Volume 50 Number 27 7 April 2014 Pages 3521–3632
ChemComm Chemical Communications
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ISSN 1359-7345
COMMUNICATION Jungbae Kim et al. A highly sensitive immunoassay using antibody-conjugated spherical mesoporous silica with immobilized enzymes
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Cite this: Chem. Commun., 2014, 50, 3546 Received 19th October 2013, Accepted 4th December 2013
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A highly sensitive immunoassay using antibodyconjugated spherical mesoporous silica with immobilized enzymes† Ji Young Eum,‡a Sang Youn Hwang,‡a Youngjun Ju,a Jong Min Shim,b Yunxian Piao,c Jinwoo Lee,b Hak-Sung Kimc and Jungbae Kim*a
DOI: 10.1039/c3cc48044e www.rsc.org/chemcomm
A highly sensitive immunoassay was developed by using antibodyconjugated spherical mesoporous silica with immobilized enzymes. The higher ratio of enzyme/antibody than conventional ELISA improved both the sensitivity and dynamic range. Especially, the use of spherical mesoporous silica could achieve a limit of detection (LOD) with a sensitivity that is 20 times more than that of ELISA using amorphous silica.
The detection method with high selectivity, sensitivity and reliability is crucial for early diagnostics in medical fields as well as point of care testing.1 Among various detection methods, enzyme linked immunosorbent assay (ELISA) is one of the most popular tools in clinical and medical fields due to the specific binding of antibodies to target analytes and the amplified signal generation catalyzed by antibody-conjugated enzymes.2 However, the detection of target analytes in a trace amount often requires further signal enhancement of conventional ELISA. As one of the potential solutions for improvement of the sensitivity of ELISA, the enzyme-immobilized nanomaterials were conjugated with antibodies, and used for the ELISA. This approach improved the sensitivity of ELISA by increasing the ratio of enzymes to antibody.3 In other words, when compared to conventional ELISA using one enzyme per antibody, a greater number of enzyme molecules are involved in the signal generation per unit binding between antigen and antibody, formulating a sensitive ELISA protocol. Recently, nanobiocatalytic approaches, using various nanomaterials for immobilizing and stabilizing enzymes, have gathered more and more attention due to their potential applications in various fields,4 such as biosensors,5 biofuel cells,6 proteomic analysis,7 and ELISA.3 Especially, mesoporous silica were used for a
Department of Chemical and Biological Engineering, Korea University, Seoul 136-713, Republic of Korea. E-mail: [email protected] b Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Gyungbuk 790-784, Korea c Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea † Electronic supplementary information (ESI) available: Fig. S1 and S2, Table S1. See DOI: 10.1039/c3cc48044e ‡ These authors contributed equally.
3546 | Chem. Commun., 2014, 50, 3546--3548
the development of a stable enzyme system with high enzyme loading via an approach of nanoscale enzyme reactors (NERs), in which enzymes were first adsorbed onto the mesoporous materials and further crosslinked to prevent the enzyme leaching from mesoporous materials.8 As an extension of NER success, antibodies were further conjugated on the surface of amine-functionalized mesoporous silica, and ELISA was performed for the multiplexed immunoassay under the name of NER-LISA (NER-linked immunosorbent assay). However, the limit of detection (LOD) of human IgG (hIgG) was 10 ng mL 1,3d which needs to be rigorously improved for the successful application of NER-LISA. As a potential explanation for this poor LOD, we suspect that the amorphous shape of mesoporous silica particles might lead to inefficient or no binding of target analytes with antibodies conjugated on the concave surface of amorphous mesoporous silica particles, due to steric hindrance. In the present work, we report a highly sensitive immunoassay method by using mesoporous silica in a spherical form (spherical mesoporous silica, S-MPS) with highly-loaded glucose oxidase (GO). When mesoporous silica in an amorphous form is used, there is a chance for antibodies conjugated on the concave outer surface not to bind to the target analytes effectively. On the other hand, the use of S-MPS (Fig. S1a, ESI†) would enable more efficient binding between conjugated antibodies and the target analytes, than the amorphous mesoporous silica (Fig. S1b, ESI†) in the former study,3d with an outer surface that is convex with no concavity. Aminefunctionalized S-MPS was used for the preparation of NERs with GO (NER-GO) by adsorbing GO and crosslinking adsorbed GO molecules via glutaraldehyde treatment. As the control samples, we also prepared simply-adsorbed GO (ADS-GO) and covalently-attached GO (CA-GO) in S-MPS. NER-GO in S-MPS were further conjugated with anti-human IgG antibody (anti-hIgG antibody), and the performance of NER-LISA in the detection of hIgG was compared to those of conventional ELISA and NER-LISA with amorphous silica.3d Fig. 1 schematically shows the proposed hypothesis for the potential enhancement of ELISA signals by using antibodyconjugated NERs in S-MPS. When compared to the conventional ELISA using one enzyme per antibody (Fig. 1a), a lot more enzymes
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Fig. 1 Schematic for the detection of hIgG based on the sandwich immunoassay by using antibody-conjugated enzyme (a), amorphous NER (b), and spherical NER (c). See Fig. S1 (ESI†) for the SEM images of spherical and amorphous mesoporous silica.
in S-MPS would generate an enhanced signal, which would lead to the improvement of the sensitivity of NER-LISA. Another control is the NER-LISA using amorphous silica (Fig. 1b), in which the antibodies conjugated on the concave surface would be useless due to the steric hindrance against their binding to the target analyte (hIgG). To demonstrate this hypothetical effect of NER-LISA using spherical mesoporous silica, NER-GO was prepared via a simple two-step process: GO adsorption onto S-MPS and crosslinking. Then, NER-GO was further conjugated with anti-hIgG antibody, and NER-LISA was performed for the detection of hIgG. The GO activity was measured by the peroxidase-catalyzed oxidation of dyes (o-dianisidine), which uses hydrogen peroxide being generated by the GO-catalyzed glucose oxidation. The initial activity of ADS-GO, CA-GO and NER-GO was 5.0, 9.4 and 12.3 A500 per min per mg amino-S-MPS, respectively (Fig. 2a). NER-GO exhibited 2.5 and 1.5 times higher activity than ADS-GO and CA-GO, respectively. The higher activity of NER-GO than that of ADS-GO can be attributed to the prevention of enzyme leaching due to effective covalent linkages among fully-packed GO molecules and amino-S-MPS. The lower activity of CA-GO than that of NER-GO suggests that only the inner surface rather than all the pore volume of amino-S-MPS is used for the immobilization of CA-GO. Fig. 2b shows the stability of ADS-GO, CA-GO, and NER-GO under shaking (200 rpm) at room temperature. NER-GO maintained 98% of its initial activity after 26 days, while ADS-GO and CA-GO retained 20% and 49% of their initial activities, respectively, under the same conditions. The high enzyme stability of NER-GO can be attributed to both the covalent linkages on the surface of GO molecules, inhibiting enzyme denaturation, and covalent linkages among crosslinked GO and amino-S-MPS, preventing the leaching of GO molecules in an effective way.4 Highly stable and active NER-GO was further conjugated with anti-hIgG antibody for the detection of hIgG via NER-LISA. It is anticipated that most antibodies would be conjugated on the surface of spherical mesoporous silica because the internal mesopores must be filled with crosslinked GO molecules. To determine the quantitative detection of hIgG via NER-LISA, various concentrations of hIgG (0–10 mg mL 1) were captured on an antibody-conjugated well
This journal is © The Royal Society of Chemistry 2014
Fig. 2 (a) Initial activities of ADS-GO, CA-GO and NER-GO, (b) stabilities of ADS-GO, CA-GO, and NER-GO at room temperature under shaking (200 rpm). The relative activity represents the percentage ratio of residual activity at each time point to the initial activity of each sample.
plate, and incubated with antibody-conjugated NER-GO for sandwich immunoassay. As a control experiment, antibodyconjugated GO was also prepared and used for conventional ELISA under the same conditions. The absorbance signal was measured by peroxidase-catalyzed oxidation of amplex red that uses hydrogen peroxide generated by GO-catalyzed glucose oxidation. Fig. 3a shows the correlation between the hIgG concentration and the signals from both conventional ELISA and NER-LISA. The signal of NER-LISA with 1 mg mL 1 of hIgG was 2 times higher than that of conventional ELISA under the same conditions. The coefficients of variations (CVs) of background signal with conventional ELISA and NER-ELISA were 4.6% and 12.2%, respectively, indicating that the CV of background signal with NER-LISA is bigger than that with ELISA. The LOD of NER-LISA was estimated to be B0.5 ng mL 1 of hIgG (3.3 pM) while the LOD of conventional ELISA was B10 ng mL 1 of hIgG (67 pM) (Fig. 3a). In other words, the sensitivity of NER-LISA was about 20 times higher than that of conventional immunoassay. This result suggests that antibodyconjugated NER-GO with a higher enzyme/antibody ratio enhances the signal when compared to the conventional ELISA based on oneto-one ratio of enzyme and antibody. The dynamic ranges of conventional ELISA and NER-LISA were 10–1000 ng mL 1 and 0.1–1000 ng mL 1, respectively (Fig. 3a and Fig. S2, ESI†), indicating that the dynamic range of NER-LISA is two-orders wider than that of conventional ELISA. Interestingly, the upper side of the
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Fig. 3 (a) Calibration curves of the sandwich immunoassay for the detection of hIgG at various concentrations from 0.1 to 10 000 ng mL 1 by using antibody-conjugated GO (conventional immunoassay) and antibodyconjugated spherical NER-GO (NER-LISA). (b) The specificity of NER-LISA in the detection of hIgG (1 mg mL 1). The samples of rIgG (1 mg mL 1) and mIgG (1 mg mL 1) were used as controls. The error bars represent the standard deviation from the triplicate experiments.
dynamic range was the same, but the lower side of the dynamic range was extended from 10 ng mL 1 of ELISA to 0.1 ng mL 1 of NER-LISA. This suggests that the detection of hIgG in a trace amount became more feasible due to the higher ratio of enzyme to antibody in the approach of NER-LISA. NER-LISA in this study is based on the use of spherical mesoporous silica (S-MPS), and the results can be compared to those of a former study using amorphous (irregular shape) mesoporous silica (Table S1, ESI†).3d First of all, due to the rapid increase of the reaction signal with NER-LISA in S-MPS, two different reaction times were used for the signal measurements: 5 min for unsaturated signals and 30 min for the comparison with the result of amorphous silica in the former study.3d NER-LISA using S-MPS improved the sensitivity (lower LOD) regardless of incubation times when compared to the former study using amorphous mesoporous silica. This signal enhancement can be explained by the use of spherical-shaped mesoporous silica that facilitates the binding of target analytes to well-exposed antibodies on S-MPS. Fig. 3b shows the specificity of NER-LISA to the target antigen (hIgG) together with the results of negative controls, rIgG and
3548 | Chem. Commun., 2014, 50, 3546--3548
Communication
mIgG. The signals with the control samples (rIgG and mIgG) were much lower than the signal with the target antigen (hIgG), suggesting a good specificity of NER-LISA. It has been demonstrated that the approach of NER-LISA using spherical mesoporous silica is highly sensitive and specific with a broad detection range in detecting the model analyte of hIgG. The use of spherical mesoporous silica allows for high enzyme loading in their mesopores and facile conjugation of antibodies, which has renovated the approach of NER-LISA by improving the LOD (sensitivity) based on the efficient utilization of conjugated antibodies for the specific binding of target analytes. Especially, NER-LISA using S-MPS could detect B0.5 ng mL 1 of hIgG (3.3 pM) as a limit of detection, which is about 20-fold more sensitive than NER-LISA using amorphous mesoporous silica. It is anticipated that the success of NER-LISA in improving the sensitivity of hIgG detection can be easily extended to the detection of any other analytes in medical diagnostics and point-of-care testing. When compared to the conventional ELISA, the high enzyme stability of NER-GO can also guarantee the improved performance stability of NER-LISA, which would be valuable for the successful point-of-care testing as well as immunoassay in field applications. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government Ministry of Science, ICT and Future Planning (No. 2013K000229 and ’2013, University-Institute Cooperation Program) and by the ‘International Collaborative R&D Program’ of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea government Ministry of Trade, Industry & Energy (No. 20118510020020).
Notes and references 1 (a) J. F. Rusling, C. V. Kumar, J. S. Gutkind and V. Patel, Analyst, 2010, 135, 2496–2511; (b) V. Gubala, L. F. Harris, A. J. Ricco, M. X. Tan and D. E. Williams, Anal. Chem., 2012, 84, 487–515. 2 (a) E. Engvall and P. Perlmann, Immunochemistry, 1971, 8, 871–874; (b) B. Nilsson, Curr. Opin. Immunol., 1989, 2, 898–904. 3 (a) J. Wang, G. Liu and M. R. Jan, J. Am. Chem. Soc., 2004, 126, 3010–3011; (b) B. Munge, G. Liu, G. Collins and J. Wang, Anal. Chem., 2005, 77, 4662–4666; (c) D. Tang and J. Ren, Anal. Chem., 2008, 80, 8064–8070; (d) Y. Piao, D. Lee, J. Lee, T. Hyeon, J. Kim and H.-S. Kim, Biosens. Bioelectron., 2009, 25, 906–912; (e) D. Tang, B. Su, J. Tang, J. Ren and G. Chen, Anal. Chem., 2010, 82, 1527–1534; ( f ) B. S. Munge, A. L. Coffey, J. M. Doucette, B. K. Somba, R. Malhotra, V. Patel, J. S. Gutkind and J. F. Rusling, Angew. Chem., Int. Ed., 2011, 50, 7915–7918; ( g) Y. Piao, Z. Jin, D. Lee, H. J. Lee, H. B. Na, T. Hyeon, M. K. Oh, J. Kim and H. S. Kim, Biosens. Bioelectron., 2011, 26, 3192–3199; (h) H. J. Lee, B. C. Kim, M. K. Oh and J. Kim, Chem. Commun., 2012, 48, 5971–5973. 4 J. Kim, J. W. Grate and P. Wang, Trends Biotechnol., 2008, 26, 639–646. 5 J. Wang, Electroanalysis, 2005, 17, 7–14. 6 (a) J. Kim, H. Jia and P. Wang, Biotechnol. Adv., 2006, 24, 296–308; (b) S. D. Minteer, B. Y. Liaw and M. J. Cooney, Curr. Opin. Biotechnol., 2007, 18, 228–234. 7 J. Kim, B. C. Kim, D. Lopez-Ferrer, K. Petritis and R. D. Smith, Proteomics, 2010, 10, 687–699. 8 (a) J. Lee, J. Kim, J. Kim, H. Jia, M. I. Kim, J. H. Kwak, S. Jin, A. Dohnalkova, H. G. Park, H. N. Chang, P. Wang, J. W. Grate and T. Hyeon, Small, 2005, 1, 744–753; (b) J. Kim, J. W. Grate and P. Wang, Chem. Eng. Sci., 2006, 61, 1017–1026.
This journal is © The Royal Society of Chemistry 2014
ChemComm Chemical Communications
www.rsc.org/chemcomm
ISSN 1359-7345
COMMUNICATION Jungbae Kim et al. A highly sensitive immunoassay using antibody-conjugated spherical mesoporous silica with immobilized enzymes
ChemComm
Published on 04 December 2013. Downloaded by Monash University on 25/10/2014 10:34:23.
COMMUNICATION
Cite this: Chem. Commun., 2014, 50, 3546 Received 19th October 2013, Accepted 4th December 2013
View Article Online View Journal | View Issue
A highly sensitive immunoassay using antibodyconjugated spherical mesoporous silica with immobilized enzymes† Ji Young Eum,‡a Sang Youn Hwang,‡a Youngjun Ju,a Jong Min Shim,b Yunxian Piao,c Jinwoo Lee,b Hak-Sung Kimc and Jungbae Kim*a
DOI: 10.1039/c3cc48044e www.rsc.org/chemcomm
A highly sensitive immunoassay was developed by using antibodyconjugated spherical mesoporous silica with immobilized enzymes. The higher ratio of enzyme/antibody than conventional ELISA improved both the sensitivity and dynamic range. Especially, the use of spherical mesoporous silica could achieve a limit of detection (LOD) with a sensitivity that is 20 times more than that of ELISA using amorphous silica.
The detection method with high selectivity, sensitivity and reliability is crucial for early diagnostics in medical fields as well as point of care testing.1 Among various detection methods, enzyme linked immunosorbent assay (ELISA) is one of the most popular tools in clinical and medical fields due to the specific binding of antibodies to target analytes and the amplified signal generation catalyzed by antibody-conjugated enzymes.2 However, the detection of target analytes in a trace amount often requires further signal enhancement of conventional ELISA. As one of the potential solutions for improvement of the sensitivity of ELISA, the enzyme-immobilized nanomaterials were conjugated with antibodies, and used for the ELISA. This approach improved the sensitivity of ELISA by increasing the ratio of enzymes to antibody.3 In other words, when compared to conventional ELISA using one enzyme per antibody, a greater number of enzyme molecules are involved in the signal generation per unit binding between antigen and antibody, formulating a sensitive ELISA protocol. Recently, nanobiocatalytic approaches, using various nanomaterials for immobilizing and stabilizing enzymes, have gathered more and more attention due to their potential applications in various fields,4 such as biosensors,5 biofuel cells,6 proteomic analysis,7 and ELISA.3 Especially, mesoporous silica were used for a
Department of Chemical and Biological Engineering, Korea University, Seoul 136-713, Republic of Korea. E-mail: [email protected] b Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Gyungbuk 790-784, Korea c Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea † Electronic supplementary information (ESI) available: Fig. S1 and S2, Table S1. See DOI: 10.1039/c3cc48044e ‡ These authors contributed equally.
3546 | Chem. Commun., 2014, 50, 3546--3548
the development of a stable enzyme system with high enzyme loading via an approach of nanoscale enzyme reactors (NERs), in which enzymes were first adsorbed onto the mesoporous materials and further crosslinked to prevent the enzyme leaching from mesoporous materials.8 As an extension of NER success, antibodies were further conjugated on the surface of amine-functionalized mesoporous silica, and ELISA was performed for the multiplexed immunoassay under the name of NER-LISA (NER-linked immunosorbent assay). However, the limit of detection (LOD) of human IgG (hIgG) was 10 ng mL 1,3d which needs to be rigorously improved for the successful application of NER-LISA. As a potential explanation for this poor LOD, we suspect that the amorphous shape of mesoporous silica particles might lead to inefficient or no binding of target analytes with antibodies conjugated on the concave surface of amorphous mesoporous silica particles, due to steric hindrance. In the present work, we report a highly sensitive immunoassay method by using mesoporous silica in a spherical form (spherical mesoporous silica, S-MPS) with highly-loaded glucose oxidase (GO). When mesoporous silica in an amorphous form is used, there is a chance for antibodies conjugated on the concave outer surface not to bind to the target analytes effectively. On the other hand, the use of S-MPS (Fig. S1a, ESI†) would enable more efficient binding between conjugated antibodies and the target analytes, than the amorphous mesoporous silica (Fig. S1b, ESI†) in the former study,3d with an outer surface that is convex with no concavity. Aminefunctionalized S-MPS was used for the preparation of NERs with GO (NER-GO) by adsorbing GO and crosslinking adsorbed GO molecules via glutaraldehyde treatment. As the control samples, we also prepared simply-adsorbed GO (ADS-GO) and covalently-attached GO (CA-GO) in S-MPS. NER-GO in S-MPS were further conjugated with anti-human IgG antibody (anti-hIgG antibody), and the performance of NER-LISA in the detection of hIgG was compared to those of conventional ELISA and NER-LISA with amorphous silica.3d Fig. 1 schematically shows the proposed hypothesis for the potential enhancement of ELISA signals by using antibodyconjugated NERs in S-MPS. When compared to the conventional ELISA using one enzyme per antibody (Fig. 1a), a lot more enzymes
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 04 December 2013. Downloaded by Monash University on 25/10/2014 10:34:23.
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ChemComm
Fig. 1 Schematic for the detection of hIgG based on the sandwich immunoassay by using antibody-conjugated enzyme (a), amorphous NER (b), and spherical NER (c). See Fig. S1 (ESI†) for the SEM images of spherical and amorphous mesoporous silica.
in S-MPS would generate an enhanced signal, which would lead to the improvement of the sensitivity of NER-LISA. Another control is the NER-LISA using amorphous silica (Fig. 1b), in which the antibodies conjugated on the concave surface would be useless due to the steric hindrance against their binding to the target analyte (hIgG). To demonstrate this hypothetical effect of NER-LISA using spherical mesoporous silica, NER-GO was prepared via a simple two-step process: GO adsorption onto S-MPS and crosslinking. Then, NER-GO was further conjugated with anti-hIgG antibody, and NER-LISA was performed for the detection of hIgG. The GO activity was measured by the peroxidase-catalyzed oxidation of dyes (o-dianisidine), which uses hydrogen peroxide being generated by the GO-catalyzed glucose oxidation. The initial activity of ADS-GO, CA-GO and NER-GO was 5.0, 9.4 and 12.3 A500 per min per mg amino-S-MPS, respectively (Fig. 2a). NER-GO exhibited 2.5 and 1.5 times higher activity than ADS-GO and CA-GO, respectively. The higher activity of NER-GO than that of ADS-GO can be attributed to the prevention of enzyme leaching due to effective covalent linkages among fully-packed GO molecules and amino-S-MPS. The lower activity of CA-GO than that of NER-GO suggests that only the inner surface rather than all the pore volume of amino-S-MPS is used for the immobilization of CA-GO. Fig. 2b shows the stability of ADS-GO, CA-GO, and NER-GO under shaking (200 rpm) at room temperature. NER-GO maintained 98% of its initial activity after 26 days, while ADS-GO and CA-GO retained 20% and 49% of their initial activities, respectively, under the same conditions. The high enzyme stability of NER-GO can be attributed to both the covalent linkages on the surface of GO molecules, inhibiting enzyme denaturation, and covalent linkages among crosslinked GO and amino-S-MPS, preventing the leaching of GO molecules in an effective way.4 Highly stable and active NER-GO was further conjugated with anti-hIgG antibody for the detection of hIgG via NER-LISA. It is anticipated that most antibodies would be conjugated on the surface of spherical mesoporous silica because the internal mesopores must be filled with crosslinked GO molecules. To determine the quantitative detection of hIgG via NER-LISA, various concentrations of hIgG (0–10 mg mL 1) were captured on an antibody-conjugated well
This journal is © The Royal Society of Chemistry 2014
Fig. 2 (a) Initial activities of ADS-GO, CA-GO and NER-GO, (b) stabilities of ADS-GO, CA-GO, and NER-GO at room temperature under shaking (200 rpm). The relative activity represents the percentage ratio of residual activity at each time point to the initial activity of each sample.
plate, and incubated with antibody-conjugated NER-GO for sandwich immunoassay. As a control experiment, antibodyconjugated GO was also prepared and used for conventional ELISA under the same conditions. The absorbance signal was measured by peroxidase-catalyzed oxidation of amplex red that uses hydrogen peroxide generated by GO-catalyzed glucose oxidation. Fig. 3a shows the correlation between the hIgG concentration and the signals from both conventional ELISA and NER-LISA. The signal of NER-LISA with 1 mg mL 1 of hIgG was 2 times higher than that of conventional ELISA under the same conditions. The coefficients of variations (CVs) of background signal with conventional ELISA and NER-ELISA were 4.6% and 12.2%, respectively, indicating that the CV of background signal with NER-LISA is bigger than that with ELISA. The LOD of NER-LISA was estimated to be B0.5 ng mL 1 of hIgG (3.3 pM) while the LOD of conventional ELISA was B10 ng mL 1 of hIgG (67 pM) (Fig. 3a). In other words, the sensitivity of NER-LISA was about 20 times higher than that of conventional immunoassay. This result suggests that antibodyconjugated NER-GO with a higher enzyme/antibody ratio enhances the signal when compared to the conventional ELISA based on oneto-one ratio of enzyme and antibody. The dynamic ranges of conventional ELISA and NER-LISA were 10–1000 ng mL 1 and 0.1–1000 ng mL 1, respectively (Fig. 3a and Fig. S2, ESI†), indicating that the dynamic range of NER-LISA is two-orders wider than that of conventional ELISA. Interestingly, the upper side of the
Chem. Commun., 2014, 50, 3546--3548 | 3547
View Article Online
Published on 04 December 2013. Downloaded by Monash University on 25/10/2014 10:34:23.
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Fig. 3 (a) Calibration curves of the sandwich immunoassay for the detection of hIgG at various concentrations from 0.1 to 10 000 ng mL 1 by using antibody-conjugated GO (conventional immunoassay) and antibodyconjugated spherical NER-GO (NER-LISA). (b) The specificity of NER-LISA in the detection of hIgG (1 mg mL 1). The samples of rIgG (1 mg mL 1) and mIgG (1 mg mL 1) were used as controls. The error bars represent the standard deviation from the triplicate experiments.
dynamic range was the same, but the lower side of the dynamic range was extended from 10 ng mL 1 of ELISA to 0.1 ng mL 1 of NER-LISA. This suggests that the detection of hIgG in a trace amount became more feasible due to the higher ratio of enzyme to antibody in the approach of NER-LISA. NER-LISA in this study is based on the use of spherical mesoporous silica (S-MPS), and the results can be compared to those of a former study using amorphous (irregular shape) mesoporous silica (Table S1, ESI†).3d First of all, due to the rapid increase of the reaction signal with NER-LISA in S-MPS, two different reaction times were used for the signal measurements: 5 min for unsaturated signals and 30 min for the comparison with the result of amorphous silica in the former study.3d NER-LISA using S-MPS improved the sensitivity (lower LOD) regardless of incubation times when compared to the former study using amorphous mesoporous silica. This signal enhancement can be explained by the use of spherical-shaped mesoporous silica that facilitates the binding of target analytes to well-exposed antibodies on S-MPS. Fig. 3b shows the specificity of NER-LISA to the target antigen (hIgG) together with the results of negative controls, rIgG and
3548 | Chem. Commun., 2014, 50, 3546--3548
Communication
mIgG. The signals with the control samples (rIgG and mIgG) were much lower than the signal with the target antigen (hIgG), suggesting a good specificity of NER-LISA. It has been demonstrated that the approach of NER-LISA using spherical mesoporous silica is highly sensitive and specific with a broad detection range in detecting the model analyte of hIgG. The use of spherical mesoporous silica allows for high enzyme loading in their mesopores and facile conjugation of antibodies, which has renovated the approach of NER-LISA by improving the LOD (sensitivity) based on the efficient utilization of conjugated antibodies for the specific binding of target analytes. Especially, NER-LISA using S-MPS could detect B0.5 ng mL 1 of hIgG (3.3 pM) as a limit of detection, which is about 20-fold more sensitive than NER-LISA using amorphous mesoporous silica. It is anticipated that the success of NER-LISA in improving the sensitivity of hIgG detection can be easily extended to the detection of any other analytes in medical diagnostics and point-of-care testing. When compared to the conventional ELISA, the high enzyme stability of NER-GO can also guarantee the improved performance stability of NER-LISA, which would be valuable for the successful point-of-care testing as well as immunoassay in field applications. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government Ministry of Science, ICT and Future Planning (No. 2013K000229 and ’2013, University-Institute Cooperation Program) and by the ‘International Collaborative R&D Program’ of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea government Ministry of Trade, Industry & Energy (No. 20118510020020).
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