Measurement of Intracellular pH Changes Based on DNA-Templated

Jul 22, 2014 - cowpea chlorotic mottle virus (CCMV) coat protein. Moreover, because the pH sensor can be translocated into cells without any...
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Measurement of Intracellular pH Changes Based on DNA-Templated Capsid Protein Nanotubes Limin Ning,† Xiaoxi Li,† Dawei Yang,† Peng Miao,† Zonghuang Ye,† and Genxi Li*,†,‡ †

Department of Biochemistry and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 22 Hankou Road, Nanjing 210093, China ‡ Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, 99 Shangda Road, Shanghai 200444, China S Supporting Information *

ABSTRACT: Intracellular pH (pHi) is a fundamental modulator of cell function. Minute changes in pHi may cause great effects in many cellular activities such as metabolism and signal transduction. Herein we report an electrochemical pHi sensor based on viral-coat proteins− DNA nanotubes modified gold electrode. The sensor is pH-sensitive as a result of the pH-dependent electrochemical property of methylene blue (MB) and cell permeable owing to the polyarginine domain of the cowpea chlorotic mottle virus (CCMV) coat protein. Moreover, because the pH sensor can be translocated into cells without any further operations, the measurement of pHi changes can be greatly simplified. The pH sensor has a broad pH spectrum in the pH range from 4.0 to 9.0 and responds rapidly to the pH changes of cells, so it may hold great potential to be a valuable tool to study pH-dependent biological and pathological processes in the future.

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increasing pH.10,13,14 Meanwhile, as a dye for cell staining, MB can bind strongly to DNA via intercalation,15 so the electrochemical signal of MB inside cells may be obtained with DNA as the electron transfer mediator, and therefore, the pH value inside cells can be sensed by MB bound to DNA. Furthermore, to deliver the pH sensor into cells, components with cell permeability have been introduced in this work. In our previous report, Ca2+ has been used as the transfection agent to transfect DNA into cells.10 However, the operations to transfect DNA into cells are laborious, and the delivery into cells cannot be achieved gently and simply. In this respect, it may be of great importance to develop a pH sensor with both pH sensitivity and cell permeability without introducing transfection agents. Arginine-rich peptides have a unique ability to cross the plasma membrane of cells.16−20 They are of low cytotoxicity and are able to be translocated into cells simply by adding them to the tissue-culture medium, thus they have been used to facilitate the uptake of a variety of biopolymers and small molecules into cells.21 The arginine-rich peptides are usually derived from virus proteins and are used by a variety of RNA-binding proteins to recognize specific RNA hairpins.22 Without any covalent attachment and additional peptide synthesis, the recognition of arginine-rich peptides to RNA can simplify the polyargininemediated transduction. Therefore, we propose that argininerich peptide may be an excellent candidate to translocate the pH sensor into cells, if it can also form a complex with DNA,

ntracellular pH (pHi), known as the pH value of intracellular fluid, plays a vital role in cell function, including proliferation and apoptosis, multidrug resistance, ion transport, and endocytosis.1 The connections between the cell function and pHi are so intimate that even slight changes of pHi may cause great impact on many cellular activities. Therefore, abnormal pHi is closely associated with inappropriate cell function, growth, and division, and the measurement of pHi changes can provide critical information for studying physiological and pathological processes.2 So far, several strategies have been proposed for the detection of pHi with different techniques including electrochemistry,3,4 nuclear magnetic resonance (NMR),5,6 absorbance spectroscopy,7,8 and fluorescence spectroscopy.2 Among these strategies, detection with fluorescence spectroscopy has attracted much interest with respect to spatial and temporal observation of pHi changes. However, fluorescent dyes are often delivered into cells by ways that may perturb the cell’s resting-state physiology. Therefore, it is highly and urgently required to develop new methods for the measurement of pHi. Electrochemical techniques have now attracted more and more attention and have been extensively employed for the study of small molecules and biological macromolecules.9 Meanwhile, it is also an efficient way to study species inside cells.10−12 Therefore, it may hold great promise to develop an electrochemical pH sensor to indicate the pH value inside cells. So, in this work, we have made use of methylene blue (MB) to develop an electrochemical sensor for the measurement of pHi, because the peak potentials of both oxidation and reduction peaks of MB can sensitively shift in a negative direction with © 2014 American Chemical Society

Received: January 13, 2014 Accepted: July 22, 2014 Published: July 22, 2014 8042

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immobilized on the surface of the electrode via gold−sulfur chemistry by incubating the electrode with 1 μM thiolated DNA 1 for 16 h. Then, the electrode was immersed in an aqueous solution of 1 mM spacer thiol molecules, MCH, for 1 h to obtain well-aligned DNA monolayers followed by the hybridization with 1 μM complementary DNA 2 for another 1 h at 37 °C. After that, cyclic voltammograms (CVs) and differential pulse voltammograms (DPVs) were obtained at the DNA modified electrode in a 10 mM phosphate-buffered saline (PBS) (pH 4.0−9.0) containing 25 μM MB, so as to study the pH sensitivity of MB bound to DNA on the electrode surface. Fabrication of pHi Sensor Based on CPs−DNA Modified Electrode. CCMV CPs were employed to translocate the sensor into cells by forming a complex with DNA. CPs−DNA nanotubes formed spontaneously after incubating the DNA modified gold electrode with CPs in Tris-HCl buffer. The assembly reactions have also been examined by differential pulse voltammetry with a test solution containing 1 M KNO3 and 5 mM [Fe(CN)6]3−/4−. Finally, the electrochemistry of the MB bound to CPs−DNA nanotubes modified electrode has also been examined by recording CVs and DPVs with a 10 mM phosphate-buffered saline (PBS) (pH 4.0−9.0) containing 25 μM MB, so as to study the pH sensitivity of MB bound to CPs−DNA. Measurement of pHi Changes. Embryonic kidney 293T cells and A549 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin), at 37 °C in a water-saturated incubator with 5% CO2. Before the measurement of pHi changes, the cells were washed with phosphate-buffered saline (PBS) twice and detached by Trypsin-EDTA for 1−3 min. The cells were then harvested by centrifugation (1000 rpm, 5 min) and resuspended in the same culture medium with the concentration of 5 × 105 cell/mL. After that, MB stocked in PBS with a concentration of 1 mM was added to the cells suspension with 25 μM as the final concentration. Subsequently, 20 μL of cell suspension was dripped on the surface of the CPs−DNA modified electrode and incubated for 2 h at 37 °C to achieve cell adhesion on the electrode. After that, DPVs were obtained in 10 mM phosphate buffered saline (PBS) (pH 7.40) containing 25 μM MB. To induce intracellular acidosis, cells were incubated in a 20 mM ammonium chloride (NH4Cl) solution for various periods (0− 5 h). Thereafter, the cells were washed twice in PBS, and the pHi changes were monitored with the method we proposed in this work. Besides, cell culture medium and cell debris were employed as controls. After cells were dyed with 25 μM MB for 2 h, the supernatant was collected as cell culture medium by centrifugation (1000 rpm, 5 min). The cell debris was prepared by sonicating the cell suspension which has been dyed with 25 μM MB for 2 h. Electrochemical Experiments. Cycle voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy were performed on a model 660C electrochemical analyzer (CH Instruments) at room temperature around 25 °C. For all the electrochemical measurements, a three-electrode system consisting of the modified gold electrode as the working electrode, a saturated calomel reference electrode (SCE), and a platinum auxiliary electrode was used.

similar to RNA. Fortunately, Zlotnick et al. have reported a synthetic tubular nanostructure, prepared from the selfassembling coat proteins (CPs) of cowpea chlorotic mottle virus (CCMV) templated by double-stranded DNA (dsDNA).23 In the nanotubes, CPs can interact with DNA as they do with virus RNA. The 17 nm CPs−DNA tubes can be stable and highly uniform. Meanwhile, the CCMV Gag (7−25), a part of CCMV coat proteins, also shows a moderate degree of translocation.21 So, it should be possible for us to develop an electrochemical pHi sensor with cell permeability. First, DNA molecules can be easily immobilized onto an electrode surface. Second, CCMV CPs can form a complex with the DNA molecules that have been previously immobilized on the electrode surface. Consequently, with the help of CCMV CPs, the pH sensor can be incorporated into the target cells. Therefore, an electrochemical pHi sensor with cell permeability has been developed in this work, and the changes of pHi can be monitored by using this pH sensor.

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EXPERIMENTAL SECTION Materials and Chemicals. CCMV CPs were purchased from Shanghai YouLong Biotechnology Co., Ltd. The thiolated DNA 1, 5′-HS-(CH2)6-AAA CTG TGC AGC AGT GCC CGA TGG GAG GGG GTT GCC ACG GGA CTC AGG GGA GA3′, and its complementary DNA 2, TCT CCC CTG AGT CCC GTG GCA ACC CCC TCC CAT CGG GCA CTG CTG CAC AG, were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd. Methylene blue (MB), trypsin, mercaptohexanol (MCH), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), ammonium chloride (NH4Cl), and ethylenediaminetetraacetic acid (EDTA) were obtained from Sigma. Fetal bovine serum (FBS) and Dulbecco’s modified Eagle medium (DMEM) were obtained from Nanjing Punuo En Biotechnology Co., Ltd. The cell lines, 293T cells (HEK epithelial cell), and A549 cells were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. All other chemicals were of analytical grade and used as received. All solutions were prepared with doubly distilled water, which was purified with a Milli-Q purification system (Barnstead) to a specific resistance of >18 MΩ cm. The buffer solutions used in this work were as follows. DNA immobilization buffer: 10 mM Tris-HCl with 1 mM EDTA, 10 mM TCEP and 100 mM NaCl (pH 7.40); Hybridization buffer: 10 mM phosphate buffered saline (PBS, pH 7.40) with 1 M NaCl; CPs binding buffer solution: 20 mM Tris-HCl with 150 mM NaCl (pH 7.40). Fabrication of pH Sensor Based on DNA-Modified Electrode. The substrate gold electrode (3 mm diameter) was pretreated as follows. First, the electrode was soaked in piranha solution (98% H2SO4:30% H2O2 = 3:1) for 5 min. Then, it was polished carefully on P3000 silicon carbide paper and alumina slurry (1 μm, 0.3 μm, 0.05 μm), respectively. After that, it was thoroughly washed by sonicating in both ethanol and doubledistilled water for about 5 min. After the above pretreatment, the electrode was soaked in nitric acid (50%) for 30 min and then electrochemically cleaned with cyclic voltammetry, scanning from 0 to 1.6 V for 20 cycles in 0.5 M H2SO4 to remove any remaining impurities. Finally, the electrode was thoroughly rinsed with pure water and dried with nitrogen, and it was ready for further experiment. The behavior of MB bound to a DNA modified electrode at various pH values was investigated in this work. First, DNA was

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RESULTS AND DISCUSSION The strategy in this work to monitor the changes of pHi by electrochemical technique based on DNA-templated capsid 8043

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Scheme 1. Schematic Illustration of the Measurement of pHi Changesa

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CCMV CPs nanotubes are assembled on the electrode surface with ds-DNA as the template. The nanotubes can interact with the cells, and thus the pH sensor is assimilated into the cells. The electrochemical signal from MB molecules is transferred out of the cells along the DNA chains, and the change of the intracellular pH can be shown by the shift of the peak potential of MB.

examined. The plateau achieved in Figure S-3 demonstrates that 30 min is enough for the combination of CPs and DNA. Because CCMV CPs have been introduced onto the electrode, the pH sensitivity of MB should be rechecked. Figure 1 displays the DPVs obtained at the CPs−DNA

protein nanotubes has been illustrated in Scheme 1. First, the CPs−DNA nanotubes are fabricated on the electrode surface by incubating CPs solution with ds-DNA modified electrode. In the meantime, the cells are pretreated with MB and then dripped onto the electrode surface. Consequently, the CPs− DNA nanotubes are transferred into the cells with the help of CPs, and the MB molecules inside the cells bind to the DNA in the CPs−DNA nanotubes. Because the signal of pH-sensitive MB can be transferred across the cell membrane with DNA as the electron transfer mediator, the pH value of intracellular fluid can be sensed, and the changes of pHi can be monitored. pH Sensitivity of MB Bound to DNA. The electrochemical behavior of MB bound to a DNA modified electrode in various pH mediums has been examined first. As shown by the CVs in Figure S-1A, a couple of redox peaks can be observed after the DNA modified electrode is placed in the buffer solution containing 25 μM MB in the pH range from 4.0 to 9.0. Moreover, the potentials of both oxidation and reduction peaks shift in a negative direction as the pH value increases, indicating that MB bound to DNA on the electrode surface can be pH sensitive. The reason has been clear, because the number of H+ participating in the electrode process is related to the pH of the test solution. To enhance the effectiveness of the pH sensor, a more sensitive electrochemical technique, differential pulse voltammetry, has been utilized to obtain a stronger electrochemical signal (Figure S-1B). pH Sensitivity of MB Bound to CPs−DNA. The assembly of CPs−DNA on the electrode surface has been demonstrated to be very simple. The CPs−DNA nanotubes can form spontaneously after the incubation of the DNA modified electrode with CPs solution. The optimum experimental conditions have been investigated. First, the required concentration of CCMV CPs has been checked. Experimental results reveal that 7.5 μg/mL CCMV CPs are needed to form CPs−DNA complex on the electrode surface (Figure S-2). Second, the required time for the incubation of the DNA modified electrode with the CCMV CPs solution has been

Figure 1. DPVs obtained at the CPs−DNA modified electrode for 25 μM MB solution with different pH values. Inset shows the linear relationship between the peak potential and pH value.

modified electrode for different pH values. As shown in Figure 1, both the peak current and the peak potential of MB are pHdependent. As the pH increases, the peak current increases and the peak potential shifts in a negative direction. To further investigate the pH-dependent property of the sensor, DMEM with and without FBS are introduced. As shown in Figure S-4, the peak current is significantly different in different buffer solutions with the same pH value, whereas for the peak potential, no shift can be observed. So, the pH-dependent electrochemical potential difference versus a saturated calomel reference electrode is used to indicate pH values. As shown in the inset of Figure 1, as the pH increases, the peak potential shifts linearly in a negative direction with a change in slope at the calculated pH 7.65. The regression equation is y1 = 8044

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−32.46x1 + 4.45 for lower pH ranging from 4.0 to 7.65 and y2 = 10.22x2 − 165.96 for higher pH ranging from 7.65 to 9.0, where y is the peak potential, mV; x is the pH value; n = 5; r1 = 0.9986; r2 = 0.9788. The relative standard deviations (RSDs) across five independent experimental results are calculated to be within 2.28%. The difference of the peak potentials in DPVs is larger at lower pH compared with that at higher pH, which is ascribed to different number of the H+ participating in the electrode process at various pH.13 Fortunately, the difference of the peak potentials is appreciable at the expected pH range for cells, so the sensor can be used to discriminate the minute change of intracellular pH. Measurement of Intracellular pH Changes. After the electrochemical pH sensor with both pH sensitivity and cell permeability has been fabricated, we have tried to examine whether this pH sensor can be used for pHi measurement. Before that, electrochemical impedance spectroscopy is used to demonstrate whether the cells can be assembled onto the electrode surface with the help of CCMV CPs. The impedance spectra contain a semicircle portion at higher frequencies relating to the electron transfer limited process and a linear part at lower frequencies corresponding to diffusion. So, the semicircle diameter may reflect the interfacial charge transfer resistance. As shown in Figure 2, after the immobilization of the

To further investigate whether the CPs−DNA nanostructure can enter cells efficiently, experiments with differential pulse voltammetry have been conducted. Comparing curve a with curve b in Figure 3A, we can observe a positive shift of the peak potential after the CPs−DNA modified electrode has been treated with cells. To further examine whether the shift comes from the pH gradient across the cell membrane, series of control experiments have been conducted. First, the electrode modified with ds-DNA alone is introduced to show the effect of CCMV CPs. As shown by curve c and curve d in Figure 3A, for the DNA alone modified electrode, the peak potential is nearly unchanged after the electrode has been treated with cells. So the shift is induced by the employment of CPs which can transfer the pH sensor into cells. Second, to eliminate the factors that may affect the shift of the peak potentials, cell culture medium and cells debris are introduced as controls. As shown in Figure 3B, no shifts can be observed after the CPs− DNA modified electrode has been treated with cell culture medium or cells debris. Therefore, the pH sensor can be transferred into cells with the help of CPs, and the electrochemical signal of pH-sensitive MB inside cells can be obtained. To better monitor the changes of intracellular pH, 20 mM ammonium chloride (NH4Cl) solution is added to induce intracellular acidosis by promptly acidifying intracellular compartments because of rapid NH3 transport and NH3/ NH4+ equilibration. Figure 4A shows the obvious shifts of the peak potential after the 293T cells are treated with NH4Cl for various periods. Therefore, after treating cells with NH4Cl for 3 h, an obvious positive shift can be observed due to intracellular acidification. With the incubation time prolonged to 5 h, the peak potential shifts further in a positive direction due to the decreased pHi, although the potential shift is slight. So, the electrochemical pHi sensor we have fabricated can show the pHi changes. It should be noticed that the potential shift cannot keep changing in great scale, although the peak current is greatly decreased, as the acidification time is prolonged to 5 h. The reason is that the pH value inside the cells will no longer be changed significantly with the increased time duration of acidification, but the peak current can be easily affected by ions, small molecules inside the cells (Figure S-4). To further verify the feasibility of the pH sensor, we have employed another cell line (A549 cells) for this study. As shown in Figure 4B, the sensor can also measure the intracellular pH changes after A549 cells being acidized for different time intervals.

Figure 2. Nyquist plots corresponding to (a) ds-DNA modified electrode, (b) CPs−DNA modified electrode, (c) CPs−DNA modified electrode after treatment with 293T cells, (d) the electrode further treated with cell lysate. Test solution: 1 M KNO3 containing 5 mM [Fe(CN)6]3−/4−. Biasing potential: 0.227 V. Amplitude: 5 mV. Frequency range: 0.1 Hz−100 kHz.

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CONCLUSIONS Taking advantage of the pH-dependent property of MB and cell permeability of CCMV CPs, we have developed an electrochemical pHi sensor for the measurement of pHi changes. The fabrication of this sensor is very simple, and it is not necessary to inject the sensor into cells for the measurements. Meanwhile, the sensor is biologically compatible because its surface is entirely made up of protein and DNA. Compared with the previous strategies in which cell permeable fluorescence compounds are imported using concepts similar to the “prodrug approach”, chemical modification is needless and the abnormal cellular events induced by hydrolysis products of fluorescence dyes can be avoided in this work. Moreover, with the pH-dependent peak potential of MB as the signal readout, the photobleaching of fluorescent dyes can be effectively avoided. The restrictions of pKa values of fluorescence dyes on pH spectrum can also be overcome, so our sensor may deliver a

thiolated DNA 1 and its complementary DNA 2 onto the electrode surface, a semicircle can be observed due to the formation of a negatively charged interface, which may electrostatically repel the also negatively charged redox species [Fe(CN)6]3−/4−. However, after the electrode is further treated with CCMV CPs, the interfacial electron transfer resistance decreases sharply, since CCMV CPs are positively charged which can significantly facilitate [Fe(CN)6]3−/4− to get access to the electrode surface. It can be also observed that the impedance increases after the further incubation of the electrode with 293T cells, because cell adhesion has been achieved on the electrode surface, which will hinder [Fe(CN)6]3−/4− getting access to the electrode surface. Nevertheless, after treating with cell lysis buffer, the resistance jumps back due to the breakdown of cells and the dissociation of CPs from CPs−DNA. 8045

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Figure 3. (A) DPVs obtained at CPs−DNA modified electrode (a) before and (b) after cell treatment, curve (c) and (d) are the cases by using dsDNA modified electrode before and after cell treatment. (B) DPVs obtained at CPs−DNA modified electrode (a) before and after the treatment of (b) cell culture medium, (c) cell debris, and (d) cell suspension.

Figure 4. Measurement of pHi changes in (A) 293T cells and (B) A549 cells. DPVs obtained at CPs−DNA modified electrode (a) before and (b) after treatment with cells. Curve (c) and curve (d) are the cases that the cells are further acidized by NH4Cl for 3 and 5 h. (2) Han, J.; Burgess, K. Chem. Rev. 2010, 110, 2709−2728. (3) Ellis, D.; Thomas, R. C. Nature 1976, 262, 224−225. (4) Carter, N. W.; Rector, F. C., Jr.; Campion, D. S.; Seldin, D. W. J. Clin. Invest. 1967, 46, 920−933. (5) Foyer, C.; Walker, D.; Spencer, C.; Mann, B. Biochem. J. 1982, 202, 429−434. (6) Gillies, R. J.; Ugurbil, K.; den Hollander, J. A.; Shulman, R. G. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 2125−2129. (7) LaManna, J. C. Metab. Brain Dis. 1987, 2, 167−182. (8) Zhang, R. G.; Kelsen, S. G.; LaManna, J. C. J. Appl. Physiol. 1990, 68, 1101−1106. (9) Li, G. X.; Miao, P. Electrochemical Analysis of Proteins and Cells; Springer: Berlin, Germany, 2012. (10) Meng, F. B.; Yang, J. H.; Liu, T.; Zhu, X. L.; Li, G. X. Anal. Chem. 2009, 81, 9168−9171. (11) Liu, J.; Zhou, H.; Xu, J. J.; Chen, H. Y. Chem. Commun. 2011, 47, 4388−4390. (12) Ning, L.; Li, X.; Ding, X.; Yin, Y.; Li, G. Int. J. Mol. Sci. 2012, 13, 10424−10431. (13) Ju, H. X.; Zhou, J.; Cai, C. X.; Chen, H. Y. Electroanal. 1995, 7, 1165−1170. (14) Li, G. X.; Long, Y. T.; Chen, H. Y.; Zhu, D. X. Fresenius J. Anal. Chem. 1996, 356, 359−360. (15) Tuite, E.; Norden, B. J. Am. Chem. Soc. 1994, 116, 7548−7556. (16) Diaz-Mochon, J. J.; Bialy, L.; Watson, J.; Sanchez-Martin, R. M.; Bradley, M. Chem. Commun. 2005, 3316−3318. (17) Rothbard, J. B.; Jessop, T. C.; Lewis, R. S.; Murray, B. A.; Wender, P. A. J. Am. Chem. Soc. 2004, 126, 9506−9507. (18) Futaki, S. Adv. Drug Delivery Rev. 2005, 57, 547−558.

broader pH spectrum, better matched to the physiological pH range. Therefore, our pH sensor may hold great promise in studying pH-dependent biological and pathological processes, such as cell death, cell proliferation, and tumor growth, in the future.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. This material is available free of charge via the Internet at http:http://pubs.acs. org/.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86 25 83592510. Tel.: +86 25 83593596. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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. 21235003).

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REFERENCES

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