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Covalent grafting folate on Au electrode via click chemistry
Electrochemistry Communications 23 (2012) 98–101
Contents lists available at SciVerse ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
Covalent grafting folate on Au electrode via click chemistry Yuehong Pang ⁎, Zhaoqiang Ge, Yong Liu, Xiaofang Shen State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
a r t i c l e
i n f o
Article history: Received 2 July 2012 Accepted 17 July 2012 Available online 24 July 2012 Keywords: Click reaction Folate Cancer cells Electrochemistry
a b s t r a c t Covalently conjugating folate on Au surface was achieved via click chemistry, copper (I)-catalyzed azide-alkyne cycloaddition reaction. Human leukemia K562 cells were used as a model system to investigate capturing cancer cells by folate-targeted electrode. Scanning optical microscopy and electrochemical method were used to monitor the capture of K562 cells on folate-clicked electrode. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Cancer cells can produce an abnormal quantity of proteins or specific receptors on their surface or within the cell. These over-expressed components can be detected and bound in order to detect, isolate, quantify and destroy cancer cells. While antibody targeted drugs have been the first to enter the clinic, recent studies demonstrate that folate can also be used to deliver attached imaging and therapeutic agents selectively to malignant cells in both animal tumor models and human cancer patients [1,2]. Folate is a vitamin that is essential for the proliferation and maintenance of all cells. Because most mammalian cells obtain their normal folate requirement via a low affinity reduced folate carrier or proton-coupled folate transporter, accessible folate receptors (FR) are normally expressed in significant numbers only on cancer cells, activated macrophages, and the proximal tubule cells of the kidney. FR, also known as the folate binding protein, is a glycosylphosphatidylinositolanchored protein that binds both folate and folate-linked drugs with high affinity (Kd ~100 pM) [3]. The high affinity between folate and FR makes folate targets tumors in a manner similar to monoclonal antibodies. Based on this strategy, folate-targeted cancer therapeutics and imaging agents are currently in human clinical trials [3]. Electrochemical methods have been known to be simple, rapid, sensitive and convenient. Electrochemical detection can be carried out for overproduced proteins at the surface of cancer cells [4,5]. The improvement of the selectivity of electrochemical techniques using antibodies as well as other specific ligands is being carefully considered. While most attention has been paid on the electrochemical behaviors and determination of folate in foods, some pioneer
⁎ Corresponding author. Fax: +86 510 85329081. E-mail address: [email protected] (Y. Pang). 1388-2481/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.elecom.2012.07.018
researchers have explored using folate to selectively capture and detect cancer cells [5–9]. Conjugating folate on electrode is essential when using folatetargeted electrochemical sensing. Click chemistry provides a molecular approach that uses only the most practical and reliable chemical reactions to connect a diversity of structures bearing a wide variety of functional groups. Surface modification using click reaction offers a fast, mild reaction conditions, high chemoselectivity, high reaction yield and no side products strategy [6,7]. Herein, our primary focus is to propose a new method to covalently modify Au electrode with folate via click chemistry. K562 cells were used as a model to investigate capturing and immobilizing cancer cells by folate-targeted electrode (Scheme 1). 2. Experimental 2.1. Materials Folate was purchased from Aladdin (Shanghai, China). 6-thiolhexanol was obtained from Sigma-Aldrich (St.Louis, Missouri). Leukemia K562 cell line was kindly provided from Shanghai Institute of Cellular Biology of Chinese Academy of Sciences. Azidoundecanethiol [8] and propargyl folate [9] were synthesized according to the literature procedure. Other chemicals were of analytical grade and used without further purification. 2.2. Instrumentation The Fourier-transform infrared (FTIR) spectra of products were analyzed using a Nicolet, Nexus 470 FTIR spectrometer (Thermo Forma, USA). Nuclear magnetic resonance (NMR) spectra were measured with an advanced III 400 MHz Digital NMR spectrometer (Bruker, Switzerland). Electrochemical Measurements were performed on a
Y. Pang et al. / Electrochemistry Communications 23 (2012) 98–101
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Scheme 1. The immobilization folate onto Au electrode surface via click chemistry and its capture of cancer cell.
CHI 660c electrochemical analyzer (CH Instruments, Chenhua, China). Cancer cells were observed using AM-413 T5 scanning optical microscope (Dino-lite, China). 2.3. Procedures 2.3.1. Formation of mixed azide-terminated SAMs The surface of the substrate Au electrode was first polished with 0.5 μm Al2O3 powder, and washed ultrasonically in absolute alcohol and deionized water for 3 min, respectively. Then, the Au electrode was placed in dilute ethanolic solutions with 0.8 mM azidoundecanethiol (N3(CH2)11SH) and 0.2 mM dilute thiol (the total thiol concentration is 1.0 mM) for 24 h to form mixed azide-terminated self-assembled monolayers (SAMs). After SAMs, the electrode was rinsed with ethanol and dried under nitrogen. 2.3.2. Covalent grafting folate on Au surface via click chemistry The mixed azide-terminated SAMs modified electrode was immersed in propargyl folate in 1:1 water/DMSO aqueous solutions. Then, 1.0 mol% copper (II) sulfate pentahydrate and 5.0 mol% sodium ascorbate were added as catalysts at room temperature. After incubation for 24 h, the electrodes were washed with DMSO and twice distilled water to remove excess adsorbate and then dried under nitrogen.
2.3.3. Cell culture Leukemia K562 cells were cultured in folate-free RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 0.5% (v/v) Penicillin-Streptomycin solution at 37 °C in a humidified atmosphere containing 5% CO2. The cell line was washed with cold PBS (pH = 7.4) and pelleted by centrifugation (800 rpm, 5 min). After removing the supernatant, cells were resuspended in cold (4 °C) PBS solution (2 mL) followed by centrifugation. After washing by suspension/centrifugation process three times, Leukemia K562 cells were dispersed in PBS solution. Capturing the K562 cells onto folate-clicked Au electrode was achieved by treating cells with the electrode for 0, 20, 40, 60 min. At the end of these times, the process was considered complete as indicated by the massive attachment of cells to the electrode. 3. Results and discussion 3.1. Alkyne-functionalized folate Tumor-targeting folate conjugates covalently linked via folate's γ-carboxyl moiety maintain a high affinity for the FRs, and the mechanism of cellular uptake of folate conjugates by FRs is as effective as that displayed by folate in its free form [10]. Therefore, alkyne was designed to bound with folate on its γ-carboxyl in this paper, as shown in Fig. 1A. The FTIR spectra of folate and alkyne-folate are
Fig. 1. (A) Synthesis route of alkyne-functionalized folate (B) FT-IR spectra of folate (a) and alkyne-functionalized folate (b); (C) 1H NMR spectra of alkyne-functionalized folate.
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Y. Pang et al. / Electrochemistry Communications 23 (2012) 98–101
shown in Fig. 1B. The characteristic absorption of the carbon-carbon triple bond (C`C) was observed at 2119 cm−1 and 661 cm−1. The data obtained by 1HNMR (Fig. 1C) also confirmed that alkynesubstituted folate was formed. 1 H NMR (DMSO-d6, ppm): 8.64 (s, 1 H), 8.29–8.24 (d, 1 H), 8.04– 8.02 (d, 1 H), 7.67–7.65(d, 2 H), 6.93 (s, 2 H), 6.65–6.63 (d, 2 H), 4.49–4.48 (d, 2 H), 4.32–4.30 (m, 1 H), 3.84–3.81 (m, 2 H), 3.07–3.05 (t, 1 H), 2.88 (s, 1 H), 2.72(s, 1 H), 2.31–2.20 (m, 2 H), 1.98–1.96 (m, 1 H), 1.87–1.85 (m, 1 H). 3.2. Electrochemical characterization Self-assembled monolayers (SAMs) on gold surfaces offer a wellcharacterized platform on which electrontransfer processes can be studied. Hydrophobic SAMs are known to be more stable in polar aqueous solvent systems. Hence, we prepared mixed SAMs by depositing a mixed solution of two different thiols, an azide-terminated alkyl thiol (linker) and a 6-thiol-hexanol (diluent). After deposition, the excess reagents were removed by rinsing the surfaces with ethanol. The modified Au electrode was characterized by cyclic voltammogram and typical electrochemical impedance spectrum after each step of the fabrication process (Fig. 2). A pair of reversible redox peaks corresponding to Fe(CN)63−/Fe(CN)64− couple were observed at bare gold electrode (Fig. 2A-a). Azide-terminated mixed SAMs passivated the electrode and effectively blocked the charge transfer between the redox couple in the solution and the Au electrode, thus a decrease in the current response has been observed (Fig. 2A-b). After click reaction with alkyne-functionalized folate, the charge transfer ability of the modified Au electrode was further reduced, accordingly exhibiting a continuing decrease in the current response (Fig. 2A-c). Electrochemical impedance spectroscopy can provide useful information on the impedance changes of the electrode surface during the fabrication process. The impedance measurement was carried out covering the 105- 0.1 Hz frequency intervals using a potential of 0.21 V. The bare Au electrode shows very low ET resistance (Rct =297 Ω, Fig. 2B-a).
Fig. 2. Cyclic voltammograms (A) and electrochemical impedance spectra (B) for bare Au electrode (a), azide-terminated mixed SAMs (b), after click reaction with alkyne-functionalized folate(c). Electrolyte solution: 2.5 mM K4Fe(CN)6 and 2.5 mM K3Fe(CN)6 containing 0.1 mM KCl; scan rate 50 mV/s. Inset in figure B is the enlargement of EIS of bare Au electrode.
SAMs formed from N3(CH2)11SH and decanethiol lead to an obviously increment of interfacial ET resistance (Rct =9256 Ω, Fig. 2B-b). After the click reaction of alkyne-functionalized folate with azide-bearing mixed SAMs, the negatively charged carboxyl group in folate and Fe(CN)63−/ Fe(CN)64− forms the electrostatic repulsion, which block further the access of electrode surface (Rct =5.355×104 Ω, Fig. 2B-c). Click chemistry is superior to other coupling reactions on surfaces because of its chemoselectity. The precursors, azide and alkyne are inert to reaction with most other functional groups. This process uses mild conditions and leads to a single product. It is usually quantitative and occurs under aqueous conditions at room temperature. Upon click chemistry, the resulting linker between the SAM and the redox-active species is a 1,2,3-triazole, which is very robust to hydrolysis or redox reactions. Triazoles provide electronic coupling comparable to that reported with other linkers [11]. Cyclic voltammogram of azide-terminated mixed SAMs film in alkyne-functionalized folate solution 24 h without Cu (I) catalyst has no redox peak. And it is clearly aware that the click reaction cannot perform without the Cu(I) catalyst. After the click reaction, a well-defined anodic peak has been observed, which indicates that folate group is successfully grafted onto Au electrode. The peak potential is around 0.60 V, which is considerably more negative than that of free folate at the multi-walled carbon nanotubes/Au electrode (0.83 V) [12]. It is due to the conjugation effects of the triazole ring, which increases the
Fig. 3. Scanning optical microscopy photos (A) and electrochemical impedance spectra (B) for bare Au electrode (a), covalent grafting folate on Au electrode incubated in leukemia cell K562 for 20 min (b), 40 min (c) and 60 min (d). Cell density: 2.2×105 cell/mL. Inset in figure B is the enlargement of EIS of bare gold electrode.
Y. Pang et al. / Electrochemistry Communications 23 (2012) 98–101
electron density of the folate group and makes the oxidation of the folate unit easier. 3.3. Monitoring of Leukemia K562 cells capture on FA-Functionalized Au electrode surface Folate-conjugates can enter cancer cells by FR-mediated endocytosis and move through many organelles supplying transported materials to cell cytoplasm. The Folate-targeted drug is then released in the endosomes/lysosomes mainly by enzymatic cleavage. Due to recycling of the unligated FR back to the cell surface, the uptake process can be reiterated, allowing continuous supply of folate-linked drugs into the cell [10]. However, the folate was linked with a Au electrode but not a drug molecule or nanoparticles, which is much smaller than cells. When the folate conjugated with FR, endocytosis cannot happen. On the contrary, the cell was captured on the electrode. As seen from Fig. 3A, K562 cells were capable of immobilizing to FA functional Au electrode. With an increasing incubation time, the density of cells captured on the film increased (photo b–d, Fig. 3A). After incubation for 60 min, K562 cells apparently spread evenly over the entire surface (photo d, Fig. 3A). These observations could be monitored with impedance measurements. The bare gold electrode shows very low ET resistance (Rct = 297 Ω, Fig. 3B-a). When Leukemia K562 cells suspension is employed, due to the interaction between folate and FR and the consequent immobilization of cell on the electrode surface, the electrochemical communication between the electrode surface and the electroactive probes, K3[Fe(CN)6]/K4[Fe(CN)6] will be greatly blocked, which makes the increment of resistance. With an increasing incubation time, Rct increased from 9.35× 104 Ω to 6.74× 105 Ω (curves b–d, Fig. 3B), resulting from more cells capture on the electrode, which introduced a barrier for electrochemical process. These results were consistent with the observation from scanning optical microscopy.
101
4. Conclusion Folate group was successfully grafted on Au electrode surface via click chemistry. Leukemia K562 cells can be captured and immobilized on the electrode duo to interaction between folate and FR. Such strategy provides a simple and convenient way for assembling covalently folate surface functionalization and presents potential applications in cancer cells biosensor.
Acknowledgements This work was supported by the National Natural Science Foundation of China (21005032), the Major State Basic Research Development Program of China (973 Program, 2012CB720806), and the National Science and Technology Support Project (2011BAK10B03).
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
W. Xia, P.S. Low, Journal of Medicinal Chemistry 53 (2010) 6811–6824. P.S. Low, S.A. Kularatne, Current Opinion in Chemical Biology 13 (2009) 256–262. E.I. Sega, P.S. Low, Cancer and Metastasis Reviews 27 (2008) 655–664. M. Perfezou, A. Turner, A. Merkoci, Chemical Society Reviews 41 (2012) 2606–2622. L. Liu, X.L. Zhu, D.M. Zhang, J.Y. Huang, G.X. Li, Electrochemistry Communications 9 (2007) 2547–2550. Y.H. Lau, P.J. Rutledge, M. Watkinson, M.H. Todd, Chemical Society Reviews 40 (2011) 2848–2866. S.K. Mamidyala, M.G. Finn, Chemical Society Reviews 39 (2010) 1252–1261. J.P. Collman, N.K. Devaraj, C.E.D. Chidsey, Langmuir 20 (2004) 1051–1053. P. De, S.R. Gondi, B.S. Sumerlin, Biomacromolecules 9 (2008) 1064–1070. A. Guaragna, A. Chiaviello, C. Paolella, D. D'Alonzo, G. Palumbo, G. Palumbo, Bioconjugate Chemistry 23 (2012) 84–96. R.A. Decreau, J.P. Collman, A. Hosseini, Chemical Society Reviews 39 (2010) 1291–1301. S.H. Wei, F.Q. Zhao, Z.Y. Xu, B.Z. Zeng, Microchimica Acta 152 (2006) 285–290.
Contents lists available at SciVerse ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
Covalent grafting folate on Au electrode via click chemistry Yuehong Pang ⁎, Zhaoqiang Ge, Yong Liu, Xiaofang Shen State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
a r t i c l e
i n f o
Article history: Received 2 July 2012 Accepted 17 July 2012 Available online 24 July 2012 Keywords: Click reaction Folate Cancer cells Electrochemistry
a b s t r a c t Covalently conjugating folate on Au surface was achieved via click chemistry, copper (I)-catalyzed azide-alkyne cycloaddition reaction. Human leukemia K562 cells were used as a model system to investigate capturing cancer cells by folate-targeted electrode. Scanning optical microscopy and electrochemical method were used to monitor the capture of K562 cells on folate-clicked electrode. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Cancer cells can produce an abnormal quantity of proteins or specific receptors on their surface or within the cell. These over-expressed components can be detected and bound in order to detect, isolate, quantify and destroy cancer cells. While antibody targeted drugs have been the first to enter the clinic, recent studies demonstrate that folate can also be used to deliver attached imaging and therapeutic agents selectively to malignant cells in both animal tumor models and human cancer patients [1,2]. Folate is a vitamin that is essential for the proliferation and maintenance of all cells. Because most mammalian cells obtain their normal folate requirement via a low affinity reduced folate carrier or proton-coupled folate transporter, accessible folate receptors (FR) are normally expressed in significant numbers only on cancer cells, activated macrophages, and the proximal tubule cells of the kidney. FR, also known as the folate binding protein, is a glycosylphosphatidylinositolanchored protein that binds both folate and folate-linked drugs with high affinity (Kd ~100 pM) [3]. The high affinity between folate and FR makes folate targets tumors in a manner similar to monoclonal antibodies. Based on this strategy, folate-targeted cancer therapeutics and imaging agents are currently in human clinical trials [3]. Electrochemical methods have been known to be simple, rapid, sensitive and convenient. Electrochemical detection can be carried out for overproduced proteins at the surface of cancer cells [4,5]. The improvement of the selectivity of electrochemical techniques using antibodies as well as other specific ligands is being carefully considered. While most attention has been paid on the electrochemical behaviors and determination of folate in foods, some pioneer
⁎ Corresponding author. Fax: +86 510 85329081. E-mail address: [email protected] (Y. Pang). 1388-2481/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.elecom.2012.07.018
researchers have explored using folate to selectively capture and detect cancer cells [5–9]. Conjugating folate on electrode is essential when using folatetargeted electrochemical sensing. Click chemistry provides a molecular approach that uses only the most practical and reliable chemical reactions to connect a diversity of structures bearing a wide variety of functional groups. Surface modification using click reaction offers a fast, mild reaction conditions, high chemoselectivity, high reaction yield and no side products strategy [6,7]. Herein, our primary focus is to propose a new method to covalently modify Au electrode with folate via click chemistry. K562 cells were used as a model to investigate capturing and immobilizing cancer cells by folate-targeted electrode (Scheme 1). 2. Experimental 2.1. Materials Folate was purchased from Aladdin (Shanghai, China). 6-thiolhexanol was obtained from Sigma-Aldrich (St.Louis, Missouri). Leukemia K562 cell line was kindly provided from Shanghai Institute of Cellular Biology of Chinese Academy of Sciences. Azidoundecanethiol [8] and propargyl folate [9] were synthesized according to the literature procedure. Other chemicals were of analytical grade and used without further purification. 2.2. Instrumentation The Fourier-transform infrared (FTIR) spectra of products were analyzed using a Nicolet, Nexus 470 FTIR spectrometer (Thermo Forma, USA). Nuclear magnetic resonance (NMR) spectra were measured with an advanced III 400 MHz Digital NMR spectrometer (Bruker, Switzerland). Electrochemical Measurements were performed on a
Y. Pang et al. / Electrochemistry Communications 23 (2012) 98–101
99
Scheme 1. The immobilization folate onto Au electrode surface via click chemistry and its capture of cancer cell.
CHI 660c electrochemical analyzer (CH Instruments, Chenhua, China). Cancer cells were observed using AM-413 T5 scanning optical microscope (Dino-lite, China). 2.3. Procedures 2.3.1. Formation of mixed azide-terminated SAMs The surface of the substrate Au electrode was first polished with 0.5 μm Al2O3 powder, and washed ultrasonically in absolute alcohol and deionized water for 3 min, respectively. Then, the Au electrode was placed in dilute ethanolic solutions with 0.8 mM azidoundecanethiol (N3(CH2)11SH) and 0.2 mM dilute thiol (the total thiol concentration is 1.0 mM) for 24 h to form mixed azide-terminated self-assembled monolayers (SAMs). After SAMs, the electrode was rinsed with ethanol and dried under nitrogen. 2.3.2. Covalent grafting folate on Au surface via click chemistry The mixed azide-terminated SAMs modified electrode was immersed in propargyl folate in 1:1 water/DMSO aqueous solutions. Then, 1.0 mol% copper (II) sulfate pentahydrate and 5.0 mol% sodium ascorbate were added as catalysts at room temperature. After incubation for 24 h, the electrodes were washed with DMSO and twice distilled water to remove excess adsorbate and then dried under nitrogen.
2.3.3. Cell culture Leukemia K562 cells were cultured in folate-free RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 0.5% (v/v) Penicillin-Streptomycin solution at 37 °C in a humidified atmosphere containing 5% CO2. The cell line was washed with cold PBS (pH = 7.4) and pelleted by centrifugation (800 rpm, 5 min). After removing the supernatant, cells were resuspended in cold (4 °C) PBS solution (2 mL) followed by centrifugation. After washing by suspension/centrifugation process three times, Leukemia K562 cells were dispersed in PBS solution. Capturing the K562 cells onto folate-clicked Au electrode was achieved by treating cells with the electrode for 0, 20, 40, 60 min. At the end of these times, the process was considered complete as indicated by the massive attachment of cells to the electrode. 3. Results and discussion 3.1. Alkyne-functionalized folate Tumor-targeting folate conjugates covalently linked via folate's γ-carboxyl moiety maintain a high affinity for the FRs, and the mechanism of cellular uptake of folate conjugates by FRs is as effective as that displayed by folate in its free form [10]. Therefore, alkyne was designed to bound with folate on its γ-carboxyl in this paper, as shown in Fig. 1A. The FTIR spectra of folate and alkyne-folate are
Fig. 1. (A) Synthesis route of alkyne-functionalized folate (B) FT-IR spectra of folate (a) and alkyne-functionalized folate (b); (C) 1H NMR spectra of alkyne-functionalized folate.
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Y. Pang et al. / Electrochemistry Communications 23 (2012) 98–101
shown in Fig. 1B. The characteristic absorption of the carbon-carbon triple bond (C`C) was observed at 2119 cm−1 and 661 cm−1. The data obtained by 1HNMR (Fig. 1C) also confirmed that alkynesubstituted folate was formed. 1 H NMR (DMSO-d6, ppm): 8.64 (s, 1 H), 8.29–8.24 (d, 1 H), 8.04– 8.02 (d, 1 H), 7.67–7.65(d, 2 H), 6.93 (s, 2 H), 6.65–6.63 (d, 2 H), 4.49–4.48 (d, 2 H), 4.32–4.30 (m, 1 H), 3.84–3.81 (m, 2 H), 3.07–3.05 (t, 1 H), 2.88 (s, 1 H), 2.72(s, 1 H), 2.31–2.20 (m, 2 H), 1.98–1.96 (m, 1 H), 1.87–1.85 (m, 1 H). 3.2. Electrochemical characterization Self-assembled monolayers (SAMs) on gold surfaces offer a wellcharacterized platform on which electrontransfer processes can be studied. Hydrophobic SAMs are known to be more stable in polar aqueous solvent systems. Hence, we prepared mixed SAMs by depositing a mixed solution of two different thiols, an azide-terminated alkyl thiol (linker) and a 6-thiol-hexanol (diluent). After deposition, the excess reagents were removed by rinsing the surfaces with ethanol. The modified Au electrode was characterized by cyclic voltammogram and typical electrochemical impedance spectrum after each step of the fabrication process (Fig. 2). A pair of reversible redox peaks corresponding to Fe(CN)63−/Fe(CN)64− couple were observed at bare gold electrode (Fig. 2A-a). Azide-terminated mixed SAMs passivated the electrode and effectively blocked the charge transfer between the redox couple in the solution and the Au electrode, thus a decrease in the current response has been observed (Fig. 2A-b). After click reaction with alkyne-functionalized folate, the charge transfer ability of the modified Au electrode was further reduced, accordingly exhibiting a continuing decrease in the current response (Fig. 2A-c). Electrochemical impedance spectroscopy can provide useful information on the impedance changes of the electrode surface during the fabrication process. The impedance measurement was carried out covering the 105- 0.1 Hz frequency intervals using a potential of 0.21 V. The bare Au electrode shows very low ET resistance (Rct =297 Ω, Fig. 2B-a).
Fig. 2. Cyclic voltammograms (A) and electrochemical impedance spectra (B) for bare Au electrode (a), azide-terminated mixed SAMs (b), after click reaction with alkyne-functionalized folate(c). Electrolyte solution: 2.5 mM K4Fe(CN)6 and 2.5 mM K3Fe(CN)6 containing 0.1 mM KCl; scan rate 50 mV/s. Inset in figure B is the enlargement of EIS of bare Au electrode.
SAMs formed from N3(CH2)11SH and decanethiol lead to an obviously increment of interfacial ET resistance (Rct =9256 Ω, Fig. 2B-b). After the click reaction of alkyne-functionalized folate with azide-bearing mixed SAMs, the negatively charged carboxyl group in folate and Fe(CN)63−/ Fe(CN)64− forms the electrostatic repulsion, which block further the access of electrode surface (Rct =5.355×104 Ω, Fig. 2B-c). Click chemistry is superior to other coupling reactions on surfaces because of its chemoselectity. The precursors, azide and alkyne are inert to reaction with most other functional groups. This process uses mild conditions and leads to a single product. It is usually quantitative and occurs under aqueous conditions at room temperature. Upon click chemistry, the resulting linker between the SAM and the redox-active species is a 1,2,3-triazole, which is very robust to hydrolysis or redox reactions. Triazoles provide electronic coupling comparable to that reported with other linkers [11]. Cyclic voltammogram of azide-terminated mixed SAMs film in alkyne-functionalized folate solution 24 h without Cu (I) catalyst has no redox peak. And it is clearly aware that the click reaction cannot perform without the Cu(I) catalyst. After the click reaction, a well-defined anodic peak has been observed, which indicates that folate group is successfully grafted onto Au electrode. The peak potential is around 0.60 V, which is considerably more negative than that of free folate at the multi-walled carbon nanotubes/Au electrode (0.83 V) [12]. It is due to the conjugation effects of the triazole ring, which increases the
Fig. 3. Scanning optical microscopy photos (A) and electrochemical impedance spectra (B) for bare Au electrode (a), covalent grafting folate on Au electrode incubated in leukemia cell K562 for 20 min (b), 40 min (c) and 60 min (d). Cell density: 2.2×105 cell/mL. Inset in figure B is the enlargement of EIS of bare gold electrode.
Y. Pang et al. / Electrochemistry Communications 23 (2012) 98–101
electron density of the folate group and makes the oxidation of the folate unit easier. 3.3. Monitoring of Leukemia K562 cells capture on FA-Functionalized Au electrode surface Folate-conjugates can enter cancer cells by FR-mediated endocytosis and move through many organelles supplying transported materials to cell cytoplasm. The Folate-targeted drug is then released in the endosomes/lysosomes mainly by enzymatic cleavage. Due to recycling of the unligated FR back to the cell surface, the uptake process can be reiterated, allowing continuous supply of folate-linked drugs into the cell [10]. However, the folate was linked with a Au electrode but not a drug molecule or nanoparticles, which is much smaller than cells. When the folate conjugated with FR, endocytosis cannot happen. On the contrary, the cell was captured on the electrode. As seen from Fig. 3A, K562 cells were capable of immobilizing to FA functional Au electrode. With an increasing incubation time, the density of cells captured on the film increased (photo b–d, Fig. 3A). After incubation for 60 min, K562 cells apparently spread evenly over the entire surface (photo d, Fig. 3A). These observations could be monitored with impedance measurements. The bare gold electrode shows very low ET resistance (Rct = 297 Ω, Fig. 3B-a). When Leukemia K562 cells suspension is employed, due to the interaction between folate and FR and the consequent immobilization of cell on the electrode surface, the electrochemical communication between the electrode surface and the electroactive probes, K3[Fe(CN)6]/K4[Fe(CN)6] will be greatly blocked, which makes the increment of resistance. With an increasing incubation time, Rct increased from 9.35× 104 Ω to 6.74× 105 Ω (curves b–d, Fig. 3B), resulting from more cells capture on the electrode, which introduced a barrier for electrochemical process. These results were consistent with the observation from scanning optical microscopy.
101
4. Conclusion Folate group was successfully grafted on Au electrode surface via click chemistry. Leukemia K562 cells can be captured and immobilized on the electrode duo to interaction between folate and FR. Such strategy provides a simple and convenient way for assembling covalently folate surface functionalization and presents potential applications in cancer cells biosensor.
Acknowledgements This work was supported by the National Natural Science Foundation of China (21005032), the Major State Basic Research Development Program of China (973 Program, 2012CB720806), and the National Science and Technology Support Project (2011BAK10B03).
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
W. Xia, P.S. Low, Journal of Medicinal Chemistry 53 (2010) 6811–6824. P.S. Low, S.A. Kularatne, Current Opinion in Chemical Biology 13 (2009) 256–262. E.I. Sega, P.S. Low, Cancer and Metastasis Reviews 27 (2008) 655–664. M. Perfezou, A. Turner, A. Merkoci, Chemical Society Reviews 41 (2012) 2606–2622. L. Liu, X.L. Zhu, D.M. Zhang, J.Y. Huang, G.X. Li, Electrochemistry Communications 9 (2007) 2547–2550. Y.H. Lau, P.J. Rutledge, M. Watkinson, M.H. Todd, Chemical Society Reviews 40 (2011) 2848–2866. S.K. Mamidyala, M.G. Finn, Chemical Society Reviews 39 (2010) 1252–1261. J.P. Collman, N.K. Devaraj, C.E.D. Chidsey, Langmuir 20 (2004) 1051–1053. P. De, S.R. Gondi, B.S. Sumerlin, Biomacromolecules 9 (2008) 1064–1070. A. Guaragna, A. Chiaviello, C. Paolella, D. D'Alonzo, G. Palumbo, G. Palumbo, Bioconjugate Chemistry 23 (2012) 84–96. R.A. Decreau, J.P. Collman, A. Hosseini, Chemical Society Reviews 39 (2010) 1291–1301. S.H. Wei, F.Q. Zhao, Z.Y. Xu, B.Z. Zeng, Microchimica Acta 152 (2006) 285–290.