus immunoassay, Ultrasensitive multiplexed detection


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INTRODUCTION With the development of science and nanotechnology, single-molecule or single-cell detection has attracted wide attention. Compared with conventional methods, single-molecule detection (SMD) can explore the physicochemical characteristics of objects at the molecular level.1 The ability to specifically and accurately detect single biomolecules is important for clinical medicine and early diagnosis of diseases.2 However, a method with high sensitivity and spatial resolution is required to collect and amplify weak signals for SMD.3,4 In 1961, Rotman first embedded single β-galactosidase molecules into emulsion drops and used its fluorescence products to realize SMD.5 After that, Rissin et al. used microfabrication technology to prepare femtoliter arrays with uniform size and achieved attomole detection limits via enzyme amplification.6 Guan et al. also realized SMD based on a droplet microfluidic chip and the catalysis of single β-galactosidase molecules.7 Considering its high efficiency, the use of enzyme can catalyze the production of lots of fluorescence products and effectively amplify signals for single-molecule fluorescence detection.8−10 However, the background fluorescence of matrix restrict its practical application. To solve the problem, Rissin et al. used 2.7 μm magnetic beads to capture low-abundance proteins from blood.11 Magnetic beads can eliminate the interference of complex matrix, but the presence of magnetic beads may influence the intensity of fluorescence imaging and fluorescence photobleaching is also a problem to be considered, especially for single-molecule fluorescence detection. The development of fluorescent magnetic multifunctional nanospheres may bring new 3 ACS Paragon Plus Environment


breakthroughs. Today, threats to biological security require methods with the ability to detect multiplex targets with high sensitivity and fast speed.12 Single-component detection cannot provide sufficient accurate information for the diagnosis of diseases. Simultaneous detection of multicomponent targets can effectively reduce cost, save time, timely distinguish different pathogens causing similar symptoms and realize high-throughput screening.13−17 Fluorescence method is easy to realize multiplexed detection because fluorescence signals of different emission wavelengths can be collected simultaneously under the excitation of a single light.18,19 Fluorescent dye-labeled microbeads have been widely used for high-throughput detection,20 but the narrow excitation spectra and photobleaching of dyes limit its wide application. The strong fluorescence intensity, photostability, size and composition-dependent optical properties of quantum dots (QDs) enable them great advantages in optical encoding and single-molecule fluorescence detection.21−25 Currently, embedding or assembly method has been developed to incorporate QDs inside or assembled onto the surface of nanospheres to achieve optical encoding. The prepared nanospheres have faster reaction kinetics, stronger fluorescence intensity and optical stability, which have been widely used in bacteria, cell, virus, and nucleic acid detection.26−30 The excellent properties of nanospheres make them have a broad application prospect in SMD. Here, fluorescent magnetic multifunctional nanospheres were prepared by assembling magnetic nanoparticles and QDs with different emission wavelengths on 4 ACS Paragon Plus Environment

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the surface of copolymer nanospheres. The prepared nanospheres maintained the fast magnetic response ability of magnetic nanoparticles and the strong fluorescence of QDs, which can not only separate rare targets from complex samples without sample pretreatment, but also enable multiplexed detection with high sensitivity. By using the nanospheres as both capture carriers and signal labels, a digital single virus immunoassay was developed for simultaneous detection and separation of multiplex viruses. Antibodies against H9N2, H1N1 and H7N9 AIV were conjugated to the green, yellow and red fluorescent magnetic nanospheres (GMNs, YMNs and RMNs) respectively. By controlling the concentration ratio of nanospheres to target virus, at most one virus was conjugated on the surface of each nanosphere. After sandwich immunoreaction in antibody modified micropore arrays, the fluorescence images of 500 micropores were obtained under an inverted fluorescence microscope. Based on digital analysis, the micropores with or without fluorescence were denoted as “1” or “0” respectively. Therefore, the concentration of H9N2, H1N1 and H7N9 AIV could be calculated by the “1” probability of GMNs, YMNs and RMNs respectively, as shown in Scheme 1. The integration of digital analysis provided an ultrasensitive quantitative method.



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Scheme 1. Schematic diagram of the digital single virus immunoassay for multiplex avian influenza virus detection.

EXPERIMENTAL SECTION Reagents and Instruments. Inactivated H9N2 AIV, H1N1 AIV and H7N9 AIV were obtained from Wuhan Institute of Virology, Chinese Academy of Sciences. Monoclonal antibody (mAb) and polyclonal antibody (pAb) of anti-H9N2, anti-H1N1 and anti-H7N9 hemagglutinin were purchased from Sino Biological Inc in Beijing. 3-aminopropyltriethoxysilane (APTES) was purchased from Sinopharm Chemical Reagent Co., Ltd in Shanghai. Fluorescence images were obtained with an inverted fluorescence microscope (Nikon Ti-U) which equipped with a CCD camera (Nikon DS-Ri1). Fluorescence emission spectra were collected on a Fluorolog-3 fluorescence spectrometer (HORIBA JOBIN YVON). Antibodies

Modification

of

Multifunctional

Nanospheres.

Using

poly(styrene/acrylamide) nanospheres (Pst-AAm-COOH) as template, γ-Fe2O3 nanoparticles (four layers) and CdSe/ZnS QDs (three layers) with different emission wavelengths were gradually assembled onto Pst-AAm-COOH surface to fabricate 6 ACS Paragon Plus Environment

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GMNs, YMNs and RMNs, respectively. To modify antibodies on the nanospheres, 10 μL GMNs, 7 μL YMNs and 5 μL RMNs were reacted with 200 μL phosphate buffer solution (PBS, 0.01 M pH 6.1) containing 5 mM EDC/NHS at 37 °C for 30 min, respectively. Then GMNs, YMNs and RMNs were reacted with 100 μL 0.1 mg/mL antibodies anti-H9N2, anti-H1N1 and anti-H7N9, respectively. Finally, the antibodies modified nanospheres (IGMNs, IYMNs and IRMNs) were obtained and stored at 4 °C. Preparation of Antibody Modified Micropore Arrays. The 24×24 mm2 cover glass slides were used as substrates and amino modified according to our previous work.31 Subsequently, 300 μL pH 7.2 PBS containing 1 mM succinic anhydride was added and reacted for 30 min. After activation with EDC/NHS, the glass slides were placed into 300 μL pH 7.2 PBS containing three kinds of antibodies (anti-H9N2, anti-H1N1 and anti-H7N9) simultaneously to prepare mAb modified glass slides. Then the glass slides were blocked with 1% BSA and stored at 4 °C. At the same time, 20×24 mm2 PDMS membrane was obtained and punched with 800 μm needle to get 500 micropores according to our previous work.28 Manual operation was used to punch holes in the PDMS membrane. The PDMS membrane with 500 micropores was bonded to the glass slide to fabricate mAb modified micropore arrays. The micropore arrays weren’t reusable to ensure the accuracy of the experimental results. Detection Procedure of the Digital Single Virus Immunoassay. As illustrated in Scheme 1, antibody modified multifunctional nanospheres were added into the sample 7 ACS Paragon Plus Environment


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and reacted at 37 °C for 30 min. Then the target/nanosphere complexes were dispersed into 300 μL PBS. Each 0.5 μL target/nanosphere solution was added into mAb modified micropore arrays by manual pipetting. After sandwich immunoreaction at 37 °C for 30 min and washing with PBS, the fluorescence images of each individual micropore under different virus concentrations were collected and then the distribution of encoded nanospheres in 500 micropores could be obtained by combining above images together. The micropores with or without fluorescence nanospheres were denoted as “1” or “0”, respectively. Finally, the “1” probabilities of GMNs, YMNs and RMNs among 500 micropores were used to calculate the concentration of H9N2, H1N1 and H7N9 AIV, respectively. Single-Component Virus Detection. Three kinds of encoded nanospheres were prepared based on layer-by-layer (LBL) assembly method, including GMNs (525 nm), YMNs (565 nm) and RMNs (615 nm). Different concentrations of single-component virus (H9N2 AIV, H1N1 AIV, or H7N9 AIV) were detected. The fluorescence

images

of

500

micropores

were

collected

after

sandwich

immunoreaction. And the “1” probability of GMNs, YMNs and RMNs was obtained respectively for H9N2, H1N1 and H7N9 AIV counting. Simultaneous Detection of Multiplex Virus. IMNs, IYMNs and IRMNs were simultaneously added to the multiplex virus sample at 37 °C for 30 min with gentle shaking. Then the complexes were individually added into antibody modified micropore arrays to conduct immunoreaction. After washing with PBS, the “1” probability of GMNs, YMNs and RMNs were obtained simultaneously for multiplex 8 ACS Paragon Plus Environment

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virus detection.

RESULTS AND DISCUSSION Characterization of Multifunctional Nanospheres. Using Pst-AAm-COOH as templates,32,33 the coordination between polymer and metal elements on γ-Fe2O3 and QDs surface is used to realize LBL assembly (Figure 1A). This method has good controllability and repeatability, and can accurately construct optical or magnetic encoding.34 Here, three kinds of encoded nanospheres were prepared by assembling magnetic nanoparticles and QDs with different emission wavelengths on the surface of Pst-AAm-COOH, respectively. As shown in Figure 1B, the nanospheres were uniform and dispersed enough to ensure the reliability of parallel experiment results. The average diameter of GMNs, YMNs and RMNs were 324±7 nm, 343±11 nm and 371±10 nm respectively by randomly counting 200 nanospheres (Figure 1C). By LBL assembly method, multiple QDs can be loaded onto the surface of each nanosphere. The detection sensitivity can be improved effectively by using the encoded nanospheres as signal labels.



Fig. 1. (A) Fabrication process of multifunctional nanospheres. TEM images (B) and histogram for the size distribution (C) of GMNs, YMNs and RMNs, respectively. From the fluorescence images in Figure 2A, we can see that the nanospheres have good photostability, dispersibility, and the green, yellow and red fluorescence images can be clearly distinguished. The fluorescence of fluorescent magnetic nanospheres was strong and the fluorescence of individual GMNs, YMNs and RMNs can be observed under an inverted fluorescence microscope. The application of these encoded nanospheres can solve the problem of photobleaching in fluorescence detection and realize multiplexed detection. The fluorescence spectra of GMNs, YMNs and RMNs have little overlap (Figure S1A, Supporting Information), indicating less interference with each other in the simultaneous detection. Furthermore, the fluorescence spectra of RMNs, YMNs and GMNs are in good agreement with QDs and only a few nanometers shift at the position of the peak, proving that the nanospheres keep the fluorescence property of QDs well (Figure 10 ACS Paragon Plus Environment

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S1B-D, Supporting Information).35

Figure 2. Fluorescence images (A) and capture efficiencies (B) of GMNs, YMNs and RMNs, respectively. The content of target substance in clinical diagnosis is low and the interference of complex matrix is high. Samples need to be pretreated before testing. Magnetic nanoparticles have many unique advantages in the efficient capture and separation of targets,

including

large

specific

surface

area,

superparamagnetism,

good

biocompatibility and controllability.36,37 Magnetic nanoparticles can be simply operated by using a magnet. Here, the fluorescent magnetic nanospheres prepared by LBL assembly method are fully combined the excellent properties of QDs with magnetic nanoparticles. Almost 100% nanospheres were captured by an external magnet within 150 s (Figure 2B). Therefore, 150 s was selected as the adsorption time of nanospheres on the magnetic scaffold. From the hysteresis loops of GMNs, YMNs and RMNs (Figure S2, Supporting Information), their saturation magnetizations were 14.83, 12.16 and 16.21 emu/g respectively, showing superparamagnetism. The use of multifunctional nanospheres as capture carriers can realize fast capture and separation 11 ACS Paragon Plus Environment


of targets, save time and simplify operation. In view of their large specific surface area and abundant functional groups, polyclonal antibody (pAb) was further modified to the surface of multifunctional nanospheres. After pAb modification, the zeta potentials of GMNs, YMNs and RMNs changed from −28.1, −23.8 and −24.2 mV to −16.7, −17.2 and −14.9 mV respectively (Figure S3, Supporting Information). Correspondingly, the hydrated size increased from 363.1, 384.9 and 409.1 nm to 404.5, 420.3 and 451.1 nm respectively with the dispersive index as low as 0.029, demonstrating the successful modification of pAb and good dispersion in aqueous solution. The same concentration of RMNs and antibody modified RMNs (IRMNs) were reacted with Dylight Fluor 488-labeled goat anti-rabbit IgG and observed under a fluorescence microscope. As shown in Figure 3A, B, the RMNs sample can only see red fluorescence of the nanospheres itself, while yellow-green fluorescence can be seen in IRMNs sample, further indicating the successful modification of pAb. The capture efficiency of virus under different pAb concentrations was investigated. Capture efficiency increased with pAb concentrations and tended to 100% when the concentration reached 0.1 mg.mL-1 (Figure S4, Supporting Information). In order to further characterize the number of pAb on nanospheres surface, the linear curve between nanosphere concentrations and A600nm was obtained based on the UV-vis absorption peak of nanospheres at 600 nm. Accordingly, the linear curve between Dylight Fluor 488-labeled IgG concentrations and fluorescence intensity was obtained based on the fluorescence emission peak of Dylight-labeled IgG (Figure S5, 12 ACS Paragon Plus Environment

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Supporting Information). A certain amount of nanospheres reacted with antibodies and then Dylight-labeled IgG to obtain a sample. According to the absorbance of the sample at 600 nm and its fluorescence intensity, the concentrations of nanospheres and Dylight-labeled IgG could be calculated by above two linear curves. Dylight-labeled IgG concentration related to antibody concentration. Thus, the number of antibody on the surface of each nanosphere could be obtained. To ensure the accuracy of the results, three parallel tests were carried out for each data. The number of pAb conjugated on each RMNs was calculated to be about 142, indicating that antibody modified nanospheres could realize efficient capture and separation of targets from complex matrix. Moreover, the fluorescence intensity of multifunctional nanospheres and the biological activity of pAb on the surface of nanospheres kept stable after 6 weeks storage, demonstrating its good storage stability and photostability (Figure S6, Supporting Information).

Figure 3. Fluorescence images of RMNs (A) and IRMNs (B) after reaction with Dylight Fluor 488-labeled IgG. (C) TEM image of virus/nanosphere complexes. Here, single virus immunoassay was achieved mainly by regulating the concentration ratio between multifunctional nanospheres and target virus. A large number of TEM images have been calculated, and the conclusion was that when the concentration ratio of nanospheres to virus was greater than 5:1, at most one virus was 13 ACS Paragon Plus Environment


captured on the surface of each nanosphere (Figure 3C).11,31 Conversely, when the concentration ratio was

Digital Single Virus Immunoassay for Ultrasensitive Multiplex Avian

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