Robust and Highly Sensitive Fluorescence Approach for Point-of

(2) So far, the acknowledged standard detection method of AIV is virus isolation(3) in .... H9N2 viruses were stained with a lipophilic membrane dye D...
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Robust and Highly Sensitive Fluorescence Approach for Point-ofCare Virus Detection Based on Immunomagnetic Separation Wei Zhao,† Wan-Po Zhang,‡,§ Zhi-Ling Zhang,† Rui-Li He,† Yi Lin,† Min Xie,† Han-Zhong Wang,*,§ and Dai-Wen Pang*,† †

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Research Center for Nanobiology and Nanomedicine (MOE 985 Innovative Platform) and State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan, 430072, P. R. China ‡ College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, P. R. China § State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, P. R. China S Supporting Information *

ABSTRACT: In this work, robust approach for a highly sensitive pointof-care virus detection was established based on immunomagnetic nanobeads and fluorescent quantum dots (QDs). Taking advantage of immunomagnetic nanobeads functionalized with the monoclonal antibody (mAb) to the surface protein hemagglutinin (HA) of avian influenza virus (AIV) H9N2 subtype, H9N2 viruses were efficiently captured through antibody affinity binding, without pretreatment of samples. The capture kinetics could be fitted well with a first-order bimolecular reaction with a high capturing rate constant kf of 4.25 × 109 (mol/L)−1 s−1, which suggested that the viruses could be quickly captured by the well-dispersed and comparable-size immunomagnetic nanobeads. In order to improve the sensitivity, high-luminance QDs conjugated with streptavidin (QDs-SA) were introduced to this assay through the high affinity biotin-streptavidin system by using the biotinylated mAb in an immuno sandwich mode. We ensured the selective binding of QDs-SA to the available biotin-sites on biotinylated mAb and optimized the conditions to reduce the nonspecific adsorption of QDs-SA to get a limit of detection low up to 60 copies of viruses in 200 μL. This approach is robust for application at the point-of-care due to its very good specificity, precision, and reproducibility with an intra-assay variability of 1.35% and an interassay variability of 3.0%, as well as its high selectivity also demonstrated by analysis of synthetic biological samples with mashed tissues and feces. Moreover, this method has been validated through a double-blind trial with 30 throat swab samples with a coincidence of 96.7% with the expected results.

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Thus, for clinical samples, recognition and separation of targets are essential and key steps for accurate detection. Polymer micro/nanobeads especially consisting of maghemite (γ-Fe2O3) or magnetite (Fe3O4) as superparamagnetic beads have unique advantages for this usage: (1) their high surface-tovolume ratio makes beads easily bound to analytes; (2) the well dispersed beads tend to have fast kinetics; (3) the beads are convenient to be manipulated due to their magnetic property. Superparamagnetic beads conjugated with recognition units are widely used either for detection10,11 or separation.12,13 As for virus detection, magnetic beads-based sensitive methods with advanced analytical instrument such as flow cytometry,14 mass spectrometry,15,16 mid-infrared spectrometry,17 and two-dimensional high-performance liquid chromatography (2D-HPLC)18 have been successfully performed. In the past decade, new nanoscale materials with some attractive properties have been popularly employed in pathogen detection to amplify bioassay

ensitive and reliable viral pathogen detection is becoming greatly important in various areas such as food and environmental safety, clinical diagnosis, and antibioterrism.1 Although new technologies have markedly improved the ways of pathogen detections, there still exits a general need for robust, sensitive, and fast methods for clinics at the point-ofcare. Taking avian influenza viruses (AIV), for example, they are type A influenza viruses which have caused several worldwide outbreaks of influenza-like illness in the recent decade.2 So far, the acknowledged standard detection method of AIV is virus isolation3 in cultured cells, eggs, or laboratory animals, subsequently with the serological methods for final identification, but the outcomes may take 1−2 weeks. Other commercial and widely used methods in lab diagnoses are enzyme-linked immunosorbent assays (ELISAs)4 and polymerase chain reaction (PCR) methods.5−8 However, detections at the point-of-care are met with more severe technical challenges due to low concentration of target organisms in a complex biological sample.9 ELISA methods are not typically viewed as high-sensitivity methods. Also, the applications of PCR methods may not be stable with nonspecific stains, degrading nucleases, and cellular debris existing in clinical samples. © 2012 American Chemical Society

Received: November 24, 2011 Accepted: February 1, 2012 Published: February 1, 2012 2358

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signals and improve the sensitivity.19 Their distinct sizedependent properties and high surface-to-volume ratios suggest that target-binding events are able to transduce into detectable signals. One example was gold nanoparticles which have been applied in a variety of virus detection methodologies such as surface plasmon resonance (SPR),20 electrochemistry,21 and surface-enhanced Raman scattering (SERS).22 Another promising nanomaterial is semiconductor nanocrystal quantum dots (QDs) and their utility as fluorescent probes have been most highlighted due to its high sensitivity and selectivity. Recent research has shown that QD-based probes can be successfully functionalized with biorecognition molecules such as peptides, antibodies, or nucleic acids.23,24 Compared with traditional fluorescence dyes, QDs exhibit their unique optical advantages such as wide excitation spectrum, size- and composition-tunable fluorescence emission, high quantum yields, and less photobleaching. Taking advantage of these properties, QDs are well suitable for fluorescence-based biological applications,25,26 and QDs have been used in a variety of laboratory detection applications such as cancer cells, 27,28 bacteria,29,30 or viruses.31,32 However, robustness and reproducibility of nanoparticle-based methods are still needed to be improved for clinical applications. The approach of combining immunomagnetic micro/nanobeads and fluorescence QDs for target pathogen detection is promising for on-site application at the point-of-care due to its high sensitivity and selectivity, easy manipulation, versatility, no pretreatment of samples, and no need for well-trained staff. However, applications of this approach for virus detection at the point-of-care have not been reported. In this work, the approach of combining immunomagnetic nanobeads and fluorescent QDs was first used for highly sensitive virion detection, and the AIV H9N2 virus which is classified by the viral surface protein of hemagglutinin (HA) and neuraminidase (NA) was chosen as a model virus. The superparamagnetic nanobeads modified with monoclonal antiHA antibody (mAb) to surface protein HA of H9N2 enable capture and separation of H9N2 AIV without the pretreatment of samples. The kinetics between the immunomagnetic nanobeads and viruses were studied to understand the nanoscale-particles reaction as a first-order bimolecular reaction. High-luminance QDs conjugated with streptavidin (QDs-SA) were introduced to this assay through the high affinity biotin-streptavidin system by the biotinylated mAb in an immuno sandwich mode as shown in Scheme 1. We ensured

the selective binding of QDs-SA to the available biotin-sites on biotinylated mAb and optimized the method to get a low limit of detection with up to 60 copies of viruses in 200 μL. The robustness of this approach is demonstrated by its very good specificity, precision, reproducibility, as well as its high selectively also in analysis of synthetic samples with mashed chicken tissues and feces. What’s more, this method has been validated by a double-blind trial with 30 throat swab samples without pretreatment with a high coincidence of 96.7% with the expected results. Therefore, this method is promising in the ultrasensitive detection of viruses at the point-of-care.

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EXPERIMENTAL SECTION Materials and Reagents. H9N2 AIV, the inactivated H5N1 AIV, Egg Drop Syndrome (EDS), Infectious Bursal Disease Virus (IBDV), Infectious Bronchitis Virus (IBV), Newcastle Disease Virus (NDV), and the monoclonal anti-HA antibody (mAb) were obtained from Wuhan Institute of Virolog, Chinese Academy of Sciences. The mAb was biotinylated with an EZ-link sulfo-NHS-LC-biotinylation kit purchased from Pierce Biotechnology (Rockford, IL). Superparamagnetic Amino-Adembeads (200 nm) were purchased from Ademtech SA (Pessac, France). N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and streptavidin labeled with fluorescein isothiocyanate (SA-FITC) were purchased from Sigma-Aldrich (St. Louis, MO). Quantum dotsstreptavidin conjugates (QDs-SA, 602 nm) were purchased from Wuhan Jiayuan Quantum Dots Co. Ltd. (Wuhan, China). The dyes of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) and 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) for virus staining were purchased from Beyotime Institute of Biotechnology (Jiangsu China). All other chemical reagents were purchased from Shanghai Chemical Reagent Company. Fabrication of Immunomagnetic Nanobeads. The immunomagetic nanobeads were obtained by incubating the superparamagnetic nanobeads (0.1 mg/mL) with 5 μg of EDCactivated anti-HA mAb (0.01 mg/mL) in 500 μL of 0.1 mol/ mL phosphate buffer solution (PBS, pH 7.2) for 2 h at 37 °C with a gentle shaking of 120 rounds/min, then blocked with 1% (m/v) BSA for 0.5 h at 37 °C. The immunomagnetic nanobeads were separated from the free mAb with a magnetic scaffold and resuspended in 500 μL of PBS, then stored at 4 °C for further use. Targeted Virus Detection Protocol. The content of H9N2 AIV was quantified by the H9N2 AIV real-time PCR kit (Shenzhen PG Biotech.) (see Figure S5 in the Supporting Information). The allantoic fluid samples of H9N2 AIV containing a certain content of viruses were mixed with 0.05 mg/mL immunomagnetic nanobeads in 200 μL of PBS (pH 7.2, 0.1 mol/mL) for 15 min incubation at 37 °C with gentle shaking of 120 rounds/min. The bead-virus composites were separated with a magnetic scaffold to remove the suspension and then resuspended in 100 μL of PBS. After incubation with 1 μg of biotinylated mAb for 15 min at 37 °C, the composites of bead-virus-bintinylated mAb were washed twice using 0.01% (v/v) tween in PBS. Then the composites of bead-virusbintinylated mAb suspended in 200 μL of PBS was added with 1 μL of 10−7 mol/L QDs-SA for 15 min incubation at 37 °C. After washing three times, the fluorescence spectrum of the sample suspended in 150 μL of PBS was analyzed by a fluorescence spectrometer (Shimadzu). The transmission electron microscopy (TEM) image of bead-virus composites

Scheme 1. Principle of the Fluorescence Approach for Virus Detection Based on Immunomagnetic Separation

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of Target Virus. The conjugation of nanobeads with the mAb was demonstrated by the changes in both the surface charge (ζ potential changed from 36.4 to −34.1 mV, data not shown) and the hydrated particle size from 197 to 343 nm (see Figure S1 in the Supporting Information) of the nanobeads. The molecule number of mAb conjugated to each immunomagnetic nanobead was evaluated by the fluorescence intensity of the fluorescein-labeled IgG conjugated to nanobeads33 and was found to be about 700−1000 protein molecules on an immunomagnetic nanobead from the amount of fluoresceinlabeled IgG of 20−25 μg per mg of nanobeads (see Figure S2 in the Supporting Information). The amount of 700−1000 protein molecules per bead implied a high coupling efficiency and a dense coverage of mAb with sufficient binding sites to the target virus to ensure the capture capacity of nanobeads. The capture of the viruses with the beads is shown in Figure 1. Evidently, the blackish spots are nanobeads, while the whitish

was obtained by a Hitachi H-7000FA electron microscope. The fluorescence images of the samples were obtained by a confocal microscope as described below. Control experiments were carried out as outlined above, except H9N2 AIV was replaced with other common avian viruses of inactivated H5N1, EDS, IBV, IBDV, and NDV. Confocal Microscopy Assay. The colocalization analysis of nanobeads, H9N2 AIV, and fluorescent QDs was performed in two parallel experiments. One was stained H9N2 viruses before the capture. A H9N2 sample of 105 copies/mL were incubated with 0.05 mg/mL lipophilic membrane dye DiO (which has a excitation wavelength at 484 nm and a maximum emission wavelength at 501 nm) in 100 μL of PBS (pH 7.2, 0.1 mol/mL) for 30 min, then separated from the free dye by using a desalting NAP-5 column (GE). The stained viruses were treated with the immunomagnetic nanobeads and QDs-SA as the protocol described above. Another parallel experiment was performed as staining the bead-virus composites after being captured by 0.05 mg/mL DiO for 30 min. The stained beadvirus composites were washed two times and then reacted with biotinylated mAb and QDs-SA. Fluorescence images of the two H9N2 samples were obtained by an Andor Revolution XD confocal system containing an Olympus IX 81 microscope with a Nipkow disk type confocal unit (CSU 22, Yokogawa) and an EMCCD (Andor iXon DV885K single photon detector). The 488 laser (DPSS, USA) was used to excite DiO, and the 561 nm laser was used to excite the QDs, respectively. The fluorescence signals from the two fluorophores were separated by using a 525/50 nm bandpass filter (Chroma) and a 605/20 nm bandpass filter (Chroma) and imaged alternately onto the EMCCD by separate channels. Kinetics Study. H9N2 viruses of 5 × 106 copies/mL in 500 μL of PBS were incubated with 10 μL of 0.5 mg/mL dye DiI in DMSO for 30 min at 37 °C. Free dye was removed by a desalting NAP-5 column (GE), and the fluorescence intensity of purified DiI-stained viruses of 106 copies/mL was recorded by a Fluorolog-3 fluorescence spectrometer (Horbia Jobin Yvon). Stained viruses of 106 copies/mL were incubated with 0.05 mg/mL immunomagnetic nanobeads in 200 μL of PBS (pH 7.2, 0.1 mol/mL), and the reaction was frozen in the times of 15 s, 30 s, 45 s, 1 min, 2 min, 3 min, 4 min, 5 min, 8 min, 10 min, 15 min, and 20 min, respectively, by adding 200 μL of 0.1 mol/mL pH 2 Na2HPO4-citric acid and cooling them in an ice bath. Meanwhile, a magnet was prepared to separate the nanobeads at once. Then the fluorescence of the suspensions under different reaction times was recorded. The capture efficiencies were calculated by the decreasing of the fluorescence intensities of the suspensions. The same experiments were performed as outlined above with different amounts of the immunomagnetic nanobeads (0.045, 0.03, and 0.02 mg/mL) and stained viruses of the same quantity. Detection in Synthetic Samples with Mashed Chicken Tissues. Fresh liver, lung, and feces from the healthy chicken were mixed with the H9N2 AIV samples, respectively. The tissues and feces were crushed, and then the supernatant was separated by centrifuging. The supernatant samples were reacted with the immunomagnetic nanobeads and QDs-SA as the protocol described above, and the control experiments were performed the same except no H9N2 AIV was added.

Figure 1. TEM image of bead-virus composites. The blackish spots are nanobeads, while the whitish spots around the nanobeads characteristic of virion morphology are viruses.

spots are characteristic of virion morphology around the nanobeads. The capture was also suggested by the increase in the hydrated particle size of immunomagenetic nanobeads from 343 to 500 nm (see Figure S1 in the Supporting Information). Furthermore, an expected 304 bp fragment related to H9N2 (Figure S3 in the Supporting Information) by reversetranscription (RT) PCR in the decomposer from the nanobeads has been found, reconfirming that target H9N2 had been captured by the nanobeads. The identification of H9N2 AIV by QDs-SA was proved by a colocalization analysis of nanobeads, H9N2 AIV, and fluorescent QDs. H9N2 viruses were stained with a lipophilic membrane dye DiO whose fluorescence could be separated from the QDs’ fluorescence by different bandpass filters in the confocal microscope. The fluorescence of DiO from the stained viruses (green spots in Figure 2b) and fluorescent QDs (red spots in Figure 2c) were merged (orange spots in Figure 2d) to show the colocalization. The good match demonstrated that H9N2 AIV were captured by the immunomagnetic nanobeads and then identified with QDs-SA. Moreover, we stained the bead-virus composites with dye DiO after viruses were captured to confirm the capture. The fluorescent spots of DiO (green spots in Figure 2f) from the stained viruses on the beads were

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RESULTS AND DISCUSSION Efficient Capture of Target Virus. Coupling the Antibody to Superparamagnetic Nanobeads and Capture 2360

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Figure 2. Confocal microscopy images of fluorescent QDs, immunomagnetic nanobeads, and target virus. The images displayed in the upside row (from a to d) and the downside row (from e to h) presents the DiO dye stain assay before and after viruses being captured. The nanobeads in brightfield (black spots in parts a and e), the fluorescence of DiO from the stained virus (the green spots in parts b and f), and the fluorescence of QDs (the red spots in parts c and g) are matched well(the orange spots in parts d and h).

constant due to the excessive amount, and the preceding form to eq 2 is given by

also matched well with immunomagnetic nanobeads (black spots in Figure 2e) and fluorescent QDs (red spots in Figure 2g). A comparison of the images of the detecting results of the H9N2 AIV sample, control sample of inactive H5N1 AIV, and the buffer blank sample were shown in Figure 3 to demonstrate the high selectivity of this method. The images of the H9N2 AIV sample showed the QDs’ fluorescence (bright spots in Figure 3a) in the place where the immunomagnetic nanobeads existed (Figure 3b of the corresponding areas in the brightfield). Meanwhile, the inactive H5N1 AIV as the control sample and the blank sample exhibited no fluorescence (Figure 3c,d) in the locations where the immunomagnetic nanobeads existed (Figure 3e,f). Capture Kinetics and Capture Efficiency. We found that the capture kinetics could be perfectly fitted with a first-order bimolecular reaction when the concentration of the nanobeads was greatly exceeded. We assumed that one bead would bind only one virus in most cases due to the 103 times larger amount of the immunomagnetic nanobeads, and one virus would not bind two or more nanobeads due to the steric hindrance. So the reaction kinetics of the nanobeads and viruses, with a first-order association rate constant kf and a dissociation rate constant kr, could be represented by following equations:34,35

c b(t ) =

c b(t ) ≈ c virus(1 − e−kt )

(4)

A typical example of the time-dependent change of capture efficiency is depicted in Figure 4a, with the concentration of beads of 8.3 × 10−12 mol/L (5 × 109 particles/mL, 0.05 mg/ mL) and the virus concentration of 106 copies/mL. It could be perfectly fitted to eq 4 with the mathematical simulation fits R2 = 0.9983, and the apparent rate constant k is derived to be 0.036 18 s−1, which is similar to the apparent rate constant k value for antibody−antigen binding systems determined by other researchers.36,37 The association rate constant kf is calculated to be 4.25 × 109 (mol/L)−1 s−1 (as shown in Figure 4b) according to the equation k = kfc + kr, with different apparent rate constants k at the different concentrations of immunomagnetic beads of 7.47 × 10−12, 4.98 × 10−12, and 3.32 × 10−12 mol/L (see Figure S4 in the Supporting Information). The capture efficiency with different concentrations of target virus under 15 min incubation was further reconfirmed by estimating the decrease of free viruses in the solution using the real-time PCR method. As shown in Figure S5 in the Supporting Information, the capture efficiency reached 86 ± 1% at a low concentration of 1000 copies/mL and kept a value around 91% when the concentrations of viruses were more than 104 copies/mL.

kf

dc b(t )/dt = k f c(c virus − c b) − k rc b

(3)

where the apparent rate constant of the binding process was given as k = kfc + kr. As kfc ≫ kr, the eq 3 was expressed by the following form:

bead + virus ↔ B−V kr

k f cc virus(1 − e−(k f c + k r)t ) kf c + kr

(1) (2)

In eq 1, bead, virus, and B−V represented the immunomagnetic nanobeads, free viruses, and the bound bead-virus composites. Their concentrations were represented as c, cvirus, and cb, respectively, as show in eq 2. The c was considered 2361

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Figure 4. (a) A plot of time-dependent change of capture efficiency at the immunomagnetic nanobead concentration of 8.3 × 10−12 mol/L, which could be fitted well with the first-order bimolecular reaction analysis (in the insert) and (b) a plot of the apparent rate constant k versus different normalized concentrations of immunomagnetic nanobeads of 8.3 × 10−12 mol/L, 7.47 × 10−12 mol/L, 4.98 × 10−12 mol/L, and 3.32 × 10−12 mol/L with the same virus amount (106 copies/mL).

(curve e), while the fluorescence intensity of the sample (curve f) for the negative control stayed unchanged. Thus, 15 min of incubation of QDs-SA with bead-virus-biotinylated mAb composites was enough for the detection. Other reasons that may introduce the nonspecific adsorption of QDs are hydrophobic and electrostatic interactions between capping molecules on the surface of QDs and biomolecules in this polydispersity system. So we also optimized the pH value and salt concentration of the reaction buffer to reduce the nonspecific adsorption of QDs and to improve the sensitivity. Here, detection in PBS pH 7.2 at different salt concentrations of 0.01 mol/L, 0.1 mol/L, and 0.5 mol/L were carried out to find the optimal ionic intensity (see Figure S8a in the Supporting Information). We found that the biggest ratio of positive versus negative (P/N) was obtained in PBS (pH 7.2, 0.1 mol/L), while at the lower salt concentration of 0.01 mol/L, the nonspecific adsorption of QDs-SA increased and at the higher salt concentration of 0.5 mol/L the polydispersity stability was broken, which could be seen by the quick precipitation of the nanobeads. For the optimization of the salt concentration, PBS at the pH values of pH 5, pH 6, pH 7.2, pH 8, and pH 9 were used as the reaction buffer solution. The biggest ratio of P/N was obtained in PBS (pH 7.2, 0.1 mol/L) with weak nonspecific adsorption of QDs (see Figure S8c in the Supporting Information). HA proteins have been found more stable at high pH than at the low pH,39 but the high pH might influence the immune response which was suggested by the decreased positive signals. Thus, the most proper reacting condition was found to be PBS (pH 7.2, 0.1 mol/L). Robustness for Point-of-Care Detection. Specificity, Precision, and Reproducibility. After optimization, the

Figure 3. Confocal microscopy fluorescence images of AIV H9N2 (a) sample, control sample H5N1 (c), blank sample (e), and the brightfield of the beads from the corresponding areas of H9N2 sample (b), H5N1 sample (d), and water blank (f), respectively.

Reliable and Sensitive Detection Strategy. Here, the biotinylated mAb was achieved by an amidation reaction with an activated biotin-reagent by N-hydroxysuccinimide (NHS) ester. However, the nonspecific absorption of QDs-SA may be introduced by the excessive and unreacted activated biotinreagents with the NHS ester, which would react with any possible amine groups such as amine groups of beads or of nontarget viruses in control samples. So an HPLC system (see the discussion of Figure S6 in the Supporting Information) based on size exclusion was used for purification. A complete separation was achieved by the different retention times of the biotinylated mAb and free biotin-reagents. The purified biotinylated mAb was proved to have 0.8−1 available biotin sites for QDs-SA labeling by the method of monitoring the fluorescence enhancement of streptavidin labeled with fluorescein isothiocyanate (streptavidin-FITC) by biotinylated antibody38 (see Figure S6 in the Supporting Information). The incubation time of QDs-SA with the composite of bead-virusbiotinylated mAb was optimized by comparing the changes of fluorescence intensities of QDs-SA bound to bead-virusbiotinylated mAb composites with increasing incubation time. As shown in Figure S7 in the Supporting Information, the fluorescence intensity reached a plateau after 15 min of incubation in all the cases with different concentrations of viruses from 107 copies/mL (curve a) to 1000 copies/mL 2362

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sample in different dilution ratios, respectively. The interassay variability of 3.30% suggested an acceptable variability of different batches of immunomagnetic nanobeads. Quantitative Analysis and Detection Limit. Under the optimal conditions, this fluorescence method was employed for the detection of H9N2 AIV. The immunomagnetic nanobeads were incubated with H9N2 solution that were 2-fold serial diluted to the concentrations ranging from 4 × 106 copies in 200 μL to 60 copies in 200 μL. Fluorescence intensities with different virus concentrations were collected. As shown in Figure 6, a linear range was exhibited at a very low

methods showed perfect specificity by detecting several common avian viruses such as inactive H5N1, EDS, IBV, IBDV, and NDV as control samples. Typical fluorescence spectra were shown in the insert of Figure 5. The QDs emission

Figure 5. Histogram for detecting results of the specificity of this method with H9N2 samples and several other common avian viruses of inactive H5N1, EDS, IBV, IBDV, and NDV as negative samples with an insert of a typical fluorescence emission spectra of detecting results of AIV H9N2, negative inactive H5N1, and a blank sample.

peak was easily detected in the positive sample, while no obvious QDs peaks were found in the negative samples of inactive AIV H5N1 and buffer blank. In this assay, we established a threshold as the mean fluorescence intensities of the QDs at 602 nm of 100 blank samples plus 3 times the standard deviation (mean + 3SD). A sample would be considered positive when its QDs signal intensity is greater than the threshold value. The results of the specificity detection were shown in Figure 5. Except the H9N2 samples, all other avian viruses samples were considered negative due to their fluorescence intensity were below the threshold value. Good precision and reproducibility are also important criteria for further application of the method at the point-of-care. The precision of this method was presented by the intra-assay variability, which was the variability of the same sample parallelly analyzed several times. Here, samples under several random dilution ratios of a fixed virus concentration were detected five times with the same batch of immunomagnetic nanobeads, and the intra-assay variability (Table 1) was

Figure 6. Plots of the QDs FL intensity versus the virus concentration. Insert: The linear relationship of fluorescence versus virus concentration from 60 copies to 1000 copies in 200 μL.

concentration from 60 copies to 1000 copies in 200 μL with acceptable error bars of signals around 5% and the growth of fluorescence intensity went flattened with the increasing of H9N2 concentration. According to the established positive threshold, the detection limit of this method was low up to 60 copies in 200 μL. The detection limit is similar to the real-time PCR method,40,41 which now has been the most sensitive method. Selective Detection in Synthetic Samples and Throat Swab Samples. AIV were found to replicate in the organs of infected human or animals,42,43 so methods should meet the requirements of reliable detection of samples of a complex composition with tissues and feces. Here, we presented the capability of this method in complex samples. We synthesized H9N2 samples with mashed fresh chicken lung, liver, and feces and synthesized negative samples in the same way with no H9N2 added. Selective detection was shown in Figure 7 that all

Table 1. Intra- and Interassay Variability of This Method intra-assay dilution of standard 4.0 × 106 1.0 × 105 5.0 × 104 1.0 × 103

mean

SD

CV (%) (n = 5)

8 430 139 1.65 11 216 1.83 832 16 174 1.03 917 34 300 0.87 522 intra-assay variability 1.35%

interassay mean

SD

CV (%) (n = 5)

8 378 134 1.60 11 227 1.94 682 15 966 6.12 792 34 1219 3.52 624 interassay variability 3.30%

calculated by the mean of the coefficient of variation (CV % = SD/mean) of the parallel results. The intra-assay variability of 1.35% proved the good precision and the good reproducibility of this method. To further confirm the reproducibility, the interassay variability was tested as in the same way but using five different batches of immunomagnetic nanobeads for each

Figure 7. Histogram for fluorescence intensities of synthetic samples with different mashed chicken tissues and feces. 2363

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positive samples with different tissues and feces presented the obvious QDs signals, while signals of negative samples were all below the threshold. The results suggested that this fluorescence method has selective detection in complex biological samples. Even with mashed tissues and feces, the immunomagnetic nanobeads had the capture capacity of target viruses and the QDs-SA can make a specific identification. What’s more, to further validate the reliability and possibility of this fluorescence method for clinical sample detection, a double-blind trial of 30 throat swab samples of chicken without pretreatment was performed. Here, 30 throat swab samples randomly assigned were detected with our method. The results turned out to be 25 samples as positive and 5 samples as negative. This result had a high coincidence of 96.7% with the expected results, which were also affirmed by the real-time PCR method as 26 positive samples and 4 negative samples. The only one sample with different results of this fluorescence method proved the reliability and the prospect for serving at the point-of-care.

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CONCLUSIONS In summary, we reported a robust and highly sensitive detection strategy for virion detection without pretreatment of samples using immunomagnetic nanobeads for target virus capturing and fluorescent QDs-SA as the detection signal. This method is demonstrated to be robust and sensitive enough for the point-of-care detection with its superior properties of high sensitivity, selectivity, and reproducibility. What’s more, this method is easy to be manipulated, and the time-consumption is less than 50 min. The reliable detection of synthetic biological samples and throat swab samples have validated the promising prospect of this method for sensitive point-of-care detection, and the detection principle can be expanded to other viral pathogen detections.

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ASSOCIATED CONTENT

* Supporting Information S

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Han-Zhong Wang: phone and fax, (+86)-27-87199239; email, [email protected]. Dai-Wen Pang: phone, (+86)-2768756759; fax, (+86)-27-68754067; e-mail, [email protected]. cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Wei Zhao and Wan-Po Zhang contributed equally to this work. This work was supported by the National Basic Research Program of China (973 Program, Grant No. 2011CB933600), the Science Fund for Creative Research Groups of NSFC (Grant No. 20921062), the National Natural Science Foundation of China (Grants 20833006, 21175100, 20875071, 21005056, and 81071227), and the “3551 Talent Program” of the Administrative Committee of East Lake HiTech Development Zone (Grant [2011]137).

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