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SiO2 nanocomposites as labels
Author’s Accepted Manuscript Highly sensitive enzyme-free immunosorbent assay for porcine circovirus type 2 antibody using AuPt/SiO2 nanocomposites as labels Long Wu, Wenmin Yin, Kun Tang, Kang Shao, Qin Li, Pan Wang, Yunpeng Zuo, Xiaomin Lei, Zhicheng Lu, Heyou Han www.elsevier.com/locate/bios
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S0956-5663(16)30276-7 http://dx.doi.org/10.1016/j.bios.2016.04.001 BIOS8588
To appear in: Biosensors and Bioelectronic Received date: 20 January 2016 Revised date: 18 March 2016 Accepted date: 2 April 2016 Cite this article as: Long Wu, Wenmin Yin, Kun Tang, Kang Shao, Qin Li, Pan Wang, Yunpeng Zuo, Xiaomin Lei, Zhicheng Lu and Heyou Han, Highly sensitive enzyme-free immunosorbent assay for porcine circovirus type 2 antibody using Au-Pt/SiO 2 nanocomposites as labels, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highly sensitive enzyme-free immunosorbent assay for porcine circovirus type 2 antibody using Au-Pt/SiO2 nanocomposites as labels Long Wu1, Wenmin Yin1, Kun Tang, Kang Shao, Qin Li, Pan Wang, Yunpeng Zuo, Xiaomin Lei, Zhicheng Lu, Heyou Han* State Key Laboratory of Agricultural Microbiology, College of Food Science and Technology, College of Science, Huazhong Agricultural University, Wuhan 430070, PR China. *Corresponding Author. E-mail: [email protected] Abstract Improving the performance of conventional enzyme-linked immunosorbent assay (ELISA) is of great importance to meet the demand of early clinical diagnosis of various diseases. Herein, we report a feasible enzyme-free immunosorbent assay (EFISA) system using antibody conjugated Au-Pt/SiO2 nanocomposites (APS NCs) as labels. In this system, Au-Pt/SiO2 nanaospheres (APS NPs) were first synthesized by wet chemical method and exhibited intrinsic peroxidase and catalase-like activity with excellent water-solubility. Then APS NCs were utilized as labels to replace HRP conjugated antibody, and Fe3O4 magnetic beads (MBs) to entrap the analyte. To discuss the performance of EFISA system, Human IgG was served as a model analyte, and porcine circovirus type 2 (PCV2) serums as real samples. The system boosted the detection limit of HIgG to 75 pg mL−1 with a RSD below 5%, a 264-fold improvement as compared with conventional ELISA. This is the first time that APS NCs have been
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Equal contribution. 1
used and successfully optimized for the sensitive dilution detection of PCV2 antibody (5:107) in ELISA. Besides, APS NCs have advantages related to low cost, easy preparation, good stability and tunable catalytic activity, which make them a potent enzyme mimetic candidate and may find potential applications in bioassays and clinical diagnostics. Keywords: Enzyme-free immunosorbent assay; Au-Pt/SiO2 nanocomposites; Fe3O4 magnetic beads; Human IgG; Porcine circovirus type 2 1. Introduction Enzyme linked immunosorbent assay (ELISA), a widely accepted and powerful immunoassay method, has received common interests due to its simplicity, good specificity, easy operation and instrumentation (Jayasena et al., 2015; Liang et al., 2015). Since it was firstly constructed and applied for immunoglobulin assay in 1971 (Engvall and Perlman, 1971), ELISA has become the gold standard for experimental and clinical analysis, especially in the animal disease diagnosis. Porcine circovirus type 2 (PCV2) is a common virus known to infect mammals and has caused enormous economic losses in the swine industry worldwide since it was firstly discovered in 1998 (Wu et al., 2015; Opriessnig et al., 2007). Thus, it is critical to construct an accurate and sensitive method to implement the early diagnosis for PCV2 antibody. The conventional ELISA with HRP enzyme conjugated secondary antibody and coloring substrate solution (TMB) plays an important role in immunoassay (Frey et al., 2000; Ambrosi et al., 2010), but it is still a challenge to find new approaches that could improve the simplicity, selectivity, and sensitivity (Laube et al., 2011; Tang et
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al., 2010). Therefore, it is of utmost importance to establish a method with high sensitivity and low cost for the detection of PCV2 antibody. To enhance the conventional ELISA, great efforts have been made in boosting the stability, detection limit and range (Gao et al., 2015; Qu et al., 2014; Diaz-Amigo and Popping, 2013). Herein, the introduction of nanoparticles (NPs) has largely improved the performance of conventional ELISA (Jia et al., 2009), especially the sensitivity (Chen et al., 2014a). For example, a great many of NPs have been adopted as carriers for the recognition antibody and/or HRP to obtain signal amplification owing to the strong adsorption ability and high surface areas (Pérez-López and Merkoçi, 2011, Chen et al., 2014b). Besides, some NPs are used to serve as activatable fluorescence probes and chromogenic substrates (Liu et al., 2013; de la Rica and Stevens, 2012). In addition, some peroxidase- or oxidase-like NPs such as FeS nano-sheets and graphene oxide, known as mimetic peroxidase, can take place of HRP in conventional ELISA with further modification (Dai et al., 2009; Song et al., 2010). By taking advantages of good enzyme-like activity, low cost and high stability (Gao et al., 2007; Wu et al., 2014), the NPs family is expected to be a promising candidate as mimetic enzymes. Pt NPs, especially hybridized with other metals, have gained extensive attention in electrochemical catalysis (Zuo et al., 2015), catalytic hydrogenation (Bratlie et al., 2007) and air purification (Zhou et al., 2005). Theses Pt NPs possess excellent oxidase-like (Bai et al., 2011), peroxidase-like (Cai et al., 2013) and catalase-like activity. To further improve the performance of Pt NPs, the design of supported Pt has been proposed as high-efficiency catalysts. For example, Pt NPs supported on
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grapheme (Xin et al., 2011), carbon nanotubes (Zhao et al., 2007), carbon (Song et al., 2007) and TiO2 (Drew et al., 2005) have been widely employed for electrocatalytic oxidation of methanol. However, it is inconvenient to further modify the Pt NPs and apply them to biological analysis since there is no extra functional group on their surface except the high-density surfactant such as cetyltrimethylammonium bromide (CTAB). Taking the convenient surface modification into account, SiO2 nanospheres are ideal carries in the construction of biosensors coupled with other techniques and widely used in immunoassay (Mao et al., 2012; Shao et al., 2014; Huang et al., 2014). In this work, we proposed a facile enzyme-free immunosorbent assay (EFISA) system using APS NCs as labels. A simple wet chemical method was first explored to prepare SiO2 loaded Au-Pt NPs based on the previous work (Guo et al., 2008) with some changes (see Scheme 1a). Afterwards, peroxidase-like activity of APS NPs was investigated and the HRP conjugated secondary antibody (Ab2) was replaced by APS NCs (see Scheme 1b). Even without the addition of any H2O2, APS NCs could exhibit good catalytic properties to TMB in the presence of oxygen and H+. Moreover, the APS NCs could provide both the mimetic peroxidase (Au-Pt NPs) and binding sites (SiO2 nanospheres), suggesting their potentials in future ELISA application. What is more, Fe3O4 magnetic beads (MBs) were fixed on the bottom of the well plates by simply applying an external magnetic field to entrap antigens or Ab1 for convenient separation (Gao et al., 2013) (see Scheme 1b). As a proof of concept, Human IgG was detected as a model analyte, and porcine circovirus type 2 (PCV2) serums as real samples in the EFISA system. This method behaved easier operation, lower cost and
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higher sensitivity compared with previous work (Wu et al., 2015). 2. Material and methods 2.1. Chemicals and materials Human IgG antigen (HIgG, 50 mg mL−1), rabbit anti-human IgG antibody (HAb1, 1 mg mL−1), goat anti-human IgG antibody (HAb2, 2 mg mL−1), rabbit anti-pig IgG antibody (PAb2, 1 mg mL−1) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China); PCV2 antibody (PAb1), positive and negative serum of PCV packaged in the PPA-ELISA kits were from Shandong Lvdu Bio-science & Technology Co. Ltd; Recombinant PCV2 Cap 2 antigen was from Puhuashi Sci-Tech Development Co., Ltd. (Beijing, China). Human IgG ELISA kit was obtained from Wuhan Booute Biotechnology Co., Ltd. The porcine pseudorabies virus (PrV), porcine reproductive and respiratory syndrome virus (PRRSV) positive and negative serum samples (ELISA kit) were obtained from Wuhan Keqian Animal Biological Products Co.Ltd. (China). Tetraethyl orthosilicate (TEOS, 99%), 3-aminopropyl trimethoxysilane (APTMS, 97%), bovine serum albumin (BSA), glutaraldehyde solution (GA, 25%) were purchased from Sigma-Aldrich; Potassium hexachloroplatinate (K2PtCl6, AR), tetrachloroauric(III) acid hydrate (HAuCl4·4H2O, AR), cetyltrimethylammonium bromide (CTAB), poly(sodium-p-styrenesulfonate) (PSS, MW = 70 K), polyvinyl pyrrolidone (PVP, MW = 58 K), o-Phenylenediamine (OPD), sodium citrate (SCT) and other relevant reagents were obtained from Sinopharm Chemical Reagent Co. Ltd.; Fe3O4 magnetic beads with amino group (MBs-NH2, 0.5%, w/w) were from
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Tianjin Baseline Chromtech Research Centre. All chemicals and solvents were of analytical grade and used as received without further purification. Ultrapure water obtained from a Millipore water purification system (Milli-Q, Millipore, 18.2 MΩ resistivity) was used throughout the experiment. 2.2. Instrumentation Fluorescence and optical density (OD) measurements were operated on Perkin Elmer 1420 Multilabel Counter; The UV–vis absorption spectra were from Nicolet Evolution 300 UV–vis spectrometer (Thermo Nicolet, America); Fouriertransform infrared (FT-IR) spectra were acquired on a Nicolet Avatar-330 spectrometer with 4 cm−1 resolution using the KBr pellet technique; Transmission electron microscopy (TEM) images were collected by a JEM-2010 transmission electron microscope (JEOL, Japan); Hydrodynamic diameters were measured by using dynamic light scattering (DLS) technique on Malvern Zetasizer Nanoseries (Malvern, England). 2.3. Preparation of APS NPs and APS NCs labels The preparation procedures of APS NPs were illustrated in Scheme 1a. Firstly, Au NPs, SiO2 nanospheres and Au−SiO2 hybrids were synthesized according to the previous work with some modification (Wu et al., 2014; Guo et al., 2008). Then, APS NPs were simply prepared by reducing H2PtCl6 on the surface of Au−SiO2 hybrids under room temperature. To obtain APS NCs labels for the specific identification of antigen, Ab2 was conjugated to APS NPs via electrostatic interaction and Schiff base structures. The details were all described in Supplementary Information. 2.4. Kinetic analysis
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To further understand the performance of APS NPs, the catalytic oxidation reaction kinetics for the two most common coloring substrate (TMB and OPD) were studied by absorption spectra. Unless otherwise stated, the kinetic experiments were carried out at room temperature with APS NPs (1.8 mg mL−1) in 1.0 mL of PBS buffer (pH = 5.5) with different concentrations of TMB or OPD. The kinetic parameters were calculated on the basis of Michaelise−Menten equation v = Vmax×[S]/(Km + [S]), where v is the initial velocity, Vmax is the maximal reaction velocity, Km is the Michaelis constant and [S] is the concentration of substrate (He et al., 2011). 2.5. Construction of EFISA system The goal of this work is to construct an enzyme-free immunosorbent assay (EFISA) system for the highly sensitive detection of PCV2 antibody. Scheme 1b showed the Ag−Ab1−Ab2 indirect immunoassay model of EFISA system. Three main modified parts are described in the scheme: (i) APS NCs took the place of the HRP-conjugated Ab2 and acted as detection labels; (ii) Ag-conjugated Fe3O4 MBs were fixed on the bottom of plates through an external magnetic field; (iii) TMB was used in the color reaction step in the absence of H2O2. The detailed immunoassay for HIgG and PCV2 antibody was consistent with our previous work (Wu et al., 2015) in Supplementary Information. 3. Results and discussion 3.1. Characterization of the APS NPs and APS NCs FT-IR spectroscopy was first used to investigate the functionalization process of APS NPs. As shown in Fig. S1A (Supplementary Information), the wide and strong
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absorption band at 1100 cm–1 (Si-O-Si) was corresponding to the characteristic bands of silica nanospheres. Meanwhile, the absorption bands of amino-functionalized silica at 1640, 960, 800, and 560 cm–1 (N–H) confirmed the successful modification of silica nanospheres, Fe3O4 MBs, and APS NPs. As depicted in Fig. S1B, a typical UV-vis absorption spectrum of Au NPs was exhibited with a sharp peak located at 508 nm (curve a), which was consistent with the local plasmon resonance of Au NPs (~5 nm). However, the peak disappeared after the deposition of Pt on the surface of Au−SiO2 hybrids (curve b). For APS NCs, two weak absorption peaks (curve c) were observed, which can be ascribed to the amide bond (220 nm) and the tryptophan and tyrosine residues present in the protein (280 nm) (Bhainsa and D'Souza, 2006). Fig. S1C described the zeta potential of different modification steps in the synthesis of APS NCs. It is noteworthy that the zeta potential of APS NPs increased dramatically from −38.67 to −5.57 mV after combining with Ab2, which revealed that Ab2 were successfully conjugated with APS NPs. To prove the presence of Au-Pt, Fig. S1D showed the energy-dispersive X-ray spectroscopy (EDX) spectrum of the APS NPs. Two main peaks (Au and Pt) were observed (other peaks originated from the carbon-supported copper grid), indicating that the APS NPs were made up of metallic gold, platinum, and silica. TEM images of silica nanospheres, Au−SiO2 hybrids, and APS NCs were taken as shown in Fig. 1. Fig. 1A indicated that the synthesized silica nanospheres are well-distributed and uniform with an average diameter of approximately 90 nm. The representative TEM images (Fig. 1B) showed numerous and individual particles are
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dotted on SiO2 nanospheres, indicating the homogeneous distribution of Au NPs on the SiO2 nanospheres. From Fig. 1C and Fig. 1E, it could be clearly observed that each silica nanosphere is composed of small Au/Pt hybrid nanoparticles with a rough surface. These small Au/Pt hybrid nanoparticles (< 5 nm) are spread out on the surface of silica nanospheres with some vacancy left, which provides extra binding sites for Ab2. In addition, a thin layer of Ab2 conjugated shell could be recognized in Fig. 1D. Furthermore, the features could be verified through the magnified image as shown in Fig. 1F. More detailed characteristics were analyzed by XPS spectrum (Fig. S2), HRTEM image (Fig. S3), SEM image (Fig. S4) and elemental analysis (Table S1) in Supplementary Information. 3.2. Characterization of oxidase- and peroxidase-like activity of APS NPs To discuss the catalytic activity, TMB (Asati et al., 2009) and OPD (Gao et al., 2007) as substrate, were chosen to demonstrate the oxidase- /peroxidase-like activities of APS NPs in the absence and presence of H2O2, respectively. We found that APS NPs can catalyze the oxidation of TMB or OPD by dissolved oxygen in water in the acid environment, generating the typical blue color for TMB and yellow color for OPD within 10 min (Fig. S5B). Meanwhile, no color change was observed in the solutions without APS NPs. The results verified our initial presumption that APS NPs possess oxidase-like activity. Next, the effect of H2O2 on TMB and OPD oxidation was discussed. With the addition of H2O2, the solution with TMB and OPD exhibited much faster and deeper color change at the same conditions (Fig. S5A). In contrast, the solutions without APS NPs showed negligible color change. The results revealed
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the peroxidase-like activities of APS NPs toward peroxidase substrates. It is well known that Pt NPs behaved good catalytic performance for the oxygen reduction reaction (Beyhan et al., 2015) and hydrogen peroxide reduction (Katsounaros et al., 2012) in electrocatalytic processes. The oxidation pathway of oxygen or hydrogen peroxide to TMB and OPD was believed to be similar with that of the electrochemical reductions (He et al., 2011). In this work, we studied the electrochemical performance of APS NPs as shown in Fig. S6. Compared with the air-saturated solution (curve c), no reduction peak of oxygen was observed in the N2-purged solution (curve b). Also, the effects of oxygen and light on the oxidation of TMB were explored (Fig. S7). The results indicated that the dissolved oxygen is the electron acceptors for the oxidation in the absence of H2O2, which is in agreement with the previous work (He et al., 2011). 3.3. Catalytic activity dependence on surface structure and modification Three main factors, such as the size and distribution of Au-Pt dots (Fig. S8), the density of Pt on SiO2 nanospheres (Fig. S9, Fig. S10A) and the surface capping molecules (Fig. S11) were investigated on catalytic activity. Also, we explored the stability of APS NCs (Fig. S10B) and analyzed the elemental composition (Table S1). The related details were discussed in Supplementary Information. 3.4. Stability and catalytic activity of APS NPs against temperature, pH and time The ability of nanoparticles against aggregation in different harsh conditions is vital to promote their application. Herein, the three main factors, temperature, pH and time (Fig. S12), were discussed on the stability of APS NPs and APS NCs. Besides, the
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catalytic stability of APS NPs was characterized by UV-vis absorption spectra (Fig. S13). All the details were described in Supplementary Information. 3.5. The optimization of H2O2 and TMB concentrations and pH on EFISA system The effects of H2O2 and TMB were first discussed before the EFISA immunoassay. As depicted in Fig. 2A, the initial reaction rate increased with the increasing of H2O2 concentration and showed a linear relationship in range of 0 to 20 mM (see the red line). Increasing H2O2 concentration further, the increase in reaction rate slowly dropped down. The whole developing trend is similar to that of Au@Pt peroxidase mimetics (He et al., 2011). Considering the coloring time (~ 5 min) during the ELISA tests, we finally chose 10 mM of H2O2 to react with the substrate solution. Meanwhile, we also discussed the effect of TMB concentration on the reaction catalyzed by APS NCs in the presence of H2O2. Fig. 2B showed the absorbance evolution over time at several TMB concentrations, indicating that the reaction rates gradually increased with TMB concentration. It can also be observed from Fig. 2C that the reaction rate reached the maximum at a high TMB concentration (0.6 mM). Thus, the substrate solution containing 0.6 mM of TMB was used in the following assays. Fig. 2D displayed the reaction rate in the absence of H2O2 with a slower increment over the whole concentration range compared with Fig. 2C. The results revealed that the feasibility of EFISA tests in the absence of H2O2 at a higher TMB concentration. Moreover, the effect of pH on the relative reaction rate of TMB oxidation was investigated (Fig. S14). Considering the stability of Ab2, the optimal pH of PBS buffer solution was chosen as 5.5 though the APS NPs behave higher activity at a
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lower pH value. Finally, a comparison between APS NCs and HRP was made by calculating the apparent kinetic parameters for TMB oxidation in the presence and absence of H2O2 (Fig. S15). The results were discussed in Supplementary Materials and compared with other reported materials (Table S2). 3.6. Immunoassay for HIgG with EFISA system Under the above optimized conditions, we conducted the immunoassay for HIgG with Fe3O4 MBs based ELISA system (Fig. 3A) and EFISA system (Fig. 3B), respectively. Both systems exhibited good linearity between optical signal and the logarithmic value of the tested ranges of HIgG concentration. The linear regression equation in Fig. 3A was presented as Y1 = −0.2266 + 0.2098 lg X (X: pg mL−1) over the range from 100 to 30000 pg mL−1 with R2 = 0.9882, and the detection limit was 100 pg mL−1 (S/N=3). Fig. 3B exhibited a regression equation of Y2 = −0.4112 + 0.2893 lg X (X: pg mL−1) over the range from 100 to 30000 pg mL−1 with R2 = 0.9974, and showed a detection limit of 75 pg mL−1 (S/N=3). Obviously, both the two systems possessed a higher sensitivity than that of the amplified ELISA system (3.9 ng mL−1) previously reported (Wu et al., 2015). Besides, the EFISA system performed 264-fold enhancements in sensitivity than that (19.8 ng mL−1) of the conventional ELISA system (Wu et al., 2015). This level of sensitivity was comparable to that of other methods in previously published reports for HIgG determination (Table 1). Moreover, the reproducibility of the EFISA system was tested with intra-assay and inter-assay by determining one HIgG level (20 ng mL−1) for three measurements and the variation coefficients were under 5%, indicating the acceptable reproducibility of
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the proposed EFISA system. To further investigate the feasibility and specificity of the EFISA system to real samples, HIgG in human serum (Fig. 3C) and the interference such as BSA, hemoglobulin, L-cysteine and glucose (Fig. 3D) were investigated and compared with commercial kit. Fig. 3C behaved the results of HIgG in five human serum samples after 106-fold dilution. The EFISA system followed the same tendency with commercial kit but exhibited higher signal to HIgG. The facts reflected the acceptable feasibility of EFISA system in real samples. Besides, the recovery and RSD of EFISA system are in the range of 89.5–110.8% and 3.6–4.8%, respectively (Table S3), which was satisfactory for quantitative assays in biological samples. It can be seen from Fig. 3D that both the two systems behaved unobvious difference (RSD < 5%) by introducing the four interferences but EFISA system showed higher response to HIgG, suggesting the good specificity of the EFISA system. Furthermore, it was found that the EFISA system retained nearly the same original optical response (OD450) even after 6 weeks of storage at 4 °C (Fig. S10B), revealing the good stability of the system. 3.7. Detection of PCV2 antibody in swine serum samples with EFISA system In order to evaluate the performance of the EFISA system in actual swine serum samples, a series of positive serum samples with different dilution ratios were tested. As shown in Fig. 4A, the EFISA system possessed higher sensitivity and the signal was amplified by nearly 2 order of magnitude even judged by naked eye as compared with conventional ELISA. The results demonstrated that the system had an excellent capability in response to changes of the actual serum samples. After that, we discussed
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the EFISA system without H2O2, which shows comparable detection results with the presence of H2O2 (Fig. S16). Fig. 4B showed the results of PCV2 antibody in positive swine serum samples at different dilution ratios. Inset calibration curve displayed a linear regression equation Y3 = 1.150 + 0.1636 lg X (X: dilution ratio) ranging from 1:102 to 5:107 with R2 = 0.9883 (S/N=3). Compared with the results as previously reported in the conventional ELISA system (Wu et al., 2015) (range from 1:102 to 5:105), the proposed EFISA system provides higher sensitivity and wider linear range. The results indicated that the detection sensitivity of PCV2 antibody was enhanced 100-fold by the EFISA system, which were consistent with the colorimetric detection (Fig. 4A). We also investigated the probable causes of signal amplification for PCV2 antibody detection. In contrast, we discussed the indirect immunoassay in conventional ELISA (control), ELISA only with APS NCs, ELISA only with Fe3O4 MBs and the EFISA system, respectively. As shown in Fig. 4C, the signal got significantly enhanced with the integration of Fe3O4 MBs and APS NCs, while ELISA only with Fe3O4 MBs or APS NCs behaved limited signal amplification, suggesting the superiority of EFISA system. Moreover, the relatively low absorption values without PCV2 antibody also illustrated that the nonspecific binding was successfully inhibited. The facts revealed that the enhanced signal mainly caused by two factors: Fe3O4 MBs contribute to entrap the substrate effectively, and APS NCs behave as excellent mimetic peroxidase, which is consistent with our proposed strategy (Scheme 1). Furthermore, the specificity of the EFISA system for PCV2 positive serum was also
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investigated by comparing with BSA solution, porcine reproductive and respiratory syndrome (PRRS) positive serum, pseudorabies virus (PrV) positive serum, PCV2 negative serum, pooled serum (positive serum/negative serum = 1:1), respectively. As shown in Fig. 4D, the stronger optical densities were acquired for PCV2 positive serum and the pooled serum. However, nearly no signals appeared for BSA, PRRS positive serum, PrV positive serum and PCV2 negative serum. The results testified that the PCV2 antibody can be effectively recognized by the proposed EFISA system with high specificity. 4. Conclusions In summary, a facile EFISA system was constructed in this study using two steps: Fe3O4 MBs entrapped antigen as substrate and Ab2 conjugated APS NCs as labels. Verification of the indirect immunoassay indicated that the EFISA system exhibited higher sensitivity for both HIgG and PCV2 antibody than that of the conventional ELISA. The observations demonstrate that the APS NCs behaved favorable stability against temperature and pH as well as excellent peroxide-like activities towards peroxidase substrates. Besides, they can be cheaply prepared by wet chemical method at room temperature in a short time and showed tunable catalytic activity by controlling the structure. These advantages exactly overcome the shortcomings of natural enzymes. The sensitivity of HIgG is lowered to 75 pg mL−1 with a 264-fold improvement as compared to conventional ELISA. Also, a 100-fold improvement was obtained in the detection limit for PCV2 antibody immunoassay. Furthermore, the EFISA system could behave comparable detection results even without H2O2. All the
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Tao, H.; Liao, X.; Xu, M.; Xie, X.; Zhong, F.; Yi, Z., 2014. Anal. Methods 6, 2560– 2565. Wang, S.; Chen, Z.; Choo, J.; Chen, L., 2016. Anal. Bioanal. Chem. 408, 1015−1022. Wu, L.; Chen, K.; Lu, Z.; Li, T.; Shao, K.; Shao, F.; Han, H., 2014. Anal. Chim. Acta 845, 92−97. Wu, L.; Li, X.; Shao, K.; Ye, S.; Liu, C.; Zhang, C.; Han, H., 2015. Anal. Chim. Acta 887, 192−200. Xin, Y.; Liu, J.; Zhou, Y.; Liu, W.; Gao, J.; Xie, Y.; Yin, Y.; Zou, Z., 2011. J. Power Sources 196, 1012–1018. Zarei, H.; Ghourchian, H.; Eskandari, K.; Zeinali, M., 2012. Anal. Methods, 421, 446−453. Zhao, Y.; Fan, L.; Zhong, H.; Li, Y.; Yang, S., 2007. Adv. Funct. Mater. 17, 1537– 1541. Zhou, S.; Varughese, B.; Eichhorn, B.; Jackson, G.; McIlwrath, K., 2005. Angew. Chem. 117, 4615 –4619. Zuo, Y.; Wu, L.; Cai, K.; Li, T.; Yin, W.; Li, D.; Li, N.; Liu, J.; Han, H., 2015. ACS Appl. Mater. Interfaces 7, 17725−1773.
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Scheme 1. Schematic illustration of the enzyme-free immunosorbent assay system, (a) the preparation procedures of APS NPs, and (b) the immunoassay process for PCV2 antibody using APS NCs as labels.
Fig. 1. TEM images of (A) silica nanospheres, (B) Au−SiO2 hybrids, (C) APS NPs, (D) APS NCs, (E) a magnified version of APS NPs, and (F) APS NCs.
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Fig. 2. (A) Effect of H2O2 concentration on the reaction rate of TMB oxidation catalyzed by APS NCs. The straight line is a linear regression of relative reaction rate upon H2O2 (from 0 to 20 mM). The inset is the evolution of A650 value over time at different H2O2 concentrations. (B) The time-dependent absorbance (A650) of different TMB concentrations with H2O2. (C) Effect of TMB concentration on the reaction rate catalyzed by APS NCs with H2O2 and (D) without H2O2. All the error bars were calculated based on the standard deviation of three measurements.
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Fig. 3. (A) Fe3O4 MBs based ELISA system (Fe3O4-Ag−Ab1−Ab2) and (B) EFISA system (Fe3O4-Ag−Ab1−Au-Pt/SiO2-Ab2) for the detection of HIgG (Inset: linear calibration plot between optical density and CHIgG from 100 to 30000 pg mL−1). EFISA system and commercial kit for the immunoassay of (C) HIgG in five human serum samples and (D) 20 ng mL−1 HIgG + 200 ng mL−1 interference: (1) HIgG only, (2) BSA + HIgG, (3) hemoglobulin + HIgG, (4) L-cysteine + HIgG and (5) glucose + HIgG. All the error bars were calculated based on the standard deviation of at least three measurements.
Fig. 4. (A) Colorimetric ELISA detection of PCV2 antibody with (a) EFISA system and (b) conventional ELISA. (B) Amplified assay using EFISA system (Inset: the amplified results of different dilution ratios of PCV2 positive serum: 1:102, 5:103, 1:103, 5:104, 1:104, 5:105, 1:105, 5:106, 1:106, 5:107). (C) Verification of the indirect immunoassay for PCV2 antibody with EFISA system. (D) Specificity of BSA, PRRS, PrV, PCV2 negative serum, pooled serum (PCV2 positive serum/PCV2 negative serum = 1:1), and PCV2 positive serum using EFISA system. All the error bars were calculated based on the standard deviation of three measurements.
22
Table 1 Comparison of the proposed EFISA system with other methods for HIgG detection Linear range (ng
Detection limit
mL−1)
(ng mL−1)
NER-LISAa
—
∼0.5
Eum et al., 2014
ELISA-like system
0.7–100
0.3
Wang et al., 2016
Amplified ELISA
5–20000
3.9
Wu et al., 2015
NSETb method
4–220
0.83
Tao et al., 2014
CVc method
30–1000
25
Zarei et al., 2012
EFISA system
0.1– 30
0.075
This work
Detection technique
Reference
a: nanoscale enzyme reactors-linked immunosorbent assay; b: nanoparticle surface energy transfer; c: cyclic voltammograms
Highlights 1. An enzyme-free ELISA system was developed for cost-effective monitoring of PCV2. 2. Au-Pt/SiO2 nanoparticles took place of HRP with lower cost, easier preparation and better peroxidase-like activity. 3. F3O4 magnetic beads were utilized to entrap the substrate for convenient separation. 4. The system behaved comparable sensitivity with conventional ELISA even in the absence of H2O2. 5. The system exhibited a 264-fold improvement in HIgG detection as compared with conventional ELISA.
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PII: DOI: Reference:
S0956-5663(16)30276-7 http://dx.doi.org/10.1016/j.bios.2016.04.001 BIOS8588
To appear in: Biosensors and Bioelectronic Received date: 20 January 2016 Revised date: 18 March 2016 Accepted date: 2 April 2016 Cite this article as: Long Wu, Wenmin Yin, Kun Tang, Kang Shao, Qin Li, Pan Wang, Yunpeng Zuo, Xiaomin Lei, Zhicheng Lu and Heyou Han, Highly sensitive enzyme-free immunosorbent assay for porcine circovirus type 2 antibody using Au-Pt/SiO 2 nanocomposites as labels, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2016.04.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highly sensitive enzyme-free immunosorbent assay for porcine circovirus type 2 antibody using Au-Pt/SiO2 nanocomposites as labels Long Wu1, Wenmin Yin1, Kun Tang, Kang Shao, Qin Li, Pan Wang, Yunpeng Zuo, Xiaomin Lei, Zhicheng Lu, Heyou Han* State Key Laboratory of Agricultural Microbiology, College of Food Science and Technology, College of Science, Huazhong Agricultural University, Wuhan 430070, PR China. *Corresponding Author. E-mail: [email protected] Abstract Improving the performance of conventional enzyme-linked immunosorbent assay (ELISA) is of great importance to meet the demand of early clinical diagnosis of various diseases. Herein, we report a feasible enzyme-free immunosorbent assay (EFISA) system using antibody conjugated Au-Pt/SiO2 nanocomposites (APS NCs) as labels. In this system, Au-Pt/SiO2 nanaospheres (APS NPs) were first synthesized by wet chemical method and exhibited intrinsic peroxidase and catalase-like activity with excellent water-solubility. Then APS NCs were utilized as labels to replace HRP conjugated antibody, and Fe3O4 magnetic beads (MBs) to entrap the analyte. To discuss the performance of EFISA system, Human IgG was served as a model analyte, and porcine circovirus type 2 (PCV2) serums as real samples. The system boosted the detection limit of HIgG to 75 pg mL−1 with a RSD below 5%, a 264-fold improvement as compared with conventional ELISA. This is the first time that APS NCs have been
1
Equal contribution. 1
used and successfully optimized for the sensitive dilution detection of PCV2 antibody (5:107) in ELISA. Besides, APS NCs have advantages related to low cost, easy preparation, good stability and tunable catalytic activity, which make them a potent enzyme mimetic candidate and may find potential applications in bioassays and clinical diagnostics. Keywords: Enzyme-free immunosorbent assay; Au-Pt/SiO2 nanocomposites; Fe3O4 magnetic beads; Human IgG; Porcine circovirus type 2 1. Introduction Enzyme linked immunosorbent assay (ELISA), a widely accepted and powerful immunoassay method, has received common interests due to its simplicity, good specificity, easy operation and instrumentation (Jayasena et al., 2015; Liang et al., 2015). Since it was firstly constructed and applied for immunoglobulin assay in 1971 (Engvall and Perlman, 1971), ELISA has become the gold standard for experimental and clinical analysis, especially in the animal disease diagnosis. Porcine circovirus type 2 (PCV2) is a common virus known to infect mammals and has caused enormous economic losses in the swine industry worldwide since it was firstly discovered in 1998 (Wu et al., 2015; Opriessnig et al., 2007). Thus, it is critical to construct an accurate and sensitive method to implement the early diagnosis for PCV2 antibody. The conventional ELISA with HRP enzyme conjugated secondary antibody and coloring substrate solution (TMB) plays an important role in immunoassay (Frey et al., 2000; Ambrosi et al., 2010), but it is still a challenge to find new approaches that could improve the simplicity, selectivity, and sensitivity (Laube et al., 2011; Tang et
2
al., 2010). Therefore, it is of utmost importance to establish a method with high sensitivity and low cost for the detection of PCV2 antibody. To enhance the conventional ELISA, great efforts have been made in boosting the stability, detection limit and range (Gao et al., 2015; Qu et al., 2014; Diaz-Amigo and Popping, 2013). Herein, the introduction of nanoparticles (NPs) has largely improved the performance of conventional ELISA (Jia et al., 2009), especially the sensitivity (Chen et al., 2014a). For example, a great many of NPs have been adopted as carriers for the recognition antibody and/or HRP to obtain signal amplification owing to the strong adsorption ability and high surface areas (Pérez-López and Merkoçi, 2011, Chen et al., 2014b). Besides, some NPs are used to serve as activatable fluorescence probes and chromogenic substrates (Liu et al., 2013; de la Rica and Stevens, 2012). In addition, some peroxidase- or oxidase-like NPs such as FeS nano-sheets and graphene oxide, known as mimetic peroxidase, can take place of HRP in conventional ELISA with further modification (Dai et al., 2009; Song et al., 2010). By taking advantages of good enzyme-like activity, low cost and high stability (Gao et al., 2007; Wu et al., 2014), the NPs family is expected to be a promising candidate as mimetic enzymes. Pt NPs, especially hybridized with other metals, have gained extensive attention in electrochemical catalysis (Zuo et al., 2015), catalytic hydrogenation (Bratlie et al., 2007) and air purification (Zhou et al., 2005). Theses Pt NPs possess excellent oxidase-like (Bai et al., 2011), peroxidase-like (Cai et al., 2013) and catalase-like activity. To further improve the performance of Pt NPs, the design of supported Pt has been proposed as high-efficiency catalysts. For example, Pt NPs supported on
3
grapheme (Xin et al., 2011), carbon nanotubes (Zhao et al., 2007), carbon (Song et al., 2007) and TiO2 (Drew et al., 2005) have been widely employed for electrocatalytic oxidation of methanol. However, it is inconvenient to further modify the Pt NPs and apply them to biological analysis since there is no extra functional group on their surface except the high-density surfactant such as cetyltrimethylammonium bromide (CTAB). Taking the convenient surface modification into account, SiO2 nanospheres are ideal carries in the construction of biosensors coupled with other techniques and widely used in immunoassay (Mao et al., 2012; Shao et al., 2014; Huang et al., 2014). In this work, we proposed a facile enzyme-free immunosorbent assay (EFISA) system using APS NCs as labels. A simple wet chemical method was first explored to prepare SiO2 loaded Au-Pt NPs based on the previous work (Guo et al., 2008) with some changes (see Scheme 1a). Afterwards, peroxidase-like activity of APS NPs was investigated and the HRP conjugated secondary antibody (Ab2) was replaced by APS NCs (see Scheme 1b). Even without the addition of any H2O2, APS NCs could exhibit good catalytic properties to TMB in the presence of oxygen and H+. Moreover, the APS NCs could provide both the mimetic peroxidase (Au-Pt NPs) and binding sites (SiO2 nanospheres), suggesting their potentials in future ELISA application. What is more, Fe3O4 magnetic beads (MBs) were fixed on the bottom of the well plates by simply applying an external magnetic field to entrap antigens or Ab1 for convenient separation (Gao et al., 2013) (see Scheme 1b). As a proof of concept, Human IgG was detected as a model analyte, and porcine circovirus type 2 (PCV2) serums as real samples in the EFISA system. This method behaved easier operation, lower cost and
4
higher sensitivity compared with previous work (Wu et al., 2015). 2. Material and methods 2.1. Chemicals and materials Human IgG antigen (HIgG, 50 mg mL−1), rabbit anti-human IgG antibody (HAb1, 1 mg mL−1), goat anti-human IgG antibody (HAb2, 2 mg mL−1), rabbit anti-pig IgG antibody (PAb2, 1 mg mL−1) were obtained from Sangon Biotech Co., Ltd. (Shanghai, China); PCV2 antibody (PAb1), positive and negative serum of PCV packaged in the PPA-ELISA kits were from Shandong Lvdu Bio-science & Technology Co. Ltd; Recombinant PCV2 Cap 2 antigen was from Puhuashi Sci-Tech Development Co., Ltd. (Beijing, China). Human IgG ELISA kit was obtained from Wuhan Booute Biotechnology Co., Ltd. The porcine pseudorabies virus (PrV), porcine reproductive and respiratory syndrome virus (PRRSV) positive and negative serum samples (ELISA kit) were obtained from Wuhan Keqian Animal Biological Products Co.Ltd. (China). Tetraethyl orthosilicate (TEOS, 99%), 3-aminopropyl trimethoxysilane (APTMS, 97%), bovine serum albumin (BSA), glutaraldehyde solution (GA, 25%) were purchased from Sigma-Aldrich; Potassium hexachloroplatinate (K2PtCl6, AR), tetrachloroauric(III) acid hydrate (HAuCl4·4H2O, AR), cetyltrimethylammonium bromide (CTAB), poly(sodium-p-styrenesulfonate) (PSS, MW = 70 K), polyvinyl pyrrolidone (PVP, MW = 58 K), o-Phenylenediamine (OPD), sodium citrate (SCT) and other relevant reagents were obtained from Sinopharm Chemical Reagent Co. Ltd.; Fe3O4 magnetic beads with amino group (MBs-NH2, 0.5%, w/w) were from
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Tianjin Baseline Chromtech Research Centre. All chemicals and solvents were of analytical grade and used as received without further purification. Ultrapure water obtained from a Millipore water purification system (Milli-Q, Millipore, 18.2 MΩ resistivity) was used throughout the experiment. 2.2. Instrumentation Fluorescence and optical density (OD) measurements were operated on Perkin Elmer 1420 Multilabel Counter; The UV–vis absorption spectra were from Nicolet Evolution 300 UV–vis spectrometer (Thermo Nicolet, America); Fouriertransform infrared (FT-IR) spectra were acquired on a Nicolet Avatar-330 spectrometer with 4 cm−1 resolution using the KBr pellet technique; Transmission electron microscopy (TEM) images were collected by a JEM-2010 transmission electron microscope (JEOL, Japan); Hydrodynamic diameters were measured by using dynamic light scattering (DLS) technique on Malvern Zetasizer Nanoseries (Malvern, England). 2.3. Preparation of APS NPs and APS NCs labels The preparation procedures of APS NPs were illustrated in Scheme 1a. Firstly, Au NPs, SiO2 nanospheres and Au−SiO2 hybrids were synthesized according to the previous work with some modification (Wu et al., 2014; Guo et al., 2008). Then, APS NPs were simply prepared by reducing H2PtCl6 on the surface of Au−SiO2 hybrids under room temperature. To obtain APS NCs labels for the specific identification of antigen, Ab2 was conjugated to APS NPs via electrostatic interaction and Schiff base structures. The details were all described in Supplementary Information. 2.4. Kinetic analysis
6
To further understand the performance of APS NPs, the catalytic oxidation reaction kinetics for the two most common coloring substrate (TMB and OPD) were studied by absorption spectra. Unless otherwise stated, the kinetic experiments were carried out at room temperature with APS NPs (1.8 mg mL−1) in 1.0 mL of PBS buffer (pH = 5.5) with different concentrations of TMB or OPD. The kinetic parameters were calculated on the basis of Michaelise−Menten equation v = Vmax×[S]/(Km + [S]), where v is the initial velocity, Vmax is the maximal reaction velocity, Km is the Michaelis constant and [S] is the concentration of substrate (He et al., 2011). 2.5. Construction of EFISA system The goal of this work is to construct an enzyme-free immunosorbent assay (EFISA) system for the highly sensitive detection of PCV2 antibody. Scheme 1b showed the Ag−Ab1−Ab2 indirect immunoassay model of EFISA system. Three main modified parts are described in the scheme: (i) APS NCs took the place of the HRP-conjugated Ab2 and acted as detection labels; (ii) Ag-conjugated Fe3O4 MBs were fixed on the bottom of plates through an external magnetic field; (iii) TMB was used in the color reaction step in the absence of H2O2. The detailed immunoassay for HIgG and PCV2 antibody was consistent with our previous work (Wu et al., 2015) in Supplementary Information. 3. Results and discussion 3.1. Characterization of the APS NPs and APS NCs FT-IR spectroscopy was first used to investigate the functionalization process of APS NPs. As shown in Fig. S1A (Supplementary Information), the wide and strong
7
absorption band at 1100 cm–1 (Si-O-Si) was corresponding to the characteristic bands of silica nanospheres. Meanwhile, the absorption bands of amino-functionalized silica at 1640, 960, 800, and 560 cm–1 (N–H) confirmed the successful modification of silica nanospheres, Fe3O4 MBs, and APS NPs. As depicted in Fig. S1B, a typical UV-vis absorption spectrum of Au NPs was exhibited with a sharp peak located at 508 nm (curve a), which was consistent with the local plasmon resonance of Au NPs (~5 nm). However, the peak disappeared after the deposition of Pt on the surface of Au−SiO2 hybrids (curve b). For APS NCs, two weak absorption peaks (curve c) were observed, which can be ascribed to the amide bond (220 nm) and the tryptophan and tyrosine residues present in the protein (280 nm) (Bhainsa and D'Souza, 2006). Fig. S1C described the zeta potential of different modification steps in the synthesis of APS NCs. It is noteworthy that the zeta potential of APS NPs increased dramatically from −38.67 to −5.57 mV after combining with Ab2, which revealed that Ab2 were successfully conjugated with APS NPs. To prove the presence of Au-Pt, Fig. S1D showed the energy-dispersive X-ray spectroscopy (EDX) spectrum of the APS NPs. Two main peaks (Au and Pt) were observed (other peaks originated from the carbon-supported copper grid), indicating that the APS NPs were made up of metallic gold, platinum, and silica. TEM images of silica nanospheres, Au−SiO2 hybrids, and APS NCs were taken as shown in Fig. 1. Fig. 1A indicated that the synthesized silica nanospheres are well-distributed and uniform with an average diameter of approximately 90 nm. The representative TEM images (Fig. 1B) showed numerous and individual particles are
8
dotted on SiO2 nanospheres, indicating the homogeneous distribution of Au NPs on the SiO2 nanospheres. From Fig. 1C and Fig. 1E, it could be clearly observed that each silica nanosphere is composed of small Au/Pt hybrid nanoparticles with a rough surface. These small Au/Pt hybrid nanoparticles (< 5 nm) are spread out on the surface of silica nanospheres with some vacancy left, which provides extra binding sites for Ab2. In addition, a thin layer of Ab2 conjugated shell could be recognized in Fig. 1D. Furthermore, the features could be verified through the magnified image as shown in Fig. 1F. More detailed characteristics were analyzed by XPS spectrum (Fig. S2), HRTEM image (Fig. S3), SEM image (Fig. S4) and elemental analysis (Table S1) in Supplementary Information. 3.2. Characterization of oxidase- and peroxidase-like activity of APS NPs To discuss the catalytic activity, TMB (Asati et al., 2009) and OPD (Gao et al., 2007) as substrate, were chosen to demonstrate the oxidase- /peroxidase-like activities of APS NPs in the absence and presence of H2O2, respectively. We found that APS NPs can catalyze the oxidation of TMB or OPD by dissolved oxygen in water in the acid environment, generating the typical blue color for TMB and yellow color for OPD within 10 min (Fig. S5B). Meanwhile, no color change was observed in the solutions without APS NPs. The results verified our initial presumption that APS NPs possess oxidase-like activity. Next, the effect of H2O2 on TMB and OPD oxidation was discussed. With the addition of H2O2, the solution with TMB and OPD exhibited much faster and deeper color change at the same conditions (Fig. S5A). In contrast, the solutions without APS NPs showed negligible color change. The results revealed
9
the peroxidase-like activities of APS NPs toward peroxidase substrates. It is well known that Pt NPs behaved good catalytic performance for the oxygen reduction reaction (Beyhan et al., 2015) and hydrogen peroxide reduction (Katsounaros et al., 2012) in electrocatalytic processes. The oxidation pathway of oxygen or hydrogen peroxide to TMB and OPD was believed to be similar with that of the electrochemical reductions (He et al., 2011). In this work, we studied the electrochemical performance of APS NPs as shown in Fig. S6. Compared with the air-saturated solution (curve c), no reduction peak of oxygen was observed in the N2-purged solution (curve b). Also, the effects of oxygen and light on the oxidation of TMB were explored (Fig. S7). The results indicated that the dissolved oxygen is the electron acceptors for the oxidation in the absence of H2O2, which is in agreement with the previous work (He et al., 2011). 3.3. Catalytic activity dependence on surface structure and modification Three main factors, such as the size and distribution of Au-Pt dots (Fig. S8), the density of Pt on SiO2 nanospheres (Fig. S9, Fig. S10A) and the surface capping molecules (Fig. S11) were investigated on catalytic activity. Also, we explored the stability of APS NCs (Fig. S10B) and analyzed the elemental composition (Table S1). The related details were discussed in Supplementary Information. 3.4. Stability and catalytic activity of APS NPs against temperature, pH and time The ability of nanoparticles against aggregation in different harsh conditions is vital to promote their application. Herein, the three main factors, temperature, pH and time (Fig. S12), were discussed on the stability of APS NPs and APS NCs. Besides, the
10
catalytic stability of APS NPs was characterized by UV-vis absorption spectra (Fig. S13). All the details were described in Supplementary Information. 3.5. The optimization of H2O2 and TMB concentrations and pH on EFISA system The effects of H2O2 and TMB were first discussed before the EFISA immunoassay. As depicted in Fig. 2A, the initial reaction rate increased with the increasing of H2O2 concentration and showed a linear relationship in range of 0 to 20 mM (see the red line). Increasing H2O2 concentration further, the increase in reaction rate slowly dropped down. The whole developing trend is similar to that of Au@Pt peroxidase mimetics (He et al., 2011). Considering the coloring time (~ 5 min) during the ELISA tests, we finally chose 10 mM of H2O2 to react with the substrate solution. Meanwhile, we also discussed the effect of TMB concentration on the reaction catalyzed by APS NCs in the presence of H2O2. Fig. 2B showed the absorbance evolution over time at several TMB concentrations, indicating that the reaction rates gradually increased with TMB concentration. It can also be observed from Fig. 2C that the reaction rate reached the maximum at a high TMB concentration (0.6 mM). Thus, the substrate solution containing 0.6 mM of TMB was used in the following assays. Fig. 2D displayed the reaction rate in the absence of H2O2 with a slower increment over the whole concentration range compared with Fig. 2C. The results revealed that the feasibility of EFISA tests in the absence of H2O2 at a higher TMB concentration. Moreover, the effect of pH on the relative reaction rate of TMB oxidation was investigated (Fig. S14). Considering the stability of Ab2, the optimal pH of PBS buffer solution was chosen as 5.5 though the APS NPs behave higher activity at a
11
lower pH value. Finally, a comparison between APS NCs and HRP was made by calculating the apparent kinetic parameters for TMB oxidation in the presence and absence of H2O2 (Fig. S15). The results were discussed in Supplementary Materials and compared with other reported materials (Table S2). 3.6. Immunoassay for HIgG with EFISA system Under the above optimized conditions, we conducted the immunoassay for HIgG with Fe3O4 MBs based ELISA system (Fig. 3A) and EFISA system (Fig. 3B), respectively. Both systems exhibited good linearity between optical signal and the logarithmic value of the tested ranges of HIgG concentration. The linear regression equation in Fig. 3A was presented as Y1 = −0.2266 + 0.2098 lg X (X: pg mL−1) over the range from 100 to 30000 pg mL−1 with R2 = 0.9882, and the detection limit was 100 pg mL−1 (S/N=3). Fig. 3B exhibited a regression equation of Y2 = −0.4112 + 0.2893 lg X (X: pg mL−1) over the range from 100 to 30000 pg mL−1 with R2 = 0.9974, and showed a detection limit of 75 pg mL−1 (S/N=3). Obviously, both the two systems possessed a higher sensitivity than that of the amplified ELISA system (3.9 ng mL−1) previously reported (Wu et al., 2015). Besides, the EFISA system performed 264-fold enhancements in sensitivity than that (19.8 ng mL−1) of the conventional ELISA system (Wu et al., 2015). This level of sensitivity was comparable to that of other methods in previously published reports for HIgG determination (Table 1). Moreover, the reproducibility of the EFISA system was tested with intra-assay and inter-assay by determining one HIgG level (20 ng mL−1) for three measurements and the variation coefficients were under 5%, indicating the acceptable reproducibility of
12
the proposed EFISA system. To further investigate the feasibility and specificity of the EFISA system to real samples, HIgG in human serum (Fig. 3C) and the interference such as BSA, hemoglobulin, L-cysteine and glucose (Fig. 3D) were investigated and compared with commercial kit. Fig. 3C behaved the results of HIgG in five human serum samples after 106-fold dilution. The EFISA system followed the same tendency with commercial kit but exhibited higher signal to HIgG. The facts reflected the acceptable feasibility of EFISA system in real samples. Besides, the recovery and RSD of EFISA system are in the range of 89.5–110.8% and 3.6–4.8%, respectively (Table S3), which was satisfactory for quantitative assays in biological samples. It can be seen from Fig. 3D that both the two systems behaved unobvious difference (RSD < 5%) by introducing the four interferences but EFISA system showed higher response to HIgG, suggesting the good specificity of the EFISA system. Furthermore, it was found that the EFISA system retained nearly the same original optical response (OD450) even after 6 weeks of storage at 4 °C (Fig. S10B), revealing the good stability of the system. 3.7. Detection of PCV2 antibody in swine serum samples with EFISA system In order to evaluate the performance of the EFISA system in actual swine serum samples, a series of positive serum samples with different dilution ratios were tested. As shown in Fig. 4A, the EFISA system possessed higher sensitivity and the signal was amplified by nearly 2 order of magnitude even judged by naked eye as compared with conventional ELISA. The results demonstrated that the system had an excellent capability in response to changes of the actual serum samples. After that, we discussed
13
the EFISA system without H2O2, which shows comparable detection results with the presence of H2O2 (Fig. S16). Fig. 4B showed the results of PCV2 antibody in positive swine serum samples at different dilution ratios. Inset calibration curve displayed a linear regression equation Y3 = 1.150 + 0.1636 lg X (X: dilution ratio) ranging from 1:102 to 5:107 with R2 = 0.9883 (S/N=3). Compared with the results as previously reported in the conventional ELISA system (Wu et al., 2015) (range from 1:102 to 5:105), the proposed EFISA system provides higher sensitivity and wider linear range. The results indicated that the detection sensitivity of PCV2 antibody was enhanced 100-fold by the EFISA system, which were consistent with the colorimetric detection (Fig. 4A). We also investigated the probable causes of signal amplification for PCV2 antibody detection. In contrast, we discussed the indirect immunoassay in conventional ELISA (control), ELISA only with APS NCs, ELISA only with Fe3O4 MBs and the EFISA system, respectively. As shown in Fig. 4C, the signal got significantly enhanced with the integration of Fe3O4 MBs and APS NCs, while ELISA only with Fe3O4 MBs or APS NCs behaved limited signal amplification, suggesting the superiority of EFISA system. Moreover, the relatively low absorption values without PCV2 antibody also illustrated that the nonspecific binding was successfully inhibited. The facts revealed that the enhanced signal mainly caused by two factors: Fe3O4 MBs contribute to entrap the substrate effectively, and APS NCs behave as excellent mimetic peroxidase, which is consistent with our proposed strategy (Scheme 1). Furthermore, the specificity of the EFISA system for PCV2 positive serum was also
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investigated by comparing with BSA solution, porcine reproductive and respiratory syndrome (PRRS) positive serum, pseudorabies virus (PrV) positive serum, PCV2 negative serum, pooled serum (positive serum/negative serum = 1:1), respectively. As shown in Fig. 4D, the stronger optical densities were acquired for PCV2 positive serum and the pooled serum. However, nearly no signals appeared for BSA, PRRS positive serum, PrV positive serum and PCV2 negative serum. The results testified that the PCV2 antibody can be effectively recognized by the proposed EFISA system with high specificity. 4. Conclusions In summary, a facile EFISA system was constructed in this study using two steps: Fe3O4 MBs entrapped antigen as substrate and Ab2 conjugated APS NCs as labels. Verification of the indirect immunoassay indicated that the EFISA system exhibited higher sensitivity for both HIgG and PCV2 antibody than that of the conventional ELISA. The observations demonstrate that the APS NCs behaved favorable stability against temperature and pH as well as excellent peroxide-like activities towards peroxidase substrates. Besides, they can be cheaply prepared by wet chemical method at room temperature in a short time and showed tunable catalytic activity by controlling the structure. These advantages exactly overcome the shortcomings of natural enzymes. The sensitivity of HIgG is lowered to 75 pg mL−1 with a 264-fold improvement as compared to conventional ELISA. Also, a 100-fold improvement was obtained in the detection limit for PCV2 antibody immunoassay. Furthermore, the EFISA system could behave comparable detection results even without H2O2. All the
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results indicate that the proposed EFISA system can provide a promising platform in bioassays and clinical diagnostics. Acknowledgements We gratefully acknowledge the financial support from National Natural Science Foundation of China (21375043, 21175051). References Ambrosi, A.; Airo, F.; Merkoc, A., 2010. Anal. Chem. 82, 1151–1156. Asati, A.; Santra, S.; Kaittanis, C.; Nath, S.; Perez, J. M., 2009. Angew. Chem. Int. Ed. 48, 2308−2312. Bai, F.; Sun, Z.; Wu, H.; Haddad, R. E.; Xiao, X.; Fan, H., 2011. Nano Lett. 11, 3759–3762. Beyhan, S.; Sahin, N. E.; Pronier, S.; Léger, J. M.; Kadirgan, F., 2015. Electrochmi. Acta. 151, 565−573. Bhainsa, K. C.; D'Souza, S. F., 2006. Colloids Surf., B Biointerfaces 47, 160−164. Bratlie, K. M.; Lee, H.; Komvopoulos, K.; Yang, P.; Somorjai, G. A., 2007. Nano Lett. 7, 3097–3101. Cai, K.; Lv, Z.; Chen, K.; Huang, L.; Wang, J.; Shao, F.; Wang, Y.; Han, H., 2013. Chem. Commun. 49, 6024–6026. Chen, F.; Hou, S.; Li, Q.; Fan, H.; Fan, R.; Xu, Z.; Zhala, G.; Mai, X.; Chen, X.; Chen, X.; Liu, Y., 2014a. Anal. Chem., 86, 10021–10024. Chen, K.; Wu, L.; Jiang, X.; Lu, Z.; Han H., 2014b. Biosens. Bioelectron. 62, 196– 200.
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Scheme 1. Schematic illustration of the enzyme-free immunosorbent assay system, (a) the preparation procedures of APS NPs, and (b) the immunoassay process for PCV2 antibody using APS NCs as labels.
Fig. 1. TEM images of (A) silica nanospheres, (B) Au−SiO2 hybrids, (C) APS NPs, (D) APS NCs, (E) a magnified version of APS NPs, and (F) APS NCs.
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Fig. 2. (A) Effect of H2O2 concentration on the reaction rate of TMB oxidation catalyzed by APS NCs. The straight line is a linear regression of relative reaction rate upon H2O2 (from 0 to 20 mM). The inset is the evolution of A650 value over time at different H2O2 concentrations. (B) The time-dependent absorbance (A650) of different TMB concentrations with H2O2. (C) Effect of TMB concentration on the reaction rate catalyzed by APS NCs with H2O2 and (D) without H2O2. All the error bars were calculated based on the standard deviation of three measurements.
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Fig. 3. (A) Fe3O4 MBs based ELISA system (Fe3O4-Ag−Ab1−Ab2) and (B) EFISA system (Fe3O4-Ag−Ab1−Au-Pt/SiO2-Ab2) for the detection of HIgG (Inset: linear calibration plot between optical density and CHIgG from 100 to 30000 pg mL−1). EFISA system and commercial kit for the immunoassay of (C) HIgG in five human serum samples and (D) 20 ng mL−1 HIgG + 200 ng mL−1 interference: (1) HIgG only, (2) BSA + HIgG, (3) hemoglobulin + HIgG, (4) L-cysteine + HIgG and (5) glucose + HIgG. All the error bars were calculated based on the standard deviation of at least three measurements.
Fig. 4. (A) Colorimetric ELISA detection of PCV2 antibody with (a) EFISA system and (b) conventional ELISA. (B) Amplified assay using EFISA system (Inset: the amplified results of different dilution ratios of PCV2 positive serum: 1:102, 5:103, 1:103, 5:104, 1:104, 5:105, 1:105, 5:106, 1:106, 5:107). (C) Verification of the indirect immunoassay for PCV2 antibody with EFISA system. (D) Specificity of BSA, PRRS, PrV, PCV2 negative serum, pooled serum (PCV2 positive serum/PCV2 negative serum = 1:1), and PCV2 positive serum using EFISA system. All the error bars were calculated based on the standard deviation of three measurements.
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Table 1 Comparison of the proposed EFISA system with other methods for HIgG detection Linear range (ng
Detection limit
mL−1)
(ng mL−1)
NER-LISAa
—
∼0.5
Eum et al., 2014
ELISA-like system
0.7–100
0.3
Wang et al., 2016
Amplified ELISA
5–20000
3.9
Wu et al., 2015
NSETb method
4–220
0.83
Tao et al., 2014
CVc method
30–1000
25
Zarei et al., 2012
EFISA system
0.1– 30
0.075
This work
Detection technique
Reference
a: nanoscale enzyme reactors-linked immunosorbent assay; b: nanoparticle surface energy transfer; c: cyclic voltammograms
Highlights 1. An enzyme-free ELISA system was developed for cost-effective monitoring of PCV2. 2. Au-Pt/SiO2 nanoparticles took place of HRP with lower cost, easier preparation and better peroxidase-like activity. 3. F3O4 magnetic beads were utilized to entrap the substrate for convenient separation. 4. The system behaved comparable sensitivity with conventional ELISA even in the absence of H2O2. 5. The system exhibited a 264-fold improvement in HIgG detection as compared with conventional ELISA.
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