2.1 Materials and Apparatus

RSV strain was purchased from Guangzhou Bote Biological Technology Development Co., Ltd. (Guangzhou, China). The ELISA kit comprised a microtiter plate coated with anti-RSV, anti-RSV-HRP, hydrogen, TMB, 2 N sulfuric acid, a surfactant wash buffer, and RSV standard was purchased from Fengxiang Biotechnology Co., Ltd. (Shanghai, China). H9N2 avian influenza virus was obtained from Wuhan Institute of Virology, Chinese Academy of Sciences. Goat polyclonal anti-RSV biotin-conjugated antibody (Ab1, ab19986) and goat polyclonal anti-RSV antibody (Ab2, ab20745) were purchased from Abcam Inc. (Cambridge, MA). Streptavidin coated magnetic beads (MBs-SA), 4-nitrophenyl phosphate disodium salt hexahydrate (pNPP) and alkaline phosphatase (ALP) were obtained from Sigma-Aldrich (St. Louis, MO). HAuCl4·H2O was purchased from Sinopharm Group Chemical Regent Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) and trisodium citrate were obtained from Beijing Dingguo Changsheng Biotechnology Co. Ltd (Beijing, China). The phosphate buffer solution (PBS) consisted of 0.15 M NaCl, 2 mM KCl and 0.02 M phosphate buffer (pH 7.4). The diluted buffer of ALP consisted of 50% glycerol, 10 mM Tris, 5 mM MgCl2 and 0.1 mM ZnCl2 (pH 7.0). The detection buffer consisted of 50 mM Tris and 0.5 mM MgCl2 (pH 9.0). All reagents used in this experiment were of analytical grade without further purification, and Mili-Q purified water (18.2 MΩ) was used throughout the experiment.

The absorption spectra of AuNPs were recorded on a U-3010 spectrophotometer (Hitachi, Tokyo, Japan). The optical density (OD) at 405 nm was measured with a microplate reader (Biotek, Vermon, USA). JEM-2010 transmission electron microscopy (TEM) was used to identify the size and shape of gold nanoparticles. A Zetasizer Nano-ZS System (Malvern Inc.) was used for dynamic light scattering (DLS) measurements and zeta potential measurements.

2.2 Synthesis of AuNPs

AuNPs were prepared based on the classical citrate-reduction method with slight modifications [22]. Briefly, 2 mL of 1% (w/v) HAuCl4·H2O in 50 mL of ultrapure water was heated to reflux with vigorous stirring. Then, 1 mL of 5% (w/v) sodium citrate solution was rapidly introduced to react with HAuCl4, resulting in a color change of the mixture from pale yellow to gray, then purple, and finally to wine red. After 20 min, the heater was removed and the solution was left stirring and cooling down to room temperature. The product solution was stored at 4 °C for further use.

2.3 Preparation of MBs–Ab1 Complexes

Streptavidin-functionalized MBs were diluted to 0.1 mg/mL with PBS (pH 7.4). Then, 100 μL 100 μg/mL of biotinylated antibody (Bio-Ab1) was added to 1 mL of SA-MBs solution, and the mixed solution was incubated for 30 min at room temperature. Following that, the mixture was collected by applying an external magnetic field. Finally, the MBs–Ab1 was thoroughly washed three times with PBS (pH 7.4) and was resuspended into 1 mL of PBS (pH 7.4) containing 0.5% (w/v) BSA and stored at 4 °C for further usage.

2.4 Preparation of ALP–AuNPs–Ab2 Conjugates

The ALP–AuNPs–Ab2 conjugates were prepared by physical adsorption of Ab2 and ALP on AuNPs [23]. Briefly, the pH of AuNPs solution was adjusted to 9.0 with NaOH. Then, 100 μL of Ab2 (50 μg/mL) and 100 μL of ALP (180 μg/mL) were added to 1 mL of gold colloids. After gentle stirring for 2 h at room temperature, the mixture was further incubated overnight in the refrigerator to complete the reaction, which results in the adsorption of ALP and Ab2 on the surfaces of the AuNPs. Subsequently, the conjugates were centrifuged at 15,000 rpm for 20 min to remove the supernatant. The obtained precipitates (ALP–AuNPs–Ab2) were redispersed in 1 mL of PBS (pH 7.4) containing 0.5% (w/v) BSA and stored at 4 °C.

2.5 MBs-Based Immunoassay for RSV

MBs–Ab1 was used for fast target RSV collection and separation. 50 μL RSV with various concentrations and 50 μL of MBs–Ab1 suspension were successively injected into an Eppendorf tube. After incubation for 30 min at 37 °C with gentle shaking, the resulting mixture was magnetically collected and washed with PBS. Afterward, 100 μL of ALP–AuNPs–Ab2conjugates was added into the centrifuge tube and incubated for an additional 30 min under the same condition. The formed sandwich complexes between ALP–AuNPs–Ab2 and MBs–Ab1was separated and washed as the above protocol. Following that, the substrate solution (20 μL of 50 mM pNPP and 180 μL Tris–HCl) of ALP was introduced to the tube and shaken for 30 min for the color development. Subsequently, the reaction was stopped by adding 50 μL of 1 M NaOH and the optical density was recorded at 405 nm.

2.6 RSV Detection in Cell Lysate

RSV was propagated using HEp-2 human epithelial cells maintained in RPMI 1640 medium supplemented with 2% fetal bovine serum, penicillin (6 mg/mL) and streptomycin (10 mg/mL). Upon detectable cytopathic effect, RSV was harvested in serum-free medium followed by two freeze–thaws, after which the contents were collected and centrifuged at 3000g for 10 min. The virus concentration was determined by our method as described above and the standard ELISA method, respectively. The commercial ELISA kit was used in accordance with the manufacturer’s instructions.

3.1 Analytical Concept

The procedure of this immunoassay is described in Fig. 1. In this study, MB is employed as solid substrate to magnetically separate the immune complexes. Thus, as shown in Fig. 1a, the capture antibody (Ab1) is first specifically linked to the surfaces of MB via the fast and tight streptavidin–biotin interaction. For the immunoassay development, detection antibody (Ab2) and enzyme molecule (ALP) are immobilized on the citrate-protected spherical AuNP by physical adsorption to form ALP–AuNP–Ab2 conjugates (Fig. 1b). Subsequently, in the presence of RSV, the sandwiched immunocomplex between MB–Ab1 and ALP–AuNP–Ab2 can be formed following the illustration in Fig. 1c. The carried ALP molecules catalyze the dephosphorylation of non-colored pNPP to generate colored product pNP, which can be easily distinguished by the naked eye, making it suitable for detecting analytes in laboratories with fewer resources. In addition, since the amount of ALP is proportional to the concentration of RSV sandwiched through immunoreaction, the amount of RSV in the immunocomplex can be simply determined by measuring the optical intensity of the solution in a plate reader. Therefore, by employing MBs as solid substrate and AuNPs as enzyme carrier for signal amplification, this developed immunoassay would significantly improve the analytical performances with shorter time for the detection of RSV.

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3.2 Characterization of the ALP–AuNPs–Ab2 Probe

AuNPs used in this immunoassay were prepared by reducing HAuCl4 with sodium citrate, thus they are surface coated by the citrate group. The as-prepared AuNPs are red in color, with a characteristic absorption peak at 518 nm, which is ascribed to the surface plasmon resonance of the AuNPs (Fig. 2a). Meanwhile, a TEM image shows that AuNPs are highly dispersed in aqueous solution and the average size is about 13 nm (Fig. 2b). After modification with ALP and antibody, the plasmon absorption peak of AuNPs shifted from 518 to 524 nm (Fig. 2a), suggesting that the biomolecules are successfully immobilized onto the AuNPs [24]. Furthermore, dynamic light scattering (DLS) was employed to analyze the hydrodynamic diameter of bare AuNPs and labeled AuNPs (Fig. 2c, d), which was increased from 17.5 ± 0.9 to 25.0 ± 0.1 nm after labeling, indicating the successful formation of the bioconjugates. Besides, the zeta potential measurements were also performed to characterize the change of surface charge after the formation of ALP–AuNPs–Ab2 conjugates. The surface potential of the bare AuNPs was −30.3 ± 0.7 mV because of the citrate groups capping, while it was neutralized to −9.9 ± 0.9 mV after conjugation, which could be ascribed to the effect of the positively charged amine groups (–NH3 +) in antibodies and enzymes. Taken together, the above results indicated the successful formation ALP–AuNPs–Ab2 conjugates.

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To test the catalytic activity of ALP on the surfaces of AuNPs, ALP–AuNPs–Ab2 was reacted with pNPP, which is the substrate of ALP. As shown in Fig. 3, the AuNPs complex could convert the enzyme–substrate pNPP into a colored molecule pNP, which exhibited with a characteristic absorbance peak at about 405 nm. The results demonstrate that the ALP–AuNPs–Ab2 complex with catalytic activities could be further applied to the immunoassay for RSV.

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3.3 Optimization of Experimental Conditions

To improve the sensitivity and reproducibility for RSV quantification, we have also optimized the experimental conditions, including the concentration of ALP and antibody, the pH for conjugation and the concentration of ALP–AuNP–Ab2 conjugates to be used in the immunoassay.

Citrate capped AuNPs are generally acknowledged to be relatively unstable and tend to aggregate irreversibly even in presence of a small amount of salt (NaCl), accompanying by a color change from red to blue. However, when AuNPs are functionalized with biomolecule, such as DNA or protein, the particles become much more stable and resistant to high concentrations of salt. Thus, to ensure the best gold surface coverage, the gold aggregation test was preliminarily performed to judge the optimal pH value to use for the anti-RSV antibody and ALP conjugation because the conjugation process is influenced greatly by the pH which determines the charges and stability of the protein. In this work, the optimal conditions for the preparation of ALP–AuNP–Ab2 conjugates were determined by measuring the difference between the absorbance at 520 and at 680 nm and plotting it against the different pH values (Fig. 4a, b). As can be seen, the highest level of AuNPs surface coverage by the anti-RSV antibody is achieved when the complex prepared at pH 9.0, while the optimal pH at which gives the highest absorbance difference is found around 7.0–9.0. Taken together, the pH of AuNPs solution was adjusted to pH 9.0 for the conjugation of anti-RSV antibody and ALP to AuNPs. Similarly, the antibody concentration that prevents AuNPs aggregation was also determined by the gold aggregation test. The minimum antibody concentration for conjugation is 10 μg for 1 mL of AuNPs (Fig. 4c). Meanwhile, with the increasing concentration of antibody, the spectrum of AuNPs–anti-RSV conjugate solutions became more and more similar to that of bare AuNPs, indicating that the surface of AuNPs is fully covered by antibody molecules (Fig. 4d). In addition, it was found 36.5 μg/mL of ALP molecules was necessary to completely cover the AuNPs surface (Fig. 4e). Based on the principle of low cost and high sensitivity, 5 μg anti-RSV antibody and 18 μg ALP was adopted for the preparation of ALP–AuNPs–Ab2conjugates in the subsequent study.

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Furthermore, the concentration of the ALP–AuNPs–Ab2 conjugates to be used in the immunoassay was also optimized (Fig. 4f). Various dilution coefficients of 1:2, 1:5, 1:20, 1:50 and 1:100 were analyzed. As the concentration of ALP–AuNPs–Ab2 increased, the value of optical density increased accordingly. However, when a high complex concentration (1:2) was employed, the negative control (without RSV) exhibited a high nonspecific signal, which significantly lowered the signal-to-noise ratio. Taken together, the optimal dilution, with a good balance between low nonspecific signal and high sensitivity, was 1:20.

3.4 Analytical Characteristics for RSV Detection

Analytical performance characteristics of the method were determined by measuring the absorbance at 405 nm with varying concentration of RSV. Figure 5 shows the color signal and optical response produced by our method for the detection of RSV. There was a good linear relationship between the optical signal and the concentration of RSV in the range of 0.5–80 pg/mL, and the regression equation was Y = 0.0096 x + 0.0523 (the correlation coefficient R = 0.9923), where Y is the absorption intensity, and x is the concentration of RSV. The limit of detection (LOD, 3σ/k) for this immunoassay is 0.27 pg/mL, which is over sevenfold lower than that of commercially available RSV ELISA kit (2 pg/mL). Furthermore, using MBs as solid substrate to magnetically separate the immune complexes, which eliminates the multiple washing steps in conventional ELISA, the total assay time was decreased to less than 2.5 h without any pretreatment. Therefore, by taking advantage of the magnetic separation and enrichment of MBs and signal amplification of AuNPs carrier, our strategy provide an enhanced immunoassay for fast and sensitive detection of viral pathogens in clinical diagnosis. Furthermore, the performance of the novel method has been compared with other immunoassays for viral pathogens. Characteristics such as the detection limit and assay time are summarized for all of them in Table 1. As can be observed, the detection limit of the developed immunoassay is competitive with the most sensitive viral detection techniques and the assay time by employing MBs as solid support is much lower than those of other microplate-based immunoassays. The reason for sensitivity enhancement is attributed to the fact that AuNPs can carry multi-ALP molecules, and they gave a higher catalytic efficiency, which therefore, amplify the enzymatic signal. On the other hand, the use of MBs not only reduces the immunoreaction time but also amplifies the signal due to an increase in the surface area and the magnetic enrichment effect.

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Table 1

Brief summary of the results reported on several immunoassays for the detection of viral pathogen

The specificity of the immunoassay played an important role in analyzing pathogens in complex biological samples in situ without any pretreatment. Therefore, to assess the specificity of the proposed immunoassay for RSV detection, other antigens such as H9N2 influenza virus, Gram-negative bacteria Escherichia coli (E. coli), Gram-positive bacteria Staphylococcus aureus (S. aureus), human epithelial type 2 (HEp-2) cells, fetal bovine serum (FBS), and RPMI-1640 medium were employed to investigate following the same procedure. As indicated clearly in Fig. 6, obviously higher optical intensity can be observed with the target virus than that of other components, demonstrating the high specificity of the developed immunoassay. In addition, the reproducibility of the immunoassay for RSV was investigated with intra-assay and interassay precision. The intra-assay precision was performed by analyzing one RSV level for five repetitive measurements, while the interassay precision was carried out by determining one RSV level with five times independently. The intra- and interassay variation coefficients obtained from 20 pg/mL RSV were 5.2 and 7.1%, respectively, indicating that the immunoassay possessed acceptable reproducibility.

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