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Simultaneous Point-of-Care Detection of Enterovirus 71 and Coxsackievirus B3 Jia-Jia Wang,† Yong-Zhong Jiang,†,‡ Yi Lin,† Li Wen,† Cheng Lv,† Zhi-Ling Zhang,† Gang Chen,†,§ and Dai-Wen Pang*,† †

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 ‡ Hubei Provincial Center for Disease Control and Prevention, Wuhan, 430072, People’s Republic of China § Key Laboratory of Oral Biomedicine (Ministry of Education) and Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, Wuhan, 430079, People’s Republic of China S Supporting Information *

ABSTRACT: Human enterovirus 71 (EV71) is one of the pathogens that causes hand, foot, and mouth disease (HFMD), which generally leads to neurological diseases and fatal complications among children. Since the early clinical symptoms from EV71 infection are very similar to those from Coxsackievirus B3 (CVB3) infection, a robust and sensitive detection method that can be used to distinguish EV71 and CVB3 is urgently needed for prompting medical treatment of related diseases. Herein, based on immunomagnetic nanobeads and fluorescent semiconductor CdSe quantum dots (QDs), a method for simultaneous point-of-care detection of EV71 and CVB3 is proposed. The synchronous detection of EV71 and CVB3 virions was achieved within 45 min with high specificity and repeatability. The limits of detection are 858 copies/500 μL for EV71 and 809 copies/500 μL for CVB3.This proposed method was further validated with 20 human throat swab samples obtained from EV71 or CVB3 positive cases, with results 93.3% consistent with those by the real-time PCR method, demonstrating the potential of this method for clinical quantification of EV71 and CVB3. The method may also facilitate the prevention and treatment of the diseases.

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over the years, including multiplex PCR assay,14,15 reverse transcription loop-mediated isothermal amplification,16 and dual-color upconversion fluorescent method.17 Nevertheless, these methods require extraction of nucleic acid and are timeconsuming, hindering their application in point-of-care multiple viruses detection. Therefore, development of a convenient, rapid and sensitive approach for simultaneous virus determination is still urgently needed. The rapid development of nanomaterial offers opportunities for sensitive virus detection. A fantastic nanomaterial is fluorescent quantum dots (QDs), which has been intensively used for such as biodetection18,19 or bioimaging20,21 due to their unique properties such as broad absorption, sharp and symmetric emission spectra,22,23 size-tunable photoluminescence,24 and photobleaching resistance.25 Taking full advantage of these properties, virus detection probes based on QDs have also been constructed in many laboratories. Zhang et al.26 used CdTe QDs to establish a convenient fluorescent method for the

ecently, diseases caused by infectious viruses are emerging in an endless stream, leading to severe threat to human health. Enterovirus, a family of small, nonenveloped and positive strands of RNA viruses with a diameter of about 30 nm, is the primary etiologic agent that can cause seasonal epidemics to children.1,2 One of the serious diseases caused by Enterovirus is hand, foot, and mouth disease (HFMD), which generally infects infants and children.3,4 HFMD frequently associated with Enterovirus 71 (EV71) infection usually leads to neurological diseases and many fatal injuries.5,6 The early clinical symptoms of EV71 infection are very similar to those of Coxsackievirus B3 (CVB3), which mainly causes myocarditis, pericarditis and viral encephalitis.7 Therefore, there is an urgent need to establish a robust and sensitive method to distinguish and quantify these two kinds of viruses simultaneously for further improvement of the therapeutic efficiency. Conventional methods for detection of viruses mainly include virus isolation culture,8 enzyme-linked immunosorbent assays (ELISAs),9 loop-mediated isothermal amplification,10 and polymerase chain reaction (PCR).11−13 However, these methods are unable to quantifying multiplex viruses simultaneously and rapidly. Novel methods with regard to multiple Enterovirus detection in single specimen have been developed © XXXX American Chemical Society

Received: August 24, 2015 Accepted: October 13, 2015

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Analytical Chemistry sensitive detection of H9N2 virus. Chen et al.27 prepared a plasmonic resonator based on hybrid nanoclusters of QDs and Au for specific detection of hepatitis B virus. He et al.28 has developed a fluorescent probe based on CdTe:Zn2+ QDs with Ru-(bpy)2 (mcbpy-O-Su-ester) (PF6)2− for determination of EV71 virus. They also proposed a method by quenching multicolor QDs fluorescence with graphene oxide for the simultaneous determination of EV71 and CVB3.29 All these studies show that QDs are well suitable for the sensitive detection of virus. Magnetic nanobeads, another fantastic nanomaterial, have also been widely used for biodetection30−33 and bioseparation34−37 due to their superior magnetic separation characteristics, good maneuverability, fast binding kinetics, and biological targeting properties.38 For example, Liu et al.39 used magnetic nanobeads as carrier for AChE and realized sensitive colorimetric detection of EV71. Zhang et al.40 fabricated an immunomagnetic separation-based electrochemical method for determination of H9N2 virus. The approach that combine QDs fluorescence with immunomagnetic separation can realize sensitive and strong interference virus detection. Our previous studies have also established several such virus detection methods, including highly sensitive fluorescence approaches41−43 and microfluidic immunomagnetic fluorescence assay for H9N2.44 However, method based on the combination of magnetic nanobeads and multicolor fluorescence QDs for convenient multiplex detection of viruses has not yet been reported. In the present study, a novel method is proposed for simultaneous detection of CVB3 and EV71 based on immunomagnetic bioseparation and QDs fluorescence recognition. As shown in Scheme 1, magnetic nanobeads modified

human throat swab samples and has an excellent coincidence with PCR. The investigation indicated that our approach had a wide range of potential applications in effective virus surveillance, high throughput screening test, and early diagnosis in remote areas.

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EXPERIMENTAL SECTION Materials and Instruments. The inactivated EV71 were obtained from Hubei Provincial Center for Disease Control and Prevention. The inactivated CVB3 and EV71 mAb was obtained from Wuhan Institute of Virology, Chinese Academy of Sciences. CVB3 mAb was purchased from Millipore (MA, U.S.A.). Super paramagnetic carboxyl-adembeads (500 nm) were purchased from Ademtech SA (Pessac, France). N-(3(Dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (MA, U.S.A.). Quantum dots (QDs 525 and QDs 605) were purchased from Wuhan Jiayuan Quantum Dots Co. Ltd. (Wuhan, China). All other chemical reagents were purchased from Shanghai Chemical Reagent Company. All solutions were prepared using ultrapure water (Millipore). Fluorescence spectrum was collected with Fluorolog-3 (Horiba Jobin Yvon) fluorescence spectrometer. UV−vis spectra were recorded by a UV-2550 spectrophotometer (Shimadzu, Tokyo, Japan). The transmission-electron microscopy (TEM) images were obtained with the Hitachi H-7000FA electron microscope. The fluorescence images were obtained with an ECLIPSE TE2000-U microscopy (Nikon). The hydrated particle size and the surface charge dynamic light scatting were detected by dynamic light scatting (DLS; Zetasizer Nano ZS90, Malvern Instruments). All optical measurements were performed at room temperature. Preparation of MS-Abs and QDs-Abs. The immunomagnetic nanobeads were obtained by incubating the magnetic nanobeads (50 mg/mL) with EDC and NHS in 500 μL 10 mM phosphate buffer solution (PBS, pH 6.8). After activating for 30 min at 37 °C, the magnetic nanobeads were separated with a magnetic frame and washed with PBS (10 mM, pH 7.2) for three times. Then they were redispersed in 500 μL of PBS (10 mM, pH 7.2) to react with 5 μg EV71 or CVB3 mAb for 2 h at 37 °C. Then, the free mAb were separated with a magnetic scaffold and the MS-Ab were blocked with 1% BSA for 30 min at 37 °C, resuspended in 500 μL of PBS (10 mM, pH 7.2), and then stored at 4 °C for further use. Details of direct coupling of EV71 mAb with QDs 525 and CVB3 mAb with QDs 605 can be found in the Supporting Information. Detection of Virus. The concentration of EV71 and CVB3 were quantified by real-time PCR (CFX Connect RT system, BIO-RAD, U.S.A.). EV71 or CVB3 samples were mixed with 0.1 mg/mL immunomagnetic nanobeads in 500 μL of 10 mM PBS (pH 7.2) and incubated for 15 min at 37 °C. Bead−virus complexes were separated with a magnetic scaffold to remove the excess and resuspended in 200 μL of PBS. Then, 1 μL of 10−7 mol/L QDs-Ab was added and incubated for another 25 min at 37 °C. After washing three times using PBS with 1% BSA, the sample was suspended in 100 μL of PBS and analyzed with a fluorescence spectrometer. Transmission electron microscopy (TEM) was used to characterize the bead-virus complexes, and the fluorescence images of the samples were obtained with microscopy. Inactivated viruses, including H9N2, PrV, and NDV, were used instead of EV71 or CVB3 in control experiments. Furthermore, MS-Abs and QDs-Abs were added to a human throat swab obtained from EV71 and CVB3

Scheme 1. Schematic Principle of Simultaneous Detection of EV71 and CVB3

with EV71 monoclonal antibody (MS-Ab1) and CVB3 monoclonal antibody (MS-Ab2) are used to capture and separate the two viruses without any pretreatment of the complex samples. Meanwhile, monoclonal antibodies labeled with two colored QDs that can be excited with a single excitation were also used to form sandwich structures that could quantify EV71 and CVB3. The sensitivity, reliability, reproducibility, and time consumption of the immunosensor were evaluated. The whole immunomagnetic capture and QDs labeling process could be accomplished within 45 min. Moreover, the proposed method was further validated with B

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Figure 1. Efficient capture of EV71 virions with immunomagnetic nanobeads. (A, B) Typical TEM images of EV71 virions (A) and immunomagnetic nanobead-EV71 complex (B); The blackish sphere is one nanobead, while the whitish spheres around the nanobead characteristic of virion morphology are EV71 virions. (C) Agarose gel electrophoresis of RT-PCR product obtained from the immunomagnetic bead-EV71 virions complex. Lanes 4−6 correspond to EV71 of 7.5 × 103, 1.5 × 104 and 3 × 105 copies/mL, respectively. Lane 2 is the positive control (6 × 106 copies/ mL), and lane 3 is the negative control. Lane 1 is the molecular weight marker. (D) Capture efficiencies of the immunomagnetic nanobeads incubated with EV71 virions of different concentrations for 15 min.

nanobead-EV71 complex after MS-Abs capturing were shown in Figure 1A,B. Obviously, EV71 virions were successfully captured with the immunomagnetic beads. As shown in Figure 1C, the capture of EV71 virions with as-prepared immunomagnetic nanobeads was also validated with PCR. The expected 220 bp fragment related to EV71 from the beads has been found, further confirming that target EV71 virions were captured by the beads. The capture efficiency of EV71 virus was also evaluated using real-time PCR method (details can be found in Supporting Information). As shown in Figure 1D, the average capture efficiency was 82% at a low concentration of 2000 copies/mL and around 87% when virus concentrations was 3 × 105 copies/mL. The results suggested that high efficiency and specific capture of virus were achieved with the method. Identification of Viruses with QDs-Abs. The identification of EV71 virions by QDs-Abs was proved by the microscopy fluorescence images of EV71 sample, control sample of CVB3 and the blank in Figure 2 (similar result of CVB3 was shown in Figure S4). The images of EV71 sample showed the fluorescence of QDs 525 (green spots in Figure 2A) in the place where the immunomagnetic nanobeads existed (the corresponding areas in the bright field in Figure 2B). Meanwhile, the blank and CVB3 as the control samples exhibited no fluorescence (Figure 2C,E) in the position where

positive cases following the procedure described above, and then the obtained results were compared with the analytical results by real-time PCR method.

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RESULTS AND DISCUSSION Characterization of MS-Abs and QDs-Abs. Hydrated particle sizes and surface charges of beads were detected with DLS to demonstrate the conjugation of magnetic nanobeads with mAbs. Hydrated particle size was found to change from 411 to 439 nm, while the surface charge varied from −27.5 to −43.6 mV (Figure S1). In particular, the use of multicolor QDs probes in immunodetection is considered one of the most promising and clinically relevant applications. Here, we describe the preparation of stable QDs probes which were achieved by coupling the monoclonal antibody Fab fragment to the surface of QDs. The fluorescence spectra and UV−vis spectrum were almost identical before and after QDs conjugation with Abs (Supporting Information, Figure S2). Agarose gel electrophoresis was used to evaluate the difference in the electrophoretic migration rate after coupling. The migration of QDs was slightly faster than QDs coupled with Abs, suggesting the conjugation of Abs to QDs. Efficient Capture and Identification of Viruses. Capture of Viruses with MS-Abs and the Capture Efficiency. Typical TEM images of EV71 virions and immunomagnetic C

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by our previous investigation.41 The equilibrium of immunomagnetic bead-virus complex with QDs-Ab1 was reached within 25 min, which is a little later than the first step. It could be attributed to the increased particle size after antibody immobilization on the QDs surface; thus, the steric hindrance increased and the reaction rate reduced accordingly. Total time of the detection process with this method is less than 45 min, which is far less than traditional PCR method, providing the opportunity toward point-of-care detection of virus. Other factors such as pH and salt concentration that may affect the detection results and introduce nonspecific adsorption are hydrophobic and electrostatic interactions between QDs and biomolecules in this system. So the pH and salt concentration of the reaction buffer were also optimized (Figure S5). Results showed that the highest ratio of positive divide negative (P/N) was obtained in 10 mM PBS (pH 7.2) without NaCl. The higher the NaCl concentration, the lower the P/N. High NaCl concentration may even lead to the precipitation of the QDs-Ab. For the optimization of the pH, the highest P/N was obtained in 10 mM PBS (pH 7.2). Unfavorable pH might influence the immunoreactivity, which was demonstrated by the decreased signals. Therefore, 10 mM PBS (pH 7.2) was used as the reaction buffer solution in all the experiments. Quantification of EV71 and CVB3. Under the optimum conditions, EV71 and CVB3 can be determined by the method, respectively. Fluorescence spectra of EV71 and CVB3 virions with different virus concentrations captured by MS-Abs and quantified by QD-Abs with our method were obtained within the range of 1.0 × 103 copies/500 μL to 1.2 × 105 copies/500 μL. As shown in Figure 4, fluorescence intensity increased linearly with the EV71 virus concentration in the range between 1.0 × 103 copies/500 μL and 1.5 × 104 copies/500 μL with a detection limit of 594 copies in 500 μL based on 3× the standard deviation of the blanks to the slope of the linear equation. Likewise, for the quantification of CVB3, a linear relationship was obtained when the virus concentration was between 1.0 × 103 copies/500 μL to 1.5 × 104 copies/500 μL, with a detection limit of 526 copies in 500 μL. The high sensitivity and low detection limit of this method in the detection of EV71 and CVB3 indicated that this method is

Figure 2. Fluorescence images of EV71 (A), blank (C), control CVB3 (E), and the corresponding bright field images (B, D, F).

the immunomagnetic beads presence (Figure 2D,F). All the above results indicated that this method could be used to specifically identify and capture target virions. Sensitive and Rapid Detection Strategy. Total detection time is critical for the application of a point-of-care detection. Here, the incubation time of the two steps included in EV71 detection was optimized. As shown in the fluorescence responses of the EV71-QDs complex as a function of capture time of MS-Abs1 with EV71 virions (Figure 3A), the fluorescence intensity reached the platform within 15 min, which is similar to the rate of antibody−antigen binding studied

Figure 3. Optimizing the incubation time. (A) Fluorescence responses of the EV71-QDs complex as a function of capture time by 8 × 10−2 g/L immunomagnetic nanobeads in the presence (red curve) and absence (black curve) of 3 × 105 copies/mL EV71. (B) Time-dependent fluorescence responses of the EV71-QDs complex during 0.3 μM QDs-Ab labeling in the presence (red curve) and absence (black curve) of 3 × 105 copies/mL EV71. Excitation wavelength = 380 nm. D

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Figure 4. (A) Fluorescence spectra of EV71-QDs complex upon the increasing of EV71 concentration. (B) Plot of fluorescence intensity of EV71QDs complex as a function of EV71 concentration. Inset: The calibration curve of EV71 quantification. (C) Fluorescence spectra of CVB3-QDs complex upon the increasing of CVB3 concentration. (D) Plot of fluorescence intensity of CVB3-QDs complex as a function of CVB3 concentration. Inset: The calibration curve of CVB3 quantification. Concentrations: immunomagnetic bead, 8 × 10−2 mg/mL; QDs-Ab, 0.3 μM; excitation wavelength = 380 nm.

Figure 5. Selectivity of EV71 (A) and CVB3 (B) detection. PrV, H9N2, and NDV were used as negative samples. MS means the treatment with magnetic beads.

EV71 or CVB3 virions, no obvious fluorescence signal of QDs was found (Figure 5A,B), indicating that the nonspecific binding was negligible and the detection method had very good specificity. In this method, we set the negative threshold as the mean fluorescence intensities of the QDs 525 or QDs 605 of 30 blank samples plus 3× the standard deviation (mean + 3SD). For simultaneous detection of EV71 and CVB3, it is crucial to validate whether there is cross-reaction. As shown in Figure S6, no cross-reaction between the two antibodies was observed in

more sensitive than the traditional ELISA method and has the same magnitude with real-time PCR39 method. Simultaneous Point-of-Care Detection of Both EV71 and CVB3. Selectivity and Reliability. Several other viruses such as PrV, H9N2, and NDV were detected with this method to verify the selectivity. As shown in Figure 5, the characteristic QDs fluorescence emission spectrum could be observed only when the target virus was presented. Similarly, when MS without antibodies modification were used to incubate with E

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Analytical Chemistry our experiments. When only EV71 virus exists, there was just one corresponding fluorescence peak of QDs 525. Likewise, the fluorescence peak of QDs 605 appeared only when CVB3 virions existed. Thus, this method can be employed for the simultaneous detection of EV71 and CVB3. The microscopy fluorescence images of EV71 and CVB3 (Figure 2 and Figure S4) also demonstrated the high selectivity of this method. Thus, it can be concluded that MS-Abs and QDs-Abs specifically identified and captured their target viruses. The precision and reproducibility of this method were evaluated by the intra-assay and inter-assay variability.41 Table 1 showed the coefficient of variation for EV71 and CVB3. The intra-assay variability and interassay variability proved the perfect precision and reproducibility of this method. Table 1. Intra- and Inter-Assay Variability of EV71 and CVB3 with This Method intra-assay EV71 virion concentration (copies/mL) 4.0 × 103 3.0 × 104

CVB3 virion concentration (copies/mL) 4.0 × 103 3.0 × 104

mean

SD

inter-assay CV (%) (n = 5)

56464 1293 2.3 98568 3469 3.5 intra-assay variability 2.9% intra-assay

mean

SD

CV (%) (n = 5)

63986 1439 2.2 117663 3671 3.1 intra-assay variability 2.7%

mean

SD

CV (%) (n = 5)

56574 1725 3.1 98501 3959 4.0 inter-assay variability 3.5% inter-assay

mean

SD

CV (%) (n = 5)

63114 1590 2.5 128949 5029 3.9 inter-assay variability 3.2%

Simultaneous Detection of Both EV71 and CVB3. All results above provide a guarantee for testing EV71 and CVB3 simultaneously. EV71 and CVB3 were added to the mixture solution of MS-Ab1 and MS-Ab2, then QDs-Ab1 and QDs-Ab2 that can be excited by a single excitation light were added. The detection results were shown in Figure 6, with the increased concentration of EV71 and CVB3, the fluorescence intensities of QDs 525 and QDs 605 increased with an excellent linear correlation, respectively. The detection limit of EV71 was 858 copies in 500 μL with a linear range of 1.5 × 103 to 2.0 × 104 copies/500 μL, whereas CVB3 can be measured at 809 copies per 500 μL with a linear range of 1.0 × 103 to 1.5 × 104 copies per 500 μL. Obviously, multiplex viruses detection had some influence on the sensitivity and the detection limit of EV71 is a little higher than CVB3. This can be attributed to the lower quantum yield of QDs 525 and the stronger scattering of magnetic beads at the shorter wavelength. Through this simultaneous determination method, virus differentiation and quantification could be realized at the same time with good selectivity and sensitivity. Furthermore, compared with RTPCR and ELISA,39 the detection time was distinctly shortened Clinical Sample detection and Analysis. It is important to further demonstrate the reliability and possibility of this fluorescence method for complex and clinical sample detection. The potential applications of this method were validated with synthesized complex samples mixed with both EV71 and CVB3 in DMEM, serum or blood, and with synthesized negative samples in the same way without any virus. As shown in Figure 7, all positive samples presented obvious QDs signals, while negative samples were all under the threshold in signal. Results

Figure 6. Simultaneous detection of EV71 and CVB3. (A) Fluorescence spectra of EV71 and CVB3 with the increasing virion concentration. (B) Linear relationship of fluorescence intensity vs EV71 concentration. (C) Linear relationship of fluorescence intensity vs CVB3 concentration.

suggest that this method has excellent selectivity for complex samples and might be used to detect clinical samples. Subsequently, 20 human throat swabs from EV71 and CVB3 positive cases were found to be 15 positive and 5 negative, 93.3% consistent with those by the real-time PCR method, demonstrating the potential of this method for simultaneous quantification of EV71 and CVB3 in clinical samples. F

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Figure 7. Histogram for fluorescence intensities of EV71 (A) and CVB3 (B) detection in different media.

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CONCLUSIONS In summary, a specific and highly sensitive method is developed for multiplex detection of EV71 and CVB3 based on immunomagnetic nanobeads capture and dual-color fluorescent QDs-Ab recognition. By combining the efficient enrichment ability of magnetic nanobeads with excellent fluorescent properties of QDs, 858 copies/500 μL EV71 and 809 copies/ 500 μL CVB3 could be simultaneously detected within 45 min without tedious manipulations. Its successful application to clinical throat swab samples illustrates that the method is promising in point-of-care diagnosis of real samples, which will facilitate the timely treatment and control of HFMD.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03247. Supporting figures and additional experimental details (PDF). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03247.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86-27-68756759. Fax: +86-27-68754067. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program, 2011CB933600), the National Natural Science Foundation of China (21275111, 21535005), the Health Department of Hubei Province of China (JX6A05), the 111 Project (111-2-10), the China Scholarship Council, and the Collaborative Innovation Center for Chemistry and Molecular Medicine.

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REFERENCES

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DOI: 10.1021/acs.analchem.5b03247 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.5b03247 Anal. Chem. XXXX, XXX, XXX−XXX