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mesoporous silica and multienzyme functionalized mesoporous silica
Materials Science and Engineering B 176 (2011) 1474–1478
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering B journal homepage: www.elsevier.com/locate/mseb
Chemiluminescence immunoassay based on dual signal amplification strategy of Au/mesoporous silica and multienzyme functionalized mesoporous silica Jiehua Lin ∗ , Yue Zhao, Zhijing Wei, Wei Wang Key Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China
a r t i c l e
i n f o
Article history: Received 8 April 2011 Received in revised form 22 June 2011 Accepted 4 September 2011 Available online 17 September 2011 Keywords: Chemiluminescent immunoassay Mesoporous silica Horseradish peroxidase ␣-Fetoprotein
a b s t r a c t A chemiluminescent dual signal amplification strategy for the determination of ␣-fetoprotein (AFP) was proposed based on a sandwich immunoassay format. Monoclonal antibody of AFP immobilized on the gold nanoparticles doped mesoporous SiO2 (Au/SiO2 ) were prepared and used as a primary antibody. Horseradish peroxidase (HRP) and HRP-labeled secondary antibody (Ab2 ) co-immobilized into the mesoporous SiO2 nanoparticles (HRP–Ab2 /SiO2 ) were used as the labeled immunological probe. Due to the high ratio surface areas and pore volumes of the mesoporous SiO2 , not only the amount of AFP monoclonal antibody but also the amount of the modified HRP and Ab2 in HRP–Ab2 /SiO2 were largely increased. Thus the chemiluminescent signal was amplified by using the system of luminol and H2 O2 under the catalysis of HRP. Under the optimal conditions, two linear ranges for AFP were obtained from 0.01 to 0.5 ng mL−1 and 0.5 to 100 ng mL−1 with a detection limit of 0.005 ng mL−1 (3). The fabricated signal amplification strategy showed an excellent promise for sensitive detection of AFP and other tumor markers. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In the tumorous process, increased levels of tumor markers in human serum are associated in patients with certain tumors. So the determination of tumor markers plays an important role in screening for a disease, in diagnosing a disease, and in determining the prognosis of a disease [1]. Due to the highly sensitive and selective nature of the recognition between antigen and antibody, immunoassay has been considered as a major analytical method for the determination of tumor markers [2,3]. The average concentration of ␣-fetoprotein (AFP) is about 25 ng mL−1 in healthy human serum, but rises greatly in patients with liver cancer [4,5]. It is an important tumor marker in the diagnosis and management of original liver carcinoma. Great efforts have been made to enhance detection sensitivity and/or obtain low detection limits by the signal amplification technique in clinical immunoassays [6–8]. Bio-bar code assays with the PCR-like sensitivity have shown the prospect on the signal amplification for the ultrasensitive detection of proteins at very low concentration [9–14]. Recently, much attention has been focused on signal amplification by using horseradish peroxidase (HRP) or alkaline phosphatase (ALP) to improve the detection limit and sensitivity [15–18].
∗ Corresponding author. Tel.: +86 532 84023927; fax: +86 532 84023927. E-mail address: [email protected] (J. Lin). 0921-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mseb.2011.09.016
Chemiluminescent (CL) detection possesses several advantages such as no radioactive wastes, the relatively simple instrumentation required, the very low detection limit and wide dynamic range [19]. HRP is one of the most commonly enzymes used in CL detection for antibody and antigen labeling through catalyzing the luminescent luminol oxidation in the presence of hydrogen peroxide (H2 O2 ). An amplification label was prepared for highly sensitive detection of protein by immobilizing HRP-labeled secondary antibody (Ab2 ) on the surface of gold nanoparticles (Au NPs) [20,21]. In order to maximize the ratio of enzyme tags, HRP enzymes and Ab2 were co-immobilized onto carbon nanotubes [22,23] or silica nanoparticles [24]. After a sandwich immunoassay procedure, enhanced signals were obtained due to a high HRP and HRP-labeled Ab2 molar ratio. Mesoporous silica (MPS) with high ratio surface areas and pore volumes has been considered as a promising matrix for the immobilization of proteins [25–27]. Yang et al. reported an amplified electrochemical immunosensor by co-immobilizing HRP and secondary anti-human IgG antibody on the surface of MPS [28]. An amplified CL immunosensor by using HRP-functionalized MPS as labels was developed for the determination of carcinoembryonic antigen (CEA) [29]. The amplification labels were fabricated by coimmobilization of HRP and anti-CEA onto the surface of MPS using 3-aminopropyltriethoxysilane (APTES) as the linkage. The monoclonal antibody coated on the 96-well plates was used as the primary antibody. Based on a sandwich immunoassay format, the CL signal was amplified by the increased amount of HRP on the surface of MPS. In the current work, monoclonal antibody of AFP
J. Lin et al. / Materials Science and Engineering B 176 (2011) 1474–1478
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Fig. 1. Schematic diagram of flow injection CL determination of AFP with HRP–Ab2 /SiO2 as sensitive labels based on sandwich immunoassay.
(Ab1 ) immobilized on the Au NPs doped mesoporous SiO2 (Au/SiO2 ) were prepared and used as a primary antibody. HRP and HRPlabeled antibody were co-immobilizing in the amino-modified mesoporous SiO2 as the novel immunological probes (name as HRP–Ab2 /SiO2 ). Both the amount of AFP monoclonal antibody and the amount of the modified HRP in HRP–Ab2 /SiO2 were largely increased due to the high ratio surface areas of the mesoporous SiO2 . Based on a sandwich immunoassay format, the chemiluminescent signal was amplified by using the system of luminol and H2 O2 under the catalysis of HRP. The proposed immunoassay strategy showed an acceptable accuracy for the determination of AFP compared with the results obtained by an ELISA method.
2. Experimental 2.1. Reagents and chemicals The anti-AFP monoclonal antibody of AFP used as the primary antibody and AFP ELISA kits were purchased from Biocell Bioeng. Co. (Zhengzhou, China). Tetraethoxysilane (TEOS) and aminopropyltriethoxysilane (APTES) were obtained from Aldrich. EO20PO70EO20 (P123) was obtained from ACROS. Trimethylbenzene was purchased from Shanghai Kefeng Chemical Reagent Company in China. N-hydroxysuccinimide (NHS) and 1-ethyl3-dimethylaminopropyl carbodiimide hydrochloride (EDC) were acquired from Sigma. All solutions were prepared with doublydistilled water. Doubly distilled water was used throughout the experiment. Other chemicals were of analytical grade and used without further purification.
2.2. Apparatus The chemiluminescence detection was conducted on a flow injection chemiluminescence system (IFFM-E Luminescence Analyzer, Xi’an Remax Electronic Instrument Limited Co., China). Transmission electron micrograph (TEM) was recorded on JEM2000EX (JEOL, Japan). The nitrogen gas adsorption/desorption isotherms measured by a Micromeritics Tristar 3000 system. The IR spectra were recorded with a Vertex 4200 FTIR spectrometer. The CL spectra were measured on F-4500 fluorescence spectrophotometer (Hitachi, Japan) with the excitation light source being turned off.
2.3. Preparation of HRP–Ab2 /SiO2 immunological labels The preparations of amino-modified mesoporous SiO2 and Au/SiO2 were illustrated in the supporting section. The aminomodified mesoporous SiO2 nanoparticles were dispersed in 2 mL of glutaraldehyde solution (5%, w/w), and kept stirring for 1.5 h at room temperature. After the centrifugation and washing process, the glutaraldehyde-modified nanoparticles were re-dispersed in 1 mL of 0.1 mol L−1 PBS buffer containing 0.1 mg L−1 HRP and 500 L of Ab2 (working solution in AFP ELISA kit). Thus, the HRP–Ab2 /SiO2 immunological labels were prepared after blocking excess active groups by 1% BSA solution. After washing with PBS and water, the prepared HRP–Ab2 /SiO2 were dispersed in 1.0 mL PBS buffer and stored at 4 ◦ C in prior to use. Ab2 -only modified SiO2 (Ab2 /SiO2 ) or HRP-only modified SiO2 (HRP/SiO2 ) nanoparticles were also prepared for the control experiments.
J. Lin et al. / Materials Science and Engineering B 176 (2011) 1474–1478
Transmittance / %
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100 75
1662
50
100
Transmittance / %
a 1548
b
80
60 4000
1655
3000
1545
2000
1000 -1
Wave numbers (cm ) Fig. 3. FTIR spectra of (a) HRP/SiO2 and (b) HRP.
nearly the same as those obtained in the protein spectrum (1655 and 1545 cm−1 in Fig. 3b). If protein is denatured, the intensity of the characteristic bands will significantly weaken or even disappear [32]. The result suggests that the immobilized HRP retain the essential features of their native structure in the mesopores. Fig. 2. Transmission electron micrograph of the prepared mesoporous SiO2 (A) and SiO2 /Au (B).
2.4. Sandwich immunoassay for the determination of AFP As shown in Fig. 1, 100 L AFP antigen (Ag) was carried by 0.1 M pH 7.0 PBS to react with the immobilized Ab1 on Ab1 /Au/SiO2 modified glass sheet. The Ag–Ab1 complexes formed during the first step of the sandwich reaction with an incubation time of 15 min at 25 ◦ C. After washing thoroughly with PBS, 100 L of HRP–Ab2 /SiO2 suspension was injected to react with Ag–Ab1 –Au/SiO2 at 25 ◦ C for 20 min. Then, HRP–Ab2 /SiO2 was retained in the flow cell due to the formed Ab2 –Ag–Ab1 sandwich complex in the second step. After washing with 0.1 M pH 7.0 PBS to remove nonspecific adsorption of HRP–Ab2 /SiO2 , 500 L CL substrates of luminol and H2 O2 were injected. Amplified CL signal of luminol and H2 O2 was produced in the presence of the trapped HRP–Ab2 /SiO2 . After the determination, the immunoassay flowing cell was regenerated. A flow rate of 1.0 mL min−1 was used throughout the experiment.
3.2. Signal amplification of HRP–Ab2 /SiO2 label Based on a sandwich immunoassay, the signal amplification of the prepared HRP–Ab2 /SiO2 was investigated and the results are shown in Fig. 4. The blank experiment was carried by injecting a mixture of luminol (1.0 mmol L−1 ) and H2 O2 (1.0 mmol L−1 ) into the flowing cell without the sample antigen and HRP–Ab2 /SiO2 labels (curves a). The CL signal was attributed to the reaction of luminol–H2 O2 . After injecting sample AFP antigen (10 ng mL−1 ) followed by 100 L HRP-labeled Ab2 into the flow cell, the sandwich immunoconjugates of Ab2 –Ag–Ab1 was formed. It can be seen that the CL intensity of luminol–H2 O2 increased in the presence of HRPlabels (curves b). When the same amount of Ab2 was immobilized in the mesoporous SiO2 , and used as the immunological labels instead of the HRP-labeled antibody, enhanced CL signals were observed from curves c. It suggested that the aggregates of HRP-labels could amplify the CL signal. However, the CL signals were highly amplified by using HRP–Ab2 /SiO2 as labels (curves d) due to a larger
3. Results and discussion 3.1. Characterizations of the prepared mesoporous SiO2 NPs A typical N2 adsorption/desorption isotherm of the mesoporous silica is presented in Fig. S1. The isotherm is a type IV curve of mesoporous materials. A steep hysteretic loop is observed from this curve, which is typical for mesoporous materials that exhibit capillary condensation and evaporation. The specific surface area and pore volume obtained by the N2 adsorption isotherms and calculated by the Brunauer–Emmett–Teller method were 901 cm2 g−1 and 0.59 cm3 g−1 , respectively. Fig. 2 shows the TEM micrographs of the prepared mesoporous SiO2 and Au/SiO2 nanoparticles. Some lattice fringes can be observed at the edges of the spheres. The Au/SiO2 nanoparticles in Fig. 2b showed the aggregates of the trapped Au NPs. The adsorption of HRP in the mesoporous SiO2 was characterized by FTIR spectra in Fig. 3. The shapes of the amide I and amide II infrared absorbance bands of proteins can provide detailed information on the secondary structure of polypeptide chain [31]. As shown in Fig. 3a, the characteristic amide I (1662 cm−1 ) and amide II (1548 cm−1 ) bands of HRP adsorbed in mesoporous silica are
Fig. 4. CL intensity of the Ab1 /SiO2 /Au incubating with: (a) background solution of luminol–H2 O2 , (b) 10 ng mL−1 AFP and 100 L HRP-labeled Ab2 in solution, (c) 10 ng mL−1 AFP and Ab2 /SiO2 , (d) 10 ng mL−1 AFP and HRP–Ab2 /SiO2 suspension. Conditions: luminol, 1.0 mmol L−1 in 0.1 mol L−1 Tris–HCl (pH 9.0); H2 O2 , 1.0 mmol L−1 ; flow rate, 1.0 mL min−1 .
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Fig. 5. Effects of the two-steps incubation time of (a) antigen and Ab1 ; (b) HRP–SiO2 /Ab2 and Ag–Ab1 on the sandwich immunoassay procedure. Conditions: luminol, 1.0 mmol L−1 in 0.1 mol L−1 Tris–HCl (pH 9.0); H2 O2 , 1.0 mmol L−1 ; flow rate, 1.0 mL min−1 .
amount of HRP tags loaded on mesoporous SiO2 . Under the same condition, HRP–Ab2 /SiO2 and Ab1 /Au/SiO2 labels were incubated together without AFP antigen, no enhanced signal was observed. When HRP/SiO2 was incubated with AFP antigen and Ab1 /Au/SiO2 , no enhanced signal was observed. All of these results suggested that the signal amplification was from co-coated HRP and HRP-Ab2 tags. 3.3. Optimized immunoassay conditions 0.1 M Tris–HCl with a pH value of 9.0 was chosen as the buffer solution according to our previous work. The incubation time was an important parameter during the immunoassay procedure. By incubating Ab1 /Au/SiO2 with AFP antigen solution and then with HRP–Ab2 /SiO2 labels for different lengths of time, the effects of the incubation times on the sandwich immunoassay were examined. The results are shown in Fig. 5. With the increasing incubation time, the CL signal increased and trended to a maximum value at an incubation time of 15 min and 20 min, respectively. So an incubation time of 15 min for the first-step and an incubation time of 20 min for the second-step immunological reaction were selected in the current work. The amount of HRP adsorbed in mesoporous SiO2 also affected the CL intensity and were investigated in the current work (Fig. S3). The concentration of HRP in solution was optimized by using HRP solutions at different concentrations from 0.01 to 0.5 mg L−1 . HRP at higher concentration in solution could increase the absolute amount of HRP loaded per mesoporous nanoparticle, which is expected to improve the signal amplification. The CL signal increased sharply as the concentration of HRP increased from 0.01 to 0.1 mg L−1 . The CL signal increased slightly when the concentration of HRP was changed from 0.1 to 0.5 mg L−1 . When the HRP concentration was higher than 0.1 mg L−1 , most of the active sites of mesoporous SiO2 were occupied by HRP, that is to say, HRP were saturated on the surface SiO2 . Thus, the optimal concentration of HRP was selected at 0.1 mg L−1 in this work.
relative chemiluminescent intensity vs the AFP concentration. The plot shows a linear increase in the two concentration ranges from 0.01 to 0.5 and 0.5 to 100 ng mL−1 , with the correlation coefficients of 0.991 and 0.999, respectively. The detection limit was calculated to be 0.005 ng mL−1 according to 3, which was comparable with 5 pg mL−1 and 0.01 ng mL−1 reported by Yang et al. [20], Bi et al. [21] and Wu et al. [24], lower than the detection limit reported by Lin et al. [33] and Yakovleva et al. [34]. The lower detection limit was attributed to the CL amplification. Due to the co-immobilized HRP labeled Ab2 and HRP enzyme on the surface of mesoporous SiO2 , the CL signals were amplified by the increased HRP tags. It is more flexible by integrating a flow injection system, which may enable a more simple and sensitive determination of AFP and thus provide a promising platform for the development of amplification biosensors. 3.5. Stability and reproducibility When the Ab1 /Au/SiO2 modified glass sheets and HRP–Ab2 /SiO2 NPs were not in use, they were stored at 4 ◦ C. The storage stability was studied on a 20-day period in this work. At different storage periods between 20 days, the same Ab1 /Au/SiO2 modified glass
3.4. Calibration plot for the determination of AFP Under optimal conditions, typical CL signals for AFP determination were obtained based on a sandwich immunoassay format. The blank signal was measured by injecting a mixture of luminol (1.0 mmol L−1 ) and H2 O2 (1.0 mmol L−1 ) into the flow cell at a flow rate of 1.0 mL min−1 . With the increasing AFP concentration, the amount of the HRP–Ab2 /SiO2 retained in the flow cell increased due to the formed sandwich immunocomplex. Fig. 6 shows the plot of
Fig. 6. Calibration plot for the sandwich immunoassay of AFP. Inset: linear relation between relative chemiluminescent intensity and AFP concentration at (a) 0.01–0.5 ng mL−1 and (b) 0.5–100 ng mL−1 .
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sheet and HRP–Ab2 /SiO2 suspension were used to detect sample AFP with a concentration of 10 ng mL−1 . The results are shown in Fig. S4. It could be seen that the analytical performances retained 92% of its initial CL intensity after 20-day storage, indicating that the prepared Ab1 /Au/SiO2 and HRP–Ab2 /SiO2 had good stability. The reproducibility was assessed with the relative standard deviation (RSD), which was evaluated from five parallel measurements of sample AFP by using Ab1 /Au/SiO2 and HRP–Ab2 /SiO2 . The RSD obtained at the AFP concentration of 0.1 ng mL−1 and 25 ng mL−1 were 8.2% and 4.9%, respectively. The results were indicating the preparation of the Ab1 /Au/SiO2 and HRP–Ab2 /SiO2 possessed good stability.
as a new secondary antibody, the CL response was enhanced by the increased amount of HRP tags. As a result, the sensitivity for AFP determination was heightened. In addition, interference experiments indicated that the proposed method display good biospecificity and bio-selectivity. The experimental results indicated that the proposed strategy is suitable to be used as platforms in the determination of other tumor markers. Acknowledgements The work was supported by the National Natural Science Foundation of China (No. 20905041) and the Natural Science Foundation of Shandong Province (J2008B02).
3.6. Regeneration procedure Appendix A. Supplementary data Regeneration of the antibodies immobilized on the matrix is very important for the continuous detection. It was reported that the most efficient regeneration buffer was found to be 0.1 M glycine–HCl at the pH value of 2.2 [30]. It allowed fast and complete dissociation of the immuno-complex within a shorter regeneration time. We chose 0.1 M glycine–HCl (pH 2.2) as the regeneration buffer in the current work. After each regeneration procedure, Ab1 /Au/SiO2 and HRP–Ab2 /SiO2 NPs were examined to check their relative activity. The result is shown in Fig. S5. It was shown that CL intensity was 93.2% of the initial response for AFP after 15 times regeneration. After that, the CL signals showed observable decrease. 3.7. Accuracy and clinical application The determination accuracy was examined by comparing the results obtained with the proposed chemiluminescent immunoassay (CLIA) and ELISA assay. The mean AFP concentrations in 22 sera samples determined with ELISA assay and this proposed method was given in Fig. S6. The result shows that the two methods were in an acceptable agreement. 3.8. Specificity for the AFP detection Cross-reactivity is a crucial analytical parameter regarding specificity. The specificity for the AFP detection was investigated by mixing Ab1 /Au/SiO2, HRP–Ab2 /SiO2 with 10 ng mL−1 AFP with the co-existed prostate specific antigen (PSA, 10 ng mL−1 ) and CEA (10 ng mL−1 ). The result is shown in Fig. S7. There were no significant interference from the co-existed PSA and CEA. So the effect of interfering compound on the determination of AFP could be negligible under the testing conditions. 4. Conclusions
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mseb.2011.09.016. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30]
In this work, a new signal amplification immunoassay was proposed to detect AFP by using enzyme-functionalized mesoporous SiO2 nanoparticles based on the flow injection CL analysis. The monoclonal anti-AFP coated Au/SiO2 was used for recognition of its antigen in the first-step reaction. After HRP–Ab2 /SiO2 with co-immobilized HRP labeled Ab2 and HRP enzyme was used
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Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering B journal homepage: www.elsevier.com/locate/mseb
Chemiluminescence immunoassay based on dual signal amplification strategy of Au/mesoporous silica and multienzyme functionalized mesoporous silica Jiehua Lin ∗ , Yue Zhao, Zhijing Wei, Wei Wang Key Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China
a r t i c l e
i n f o
Article history: Received 8 April 2011 Received in revised form 22 June 2011 Accepted 4 September 2011 Available online 17 September 2011 Keywords: Chemiluminescent immunoassay Mesoporous silica Horseradish peroxidase ␣-Fetoprotein
a b s t r a c t A chemiluminescent dual signal amplification strategy for the determination of ␣-fetoprotein (AFP) was proposed based on a sandwich immunoassay format. Monoclonal antibody of AFP immobilized on the gold nanoparticles doped mesoporous SiO2 (Au/SiO2 ) were prepared and used as a primary antibody. Horseradish peroxidase (HRP) and HRP-labeled secondary antibody (Ab2 ) co-immobilized into the mesoporous SiO2 nanoparticles (HRP–Ab2 /SiO2 ) were used as the labeled immunological probe. Due to the high ratio surface areas and pore volumes of the mesoporous SiO2 , not only the amount of AFP monoclonal antibody but also the amount of the modified HRP and Ab2 in HRP–Ab2 /SiO2 were largely increased. Thus the chemiluminescent signal was amplified by using the system of luminol and H2 O2 under the catalysis of HRP. Under the optimal conditions, two linear ranges for AFP were obtained from 0.01 to 0.5 ng mL−1 and 0.5 to 100 ng mL−1 with a detection limit of 0.005 ng mL−1 (3). The fabricated signal amplification strategy showed an excellent promise for sensitive detection of AFP and other tumor markers. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In the tumorous process, increased levels of tumor markers in human serum are associated in patients with certain tumors. So the determination of tumor markers plays an important role in screening for a disease, in diagnosing a disease, and in determining the prognosis of a disease [1]. Due to the highly sensitive and selective nature of the recognition between antigen and antibody, immunoassay has been considered as a major analytical method for the determination of tumor markers [2,3]. The average concentration of ␣-fetoprotein (AFP) is about 25 ng mL−1 in healthy human serum, but rises greatly in patients with liver cancer [4,5]. It is an important tumor marker in the diagnosis and management of original liver carcinoma. Great efforts have been made to enhance detection sensitivity and/or obtain low detection limits by the signal amplification technique in clinical immunoassays [6–8]. Bio-bar code assays with the PCR-like sensitivity have shown the prospect on the signal amplification for the ultrasensitive detection of proteins at very low concentration [9–14]. Recently, much attention has been focused on signal amplification by using horseradish peroxidase (HRP) or alkaline phosphatase (ALP) to improve the detection limit and sensitivity [15–18].
∗ Corresponding author. Tel.: +86 532 84023927; fax: +86 532 84023927. E-mail address: [email protected] (J. Lin). 0921-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mseb.2011.09.016
Chemiluminescent (CL) detection possesses several advantages such as no radioactive wastes, the relatively simple instrumentation required, the very low detection limit and wide dynamic range [19]. HRP is one of the most commonly enzymes used in CL detection for antibody and antigen labeling through catalyzing the luminescent luminol oxidation in the presence of hydrogen peroxide (H2 O2 ). An amplification label was prepared for highly sensitive detection of protein by immobilizing HRP-labeled secondary antibody (Ab2 ) on the surface of gold nanoparticles (Au NPs) [20,21]. In order to maximize the ratio of enzyme tags, HRP enzymes and Ab2 were co-immobilized onto carbon nanotubes [22,23] or silica nanoparticles [24]. After a sandwich immunoassay procedure, enhanced signals were obtained due to a high HRP and HRP-labeled Ab2 molar ratio. Mesoporous silica (MPS) with high ratio surface areas and pore volumes has been considered as a promising matrix for the immobilization of proteins [25–27]. Yang et al. reported an amplified electrochemical immunosensor by co-immobilizing HRP and secondary anti-human IgG antibody on the surface of MPS [28]. An amplified CL immunosensor by using HRP-functionalized MPS as labels was developed for the determination of carcinoembryonic antigen (CEA) [29]. The amplification labels were fabricated by coimmobilization of HRP and anti-CEA onto the surface of MPS using 3-aminopropyltriethoxysilane (APTES) as the linkage. The monoclonal antibody coated on the 96-well plates was used as the primary antibody. Based on a sandwich immunoassay format, the CL signal was amplified by the increased amount of HRP on the surface of MPS. In the current work, monoclonal antibody of AFP
J. Lin et al. / Materials Science and Engineering B 176 (2011) 1474–1478
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Fig. 1. Schematic diagram of flow injection CL determination of AFP with HRP–Ab2 /SiO2 as sensitive labels based on sandwich immunoassay.
(Ab1 ) immobilized on the Au NPs doped mesoporous SiO2 (Au/SiO2 ) were prepared and used as a primary antibody. HRP and HRPlabeled antibody were co-immobilizing in the amino-modified mesoporous SiO2 as the novel immunological probes (name as HRP–Ab2 /SiO2 ). Both the amount of AFP monoclonal antibody and the amount of the modified HRP in HRP–Ab2 /SiO2 were largely increased due to the high ratio surface areas of the mesoporous SiO2 . Based on a sandwich immunoassay format, the chemiluminescent signal was amplified by using the system of luminol and H2 O2 under the catalysis of HRP. The proposed immunoassay strategy showed an acceptable accuracy for the determination of AFP compared with the results obtained by an ELISA method.
2. Experimental 2.1. Reagents and chemicals The anti-AFP monoclonal antibody of AFP used as the primary antibody and AFP ELISA kits were purchased from Biocell Bioeng. Co. (Zhengzhou, China). Tetraethoxysilane (TEOS) and aminopropyltriethoxysilane (APTES) were obtained from Aldrich. EO20PO70EO20 (P123) was obtained from ACROS. Trimethylbenzene was purchased from Shanghai Kefeng Chemical Reagent Company in China. N-hydroxysuccinimide (NHS) and 1-ethyl3-dimethylaminopropyl carbodiimide hydrochloride (EDC) were acquired from Sigma. All solutions were prepared with doublydistilled water. Doubly distilled water was used throughout the experiment. Other chemicals were of analytical grade and used without further purification.
2.2. Apparatus The chemiluminescence detection was conducted on a flow injection chemiluminescence system (IFFM-E Luminescence Analyzer, Xi’an Remax Electronic Instrument Limited Co., China). Transmission electron micrograph (TEM) was recorded on JEM2000EX (JEOL, Japan). The nitrogen gas adsorption/desorption isotherms measured by a Micromeritics Tristar 3000 system. The IR spectra were recorded with a Vertex 4200 FTIR spectrometer. The CL spectra were measured on F-4500 fluorescence spectrophotometer (Hitachi, Japan) with the excitation light source being turned off.
2.3. Preparation of HRP–Ab2 /SiO2 immunological labels The preparations of amino-modified mesoporous SiO2 and Au/SiO2 were illustrated in the supporting section. The aminomodified mesoporous SiO2 nanoparticles were dispersed in 2 mL of glutaraldehyde solution (5%, w/w), and kept stirring for 1.5 h at room temperature. After the centrifugation and washing process, the glutaraldehyde-modified nanoparticles were re-dispersed in 1 mL of 0.1 mol L−1 PBS buffer containing 0.1 mg L−1 HRP and 500 L of Ab2 (working solution in AFP ELISA kit). Thus, the HRP–Ab2 /SiO2 immunological labels were prepared after blocking excess active groups by 1% BSA solution. After washing with PBS and water, the prepared HRP–Ab2 /SiO2 were dispersed in 1.0 mL PBS buffer and stored at 4 ◦ C in prior to use. Ab2 -only modified SiO2 (Ab2 /SiO2 ) or HRP-only modified SiO2 (HRP/SiO2 ) nanoparticles were also prepared for the control experiments.
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Transmittance / %
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100 75
1662
50
100
Transmittance / %
a 1548
b
80
60 4000
1655
3000
1545
2000
1000 -1
Wave numbers (cm ) Fig. 3. FTIR spectra of (a) HRP/SiO2 and (b) HRP.
nearly the same as those obtained in the protein spectrum (1655 and 1545 cm−1 in Fig. 3b). If protein is denatured, the intensity of the characteristic bands will significantly weaken or even disappear [32]. The result suggests that the immobilized HRP retain the essential features of their native structure in the mesopores. Fig. 2. Transmission electron micrograph of the prepared mesoporous SiO2 (A) and SiO2 /Au (B).
2.4. Sandwich immunoassay for the determination of AFP As shown in Fig. 1, 100 L AFP antigen (Ag) was carried by 0.1 M pH 7.0 PBS to react with the immobilized Ab1 on Ab1 /Au/SiO2 modified glass sheet. The Ag–Ab1 complexes formed during the first step of the sandwich reaction with an incubation time of 15 min at 25 ◦ C. After washing thoroughly with PBS, 100 L of HRP–Ab2 /SiO2 suspension was injected to react with Ag–Ab1 –Au/SiO2 at 25 ◦ C for 20 min. Then, HRP–Ab2 /SiO2 was retained in the flow cell due to the formed Ab2 –Ag–Ab1 sandwich complex in the second step. After washing with 0.1 M pH 7.0 PBS to remove nonspecific adsorption of HRP–Ab2 /SiO2 , 500 L CL substrates of luminol and H2 O2 were injected. Amplified CL signal of luminol and H2 O2 was produced in the presence of the trapped HRP–Ab2 /SiO2 . After the determination, the immunoassay flowing cell was regenerated. A flow rate of 1.0 mL min−1 was used throughout the experiment.
3.2. Signal amplification of HRP–Ab2 /SiO2 label Based on a sandwich immunoassay, the signal amplification of the prepared HRP–Ab2 /SiO2 was investigated and the results are shown in Fig. 4. The blank experiment was carried by injecting a mixture of luminol (1.0 mmol L−1 ) and H2 O2 (1.0 mmol L−1 ) into the flowing cell without the sample antigen and HRP–Ab2 /SiO2 labels (curves a). The CL signal was attributed to the reaction of luminol–H2 O2 . After injecting sample AFP antigen (10 ng mL−1 ) followed by 100 L HRP-labeled Ab2 into the flow cell, the sandwich immunoconjugates of Ab2 –Ag–Ab1 was formed. It can be seen that the CL intensity of luminol–H2 O2 increased in the presence of HRPlabels (curves b). When the same amount of Ab2 was immobilized in the mesoporous SiO2 , and used as the immunological labels instead of the HRP-labeled antibody, enhanced CL signals were observed from curves c. It suggested that the aggregates of HRP-labels could amplify the CL signal. However, the CL signals were highly amplified by using HRP–Ab2 /SiO2 as labels (curves d) due to a larger
3. Results and discussion 3.1. Characterizations of the prepared mesoporous SiO2 NPs A typical N2 adsorption/desorption isotherm of the mesoporous silica is presented in Fig. S1. The isotherm is a type IV curve of mesoporous materials. A steep hysteretic loop is observed from this curve, which is typical for mesoporous materials that exhibit capillary condensation and evaporation. The specific surface area and pore volume obtained by the N2 adsorption isotherms and calculated by the Brunauer–Emmett–Teller method were 901 cm2 g−1 and 0.59 cm3 g−1 , respectively. Fig. 2 shows the TEM micrographs of the prepared mesoporous SiO2 and Au/SiO2 nanoparticles. Some lattice fringes can be observed at the edges of the spheres. The Au/SiO2 nanoparticles in Fig. 2b showed the aggregates of the trapped Au NPs. The adsorption of HRP in the mesoporous SiO2 was characterized by FTIR spectra in Fig. 3. The shapes of the amide I and amide II infrared absorbance bands of proteins can provide detailed information on the secondary structure of polypeptide chain [31]. As shown in Fig. 3a, the characteristic amide I (1662 cm−1 ) and amide II (1548 cm−1 ) bands of HRP adsorbed in mesoporous silica are
Fig. 4. CL intensity of the Ab1 /SiO2 /Au incubating with: (a) background solution of luminol–H2 O2 , (b) 10 ng mL−1 AFP and 100 L HRP-labeled Ab2 in solution, (c) 10 ng mL−1 AFP and Ab2 /SiO2 , (d) 10 ng mL−1 AFP and HRP–Ab2 /SiO2 suspension. Conditions: luminol, 1.0 mmol L−1 in 0.1 mol L−1 Tris–HCl (pH 9.0); H2 O2 , 1.0 mmol L−1 ; flow rate, 1.0 mL min−1 .
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Fig. 5. Effects of the two-steps incubation time of (a) antigen and Ab1 ; (b) HRP–SiO2 /Ab2 and Ag–Ab1 on the sandwich immunoassay procedure. Conditions: luminol, 1.0 mmol L−1 in 0.1 mol L−1 Tris–HCl (pH 9.0); H2 O2 , 1.0 mmol L−1 ; flow rate, 1.0 mL min−1 .
amount of HRP tags loaded on mesoporous SiO2 . Under the same condition, HRP–Ab2 /SiO2 and Ab1 /Au/SiO2 labels were incubated together without AFP antigen, no enhanced signal was observed. When HRP/SiO2 was incubated with AFP antigen and Ab1 /Au/SiO2 , no enhanced signal was observed. All of these results suggested that the signal amplification was from co-coated HRP and HRP-Ab2 tags. 3.3. Optimized immunoassay conditions 0.1 M Tris–HCl with a pH value of 9.0 was chosen as the buffer solution according to our previous work. The incubation time was an important parameter during the immunoassay procedure. By incubating Ab1 /Au/SiO2 with AFP antigen solution and then with HRP–Ab2 /SiO2 labels for different lengths of time, the effects of the incubation times on the sandwich immunoassay were examined. The results are shown in Fig. 5. With the increasing incubation time, the CL signal increased and trended to a maximum value at an incubation time of 15 min and 20 min, respectively. So an incubation time of 15 min for the first-step and an incubation time of 20 min for the second-step immunological reaction were selected in the current work. The amount of HRP adsorbed in mesoporous SiO2 also affected the CL intensity and were investigated in the current work (Fig. S3). The concentration of HRP in solution was optimized by using HRP solutions at different concentrations from 0.01 to 0.5 mg L−1 . HRP at higher concentration in solution could increase the absolute amount of HRP loaded per mesoporous nanoparticle, which is expected to improve the signal amplification. The CL signal increased sharply as the concentration of HRP increased from 0.01 to 0.1 mg L−1 . The CL signal increased slightly when the concentration of HRP was changed from 0.1 to 0.5 mg L−1 . When the HRP concentration was higher than 0.1 mg L−1 , most of the active sites of mesoporous SiO2 were occupied by HRP, that is to say, HRP were saturated on the surface SiO2 . Thus, the optimal concentration of HRP was selected at 0.1 mg L−1 in this work.
relative chemiluminescent intensity vs the AFP concentration. The plot shows a linear increase in the two concentration ranges from 0.01 to 0.5 and 0.5 to 100 ng mL−1 , with the correlation coefficients of 0.991 and 0.999, respectively. The detection limit was calculated to be 0.005 ng mL−1 according to 3, which was comparable with 5 pg mL−1 and 0.01 ng mL−1 reported by Yang et al. [20], Bi et al. [21] and Wu et al. [24], lower than the detection limit reported by Lin et al. [33] and Yakovleva et al. [34]. The lower detection limit was attributed to the CL amplification. Due to the co-immobilized HRP labeled Ab2 and HRP enzyme on the surface of mesoporous SiO2 , the CL signals were amplified by the increased HRP tags. It is more flexible by integrating a flow injection system, which may enable a more simple and sensitive determination of AFP and thus provide a promising platform for the development of amplification biosensors. 3.5. Stability and reproducibility When the Ab1 /Au/SiO2 modified glass sheets and HRP–Ab2 /SiO2 NPs were not in use, they were stored at 4 ◦ C. The storage stability was studied on a 20-day period in this work. At different storage periods between 20 days, the same Ab1 /Au/SiO2 modified glass
3.4. Calibration plot for the determination of AFP Under optimal conditions, typical CL signals for AFP determination were obtained based on a sandwich immunoassay format. The blank signal was measured by injecting a mixture of luminol (1.0 mmol L−1 ) and H2 O2 (1.0 mmol L−1 ) into the flow cell at a flow rate of 1.0 mL min−1 . With the increasing AFP concentration, the amount of the HRP–Ab2 /SiO2 retained in the flow cell increased due to the formed sandwich immunocomplex. Fig. 6 shows the plot of
Fig. 6. Calibration plot for the sandwich immunoassay of AFP. Inset: linear relation between relative chemiluminescent intensity and AFP concentration at (a) 0.01–0.5 ng mL−1 and (b) 0.5–100 ng mL−1 .
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sheet and HRP–Ab2 /SiO2 suspension were used to detect sample AFP with a concentration of 10 ng mL−1 . The results are shown in Fig. S4. It could be seen that the analytical performances retained 92% of its initial CL intensity after 20-day storage, indicating that the prepared Ab1 /Au/SiO2 and HRP–Ab2 /SiO2 had good stability. The reproducibility was assessed with the relative standard deviation (RSD), which was evaluated from five parallel measurements of sample AFP by using Ab1 /Au/SiO2 and HRP–Ab2 /SiO2 . The RSD obtained at the AFP concentration of 0.1 ng mL−1 and 25 ng mL−1 were 8.2% and 4.9%, respectively. The results were indicating the preparation of the Ab1 /Au/SiO2 and HRP–Ab2 /SiO2 possessed good stability.
as a new secondary antibody, the CL response was enhanced by the increased amount of HRP tags. As a result, the sensitivity for AFP determination was heightened. In addition, interference experiments indicated that the proposed method display good biospecificity and bio-selectivity. The experimental results indicated that the proposed strategy is suitable to be used as platforms in the determination of other tumor markers. Acknowledgements The work was supported by the National Natural Science Foundation of China (No. 20905041) and the Natural Science Foundation of Shandong Province (J2008B02).
3.6. Regeneration procedure Appendix A. Supplementary data Regeneration of the antibodies immobilized on the matrix is very important for the continuous detection. It was reported that the most efficient regeneration buffer was found to be 0.1 M glycine–HCl at the pH value of 2.2 [30]. It allowed fast and complete dissociation of the immuno-complex within a shorter regeneration time. We chose 0.1 M glycine–HCl (pH 2.2) as the regeneration buffer in the current work. After each regeneration procedure, Ab1 /Au/SiO2 and HRP–Ab2 /SiO2 NPs were examined to check their relative activity. The result is shown in Fig. S5. It was shown that CL intensity was 93.2% of the initial response for AFP after 15 times regeneration. After that, the CL signals showed observable decrease. 3.7. Accuracy and clinical application The determination accuracy was examined by comparing the results obtained with the proposed chemiluminescent immunoassay (CLIA) and ELISA assay. The mean AFP concentrations in 22 sera samples determined with ELISA assay and this proposed method was given in Fig. S6. The result shows that the two methods were in an acceptable agreement. 3.8. Specificity for the AFP detection Cross-reactivity is a crucial analytical parameter regarding specificity. The specificity for the AFP detection was investigated by mixing Ab1 /Au/SiO2, HRP–Ab2 /SiO2 with 10 ng mL−1 AFP with the co-existed prostate specific antigen (PSA, 10 ng mL−1 ) and CEA (10 ng mL−1 ). The result is shown in Fig. S7. There were no significant interference from the co-existed PSA and CEA. So the effect of interfering compound on the determination of AFP could be negligible under the testing conditions. 4. Conclusions
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mseb.2011.09.016. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30]
In this work, a new signal amplification immunoassay was proposed to detect AFP by using enzyme-functionalized mesoporous SiO2 nanoparticles based on the flow injection CL analysis. The monoclonal anti-AFP coated Au/SiO2 was used for recognition of its antigen in the first-step reaction. After HRP–Ab2 /SiO2 with co-immobilized HRP labeled Ab2 and HRP enzyme was used
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