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A “signal-on” electrochemical aptasensor for simultaneous detection of two tumor markers
Biosensors and Bioelectronics 34 (2012) 249–252
Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A “signal-on” electrochemical aptasensor for simultaneous detection of two tumor markers Jing Zhao a , Xiaolin He a , Bing Bo b , Xinjian Liu c , Yongmei Yin b,∗∗ , Genxi Li a,c,∗ a b c
Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, PR China Department of Oncology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR China Department of Biochemistry and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e
i n f o
Article history: Received 29 November 2011 Received in revised form 17 January 2012 Accepted 10 February 2012 Available online 19 February 2012 Keywords: Aptamers MUC1 VEGF165 Electrochemical aptasensor
a b s t r a c t In this paper, we report a “signal-on” electrochemical aptasensor for simultaneous determination of two tumor markers MUC1 and VEGF165 , by using a ferrocene-labeled aptamer-complementary DNA (cDNA) as probe. Since the cDNA immobilized on an electrode surface can hybridize with both MUC1 aptamer and VEGF165 aptamer to form a long double strand with ferrocene far away from the electrode surface, the probe cannot give electrochemical signal. Nevertheless, the presence of the two tumor markers will inhibit the hybridization of cDNA with the aptamers, thus the distance between ferrocene and the electrode is changed, and a “signal-on” electrochemical method to detect two tumor markers is developed. Experimental results show that the electrochemical signal increases with the addition of either tumor markers, but the biggest electrochemical signal can only be obtained when both tumor markers are present. Therefore, the proposed electrochemical aptasensor can not only detect the two markers but also distinguish their co-existence. It may also display high selectivity and sensitivity towards the detection of the tumor markers, so it might have potential clinical application in the future. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Aptamer that offers high affinity and specificity against the target may have many unique advantages, such as small size, low expense, easy for chemical modification as well as reduced immunogenicity and toxicity (Ellington and Szostak, 1990; Hermann and Patel, 2000; Li et al., 2010a), so aptamer has been widely used as a sensing element for developing biosensors, which are called aptasensors (Zhu et al., 2011). Due to the excellent portability, low cost and easy-to-operate, electrochemical aptasensors have received more and more research interests (Sassolas et al., 2009; Wang et al., 2009a; Zhang et al., 2010; Zuo et al., 2007; Lu et al., 2008a; Han et al., 2009; Huang et al., 2008). It has been known that aptamer may fold into a well-defined three-dimensional structure when binding with the target, which will alter the distance between the signal molecules labeled on aptamer and the electrode surface (Hianik and Wang, 2009). Based on the signal change induced by the structural transformation, aptasensors are divided into “signal-on” and “signal-off” biosensors, and the “signal-on” sensors may have more potential
∗ Corresponding author at: Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, PR China. Fax: +86 25 83592510. ∗∗ Corresponding author. Fax: +86 25 83710040. E-mail addresses: [email protected] (Y. Yin), [email protected] (G. Li). 0956-5663/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2012.02.016
applications because of the improved sensitivity and specificity (Sassolas et al., 2009; Xiang et al., 2011). In the meantime, the electrochemical aptasenors using aptamer-complementary DNA (cDNA) as probes are believed to be much more universal than that using surface-confined aptamers as probes (Lu et al., 2008b). In recent years, aptamers have been screened for targeting and measuring some tumor markers, which have great value in evaluating the progress of cancer, monitoring therapeutic response and predicting the recurrence (Shieh et al., 2010; Shao et al., 2010; Zhu et al., 2010; Wang et al., 2009b). Human mucin-1 (MUC1) aptamers and vascular endothelial growth factor-165 (VEGF165 ) aptamers are two known aptamers that are of great importance in the assessment of breast cancer (Borbas et al., 2007; Ferreira et al., 2006; Hasegawa et al., 2008; Ruckman et al., 1998; Ikebukuro et al., 2007). To breast cancer, elevated level of MUC1 is associated with a poor prognosis, a higher risk of recurrence and increased lymph node metastases in the breast cancer patients (Regimbald et al., 1996; Cheung et al., 2000; Rahn et al., 2001; Mukhopadhyay et al., 2011), while overexpression of VEGF is significantly correlated to poorer survival in both lymph node-negative and lymph node-positive breast cancer (Guo et al., 2003; Carpini et al., 2010; Linderholm et al., 2008; Shivakumar et al., 2009; Ghosh et al., 2008; Linderholm et al., 1998; Ferrara et al., 2003). Up to now, most electrochemical aptasensors are proposed to detect just one tumor marker (Li et al., 2011; Zhou et al., 2007), which is not reliable, since one tumor marker may yield
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false-positive result in the diagnosis of cancers. Now that most cancer diseases are associated with the presence of more than one tumor markers, developing an effective aptasensor for simultaneous measurement of co-existing tumor markers might be beneficial to improve the detection accuracy and provide more precise information on prognostic and treatment (Li et al., 2010b). Therefore, we have herein proposed a novel “signal-on” electrochemical aptasensor for simultaneous determination of two tumor markers, MUC1 and VEGF165 , by using ferrocene (Fc)-labeled cDNA as the probe. The experimental results have proven that our aptasensor can efficiently monitor the presence of the two tumor markers with high specificity, so it may have potential application in the future. 2. Experimental 2.1. Materials and chemicals MUC1 peptide (APDTRPAPG) was synthesized by Invitrogen and resuspended in PBS (10 mM, pH7.4). Recombinant Zebrafish VEGF165 purified from the insect cell line Sf21 was purchased from R&D Systems and resuspended in TBSE buffer (10 mM Tris–HCl, 100 mM NaCl, 0.1 mM EDTA, pH 7.8). Tris (hydroxymethyl) aminomethane, Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), 1-Ethyl-3-[(3-dimethylamino) propyl] carbodiimide (EDC), 6-Mercapto-1-hexanol (MCH) and NHydroxysuccinimide (NHS) were purchased from Sigma. All other chemicals were analytical reagent and the aqueous solutions were prepared with doubly distilled water which was purified with a Milli-Q purification system (Branstead, USA) to a specific resistance of >18 M cm. DNA was manufactured by Shanghai Sangon Biological Engineering Technology & Service Co., Ltd. The DNA sequences are as follows. MUC1 aptamer: 5 -GCAGTTGATCCTTTGGATACCCTGG-3 ; VEGF165 aptamer: 5 -GGGCCCGTCCGTATGGTGGGTGTGCTGGCC3 ; cDNA: 5 -SH-AGGATCAACTGCGGCCAGCACACCCAGATCCT-NH2 3 . 2.2. Preparation of Fc-labeled cDNA modified gold electrode The 5 mm disk gold electrode purchased from Shanghai Chenhua Inc. (Shanghai, China) was firstly polished on fine sand papers and alumina (particle size of about 0.05 m)/water slurry on silk. Then it was ultra-sonicated in both ethanol and doubly distilled water for about 5 min, respectively. Finally, the electrode was electrochemically cleaned to remove any remaining impurities in 0.5 M H2 SO4 . After drying with nitrogen, the gold electrode was immersed into 10 mM Tris–HCl (pH 7.4) containing 1 M cDNA, 0.1 mM EDTA, 0.1 M NaCl and 10 mM TCEP for 16 h, followed by 1 h treatment with 2 mM MCH. After thoroughly rinsed with doubly distilled water, the modified electrode was immersed in an alcohol solution containing 5 mM EDC, 25 mM NHS and 5 mM ferrocene carboxylic acid for 2 h at 37 ◦ C. The Fc-labeled cDNA modified gold electrode was thus prepared and ready for the following experiments. 2.3. Electrochemical detection of tumor markers In the hybridization reaction, Fc-labeled cDNA modified electrode was immersed in the hybridization solution (0.1 M NaCl, 10 mM Tris–HCl, pH 7.8) containing 10 nM MUC1 aptamer and 10 nM VEGF165 aptamer for 1.5 h at room temperature. For the detection of one tumor marker, different concentrations of MUC1
Fig. 1. Schematic illustration of the method to simultaneously detect two tumor markers.
or VEGF165 were incubated with the according aptamers for 1 h at room temperature before the hybridization reaction. For the detection of both tumor markers, 20 nM MUC1 and different concentrations of VEGF165 were added in the test solution to bind with their aptamers for 1 h at room temperature before the hybridization reaction. In the control experiment, 100 nM BSA instead of the tumor markers was employed. After the hybridization reaction, the electrode was then thoroughly rinsed with doubly distilled water and prepared for the electrochemical measurements. The electrochemical experiments were carried out on a CHI 660C electrochemical analyzer (CH Instruments). A three-electrode system consisting of the modified gold electrode, saturated calomel reference electrode (SCE) and platinum counter electrode was used for the measurements. Square wave voltammetry (SWV) was performed in 0.1 M KClO4 . The parameters were as follows: potential scan range from 0 to 0.6 V, the potential step of 4 mV, the amplitude of 25 mV and the frequency of 15 Hz. Electrochemical impedance spectroscopy (EIS) experiments were performed in 0.1 M PBS (pH 7.4) containing 5 mM Fe(CN)6 3−/4− and 0.1 M NaCl, with the biasing potential of 0.224 V, the amplitude of 5 mV and the frequency range from 1 Hz to 100 kHz. 3. Results and discussion Fig. 1 may illustrate the mechanism of the proposed method to simultaneously detect two tumor markers. The Fc-labeled cDNA contains six complementary base pairs at its ends and is partially complementary to both MUC1 aptamer and VEGF165 aptamer. Therefore, when immobilized on the surface of a gold electrode, cDNA can spontaneously form a loop-stem structure with Fc close to the electrode surface, so electrochemical signal can be obtained. In the absence of the two tumor markers, cDNA hybridizes with both MUC1 aptamer and VEGF165 aptamer to form a long double strand that keeps Fc molecules far away from the electrode surface, so no wave can be observed. Nevertheless, if one tumor marker (taking MUC1 as an example in Fig. 1) is present, the aptamer of this tumor marker will bind with its target, so only the other aptamer can hybridize with some of the sequences of the cDNA. Consequently, due to the flexibility of the short double-stranded structure, Fc molecule is much closer to the electrode surface than that with the long double strand, thus an electrochemical wave can be observed, although it is not very high. Furthermore, when both MUC1 and VEGF165 co-exist, both of their aptamers will bind with their targets, so the unhybridized cDNA remains the loopstem structure to exhibit very fine electrochemical wave. Since
J. Zhao et al. / Biosensors and Bioelectronics 34 (2012) 249–252
Fig. 2. Square wave voltammograms obtained at the cDNA modified electrode in the presence of different concentrations of MUC1 (from a to f: 0, 1 nM, 5 nM, 10 nM, 20 nM, 40 nM), and 10 nM MUC1 aptamer and 10 nM VEGF165 aptamer. Curve g is the case that the two aptamers are not involved in the system. The insert graph shows the relationship between the peak current and the concentration of MUC1. Error bars represent the standard deviations of three independent measurements. A baseline correction of the resulting voltammogram has been performed using the linear baseline correction mode of the CHI 660C software. Electrolyte: 0.1 M KClO4 ; potential step: 4 mV; amplitude: 25 mV; frequency: 15 Hz.
the oxidation of Fc is highly distance-dependent (Yu et al., 2009), the altered distance between Fc and the electrode may lead to the changes of electrochemical signals, thus the presence of one tumor marker or both can be specifically detected. 3.1. Electrochemical detection of one tumor marker by SWV Herein, we have employed a sensitive electrochemical technique, square wave voltammetry, to monitor the distance change between the signal molecule Fc and the electrode surface so as to detect the tumor markers. As shown in Fig. 2, a well-defined Fc oxidation peak can be obtained at 0.32 V at the cDNA modified electrode, ascribing to the close approach of Fc to the electrode surface (Fig. 2g). In contrast, nearly no electrochemical response can be observed after cDNA hybridizes with both MUC1 aptamer and VEGF165 aptamer (Fig. 2a), as the newly-formed long double strand has hindered the electron transfer between Fc and the electrode. Nevertheless, if one tumor marker is present, here taking MUC1 as an example, the aptamer of MUC1 will combine with its target, thus only the other aptamer, i.e. the aptamer of VEGF165 , can hybridize with the cDNA, thus a short double strand is formed. The short double strand formed by the hybridization of cDNA and VEGF165 aptamer may decrease the distance between Fc and the electrode, so the peak current of Fc can be obtained, which increases with the concentration of MUC1 (Fig. 2b–f). The highest electrochemical response can be obtained in the presence of 20 nM MUC1. Meanwhile, MUC1 has been found to be detected in a linear range from 1 nM to 20 nM. The detection limit is 0.33 nM (3 times signal-tonoise ratio), which is much lower than that in the previous reports (Cheng et al., 2009). So, this “signal-on” electrochemical aptasensor is highly sensitive.
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Fig. 3. Square wave voltammograms obtained at the cDNA modified electrode in the presence of 20 nM MUC1 and different concentrations of VEGF165 (from a to e: 0, 1 nM, 5 nM, 10 nM, 20 nM). Curve f is the case that the two aptamers are not involved in the system. The insert graph shows the relationship between the peak current and the total concentration of MUC1 and VEGF165 . Others same as in Fig. 2.
studied the cases that both 20 nM MUC1 and different concentrations of VEGF165 are present. Since the aptamers of both MUC1 and VEGF165 would preferentially bind with their targets instead of hybridizing with the cDNA, the cDNA may result in a much higher electrochemical response in the presence of both tumor markers (Fig. 3). The electrochemical response is also found to be concentration-dependent to VEGF165 , and the peak current increases linearly with the concentration of VEGF165 in the range from 1 nM to 20 nM (Fig. 3, Inset). The biggest electrochemical signal can be observed in the presence of not only 20 nM MUC1 but also 20 nM VEGF165 , which is almost as high as the peak obtained at the cDNA modified electrode. 3.3. Control experiments for the detection of tumor markers In order to better define the usage of our aptasensor, we have further compared the electrochemical signals obtained by the control experiments. As shown in Fig. 4, an intense electrochemical signal can be observed when 20 nM MUC1 and 20 nM VEGF165 are simultaneously present, while the electrochemical response is about the half if only one tumor marker (40 nM MUC1 or 40 nM VEGF165 ) is present, although the total concentration of the tumor markers are the same (40 nM). So, our “signal-on” electrochemical
3.2. Electrochemical detection of both tumor marker by SWV Fig. 2 also shows that the highest peak current obtained in the presence of MUC1 can only be approximately half of that obtained at cDNA modified electrode, which is also reasonable, since Fc molecule within the short double-stranded structure is not close enough to the electrode surface. Therefore, we have further
Fig. 4. Square wave voltammograms obtained at the cDNA modified electrode in the presence of 40 nM MUC1, or 40 nM VEGF165 , or 20 nM MUC1 plus 20 nM VEGF165 , or 100 nM BSA. Others same as in Fig. 2.
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determination of two tumor markers, MUC1 and VEGF165 . The aptasensor can not only sensitively detect the two tumor markers, but also efficiently distinguish their co-existence. Since cancer diagnosis based on the recognition of two tumor markers will be more reliable and accurate than that with only one tumor marker detected, our detection may provide more useful information for clinical applications. Moreover, by altering the related aptamers, this method can be expanded to detect more kinds of tumor markers in accordance with the clinic demands, so this work may have very high and potential values for basic researches as well as clinical applications in the future. Acknowledgments
Fig. 5. Electrochemical impedance spectra (Nyquist plots) of the bare gold electrode, cDNA modified electrode, and the modified electrode with the presence of different concentrations of tumor markers (0, 20 nM MUC1, 20 nM MUC1 and 20 nM VEGF165 ), in a 0.1 M PBS (pH 7.4) containing 5 mM Fe(CN)6 3−/4− and 0.1 M NaCl, with the biasing potential of 0.224 V, the amplitude of 5 mV and the frequency range from 1 Hz to 100 kHz.
aptasensor can availably distinguish the co-existence of the two tumor markers from that only one tumor marker exists. In addition, Fig. 4 has also shown that no distinct electrochemical response can be observed even when 100 nM BSA is employed, which has proven the high selectivity of our aptasensor. Certainly, it should be mentioned that for the case of 10 nM MUC1 and 10 nM VEGF165 , it may have the same response for the case of 20 nM MUC1 only. Therefore, the highest electrochemical signal can only be observed when 20 nM MUC1 and 20 nM VEGF165 are simultaneously present. In other words, to distinguish the coexistence of the two tumor markers, we should first of all obtain the highest response of one tumor marker, and then get to know the highest response when both co-exist. 3.4. Electrochemical detection of tumor markers by EIS We have also conducted EIS experiments to confirm the availability of the detection of the two tumor markers by using our aptasensor. As is shown in Fig. 5, the modification of cDNA onto the gold electrode surface may significantly increase the electrontransfer resistance due to the steric hindrance, since the loop-stem structure of cDNA will prohibit [Fe(CN)6 ]3−/4− molecule reaching to the gold electrode and cDNA will electrostatically repel [Fe(CN)6 ]3−/4− . When both the tumor markers are absent, cDNA hybridizes with the aptamers of both tumor markers to form a rigid long double strand, which makes cDNA stand up off the surface of the electrode, resulting in availability of [Fe(CN)6 ]3−/4− to the surface, so a decreased impedance can be observed. Nevertheless, when 20 nM MUC1 is present, cDNA hybridizes with only VEGF165 aptamer to form a short double strand, which is much closer to the electrode surface, thus the impedance is bigger than that with a long double strand. Furthermore, when both 20 nM MUC1 and 20 nM VEGF165 are simultaneously present, the impedance value is almost as big as that obtained at the cDNA modified electrode, because the aptamers of the two tumor markers will combine with their targets instead of hybridizing with the cDNA. Therefore, similar to the SWV results, EIS has also illustrated that the presence of one and two tumor markers can be differentiated by our aptasensor. 4. Conclusions In summary, we have fabricated a simple, effective and selective “signal-on” electrochemical aptasensor for simultaneous
This work is supported by the National Science Fund for Distinguished Young Scholars (grant no. 20925520), the National Natural Science Foundation of China (grant no. 81172503), and the Leading Academic Discipline Project of Shanghai Municipal Education Commission (grant no. J50108). References Borbas, K.E., Ferreira, C.S.M., Perkins, A., Bruce, J.I., Missailidis, S., 2007. Bioconjug. Chem. 18 (4), 1205–1212. Carpini, J.D., Karam, A.K., Montgomery, L., 2010. Angiogenesis 13 (1), 43–58. Cheng, A.K.H., Su, H., Wang, Y.A., Yu, H.Z., 2009. Anal. Chem. 81 (15), 6130–6139. Cheung, K.L., Graves, C.R.L., Robertson, J.F.R., 2000. Cancer Treat. Rev. 26 (2), 91–102. Ellington, A.D., Szostak, J.W., 1990. Nature 346 (6287), 818–822. Ferrara, N., Gerber, H.P., LeCouter, J., 2003. Nat. Med. 9 (6), 669–676. Ferreira, C.S.M., Matthews, C.S., Missailidis, S., 2006. Tumour Biol. 27 (6), 289–301. Ghosh, S., Sullivan, C.A., Zerkowski, M.P., Molinaro, A.M., Rimm, D.L., Camp, R.L., Chung, G.G., 2008. Hum. Pathol. 39 (12), 1835–1843. Guo, P., Fang, Q., Tao, H.Q., Schafer, C.A., Fenton, B.M., Ding, I., Hu, B., Cheng, S.Y., 2003. Cancer Res. 63 (15), 4684–4691. Han, K., Chen, L., Lin, Z., Li, G., 2009. Electrochem. Commun. 11 (1), 157–160. Hasegawa, H., Sode, K., Ikebukuro, K., 2008. Biotechnol. Lett. 30 (5), 829–834. Hermann, T., Patel, D.J., 2000. Science 287 (5454), 820–825. Hianik, T., Wang, J., 2009. Electroanalysis 21 (11), 1223–1235. Huang, Y., Ge, B., Sen, D., Yu, H., 2008. J. Am. Chem. Soc. 130 (25), 8023–8029. Ikebukuro, K., Hasegawa, H., Sode, K., 2007. Nucleic Acids Symp. Ser. (Oxf). 51 (1), 399–400. Li, D., Song, S., Fan, C., 2010a. Acc. Chem. Res. 43 (5), 631–641. Li, T., Fan, Q., Liu, T., Zhu, X., Zhao, J., Li, G., 2010b. Biosens. Bioelectron. 25 (12), 2686–2689. Li, Y., Bao, J., Han, M., Dai, Z., Wang, H., 2011. Biosens. Bioelectron. 26 (8), 3531–3535. Linderholm, B., Tavelin, B., Grankvist, K., Henriksson, R., 1998. J. Clin. Oncol. 16 (9), 3121–3128. Linderholm, B.K., Gruvberger-Saal, S., Fernö, M., Bendahl, P.O., Malmström, P., 2008. Breast 17 (5), 484–491. Lu, Y., Li, X., Zhang, L., Yu, P., Su, L., Mao, L., 2008a. Anal. Chem. 80 (6), 1883–1890. Lu, Y., Zhu, N., Yu, P., Mao, L., 2008b. Analyst 133 (9), 1256–1260. Mukhopadhyay, P., Chakraborty, S., Ponnusamy, M.P., Lakshmanan, I., Jain, M., Batr, S.K., 2011. Biochim. Biophys. Acta 1815 (2), 224–240. Rahn, J.J., Dabbagh, L., Pasdar, M., Hugh, J.C., 2001. Cancer 91 (11), 1973–1982. Regimbald, L.H., Pilarski, L.M., Longenecker, B.M., Reddish, M.A., Zimmermann, G., Hugh, J.C., 1996. Cancer Res. 56 (18), 4244–4249. Ruckman, J., Green, L.S., Beeson, J., Waugh, S., Gillette, W.L., Henninger, D.D., ´ N., 1998. J. Biol. Chem. 273 (32), 20556–20567. Claesson-Welsh, L., Janjic, Sassolas, A., Blum, L.J., Leca-Bouvier, B.D., 2009. Electroanalysis 21 (11), 1237–1250. Shao, Z., Li, Y., Yang, Q., Wang, J., Li, G., 2010. Anal. Bioanal. Chem. 398 (7–8), 2963–2967. Shieh, Y.A., Yang, S.J., Wei, M.F., Shieh, M.J., 2010. ACS Nano 4 (3), 1433–1442. Shivakumar, S., Prabhakar, B.T., Jayashree, K., Rajan, M.G.R., Salimath, B.P., 2009. J. Cancer Res. Clin. Oncol. 135 (4), 627–636. Wang, J., Cao, Y., Chen, G., Li, G., 2009a. Chembiochem 10 (13), 2171–2176. Wang, J., Meng, W., Zheng, X., Huang, J., Li, G., 2009b. Biosens. Bioelectron. 24 (6), 1598–1602. Xiang, Y., Qian, X., Jiang, B., Chai, Y., Yuan, R., 2011. Chem. Commun. 47 (16), 4733–4735. Yu, B., Hu, H., Wang, D., Huck, W.T.S., Zhou, F., Liu, W., 2009. J. Mater. Chem. 19 (43), 8129–8134. Zhang, K., Zhu, X., Wang, J., Xu, L., Li, G., 2010. Anal. Chem. 82 (8), 3207–3211. Zhou, L., Ou, L.J., Chu, X., Shen, G.L., Yu, R.Q., 2007. Anal. Chem. 79 (19), 7492–7500. Zhu, X., Cao, Y., Liang, Z., Li, G., 2010. Protein Cell 1 (9), 842–846. Zhu, X., Zhao, J., Wu, Y., Shen, Z., Li, G., 2011. Anal. Chem. 83 (11), 4085–4089. Zuo, X., Song, S., Zhang, J., Pan, D., Wang, L., Fan, C., 2007. J. Am. Chem. Soc. 129 (5), 1042–1043.
Contents lists available at SciVerse ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A “signal-on” electrochemical aptasensor for simultaneous detection of two tumor markers Jing Zhao a , Xiaolin He a , Bing Bo b , Xinjian Liu c , Yongmei Yin b,∗∗ , Genxi Li a,c,∗ a b c
Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, PR China Department of Oncology, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, PR China Department of Biochemistry and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e
i n f o
Article history: Received 29 November 2011 Received in revised form 17 January 2012 Accepted 10 February 2012 Available online 19 February 2012 Keywords: Aptamers MUC1 VEGF165 Electrochemical aptasensor
a b s t r a c t In this paper, we report a “signal-on” electrochemical aptasensor for simultaneous determination of two tumor markers MUC1 and VEGF165 , by using a ferrocene-labeled aptamer-complementary DNA (cDNA) as probe. Since the cDNA immobilized on an electrode surface can hybridize with both MUC1 aptamer and VEGF165 aptamer to form a long double strand with ferrocene far away from the electrode surface, the probe cannot give electrochemical signal. Nevertheless, the presence of the two tumor markers will inhibit the hybridization of cDNA with the aptamers, thus the distance between ferrocene and the electrode is changed, and a “signal-on” electrochemical method to detect two tumor markers is developed. Experimental results show that the electrochemical signal increases with the addition of either tumor markers, but the biggest electrochemical signal can only be obtained when both tumor markers are present. Therefore, the proposed electrochemical aptasensor can not only detect the two markers but also distinguish their co-existence. It may also display high selectivity and sensitivity towards the detection of the tumor markers, so it might have potential clinical application in the future. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Aptamer that offers high affinity and specificity against the target may have many unique advantages, such as small size, low expense, easy for chemical modification as well as reduced immunogenicity and toxicity (Ellington and Szostak, 1990; Hermann and Patel, 2000; Li et al., 2010a), so aptamer has been widely used as a sensing element for developing biosensors, which are called aptasensors (Zhu et al., 2011). Due to the excellent portability, low cost and easy-to-operate, electrochemical aptasensors have received more and more research interests (Sassolas et al., 2009; Wang et al., 2009a; Zhang et al., 2010; Zuo et al., 2007; Lu et al., 2008a; Han et al., 2009; Huang et al., 2008). It has been known that aptamer may fold into a well-defined three-dimensional structure when binding with the target, which will alter the distance between the signal molecules labeled on aptamer and the electrode surface (Hianik and Wang, 2009). Based on the signal change induced by the structural transformation, aptasensors are divided into “signal-on” and “signal-off” biosensors, and the “signal-on” sensors may have more potential
∗ Corresponding author at: Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, PR China. Fax: +86 25 83592510. ∗∗ Corresponding author. Fax: +86 25 83710040. E-mail addresses: [email protected] (Y. Yin), [email protected] (G. Li). 0956-5663/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2012.02.016
applications because of the improved sensitivity and specificity (Sassolas et al., 2009; Xiang et al., 2011). In the meantime, the electrochemical aptasenors using aptamer-complementary DNA (cDNA) as probes are believed to be much more universal than that using surface-confined aptamers as probes (Lu et al., 2008b). In recent years, aptamers have been screened for targeting and measuring some tumor markers, which have great value in evaluating the progress of cancer, monitoring therapeutic response and predicting the recurrence (Shieh et al., 2010; Shao et al., 2010; Zhu et al., 2010; Wang et al., 2009b). Human mucin-1 (MUC1) aptamers and vascular endothelial growth factor-165 (VEGF165 ) aptamers are two known aptamers that are of great importance in the assessment of breast cancer (Borbas et al., 2007; Ferreira et al., 2006; Hasegawa et al., 2008; Ruckman et al., 1998; Ikebukuro et al., 2007). To breast cancer, elevated level of MUC1 is associated with a poor prognosis, a higher risk of recurrence and increased lymph node metastases in the breast cancer patients (Regimbald et al., 1996; Cheung et al., 2000; Rahn et al., 2001; Mukhopadhyay et al., 2011), while overexpression of VEGF is significantly correlated to poorer survival in both lymph node-negative and lymph node-positive breast cancer (Guo et al., 2003; Carpini et al., 2010; Linderholm et al., 2008; Shivakumar et al., 2009; Ghosh et al., 2008; Linderholm et al., 1998; Ferrara et al., 2003). Up to now, most electrochemical aptasensors are proposed to detect just one tumor marker (Li et al., 2011; Zhou et al., 2007), which is not reliable, since one tumor marker may yield
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false-positive result in the diagnosis of cancers. Now that most cancer diseases are associated with the presence of more than one tumor markers, developing an effective aptasensor for simultaneous measurement of co-existing tumor markers might be beneficial to improve the detection accuracy and provide more precise information on prognostic and treatment (Li et al., 2010b). Therefore, we have herein proposed a novel “signal-on” electrochemical aptasensor for simultaneous determination of two tumor markers, MUC1 and VEGF165 , by using ferrocene (Fc)-labeled cDNA as the probe. The experimental results have proven that our aptasensor can efficiently monitor the presence of the two tumor markers with high specificity, so it may have potential application in the future. 2. Experimental 2.1. Materials and chemicals MUC1 peptide (APDTRPAPG) was synthesized by Invitrogen and resuspended in PBS (10 mM, pH7.4). Recombinant Zebrafish VEGF165 purified from the insect cell line Sf21 was purchased from R&D Systems and resuspended in TBSE buffer (10 mM Tris–HCl, 100 mM NaCl, 0.1 mM EDTA, pH 7.8). Tris (hydroxymethyl) aminomethane, Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), 1-Ethyl-3-[(3-dimethylamino) propyl] carbodiimide (EDC), 6-Mercapto-1-hexanol (MCH) and NHydroxysuccinimide (NHS) were purchased from Sigma. All other chemicals were analytical reagent and the aqueous solutions were prepared with doubly distilled water which was purified with a Milli-Q purification system (Branstead, USA) to a specific resistance of >18 M cm. DNA was manufactured by Shanghai Sangon Biological Engineering Technology & Service Co., Ltd. The DNA sequences are as follows. MUC1 aptamer: 5 -GCAGTTGATCCTTTGGATACCCTGG-3 ; VEGF165 aptamer: 5 -GGGCCCGTCCGTATGGTGGGTGTGCTGGCC3 ; cDNA: 5 -SH-AGGATCAACTGCGGCCAGCACACCCAGATCCT-NH2 3 . 2.2. Preparation of Fc-labeled cDNA modified gold electrode The 5 mm disk gold electrode purchased from Shanghai Chenhua Inc. (Shanghai, China) was firstly polished on fine sand papers and alumina (particle size of about 0.05 m)/water slurry on silk. Then it was ultra-sonicated in both ethanol and doubly distilled water for about 5 min, respectively. Finally, the electrode was electrochemically cleaned to remove any remaining impurities in 0.5 M H2 SO4 . After drying with nitrogen, the gold electrode was immersed into 10 mM Tris–HCl (pH 7.4) containing 1 M cDNA, 0.1 mM EDTA, 0.1 M NaCl and 10 mM TCEP for 16 h, followed by 1 h treatment with 2 mM MCH. After thoroughly rinsed with doubly distilled water, the modified electrode was immersed in an alcohol solution containing 5 mM EDC, 25 mM NHS and 5 mM ferrocene carboxylic acid for 2 h at 37 ◦ C. The Fc-labeled cDNA modified gold electrode was thus prepared and ready for the following experiments. 2.3. Electrochemical detection of tumor markers In the hybridization reaction, Fc-labeled cDNA modified electrode was immersed in the hybridization solution (0.1 M NaCl, 10 mM Tris–HCl, pH 7.8) containing 10 nM MUC1 aptamer and 10 nM VEGF165 aptamer for 1.5 h at room temperature. For the detection of one tumor marker, different concentrations of MUC1
Fig. 1. Schematic illustration of the method to simultaneously detect two tumor markers.
or VEGF165 were incubated with the according aptamers for 1 h at room temperature before the hybridization reaction. For the detection of both tumor markers, 20 nM MUC1 and different concentrations of VEGF165 were added in the test solution to bind with their aptamers for 1 h at room temperature before the hybridization reaction. In the control experiment, 100 nM BSA instead of the tumor markers was employed. After the hybridization reaction, the electrode was then thoroughly rinsed with doubly distilled water and prepared for the electrochemical measurements. The electrochemical experiments were carried out on a CHI 660C electrochemical analyzer (CH Instruments). A three-electrode system consisting of the modified gold electrode, saturated calomel reference electrode (SCE) and platinum counter electrode was used for the measurements. Square wave voltammetry (SWV) was performed in 0.1 M KClO4 . The parameters were as follows: potential scan range from 0 to 0.6 V, the potential step of 4 mV, the amplitude of 25 mV and the frequency of 15 Hz. Electrochemical impedance spectroscopy (EIS) experiments were performed in 0.1 M PBS (pH 7.4) containing 5 mM Fe(CN)6 3−/4− and 0.1 M NaCl, with the biasing potential of 0.224 V, the amplitude of 5 mV and the frequency range from 1 Hz to 100 kHz. 3. Results and discussion Fig. 1 may illustrate the mechanism of the proposed method to simultaneously detect two tumor markers. The Fc-labeled cDNA contains six complementary base pairs at its ends and is partially complementary to both MUC1 aptamer and VEGF165 aptamer. Therefore, when immobilized on the surface of a gold electrode, cDNA can spontaneously form a loop-stem structure with Fc close to the electrode surface, so electrochemical signal can be obtained. In the absence of the two tumor markers, cDNA hybridizes with both MUC1 aptamer and VEGF165 aptamer to form a long double strand that keeps Fc molecules far away from the electrode surface, so no wave can be observed. Nevertheless, if one tumor marker (taking MUC1 as an example in Fig. 1) is present, the aptamer of this tumor marker will bind with its target, so only the other aptamer can hybridize with some of the sequences of the cDNA. Consequently, due to the flexibility of the short double-stranded structure, Fc molecule is much closer to the electrode surface than that with the long double strand, thus an electrochemical wave can be observed, although it is not very high. Furthermore, when both MUC1 and VEGF165 co-exist, both of their aptamers will bind with their targets, so the unhybridized cDNA remains the loopstem structure to exhibit very fine electrochemical wave. Since
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Fig. 2. Square wave voltammograms obtained at the cDNA modified electrode in the presence of different concentrations of MUC1 (from a to f: 0, 1 nM, 5 nM, 10 nM, 20 nM, 40 nM), and 10 nM MUC1 aptamer and 10 nM VEGF165 aptamer. Curve g is the case that the two aptamers are not involved in the system. The insert graph shows the relationship between the peak current and the concentration of MUC1. Error bars represent the standard deviations of three independent measurements. A baseline correction of the resulting voltammogram has been performed using the linear baseline correction mode of the CHI 660C software. Electrolyte: 0.1 M KClO4 ; potential step: 4 mV; amplitude: 25 mV; frequency: 15 Hz.
the oxidation of Fc is highly distance-dependent (Yu et al., 2009), the altered distance between Fc and the electrode may lead to the changes of electrochemical signals, thus the presence of one tumor marker or both can be specifically detected. 3.1. Electrochemical detection of one tumor marker by SWV Herein, we have employed a sensitive electrochemical technique, square wave voltammetry, to monitor the distance change between the signal molecule Fc and the electrode surface so as to detect the tumor markers. As shown in Fig. 2, a well-defined Fc oxidation peak can be obtained at 0.32 V at the cDNA modified electrode, ascribing to the close approach of Fc to the electrode surface (Fig. 2g). In contrast, nearly no electrochemical response can be observed after cDNA hybridizes with both MUC1 aptamer and VEGF165 aptamer (Fig. 2a), as the newly-formed long double strand has hindered the electron transfer between Fc and the electrode. Nevertheless, if one tumor marker is present, here taking MUC1 as an example, the aptamer of MUC1 will combine with its target, thus only the other aptamer, i.e. the aptamer of VEGF165 , can hybridize with the cDNA, thus a short double strand is formed. The short double strand formed by the hybridization of cDNA and VEGF165 aptamer may decrease the distance between Fc and the electrode, so the peak current of Fc can be obtained, which increases with the concentration of MUC1 (Fig. 2b–f). The highest electrochemical response can be obtained in the presence of 20 nM MUC1. Meanwhile, MUC1 has been found to be detected in a linear range from 1 nM to 20 nM. The detection limit is 0.33 nM (3 times signal-tonoise ratio), which is much lower than that in the previous reports (Cheng et al., 2009). So, this “signal-on” electrochemical aptasensor is highly sensitive.
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Fig. 3. Square wave voltammograms obtained at the cDNA modified electrode in the presence of 20 nM MUC1 and different concentrations of VEGF165 (from a to e: 0, 1 nM, 5 nM, 10 nM, 20 nM). Curve f is the case that the two aptamers are not involved in the system. The insert graph shows the relationship between the peak current and the total concentration of MUC1 and VEGF165 . Others same as in Fig. 2.
studied the cases that both 20 nM MUC1 and different concentrations of VEGF165 are present. Since the aptamers of both MUC1 and VEGF165 would preferentially bind with their targets instead of hybridizing with the cDNA, the cDNA may result in a much higher electrochemical response in the presence of both tumor markers (Fig. 3). The electrochemical response is also found to be concentration-dependent to VEGF165 , and the peak current increases linearly with the concentration of VEGF165 in the range from 1 nM to 20 nM (Fig. 3, Inset). The biggest electrochemical signal can be observed in the presence of not only 20 nM MUC1 but also 20 nM VEGF165 , which is almost as high as the peak obtained at the cDNA modified electrode. 3.3. Control experiments for the detection of tumor markers In order to better define the usage of our aptasensor, we have further compared the electrochemical signals obtained by the control experiments. As shown in Fig. 4, an intense electrochemical signal can be observed when 20 nM MUC1 and 20 nM VEGF165 are simultaneously present, while the electrochemical response is about the half if only one tumor marker (40 nM MUC1 or 40 nM VEGF165 ) is present, although the total concentration of the tumor markers are the same (40 nM). So, our “signal-on” electrochemical
3.2. Electrochemical detection of both tumor marker by SWV Fig. 2 also shows that the highest peak current obtained in the presence of MUC1 can only be approximately half of that obtained at cDNA modified electrode, which is also reasonable, since Fc molecule within the short double-stranded structure is not close enough to the electrode surface. Therefore, we have further
Fig. 4. Square wave voltammograms obtained at the cDNA modified electrode in the presence of 40 nM MUC1, or 40 nM VEGF165 , or 20 nM MUC1 plus 20 nM VEGF165 , or 100 nM BSA. Others same as in Fig. 2.
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determination of two tumor markers, MUC1 and VEGF165 . The aptasensor can not only sensitively detect the two tumor markers, but also efficiently distinguish their co-existence. Since cancer diagnosis based on the recognition of two tumor markers will be more reliable and accurate than that with only one tumor marker detected, our detection may provide more useful information for clinical applications. Moreover, by altering the related aptamers, this method can be expanded to detect more kinds of tumor markers in accordance with the clinic demands, so this work may have very high and potential values for basic researches as well as clinical applications in the future. Acknowledgments
Fig. 5. Electrochemical impedance spectra (Nyquist plots) of the bare gold electrode, cDNA modified electrode, and the modified electrode with the presence of different concentrations of tumor markers (0, 20 nM MUC1, 20 nM MUC1 and 20 nM VEGF165 ), in a 0.1 M PBS (pH 7.4) containing 5 mM Fe(CN)6 3−/4− and 0.1 M NaCl, with the biasing potential of 0.224 V, the amplitude of 5 mV and the frequency range from 1 Hz to 100 kHz.
aptasensor can availably distinguish the co-existence of the two tumor markers from that only one tumor marker exists. In addition, Fig. 4 has also shown that no distinct electrochemical response can be observed even when 100 nM BSA is employed, which has proven the high selectivity of our aptasensor. Certainly, it should be mentioned that for the case of 10 nM MUC1 and 10 nM VEGF165 , it may have the same response for the case of 20 nM MUC1 only. Therefore, the highest electrochemical signal can only be observed when 20 nM MUC1 and 20 nM VEGF165 are simultaneously present. In other words, to distinguish the coexistence of the two tumor markers, we should first of all obtain the highest response of one tumor marker, and then get to know the highest response when both co-exist. 3.4. Electrochemical detection of tumor markers by EIS We have also conducted EIS experiments to confirm the availability of the detection of the two tumor markers by using our aptasensor. As is shown in Fig. 5, the modification of cDNA onto the gold electrode surface may significantly increase the electrontransfer resistance due to the steric hindrance, since the loop-stem structure of cDNA will prohibit [Fe(CN)6 ]3−/4− molecule reaching to the gold electrode and cDNA will electrostatically repel [Fe(CN)6 ]3−/4− . When both the tumor markers are absent, cDNA hybridizes with the aptamers of both tumor markers to form a rigid long double strand, which makes cDNA stand up off the surface of the electrode, resulting in availability of [Fe(CN)6 ]3−/4− to the surface, so a decreased impedance can be observed. Nevertheless, when 20 nM MUC1 is present, cDNA hybridizes with only VEGF165 aptamer to form a short double strand, which is much closer to the electrode surface, thus the impedance is bigger than that with a long double strand. Furthermore, when both 20 nM MUC1 and 20 nM VEGF165 are simultaneously present, the impedance value is almost as big as that obtained at the cDNA modified electrode, because the aptamers of the two tumor markers will combine with their targets instead of hybridizing with the cDNA. Therefore, similar to the SWV results, EIS has also illustrated that the presence of one and two tumor markers can be differentiated by our aptasensor. 4. Conclusions In summary, we have fabricated a simple, effective and selective “signal-on” electrochemical aptasensor for simultaneous
This work is supported by the National Science Fund for Distinguished Young Scholars (grant no. 20925520), the National Natural Science Foundation of China (grant no. 81172503), and the Leading Academic Discipline Project of Shanghai Municipal Education Commission (grant no. J50108). References Borbas, K.E., Ferreira, C.S.M., Perkins, A., Bruce, J.I., Missailidis, S., 2007. Bioconjug. Chem. 18 (4), 1205–1212. Carpini, J.D., Karam, A.K., Montgomery, L., 2010. Angiogenesis 13 (1), 43–58. Cheng, A.K.H., Su, H., Wang, Y.A., Yu, H.Z., 2009. Anal. Chem. 81 (15), 6130–6139. Cheung, K.L., Graves, C.R.L., Robertson, J.F.R., 2000. Cancer Treat. Rev. 26 (2), 91–102. Ellington, A.D., Szostak, J.W., 1990. Nature 346 (6287), 818–822. Ferrara, N., Gerber, H.P., LeCouter, J., 2003. Nat. Med. 9 (6), 669–676. Ferreira, C.S.M., Matthews, C.S., Missailidis, S., 2006. Tumour Biol. 27 (6), 289–301. Ghosh, S., Sullivan, C.A., Zerkowski, M.P., Molinaro, A.M., Rimm, D.L., Camp, R.L., Chung, G.G., 2008. Hum. Pathol. 39 (12), 1835–1843. Guo, P., Fang, Q., Tao, H.Q., Schafer, C.A., Fenton, B.M., Ding, I., Hu, B., Cheng, S.Y., 2003. Cancer Res. 63 (15), 4684–4691. Han, K., Chen, L., Lin, Z., Li, G., 2009. Electrochem. Commun. 11 (1), 157–160. Hasegawa, H., Sode, K., Ikebukuro, K., 2008. Biotechnol. Lett. 30 (5), 829–834. Hermann, T., Patel, D.J., 2000. Science 287 (5454), 820–825. Hianik, T., Wang, J., 2009. Electroanalysis 21 (11), 1223–1235. Huang, Y., Ge, B., Sen, D., Yu, H., 2008. J. Am. Chem. Soc. 130 (25), 8023–8029. Ikebukuro, K., Hasegawa, H., Sode, K., 2007. Nucleic Acids Symp. Ser. (Oxf). 51 (1), 399–400. Li, D., Song, S., Fan, C., 2010a. Acc. Chem. Res. 43 (5), 631–641. Li, T., Fan, Q., Liu, T., Zhu, X., Zhao, J., Li, G., 2010b. Biosens. Bioelectron. 25 (12), 2686–2689. Li, Y., Bao, J., Han, M., Dai, Z., Wang, H., 2011. Biosens. Bioelectron. 26 (8), 3531–3535. Linderholm, B., Tavelin, B., Grankvist, K., Henriksson, R., 1998. J. Clin. Oncol. 16 (9), 3121–3128. Linderholm, B.K., Gruvberger-Saal, S., Fernö, M., Bendahl, P.O., Malmström, P., 2008. Breast 17 (5), 484–491. Lu, Y., Li, X., Zhang, L., Yu, P., Su, L., Mao, L., 2008a. Anal. Chem. 80 (6), 1883–1890. Lu, Y., Zhu, N., Yu, P., Mao, L., 2008b. Analyst 133 (9), 1256–1260. Mukhopadhyay, P., Chakraborty, S., Ponnusamy, M.P., Lakshmanan, I., Jain, M., Batr, S.K., 2011. Biochim. Biophys. Acta 1815 (2), 224–240. Rahn, J.J., Dabbagh, L., Pasdar, M., Hugh, J.C., 2001. Cancer 91 (11), 1973–1982. Regimbald, L.H., Pilarski, L.M., Longenecker, B.M., Reddish, M.A., Zimmermann, G., Hugh, J.C., 1996. Cancer Res. 56 (18), 4244–4249. Ruckman, J., Green, L.S., Beeson, J., Waugh, S., Gillette, W.L., Henninger, D.D., ´ N., 1998. J. Biol. Chem. 273 (32), 20556–20567. Claesson-Welsh, L., Janjic, Sassolas, A., Blum, L.J., Leca-Bouvier, B.D., 2009. Electroanalysis 21 (11), 1237–1250. Shao, Z., Li, Y., Yang, Q., Wang, J., Li, G., 2010. Anal. Bioanal. Chem. 398 (7–8), 2963–2967. Shieh, Y.A., Yang, S.J., Wei, M.F., Shieh, M.J., 2010. ACS Nano 4 (3), 1433–1442. Shivakumar, S., Prabhakar, B.T., Jayashree, K., Rajan, M.G.R., Salimath, B.P., 2009. J. Cancer Res. Clin. Oncol. 135 (4), 627–636. Wang, J., Cao, Y., Chen, G., Li, G., 2009a. Chembiochem 10 (13), 2171–2176. Wang, J., Meng, W., Zheng, X., Huang, J., Li, G., 2009b. Biosens. Bioelectron. 24 (6), 1598–1602. Xiang, Y., Qian, X., Jiang, B., Chai, Y., Yuan, R., 2011. Chem. Commun. 47 (16), 4733–4735. Yu, B., Hu, H., Wang, D., Huck, W.T.S., Zhou, F., Liu, W., 2009. J. Mater. Chem. 19 (43), 8129–8134. Zhang, K., Zhu, X., Wang, J., Xu, L., Li, G., 2010. Anal. Chem. 82 (8), 3207–3211. Zhou, L., Ou, L.J., Chu, X., Shen, G.L., Yu, R.Q., 2007. Anal. Chem. 79 (19), 7492–7500. Zhu, X., Cao, Y., Liang, Z., Li, G., 2010. Protein Cell 1 (9), 842–846. Zhu, X., Zhao, J., Wu, Y., Shen, Z., Li, G., 2011. Anal. Chem. 83 (11), 4085–4089. Zuo, X., Song, S., Zhang, J., Pan, D., Wang, L., Fan, C., 2007. J. Am. Chem. Soc. 129 (5), 1042–1043.