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ZnO quantum dot labeled immunosensor for carbohydrate antigen 19-9
Biosensors and Bioelectronics 26 (2011) 2720–2723
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Short communication
ZnO quantum dot labeled immunosensor for carbohydrate antigen 19-9 Baoxiang Gu a , Chunxiang Xu a,∗ , Chi Yang a , Songqin Liu b,∗∗ , Mingliang Wang b a b
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, PR China School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China
a r t i c l e
i n f o
Article history: Received 19 June 2010 Received in revised form 3 September 2010 Accepted 18 September 2010 Available online 25 September 2010 Keywords: ZnO quantum dots CA 19-9 Immunosensor Stripping voltammetry
a b s t r a c t Using ZnO quantum dots as electrochemical and fluorescent labels, a sandwich-type sensitive immunoassay was developed to detect carbohydrate antigen 19-9 (CA 19-9) which is a preferred label for pancreatic cancer. The immobilization process was mainly carried out through the electrostatic adsorption based on the high isoelectric point of ZnO, and the sandwich structure was built through the immunoreaction of CA 19-9 antibodies and antigens. The immunological recognition of CA 19-9 was converted into detection of the amplified signals of the square wave stripping voltammetry (SWV) and intrinsic photoluminescence of the labeled quantum dots. The electrochemical assay demonstrated a dynamic range of 0.1–180 U/ml with detection limit of 0.04 U/ml, while the optical spectral detection revealed 1–180 U/ml with detection limit of 0.25 U/ml. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Carbohydrate antigen 19-9 (CA 19-9) is a preferred label for pancreatic cancer, which is a highly lethiferous sarcomata and difficult to be diagnosed early in current clinical medicine. In present clinic, CA 19-9 immunoassay has become a gold standard for pancreatic cancer diagnosis (Barbara et al., 2000; Gold et al., 2006). So far, various methods have been employed to detect CA 19-9, such as electric field-driven assay (Wu et al., 2008), immobilized horseradish peroxidase assay (Du et al., 2007), and chemiluminescent multiplex assay (Fu et al., 2007). However, the sensitivity and specificity of the biomarker are not high enough (Zhao et al., 2006), while the expensive equipment and complex processes are barriers in practice. ZnO as a functional semiconductor possesses much advantages for biosensor, such as visible light transparency, environmental and electrical stability (Brayner et al., 2006), nontoxicity (Tian et al., 2002), and abundance in nature (Park et al., 2009). Especially, the high isoelectric point (IEP, 9.5) (Gu et al., 2009) of ZnO is beneficial to immobilize the biomolecules with low IEP through electrostatic attraction. ZnO quantum dots (QDs) offer a considerable promise as quantitative labels for biological assays based on their high aspect ratio, substantial optical and electronic signal amplification and unique coding capabilities (Rosi and Mirkin, 2005; Wang, 2005; Penn et al., 2003).
∗ Corresponding author. Tel.: +86 25 83790755. ∗∗ Corresponding author. Tel.: +86 25 52090613. E-mail addresses: [email protected] (C. Xu), [email protected] (S. Liu). 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.09.031
In this communication, ZnO QDs were fabricated by a simple and facile colloid method, and then developed into a low cost immunosensor to detect CA 19-9 based on the exploration of the photoluminescence and stripping voltammetric signals. The immunosensors presented high sensitivity, stability, selectivity, and good reproducibility. The assembly process of the antibodies of CA 19-9, immunoreaction between the antibody (Ab) and antigen (Ag), and sensing characteristics and optimization procedure are reported in detail.
2. Materials and methods CA 19-9 Ag and Ab, latent membrane protein (LMP) and glycine were purchased from Nanjing Sunshine Biotechnology Company, China. Bovine serum albumin (BSA) and 3-aminopropyltriethoxysilane (APTS) were purchased from SigmaAldrich, USA. Zn (CH3 COO)2 ·2H2 O, LiOH·H2 O, HgCl2 and H2 O2 were purchased from Shanghai Chemical Plant, China. Doubly distilled water was used throughout the experiments. Phosphate buffer solution (PBS) was prepared by mixing the stock solution of KH2 PO4 and K2 HPO4 . Briefly, ZnO QDs were prepared through a colloidic procedure (Lubomir and Marc, 1991). Zn(CH3 COO)2 ·2H2 O and absolute ethanol were mixed and stirred for 2 h at 80 ◦ C into a solution with 0.05 M Zn2+ . Then the equal volume of absolute ethanol containing 0.1 M LiOH·H2 O was added and stirred for about 120 min, and then cooled down to room temperature. The ZnO QDs were collected by centrifuge at 12,000 r/min, and washed with deionized water three times, and then dispersed in 500 l pH 7.4 PBS for further use.
B. Gu et al. / Biosensors and Bioelectronics 26 (2011) 2720–2723
Scheme 1. Schematic protocol of sandwich-immunosensor.
Electrochemical experiments, including stripping voltammetry and square wave voltammetry (SWV) were performed with a CHI 660 electrochemical workstation (Shanghai, China). All electrochemical measurement was operated in a three-electrode system which is composed of an in situ plated mercury film on a glassy carbon electrode (GCE) as working electrode, a platinum wire as the auxiliary electrode, and a saturated calomel electrode as the reference electrode. The morphology and size of the ZnO QDs were analyzed with a transmission electron microscope (TEM, JEM-100S, JEOL, Japan). The ultraviolet–visible absorption (UV–vis), photoluminescence (PL) and Fourier transform infrared (FTIR) spectroscopy were detected with photometers of Hitachi UV-3600, Edinburgh FLS 920, and Nicolet 380, respectively. The -potential of ZnO QDs with and without CA 19-9 modification were analyzed by Zetasizer 4 (Malvern Instruments). As described previously, the ZnO QDs and proteins can form stable protein–QD bioconjugates through a self-assembly strategy (Emmanuel et al., 2001; Igor et al., 2005). In our experiment, the bioconjugated complexes were prepared by simply immerging the QDs into the Ab solution. 200 l 10 mM pH 7.4 PBS containing 10 g/ml Ab was added into 500 l 10 mM pH 7.4 PBS containing 20 g ZnO QDs, and shook at 4 ◦ C for 2 h, then the modified quantum dots (Ab-QDs) were centrifugally separated and washed three times with 10 mM pH 7.4 PBS. In this case, the Ab was successfully immobilized on ZnO QDs surfaces based on the electrostatic attraction. The process of immunosensor construction was shown in Scheme 1. A small silicon wafer was first functionized into an aldehydeactivated surface for next biomolecular immobilization. The Si substrate was oxidized at 1100 ◦ C for 3 h in a tube furnace to coat a layer of SiO2 , cleaned by ultra sonication in acetone, ethanol, and water, and then treated in a solution of NH4 OH:H2 O2 :H2 O with ratio of 1:1:7 to form a hydroxyl rich silica surface at 80 ◦ C for 5 min (Braun et al., 2005). The hydrophilic substrates were thoroughly rinsed with deionized water, then incubated in a closed Petri dish containing 5% APTS ethanol solution for 5 h at room temperature to form an alkylaminino silane derivatized surface (Soumya et al., 2006). The silanized sample reacted with 2% gluteraldehyde in
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pH 7.4 PBS shaken for 12 h at 4 ◦ C, and then washed thoroughly in deionized water to remove the excess gluteraldehyde (Penn et al., 2003). In the next step, an Ab monolayer was covalently assembled on the aldehyde-activated surface by soaking the sample area in 30 l Ab (10 g/ml, pH 7.4) PBS in a closed Petri dish. After reaction for 30 min at 37 ◦ C, the sample was rinsed with pH 7.4 PBS and then the substrate surface was blocked by BSA in 50 l blocking buffer solution (1% BSA in pH 7.4 PBS) for 2 h followed by water washing again. Then 30 l 180 U/ml CA 19-9 was infused into the cell and incubated for 30 min at 37 ◦ C. In this situation, the CA 19-9 molecules were linked with their antibodies through immunoreaction. Under the similar procedures, a series samples with various CA 19-9 concentration from 0.1 to 180 U/ml were fabricated. As a parallel step, the CA 19-9 antibodies were immobilized on ZnO QDs to form Ab-QDs as mentioned above. Finally, sufficient Ab-QD solution was dropped on the above substrates and maintained at 37 ◦ C for 30 min. In this case, the adequate ZnO QDs were bridge-linked onto the substrate through the Ab–Ag immunoreaction. These samples were washed twice with 30 l of pH 7.4 PBS and twice with 30 l of deionized water to remove the uncombined ZnO QDs for sensing characterization. Before each electrochemical test, the working GCE was polished with alumina (particle size of about 0.03 mm)/water slurry and washed thoroughly with water, and then a mercury film was electroplated on it in 100 mM KCl solution containing 1.2 mM HNO3 and 0.7 mM HgCl2 at −1.3 V for 300 s. The ZnO QDs linked on the substrate were dissolved by adding HCl (50 l, 0.01 M) solution with sonication for 10 min, and then washed thrice with 100 l 50 mM pH 7.0 PBS. Both of the reaction and washed solutions were collected, mixed into 50 mM pH 7.0 PBS, and then adjusted to 3 ml by 50 mM pH 7.0 PBS to obtain so called pending-solution containing Zn2+ ions. The pending-solution was treated at 0.6 V for 1 min, and accumulated at −1.3 V with stirring for 3 min and without stirring for 2 min. Finally, the SWV of the pending-solution was performed from −1.6 to −0.9 V with step potential of 4 mV, amplitude of 20 mV, and frequency of 15 Hz.
3. Results and discussion As shown the inserted TEM image in Scheme 1, the ZnO QDs present uniform size and well dispersion. The size statistic (see Fig. S1 in supplementary materials) demonstrates that the diameter of QDs are main distributed in 4.5–5.5 nm with an average value of 4.8 nm. The inserted high resolution TEM image clearly exhibits the lattice fringes, in which the d-spacing of 0.26 nm matches that of (0 0 0 1) planes of the wurtzite structural ZnO (Xu et al., 2005). It indicates the high crystalline quality of ZnO QDs (Zeng et al., 2008). The FTIR spectrum of purified and dried Ab-QDs was given in Fig. S2 in the supplementary materials. The peaks at 1038 cm−1 and 3361 cm−1 are assigned, respectively to the absorption of alkyl in Ab and hydroxyl. The characteristic absorption peaks at 1575 cm−1 and 1335 cm−1 are assigned to (N–H) and (C–N), respectively. The FTIR spectrum illuminates that the Ab molecules were immobilized on the surfaces of the ZnO QDs. This result was also confirmed by the -potential measurement. The -potential was changed from +20 mV for ZnO QDs into −19.2 mV for Ab-QDs. This effective bioconjugation process is associated with electrostatic attraction at the “nano-bio” interface due to the high IEP of ZnO (Andre et al., 2009). The sandwich-immunosensing can also be characterized by monitoring the intrinsic emission of ZnO QDs. Fig. 1 shows the spectral variation of the intrinsic bandedge emission of the ZnO QDs in the sandwich-immunosensors with a series of CA 19-9 concentrations under the excitation of 325 nm. The emission peaks at 372 nm
B. Gu et al. / Biosensors and Bioelectronics 26 (2011) 2720–2723
7500
180 U/ml 120 U/ml 60 U/ml 1 U/ml 0 U/ml
6000
Intensity (a. u.)
Log(intensity)
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0.0
4500
0.5 1.0 1.5 2.0 Log(CCA19-9 (U/ml))
3000 1500 350
360
370
380
390
400
Wavelength (nm) Fig. 1. The PL spectra of the immunosensors with a series concentration of CA 19-9 inserted with plots of the spectral intensity vs. CA 19-9 concentration in logarithmic style.
reflect the extension of the energy bandgap caused by the size effect (Lu et al., 2006; Wang et al., 2004). It can be seen that the peaks enhance with increase of CA 19-9 concentration. The inset plots the calibrated relationship between the PL intensity and CA 19-9 concentration in logarithm manners. It exhibits a linear response range from 1 U/ml to 180 U/ml for CA 19-9 detection. The linear regression equation is log (intensity) = 2.62059 + 0.49405 × log (C (U/ml)), with a correlation coefficient of 0.9963. The detection limit is estimated as 0.25 U/ml according to the 3 (standard deviation) based on the slope. This detection limit and linear response range have been improved compared with the previous reported values of 2 U/ml and 5–150 U/ml (Fu et al., 2007). The repeatability of optical detection was estimated from a series of five repetitive measurements of samples containing 1, 100, and 180 U/ml Ag that yielded the relative standard deviations (RSD) of 6.8, 3.1, and 1.4%, respectively. Fig. 2 shows the SWV curves of the series pending-solutions. It can be seen that the peak intensity of SWV rises with increase of CA 19-9 concentrations. The inset shows a broad linear response range from 0.1 U/ml to 180 U/ml in logarithmic styles. The linear regression equation is log (I (A)) = 0.27659 + 0.49979 × log (C (U/ml)) with a correlation coefficient of 0.99841. The detection limit is estimated as 0.04 U/ml according to the 3 (standard deviation) based on the slope. The sensitivity is estimated as 0.47 A/U/ml. The detection limit is lower, and the linear range is broader compared with previous reported values of 0.1 U/ml and 0.16–15 U/ml (Wu et al., 2008) and of 1.37 U/ml and 2–30 U/ml (Du et al., 2007), respectively. The biosening performance in both optical and electrochemical assays is mainly due to the uniform
0 0.1 0.5
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-8
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size and well dispersion of the QDs, stable immobilization of the biomolecules, and good biocompatibility of ZnO. The repeatability of the electrochemical detection was estimated from a series of five repetitive measurements of samples containing 0.1, 100, and 180 U/ml CA 19-9 that yielded the RSD of 7.8, 3.7, and 2.4%, respectively. Specificity is an important criterion for any analytical tool. The potential interference of coexisting species toward CA 19-9 detection was studied with the immunosensor. Three type samples were fabricated in the similar procedures as mentioned above except some bio-assembly in Scheme 1 was changed correspondingly: no Ab was connected on Si substrate for type 1 sample; no CA 19-9 was used for type 2; and 10 ng/ml LMP was used instead of CA 19-9 for type 3. As shown in the SWV signals of these type samples in Fig. S3 in the supplementary materials, no significant currents peak is observed in type 1–3 samples. The response of the immunosensor with 0.1 U/ml CA 19-9 is about 8 times of type 1 and 2 samples. The response of type 3 sample is less than that of immunosensor to 0.1 U/ml CA 19-9. The value of 0.1 U/ml CA 19-9 is far less than the cutoff concentration in clinical diagnosis value of 35 U/ml (Fu et al., 2007), and 10 ng/ml is a high concentration in LMP immunoassay. This illustrates that ZnO QD-based immunosensor has a good specificity for CA 19-9 detection. To test the regeneration, the sandwich-immunosensor with 180 U/ml CA 19-9 was rinsed with stripping buffer of 0.1 M pH 2.2 glycine-hydrochloride to remove the CA 19-9 and dissolved ZnO QDs to Zn2+ ions. The rinsed-buffer was mixed in 50 mM pH 7.0 PBS to 3 ml. The functionalized wafer reacted again with 180 U/ml CA 19-9 and Ab-QDs. The same immobilization and dissolution procedures as mentioned above were repeated eight times, and every pending-solution were employed for SWV detection. The SWV current signal restored 97.5% of the initial value after eight time assays, and no significant fluctuation was observed on their calibrated relationship plot (see Fig. S4 in supplementary materials). It revealed that the sandwich-immunosensor has an excellent regeneration. The stability of the sandwich-immunosensor was examined after storing the samples in pH 7.4 PBS at 4 ◦ C for 2 weeks. No obvious change was observed. This result indicates the good stability of the sandwich-immunsensors. 4. Conclusions Based on the bioconjugation of ZnO QDs and CA 19-9 Ab–Ag immunoreaction, the sandwich-immunosensors were assembled on the functionized Si substrate for sensitive CA19-9 detection through both electrochemical and optical techniques. The optical immunoassay based on PL of ZnO QDs demonstrated the detection limit of 0.25 U/ml and the linear response range of 1–180 U/ml. Electrochemical measurement offered higher sensitivity of 0.47 A/U/ml, broader linear range of 0.1–180 U/ml, and lower detection limit of 0.04 U/ml. The sensors also presented good stability, reliability and reproducibility. The uniform small size of the ZnO QDs, stable immobilization based on high IEP of ZnO could be response to the good performance. Based on the similar immobilization and examination techniques, the ZnO QDs are also expected to build up other amperometric immunosensors to detect other important antigens based on the efficient assembly of antibodies. Acknowledgments
180
C CA19-9 (U/ml)
Fig. 2. SWV curves of the pending-solution with a series concentration of CA 19-9 inserted with the plot of peak current vs. CA 19-9 concentration in logarithmic style.
This work was supported by NSFC (10674023 and 60725413), and NSFJ (BK2008285). The authors gratefully acknowledge professor J.J. Zhu in Nanjing University (Nanjing, China) for measurement of -potential.
B. Gu et al. / Biosensors and Bioelectronics 26 (2011) 2720–2723
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2010.09.031. References Andre, E.N., Lutz, M., Darrell, V., Tian, X., Eric, M.V.H., Ponisseril, S., Fred, K., Vince, C., Mike, T., 2009. Nat. Mater. 8, 543–557. Barbara, S., Antonina, H.S., Witold, K., Pawel, S., Arievan, D., Roland, E., 2000. Cancer 89, 83–88. Braun, G., Inagaki, K., Estabrook, R.A., Wood, D.K., Levy, E., Cleland, A.N., Strouse, G.F., Reich, N.O., 2005. Langmuir 21, 10699–10701. Brayner, R., Ferrari, I.R., Brivois, N., Djediat, S., Benedetti, M.F., Fievet, F., 2006. Nano Lett. 6, 866–869. Du, D., Xu, X.X., Wang, S.F., Zhang, A.D., 2007. Talanta 71, 1257–1262. Emmanuel, T., Anthony, E.G.C., Brian, O.R., James, R.D., 2001. J. Electroanal. Chem. 517, 20–27. Fu, Z.F., Yang, Z.J., Tang, J.H., Liu, H., Yan, F., Ju, H.X., 2007. Anal. Chem. 79, 7376–7382. Gold, D.V., Modrak, D.E., Ying, Z.L., Cardillo, T.M., Sharkey, R.M., Goldenberg, D.M., 2006. J. Clin. Oncol. 24, 252–258.
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Gu, B.X., Xu, C.X., Zhu, G.P., Liu, S.Q., Chen, L.Y., Li, X.S., 2009. J. Phys. Chem. B 113, 377–381. Igor, L.M., Tetsuo, H.U., Ellen, R.G., 2005. Nat. Mater. 4, 435–438. Lu, J.G., Ye, Z.Z., Zhang, Y.Z., Liang, Q.L., Fujita, S.Z., Wang, Z.L., 2006. Appl. Phys. Lett. 89, 023122. Lubomir, S., Marc, A.A., 1991. J. Am. Chem. Soc. 113, 2826. Park, H.Y., Go, H.Y., Satish, K., Rajaram, S.M., Han, S.H., Yoon, M.Y., 2009. Anal. Chem. 81, 4280–4284. Penn, S.G., He, L., Natan, M.J., 2003. Curr. Opin. Chem. Biol. 7, 609–612. Rosi, N.L., Mirkin, C.A., 2005. Chem. Rev. 105, 1547. Soumya, K., Thaleia, B., Emmanouil, Z., George, P.C., Agis, A.I., 2006. Biosens. Bioelectron. 22, 707–714. Tian, Z.R., Voigt, J.A., Liu, J., Mckenzie, B., Mcdermott, M.J., 2002. J. Am. Chem. Soc. 124, 12954. Wang, J., 2005. Small 1, 1036. Wang, X.D., Summers, C.J., Wang, Z.L., 2004. Nano Lett. 4, 423–426. Wu, J., Yan, Y.T., Yan, F., Ju, H.X., 2008. Anal. Chem. 80, 6072–6077. Xu, C.X., Sun, X.W., Yuen, C., Chen, B.J., Yu, S.F., Dong, Z.L., 2005. Appl. Phys. Lett. 86, 011118. Zeng, H.B., Cai, W.P., Liu, P.S., Xu, X.X., Zhou, H.J., Klingshirn, C., Kalt, H., 2008. ACS Nano. 2, 1661–1670. Zhao, J., Simeone, D.M., Heidt, D., Anderson, M.A., Lubman, D.M., 2006. J. Proteome Res. 5, 1792–1802.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
ZnO quantum dot labeled immunosensor for carbohydrate antigen 19-9 Baoxiang Gu a , Chunxiang Xu a,∗ , Chi Yang a , Songqin Liu b,∗∗ , Mingliang Wang b a b
State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, PR China School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China
a r t i c l e
i n f o
Article history: Received 19 June 2010 Received in revised form 3 September 2010 Accepted 18 September 2010 Available online 25 September 2010 Keywords: ZnO quantum dots CA 19-9 Immunosensor Stripping voltammetry
a b s t r a c t Using ZnO quantum dots as electrochemical and fluorescent labels, a sandwich-type sensitive immunoassay was developed to detect carbohydrate antigen 19-9 (CA 19-9) which is a preferred label for pancreatic cancer. The immobilization process was mainly carried out through the electrostatic adsorption based on the high isoelectric point of ZnO, and the sandwich structure was built through the immunoreaction of CA 19-9 antibodies and antigens. The immunological recognition of CA 19-9 was converted into detection of the amplified signals of the square wave stripping voltammetry (SWV) and intrinsic photoluminescence of the labeled quantum dots. The electrochemical assay demonstrated a dynamic range of 0.1–180 U/ml with detection limit of 0.04 U/ml, while the optical spectral detection revealed 1–180 U/ml with detection limit of 0.25 U/ml. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Carbohydrate antigen 19-9 (CA 19-9) is a preferred label for pancreatic cancer, which is a highly lethiferous sarcomata and difficult to be diagnosed early in current clinical medicine. In present clinic, CA 19-9 immunoassay has become a gold standard for pancreatic cancer diagnosis (Barbara et al., 2000; Gold et al., 2006). So far, various methods have been employed to detect CA 19-9, such as electric field-driven assay (Wu et al., 2008), immobilized horseradish peroxidase assay (Du et al., 2007), and chemiluminescent multiplex assay (Fu et al., 2007). However, the sensitivity and specificity of the biomarker are not high enough (Zhao et al., 2006), while the expensive equipment and complex processes are barriers in practice. ZnO as a functional semiconductor possesses much advantages for biosensor, such as visible light transparency, environmental and electrical stability (Brayner et al., 2006), nontoxicity (Tian et al., 2002), and abundance in nature (Park et al., 2009). Especially, the high isoelectric point (IEP, 9.5) (Gu et al., 2009) of ZnO is beneficial to immobilize the biomolecules with low IEP through electrostatic attraction. ZnO quantum dots (QDs) offer a considerable promise as quantitative labels for biological assays based on their high aspect ratio, substantial optical and electronic signal amplification and unique coding capabilities (Rosi and Mirkin, 2005; Wang, 2005; Penn et al., 2003).
∗ Corresponding author. Tel.: +86 25 83790755. ∗∗ Corresponding author. Tel.: +86 25 52090613. E-mail addresses: [email protected] (C. Xu), [email protected] (S. Liu). 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.09.031
In this communication, ZnO QDs were fabricated by a simple and facile colloid method, and then developed into a low cost immunosensor to detect CA 19-9 based on the exploration of the photoluminescence and stripping voltammetric signals. The immunosensors presented high sensitivity, stability, selectivity, and good reproducibility. The assembly process of the antibodies of CA 19-9, immunoreaction between the antibody (Ab) and antigen (Ag), and sensing characteristics and optimization procedure are reported in detail.
2. Materials and methods CA 19-9 Ag and Ab, latent membrane protein (LMP) and glycine were purchased from Nanjing Sunshine Biotechnology Company, China. Bovine serum albumin (BSA) and 3-aminopropyltriethoxysilane (APTS) were purchased from SigmaAldrich, USA. Zn (CH3 COO)2 ·2H2 O, LiOH·H2 O, HgCl2 and H2 O2 were purchased from Shanghai Chemical Plant, China. Doubly distilled water was used throughout the experiments. Phosphate buffer solution (PBS) was prepared by mixing the stock solution of KH2 PO4 and K2 HPO4 . Briefly, ZnO QDs were prepared through a colloidic procedure (Lubomir and Marc, 1991). Zn(CH3 COO)2 ·2H2 O and absolute ethanol were mixed and stirred for 2 h at 80 ◦ C into a solution with 0.05 M Zn2+ . Then the equal volume of absolute ethanol containing 0.1 M LiOH·H2 O was added and stirred for about 120 min, and then cooled down to room temperature. The ZnO QDs were collected by centrifuge at 12,000 r/min, and washed with deionized water three times, and then dispersed in 500 l pH 7.4 PBS for further use.
B. Gu et al. / Biosensors and Bioelectronics 26 (2011) 2720–2723
Scheme 1. Schematic protocol of sandwich-immunosensor.
Electrochemical experiments, including stripping voltammetry and square wave voltammetry (SWV) were performed with a CHI 660 electrochemical workstation (Shanghai, China). All electrochemical measurement was operated in a three-electrode system which is composed of an in situ plated mercury film on a glassy carbon electrode (GCE) as working electrode, a platinum wire as the auxiliary electrode, and a saturated calomel electrode as the reference electrode. The morphology and size of the ZnO QDs were analyzed with a transmission electron microscope (TEM, JEM-100S, JEOL, Japan). The ultraviolet–visible absorption (UV–vis), photoluminescence (PL) and Fourier transform infrared (FTIR) spectroscopy were detected with photometers of Hitachi UV-3600, Edinburgh FLS 920, and Nicolet 380, respectively. The -potential of ZnO QDs with and without CA 19-9 modification were analyzed by Zetasizer 4 (Malvern Instruments). As described previously, the ZnO QDs and proteins can form stable protein–QD bioconjugates through a self-assembly strategy (Emmanuel et al., 2001; Igor et al., 2005). In our experiment, the bioconjugated complexes were prepared by simply immerging the QDs into the Ab solution. 200 l 10 mM pH 7.4 PBS containing 10 g/ml Ab was added into 500 l 10 mM pH 7.4 PBS containing 20 g ZnO QDs, and shook at 4 ◦ C for 2 h, then the modified quantum dots (Ab-QDs) were centrifugally separated and washed three times with 10 mM pH 7.4 PBS. In this case, the Ab was successfully immobilized on ZnO QDs surfaces based on the electrostatic attraction. The process of immunosensor construction was shown in Scheme 1. A small silicon wafer was first functionized into an aldehydeactivated surface for next biomolecular immobilization. The Si substrate was oxidized at 1100 ◦ C for 3 h in a tube furnace to coat a layer of SiO2 , cleaned by ultra sonication in acetone, ethanol, and water, and then treated in a solution of NH4 OH:H2 O2 :H2 O with ratio of 1:1:7 to form a hydroxyl rich silica surface at 80 ◦ C for 5 min (Braun et al., 2005). The hydrophilic substrates were thoroughly rinsed with deionized water, then incubated in a closed Petri dish containing 5% APTS ethanol solution for 5 h at room temperature to form an alkylaminino silane derivatized surface (Soumya et al., 2006). The silanized sample reacted with 2% gluteraldehyde in
2721
pH 7.4 PBS shaken for 12 h at 4 ◦ C, and then washed thoroughly in deionized water to remove the excess gluteraldehyde (Penn et al., 2003). In the next step, an Ab monolayer was covalently assembled on the aldehyde-activated surface by soaking the sample area in 30 l Ab (10 g/ml, pH 7.4) PBS in a closed Petri dish. After reaction for 30 min at 37 ◦ C, the sample was rinsed with pH 7.4 PBS and then the substrate surface was blocked by BSA in 50 l blocking buffer solution (1% BSA in pH 7.4 PBS) for 2 h followed by water washing again. Then 30 l 180 U/ml CA 19-9 was infused into the cell and incubated for 30 min at 37 ◦ C. In this situation, the CA 19-9 molecules were linked with their antibodies through immunoreaction. Under the similar procedures, a series samples with various CA 19-9 concentration from 0.1 to 180 U/ml were fabricated. As a parallel step, the CA 19-9 antibodies were immobilized on ZnO QDs to form Ab-QDs as mentioned above. Finally, sufficient Ab-QD solution was dropped on the above substrates and maintained at 37 ◦ C for 30 min. In this case, the adequate ZnO QDs were bridge-linked onto the substrate through the Ab–Ag immunoreaction. These samples were washed twice with 30 l of pH 7.4 PBS and twice with 30 l of deionized water to remove the uncombined ZnO QDs for sensing characterization. Before each electrochemical test, the working GCE was polished with alumina (particle size of about 0.03 mm)/water slurry and washed thoroughly with water, and then a mercury film was electroplated on it in 100 mM KCl solution containing 1.2 mM HNO3 and 0.7 mM HgCl2 at −1.3 V for 300 s. The ZnO QDs linked on the substrate were dissolved by adding HCl (50 l, 0.01 M) solution with sonication for 10 min, and then washed thrice with 100 l 50 mM pH 7.0 PBS. Both of the reaction and washed solutions were collected, mixed into 50 mM pH 7.0 PBS, and then adjusted to 3 ml by 50 mM pH 7.0 PBS to obtain so called pending-solution containing Zn2+ ions. The pending-solution was treated at 0.6 V for 1 min, and accumulated at −1.3 V with stirring for 3 min and without stirring for 2 min. Finally, the SWV of the pending-solution was performed from −1.6 to −0.9 V with step potential of 4 mV, amplitude of 20 mV, and frequency of 15 Hz.
3. Results and discussion As shown the inserted TEM image in Scheme 1, the ZnO QDs present uniform size and well dispersion. The size statistic (see Fig. S1 in supplementary materials) demonstrates that the diameter of QDs are main distributed in 4.5–5.5 nm with an average value of 4.8 nm. The inserted high resolution TEM image clearly exhibits the lattice fringes, in which the d-spacing of 0.26 nm matches that of (0 0 0 1) planes of the wurtzite structural ZnO (Xu et al., 2005). It indicates the high crystalline quality of ZnO QDs (Zeng et al., 2008). The FTIR spectrum of purified and dried Ab-QDs was given in Fig. S2 in the supplementary materials. The peaks at 1038 cm−1 and 3361 cm−1 are assigned, respectively to the absorption of alkyl in Ab and hydroxyl. The characteristic absorption peaks at 1575 cm−1 and 1335 cm−1 are assigned to (N–H) and (C–N), respectively. The FTIR spectrum illuminates that the Ab molecules were immobilized on the surfaces of the ZnO QDs. This result was also confirmed by the -potential measurement. The -potential was changed from +20 mV for ZnO QDs into −19.2 mV for Ab-QDs. This effective bioconjugation process is associated with electrostatic attraction at the “nano-bio” interface due to the high IEP of ZnO (Andre et al., 2009). The sandwich-immunosensing can also be characterized by monitoring the intrinsic emission of ZnO QDs. Fig. 1 shows the spectral variation of the intrinsic bandedge emission of the ZnO QDs in the sandwich-immunosensors with a series of CA 19-9 concentrations under the excitation of 325 nm. The emission peaks at 372 nm
B. Gu et al. / Biosensors and Bioelectronics 26 (2011) 2720–2723
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Wavelength (nm) Fig. 1. The PL spectra of the immunosensors with a series concentration of CA 19-9 inserted with plots of the spectral intensity vs. CA 19-9 concentration in logarithmic style.
reflect the extension of the energy bandgap caused by the size effect (Lu et al., 2006; Wang et al., 2004). It can be seen that the peaks enhance with increase of CA 19-9 concentration. The inset plots the calibrated relationship between the PL intensity and CA 19-9 concentration in logarithm manners. It exhibits a linear response range from 1 U/ml to 180 U/ml for CA 19-9 detection. The linear regression equation is log (intensity) = 2.62059 + 0.49405 × log (C (U/ml)), with a correlation coefficient of 0.9963. The detection limit is estimated as 0.25 U/ml according to the 3 (standard deviation) based on the slope. This detection limit and linear response range have been improved compared with the previous reported values of 2 U/ml and 5–150 U/ml (Fu et al., 2007). The repeatability of optical detection was estimated from a series of five repetitive measurements of samples containing 1, 100, and 180 U/ml Ag that yielded the relative standard deviations (RSD) of 6.8, 3.1, and 1.4%, respectively. Fig. 2 shows the SWV curves of the series pending-solutions. It can be seen that the peak intensity of SWV rises with increase of CA 19-9 concentrations. The inset shows a broad linear response range from 0.1 U/ml to 180 U/ml in logarithmic styles. The linear regression equation is log (I (A)) = 0.27659 + 0.49979 × log (C (U/ml)) with a correlation coefficient of 0.99841. The detection limit is estimated as 0.04 U/ml according to the 3 (standard deviation) based on the slope. The sensitivity is estimated as 0.47 A/U/ml. The detection limit is lower, and the linear range is broader compared with previous reported values of 0.1 U/ml and 0.16–15 U/ml (Wu et al., 2008) and of 1.37 U/ml and 2–30 U/ml (Du et al., 2007), respectively. The biosening performance in both optical and electrochemical assays is mainly due to the uniform
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size and well dispersion of the QDs, stable immobilization of the biomolecules, and good biocompatibility of ZnO. The repeatability of the electrochemical detection was estimated from a series of five repetitive measurements of samples containing 0.1, 100, and 180 U/ml CA 19-9 that yielded the RSD of 7.8, 3.7, and 2.4%, respectively. Specificity is an important criterion for any analytical tool. The potential interference of coexisting species toward CA 19-9 detection was studied with the immunosensor. Three type samples were fabricated in the similar procedures as mentioned above except some bio-assembly in Scheme 1 was changed correspondingly: no Ab was connected on Si substrate for type 1 sample; no CA 19-9 was used for type 2; and 10 ng/ml LMP was used instead of CA 19-9 for type 3. As shown in the SWV signals of these type samples in Fig. S3 in the supplementary materials, no significant currents peak is observed in type 1–3 samples. The response of the immunosensor with 0.1 U/ml CA 19-9 is about 8 times of type 1 and 2 samples. The response of type 3 sample is less than that of immunosensor to 0.1 U/ml CA 19-9. The value of 0.1 U/ml CA 19-9 is far less than the cutoff concentration in clinical diagnosis value of 35 U/ml (Fu et al., 2007), and 10 ng/ml is a high concentration in LMP immunoassay. This illustrates that ZnO QD-based immunosensor has a good specificity for CA 19-9 detection. To test the regeneration, the sandwich-immunosensor with 180 U/ml CA 19-9 was rinsed with stripping buffer of 0.1 M pH 2.2 glycine-hydrochloride to remove the CA 19-9 and dissolved ZnO QDs to Zn2+ ions. The rinsed-buffer was mixed in 50 mM pH 7.0 PBS to 3 ml. The functionalized wafer reacted again with 180 U/ml CA 19-9 and Ab-QDs. The same immobilization and dissolution procedures as mentioned above were repeated eight times, and every pending-solution were employed for SWV detection. The SWV current signal restored 97.5% of the initial value after eight time assays, and no significant fluctuation was observed on their calibrated relationship plot (see Fig. S4 in supplementary materials). It revealed that the sandwich-immunosensor has an excellent regeneration. The stability of the sandwich-immunosensor was examined after storing the samples in pH 7.4 PBS at 4 ◦ C for 2 weeks. No obvious change was observed. This result indicates the good stability of the sandwich-immunsensors. 4. Conclusions Based on the bioconjugation of ZnO QDs and CA 19-9 Ab–Ag immunoreaction, the sandwich-immunosensors were assembled on the functionized Si substrate for sensitive CA19-9 detection through both electrochemical and optical techniques. The optical immunoassay based on PL of ZnO QDs demonstrated the detection limit of 0.25 U/ml and the linear response range of 1–180 U/ml. Electrochemical measurement offered higher sensitivity of 0.47 A/U/ml, broader linear range of 0.1–180 U/ml, and lower detection limit of 0.04 U/ml. The sensors also presented good stability, reliability and reproducibility. The uniform small size of the ZnO QDs, stable immobilization based on high IEP of ZnO could be response to the good performance. Based on the similar immobilization and examination techniques, the ZnO QDs are also expected to build up other amperometric immunosensors to detect other important antigens based on the efficient assembly of antibodies. Acknowledgments
180
C CA19-9 (U/ml)
Fig. 2. SWV curves of the pending-solution with a series concentration of CA 19-9 inserted with the plot of peak current vs. CA 19-9 concentration in logarithmic style.
This work was supported by NSFC (10674023 and 60725413), and NSFJ (BK2008285). The authors gratefully acknowledge professor J.J. Zhu in Nanjing University (Nanjing, China) for measurement of -potential.
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