A virus-MIPs fluorescent sensor based on FRET for highly sensitive detection of JEV

Author’s Accepted Manuscript A Virus-MIPs Fluorescent Sensor Based on FRET for Highly Sensitive Detection of JEV Caishuang Liang, Huan Wang, Kui He, Chunyan Chen, Xiaoming Chen, Hang Gong, Changqun Cai www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(16)30427-1 http://dx.doi.org/10.1016/j.talanta.2016.06.010 TAL16640

To appear in: Talanta Received date: 9 March 2016 Revised date: 30 May 2016 Accepted date: 5 June 2016 Cite this article as: Caishuang Liang, Huan Wang, Kui He, Chunyan Chen, Xiaoming Chen, Hang Gong and Changqun Cai, A Virus-MIPs Fluorescent Sensor Based on FRET for Highly Sensitive Detection of JEV, Talanta, http://dx.doi.org/10.1016/j.talanta.2016.06.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A Virus-MIPs Fluorescent Sensor Based on FRET for Highly Sensitive Detection of JEV Caishuang Liang, HuanWang, Kui He, Chunyan Chen, Xiaoming Chen, Hang Gong, Changqun Cai*

Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan, Hunan 411105, China. Tel: 86-731-58292205, 86-15273219560 E-mail addresses:[email protected] (C. Cai). Abstract Major stumbling blocks in the recognition and detection of virus are the unstable biological recognition element or the complex detection means. Here a fluorescent sensor based on virus-molecular imprinted polymers (virus-MIPs) was designed for specific recognition and highly sensitive detection of Japanese encephalitis virus (JEV). The virus-MIPs were anchored on the surface of silica microspheres modified by fluorescent dye, pyrene-1-carboxaldehyde (PC). The fluorescence intensity of PC can be enhanced by the principle of fluorescence resonance energy transfer (FRET), where virus acted as energy donor and PC acted as energy acceptor. The enhanced fluorescence intensity was proportional to the concentration of virus in the range of 24-960 pM, with a limit of detection (LOD, 3σ) of 9.6 pM, and the relative standard deviation was 1.99%. In additional, the specificity study confirmed the resultant MIPs has high-selectivity for JEV. This sensor would become a new key for the detection of virus because of its high sensitive, simple operation, high stability and low cost.

Keywords: Fluorescent sensor, Virus-molecular imprinted polymers (virus-MIPs), Fluorescence resonance energy transfer (FRET), Pyrene-1-carboxaldehyde (PC), Japanese encephalitis virus (JEV) 1. Introduction Virus are pathogens that cause acute disease, which sometimes lead to significant morbidity and mortality, such as SARS [1], Ebola [2], Avian Influenza(AI) [3] and the Acquired Immunodeficiency Syndrome (AIDS) [4]. In the fight against virus, the accurate early detection of virus will be particularly important in control and prevention of pandemics [5]. At present, several approaches have been developed to detect virus, such as quartz crystal microbalance method (QCM) [6, 7], enzyme-linked immunosorbent assay (ELISA) [8], electrochemical processes [9], and photochemical method [10]. Most of these approaches require biomolecules as recognition elements, such as enzymes and antibodies, which are often produced by complex protocols. These recognition elements require specific handling conditions for their poor stabilities, and make a high cost [11, 12]. Therefore, stable and low-cost artificial antibodies are in urgent need of being developed. There have been some attempts to prepare these kinds of antibodies [13-15], in which molecularly imprinted polymers(MIPs) exhibit unique advantage in the field of biological macromolecules recognition, due to their strong affinity, high selectivity, high stability to harsh physical and chemical conditions etc.[16,17]. It is well known, the molecular imprinting technology against small molecules and some large molecules such as proteins and DNA have been well-established [18]. The

development of MIPs for virus, however, is still a great challenge due to its much larger size, more complex surface and spatial structure, which lead to highly cross-linked polymer networks and poor rebinding efficiency. Despite of these difficulties, the developments of virus imprinting were continuously updated [19-27]. But, the use of viral molecular imprinting to achieve the detection of virus is really rare. Dickert et al. reported a MIPs-based QCM technique for the detection of tobacco mosaic viruses [19-20]. Shahgaldian et al. synthesized organic/inorganic imprinted polymers to recognize non-enveloped icosahedral viruses by ELISA assay [21]. Altintas et al. immobilized the MIPs on a surface plasmon resonance sensor for specific recognition of waterborne viruses [22, 23]. While, some strict conditions were required by using above-mentioned strategies to detect virus, such as a strictly stable detection environment, a very low environmental refractive index and a necessary of processing-further. Based on the above advanced work of MIPs, a fluorescent strategy for the specific recognition and high-sensitivity detection of Japanese encephalitis virus (JEV) was proposed based on MIPs, which with the advantages of environmental adaptability, low cost and simple operate. To the best of our knowledge, no MIPs-based fluorescent sensor for virus detection was reported so far. Japanese encephalitis virus (JEV), a mosquito-borne zoonotic pathogen is of grave concern because it causes a neurotrophic killer disease Japanese Encephalitis (JE) which, in turn, is responsible globally for viral acute encephalitis syndrome (AES) [28, 29]. Therefore, the specific recognition and high-sensitivity detection of JEV are crucial for the diagnosis of JEV infection [30].

In our report, a MIPs-based fluorescent sensor was developed, in which, JEV was used as the template of the virus-MIPs, and the MIPs layer was anchored on the surface of fluorescent silica microspheres. Pyrene-1-carboxaldehyde (PC), a fluorescent dye, was used to modify the silica microspheres. Virus was detected based on fluorescence resonance energy transfer (FRET), and the fluorescence intensity of PC can be enhanced by the virus, where the virus and PC were used as energy donor and energy acceptor, respectively [31, 32]. Most of the FRET strategy reported was fluorescence quenching resulting from the common quenching material [33, 34], while it would be led to the fluorescence enhancement and achieved a higher sensitivity in this research. In addition, the direct measurement methods without handling-further would greatly simplify the detection procedure. 2. Experimental 2.1. Reagents and Instruments All reagents used were of at least analytical grade. (3-aminopropyl) triethoxysilane (APTES)

and

tetraethoxysilicane

(TEOS)

were

purchased

from

Aladdin.

Pyrene-1-carboxaldehyde (PC) was purchased from Energy Chemical. Ammonium hydroxide (NH3.H2O), acetic acid, anhydrous methanol and anhydrous ethanol were purchased from Tianjin Fuyu Fine Chemical Co., Ltd. A vaccine that was attenuated or inactivated and retains the shell shape of the virus was used instead of the virus. Japanese encephalitis virus (JEV, Japanese Encephalitis Vaccine (Vero Cell), Freeze-dried) was obtained from Beijing Tiantan Biological Products Co., Ltd. Hepatitis A virus (HAV, Hepatitis A (Live) Vaccine, Freeze-dried) was obtained from Changchun Changsheng Life Sciences Ltd. Rabies virus

(Rabies Vaccine (Vero Cell) for Human Use, Freeze-dried) was purchased from Guangzhou Promise Biological Products Co., Ltd. Leprosy virus (LV, Leprosy virus (Vero Cell), Freeze-dried) was obtained from Beijing Tiantan Biological Products Co., Ltd., Ultrapure water was homemade form lab.

All fluorescence measurements were performed on a RF-5301 spectrofluoeometer (Shimadzu, Japan). Fourier transform infrared (FT-IR) spectra (4000-500 cm-1) in KBr were recorded on a Nicolet 6700 Fourier transform infrared spectrometer (Thermo Fisher Scientific, USA). A JSM-6700F field emission scanning electron microscopy (JEOL, Japan) was employed to determine the size and morphology of the samples. 2.2. Preparation and amino-function of silica microspheres The silica microspheres were prepared by the Stöber method [35] and subsequently modified with APTES to form the amino-functioned silica microspheres. Specifically: 9 mL NH3.H2O, 16.3 mL ethanol, 24.5 mL ultrapure water were added to a 100 mL round-bottomed flask with a magnetic stirring at 1100 rpm. Then a mixed solution of 4.5 mL TEOS, 45.5 mL ethanol was added to the flask. The mixed solution was stirred at 1100 rpm for 1 min at room temperature, and then stirred at 400 rpm for 2 h continuously. Then 375 µL APTES was added to the mixed solution and kept under stirring for an additional 2 h. The amino-functionalized silica microspheres were centrifuged and washed with ethanol for three times at least before dried under vacuum. 2.3. Synthesis of PC dye-modified NH2-SiO2 microspheres

0.3 g NH2-SiO2 microspheres and 30 mL ethanol were added to a 100 mL round-bottomed flask, and then treated with ultrasonic for 5 min, followed by high speed mechanical stirring until dispersed well. Then 40 mL 2.5 mM PC dye solution dissolved in ethanol was added to the mixed solution. Kept it under mechanical stirred for 4 h at room temperature. The PC dye-modified microspheres were centrifuged and washed with ethanol for five times, then dried under vacuum. 2.4. Synthesis of virus-MIPs on the fluorescent microspheres. Typical virus-MIPs were prepared by adapting a method from the literature [21, 36]. In brief, the resultant PC dye-modified NH2-SiO2 microspheres 30 mg, acted as fluorescent carrier, were dissolved in 10 mL ultrapure water, and then ultrasonic treated for 5 min to make it dispersed, high speed mechanical stirred for 30 min. Then 2 mL 2mg mL-1 virus vaccines solution was added to the mixture. After which, the stirring speed was decreased to 160 rpm, and stirred for 1 h. Then 30 µL APTES was added. 0.5 h later, 20 µL TEOS and 20 µL NH3·H2O were added and stirred for 12 h. The non-imprinted polymer (NIPs)-capped carrier was synthesized in ultrapure water but without addition of virus. The resultant MIPs-capped and NIPs-capped carrier were centrifuged, and washed with template removal agent (methanol and acetic acid, 19:1, v: v) for eight times until there was no virus measured with fluorescence spectrophotometer, and then washed with ultrapure water for two times. Finally, the precipitate was dried under vacuum to obtain the virus-MIPs which owned specific imprinted binding sites. 2.5 Measurement for fluorescence detection of virus by virus-MIPs.

The fluorescence spectrum was measured with a fluorescence spectrophotometer (model RF-5301, Shimadzu, Japan). The excitation wavelength at 338 nm with the slit widths of excitation and emission were fixed at 5 nm and 10 nm respectively, and the intensities of fluorescence emission at 382 nm were used for quantification, 25 °C. 3. Results and discussion 3.1. Preparation and characterization of virus-MIPs and NIPs. The virus-MIPs were prepared via a multistep procedure (Schemes 1). Silica has been widely used as substrate materials due to its chemical stability and the property of easy modification [37]. In this strategy, the silica microspheres were prepared by the Stöber method [35] and subsequently modified with APTES to form the amino-functioned silica microspheres. Then the amino-functioned silica microspheres were modified by PC dye, which acted as the carrier and provided amino groups for conjugating with virus. Next, the PC-modified silica microspheres (carrier), virus (template), APTES (functional monomer), and TEOS (cross-linking agent) formed polymeric network around the template [21, 36]. Finally, the virus-MIPs that owned specific imprinted binding sites were obtained after the removal of embedded template virus. FT-IR spectra of PC dye, amino-silica microspheres, PC-modified silica microspheres and virus-MIPs were shown (Fig. 1). The strong peak at around 1103 cm-1 indicated the Si-O-Si asymmetric stretching. The moderate peak at around 798 cm-1 and 941 cm-1 were assigned to the Si-O vibration. The band at around 2958 cm-1 was the C-H stretching bands. The peaks at 3421 cm-1 and 1639 cm-1 were on behalf

of N-H stretching band, suggesting the presence of APTES on the silica microspheres (Fig. 1, curve b). The peak at 1387 cm-1 and 1527 cm-1 in the spectrum of PC-modified silica microspheres due to the vibration of CO-NH (Fig. 1, curve c). Because there were no new functional groups, compared with carriers, there was no obvious change in the spectrum of virus-MIPs (Fig. 1, curve d). The FESEM images of carriers, virus-MIPs and NIPs were shown in Fig. 2. All materials consisted of individual particles. The carriers composited of PC dye-modified NH2-SiO2 microspheres revealed a mean diameter of 270 nm (Fig. 2a). And JEV is an average diameter of 40 nm. The surface of virus-MIPs was rougher than that of NIPs, despite their similar physicochemical properties. It might indicate that there were more binding sites on the surface of virus-MIPs than that of NIPs due to imprinted sites left after the removal of template molecules from the virus-MIPs. These binding sites retained the memory of the virus and could rebind it. Also, the mean diameters of 290 nm for the virus-MIPs and 283 nm for the NIPs (Fig. 2b and Fig. 2c), which indicated the imprinting layer about of 10 nm and the non-imprinting layer less than 10 nm. 3.2. Fluorescence detection of virus by virus-MIPs. The emission spectrum of the virus solution was indicated in the range from 295 nm to 500 nm (Fig. 3, curve a). The virus-MIPs displayed the adsorption range of the fluorescent dye PC, between 220 nm and 440 nm (Fig. 3, curve b). The emission spectrum of virus-MIPs was obtained with λmax at 383 nm (Fig. 3, curve c), which was identical to the emission spectra of the fluorescent dye PC. There was overlap

between the emission of virus and the adsorption of PC, where virus acted as the donor, and PC dye acted as the acceptor. It could be predicted that the energy can transfer from virus to PC dye, which led the enhancing fluorescence intensity of PC dye after adding virus [31, 32]. The fluorescence intensity of the virus-MIPs solution was stable while it was dispersed in ultrapure water. The rebinding tests were performed by connecting virus to the virus-MIPs or NIPs at various concentrations from 0.24 pM to 1.2 nM (Fig. 4). The fluorescence response of virus-MIPs and NIPs increased with the increasing concentration of virus solution. The virus-MIPs exhibited significantly binding capacity with virus than that of NIPs at the same concentration. The maximum static adsorption concentration of virus-MIPs and NIPs for virus were 960 pM and 480 pM, respectively. In general, the virus-MIPs possess both specific and nonspecific binding sites while the NIPs only have nonspecific binding sites. Therefore, the virus-MIPs could capture more virus than NIPs. By changing the adsorption time from 0 min to 60 min, the adsorption kinetics was measured at an initial concentration of 480 pM virus (Fig. 5). There were two phases for the adsorption of virus on the virus-MIPs. The fluorescence response of the virus-MIPs to virus increased with time in the first 40 min, and then tended to be equilibrium with the time extension. It was reasonable to assume that this adsorption equilibrium was due to the smaller diffusion barrier with the thin imprinting layer and the hydrogen-bond between virus and APTES. The adsorption kinetic result showed

that the fluorescence would be measured after 40 min for rebinding the virus to the virus-MIPs. The effect of temperature on the virus-MIPs was studied at 14 °C, 25 °C, 40 °C and 50 °C respectively (Fig. 6). The rebinding tests were performed at different temperature for 4h after rebinding virus to the virus-MIPs, and then the fluorescence intensity was measured. The fluorescence response represented with F/Fo, the highest fluorescence values occurred at 25 °C (Fig. 6). And this temperature normally is the ambient temperature, thus the subsequent adsorption reaction was performed at 25 °C. Under the optimized conditions, the fluorescence intensity changing with virus concentration was investigated (Fig. 7). It can be seen that the fluorescence intensity of the virus-MIPs was enhanced gradually with the increasing concentration of virus. The fluorescence enhancing depended on the adsorptive affinity of the polymers. In the case of the virus-MIPs, the fluorescence enhancing was mainly achieved by the affinity of the imprinted binding sites for virus because of the specific interactions. A comparison of the virus-MIPs and NIPs clearly indicated that the increase of fluorescence intensity of the virus-MIPs was much larger than that of NIPs. To evaluate the analytical performance of the virus-MIPs further, the detection limit and linear range were examined. The linear range was 24-960 pM, with R (a correlation coefficient) of 0.9983 for virus (Fig. 7A). The detection limit, calculated as the concentration corresponding to a signal that was three times the standard deviation of the blank, was found to be 9.6 pM. The precision for the eleven-replicate detection of the virus-MIPs was 1.99% (relative standard deviation, RSD). The sensor was

demonstrated to be highly sensitive and highly reliable for the detection of virus. The ratio of K of the virus-MIPs and NIPs were defined as the imprinting factor (IF) to evaluate the selectivity of this sensor. For this sensor, IF of the virus-MIPs was 2.12, which indicated that the virus-MIPs can recognize the virus specifically. 3.3 Selectivity evaluation In order to estimate the selectivity of the virus-MIPs sensor, several compounds, including Japanese encephalitis virus (JEV) (the template), Hepatitis A virus (HAV), Leprosy virus (LV), and Rabies virus (RV) were tested respectively. The virus-MIPs sensor displayed fluorescence enhancement toward these virus, but the degree of fluorescence enhancement of JEV was much larger than its analogs (Fig. 8). This phenomenon could be explained from the following aspects. As for LV and RV, the rod like shape and the large size made it hard to match the molecular imprinting binding sites, therefore, they are difficult to be captured by the imprinted binding sites; and for HAV, the smaller size made them more easily enter into the imprinted binding sites but also easier to disengage from the binding sites, so a smaller degree of fluorescence enhancement occurred. In a word, the size and morphology of virus were the two main factors for selective evaluation. Therefore, the JEV-MIPs demonstrated a preferential affinity, based on the size and morphology, to the target virus compared to the non-target virus. 3.4. Application to real samples analysis To investigate the feasibility of the proposed method for real sample analysis, the added JEV in the 2000-fold dilution of human serum samples solution was selectively

captured and detected by the proposed method. The human serum samples, obtained from Xiangtan University Hospital, were stored at 0–4 °C and diluted 2000-fold with deionized water just before the determination. The determination results and recovery rates are shown in Table. 1. The data exhibited good recovery, thereby indicating that the JEV content could be satisfactorily determined by this method. 4. Conclusions In this study, a strategy was constructed to detect JEV by combining sensitive fluorescent method with specific molecular imprinted technology. Such a fluorescent molecularly imprinted sensor has not been reported. This sensor exhibited an ideal detection range and the detection limit can be as low as pM level. The potential advantage of this fluorescent sensor based on MIPs including simple operation, high stability and low cost will attract more and more investigation in improving the detection of virus in the near future. Acknowledgments This work was supported by the National Natural Science Foundation of China (21305118, 21505112, and 21402168), Science and Technology Department of Hunan Province (2013SK2021). References [1] H. Pearson, T. Clarke, A. Abbott, J. Knight, D. Cyranoski, SARS: What have we learned? Nature 424 (2003) 121-126. [2] E. Simon-Loriere, O. Faye, O. Faye, L. Koivogui, N. Magassouba, S. Keita, J.-M. Thiberge, L. Diancourt, C. Bouchier, M. Vandenbogaert, V. Caro, G. Fall, J.P. Buchmann, C.B. Matranga, P.C. Sabeti, J.-C. Manuguerra, E.C. Holmes, A.A. Sall, Distinct lineages of Ebola virus in Guinea during the 2014 West African epidemic, Nature 524 (2015) 102-104.

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Human serum

The concentration of JEV (pM) Spiked

Measured

Recovery (%)

RSD (%)

0.00

(n=3)

50.00

50.25

100.5

2.8

100.00

97.68

97.7

2.3

500.00

490. 16

98.0

2.5

-1

Condition: virus-MIPs dosage: 12.5 μg∙mL , temperature: 25 °C.

Highlights



A virus-MIPs fluorescent assay for the detection of virus was reported firstly.



The target could be captured specifically by JEV-MIPs via the recognition cavities.



A fluorescence resonance energy transfer (FRET) enhancement strategy for

the specific recognition of virus was obtained to achieve a higher sensitivity. 

Highly sensitive detection of JEV in water was offered with wide linear range.

Schemes 1. Principle of preparation of the virus-MIPs and detection of virus.

Fig. 1. FT-IR spectrum of (a) PC dye, (b) amino-silica microspheres, (c) PC-modified silica microspheres and (d) virus-MIPs.

Fig. 2. FESEM images of (a) PC dye-modified NH2-SiO2 micro-spheres, (b) virus-MIPs and (c) NIPs. (d), (e) and (f) are the enlarged FESEM images of (a), (b) and (c), respectively.

Fig. 3. (a) The emission spectrum of virus that was exited at 280 nm, (b) the absorption spectrum of PC dye, (c) the emission spectrum of virus-MIPs that was exited at 338 nm.

Fig. 4. Adsorption isotherms of virus-MIPs and NIPs. Experimental condition: 4.0 mL of 0-1.2 nM virus, 12.5 mg L-1 virus-MIPs and NIPs; incubated at room temperature for 40 min.

Fig. 5. The adsorption kinetic of virus-MIPs. Experimental condition: 2.1nM virus, 12.5 mg L-1 virus-MIPs; incubated at room temperature.

Fig. 6. Fluorescence response temperature of the virus-MIPs for the influence of

temperature. Experimental condition: 480 pM virus, 12.5 mg L-1 virus-MIPs; incubated for 40 min.

Fig. 7 Fluorescence spectra of A) the virus-MIPs and B) the NIPs with the increasing

concentrations of virus. Inset: plot of ΔIF as a function of the concentration of virus. Experimental condition: 12.5 mg L-1 virus-MIPs; incubated at room temperature for 40 min.

Fig. 8. Fluorescence response of four kinds of virus. Experimental condition: 480 pM virus, 12.5 mg L-1 virus-MIPs; incubated at room temperature for 40 min.

Graphical abstract