Biosensors and Bioelectronics 66 (2015) 84–88
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Silver nanowires-based signal amplification for CdSe quantum dots electrochemiluminescence immunoassay Tingyu Huang a, Qingmin Meng b, Guifen Jie b,n a b
Linyi University, Linyi 276000, PR China Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, Qingdao University of Science and Technology, Qingdao 266042, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 19 August 2014 Received in revised form 17 October 2014 Accepted 6 November 2014 Available online 10 November 2014
A novel silver–cysteine hybrid nanowires (SCNWs) with many reactive carboxyl and amine groups were prepared, which enable them to be used as idea signal amplifying labels in bioassays. A large number of CdSe quantum dots (QDs) were loaded on the SCNWs to develop amplified SCNWs–QDs electrochemiluminescence (ECL) signal probe. The PAMAM dendrimer-SCNWs nanohybrids covered on the electrode constructed an effective antibody immobilization matrix and made the immobilized biomolecules hold high stability and bioactivity. Based on the specific sandwich immunoreaction strategy, the detection antibody (Ab2)-SCNWs–QDs ECL signal probe was applied to the sensitive signal-on ECL immunoassay of human IgG. The SCNWs–QDs ECL not only opens promising new ECL emitting species, but also promotes the development of novel ECL signal-transition platforms for biosensing devices. & Elsevier B.V. All rights reserved.
Keywords: Silver–cysteine Hybrid nanowires SCNWs–QDs Electrochemiluminescence Immunoassay
1. Introduction Electrochemiluminescence (ECL) has grown significantly as a highly sensitive and selective analytical and diagnostic method in recent years (Deng and Ju, 2013). This method covers advantages of controllability from electrochemistry and low background from chemiluminescence (Miao, 2008; Richter, 2004). Much effort has been devoted to explore the nanomaterial-based new kind of ECL emitter, and finding new luminophores with high ECL efficiency for bioanalysis is the constant driving force of this area. Recently, a series of semiconductor nanocrystals (or QDs) (Sun et al., 2009; Hu et al., 2010) have been reported to show ECL emission properties, which are promising for construction of ECL biosensors (Wang et al., 2011). However, ECL signal of pure QDs is usually lower than that of luminal or Ru(bpy)23+, which limits the applications of QDs ECL in bioassays. The development of novel QDs nanostructure to enhance ECL is of great significance in ECL bioassays. Gold nanoparticles and carbon nanotubes were reported to enhance QDs ECL (Jie et al., 2009; Jie et al., 2008a), the dendrimer/CdSe–ZnS–QDs nanocluster was used as an ECL probe for assays of cancer cells (Jie et al., 2011). In addition, inorganic–organic hybrid materials as a class of novel materials (Yao et al., 2010) have nearly penetrated every scientific field, such as catalysis, optics, electronics, molecular recognition, sensors, biology, etc. (Yaghi et al., 2003; Bruce n
Corresponding author. E-mail address: [email protected] (G. Jie).
http://dx.doi.org/10.1016/j.bios.2014.11.011 0956-5663/& Elsevier B.V. All rights reserved.
et al., 2008; Hatchett and Josowicz, 2008; Park, et al., 2004; Wang et al., 2005). Silver nanowires have received much attention and inspired intensive research efforts for their intriguing electrical and optical properties (Fang et al., 2011; Netzer et al., 2011). It is reported that the Ag–cysteine hybrid nanowires were used as amplified signal labels in the chemiluminescent detection of cancer biomarkers (Chen, et al., 2013). So far, the SCNWs–QDs nanostructure has not been reported to develop ECL probe, let alone be applied to signal-on ECL biosensing. In this paper, we synthesized novel silver nanowires and fabricated an amplified SCNWs–QDs ECL signal probe. The unique PAMAM-SCNWs were used as promising platform for assembling large numbers of antibody molecules, we report a SCNWs-amplified QDs ECL system for sensitive signal-on ECL immunoassay of human IgG for the first time.
2. Experimental section 2.1. Synthesis of SCNWs SCNWs were prepared according to the literature (Chen, et al., 2013). AgNO3 (0.1 mol L 1) and L-cysteine (0.2 mol L 1) were dissolved in 10 mL of distilled water. Under vigorous stirring, the pH of the solution was rapidly adjusted to 9.0. Then the solution turned transparent followed by sudden appearance of white precipitates. After polymerization at RT for 24 h, the white precipitates (SCNWs) were separated by centrifugation for 10 min.
T. Huang et al. / Biosensors and Bioelectronics 66 (2015) 84–88
Then the SCNWs were washed with doubly distilled water. The appearance of the precipitate is done by centrifugation every time until pH became neutral. Finally, the SCNWs were dried at 60 °C under vacuum. 0.5 mg of SCNWs powder was dissolved in 5 mL of doubly distilled water to obtain 0.1 mg mL 1 stock solution. 2.2. Preparation of the SCNWs–QDs ECL probe Colloidal CdSe QDs were prepared as follows. 0.05 g of selenium powder and 0.037 g of sodium borohydride were added to a small flask, then 4 mL of ultrapure water was added. The solution was refilled with nitrogen for 30 min, and heated to 80 °C. After the selenium powder disappeared completely, the clear NaHSe of 0.1 mol L 1 was obtained. NaHSe solution (0.1 mol L 1) was added to 1.25 mmol L 1 N2saturated CdCl2 solution, and the pH was adjusted to 11, then 200 μL of mercaptoacetic acid (MTA) was added. The molar ratio of Cd2 þ /MTA/NaHSe was fixed at 1:2.4:0.5. After the mixture was vigorously stirred for 10 min, it was refluxed for 3 h. The MTA-QDs were extracted by centrifugation at 6000 rpm, purified twice with ethanol, and finally dissolved in 10 mL water of pH 8. EDC (0.1 mol L 1, 10 μL) and NHS (0.025 mol L 1, 10 μL) were added to 200 μL of the CdSe QDs solution and incubated for 30 min, then 100 μL of 10 5 M DNA was added and reacted at 37 °C for 12 h with gentle shaking, and then centrifugated, resuspended in 200 μL of buffer.
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1 mL of diluted SCNWs solution was activated with EDC (20 μL, 0.1 mol L 1) and NHS (20 μL, 0.025 mol L 1) for 30 min. After centrifugation at 5000 rpm, the activated SCNWs were dispersed in 1.0 mL of PBS, then 10 μL of 5 mg mL 1 Ab2 was added and reacted at 25 °C for 12 h under mild shaking. After centrifugation at 15 °C for 5 min to remove excess reagents. The obtained SCNWs–Ab2 were washed with PBS containing 0.05% Tween-20 three times and resuspended in 500 μL of buffer. After 500 μL of SCNWs–Ab2 solution was activated with EDC and NHS for 30 min, 100 μL of SH-DNA-QDs was added and reacted at 4 °C for 15 h with gentle shaking. The resulting SCNWs– Ab2–QDs probe was then centrifugated, washed twice, and resuspended in 500 μL of buffer. 2.3. Preparation of the ECL immunosensor 1 mL of diluted SCNWs solution were activated with EDC (0.1 mol L 1, 10 μL) and NHS (0.025 mol/L, 10 μL) for 30 min, then 1 mL of 0.1%PAMAM solution were added and reacted overnight. Excess reagents were removed by subsequent centrifugation and redispersion in water. 7 μL of PAMAM-SCNWs was dropped on the cleaned electrodes and dried, then the electrodes were immersed in the solution containing 0.1 mol L 1 EDC and 0.025 mol L 1 NHS for 30 min, followed by dropping 0.28 mg mL 1 Ab1 solution on the electrodes, and incubated at 4 °C in a moisture atmosphere for at least 12 h. After incubating in 2 wt% BSA at 37 °C for 1 h, the Ab1-
Fig. 1. Transmission electron microscopy (TEM) image (A), and photoluminescence (PL) spectra (B) of the CdSe QDs; TEM images of the SCNWs (C), and SCNWs–QDs signal probe (D, E).
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modified electrodes were incubated in human IgG (Ag) of different concentrations at 37 °C for 50 min, then in SCNWs–Ab2–QDs probe for 50 min, and washed with pH 7.4 PBS for ECL measurements. The modified electrodes above were in contact with 0.1 mol L 1 PBS (pH 7.4) containing 0.1 mol L 1 K2S2O8 and 0.1 mol L 1 KCl and scanned from 0 to –1.5 V. ECL signals related to the IgG concentrations were measured.
3. Results and discussion 3.1. Characterization of the QDs, SCNWs and Ab2–SCNWs–QDs signal probe Fig. 1A showed the transmission electron microscopy (TEM) image of the CdSe QDs, the average diameter was about 10 nm. Fig. 1B presented the photoluminescence (PL) spectra of the CdSe QDs, the PL emission peak was at 610 nm, and the intensity was high, indicating the CdSe QDs possess good luminescent property.
The morphology of the SCNWs was characterized by TEM image (Fig. 1C). The average diameter of the nanowires is ∼150 nm, and the length is more than 10 μm. There are six edges on the surfaces of the nanowires parallel to the major axis, inferring the nanowires are approximatively hexagonal prisms. Large number of nanoparticles were observed on the SCNWs, suggesting that nanosilver (Ag þ ) exist in a steady state, the SCNWs can be used as idea amplifying framework to assemble QDs, which showed promising advantages for ECL immunosensing. Therefore CdSe QDs were loaded on the SCNWs to develop amplified Ab2–SCNWs–QDs ECL signal probe, the morphology was further characterized by TEM image (Fig. 1D, E). The probe was not regular nanowires (Fig. 1E), which was obviously different from that of the SCNWs. From the amplified TEM image (Fig. 1D), many QDs were clearly observed on the SCNWs, indicating that the QDs were efficiently attached to the SCNWs. In addition, the SCNWs with many reactive carboxyl and amine groups were characterized by using FT-IR spectra and XRD pattern. FT-IR spectra (Fig. S1 and Table S1) show that the SCNWs have
Scheme 1. The fabrication principle of the SCNWs–QDs signal probe (A), and ECL immunosensor (B).
T. Huang et al. / Biosensors and Bioelectronics 66 (2015) 84–88
characteristic IR peaks of COO , CH, CH2 and C S (Wolpert and Hellwig, 2006; Li, et al., 1992). By comparison, the characteristic absorption peak of S H in L-cysteine at 2553 cm 1 is absent in SCNWs, implying that S Ag was formed in the SCNWs. There is a broad and strong peak of CON R at 3418 cm 1, suggesting the formation of CONR in the SCNWs. The characteristic IR peaks of peptide (1626 cm 1 for C ¼O, 1502 cm 1 and 1582 cm 1 for N H and C N) were found, and a weak stretching vibration of NH3 þ group was around 2087 cm 1, confirming that many NH2 groups have been used to form peptide linkages in the SCNWs. Fig. S2 showed the XRD pattern of the SCNWs, it was found that the reflection peaks were different from those of the orthorhombic L-cysteine (Inset A) (Kerr and Ashmore, 1973), suggesting that the as prepared SCNWs have different crystal structures compared to L-cysteine, and the silver–L–cysteine hybrid nanomaterials were successfully formed. 3.2. CdSe QDs ECL immunoassay by SCNWs-based signal amplification The principle for QDs ECL immunoassay by SCNWs-based signal amplification is depicted in Scheme 1. The SCNWs-amplified QDs signal probe was firstly fabricated as follows. The antibody (Ab2) were covalently conjugated to the SCNWs, while the aminogroup functionalized DNA was linked to the CdSe QDs, then large number of DNA-QDs were assembled on the SCNWs–Ab2 by Ag–S bond. Based on the sandwich-type immunoassay format, after the PAMAM-SCNWs nanocomposites were assembled on the electrode, the capture antibody (Ab1) was covalently linked to the nanocomposites, then IgG was bound to Ab1 after blocking with
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BSA, followed by immunoreaction with Ab2–SCNWs–QDs signal probe. Thus the signal-on ECL immunosensor was developed. Electrochemical impedance spectroscopy (EIS) was used to monitor the feature of the electrode surface. Fig. 2 A shows the EIS plots for the electrode at different stages. When PAMAM-SCNWs nanocomposites were firstly immobilized on the electrode, the electron transfer resistance obviously increased (curve b) compared with that at the bare Au electrode (curve a). Subsequently, with the step-by-step immobilization of Ab1, BSA and Ag on the electrode, the electron transfer resistance greatly increased (curve c) because of the insulating protein layer. Finally, the Ab2–SCNWs– QDs signal probe was linked to the electrode, the EIS became much larger (curve d). Therefore, EIS results confirm that the ECL immunosensor was successfully fabricated. The feasibility of the ECL immunosensor was investigated. As shown in Fig. 2B, in the absence of target IgG, the ECL signal was very low (curve a), indicating that little unspecific binding occurs. In the presence of target Ag, after the immunoreaction of Ab2– SCNWs–QDs signal probe with IgG, the electrode displayed obvious higher ECL signal (curve b), suggesting the fabrication Table 1 Comparison of human serum IgG levels by the ECL Immunosensor and ELISA. Serum samples ECL immunosensor [pg/mL] ELISA [pg/mL] relative deviation [%] a
a
1
2
3
5.0 4.8 4.2
21.9 20.6 6.3
50.8 53.6 5.2
Average value from five successive measurements.
Fig. 2. (A) EIS plots for the electrode at different stages; (B) ECL signal responses upon different concentrations of IgG (b–h); (C) Standard calibration curve for IgG detection; (D) Specificity for the determination of human IgG using the ECL immunosensor: (a) IgG; (b) BSA, (c) thrombin, (d) lysozyme.
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success of the ECL immunosensor. Fig. 2B (curve b–h) showed the ECL signal responses upon different concentrations of target Ag. When Ag concentrations increased, the ECL peak signal gradually increased, indicating that the ECL immunosensor was applied to the signal-on ECL detection of Ag concentration. Fig. 2C displays the standard calibration curve for IgG detection. The ECL signal was logarithmically related to the IgG concentrations in the range from 1.0 10 12 to 5.0 10 10 g mL 1 (R ¼0.992) with a detection limit of 1.0 10 12 g mL 1 at 3s, which is comparable to other nanomaterials-based ECL immunosensor (Jie et al., 2009; Jie et al., 2008b; Tang, et al., 2008). According to the linear equation, the IgG concentration was quantitatively measured. A series of five duplicate measurements of 1.0 10 11 g mL 1 were used for estimating the precision, and the relative standard deviation (RSD) was 6.3%, showing good reproducibility of the ECL method. To assess the specificity of the ECL immunosensor for IgG detection, the influences of some other proteins such as BSA, thrombin, and lysozyme were examined by analyzing the IgG solutions containing interfering substances three times per run in 3 h. Fig. 2D shows that none of these proteins caused obvious ECL change even with a concentration as 5.0 10 11 g mL 1, while only 1.0 10 11 g mL 1 of IgG resulted in significant ECL enhancement, indicating those proteins did not interfere with the ECL assay of IgG. The results demonstrated that the proposed ECL immunosensor exhibited good specificity for IgG assay. After the immunosensor was stored in pH 7.4 PBS at 4 °C over 2 weeks days, the analytical performances did not show an obvious decline, demonstrating that the immunosensor had good stability. The reproducibility of the immunosensor was estimated by determining 1.0 10 11 g mL 1 IgG with four immunosensors made at the same electrode. Four measurements from the batch resulted in a relative standard deviation of 7.1%, indicating good reproducibility of the developed immunosensor. 3.2.1. Application of the immunosensor in human IgG levels The feasibility of the immunosensor for the clinical applications was investigated by analyzing IgG content in human serum. The assay results using the immunosensor were compared with reference values by the ELISA method (Table 1). It obviously indicates that there is no significant difference between the results and ELISA method. Thus, the developed immunosensor could be satisfactorily applied to the clinical determination of IgG levels in human serum.
4. Conclusions In summary, we have prepared a novel silver–cysteine hybrid nanowires with good reactivity and high load capacity. A large number of QDs were assembled on the nanowires to develop the
amplified Ab2–SCNWs–QDs ECL signal probe. The PAMAM dendrimer-SCNWs nanohybrids constructed an amplified immobilization matrix for antibody molecules. Based on the specific sandwich immunoreaction, sensitive detection of IgG was achieved by using the SCNWs–QDs signal probe. This is the first report on the SCNWs-amplified QDs ECL probe, which opened a new kind of QDs nanocomposites in ECL bioassay, and provided a promising alternative tool for detection of protein in clinical laboratory.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21175078), and the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT).
Appendix A. Suplementary Information Supplementary data associated with this article can be found in the online version at.
References Bruce, P.G., Scrosati, B., Tarascon, J.M., 2008. Angew. Chem. Int. Ed. 47, 2930–2946. Chen, W.J., Zheng, L.N., Wang, M.L., Chi, Y.W., Chen, G.N., 2013. Anal. Chem. 85, 9655–9663. Deng, S.Y., Ju, H.X., 2013. Analyst 138, 43–61. Fang, J., Lebedkin, S., Yang, S., Hahn, H., 2011. Chem. Commun. 47, 5157–5159. Hatchett, D.W., Josowicz, M., 2008. Chem. Rev. 108, 746–769. Hu, X., Han, H., Hua, L., Sheng, Z., 2010. Biosens. Bioelectron. 25, 1843–1846. Jie, G.F., Li, L.L., Chen, C., Xuan, J., Zhu, J.J., 2009. Biosens. Bioelectron. 24, 3352–3358. Jie, G.F., Zhang, J.J., Wang, D.C., Cheng, C., Chen, H.Y., Zhu, J.J., 2008a. Anal. Chem. 80, 4033–4039. Jie, G.F., Huang, H.P., Sun, X.L., Zhu, J.J., 2008b. Biosens. Bioelectron. 23, 1896–1899. Jie, G.F., Wang, L., Yuan, J.X., Zhang, S.S., 2011. Anal. Chem. 83, 3873–3880. Kerr, K.A., Ashmore, J.P., 1973. Acta Crystallogr. B29, 2124–2127. Li, H.M., Wurrey, C.J., Thomas, G.J., 1992. J. Am. Chem. Soc. 114, 7463–7469. Miao, W., 2008. Chem. Rev. 108, 2506–2553. Netzer, N.L., Qiu, C., Zhang, Y., Lin, C., Zhang, L., Fong, H., Jiang, C., 2011. Chem. Commun. 47, 9606–9608. Park, S., Chung, S.W., Mirkin, C.A., 2004. J. Am. Chem. Soc. 126, 11772–11773. Richter, M.M., 2004. Chem. Rev. 104, 3003–3036. Sun, L., Bao, L., Hyun, B.-R., Bartnik, A.C., Zhong, Y.-W., Reed, J.C., Pang, D.-W., Abruna, H.D., Malliaras, G.G., Wise, F.W., 2009. Nano Lett. 9, 789–793. Tang, D.P., Yuan, R., Chai, Y.Q., 2008. Anal. Chem. 80, 1582–1588. Wang, J., Shan, Y., Zhao, W.W., Xu, J.J., Chen, H.Y., 2011. Anal. Chem. 83, 4004–4011. Wang, Y., Tang, Z.Y., Tan, S.S., Kotov, N.A., 2005. Nano Lett. 5, 243–248. Wolpert, M., Hellwig, P., 2006. Spectrochim. Acta A 64, 987–1001. Yao, H.B., Gao, M.R., Yu, S.H., 2010. Nanoscale 2, 323–334. Yaghi, O.M., O'Keeffe, M., Ockwig, N.W., Chae, H.K., Eddaoudi, M., Kim, J., 2003. Nature 423, 705–714.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Silver nanowires-based signal amplification for CdSe quantum dots electrochemiluminescence immunoassay Tingyu Huang a, Qingmin Meng b, Guifen Jie b,n a b
Linyi University, Linyi 276000, PR China Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, Qingdao University of Science and Technology, Qingdao 266042, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 19 August 2014 Received in revised form 17 October 2014 Accepted 6 November 2014 Available online 10 November 2014
A novel silver–cysteine hybrid nanowires (SCNWs) with many reactive carboxyl and amine groups were prepared, which enable them to be used as idea signal amplifying labels in bioassays. A large number of CdSe quantum dots (QDs) were loaded on the SCNWs to develop amplified SCNWs–QDs electrochemiluminescence (ECL) signal probe. The PAMAM dendrimer-SCNWs nanohybrids covered on the electrode constructed an effective antibody immobilization matrix and made the immobilized biomolecules hold high stability and bioactivity. Based on the specific sandwich immunoreaction strategy, the detection antibody (Ab2)-SCNWs–QDs ECL signal probe was applied to the sensitive signal-on ECL immunoassay of human IgG. The SCNWs–QDs ECL not only opens promising new ECL emitting species, but also promotes the development of novel ECL signal-transition platforms for biosensing devices. & Elsevier B.V. All rights reserved.
Keywords: Silver–cysteine Hybrid nanowires SCNWs–QDs Electrochemiluminescence Immunoassay
1. Introduction Electrochemiluminescence (ECL) has grown significantly as a highly sensitive and selective analytical and diagnostic method in recent years (Deng and Ju, 2013). This method covers advantages of controllability from electrochemistry and low background from chemiluminescence (Miao, 2008; Richter, 2004). Much effort has been devoted to explore the nanomaterial-based new kind of ECL emitter, and finding new luminophores with high ECL efficiency for bioanalysis is the constant driving force of this area. Recently, a series of semiconductor nanocrystals (or QDs) (Sun et al., 2009; Hu et al., 2010) have been reported to show ECL emission properties, which are promising for construction of ECL biosensors (Wang et al., 2011). However, ECL signal of pure QDs is usually lower than that of luminal or Ru(bpy)23+, which limits the applications of QDs ECL in bioassays. The development of novel QDs nanostructure to enhance ECL is of great significance in ECL bioassays. Gold nanoparticles and carbon nanotubes were reported to enhance QDs ECL (Jie et al., 2009; Jie et al., 2008a), the dendrimer/CdSe–ZnS–QDs nanocluster was used as an ECL probe for assays of cancer cells (Jie et al., 2011). In addition, inorganic–organic hybrid materials as a class of novel materials (Yao et al., 2010) have nearly penetrated every scientific field, such as catalysis, optics, electronics, molecular recognition, sensors, biology, etc. (Yaghi et al., 2003; Bruce n
Corresponding author. E-mail address: [email protected] (G. Jie).
http://dx.doi.org/10.1016/j.bios.2014.11.011 0956-5663/& Elsevier B.V. All rights reserved.
et al., 2008; Hatchett and Josowicz, 2008; Park, et al., 2004; Wang et al., 2005). Silver nanowires have received much attention and inspired intensive research efforts for their intriguing electrical and optical properties (Fang et al., 2011; Netzer et al., 2011). It is reported that the Ag–cysteine hybrid nanowires were used as amplified signal labels in the chemiluminescent detection of cancer biomarkers (Chen, et al., 2013). So far, the SCNWs–QDs nanostructure has not been reported to develop ECL probe, let alone be applied to signal-on ECL biosensing. In this paper, we synthesized novel silver nanowires and fabricated an amplified SCNWs–QDs ECL signal probe. The unique PAMAM-SCNWs were used as promising platform for assembling large numbers of antibody molecules, we report a SCNWs-amplified QDs ECL system for sensitive signal-on ECL immunoassay of human IgG for the first time.
2. Experimental section 2.1. Synthesis of SCNWs SCNWs were prepared according to the literature (Chen, et al., 2013). AgNO3 (0.1 mol L 1) and L-cysteine (0.2 mol L 1) were dissolved in 10 mL of distilled water. Under vigorous stirring, the pH of the solution was rapidly adjusted to 9.0. Then the solution turned transparent followed by sudden appearance of white precipitates. After polymerization at RT for 24 h, the white precipitates (SCNWs) were separated by centrifugation for 10 min.
T. Huang et al. / Biosensors and Bioelectronics 66 (2015) 84–88
Then the SCNWs were washed with doubly distilled water. The appearance of the precipitate is done by centrifugation every time until pH became neutral. Finally, the SCNWs were dried at 60 °C under vacuum. 0.5 mg of SCNWs powder was dissolved in 5 mL of doubly distilled water to obtain 0.1 mg mL 1 stock solution. 2.2. Preparation of the SCNWs–QDs ECL probe Colloidal CdSe QDs were prepared as follows. 0.05 g of selenium powder and 0.037 g of sodium borohydride were added to a small flask, then 4 mL of ultrapure water was added. The solution was refilled with nitrogen for 30 min, and heated to 80 °C. After the selenium powder disappeared completely, the clear NaHSe of 0.1 mol L 1 was obtained. NaHSe solution (0.1 mol L 1) was added to 1.25 mmol L 1 N2saturated CdCl2 solution, and the pH was adjusted to 11, then 200 μL of mercaptoacetic acid (MTA) was added. The molar ratio of Cd2 þ /MTA/NaHSe was fixed at 1:2.4:0.5. After the mixture was vigorously stirred for 10 min, it was refluxed for 3 h. The MTA-QDs were extracted by centrifugation at 6000 rpm, purified twice with ethanol, and finally dissolved in 10 mL water of pH 8. EDC (0.1 mol L 1, 10 μL) and NHS (0.025 mol L 1, 10 μL) were added to 200 μL of the CdSe QDs solution and incubated for 30 min, then 100 μL of 10 5 M DNA was added and reacted at 37 °C for 12 h with gentle shaking, and then centrifugated, resuspended in 200 μL of buffer.
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1 mL of diluted SCNWs solution was activated with EDC (20 μL, 0.1 mol L 1) and NHS (20 μL, 0.025 mol L 1) for 30 min. After centrifugation at 5000 rpm, the activated SCNWs were dispersed in 1.0 mL of PBS, then 10 μL of 5 mg mL 1 Ab2 was added and reacted at 25 °C for 12 h under mild shaking. After centrifugation at 15 °C for 5 min to remove excess reagents. The obtained SCNWs–Ab2 were washed with PBS containing 0.05% Tween-20 three times and resuspended in 500 μL of buffer. After 500 μL of SCNWs–Ab2 solution was activated with EDC and NHS for 30 min, 100 μL of SH-DNA-QDs was added and reacted at 4 °C for 15 h with gentle shaking. The resulting SCNWs– Ab2–QDs probe was then centrifugated, washed twice, and resuspended in 500 μL of buffer. 2.3. Preparation of the ECL immunosensor 1 mL of diluted SCNWs solution were activated with EDC (0.1 mol L 1, 10 μL) and NHS (0.025 mol/L, 10 μL) for 30 min, then 1 mL of 0.1%PAMAM solution were added and reacted overnight. Excess reagents were removed by subsequent centrifugation and redispersion in water. 7 μL of PAMAM-SCNWs was dropped on the cleaned electrodes and dried, then the electrodes were immersed in the solution containing 0.1 mol L 1 EDC and 0.025 mol L 1 NHS for 30 min, followed by dropping 0.28 mg mL 1 Ab1 solution on the electrodes, and incubated at 4 °C in a moisture atmosphere for at least 12 h. After incubating in 2 wt% BSA at 37 °C for 1 h, the Ab1-
Fig. 1. Transmission electron microscopy (TEM) image (A), and photoluminescence (PL) spectra (B) of the CdSe QDs; TEM images of the SCNWs (C), and SCNWs–QDs signal probe (D, E).
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modified electrodes were incubated in human IgG (Ag) of different concentrations at 37 °C for 50 min, then in SCNWs–Ab2–QDs probe for 50 min, and washed with pH 7.4 PBS for ECL measurements. The modified electrodes above were in contact with 0.1 mol L 1 PBS (pH 7.4) containing 0.1 mol L 1 K2S2O8 and 0.1 mol L 1 KCl and scanned from 0 to –1.5 V. ECL signals related to the IgG concentrations were measured.
3. Results and discussion 3.1. Characterization of the QDs, SCNWs and Ab2–SCNWs–QDs signal probe Fig. 1A showed the transmission electron microscopy (TEM) image of the CdSe QDs, the average diameter was about 10 nm. Fig. 1B presented the photoluminescence (PL) spectra of the CdSe QDs, the PL emission peak was at 610 nm, and the intensity was high, indicating the CdSe QDs possess good luminescent property.
The morphology of the SCNWs was characterized by TEM image (Fig. 1C). The average diameter of the nanowires is ∼150 nm, and the length is more than 10 μm. There are six edges on the surfaces of the nanowires parallel to the major axis, inferring the nanowires are approximatively hexagonal prisms. Large number of nanoparticles were observed on the SCNWs, suggesting that nanosilver (Ag þ ) exist in a steady state, the SCNWs can be used as idea amplifying framework to assemble QDs, which showed promising advantages for ECL immunosensing. Therefore CdSe QDs were loaded on the SCNWs to develop amplified Ab2–SCNWs–QDs ECL signal probe, the morphology was further characterized by TEM image (Fig. 1D, E). The probe was not regular nanowires (Fig. 1E), which was obviously different from that of the SCNWs. From the amplified TEM image (Fig. 1D), many QDs were clearly observed on the SCNWs, indicating that the QDs were efficiently attached to the SCNWs. In addition, the SCNWs with many reactive carboxyl and amine groups were characterized by using FT-IR spectra and XRD pattern. FT-IR spectra (Fig. S1 and Table S1) show that the SCNWs have
Scheme 1. The fabrication principle of the SCNWs–QDs signal probe (A), and ECL immunosensor (B).
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characteristic IR peaks of COO , CH, CH2 and C S (Wolpert and Hellwig, 2006; Li, et al., 1992). By comparison, the characteristic absorption peak of S H in L-cysteine at 2553 cm 1 is absent in SCNWs, implying that S Ag was formed in the SCNWs. There is a broad and strong peak of CON R at 3418 cm 1, suggesting the formation of CONR in the SCNWs. The characteristic IR peaks of peptide (1626 cm 1 for C ¼O, 1502 cm 1 and 1582 cm 1 for N H and C N) were found, and a weak stretching vibration of NH3 þ group was around 2087 cm 1, confirming that many NH2 groups have been used to form peptide linkages in the SCNWs. Fig. S2 showed the XRD pattern of the SCNWs, it was found that the reflection peaks were different from those of the orthorhombic L-cysteine (Inset A) (Kerr and Ashmore, 1973), suggesting that the as prepared SCNWs have different crystal structures compared to L-cysteine, and the silver–L–cysteine hybrid nanomaterials were successfully formed. 3.2. CdSe QDs ECL immunoassay by SCNWs-based signal amplification The principle for QDs ECL immunoassay by SCNWs-based signal amplification is depicted in Scheme 1. The SCNWs-amplified QDs signal probe was firstly fabricated as follows. The antibody (Ab2) were covalently conjugated to the SCNWs, while the aminogroup functionalized DNA was linked to the CdSe QDs, then large number of DNA-QDs were assembled on the SCNWs–Ab2 by Ag–S bond. Based on the sandwich-type immunoassay format, after the PAMAM-SCNWs nanocomposites were assembled on the electrode, the capture antibody (Ab1) was covalently linked to the nanocomposites, then IgG was bound to Ab1 after blocking with
87
BSA, followed by immunoreaction with Ab2–SCNWs–QDs signal probe. Thus the signal-on ECL immunosensor was developed. Electrochemical impedance spectroscopy (EIS) was used to monitor the feature of the electrode surface. Fig. 2 A shows the EIS plots for the electrode at different stages. When PAMAM-SCNWs nanocomposites were firstly immobilized on the electrode, the electron transfer resistance obviously increased (curve b) compared with that at the bare Au electrode (curve a). Subsequently, with the step-by-step immobilization of Ab1, BSA and Ag on the electrode, the electron transfer resistance greatly increased (curve c) because of the insulating protein layer. Finally, the Ab2–SCNWs– QDs signal probe was linked to the electrode, the EIS became much larger (curve d). Therefore, EIS results confirm that the ECL immunosensor was successfully fabricated. The feasibility of the ECL immunosensor was investigated. As shown in Fig. 2B, in the absence of target IgG, the ECL signal was very low (curve a), indicating that little unspecific binding occurs. In the presence of target Ag, after the immunoreaction of Ab2– SCNWs–QDs signal probe with IgG, the electrode displayed obvious higher ECL signal (curve b), suggesting the fabrication Table 1 Comparison of human serum IgG levels by the ECL Immunosensor and ELISA. Serum samples ECL immunosensor [pg/mL] ELISA [pg/mL] relative deviation [%] a
a
1
2
3
5.0 4.8 4.2
21.9 20.6 6.3
50.8 53.6 5.2
Average value from five successive measurements.
Fig. 2. (A) EIS plots for the electrode at different stages; (B) ECL signal responses upon different concentrations of IgG (b–h); (C) Standard calibration curve for IgG detection; (D) Specificity for the determination of human IgG using the ECL immunosensor: (a) IgG; (b) BSA, (c) thrombin, (d) lysozyme.
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success of the ECL immunosensor. Fig. 2B (curve b–h) showed the ECL signal responses upon different concentrations of target Ag. When Ag concentrations increased, the ECL peak signal gradually increased, indicating that the ECL immunosensor was applied to the signal-on ECL detection of Ag concentration. Fig. 2C displays the standard calibration curve for IgG detection. The ECL signal was logarithmically related to the IgG concentrations in the range from 1.0 10 12 to 5.0 10 10 g mL 1 (R ¼0.992) with a detection limit of 1.0 10 12 g mL 1 at 3s, which is comparable to other nanomaterials-based ECL immunosensor (Jie et al., 2009; Jie et al., 2008b; Tang, et al., 2008). According to the linear equation, the IgG concentration was quantitatively measured. A series of five duplicate measurements of 1.0 10 11 g mL 1 were used for estimating the precision, and the relative standard deviation (RSD) was 6.3%, showing good reproducibility of the ECL method. To assess the specificity of the ECL immunosensor for IgG detection, the influences of some other proteins such as BSA, thrombin, and lysozyme were examined by analyzing the IgG solutions containing interfering substances three times per run in 3 h. Fig. 2D shows that none of these proteins caused obvious ECL change even with a concentration as 5.0 10 11 g mL 1, while only 1.0 10 11 g mL 1 of IgG resulted in significant ECL enhancement, indicating those proteins did not interfere with the ECL assay of IgG. The results demonstrated that the proposed ECL immunosensor exhibited good specificity for IgG assay. After the immunosensor was stored in pH 7.4 PBS at 4 °C over 2 weeks days, the analytical performances did not show an obvious decline, demonstrating that the immunosensor had good stability. The reproducibility of the immunosensor was estimated by determining 1.0 10 11 g mL 1 IgG with four immunosensors made at the same electrode. Four measurements from the batch resulted in a relative standard deviation of 7.1%, indicating good reproducibility of the developed immunosensor. 3.2.1. Application of the immunosensor in human IgG levels The feasibility of the immunosensor for the clinical applications was investigated by analyzing IgG content in human serum. The assay results using the immunosensor were compared with reference values by the ELISA method (Table 1). It obviously indicates that there is no significant difference between the results and ELISA method. Thus, the developed immunosensor could be satisfactorily applied to the clinical determination of IgG levels in human serum.
4. Conclusions In summary, we have prepared a novel silver–cysteine hybrid nanowires with good reactivity and high load capacity. A large number of QDs were assembled on the nanowires to develop the
amplified Ab2–SCNWs–QDs ECL signal probe. The PAMAM dendrimer-SCNWs nanohybrids constructed an amplified immobilization matrix for antibody molecules. Based on the specific sandwich immunoreaction, sensitive detection of IgG was achieved by using the SCNWs–QDs signal probe. This is the first report on the SCNWs-amplified QDs ECL probe, which opened a new kind of QDs nanocomposites in ECL bioassay, and provided a promising alternative tool for detection of protein in clinical laboratory.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21175078), and the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT).
Appendix A. Suplementary Information Supplementary data associated with this article can be found in the online version at.
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