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A nonenzymatic optical immunoassay strategy for detection of Salmonella infection based on blue silica nanoparticles
Analytica Chimica Acta 898 (2015) 109e115
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A nonenzymatic optical immunoassay strategy for detection of Salmonella infection based on blue silica nanoparticles Qian Sun, Guangying Zhao, Wenchao Dou* Food Safety Key Laboratory of Zhejiang Province, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Blue silica nanoparticle based probes were synthesized. Immune blue silica nanoparticle showed highly sensitive and selective to Salmonella pullorum and Salmonella gallinarum. A double-probe based immunoassay for Anti-S. pullorum and S. gallinarum was developed. The advantages of the assay were rapidity, higher sensitivity and simple sample pretreatment.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 June 2015 Received in revised form 18 September 2015 Accepted 19 September 2015 Available online 1 October 2015
A novel nonenzymatic optical immunoassay strategy was for the first time designed and utilized for sensitive detection of antibody to Salmonella pullorum and Salmonella gallinarum (S. pullorum and S. gallinarum) in serum. The optical immunoassay strategy was based on blue silica nanoparticles (BlueSiNps) and magnetic beads (MB). To construct such an optical immunoassay system, the Blue-SiNPs were first synthesized by inverse microemulsion method, characterized by SEM, Zeta potential and FTIR. Two nanostructures including Blue-SiNPs and MB were both functionalized with antibody against S. pullorum and S. gallinarum (anti-PG) without using enzyme labeled antibody. Anti-PG functionalized blue silica nanoparticles (IgG-Blue-SiNps) were used as signal transduction labels, while anti-PG functionalized magnetic beads (IgG-MB) were selected to separate and enrich the final sandwich immune complexes. In the process of detecting negative serum, a sandwich immunocomplex is formed between the IgG-MB and IgG-Blue-SiNPs. With the separation of the immunocomplex using an external magnetic field, the final plaque displayed bright blue color. While in the detection of infected serum, IgG-MB and anti-PG formed sandwich immunocomplexes, IgG-Blue-SiNPs were unable to bind to the limited sites of the antigen, and a light brown plaque was displayed in the bottom of microplate well. Stable results were obtained with an incubation time of 60 min at room temperature, and different colors corresponding to different results can be directly detected with naked eye. The reaction of IgG-Blue-SiNPs with S. pullorum was inhibited by 1:100 dilution of positive chicken serum. Such a simple immunoassay holds great potential as sensitive, selective and point-of-care (POC) tool for diagnosis of other biological molecules. © 2015 Elsevier B.V. All rights reserved.
Keywords: Optical immunoassay Blue silica nanoparticles Magnetic beads Antibody Salmonella infection
1. Introduction
* Corresponding author. E-mail address: [email protected] (W. Dou). http://dx.doi.org/10.1016/j.aca.2015.09.041 0003-2670/© 2015 Elsevier B.V. All rights reserved.
Salmonella infections are significant public health concerns throughout the world owing to their contribution to morbidity and
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mortality in humans and other animal species. Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum (Salmonella pullorum and Salmonella gallinarum) cause fowl typhoid and pullorum disease in chickens, turkeys, and some other avian species, respectively [1]. Multilocus enzyme electrophoresis and relative sequence analysis showed S. pullorum and S. gallinarum owned the same O1, O9, O12 antigens. It was difficult and unnecessary to differentiate S. pullorum and S. gallinarum strictly, so they were often detected together [2]. Chicken infection by S. pullorum and S. gallinarum remains a problem in many developing countries of Asia, Central, Africa and South America [3]. Infections with S. pullorum and S. gallinarum can result in acute systemic disease and a high incidence of mortality in young poultry [4]. Vertical transmission of S. pullorum and S. gallinarum from infected parent flocks, magnified by horizontal transmission in the hatcher, can cause economically devastating losses among chicks or poults [5]. In 1935, the National Poultry Improvement Plan (NPIP) was inaugurated in the U.S. The NPIP program is based principally on the detection of infected breeding flocks before they begin producing eggs to prevent disease transmission to progeny. Breeding flocks are screened for specific serum antibodies, using a rapid wholeblood plate agglutination test, at about 16 wk of age. This largescale control program against S. pullorum and S. gallinarum is based on a commercially available stained S. pullorum and S. gallinarum antigen preparation and can be performed relatively quickly in poultry housing facilities. However, in naturally infected flocks, far fewer birds exhibit high, easily detectable antibody titers at any one time. The plate agglutination test thus appears to miss some infected birds. Hence, it would be advantageous to explore new immunoassay protocols with high sensitivity to meet the demand of early infection diagnosis. To solve the urgent need for improved diagnostic tools of antibody, optical immunosensor offers an exciting alternative, especially in poorer populations. Optical immunosensor is a simple, rapid, cost-effective, and high-sample-throughput and general analytical strategy that can be handled for unskilled personnel at the poultry farm. A key challenge for the development of high sensitive optical immunoassay is to exploit highly efficient signal transduction labels, which efficiently transform the detection event into color change [6e8]. Over the past decades, increasing attention has been paid to the design of different kinds of enzyme labels for improving the optical signal [9e11]. Unfavorably, the bioconjugate techniques between antibodies and enzymes often involve in additional conjugation reactions, which may decrease the enzymatic bioactivity to some extent [12]. In addition, natural enzymes are expensive and easily denatured by high temperature, high humidity, and enzyme inhibitors [13]. In contrast, silica nanoparticles are usually cheaper and more stable against environmental change than bio-enzymes. Colored silica nanoparticles are synthesized by modifying silica nanoparticles with organic dye, they have rich colors, good stability, and don't fade in the harsh conditions [14]. Colored silica nanoparticles are a good candidate optical label in the biotechnological systems due to its inherent advantages, such as bright color, easy preparation and good biocompatibility. In our previous work, we had developed an agglutination test for simultaneous detection of two different pathogenic bacteria using the colored silica nanoparticles as the carrier [15]. For complex biological sample, separation and enrichment of targets are essential and key steps for accurate detection. Magnetic beads (MB) as superparamagnetic beads have unique advantages for this usage, MB are conveniently manipulated by a magnet, high surface-to-volume ratios, and fast kinetics in solution [16]. MB were decorated with antibody and protein after surface modification and widely applied to rapid, efficient, and specific separate and enrich target bacteria from the original
samples [17e20]. In this study, we aimed to develop a highly specific, sensitive, and economical tool for diagnosis of S. pullorum and S. gallinarum infection in serum. We used C.I. Reactive Blue 21 to prepare blue silica nanoparticles (Blue-SiNPs) by inverse microemulsion strategy. Herein, Blue-SiNPs and MB were respectively modified with antibodies against S. pullorum and S. gallinarum (Anti-PG) to construct immune Blue-SiNps (IgG-Blue-SiNp) and immune MB (IgG-MB). Anti-PG is immuno-assayed using the competitive immune reactions of S. pullorum and S. gallinarum with Anti-PG and antibody-coated Blue-SiNps (IgG-Blue-SiNps). The IgG-Blue-SiNps were reaction indicators in this method, and the eye-catching color difference was used to identify the result. 2. Experimental section 2.1. Chemicals and reagents Triton X-100, cyclohexane, hexanol, glutaraldehyde (GLU), ammonia (25e28 wt%) were obtained from Chengdu Kelong Chemical Reagent Co., Ltd. (Chengdu, China). 3-[2-(2-aminoethylamino)ethylamino]propyl-Trimethoxysilane (APTMS), tetraethyl orthosilicate (TEOS) were obtained from Aladdin Industrial Inc. (Shanghai, China). C.I. Reactive Blue 21 was supplied by Zhejiang Shunlong Chemical Co., Ltd (Zhejiang, China). Magnetic beads were obtained from Enriching Biotechnology Co., Ltd. (Shanghai, China). Stained S. pullorum and S. gallinarum antigen, polyclonal Antibodies against S. pullorum and S. gallinarum (anti-PG), antibodies against newcastle disease virus (Anti-NDV), antibodies against egg drop syndrome virus (Anti-EDS) and positive serum to S. pullorum and S. gallinarum were purchased from China Institute of Veterinary Drugs Control (Beijing, China). Bovine serum albumin (BSA) was obtained from Beijing Dingguo Biotechnolgy Co., Ltd. (Beijing, China). S. pullorum and S. gallinarum were purchased from China Center of Industrial Culture Collection (China, Beijing) and conserved in the laboratory of the authors. Other reagents were all of analytical grade and were used as received without further purification. The water used was doubly distilled. 2.2. Characterization Hitachi SU-70 Scanning electron microscopy (SEM) was purchased from Hitachi Inc. (Tokyo, Japan); Malvern Nano 2S potential laser particle analyzer was provided by Malvern Instruments Co., Ltd. (Worcestershire, UK); Nicolet 380 Fourier transform infrared spectrometer (FTIR) was provided by Thermo Fisher Scientific Co. (Shanghai, China); 3e18 K high speed refrigerated centrifuge was purchased from Sigma Laborzentrifugen GmbH (Osterode, Germany); All electrochemical experiments were performed at room temperature (25 ± 1 C). 2.3. Synthesis of Blue-SiNps Blue-SiNps were synthesized by inverse microemulsion method according to the literature [21]. The details of the procedure are described in the following: water-in-oil (W/O) microemulsion was prepared firstly by mixing 2 mL of Trition X-100, 8 mL of cyclohexane, 2 mL of n-hexanol, 150 mL of C.I. Reactive Blue 21 (1 mg/mL) and 400 mL of water was mixed and stirred for 15 min room temperature, forming a uniform W/O microemulsion. In the presence of 100 mL of TEOS and 20 mL of APTMS, a polymerization reaction was initiated by adding l00 mL of NH3$H2O (25e28 wt.%). The reaction was allowed to continue for 48 h. After the reaction was completed, the Blue-SiNps were isolated by acetone, followed by centrifuging, ultrasonicating and washing with ethanol and water several times
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to remove any residual surfactant molecules or any physically adsorbed C.I. Reactive Blue 21 from the surface of the particles. Finally, the required the Blue-SiNps were obtained. The Blue-SiNps were suspended in 10 mL ethanol by ultrasonication for the following experiments. 2.4. Covalent immobilization of antibody on Blue-SiNps 30 mg Blue-SiNps were ultrasonically suspended in the mixed solution of 15 mL ethanol and 40 mL APTMS, The APTMS was allowed to hydrolyze under stirring for 12 h at room temperature. After hydrolysis reaction, amino groups were introduced onto the surface of Blue-SiNps. The amino-modified Blue-SiNps (BlueSiNps-NH2) were isolated by centrifugation at 8000 rpm for 15 min and washed three times with ethanol and 0.01 M Phosphate buffered saline solution (PBS, pH 7.3). 10 mL Blue-SiNps-NH2 (1 mg/ml) aqueous solution reacted with 5 mL GLU (2.5%) under stirring for 3 h at room temperature, and then centrifuged and washed with PBS. The above aldehydeactivated Blue-SiNps were suspended in 5 mL of PBS, followed by the addition of 50 mL anti-PG solution (100 mg/mL). After incubating at room temperature for 2 h, the free anti-PG was removed by centrifugation at 8000 rpm for 10 min and washed with PBS. Antibody modified Blue-SiNps (IgG-Blue-SiNps) were suspended in 10 mL of PBS containing 1% BSA to block non-specific adsorption sites on the nanoparticles. The final product IgG-Blue-SiNps were washed, centrifuged, and suspended in 10 mL of PBS (pH 7.3) buffer, either used immediately for test or stored at 4 C for later usage. 2.5. Immobilization of antibody on MB Anti-PG was covalently conjugated to carboxyl-modified MB according to the manufacturer's instructions. 100 mL of 10 mg/mL MB were mixed with 50 mL of anti-PG (100 mg/mL). The reaction was allowed to proceed at 4 C overnight. The mixture was washed for three times with washing buffer in a magnetic field. Unreacted active groups on the MB were blocked with 1% BSA. Finally, the antibody modified MB (IgG-MB) were diserpsed in 1 mL of PBS and stored at 4 C before use. 2.6. Bacterial strains and preparation S. pullorum and S. gallinarum were used as the detection antigens for positive serum. S. pullorum and S. gallinarum were conserved by the laboratory of author and grown in Lysogeny broth (LB) medium with shaking at 37 C. Cells were harvested in late exponential growth phase by centrifugation at 6000 rpm at 4 C for 10 min and washed in triplicate using physiological saline aqueous solution and dispersed in 5 mL physiological saline aqueous solution. Concentration of the bacteria was confirmed by the colony counting (CFU/mL). The enriched bacterial were inactivated with 0.5% formaldehyde and stored at before 4 C use. 2.7. General assay procedure Fig. 1 illustrates the assay principle of the optical immunoassay toward positive serum sample. In a typical assay for the detection of positive chicken serum, 20 mL of IgG-MB were mixed with 1 mL of S. pullorum and S. gallinarum (106 CFU/mL) in centrifuge tube and incubated for 30 min at 37 C with gentle shaking, the IgG-MBS. pullorum and S. gallinarum conjugates were separated magnetically and the clear supernatant was discarded. After that, the magnet was removed from the bottom of centrifuge tube, 50 mL of PBS was added and mixed with shaking. The IgG-MB-S. pullorum and S. gallinarum complexes were separated by putting magnet
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under the centrifuge tube, and supernatant was discarded. The IgGMB-S. pullorum and S. gallinarum complexes were washed with PBS for three times. The immune complexes were dispersed in 20 mL solution and transferred into microplate well, 40 mL chicken serum sample was subjected to react for 10 min, and then 20 mL IgG-Blue SiNps (1 mg/mL) was added into the microplate wells to incubate for 10 min. Place the plate on a magnet for 1 min and then remove the liquid by forcefully inverting the plate while on the separator. After that magnet removed and 50 mL PBS was added in to the well of plate and the plate was shaken for 1 min, then the PBS discarded in the magnet field. The well was washed in magnetic field with PBS for three times to remove unbound IgG-Blue-SiNps. The final plaque was recorded by digital camera. 3. Results and discussion 3.1. Characterizations of Blue-SiNps The Blue-SiNps synthesized by inverse microemulsion method showed bright blue color and good dispersion in aqueous solution (Fig. 2 inset). The organic dye molecules were trapped inside a silica matrix to form the dye doped nanoparticles. The size and morphology of Blue-SiNps was characterized by SEM (Fig. 2). The Blue-SiNps were extremely uniform in size, with the average diameter of 50 ± 2 nm determined by SEM and the characteristics of Blue-SiNps were in accordance with descriptions by Tan et al. [22,23]. Because of the protective function of the silica matrix and the stable chemical properties of C.I. reactive 21, the nanoparticles were highly stable in acidic and basic solution. To verify the stability of the Blue-SiNps, we compared the stability of gold nanoparticles (AuNps) and Blue-SiNps in different pH solution. Negligible color change was observed for the Blue-SiNps in basic and acidic conditions. In contrast, the color of AuNps dramatically changed from wine red to black purple in basic solution, and from wine red to colorless in acidic solution (As shown in Fig. S1). This result indicates that Blue-SiNps are more stable than AuNps in acidic and basic conditions. This high stability of the nanoparticles provides a foundation for the preparation of optical immunoassay used in various conditions. The chemical groups on the surface of Blue-SiNps were confirmed by Zeta potential determination. The same concentration of silica nanoparticles (0.5 mg/mL) was used in all experiments with aqueous solution containing different amounts of HCl (0.01 M) and NaOH (0.01 M) at different experimental pH values (in the range from pH 2 to pH 12), as measured by pH electrode. All values shown in this work were average of three measurements. Fig. 3A displayed the Zeta potential change of Blue-SiNps and Blue-SiNpsNH2 as a function of pH. With the changing pH values, the experimental zeta potential values of Blue-SiNps and Blue-SiNps-NH2 were both found to be more negative at higher pH. This result could arise either from enhanced ionization of the residual surface silanol or from an increased amount of hydroxide ions in the bulk mobile phase at higher pH being adsorbed onto the silica surface. The isoelectric point (IEP) of Blue-SiNps and Blue-SiNps-NH2 is at pH 4.9 and pH 8.4 respectively, the increase of Zeta potential is attributed to the increasing number of protonated amine groups on the Blue-SiNps-NH2 surface. Chemical composition and group on the surface of Blue-SiNps and Blue-SiNps-NH2 were also examined by FTIR (Fig. 3B). Dried samples were measured using KBr pellet method in range of 400e4000 cm 1. A strong IR absorption band at 1060 cm 1, corresponding to the SieOeSi of the silica core, is found in the FTIR of both Blue-SiNps and Blue-SiNps-NH2. As shown in FTIR absorption spectra of Blue-SiNps-NH2, the 3440, 1590 and 1525 cm 1 are attributed to the NeH bonds of amino group. And the absorption
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Fig. 1. Principle of the optical assay for anti-PG detection in negative serum (up) and positive serum (down) based on IgG-Blue-SiNps and IgG-MB.
Fig. 2. The SEM image of Blue-SiNps, physical image of Blue-SiNps and SiNps.
bond at 2956 is evidences of the strong vibration of CeH bond. These results are consistent with the results of Zeta potential and indicate that the amino groups are well modified on the surface of Blue-SiNps. 3.2. Assay optimization This procedure was performed to choose the optimal concentrations of IgG-MNPs, the optimal concentrations of IgG-Blue SiNps, the optimal concentrations of antigen, and the optimal incubation time for the competitive inhibition immunomagnetic assay. IgGMNPs were diluted to 1 mg/mL, 0.2 mg/mL, 0.1 mg/mL, 0.05 mg/ mL, and 0.025 mg/mL, IgG-Blue SiNps were diluted to 10 mg/mL, 5 mg/mL, 1 mg/mL, 0.5 mg/mL, and 0.25 mg/mL. A series of antigen solution were diluted to 1 108 CFU/mL, 1 106 CFU/mL, 1 104 CFU/mL, 1 102 CFU/mL, and 1 101 CFU/mL. The incubation time of serum with S. pullorum was adjusted from 0 to 60 min to achieve an optimal analytical performance. The phenomena of positive and negative reactions depending on color
changes of two kinds of nanoparticles were showed in Fig. 4. IgGMNPs played the role of immobilizing antigen. The performance of the developed optical immunoassay could be greatly affected by IgG-MNPs concentration. With the decrease of IgG-MNPs concentration the brown plaques in the positive result significantly decreased, at the same time, the black plaques in the negative result significantly increased, which decreased the contrast between negative and positive results (Fig. 4A). IgG-Blue SiNps concentrations have a strong influence on the negative results and not on positive results. The blue plaques in negative results increased with IgG-Blue SiNps concentrations, however when the concentration of IgG-Blue SiNps was 1 mg/mL, the results were distinguished easily (Fig. 4B). The antigen concentration has a similar influence on the negative results as IgG-Blue SiNps concentrations, when the concentration of antigen was the contrast between negative and positive results was most obvious (Fig. 4C). Fig. 4D shows the result changes of this optical immunoassay with different incubation time, the positive results rapidly changed with increasing incubation time, which indicates that S. Pullorum and S. gallinarum gradually binds with the antibody via antigeneantibody reaction. After the incubation time was 30 min, the positive results was significant, we could accurately judge the results. Further increase incubation time, e.g. 60 min, had very little additional beneficial effect, The optimum concentration of IgG-MNPs, concentration of IgG-Blue SiNps, antigen concentration and incubation time were selected according to the above experiment, the IgG-MNPs were diluted to 0.1 mg/mL, 1 mg/mL was selected as optimum concentration of IgGBlue SiNps, the optimal concentration of antigen was 1 106 CFU/ mL, and 30 min incubation time was chosen in this study. 3.3. Sensitivity of the optical immunoassay In this optical method, the antibody in the positive serum was detected by a competitive inhibition assay; IgG-Blue-SiNps compete with the antibody in positive serum for the limited binding sites on S. pullorum and S. gallinarum and play as the role of signal transduction labels. If the sample solution contains enough anti-PG, the antibodies would occupy the limited binding sites of antigen, the immune reaction between IgG-Blue SiNps and S. pullorum and S. gallinarum would be inhibited, IgG-Blue SiNps
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with the naked eye. Under the optimal conditions, sensitivity of the developed optical immnunoassay was studied. The plaque color in the bottom of plate well was related to the concentration of antibody. The original positive chicken serum was diluted to different titers (1:1, 1:10, 1:100 and 1:1000) with PBS. Fig. 5 showed that brown plaque was observed in the bottom of the microplate well when the titer of serum was 1:1, 1:10, and 1:100. The color of plaque in these reactions appears brown is because there is enough anti-PG combined with immunomagnetic complexes, the antibody in serum hinder the combination of IgG-Blue-SiNps with S. pullorum and S. gallinarum. There is very little blue plaque in the well of 1:100 titers, indicating the IgG-Blue-SiNaps has formed sandwich complex with S. pullorum and S. gallinarum and IgG-MB, this weakly positive result indicates low antibody level. Blue color of Blue-SiNps was observed clearly in the well of 1:1000 dilution of serum, which was too few to prevent the binding of IgG-Blue-SiNps with S. pullorum and S. gallinarum, so the blue IgG-Blue-SiNps was captured and observed after all washed steps. The sensitivity of this optical assay is compared with the rapid serum agglutination test using commercially available stained S. pullorum and S. gallinarum antigen. As showed in Fig. 6, the stained S. pullorum and S. gallinarum was mixed with different dilution positive serum (1:1, 1:10, and 1:100), agglutination phenomena were only observed in 1:1 and 1:10 dilute serum. When serum was diluted to 1:100, there was no agglutination phenomenon. Furthermore, the agglutination phenomenon of serum agglutination test result was not obvious and not easy to distinguish compared to our optical immunoassay. The contrast proved that the developed optical immunoassay is advantageous in sensitization and obvious phenomenon.
3.4. Specificity of the optical immunoassay
Fig. 3. Zeta potential of Blue-SiNps (square), Blue-SiNps-NH3 (circle), respectively (A), IR spectrum of Blue-SiNps (down) and Blue-SiNps-NH3 (up) respectively (B).
would be removed from sample solution in washing steps, and the well of microplate appears the color of MNPs, brown. While the negative control displays a bright blue plaque. The marked color difference in the wells of microplate can be easily distinguished
We examined the specificity of the developed optical immunoassay method for anti-PG detection. The specificity test was implemented with the same experimental procedure as mentioned in Section 2.4. We challenged the system with other interfering samples including negative serum, Anti-NDV, Anti-EDS, BSA, and PBS, they were all detected by our optical competitive immunoassay method under the same condition. As shown in Fig. 7, brown signal was only observed in the well of Anti-PG, blue signals were viewed in all the rest wells. This result shows that IgG-MB and IgGBlue-SiNps have good specificity for S. pullorum and S. gallinarum and this method has good specificity to Anti-PG.
Fig. 4. The effect of different factors on the positive results (up) and negative results (down) of optical competitive inhibition immunoassay. Shown are the effect of (A) IgG-MB concentration, (B) IgG-Blue-SiNps concentration, (C) antigen concentration, and (C) incubation time.
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Fig. 5. The competitive inhibition immunoassay results of IgG-MB solution and IgG-blue-SiNps solution with positive chicken serum of different dilutions: 1:1, 1:10, 1:100 and 1:1000.
Fig. 6. The serum agglutination test results of the staining S. pullorum and S. gallinarum antigen with different dilutions of positive serum: 1:1, 1:10, and 1:100, PBS as control.
Fig. 7. Specificity result of the optical immunoassay, from left to right: negative serum, positive serum, Anti-NDV, Anti-EDS, BSA solution, PBS.
3.5. Stability of the IgG-Blue-SiNps and IgG-MB The stability of the IgG-MB and IgG-Blue-SiNps was studied by the test of the positive and negative serum sample with the developed optical immunoassay after IgG-MB and IgG-Blue-SiNps were both stored at 4 C for 7, 30, 60 and 90 days. The results were displayed in Fig. 8, the phenomena of positive and negative results did not change after 90 days of storage, this result indicated that the two kinds of immune nanoparticles could retain similar reaction activity and could be used for optical measurements after storage at 4 C for 90 days. 4. Conclusions In summary, a
Blue-SiNps
based
nonenzymatic optical
immunosensing strategy is presented in this study. This new method is a robust, sensitive, inexpensive, and user-friendly analytical method compared with classical assays for the detection of antibody to S. pullorum and S. gallinarum in serum. The BlueSiNps synthesis and the process of modifying Blue-SiNps were characterized via SEM, Zeta potential, and FTIR. This strategy combines the advantages taken from immunochemical assays, magnetic beads separation, and bright color of Blue-SiNps. It does not require sophisticated equipment or costly enzyme labeled antibody. On the basis of IgG-MB enriching the antigen effectively, the color of Blue-SiNps is great signal amplification under the assay conditions. The developed calorimetric assay using Blue-SiNps as optical indicator is more easily monitored by naked eyes owing to the distinct color difference, and can detect positive serum at a dilution factor of 1:100, which is 10 fold lower than the
Fig. 8. Positive and negative results after IgG-MB and IgG-blue-SiNps were stored for: 7days, 30 days, 60 days, 90 days.
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conventional serum agglutination test. 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 stability. What's more, this method is easy to be manipulated, and time-consumption is less than 60 min. This rapid sensing technology should have great potential in a wide range of antibody-based biosensing and diagnostic applications. Acknowledgments This project was supported by the Food Science and Engineering the most important discipline of Zhejiang province (ZYTSP20141062); Zhejiang Public Innovation Platform Analysis and testing projects (2015C37023). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.aca.2015.09.041. References [1] D.F.A. Batista, et al., Polymerase chain reaction assay based on ratA gene allows differentiation between Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum, J. Vet. Diagn. Investig. 25 (2) (2013) 259e262. [2] C. Hu, W. Dou, G. Zhao, Enzyme immunosensor based on gold nanoparticles electroposition and Streptavidin-biotin system for detection of S. pullorum & S. gallinarum, Electrochimica Acta 117 (2014) 239e245. [3] M.-S. Kang, et al., Differential identification of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum and the biovar Gallinarum live vaccine strain 9R, Vet. Microbiol. 160 (3e4) (2012) 491e495. [4] R.K. Gast, Detecting infections of chickens with recent Salmonella pullorum isolates using standard serological methods, Poult. Sci. 76 (1) (1997) 17e23. [5] S. Geng, et al., Virulence determinants of Salmonella Gallinarum biovar Pullorum identified by PCR signature-tagged mutagenesis and the spiC mutant as a candidate live attenuated vaccine, Vet. Microbiol. 168 (2e4) (2014) 388e394. [6] Z. Gao, et al., Urchin-like (gold core)@(platinum shell) nanohybrids: A highly efficient peroxidase-mimetic system for in situ amplified colorimetric
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
A nonenzymatic optical immunoassay strategy for detection of Salmonella infection based on blue silica nanoparticles Qian Sun, Guangying Zhao, Wenchao Dou* Food Safety Key Laboratory of Zhejiang Province, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Blue silica nanoparticle based probes were synthesized. Immune blue silica nanoparticle showed highly sensitive and selective to Salmonella pullorum and Salmonella gallinarum. A double-probe based immunoassay for Anti-S. pullorum and S. gallinarum was developed. The advantages of the assay were rapidity, higher sensitivity and simple sample pretreatment.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 June 2015 Received in revised form 18 September 2015 Accepted 19 September 2015 Available online 1 October 2015
A novel nonenzymatic optical immunoassay strategy was for the first time designed and utilized for sensitive detection of antibody to Salmonella pullorum and Salmonella gallinarum (S. pullorum and S. gallinarum) in serum. The optical immunoassay strategy was based on blue silica nanoparticles (BlueSiNps) and magnetic beads (MB). To construct such an optical immunoassay system, the Blue-SiNPs were first synthesized by inverse microemulsion method, characterized by SEM, Zeta potential and FTIR. Two nanostructures including Blue-SiNPs and MB were both functionalized with antibody against S. pullorum and S. gallinarum (anti-PG) without using enzyme labeled antibody. Anti-PG functionalized blue silica nanoparticles (IgG-Blue-SiNps) were used as signal transduction labels, while anti-PG functionalized magnetic beads (IgG-MB) were selected to separate and enrich the final sandwich immune complexes. In the process of detecting negative serum, a sandwich immunocomplex is formed between the IgG-MB and IgG-Blue-SiNPs. With the separation of the immunocomplex using an external magnetic field, the final plaque displayed bright blue color. While in the detection of infected serum, IgG-MB and anti-PG formed sandwich immunocomplexes, IgG-Blue-SiNPs were unable to bind to the limited sites of the antigen, and a light brown plaque was displayed in the bottom of microplate well. Stable results were obtained with an incubation time of 60 min at room temperature, and different colors corresponding to different results can be directly detected with naked eye. The reaction of IgG-Blue-SiNPs with S. pullorum was inhibited by 1:100 dilution of positive chicken serum. Such a simple immunoassay holds great potential as sensitive, selective and point-of-care (POC) tool for diagnosis of other biological molecules. © 2015 Elsevier B.V. All rights reserved.
Keywords: Optical immunoassay Blue silica nanoparticles Magnetic beads Antibody Salmonella infection
1. Introduction
* Corresponding author. E-mail address: [email protected] (W. Dou). http://dx.doi.org/10.1016/j.aca.2015.09.041 0003-2670/© 2015 Elsevier B.V. All rights reserved.
Salmonella infections are significant public health concerns throughout the world owing to their contribution to morbidity and
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mortality in humans and other animal species. Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum (Salmonella pullorum and Salmonella gallinarum) cause fowl typhoid and pullorum disease in chickens, turkeys, and some other avian species, respectively [1]. Multilocus enzyme electrophoresis and relative sequence analysis showed S. pullorum and S. gallinarum owned the same O1, O9, O12 antigens. It was difficult and unnecessary to differentiate S. pullorum and S. gallinarum strictly, so they were often detected together [2]. Chicken infection by S. pullorum and S. gallinarum remains a problem in many developing countries of Asia, Central, Africa and South America [3]. Infections with S. pullorum and S. gallinarum can result in acute systemic disease and a high incidence of mortality in young poultry [4]. Vertical transmission of S. pullorum and S. gallinarum from infected parent flocks, magnified by horizontal transmission in the hatcher, can cause economically devastating losses among chicks or poults [5]. In 1935, the National Poultry Improvement Plan (NPIP) was inaugurated in the U.S. The NPIP program is based principally on the detection of infected breeding flocks before they begin producing eggs to prevent disease transmission to progeny. Breeding flocks are screened for specific serum antibodies, using a rapid wholeblood plate agglutination test, at about 16 wk of age. This largescale control program against S. pullorum and S. gallinarum is based on a commercially available stained S. pullorum and S. gallinarum antigen preparation and can be performed relatively quickly in poultry housing facilities. However, in naturally infected flocks, far fewer birds exhibit high, easily detectable antibody titers at any one time. The plate agglutination test thus appears to miss some infected birds. Hence, it would be advantageous to explore new immunoassay protocols with high sensitivity to meet the demand of early infection diagnosis. To solve the urgent need for improved diagnostic tools of antibody, optical immunosensor offers an exciting alternative, especially in poorer populations. Optical immunosensor is a simple, rapid, cost-effective, and high-sample-throughput and general analytical strategy that can be handled for unskilled personnel at the poultry farm. A key challenge for the development of high sensitive optical immunoassay is to exploit highly efficient signal transduction labels, which efficiently transform the detection event into color change [6e8]. Over the past decades, increasing attention has been paid to the design of different kinds of enzyme labels for improving the optical signal [9e11]. Unfavorably, the bioconjugate techniques between antibodies and enzymes often involve in additional conjugation reactions, which may decrease the enzymatic bioactivity to some extent [12]. In addition, natural enzymes are expensive and easily denatured by high temperature, high humidity, and enzyme inhibitors [13]. In contrast, silica nanoparticles are usually cheaper and more stable against environmental change than bio-enzymes. Colored silica nanoparticles are synthesized by modifying silica nanoparticles with organic dye, they have rich colors, good stability, and don't fade in the harsh conditions [14]. Colored silica nanoparticles are a good candidate optical label in the biotechnological systems due to its inherent advantages, such as bright color, easy preparation and good biocompatibility. In our previous work, we had developed an agglutination test for simultaneous detection of two different pathogenic bacteria using the colored silica nanoparticles as the carrier [15]. For complex biological sample, separation and enrichment of targets are essential and key steps for accurate detection. Magnetic beads (MB) as superparamagnetic beads have unique advantages for this usage, MB are conveniently manipulated by a magnet, high surface-to-volume ratios, and fast kinetics in solution [16]. MB were decorated with antibody and protein after surface modification and widely applied to rapid, efficient, and specific separate and enrich target bacteria from the original
samples [17e20]. In this study, we aimed to develop a highly specific, sensitive, and economical tool for diagnosis of S. pullorum and S. gallinarum infection in serum. We used C.I. Reactive Blue 21 to prepare blue silica nanoparticles (Blue-SiNPs) by inverse microemulsion strategy. Herein, Blue-SiNPs and MB were respectively modified with antibodies against S. pullorum and S. gallinarum (Anti-PG) to construct immune Blue-SiNps (IgG-Blue-SiNp) and immune MB (IgG-MB). Anti-PG is immuno-assayed using the competitive immune reactions of S. pullorum and S. gallinarum with Anti-PG and antibody-coated Blue-SiNps (IgG-Blue-SiNps). The IgG-Blue-SiNps were reaction indicators in this method, and the eye-catching color difference was used to identify the result. 2. Experimental section 2.1. Chemicals and reagents Triton X-100, cyclohexane, hexanol, glutaraldehyde (GLU), ammonia (25e28 wt%) were obtained from Chengdu Kelong Chemical Reagent Co., Ltd. (Chengdu, China). 3-[2-(2-aminoethylamino)ethylamino]propyl-Trimethoxysilane (APTMS), tetraethyl orthosilicate (TEOS) were obtained from Aladdin Industrial Inc. (Shanghai, China). C.I. Reactive Blue 21 was supplied by Zhejiang Shunlong Chemical Co., Ltd (Zhejiang, China). Magnetic beads were obtained from Enriching Biotechnology Co., Ltd. (Shanghai, China). Stained S. pullorum and S. gallinarum antigen, polyclonal Antibodies against S. pullorum and S. gallinarum (anti-PG), antibodies against newcastle disease virus (Anti-NDV), antibodies against egg drop syndrome virus (Anti-EDS) and positive serum to S. pullorum and S. gallinarum were purchased from China Institute of Veterinary Drugs Control (Beijing, China). Bovine serum albumin (BSA) was obtained from Beijing Dingguo Biotechnolgy Co., Ltd. (Beijing, China). S. pullorum and S. gallinarum were purchased from China Center of Industrial Culture Collection (China, Beijing) and conserved in the laboratory of the authors. Other reagents were all of analytical grade and were used as received without further purification. The water used was doubly distilled. 2.2. Characterization Hitachi SU-70 Scanning electron microscopy (SEM) was purchased from Hitachi Inc. (Tokyo, Japan); Malvern Nano 2S potential laser particle analyzer was provided by Malvern Instruments Co., Ltd. (Worcestershire, UK); Nicolet 380 Fourier transform infrared spectrometer (FTIR) was provided by Thermo Fisher Scientific Co. (Shanghai, China); 3e18 K high speed refrigerated centrifuge was purchased from Sigma Laborzentrifugen GmbH (Osterode, Germany); All electrochemical experiments were performed at room temperature (25 ± 1 C). 2.3. Synthesis of Blue-SiNps Blue-SiNps were synthesized by inverse microemulsion method according to the literature [21]. The details of the procedure are described in the following: water-in-oil (W/O) microemulsion was prepared firstly by mixing 2 mL of Trition X-100, 8 mL of cyclohexane, 2 mL of n-hexanol, 150 mL of C.I. Reactive Blue 21 (1 mg/mL) and 400 mL of water was mixed and stirred for 15 min room temperature, forming a uniform W/O microemulsion. In the presence of 100 mL of TEOS and 20 mL of APTMS, a polymerization reaction was initiated by adding l00 mL of NH3$H2O (25e28 wt.%). The reaction was allowed to continue for 48 h. After the reaction was completed, the Blue-SiNps were isolated by acetone, followed by centrifuging, ultrasonicating and washing with ethanol and water several times
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to remove any residual surfactant molecules or any physically adsorbed C.I. Reactive Blue 21 from the surface of the particles. Finally, the required the Blue-SiNps were obtained. The Blue-SiNps were suspended in 10 mL ethanol by ultrasonication for the following experiments. 2.4. Covalent immobilization of antibody on Blue-SiNps 30 mg Blue-SiNps were ultrasonically suspended in the mixed solution of 15 mL ethanol and 40 mL APTMS, The APTMS was allowed to hydrolyze under stirring for 12 h at room temperature. After hydrolysis reaction, amino groups were introduced onto the surface of Blue-SiNps. The amino-modified Blue-SiNps (BlueSiNps-NH2) were isolated by centrifugation at 8000 rpm for 15 min and washed three times with ethanol and 0.01 M Phosphate buffered saline solution (PBS, pH 7.3). 10 mL Blue-SiNps-NH2 (1 mg/ml) aqueous solution reacted with 5 mL GLU (2.5%) under stirring for 3 h at room temperature, and then centrifuged and washed with PBS. The above aldehydeactivated Blue-SiNps were suspended in 5 mL of PBS, followed by the addition of 50 mL anti-PG solution (100 mg/mL). After incubating at room temperature for 2 h, the free anti-PG was removed by centrifugation at 8000 rpm for 10 min and washed with PBS. Antibody modified Blue-SiNps (IgG-Blue-SiNps) were suspended in 10 mL of PBS containing 1% BSA to block non-specific adsorption sites on the nanoparticles. The final product IgG-Blue-SiNps were washed, centrifuged, and suspended in 10 mL of PBS (pH 7.3) buffer, either used immediately for test or stored at 4 C for later usage. 2.5. Immobilization of antibody on MB Anti-PG was covalently conjugated to carboxyl-modified MB according to the manufacturer's instructions. 100 mL of 10 mg/mL MB were mixed with 50 mL of anti-PG (100 mg/mL). The reaction was allowed to proceed at 4 C overnight. The mixture was washed for three times with washing buffer in a magnetic field. Unreacted active groups on the MB were blocked with 1% BSA. Finally, the antibody modified MB (IgG-MB) were diserpsed in 1 mL of PBS and stored at 4 C before use. 2.6. Bacterial strains and preparation S. pullorum and S. gallinarum were used as the detection antigens for positive serum. S. pullorum and S. gallinarum were conserved by the laboratory of author and grown in Lysogeny broth (LB) medium with shaking at 37 C. Cells were harvested in late exponential growth phase by centrifugation at 6000 rpm at 4 C for 10 min and washed in triplicate using physiological saline aqueous solution and dispersed in 5 mL physiological saline aqueous solution. Concentration of the bacteria was confirmed by the colony counting (CFU/mL). The enriched bacterial were inactivated with 0.5% formaldehyde and stored at before 4 C use. 2.7. General assay procedure Fig. 1 illustrates the assay principle of the optical immunoassay toward positive serum sample. In a typical assay for the detection of positive chicken serum, 20 mL of IgG-MB were mixed with 1 mL of S. pullorum and S. gallinarum (106 CFU/mL) in centrifuge tube and incubated for 30 min at 37 C with gentle shaking, the IgG-MBS. pullorum and S. gallinarum conjugates were separated magnetically and the clear supernatant was discarded. After that, the magnet was removed from the bottom of centrifuge tube, 50 mL of PBS was added and mixed with shaking. The IgG-MB-S. pullorum and S. gallinarum complexes were separated by putting magnet
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under the centrifuge tube, and supernatant was discarded. The IgGMB-S. pullorum and S. gallinarum complexes were washed with PBS for three times. The immune complexes were dispersed in 20 mL solution and transferred into microplate well, 40 mL chicken serum sample was subjected to react for 10 min, and then 20 mL IgG-Blue SiNps (1 mg/mL) was added into the microplate wells to incubate for 10 min. Place the plate on a magnet for 1 min and then remove the liquid by forcefully inverting the plate while on the separator. After that magnet removed and 50 mL PBS was added in to the well of plate and the plate was shaken for 1 min, then the PBS discarded in the magnet field. The well was washed in magnetic field with PBS for three times to remove unbound IgG-Blue-SiNps. The final plaque was recorded by digital camera. 3. Results and discussion 3.1. Characterizations of Blue-SiNps The Blue-SiNps synthesized by inverse microemulsion method showed bright blue color and good dispersion in aqueous solution (Fig. 2 inset). The organic dye molecules were trapped inside a silica matrix to form the dye doped nanoparticles. The size and morphology of Blue-SiNps was characterized by SEM (Fig. 2). The Blue-SiNps were extremely uniform in size, with the average diameter of 50 ± 2 nm determined by SEM and the characteristics of Blue-SiNps were in accordance with descriptions by Tan et al. [22,23]. Because of the protective function of the silica matrix and the stable chemical properties of C.I. reactive 21, the nanoparticles were highly stable in acidic and basic solution. To verify the stability of the Blue-SiNps, we compared the stability of gold nanoparticles (AuNps) and Blue-SiNps in different pH solution. Negligible color change was observed for the Blue-SiNps in basic and acidic conditions. In contrast, the color of AuNps dramatically changed from wine red to black purple in basic solution, and from wine red to colorless in acidic solution (As shown in Fig. S1). This result indicates that Blue-SiNps are more stable than AuNps in acidic and basic conditions. This high stability of the nanoparticles provides a foundation for the preparation of optical immunoassay used in various conditions. The chemical groups on the surface of Blue-SiNps were confirmed by Zeta potential determination. The same concentration of silica nanoparticles (0.5 mg/mL) was used in all experiments with aqueous solution containing different amounts of HCl (0.01 M) and NaOH (0.01 M) at different experimental pH values (in the range from pH 2 to pH 12), as measured by pH electrode. All values shown in this work were average of three measurements. Fig. 3A displayed the Zeta potential change of Blue-SiNps and Blue-SiNpsNH2 as a function of pH. With the changing pH values, the experimental zeta potential values of Blue-SiNps and Blue-SiNps-NH2 were both found to be more negative at higher pH. This result could arise either from enhanced ionization of the residual surface silanol or from an increased amount of hydroxide ions in the bulk mobile phase at higher pH being adsorbed onto the silica surface. The isoelectric point (IEP) of Blue-SiNps and Blue-SiNps-NH2 is at pH 4.9 and pH 8.4 respectively, the increase of Zeta potential is attributed to the increasing number of protonated amine groups on the Blue-SiNps-NH2 surface. Chemical composition and group on the surface of Blue-SiNps and Blue-SiNps-NH2 were also examined by FTIR (Fig. 3B). Dried samples were measured using KBr pellet method in range of 400e4000 cm 1. A strong IR absorption band at 1060 cm 1, corresponding to the SieOeSi of the silica core, is found in the FTIR of both Blue-SiNps and Blue-SiNps-NH2. As shown in FTIR absorption spectra of Blue-SiNps-NH2, the 3440, 1590 and 1525 cm 1 are attributed to the NeH bonds of amino group. And the absorption
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Fig. 1. Principle of the optical assay for anti-PG detection in negative serum (up) and positive serum (down) based on IgG-Blue-SiNps and IgG-MB.
Fig. 2. The SEM image of Blue-SiNps, physical image of Blue-SiNps and SiNps.
bond at 2956 is evidences of the strong vibration of CeH bond. These results are consistent with the results of Zeta potential and indicate that the amino groups are well modified on the surface of Blue-SiNps. 3.2. Assay optimization This procedure was performed to choose the optimal concentrations of IgG-MNPs, the optimal concentrations of IgG-Blue SiNps, the optimal concentrations of antigen, and the optimal incubation time for the competitive inhibition immunomagnetic assay. IgGMNPs were diluted to 1 mg/mL, 0.2 mg/mL, 0.1 mg/mL, 0.05 mg/ mL, and 0.025 mg/mL, IgG-Blue SiNps were diluted to 10 mg/mL, 5 mg/mL, 1 mg/mL, 0.5 mg/mL, and 0.25 mg/mL. A series of antigen solution were diluted to 1 108 CFU/mL, 1 106 CFU/mL, 1 104 CFU/mL, 1 102 CFU/mL, and 1 101 CFU/mL. The incubation time of serum with S. pullorum was adjusted from 0 to 60 min to achieve an optimal analytical performance. The phenomena of positive and negative reactions depending on color
changes of two kinds of nanoparticles were showed in Fig. 4. IgGMNPs played the role of immobilizing antigen. The performance of the developed optical immunoassay could be greatly affected by IgG-MNPs concentration. With the decrease of IgG-MNPs concentration the brown plaques in the positive result significantly decreased, at the same time, the black plaques in the negative result significantly increased, which decreased the contrast between negative and positive results (Fig. 4A). IgG-Blue SiNps concentrations have a strong influence on the negative results and not on positive results. The blue plaques in negative results increased with IgG-Blue SiNps concentrations, however when the concentration of IgG-Blue SiNps was 1 mg/mL, the results were distinguished easily (Fig. 4B). The antigen concentration has a similar influence on the negative results as IgG-Blue SiNps concentrations, when the concentration of antigen was the contrast between negative and positive results was most obvious (Fig. 4C). Fig. 4D shows the result changes of this optical immunoassay with different incubation time, the positive results rapidly changed with increasing incubation time, which indicates that S. Pullorum and S. gallinarum gradually binds with the antibody via antigeneantibody reaction. After the incubation time was 30 min, the positive results was significant, we could accurately judge the results. Further increase incubation time, e.g. 60 min, had very little additional beneficial effect, The optimum concentration of IgG-MNPs, concentration of IgG-Blue SiNps, antigen concentration and incubation time were selected according to the above experiment, the IgG-MNPs were diluted to 0.1 mg/mL, 1 mg/mL was selected as optimum concentration of IgGBlue SiNps, the optimal concentration of antigen was 1 106 CFU/ mL, and 30 min incubation time was chosen in this study. 3.3. Sensitivity of the optical immunoassay In this optical method, the antibody in the positive serum was detected by a competitive inhibition assay; IgG-Blue-SiNps compete with the antibody in positive serum for the limited binding sites on S. pullorum and S. gallinarum and play as the role of signal transduction labels. If the sample solution contains enough anti-PG, the antibodies would occupy the limited binding sites of antigen, the immune reaction between IgG-Blue SiNps and S. pullorum and S. gallinarum would be inhibited, IgG-Blue SiNps
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with the naked eye. Under the optimal conditions, sensitivity of the developed optical immnunoassay was studied. The plaque color in the bottom of plate well was related to the concentration of antibody. The original positive chicken serum was diluted to different titers (1:1, 1:10, 1:100 and 1:1000) with PBS. Fig. 5 showed that brown plaque was observed in the bottom of the microplate well when the titer of serum was 1:1, 1:10, and 1:100. The color of plaque in these reactions appears brown is because there is enough anti-PG combined with immunomagnetic complexes, the antibody in serum hinder the combination of IgG-Blue-SiNps with S. pullorum and S. gallinarum. There is very little blue plaque in the well of 1:100 titers, indicating the IgG-Blue-SiNaps has formed sandwich complex with S. pullorum and S. gallinarum and IgG-MB, this weakly positive result indicates low antibody level. Blue color of Blue-SiNps was observed clearly in the well of 1:1000 dilution of serum, which was too few to prevent the binding of IgG-Blue-SiNps with S. pullorum and S. gallinarum, so the blue IgG-Blue-SiNps was captured and observed after all washed steps. The sensitivity of this optical assay is compared with the rapid serum agglutination test using commercially available stained S. pullorum and S. gallinarum antigen. As showed in Fig. 6, the stained S. pullorum and S. gallinarum was mixed with different dilution positive serum (1:1, 1:10, and 1:100), agglutination phenomena were only observed in 1:1 and 1:10 dilute serum. When serum was diluted to 1:100, there was no agglutination phenomenon. Furthermore, the agglutination phenomenon of serum agglutination test result was not obvious and not easy to distinguish compared to our optical immunoassay. The contrast proved that the developed optical immunoassay is advantageous in sensitization and obvious phenomenon.
3.4. Specificity of the optical immunoassay
Fig. 3. Zeta potential of Blue-SiNps (square), Blue-SiNps-NH3 (circle), respectively (A), IR spectrum of Blue-SiNps (down) and Blue-SiNps-NH3 (up) respectively (B).
would be removed from sample solution in washing steps, and the well of microplate appears the color of MNPs, brown. While the negative control displays a bright blue plaque. The marked color difference in the wells of microplate can be easily distinguished
We examined the specificity of the developed optical immunoassay method for anti-PG detection. The specificity test was implemented with the same experimental procedure as mentioned in Section 2.4. We challenged the system with other interfering samples including negative serum, Anti-NDV, Anti-EDS, BSA, and PBS, they were all detected by our optical competitive immunoassay method under the same condition. As shown in Fig. 7, brown signal was only observed in the well of Anti-PG, blue signals were viewed in all the rest wells. This result shows that IgG-MB and IgGBlue-SiNps have good specificity for S. pullorum and S. gallinarum and this method has good specificity to Anti-PG.
Fig. 4. The effect of different factors on the positive results (up) and negative results (down) of optical competitive inhibition immunoassay. Shown are the effect of (A) IgG-MB concentration, (B) IgG-Blue-SiNps concentration, (C) antigen concentration, and (C) incubation time.
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Fig. 5. The competitive inhibition immunoassay results of IgG-MB solution and IgG-blue-SiNps solution with positive chicken serum of different dilutions: 1:1, 1:10, 1:100 and 1:1000.
Fig. 6. The serum agglutination test results of the staining S. pullorum and S. gallinarum antigen with different dilutions of positive serum: 1:1, 1:10, and 1:100, PBS as control.
Fig. 7. Specificity result of the optical immunoassay, from left to right: negative serum, positive serum, Anti-NDV, Anti-EDS, BSA solution, PBS.
3.5. Stability of the IgG-Blue-SiNps and IgG-MB The stability of the IgG-MB and IgG-Blue-SiNps was studied by the test of the positive and negative serum sample with the developed optical immunoassay after IgG-MB and IgG-Blue-SiNps were both stored at 4 C for 7, 30, 60 and 90 days. The results were displayed in Fig. 8, the phenomena of positive and negative results did not change after 90 days of storage, this result indicated that the two kinds of immune nanoparticles could retain similar reaction activity and could be used for optical measurements after storage at 4 C for 90 days. 4. Conclusions In summary, a
Blue-SiNps
based
nonenzymatic optical
immunosensing strategy is presented in this study. This new method is a robust, sensitive, inexpensive, and user-friendly analytical method compared with classical assays for the detection of antibody to S. pullorum and S. gallinarum in serum. The BlueSiNps synthesis and the process of modifying Blue-SiNps were characterized via SEM, Zeta potential, and FTIR. This strategy combines the advantages taken from immunochemical assays, magnetic beads separation, and bright color of Blue-SiNps. It does not require sophisticated equipment or costly enzyme labeled antibody. On the basis of IgG-MB enriching the antigen effectively, the color of Blue-SiNps is great signal amplification under the assay conditions. The developed calorimetric assay using Blue-SiNps as optical indicator is more easily monitored by naked eyes owing to the distinct color difference, and can detect positive serum at a dilution factor of 1:100, which is 10 fold lower than the
Fig. 8. Positive and negative results after IgG-MB and IgG-blue-SiNps were stored for: 7days, 30 days, 60 days, 90 days.
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conventional serum agglutination test. 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 stability. What's more, this method is easy to be manipulated, and time-consumption is less than 60 min. This rapid sensing technology should have great potential in a wide range of antibody-based biosensing and diagnostic applications. Acknowledgments This project was supported by the Food Science and Engineering the most important discipline of Zhejiang province (ZYTSP20141062); Zhejiang Public Innovation Platform Analysis and testing projects (2015C37023). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.aca.2015.09.041. References [1] D.F.A. Batista, et al., Polymerase chain reaction assay based on ratA gene allows differentiation between Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum, J. Vet. Diagn. Investig. 25 (2) (2013) 259e262. [2] C. Hu, W. Dou, G. Zhao, Enzyme immunosensor based on gold nanoparticles electroposition and Streptavidin-biotin system for detection of S. pullorum & S. gallinarum, Electrochimica Acta 117 (2014) 239e245. [3] M.-S. Kang, et al., Differential identification of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum and the biovar Gallinarum live vaccine strain 9R, Vet. Microbiol. 160 (3e4) (2012) 491e495. [4] R.K. Gast, Detecting infections of chickens with recent Salmonella pullorum isolates using standard serological methods, Poult. Sci. 76 (1) (1997) 17e23. [5] S. Geng, et al., Virulence determinants of Salmonella Gallinarum biovar Pullorum identified by PCR signature-tagged mutagenesis and the spiC mutant as a candidate live attenuated vaccine, Vet. Microbiol. 168 (2e4) (2014) 388e394. [6] Z. Gao, et al., Urchin-like (gold core)@(platinum shell) nanohybrids: A highly efficient peroxidase-mimetic system for in situ amplified colorimetric
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