- Email: [email protected]
Electrochemical detection of glutathione based on Hg2+-mediated strand displacement reaction strategy
Biosensors and Bioelectronics 85 (2016) 664–668
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Electrochemical detection of glutathione based on Hg2 þ -mediated strand displacement reaction strategy Yun Lv a, Lili Yang a, Xiaoxia Mao a, Mengjia Lu a, Jing Zhao a,n, Yongmei Yin b,n a b
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
art ic l e i nf o
a b s t r a c t
Article history: Received 18 March 2016 Received in revised form 10 May 2016 Accepted 21 May 2016 Available online 24 May 2016
Glutathione (GSH) plays an important role in numerous cellular functions, and the abnormal GSH expression is closely related with many dangerous human diseases. In this work, we have proposed a simple but sensitive electrochemical method for quantitative detection of GSH based on an Hg2 þ -mediated strand displacement reaction. Owing to the specific binding of Hg2 þ with T-T mismatches, helper DNA can bind to 3′ terminal of probe DNA 1 and initiate the displacement of probe DNA 2 immobilized on an electrode surface. However, Hg2 þ -mediated strand displacement reaction can be inhibited by the chelation of GSH with Hg2 þ , thereby leading to an obvious electrochemical response obtained from methylene blue that is modified onto the probe DNA. Our method can sensitively detect GSH in a wide linear range from 0.5 nM to 5 μM with a low detection limit of 0.14 nM, which can also easily distinguish target molecules in complex serum samples and even cell extractions. Therefore, this method may have great potential to monitor GSH in the physiological and pathological condition in the future. & 2016 Elsevier B.V. All rights reserved.
Keywords: Glutathione Hg2 þ Strand displacement reaction Electrochemical detection
1. Introduction Glutathione (GSH), which is also known as L-γ-glutamyl-L-cysteinylglycine, is a tripeptide composed of glutamic acid, cysteine and glycine (Hirrlinger and Dringen, 2010). As the most abundant biothiol in the living organisms, GSH plays a critical role in many cellular functions, including signal transduction, xenobiotic metabolism, gene regulation as well as the maintenance of intracellular redox balance (Grek et al., 2013; Raj et al., 2011; Samarasinghe et al., 2014; Yu et al., 2013). The abnormal level of cellular GSH has been found to be closely related with many dangerous human diseases, such as liver damage, heart diseases, rheumatoid arthritis, leukocyte loss, psoriasis, AIDs, and even cancers (Garai-Ibabe et al., 2013; Giustarini et al., 2011; Tew, 2016; Laborde, 2010; Ortega et al., 2011; Reid and Jahoor, 2001; Traverso et al., 2013; You and Park, 2010; Zhu et al., 2012). Besides, GSH is a well-known endogenous detoxicant, whose active thiol can easily interact with different components, including medicines, toxins and heavy metals (Cohen and Tcherpakov, 2010; Murphy, 2012; Perricone et al., 2009). And it is also a potent antioxidant, which has been widely used for anti-aging, anti-tumor and immunity enhancement (Chang and So, 2008; Hughes et al., 2015; Lu, 2014). n
Corresponding authors. E-mail addresses: [email protected] (J. Zhao), [email protected] (Y. Yin).
http://dx.doi.org/10.1016/j.bios.2016.05.069 0956-5663/& 2016 Elsevier B.V. All rights reserved.
Given the important roles of GSH in vivo and in vitro, it is still urgently needed to develop some simple but efficient methods for monitoring GSH under both physiological and pathological conditions. Over the past decades, a variety of analytical techniques have already been applied in the GSH determination, including highperformance liquid chromatography (HPLC), mass spectrometry (MS), colorimetry and fluorescent techniques (Choi et al., 2011; Gu et al., 2015; Li et al., 2011; Liu et al., 2010; Marchand and de Revel, 2010; Mandal et al., 2012; McMahon and Gunnlaugsson, 2012; Squellerio et al., 2012). Nonetheless, the existing methods always suffer from several disadvantages, such as low specificity, high cost, sophisticated instrument manipulation and time-consuming sample pretreatment. Benefiting from the advantages of low cost, rapid response, convenient operation, high sensitivity and specificity, electrochemical technique has become a popular technique in GSH detection. Miao et al. have reported an electrochemical sensing strategy by using two gold electrodes and two complementary thiolated oligonucleotides (Miao et al., 2009). Safavi et al. have presented an electrochemical method based on the complexation of Cu (II) with GSH by using a nanoscale copper hydroxide composite carbon ionic liquid electrode (Safavi et al., 2009). Shahmiri et al. have designed a voltammetric sensor by using an ethynylferrocene (EF) and NiO/MWCNT nanocomposite modified carbon paste electrode (Shahmiri et al., 2013). Yuan et al. have presented an electrocatalytic method by using
Y. Lv et al. / Biosensors and Bioelectronics 85 (2016) 664–668
Cu2O/NiOx/graphene oxide (GO) modified glassy carbon electrode (Yuan et al., 2013). However, these above-mentioned methods always need a relatively complicated modification process, whose sensitivity may also be restrained by the direct electrochemical response of GSH on the electrode surface, so more simple but sensitive electrochemical methods should be developed. DNA molecule has been a favorable element for the fabrication of an effective biosensor, which can offer many choices for molecular recognition, signal label and even signal amplification (Zhao et al., 2013a, 2015). Among them, the selective binding of metal ions with the native or artificial DNA bases has aroused increasing interests in DNA-based analysis. For example, Hg2 þ is able to interact with thymine-thymine (T-T) mismatch with high affinity, while cytosine-cytosine (C-C) pairs can exclusively capture Ag þ (Li et al., 2015; Yuan et al., 2014; Zhao et al., 2013b). Because of the high binding specificity between DNA bases and metal ions, the metal-mediated base pairs have been employed for ions detection, logic gate construction, and even triggering DNA amplification reactions (Zhao et al., 2013c; Zhad and Lai, 2014). Recently, the selective formation of T-Hg2 þ -T has been proven to act as a regulatory factor to modulate the rate of toehold-mediated DNA strand displacement reaction, providing a potential tool for the design of molecular machines (Ding et al., 2013). In this work, by making use of Hg2 þ -mediated strand displacement reaction, we have proposed a simple but efficient “signal-on” electrochemical method for GSH detection. The specific interaction between Hg2 þ and thymine bases can induce the dissociation of methylene blue (MB)-labeled double-stranded probe DNA on the electrode surface, while the high-affinity binding of GSH and heavy metal ions can inhibit Hg2 þ -mediated strand displacement reaction. Therefore, the sensitive detection of GSH in the buffer and even in the living cells can be realized by tracing the changes of electrochemical signals of the MB molecules are labeled on the double-stranded probe DNA.
2. Materials and experiment 2.1. Chemicals and Materials L-Glutathionevoxidized (GSH), L-cysteine (cys), phenylalanine (phe), glutamic acid (glu), thrombin, Mercaptoethanol (MCH), RIPA lysis buffer, protease inhibitors, N-ethyl maleimide (NEM) and Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Sigma. Bovine serum albumin (BSA) and fetal bovine serum were purchased from Beijing Dingguochangsheng biotech Co., Ltd. HeLa cell was purchased from Shanghai SunBio Biomedical technology Co., Ltd. For all experiments, Milli-Q water (4 18.0 MΩ) was used, which was purified by a Milli-Q Plus 185ultrapure water system (Millipore purification pack). DNA oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequences are as follows. Probe DNA 1:5′-SH-CCCCCTCTATACCGTACCTTTTTT-3′; Probe DNA 2:5′-GGTACGGTATAGAG- MB-3′; Helper DNA: 5′-TTTTTTGGTACGGTATAGAG-3′. The buffer solutions used in this work are as follows. DNA immobilization buffer: 10 mM Tris–HCl, 1 mM EDTA and 0.1 M NaCl (pH 7.4); buffer for strand displacement reactions: 10 mM Tris–HCl, 0.1 mM NaCl; Cell washing buffer(PBS): 137 mM NaCl, 2.7 mM KCl,10 mM Na2HPO4, 2 mM KH2PO4 (pH 7.4); RIPA lysis buffer: 150 mM NaCl, 1.0% IGEPALs CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris (pH 8.0); buffer for square wave voltammetry (SWV): 10 mM Tris–HCl (pH 7.4).
665
2.2. Preparation of probe DNA-modified gold electrode Firstly, the substrate gold electrode was polished on silk with 1 mm, 0.3 mm and 0.05 mm alumina slurry, separately. Then, the electrode was ultrasonicated in both ethanol and double-distilled water for 5 min, respectively. After being cleaned with piranha solution (H2SO4:H2O2 ¼3:1) for 5 min, and then rinsed by doubledistilled water, the electrode was electrochemically cleaned to remove any remaining impurities in 0.5 M H2SO4. After being dried with nitrogen, the electrode was prepared for DNA immobilization. For DNA hybridization, a mixture containing 500 nM probe DNA 1 and probe DNA 2 was heated to 95 °C for 5 min and then cooled down slowly to room temperature before use. Then, the DNA mixture was incubated with the pretreated gold electrode at room temperature for 16 h, followed by the treatment with 1 mM MCH for 1 h in order to obtain well-aligned double-stranded probe DNA monolayers. 2.3. GSH extraction from Hela cells The Hela cell line was grown in DMEM high glucose medium supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator (5% CO2–95% air). The cells were harvested at the logarithmic growth phase by trypsinization. Then, the cell suspension was centrifuged at 3000 rpm for 5 min, and the obtained cell precipitate was dispersed in PBS. The washing process was repeated twice. Subsequently, 100 μL of the RIPA lysis buffer and 1 μL protease inhibitors were added into the cell precipitate, which was incubated for 1 h on the ice. Afterward, the lysate was centrifuged at 12,000 rpm for 20 min at 4 °C in order to remove the cell debris, and the supernatant containing GSH was stored in 20 °C for the further use. 2.4. Electrochemical detection of GSH For the strand displacement reaction, 10 μM Hg2 þ and 300 nM Helper DNA were incubated with probe DNA modified electrode at 37 °C for 60 min. In order to detect GSH, different concentration of GSH was added into the above solution containing Hg2 þ and helper DNA before the incubation with electrode. For the control experiments, 50 μM cys, glu, phe, BSA and thrombin were separately added instead of GSH. For the detection in the complex samples, the bovine serum (1:100 diluted) was used instead of the buffer. GSH extraction from Hela cells was used as the real sample instead of GSH solution in the assay of the cells. For the thiolblocking studies, NEM was incubated with GSH before the reaction. Electrochemical measurements were carried out on a model 660c Electrochemical Analyzer (CH Instruments). A conventional three-electrode system was employed in electrochemical measurements, consisting of a gold electrode as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire as the counter electrode. All the electrolytes were thoroughly deoxygenated by bubbling high-purity nitrogen through the solutions for at least 20 min before the measurements, and a stream of nitrogen was blown gently across the surface of the solution in order to maintain the solution anaerobic throughout all the experiments.
3. Results and discussion 3.1. The principle of the method Fig. 1 may illustrate the principle of our method. Doublestranded probe DNA, derived from the hybridization between probe DNA 1 and probe DNA 2, can be self-assembled on the
666
Y. Lv et al. / Biosensors and Bioelectronics 85 (2016) 664–668
Fig. 1. Schematic illustration of electrochemical detection of glutathione based on Hg2 þ -mediated strand displacement reaction strategy.
electrode surface through the interaction between the thiol group at 5′-terminal of probe DNA 1 and the gold electrode. Hence, an obvious electrochemical signal can be induced due to the electron transfer between MB modified at 3′-terminal of probe DNA 2 and the electrode surface. In the presence of Hg2 þ ions, a short singlestranded “toehold” domain at 5′-terminal of helper DNA (TTTTTT) can attached to 3′-terminal of probe DNA 1 through the selective binding of Hg2 þ to T-T mismatches. Then, the formation of stable T-Hg2 þ -T complexes can trigger the metallo-toehold strand displacement and thus release MB-labeled probe DNA 2 from the electrode surface. However, because the complexation between GSH and Hg2 þ is more favorable than that between T-T mismatches and Hg2 þ , the chelation of GSH to Hg2 þ can inhibit Hg2 þ -mediated strand displacement by destroying T-Hg2 þ -T complexes in the presence of GSH. In this case, the well-maintained double-stranded probe DNA at the electrode surface can result in an obvious electrochemical response as mentioned above. Therefore, the quantitative determination of GSH can be achieved in a “signal-on” manner by tracing the electrochemical response of MB labeled on the probe DNA.
Fig. 2. SWV responses obtained at probe DNA modified electrode surface (curve a), and the electrode after Hg2 þ -mediated strand displacement reactions in the absence or presence of GSH (curve b and c). GSH concentration is 50 μM. Potential step: 4 mV. Amplitude: 50 mV. The reported SWV curves have been baseline correction.
3.2. The electrochemical studies of our method
3.3. The electrochemical detection of GSH
We have employed a sensitive electrochemical technique square wave voltammetry (SWV) to prove the principle of our detection. As shown in Fig. 2, a high peak current at 0.25 V can be observed at double-stranded probe DNA modified electrode (curve a), indicating the electron transfer of the signal molecule MB with the electrode surface. After the addition of Hg2 þ , a quite low electrochemical response can be observed at the electrode surface, ascribing to the release of MB modified probe DNA 2 from probe DNA 1 through Hg2 þ -mediated strand displacement reaction (curve b). The optimization experiments have further shown that Hg2 þ -mediated strand displacement is time-and concentration-dependent (Figs. S1 and S2). In the presence of GSH, the chelation of GSH to Hg2 þ can inhibit Hg2 þ -mediated strand displacement reaction, so a high electrochemical signal similar to that at double-stranded probe DNA modified electrode can be obtained, suggesting the preserved double-stranded probe DNA on the electrode surface (curve c). Therefore, SWV studies have firstly verified feasibility of the principle of our detection.
Then, we have conducted more detailed experiments to study the changes of the electrochemical signals with different concentrations of GSH. The addition of GSH can facilitate the inhibition of the toehold-mediated strand displacement by interfering with the formation of T-Hg2 þ -T complexes, so increased amount of MB-modified probe DNA 2 can be remained on the electrode surface for the produce of the electrochemical response. As a result, the peak current has been found to rise with the increase of GSH concentration as shown in Fig. 3. The inset of Fig. 3 has further revealed a relationship between the peak current and the logarithm of GSH concentrations in the range from 0.5 nM to 5 μM. The regression equation is I (μA)¼0.7647 þ0.1906 lgC (μM), R2 ¼0.998. The limit of detection (LOD) is calculated to be 0.14 nM defined at a signal-to-noise ratio of three. We have compared this method with other existing methods. As shown in Table 1, our method has displayed a lower detection limit and a wider linear range, showing the satisfactory sensitivity of our method (Ni et al., 2015; Safavi et al., 2009; Shahmiri et al., 2013; Shi et al., 2014; Yuan et al., 2013). Moreover, the measurements of
Y. Lv et al. / Biosensors and Bioelectronics 85 (2016) 664–668
667
Fig. 4. SWV responses obtained in the presence of GSH, cysteine, glutamic acid, phenylalanine, BSA and thrombin. Fig. 3. SWV responses obtained with different concentrations of GSH (From a to l: 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM). The inset of Figure shows a linear relationship between peak current and log of GSH concentration from 0.5 nM to 5 μM. Error bars represent the standard deviations of three independent measurements.
each concentration have been repeated for at least three times. The relative standard deviations (RSD) are all within 10%, and the average of the RSDs is 5.38%, which has suggested the well reproducibility of our detection. 3.4. The specificity of our detection Control experiments have been performed to prove the specificity of our detection. As shown in Fig. 4, a high peak current can be obtained in the presence of GSH, while quite low electrochemical responses can be obtained in the presence of the control molecules, no matter amino acids or the proteins (glutamic acid, phenylalanine, thrombin or BSA). The control results has also shown that cysteine with an active thiol group can be easily discriminated by our method, whose electrochemical response is much lower than that with GSH. This is consistent with the previous studies. The coordination of GSH and heavy metal ions depends on two or more sites within GSH, e.g. the thiol group and the carboxy group, which is more stable than the amino acid with only one functional group, such as cysteine (Li et al., 2011). So, the control experiments have confirmed the high specificity of our method. Moreover, the applicability of our method in the complex biological samples have been studied by taking the fetal calf serum as a contaminated example and Hela cells as a real sample. As shown in Fig. 5, the high peak currents can be obtained when taking
Fig. 5. GSH detection in HeLa cell extraction and the serum sample in the absence or presence of NEM. The cell concentration is 1 105 cell/mL, and GSH concentration in the serum is 100 nM.
either GSH in the diluted serum or GSH extraction from Hela cells as the sample. As a comparison, when the thiol-blocking agent NEM that can effectively decompose GSH has been introduced into the GSH samples, the electrochemical signals have significantly reduced. The comparisons have clearly shown that the electrochemical responses obtained in both the serum sample and cell extraction can be ascribed to the presence of GSH. The recoveries of the given concentration in the serum samples have been between 102% and 108% (Table S1), showing the good selectivity of our method. Therefore, the experiments in complex samples have
Table 1. The comparison of our method with other existing methods. Methods
LOD
Linear range
Ref.
Simultaneous electrochemical determination of glutathione and glutathione disulfide at a nanoscale copper hydroxide composite carbon ionic liquid electrode Ethynylferrocene-NiO/MWCNT nanocomposite modified carbon paste electrode as a novel voltammetric sensor for simultaneous determination of glutathione and acetaminophen Cu2O/NiOx/graphene oxide modified glassy carbon electrode for the enhanced electrochemical oxidation of reduced glutathione and nonenzyme glucose sensor Highly sensitive and selective colorimetric detection of glutathione based on Ag [I] ion -3, 3′, 5, 5′- tetramethylbenzidine (TMB) A dual-mode nanosensor based on carbon quantum dots and gold nanoparticles for discriminative detection of glutathione in human plasma Electrochemical detection of glutathione based on mercury ion mediated strand displacement reactions
30 nM
1–50 μM
Safavi et al., 2009
6 nM
0.01–200 μM
Shahmiri et al., 2013
0.3 μM
2 μM–1.3 mM
Yuan et al., 2013
0.05 μM
0.05–8 μM
Ni et al., 2015
50 nM
1–4 μM
Shi et al., 2014
0.14 nM
0.5 nM–5 μM
Our work
668
Y. Lv et al. / Biosensors and Bioelectronics 85 (2016) 664–668
not only reconfirmed the high selectivity of our methods, but also suggested the great potential of our method for the clinical use in the future.
4. Conclusions In summary, we have proposed a simple but efficient electrochemical method for GSH detection based on Hg2 þ -mediated strand displacement reaction. The coordination of metal ions and specific bases (e.g. Hg2 þ and T-T mismatches) can modulate the strand displacement reaction on the electrode surface, which can be inhibited by the high-affinity chelation of GSH to heavy metal ions. In this case, a “signal-on” electrochemical detection of GSH can be realized by tracing the electrochemical response of MB on the electrode surface. This method has been proven to be highly specific, which can effectively discriminate the target molecules in both the serum samples and the cell samples. Therefore, our method with the improved specificity and sensitivity can provide an alternative for monitoring GSH accurately in the physiological or pathological condition, which is of great clinical significance to the diagnosis and treatment of the related diseases. Compared to the existing methods, our method that takes the unique advantages of both the electrochemical technique and the strand displacement reaction is low cost and simple to operate, which may be feasible to the miniaturization and integration in the future. Moreover, our work may also provide some suggestions to the application of toehold-mediated strand displacement in DNAbased analysis, which may facilitate the improvement of the sensitivity and specificity of the biosensing.
Acknowledgements This work is supported by the National Natural Science Foundation of China (Grant No. 31200745) and the Innovation Program of Shanghai Municipal Education Commission (Grant No. 14YZ026).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.05.069.
References Cohen, P., Tcherpakov, M., 2010. Cell 143, 686–693. Chang, R.C.C., So, K.F., 2008. Cell. Mol. Neurobiol. 28, 643–652. Choi, I.Y., Lee, S.P., Denney, D.R., Lynch, S.G., 2011. Mult. Scler. 17, 289–296. Ding, W., Deng, W., Zhu, H., Liang, H., 2013. Chem. Commun. 49, 9953–9955. Grek, C.L., Zhang, J., Manevich, Y., Townsend, D.M., Tew, K.D., 2013. J. Biol. Chem. 288, 26497–26504. Garai-Ibabe, G., Saa, L., Pavlov, V., 2013. Anal. Chem. 85, 5542–5546. Giustarini, D., Dalle-Donne, I., Milzani, A., Rossi, R., 2011. Anal. Biochem. 415, 81–83. Gu, J., Hu, D., Wang, W., Zhang, Q., Meng, Z., Jia, X., Xi, K., 2015. Biosens. Bioelectron. 68, 27–33. Hirrlinger, J., Dringen, R., 2010. Brain Res. Rev. 63, 177–188. Hughes, T.B., Miller, G.P., Swamidass, S.J., 2015. Chem. Res. Toxicol. 28, 797–809. Laborde, E., 2010. Cell Death Differ. 17, 1373–1380. Lu, Y., Ye, M., Song, S., Li, L., Shaikh, F., Li, J., 2014. Appl. Biochem. Biotech. 174, 762–771. Liu, J., Bao, C., Zhong, X., Zhu, L., 2010. Chem. Commun. 46, 2971–2973. Li, Q.H., Zhang, L., Bai, J.M., Liu, Z.C., Liang, R.P., Qiu, J.D., 2015. Biosens. Bioelectron. 74, 886–894. Li, Y., Wu, P., Xu, H., Zhang, H., Zhong, X., 2011. Analyst 136, 196–200. Murphy, M.P., 2012. Antioxid. Redox Signal. 16, 476–495. Marchand, S., de Revel, G., 2010. Anal. Chim. Acta 660, 158–163. Mandal, P.K., Tripathi, M., Sugunan, S., 2012. Biochem. Biophys. Res. Commun. 417, 43–48. McMahon, B.K., Gunnlaugsson, T., 2012. J. Am. Chem. Soc. 134, 10725–10728. Miao, P., Liu, L., Nie, Y., Li, G., 2009. Biosens. Bioelectron. 24, 3347–3351. Ni, P., Sun, Y., Dai, H., Hu, J., Jiang, S., Wang, Y., Li, Z., 2015. Biosens. Bioelectron. 63, 47–52. Ortega, A.L., Mena, S., Estrela, J.M., 2011. Cancers 3, 1285–1310. Perricone, C., De Carolis, C., Perricone, R., 2009. Autoimmun. Rev. 8, 697–701. Raj, L., Ide, T., Gurkar, A.U., Foley, M., Schenone, M., Li, X., Stern, A.M., 2011. Nature 475, 231–234. Reid, M., Jahoor, F., 2001. Curr. Opin. Clin. Nutr. 4, 65–71. Samarasinghe, K.T.G., Munkanatta Godage, D.N.P., VanHecke, G.C., Ahn, Y.H., 2014. J. Am. Chem. Soc. 136, 11566–11569. Squellerio, I., Caruso, D., Porro, B., Veglia, F., Tremoli, E., Cavalca, V., 2012. J. Pharm. Biomed. 71, 111–118. Safavi, A., Maleki, N., Farjami, E., Mahyari, F.A., 2009. Anal. Chem. 81, 7538–7543. Shahmiri, M.R., Bahari, A., Karimi-Maleh, H., Hosseinzadeh, R., Mirnia, N., 2013. Sens. Actuators B: Chem. 177, 70–77. Shi, Y., Pan, Y., Zhang, H., Zhang, Z., Li, M.J., Yi, C., Yang, M., 2014. Biosens. Bioelectron. 56, 39–45. Traverso, N., Ricciarelli, R., Nitti, M., Marengo, B., Furfaro, A.L., Pronzato, M.A., Domenicotti, C., 2013. Oxidative Med. Cell. Longev. 2013, 1–10. Tew, K.D., 2016. Cancer Res. 76, 7–9. Yu, F., Li, P., Wang, B., Han, K., 2013. J. Am. Chem. Soc. 135, 7674–7680. You, B.R., Park, W.H., 2010. Toxicol. Vitr. 24, 1356–1362. Yuan, B., Xu, C., Liu, L., Zhang, Q., Ji, S., Pi, L., Huo, Q., 2013. Electrochim. Acta 104, 78–83. Yuan, Y., Li, W., Liu, Z., Nie, Z., Huang, Y., Yao, S., 2014. Biosens. Bioelectron. 61, 321–327. Zhu, R., Wang, Y., Zhang, L., Guo, Q., 2012. Hepatol. Res. 42, 741–749. Zhao, J., Zhu, L., Guo, C., Gao, T., Zhu, X., Li, G., 2013a. Biosens. Bioelectron. 49, 329–333. Zhao, J., Lv, Y., Kang, M., Wang, K., Xiang, Y., 2015. Analyst 140, 7818–7822. Zhao, J., Fan, Q., Zhu, S., Duan, A., Yin, Y., Li, G., 2013a. Biosens. Bioelectron. 39, 183–186. Zhao, J., Chen, C., Zhang, L., Jiang, J., Shen, G., Yu, R., 2013b. Analyst 138, 1713–1718. Zhad, H.R.L.Z., Lai, R.Y., 2014. Chem. Commun. 50, 8385–8387.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Electrochemical detection of glutathione based on Hg2 þ -mediated strand displacement reaction strategy Yun Lv a, Lili Yang a, Xiaoxia Mao a, Mengjia Lu a, Jing Zhao a,n, Yongmei Yin b,n a b
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
art ic l e i nf o
a b s t r a c t
Article history: Received 18 March 2016 Received in revised form 10 May 2016 Accepted 21 May 2016 Available online 24 May 2016
Glutathione (GSH) plays an important role in numerous cellular functions, and the abnormal GSH expression is closely related with many dangerous human diseases. In this work, we have proposed a simple but sensitive electrochemical method for quantitative detection of GSH based on an Hg2 þ -mediated strand displacement reaction. Owing to the specific binding of Hg2 þ with T-T mismatches, helper DNA can bind to 3′ terminal of probe DNA 1 and initiate the displacement of probe DNA 2 immobilized on an electrode surface. However, Hg2 þ -mediated strand displacement reaction can be inhibited by the chelation of GSH with Hg2 þ , thereby leading to an obvious electrochemical response obtained from methylene blue that is modified onto the probe DNA. Our method can sensitively detect GSH in a wide linear range from 0.5 nM to 5 μM with a low detection limit of 0.14 nM, which can also easily distinguish target molecules in complex serum samples and even cell extractions. Therefore, this method may have great potential to monitor GSH in the physiological and pathological condition in the future. & 2016 Elsevier B.V. All rights reserved.
Keywords: Glutathione Hg2 þ Strand displacement reaction Electrochemical detection
1. Introduction Glutathione (GSH), which is also known as L-γ-glutamyl-L-cysteinylglycine, is a tripeptide composed of glutamic acid, cysteine and glycine (Hirrlinger and Dringen, 2010). As the most abundant biothiol in the living organisms, GSH plays a critical role in many cellular functions, including signal transduction, xenobiotic metabolism, gene regulation as well as the maintenance of intracellular redox balance (Grek et al., 2013; Raj et al., 2011; Samarasinghe et al., 2014; Yu et al., 2013). The abnormal level of cellular GSH has been found to be closely related with many dangerous human diseases, such as liver damage, heart diseases, rheumatoid arthritis, leukocyte loss, psoriasis, AIDs, and even cancers (Garai-Ibabe et al., 2013; Giustarini et al., 2011; Tew, 2016; Laborde, 2010; Ortega et al., 2011; Reid and Jahoor, 2001; Traverso et al., 2013; You and Park, 2010; Zhu et al., 2012). Besides, GSH is a well-known endogenous detoxicant, whose active thiol can easily interact with different components, including medicines, toxins and heavy metals (Cohen and Tcherpakov, 2010; Murphy, 2012; Perricone et al., 2009). And it is also a potent antioxidant, which has been widely used for anti-aging, anti-tumor and immunity enhancement (Chang and So, 2008; Hughes et al., 2015; Lu, 2014). n
Corresponding authors. E-mail addresses: [email protected] (J. Zhao), [email protected] (Y. Yin).
http://dx.doi.org/10.1016/j.bios.2016.05.069 0956-5663/& 2016 Elsevier B.V. All rights reserved.
Given the important roles of GSH in vivo and in vitro, it is still urgently needed to develop some simple but efficient methods for monitoring GSH under both physiological and pathological conditions. Over the past decades, a variety of analytical techniques have already been applied in the GSH determination, including highperformance liquid chromatography (HPLC), mass spectrometry (MS), colorimetry and fluorescent techniques (Choi et al., 2011; Gu et al., 2015; Li et al., 2011; Liu et al., 2010; Marchand and de Revel, 2010; Mandal et al., 2012; McMahon and Gunnlaugsson, 2012; Squellerio et al., 2012). Nonetheless, the existing methods always suffer from several disadvantages, such as low specificity, high cost, sophisticated instrument manipulation and time-consuming sample pretreatment. Benefiting from the advantages of low cost, rapid response, convenient operation, high sensitivity and specificity, electrochemical technique has become a popular technique in GSH detection. Miao et al. have reported an electrochemical sensing strategy by using two gold electrodes and two complementary thiolated oligonucleotides (Miao et al., 2009). Safavi et al. have presented an electrochemical method based on the complexation of Cu (II) with GSH by using a nanoscale copper hydroxide composite carbon ionic liquid electrode (Safavi et al., 2009). Shahmiri et al. have designed a voltammetric sensor by using an ethynylferrocene (EF) and NiO/MWCNT nanocomposite modified carbon paste electrode (Shahmiri et al., 2013). Yuan et al. have presented an electrocatalytic method by using
Y. Lv et al. / Biosensors and Bioelectronics 85 (2016) 664–668
Cu2O/NiOx/graphene oxide (GO) modified glassy carbon electrode (Yuan et al., 2013). However, these above-mentioned methods always need a relatively complicated modification process, whose sensitivity may also be restrained by the direct electrochemical response of GSH on the electrode surface, so more simple but sensitive electrochemical methods should be developed. DNA molecule has been a favorable element for the fabrication of an effective biosensor, which can offer many choices for molecular recognition, signal label and even signal amplification (Zhao et al., 2013a, 2015). Among them, the selective binding of metal ions with the native or artificial DNA bases has aroused increasing interests in DNA-based analysis. For example, Hg2 þ is able to interact with thymine-thymine (T-T) mismatch with high affinity, while cytosine-cytosine (C-C) pairs can exclusively capture Ag þ (Li et al., 2015; Yuan et al., 2014; Zhao et al., 2013b). Because of the high binding specificity between DNA bases and metal ions, the metal-mediated base pairs have been employed for ions detection, logic gate construction, and even triggering DNA amplification reactions (Zhao et al., 2013c; Zhad and Lai, 2014). Recently, the selective formation of T-Hg2 þ -T has been proven to act as a regulatory factor to modulate the rate of toehold-mediated DNA strand displacement reaction, providing a potential tool for the design of molecular machines (Ding et al., 2013). In this work, by making use of Hg2 þ -mediated strand displacement reaction, we have proposed a simple but efficient “signal-on” electrochemical method for GSH detection. The specific interaction between Hg2 þ and thymine bases can induce the dissociation of methylene blue (MB)-labeled double-stranded probe DNA on the electrode surface, while the high-affinity binding of GSH and heavy metal ions can inhibit Hg2 þ -mediated strand displacement reaction. Therefore, the sensitive detection of GSH in the buffer and even in the living cells can be realized by tracing the changes of electrochemical signals of the MB molecules are labeled on the double-stranded probe DNA.
2. Materials and experiment 2.1. Chemicals and Materials L-Glutathionevoxidized (GSH), L-cysteine (cys), phenylalanine (phe), glutamic acid (glu), thrombin, Mercaptoethanol (MCH), RIPA lysis buffer, protease inhibitors, N-ethyl maleimide (NEM) and Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Sigma. Bovine serum albumin (BSA) and fetal bovine serum were purchased from Beijing Dingguochangsheng biotech Co., Ltd. HeLa cell was purchased from Shanghai SunBio Biomedical technology Co., Ltd. For all experiments, Milli-Q water (4 18.0 MΩ) was used, which was purified by a Milli-Q Plus 185ultrapure water system (Millipore purification pack). DNA oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequences are as follows. Probe DNA 1:5′-SH-CCCCCTCTATACCGTACCTTTTTT-3′; Probe DNA 2:5′-GGTACGGTATAGAG- MB-3′; Helper DNA: 5′-TTTTTTGGTACGGTATAGAG-3′. The buffer solutions used in this work are as follows. DNA immobilization buffer: 10 mM Tris–HCl, 1 mM EDTA and 0.1 M NaCl (pH 7.4); buffer for strand displacement reactions: 10 mM Tris–HCl, 0.1 mM NaCl; Cell washing buffer(PBS): 137 mM NaCl, 2.7 mM KCl,10 mM Na2HPO4, 2 mM KH2PO4 (pH 7.4); RIPA lysis buffer: 150 mM NaCl, 1.0% IGEPALs CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris (pH 8.0); buffer for square wave voltammetry (SWV): 10 mM Tris–HCl (pH 7.4).
665
2.2. Preparation of probe DNA-modified gold electrode Firstly, the substrate gold electrode was polished on silk with 1 mm, 0.3 mm and 0.05 mm alumina slurry, separately. Then, the electrode was ultrasonicated in both ethanol and double-distilled water for 5 min, respectively. After being cleaned with piranha solution (H2SO4:H2O2 ¼3:1) for 5 min, and then rinsed by doubledistilled water, the electrode was electrochemically cleaned to remove any remaining impurities in 0.5 M H2SO4. After being dried with nitrogen, the electrode was prepared for DNA immobilization. For DNA hybridization, a mixture containing 500 nM probe DNA 1 and probe DNA 2 was heated to 95 °C for 5 min and then cooled down slowly to room temperature before use. Then, the DNA mixture was incubated with the pretreated gold electrode at room temperature for 16 h, followed by the treatment with 1 mM MCH for 1 h in order to obtain well-aligned double-stranded probe DNA monolayers. 2.3. GSH extraction from Hela cells The Hela cell line was grown in DMEM high glucose medium supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator (5% CO2–95% air). The cells were harvested at the logarithmic growth phase by trypsinization. Then, the cell suspension was centrifuged at 3000 rpm for 5 min, and the obtained cell precipitate was dispersed in PBS. The washing process was repeated twice. Subsequently, 100 μL of the RIPA lysis buffer and 1 μL protease inhibitors were added into the cell precipitate, which was incubated for 1 h on the ice. Afterward, the lysate was centrifuged at 12,000 rpm for 20 min at 4 °C in order to remove the cell debris, and the supernatant containing GSH was stored in 20 °C for the further use. 2.4. Electrochemical detection of GSH For the strand displacement reaction, 10 μM Hg2 þ and 300 nM Helper DNA were incubated with probe DNA modified electrode at 37 °C for 60 min. In order to detect GSH, different concentration of GSH was added into the above solution containing Hg2 þ and helper DNA before the incubation with electrode. For the control experiments, 50 μM cys, glu, phe, BSA and thrombin were separately added instead of GSH. For the detection in the complex samples, the bovine serum (1:100 diluted) was used instead of the buffer. GSH extraction from Hela cells was used as the real sample instead of GSH solution in the assay of the cells. For the thiolblocking studies, NEM was incubated with GSH before the reaction. Electrochemical measurements were carried out on a model 660c Electrochemical Analyzer (CH Instruments). A conventional three-electrode system was employed in electrochemical measurements, consisting of a gold electrode as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire as the counter electrode. All the electrolytes were thoroughly deoxygenated by bubbling high-purity nitrogen through the solutions for at least 20 min before the measurements, and a stream of nitrogen was blown gently across the surface of the solution in order to maintain the solution anaerobic throughout all the experiments.
3. Results and discussion 3.1. The principle of the method Fig. 1 may illustrate the principle of our method. Doublestranded probe DNA, derived from the hybridization between probe DNA 1 and probe DNA 2, can be self-assembled on the
666
Y. Lv et al. / Biosensors and Bioelectronics 85 (2016) 664–668
Fig. 1. Schematic illustration of electrochemical detection of glutathione based on Hg2 þ -mediated strand displacement reaction strategy.
electrode surface through the interaction between the thiol group at 5′-terminal of probe DNA 1 and the gold electrode. Hence, an obvious electrochemical signal can be induced due to the electron transfer between MB modified at 3′-terminal of probe DNA 2 and the electrode surface. In the presence of Hg2 þ ions, a short singlestranded “toehold” domain at 5′-terminal of helper DNA (TTTTTT) can attached to 3′-terminal of probe DNA 1 through the selective binding of Hg2 þ to T-T mismatches. Then, the formation of stable T-Hg2 þ -T complexes can trigger the metallo-toehold strand displacement and thus release MB-labeled probe DNA 2 from the electrode surface. However, because the complexation between GSH and Hg2 þ is more favorable than that between T-T mismatches and Hg2 þ , the chelation of GSH to Hg2 þ can inhibit Hg2 þ -mediated strand displacement by destroying T-Hg2 þ -T complexes in the presence of GSH. In this case, the well-maintained double-stranded probe DNA at the electrode surface can result in an obvious electrochemical response as mentioned above. Therefore, the quantitative determination of GSH can be achieved in a “signal-on” manner by tracing the electrochemical response of MB labeled on the probe DNA.
Fig. 2. SWV responses obtained at probe DNA modified electrode surface (curve a), and the electrode after Hg2 þ -mediated strand displacement reactions in the absence or presence of GSH (curve b and c). GSH concentration is 50 μM. Potential step: 4 mV. Amplitude: 50 mV. The reported SWV curves have been baseline correction.
3.2. The electrochemical studies of our method
3.3. The electrochemical detection of GSH
We have employed a sensitive electrochemical technique square wave voltammetry (SWV) to prove the principle of our detection. As shown in Fig. 2, a high peak current at 0.25 V can be observed at double-stranded probe DNA modified electrode (curve a), indicating the electron transfer of the signal molecule MB with the electrode surface. After the addition of Hg2 þ , a quite low electrochemical response can be observed at the electrode surface, ascribing to the release of MB modified probe DNA 2 from probe DNA 1 through Hg2 þ -mediated strand displacement reaction (curve b). The optimization experiments have further shown that Hg2 þ -mediated strand displacement is time-and concentration-dependent (Figs. S1 and S2). In the presence of GSH, the chelation of GSH to Hg2 þ can inhibit Hg2 þ -mediated strand displacement reaction, so a high electrochemical signal similar to that at double-stranded probe DNA modified electrode can be obtained, suggesting the preserved double-stranded probe DNA on the electrode surface (curve c). Therefore, SWV studies have firstly verified feasibility of the principle of our detection.
Then, we have conducted more detailed experiments to study the changes of the electrochemical signals with different concentrations of GSH. The addition of GSH can facilitate the inhibition of the toehold-mediated strand displacement by interfering with the formation of T-Hg2 þ -T complexes, so increased amount of MB-modified probe DNA 2 can be remained on the electrode surface for the produce of the electrochemical response. As a result, the peak current has been found to rise with the increase of GSH concentration as shown in Fig. 3. The inset of Fig. 3 has further revealed a relationship between the peak current and the logarithm of GSH concentrations in the range from 0.5 nM to 5 μM. The regression equation is I (μA)¼0.7647 þ0.1906 lgC (μM), R2 ¼0.998. The limit of detection (LOD) is calculated to be 0.14 nM defined at a signal-to-noise ratio of three. We have compared this method with other existing methods. As shown in Table 1, our method has displayed a lower detection limit and a wider linear range, showing the satisfactory sensitivity of our method (Ni et al., 2015; Safavi et al., 2009; Shahmiri et al., 2013; Shi et al., 2014; Yuan et al., 2013). Moreover, the measurements of
Y. Lv et al. / Biosensors and Bioelectronics 85 (2016) 664–668
667
Fig. 4. SWV responses obtained in the presence of GSH, cysteine, glutamic acid, phenylalanine, BSA and thrombin. Fig. 3. SWV responses obtained with different concentrations of GSH (From a to l: 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, 5 μM, 10 μM, 25 μM, 50 μM). The inset of Figure shows a linear relationship between peak current and log of GSH concentration from 0.5 nM to 5 μM. Error bars represent the standard deviations of three independent measurements.
each concentration have been repeated for at least three times. The relative standard deviations (RSD) are all within 10%, and the average of the RSDs is 5.38%, which has suggested the well reproducibility of our detection. 3.4. The specificity of our detection Control experiments have been performed to prove the specificity of our detection. As shown in Fig. 4, a high peak current can be obtained in the presence of GSH, while quite low electrochemical responses can be obtained in the presence of the control molecules, no matter amino acids or the proteins (glutamic acid, phenylalanine, thrombin or BSA). The control results has also shown that cysteine with an active thiol group can be easily discriminated by our method, whose electrochemical response is much lower than that with GSH. This is consistent with the previous studies. The coordination of GSH and heavy metal ions depends on two or more sites within GSH, e.g. the thiol group and the carboxy group, which is more stable than the amino acid with only one functional group, such as cysteine (Li et al., 2011). So, the control experiments have confirmed the high specificity of our method. Moreover, the applicability of our method in the complex biological samples have been studied by taking the fetal calf serum as a contaminated example and Hela cells as a real sample. As shown in Fig. 5, the high peak currents can be obtained when taking
Fig. 5. GSH detection in HeLa cell extraction and the serum sample in the absence or presence of NEM. The cell concentration is 1 105 cell/mL, and GSH concentration in the serum is 100 nM.
either GSH in the diluted serum or GSH extraction from Hela cells as the sample. As a comparison, when the thiol-blocking agent NEM that can effectively decompose GSH has been introduced into the GSH samples, the electrochemical signals have significantly reduced. The comparisons have clearly shown that the electrochemical responses obtained in both the serum sample and cell extraction can be ascribed to the presence of GSH. The recoveries of the given concentration in the serum samples have been between 102% and 108% (Table S1), showing the good selectivity of our method. Therefore, the experiments in complex samples have
Table 1. The comparison of our method with other existing methods. Methods
LOD
Linear range
Ref.
Simultaneous electrochemical determination of glutathione and glutathione disulfide at a nanoscale copper hydroxide composite carbon ionic liquid electrode Ethynylferrocene-NiO/MWCNT nanocomposite modified carbon paste electrode as a novel voltammetric sensor for simultaneous determination of glutathione and acetaminophen Cu2O/NiOx/graphene oxide modified glassy carbon electrode for the enhanced electrochemical oxidation of reduced glutathione and nonenzyme glucose sensor Highly sensitive and selective colorimetric detection of glutathione based on Ag [I] ion -3, 3′, 5, 5′- tetramethylbenzidine (TMB) A dual-mode nanosensor based on carbon quantum dots and gold nanoparticles for discriminative detection of glutathione in human plasma Electrochemical detection of glutathione based on mercury ion mediated strand displacement reactions
30 nM
1–50 μM
Safavi et al., 2009
6 nM
0.01–200 μM
Shahmiri et al., 2013
0.3 μM
2 μM–1.3 mM
Yuan et al., 2013
0.05 μM
0.05–8 μM
Ni et al., 2015
50 nM
1–4 μM
Shi et al., 2014
0.14 nM
0.5 nM–5 μM
Our work
668
Y. Lv et al. / Biosensors and Bioelectronics 85 (2016) 664–668
not only reconfirmed the high selectivity of our methods, but also suggested the great potential of our method for the clinical use in the future.
4. Conclusions In summary, we have proposed a simple but efficient electrochemical method for GSH detection based on Hg2 þ -mediated strand displacement reaction. The coordination of metal ions and specific bases (e.g. Hg2 þ and T-T mismatches) can modulate the strand displacement reaction on the electrode surface, which can be inhibited by the high-affinity chelation of GSH to heavy metal ions. In this case, a “signal-on” electrochemical detection of GSH can be realized by tracing the electrochemical response of MB on the electrode surface. This method has been proven to be highly specific, which can effectively discriminate the target molecules in both the serum samples and the cell samples. Therefore, our method with the improved specificity and sensitivity can provide an alternative for monitoring GSH accurately in the physiological or pathological condition, which is of great clinical significance to the diagnosis and treatment of the related diseases. Compared to the existing methods, our method that takes the unique advantages of both the electrochemical technique and the strand displacement reaction is low cost and simple to operate, which may be feasible to the miniaturization and integration in the future. Moreover, our work may also provide some suggestions to the application of toehold-mediated strand displacement in DNAbased analysis, which may facilitate the improvement of the sensitivity and specificity of the biosensing.
Acknowledgements This work is supported by the National Natural Science Foundation of China (Grant No. 31200745) and the Innovation Program of Shanghai Municipal Education Commission (Grant No. 14YZ026).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.05.069.
References Cohen, P., Tcherpakov, M., 2010. Cell 143, 686–693. Chang, R.C.C., So, K.F., 2008. Cell. Mol. Neurobiol. 28, 643–652. Choi, I.Y., Lee, S.P., Denney, D.R., Lynch, S.G., 2011. Mult. Scler. 17, 289–296. Ding, W., Deng, W., Zhu, H., Liang, H., 2013. Chem. Commun. 49, 9953–9955. Grek, C.L., Zhang, J., Manevich, Y., Townsend, D.M., Tew, K.D., 2013. J. Biol. Chem. 288, 26497–26504. Garai-Ibabe, G., Saa, L., Pavlov, V., 2013. Anal. Chem. 85, 5542–5546. Giustarini, D., Dalle-Donne, I., Milzani, A., Rossi, R., 2011. Anal. Biochem. 415, 81–83. Gu, J., Hu, D., Wang, W., Zhang, Q., Meng, Z., Jia, X., Xi, K., 2015. Biosens. Bioelectron. 68, 27–33. Hirrlinger, J., Dringen, R., 2010. Brain Res. Rev. 63, 177–188. Hughes, T.B., Miller, G.P., Swamidass, S.J., 2015. Chem. Res. Toxicol. 28, 797–809. Laborde, E., 2010. Cell Death Differ. 17, 1373–1380. Lu, Y., Ye, M., Song, S., Li, L., Shaikh, F., Li, J., 2014. Appl. Biochem. Biotech. 174, 762–771. Liu, J., Bao, C., Zhong, X., Zhu, L., 2010. Chem. Commun. 46, 2971–2973. Li, Q.H., Zhang, L., Bai, J.M., Liu, Z.C., Liang, R.P., Qiu, J.D., 2015. Biosens. Bioelectron. 74, 886–894. Li, Y., Wu, P., Xu, H., Zhang, H., Zhong, X., 2011. Analyst 136, 196–200. Murphy, M.P., 2012. Antioxid. Redox Signal. 16, 476–495. Marchand, S., de Revel, G., 2010. Anal. Chim. Acta 660, 158–163. Mandal, P.K., Tripathi, M., Sugunan, S., 2012. Biochem. Biophys. Res. Commun. 417, 43–48. McMahon, B.K., Gunnlaugsson, T., 2012. J. Am. Chem. Soc. 134, 10725–10728. Miao, P., Liu, L., Nie, Y., Li, G., 2009. Biosens. Bioelectron. 24, 3347–3351. Ni, P., Sun, Y., Dai, H., Hu, J., Jiang, S., Wang, Y., Li, Z., 2015. Biosens. Bioelectron. 63, 47–52. Ortega, A.L., Mena, S., Estrela, J.M., 2011. Cancers 3, 1285–1310. Perricone, C., De Carolis, C., Perricone, R., 2009. Autoimmun. Rev. 8, 697–701. Raj, L., Ide, T., Gurkar, A.U., Foley, M., Schenone, M., Li, X., Stern, A.M., 2011. Nature 475, 231–234. Reid, M., Jahoor, F., 2001. Curr. Opin. Clin. Nutr. 4, 65–71. Samarasinghe, K.T.G., Munkanatta Godage, D.N.P., VanHecke, G.C., Ahn, Y.H., 2014. J. Am. Chem. Soc. 136, 11566–11569. Squellerio, I., Caruso, D., Porro, B., Veglia, F., Tremoli, E., Cavalca, V., 2012. J. Pharm. Biomed. 71, 111–118. Safavi, A., Maleki, N., Farjami, E., Mahyari, F.A., 2009. Anal. Chem. 81, 7538–7543. Shahmiri, M.R., Bahari, A., Karimi-Maleh, H., Hosseinzadeh, R., Mirnia, N., 2013. Sens. Actuators B: Chem. 177, 70–77. Shi, Y., Pan, Y., Zhang, H., Zhang, Z., Li, M.J., Yi, C., Yang, M., 2014. Biosens. Bioelectron. 56, 39–45. Traverso, N., Ricciarelli, R., Nitti, M., Marengo, B., Furfaro, A.L., Pronzato, M.A., Domenicotti, C., 2013. Oxidative Med. Cell. Longev. 2013, 1–10. Tew, K.D., 2016. Cancer Res. 76, 7–9. Yu, F., Li, P., Wang, B., Han, K., 2013. J. Am. Chem. Soc. 135, 7674–7680. You, B.R., Park, W.H., 2010. Toxicol. Vitr. 24, 1356–1362. Yuan, B., Xu, C., Liu, L., Zhang, Q., Ji, S., Pi, L., Huo, Q., 2013. Electrochim. Acta 104, 78–83. Yuan, Y., Li, W., Liu, Z., Nie, Z., Huang, Y., Yao, S., 2014. Biosens. Bioelectron. 61, 321–327. Zhu, R., Wang, Y., Zhang, L., Guo, Q., 2012. Hepatol. Res. 42, 741–749. Zhao, J., Zhu, L., Guo, C., Gao, T., Zhu, X., Li, G., 2013a. Biosens. Bioelectron. 49, 329–333. Zhao, J., Lv, Y., Kang, M., Wang, K., Xiang, Y., 2015. Analyst 140, 7818–7822. Zhao, J., Fan, Q., Zhu, S., Duan, A., Yin, Y., Li, G., 2013a. Biosens. Bioelectron. 39, 183–186. Zhao, J., Chen, C., Zhang, L., Jiang, J., Shen, G., Yu, R., 2013b. Analyst 138, 1713–1718. Zhad, H.R.L.Z., Lai, R.Y., 2014. Chem. Commun. 50, 8385–8387.