Letter pubs.acs.org/ac
Electrochemical Visualization of Intracellular Hydrogen Peroxide at Single Cells Ruiqin He, Huifen Tang, Dechen Jiang,* and Hong-yuan Chen The State Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210093, China S Supporting Information *
ABSTRACT: In this Letter, the electrochemical visualization of hydrogen peroxide inside one cell was achieved first using a comprehensive Au-luminol-microelectrode and electrochemiluminescence. The capillary with a tip opening of 1−2 μm was filled with the mixture of chitosan and luminol, which was coated with the thin layers of polyvinyl chloride/nitrophenyloctyl ether (PVC/NPOE) and gold as the microelectrode. Upon contact with the aqueous hydrogen peroxide, hydrogen peroxide and luminol in contact with the gold layer were oxidized under the positive potential resulting in luminescence for the imaging. Due to the small diameter of the electrode, the microelectrode tip was inserted into one cell and the bright luminescence observed at the tip confirmed the visualization of intracellular hydrogen peroxide. The further coupling of oxidase on the electrode surface could open the field in the electrochemical imaging of intracellular biomolecules at single cells, which benefited the single cell electrochemical detection.
T
3-aminophthalate species for the luminescence.11,12 By utilizing a charge coupled device (CCD) to record the luminescence, the intracellular hydrogen peroxide should be visible and the spatial information for the electrochemical reaction could be provided.13 Since the microelectrode (1−2 μm in diameter) had a small surface area, the traditional linkage of luminol on the microelectrode did not introduce sufficient molecules for detectable luminescence (data not shown). As a result, a novel Au-luminol-microelectrode/ECL system was needed to achieve the electrochemical visualization of hydrogen peroxide inside one cell. In our design as shown in Figure 1A, a capillary with a tip opening of 1−2 μm was filled with the mixture of chitosan and luminol, which was coated with a porous polymer layer to prevent the dissolution of chitosan/luminol into the solution. Then, a thin layer of gold was sputtered on the capillary as the electrode surface. The gold layer at the end of the capillary was connected with the electrochemical station. It was proposed that the rough surface of the chitosan-luminol/polymer at the tip did not result in the full coverage of gold on the membrane. Therefore, this imperfect gold layer induced the electrochemical oxidation of the concentrated luminol inside the capillary to produce a luminol intermediate and provided the breakage regions permitting the collision between the luminol intermediate and hydrogen peroxide outside the capillary to
he microelectrode with electrochemistry has emerged as an increasing important technique for fundamental studies of single cells.1,2 The critical dimension of the microelectrode was smaller than the size of one mammalian cell so that the microelectrode could be positioned at micrometric or submicrometric distance from the cell to detect the cellular efflux of biomolecules.3,4 The diffusional mass transport was extremely efficient on this micrometer sized electrode, and fast analysis of biomolecules from single cells was guaranteed.5,6 Most recently, the nanometer sized electrode was inserted into the cell to record the intracellular hydrogen peroxide and nitrogen monoxide.7,8 This achievement of electrochemistry inside the cells opened the field in the application of the electrodes for intracellular analysis. However, specific care was needed to maintain small current noise so that the current response at the picoamp level from the electrochemical detection was collected. Also, the bright-field imaging of the electrode in the cell might not provide the clear observation of the microelectrode tip on some cell types, and thus, it was difficult to correlate the electrochemical signal with the intracellular position detected. Therefore, one continuous development of intracellular electrochemical detection might need the visualization of the electrochemical detection, which will solidify the electrochemical analysis and give the spatial information on the detection. Electrochemiluminescence (ECL) is a kind of luminescence produced during the electrochemical reaction.9,10 For the luminol−hydrogen peroxide system, the electro-oxidation of luminol generated a luminol intermediate (diazaquinone), which reacted with hydrogen peroxide to generate the excited © XXXX American Chemical Society
Received: January 13, 2016 Accepted: January 26, 2016
A
DOI: 10.1021/acs.analchem.6b00150 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry
Figure 2A showed the typical luminescence trace from the electrodes in 10 mM phosphate saline buffer (PBS, pH 7.4)
Figure 1. (A) Schematic system used for the electrochemical imaging of intracellular hydrogen peroxide. (B) SEM image of capillary. (C) SEM image of luminol/chitosan capillary coated with PVC/NPOE.
Figure 2. (A) The typical ECL trace of luminol-Au electrode in the absence and presence of 200 μM hydrogen peroxide. (B) The correlation of luminescence intensity and the concentration of hydrogen peroxide. The error bar presented the standard deviation of the luminescence intensity from three independent experiments.
generate luminescence for the visualization. No insulation of gold layer on the capillary was needed because gold at the tip only was contacted with luminol for the electrochemical reaction. As compared with the traditional disk electrode including the insertion of a metal wire in the glass capillary and the coating of the luminol layer, this electrode design could bring sufficient luminol for the reaction. Also, the fabrication process was simple and easy for automation. No aqueous luminol was required in this system, which permitted the intracellular analysis. Due to the limited solubility of luminol and chitosan at the tip, the porous polymer layer was critical for the electrode performance, which should provide minor aqueous contact with luminol and fast diffusion of hydrogen peroxide through the layer to collide with the luminol intermediate. Although Nafion and polydimethylsiloxane (PDMS) were normally applied to assemble the electrode,14 the electrode coated with either film did not generate the stable luminescence in our study (data not shown). Since our previous result on ion-selective optode exhibited that polyvinyl chloride/nitrophenyloctyl ether (PVC/ NPOE) based membrane offered a better detection limit of hydrogen peroxide than the homogeneous detection in solution due to the lipophilicity of hydrogen peroxide,15 the porous PVC/NPOE layer was attempted as the protective layer in our electrode. The luminol-PVC-Au electrode prepared was imaged using a scanning electron microscope (SEM), as shown in Figure 1B,C. After the drying of the luminol-chitosan cocktail at the tip of the capillary and the following coating of the PVC/NPOE layer, a rough surface was observed at the tip. The rough surface permitted the electrochemiluminescence process of luminol and hydrogen peroxide at the gold layer for the generation of luminescence. Most importantly, the outside diameter (O.D.) of the electrode was kept at ∼2 μm during the whole process, which guaranteed the insertion of the electrode into the cell.
recorded on photomultiplier tube (PMT). After the application of the potential from −0.8 to 0.8 V, an obvious background luminescence was observed in trace a with the peak luminescence at 0.8 V. This observation was similar to our previous result on the aqueous luminol system, which revealed that luminol embedded in the chitosan layer was electrochemically oxidized at the gold layer for the background luminescence as proposed.16 The continuous recording of the luminescence from the electrode in the buffer exhibited the near-constant intensity in 30 min, which was long enough for the following imaging experiment (Figure S1). The phenomenon supported that our PVC/NPOE layer restricted the leakage of luminol from the electrode to the aqueous solution and, then, offered the electrode stability for the cellular analysis. When 0.2 mM hydrogen peroxide was introduced into the solution, more luminescence recorded in trace b confirmed that hydrogen peroxide was reacted with luminol intermediate on the gold layer to produce the additional luminescence. The continuous addition of hydrogen peroxide from 0.2 to 3 mM gave the further increase in the luminescence, as shown in Figure 2B, which exhibited that our microelectrode could reflect the alteration of hydrogen peroxide. The detection limit was poorer than that obtained on our macroscopic ITO electrode (∼50 nM), which might be attributed to small electrode size and the insufficient collision of hydrogen peroxide with the luminol intermediate on the electrode surface.16 A similar electrochemical detection limit on nanoelectrodes was reported supporting the aforementioned explanation.8 To test the feasibility in the visualization of hydrogen peroxide, the electrode was mounted under the microscope, as shown in Figure 3A. Following our previous potential mode for B
DOI: 10.1021/acs.analchem.6b00150 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry
cells or any interspace between the cell membrane and the electrode could induce the membrane impermeant PI into the cells to show red fluorescence. Experimentally, the electrode tip was positioned into the cell for 2 min and, then, withdrawn to leave the cell for the staining of FDA or Hoechst 33342 for 10 min, respectively. Immediately after the electrode was withdrawn, a small hole was observed at the cellular membrane and then disappeared confirming the insertion of the electrode inside the cell and the recovery of the cellular membrane after the electrode removal (Figure S3A−C). The following staining of the cells exhibited green fluorescence at FDA-stained cells and no more blue fluorescence at the Hoechst 33342 stained cell, as shown in Figure S4A−D, which confirmed the cell viability. The success rate to maintain the cellular viability through the electrode insertion was near 50%. Furthermore, to show the tight seal of the cellular membrane around the electrode, the electrode was inserted into the cells and PI was added into the medium for 10 min. After the careful washing of PI away from the cells, no red fluorescence observed at the cells in Figure S4E,F exhibited that no PI diffused into the cells. All these results supported that the insertion of the 2 μm-diameter electrode did not induce the deformation of the cellular membrane. The tight seal between the electrode and the membrane offered the feasibility for the intracellular analysis. For the electrochemical visualization of intracellular hydrogen peroxide at single cells, the cells were physically treated with 10 mM hydrogen peroxide for 5 min so that the intracellular hydrogen peroxide reached a millimolar level. After the cells were washed and reconditioned in PBS, the microelectrode was positioned near the cells and no luminescence was imaged under the potentials. Then, the electrode was inserted into the cells, as shown in Figure 4A, and
Figure 3. Images of the electrode in the presence of aqueous hydrogen peroxide. (A) Bright-field image; (B) the luminescence image with 5 mM hydrogen peroxide; (C) the overlapping image; (D) the sizes of luminescence spot with different concentrations of hydrogen peroxide.
ECL imaging,13 a switching of the potential between 0.8 (2 s) and −0.8 V (0.5 s) was continuously employed on the electrode to induce the luminescence, which was recorded using electron multiply CCD (EM-CCD). The exposure time was set as 1 min. No background luminescence was imaged when the electrode was exposed to PBS indicating that the luminescence from luminol itself was not strong enough for the imaging. After the introduction of 5 mM hydrogen peroxide, a bright spot was observed in Figure 3B. The overlapping of the bright-field and luminescence images in Figure 3C confirmed that the luminescence was generated at the tip of the electrode, which exhibited the electrochemical visualization of hydrogen peroxide. As listed in Figures 3D and S2, the size of luminescence spot was ∼10 μm in the presence of 1 mM hydrogen peroxide and became bigger with more hydrogen peroxide. The luminescence spot was larger than the electrode tip revealing the diffusion of intermediate around the electrode surface. Since the imaging system was not as sensitive as the PMT based system, the detection limit for the imaging of hydrogen peroxide was ∼1 mM. Fortunately, the intracellular hydrogen peroxide after the biological stimulation reached several millimolar, and thus, our electrode was qualified for the visualization of intracellular hydrogen peroxide. To achieve the intracellular analysis, the microelectrode needed to be positioned inside the cell and the cell viability after the electrode insertion was critical. The capillary with a 2 μm tip has been reported to achieve the microinjection in the individual cell with specific care.17,18 In our experiment, three fluorescent dyes, Hoechst 33342, fluorescein diacetate (FDA), and propidium iodide (PI), were used to investigate the cell viability and the membrane sealing around the electrode. In principle, living cells could actively convert the nonfluorescent FDA into fluorescin with green fluorescence as a sign of viability, and the Hoechst 33342 stained cell in the apoptotic process offered more blue fluorescence. Meanwhile, the dead
Figure 4. (A) The bright-field image of the microelectrode in the cell loaded with 10 mM hydrogen peroxide; (B) the luminescence image; (C) the overlapping image; (D) the bright-field image of the microelectrode in the cell stimulated with 800 ng/mL PMA; (E) the luminescence image; (F) the overlapping image.
the potentials were applied on the electrode immediately. The simultaneous recording of the luminescence for 1 min showed the bright spot inside the cells, as shown in Figure 4B. The overlapping of bright-field and luminescence images, as shown in Figure 4C, exhibited that the luminescence spot was positioned at the electrode tip. The control experiment was performed using the electrode contacted with the cell membrane. No luminescence could be visualized suggesting C
DOI: 10.1021/acs.analchem.6b00150 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry Notes
that the electrode outside the cell could not offer the visualization of the released hydrogen peroxide. Therefore, our observation of hydrogen peroxide should be ascribed to a relative high concentration of intracellular hydrogen peroxide. Besides hydrogen peroxide, it was noted that other reactive oxygen species (ROS) and reactive nitrogen species (RNS) were generated inside the cell. The typical concentration of ROS (mainly superoxide) was at the micromolar level and much lower than that of hydrogen peroxide.19 Therefore, the existence of ROS should not induce significant luminescence in our analysis. Meanwhile, in the presence of hydrogen peroxide, NO and nitrite ion inside the cell produced peroxynitrite, the main component of RNS, which had a concentration ranging from a few to a few hundred micromolar in the cell.20 It was reported that peroxynitrite could induce chemiluminescence with luminol without the application of potential.21 To investigate the contribution of RNS in the luminescence, the luminescence of the electrode after the insertion into the cell was recorded in the absence of potential. No luminescence was imaged, confirming that the intracellular peroxynitrite did not generate measurable luminescence in our cell analysis. As a result, the luminescence collected should be mainly attributed to intracellular hydrogen peroxide. To the best of our knowledge, this was the first electrochemical visualization of hydrogen peroxide inside the cells. After the physical elevation of intracellular hydrogen peroxide, the cells were stimulated by 800 ng/mL phorbol myryslate acetate (PMA) which increased the concentration of hydrogen peroxide inside the cells physiologically.22 As shown in Figure 4D−F, the luminescence was observed at the electrode surface, which exhibited that our electrode could reveal the fluctuation of hydrogen peroxide in the biological process. According to the calibration curve in Figure 3D, the intracellular hydrogen peroxide after PMA stimulation reached 5 mM which was similar to our previous result using the electrochemical method.23 In conclusion, a novel comprehensive luminol-Au microelectrode was prepared to induce the electrochemiluminescence with hydrogen peroxide that could be recorded using CCD. By the insertion of microelectrode into the cell, the luminescence imaged revealed the first electrochemical visualization of intracellular hydrogen peroxide. The further development will focus on the optimization of electrode structure to increase the collision possibility between oxygen containing species and luminol species after the oxidation and, thus, improve the detection limit. Also, the oxidases will be introduced on the electrode to image more biomolecules inside the cells electrochemically.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the 973 Program (2013 CB933800) and the National Natural Science Foundation of China (Nos. 21327902, 21135003, and 21575060).
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REFERENCES
(1) Adams, K. L.; Puchades, M.; Ewing, A. G. Annu. Rev. Anal. Chem. 2008, 1, 329−355. (2) Amatore, C.; Arbault, S.; Guille, M.; Lemaitre, F. Chem. Rev. 2008, 108, 2585−2621. (3) Travis, E. R.; Wightman, R. M. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 77−103. (4) Wightman, R. M. Science 2006, 311, 1570−1574. (5) Bard, A. J. ACS Nano 2008, 2, 2437−2440. (6) Penner, R. M.; Heben, M. J.; Longin, T. L.; Lewis, N. S. Science 1990, 250, 1118−1121. (7) Wang, Y. X.; Noel, J. M.; Velmurugan, J.; Nogala, W.; Mirkin, M. V.; Lu, C.; Collignon, M. G.; Lemaitre, F.; Amatore, C. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 11534−11539. (8) Sun, P.; Laforge, F. O.; Abeyweera, T. P.; Rotenberg, S. A.; Carpino, J.; Mirkin, M. V. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 443−448. (9) Bard, A. J. Electrogenerated Chemiluminescence; CRC Press: Boca Raton, FL, 2004. (10) Forster, R. J.; Bertoncello, P.; Keyes, T. E. Annu. Rev. Anal. Chem. 2009, 2, 359−385. (11) Miao, W. J. Chem. Rev. 2008, 108, 2506−2553. (12) Richter, M. M. Chem. Rev. 2004, 104, 3003−3036. (13) Zhou, J. Y.; Ma, G. Z.; Chen, Y.; Fang, D. J.; Jiang, D. C.; Chen, H. Y. Anal. Chem. 2015, 87, 8138−8143. (14) Hashemi, P.; Dankoski, E. C.; Petrovic, J.; Keithley, R. B.; Wightman, R. M. Anal. Chem. 2009, 81, 9462−9471. (15) Liu, Y. L.; Zhu, J. W.; Xu, Y. M.; Qin, Y.; Jiang, D. C. ACS Appl. Mater. Interfaces 2015, 7, 11141−11145. (16) Ma, G. Z.; Zhou, J. Y.; Tian, C. X.; Jiang, D. C.; Fang, D. C.; Chen, H. Y. Anal. Chem. 2013, 85, 3912−7. (17) Woods, L. A.; Gandhi, P. U.; Ewing, A. G. Anal. Chem. 2005, 77, 1819−23. (18) Lim, J.; Danuser, G. J. Visualized Exp. 2009, 30, 1325−1328. (19) Zhang, W.; Li, P.; Yang, F.; Hu, X.; Sun, C.; Zhang, W.; Chen, D.; Tang, B. J. Am. Chem. Soc. 2013, 135, 14956−9. (20) Reifenberger, M. S.; Arnett, K. L.; Gatto, C.; Milanick, M. A. Am. J. Physiol Renal Physiol. 2008, 295, F1191−8. (21) Daiber, A.; Oelze, M.; August, M.; Wendt, M.; Sydow, K.; Wieboldt, H.; Kleschyov, A. L.; Munzel, T. Free Radical Res. 2004, 38, 259−69. (22) Lundqvist, H.; Follin, P.; Khalfan, L.; Dahlgren, C. J. Leukoc. Biol. 1996, 59, 270−279. (23) Zhuang, L. H.; Zuo, H. Z.; Wu, Z. Q.; Wang, Y.; Fang, D. J.; Jiang, D. C. Anal. Chem. 2014, 86, 11517−22.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b00150. Experimental section; electrode stability; luminescence images of the electrode; electrode insertion inside the cell; cell viability (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Phone: 086-25-83594846. Fax: 086-25-83594846. E-mail: [email protected]. D
DOI: 10.1021/acs.analchem.6b00150 Anal. Chem. XXXX, XXX, XXX−XXX
Electrochemical Visualization of Intracellular Hydrogen Peroxide at Single Cells Ruiqin He, Huifen Tang, Dechen Jiang,* and Hong-yuan Chen The State Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210093, China S Supporting Information *
ABSTRACT: In this Letter, the electrochemical visualization of hydrogen peroxide inside one cell was achieved first using a comprehensive Au-luminol-microelectrode and electrochemiluminescence. The capillary with a tip opening of 1−2 μm was filled with the mixture of chitosan and luminol, which was coated with the thin layers of polyvinyl chloride/nitrophenyloctyl ether (PVC/NPOE) and gold as the microelectrode. Upon contact with the aqueous hydrogen peroxide, hydrogen peroxide and luminol in contact with the gold layer were oxidized under the positive potential resulting in luminescence for the imaging. Due to the small diameter of the electrode, the microelectrode tip was inserted into one cell and the bright luminescence observed at the tip confirmed the visualization of intracellular hydrogen peroxide. The further coupling of oxidase on the electrode surface could open the field in the electrochemical imaging of intracellular biomolecules at single cells, which benefited the single cell electrochemical detection.
T
3-aminophthalate species for the luminescence.11,12 By utilizing a charge coupled device (CCD) to record the luminescence, the intracellular hydrogen peroxide should be visible and the spatial information for the electrochemical reaction could be provided.13 Since the microelectrode (1−2 μm in diameter) had a small surface area, the traditional linkage of luminol on the microelectrode did not introduce sufficient molecules for detectable luminescence (data not shown). As a result, a novel Au-luminol-microelectrode/ECL system was needed to achieve the electrochemical visualization of hydrogen peroxide inside one cell. In our design as shown in Figure 1A, a capillary with a tip opening of 1−2 μm was filled with the mixture of chitosan and luminol, which was coated with a porous polymer layer to prevent the dissolution of chitosan/luminol into the solution. Then, a thin layer of gold was sputtered on the capillary as the electrode surface. The gold layer at the end of the capillary was connected with the electrochemical station. It was proposed that the rough surface of the chitosan-luminol/polymer at the tip did not result in the full coverage of gold on the membrane. Therefore, this imperfect gold layer induced the electrochemical oxidation of the concentrated luminol inside the capillary to produce a luminol intermediate and provided the breakage regions permitting the collision between the luminol intermediate and hydrogen peroxide outside the capillary to
he microelectrode with electrochemistry has emerged as an increasing important technique for fundamental studies of single cells.1,2 The critical dimension of the microelectrode was smaller than the size of one mammalian cell so that the microelectrode could be positioned at micrometric or submicrometric distance from the cell to detect the cellular efflux of biomolecules.3,4 The diffusional mass transport was extremely efficient on this micrometer sized electrode, and fast analysis of biomolecules from single cells was guaranteed.5,6 Most recently, the nanometer sized electrode was inserted into the cell to record the intracellular hydrogen peroxide and nitrogen monoxide.7,8 This achievement of electrochemistry inside the cells opened the field in the application of the electrodes for intracellular analysis. However, specific care was needed to maintain small current noise so that the current response at the picoamp level from the electrochemical detection was collected. Also, the bright-field imaging of the electrode in the cell might not provide the clear observation of the microelectrode tip on some cell types, and thus, it was difficult to correlate the electrochemical signal with the intracellular position detected. Therefore, one continuous development of intracellular electrochemical detection might need the visualization of the electrochemical detection, which will solidify the electrochemical analysis and give the spatial information on the detection. Electrochemiluminescence (ECL) is a kind of luminescence produced during the electrochemical reaction.9,10 For the luminol−hydrogen peroxide system, the electro-oxidation of luminol generated a luminol intermediate (diazaquinone), which reacted with hydrogen peroxide to generate the excited © XXXX American Chemical Society
Received: January 13, 2016 Accepted: January 26, 2016
A
DOI: 10.1021/acs.analchem.6b00150 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry
Figure 2A showed the typical luminescence trace from the electrodes in 10 mM phosphate saline buffer (PBS, pH 7.4)
Figure 1. (A) Schematic system used for the electrochemical imaging of intracellular hydrogen peroxide. (B) SEM image of capillary. (C) SEM image of luminol/chitosan capillary coated with PVC/NPOE.
Figure 2. (A) The typical ECL trace of luminol-Au electrode in the absence and presence of 200 μM hydrogen peroxide. (B) The correlation of luminescence intensity and the concentration of hydrogen peroxide. The error bar presented the standard deviation of the luminescence intensity from three independent experiments.
generate luminescence for the visualization. No insulation of gold layer on the capillary was needed because gold at the tip only was contacted with luminol for the electrochemical reaction. As compared with the traditional disk electrode including the insertion of a metal wire in the glass capillary and the coating of the luminol layer, this electrode design could bring sufficient luminol for the reaction. Also, the fabrication process was simple and easy for automation. No aqueous luminol was required in this system, which permitted the intracellular analysis. Due to the limited solubility of luminol and chitosan at the tip, the porous polymer layer was critical for the electrode performance, which should provide minor aqueous contact with luminol and fast diffusion of hydrogen peroxide through the layer to collide with the luminol intermediate. Although Nafion and polydimethylsiloxane (PDMS) were normally applied to assemble the electrode,14 the electrode coated with either film did not generate the stable luminescence in our study (data not shown). Since our previous result on ion-selective optode exhibited that polyvinyl chloride/nitrophenyloctyl ether (PVC/ NPOE) based membrane offered a better detection limit of hydrogen peroxide than the homogeneous detection in solution due to the lipophilicity of hydrogen peroxide,15 the porous PVC/NPOE layer was attempted as the protective layer in our electrode. The luminol-PVC-Au electrode prepared was imaged using a scanning electron microscope (SEM), as shown in Figure 1B,C. After the drying of the luminol-chitosan cocktail at the tip of the capillary and the following coating of the PVC/NPOE layer, a rough surface was observed at the tip. The rough surface permitted the electrochemiluminescence process of luminol and hydrogen peroxide at the gold layer for the generation of luminescence. Most importantly, the outside diameter (O.D.) of the electrode was kept at ∼2 μm during the whole process, which guaranteed the insertion of the electrode into the cell.
recorded on photomultiplier tube (PMT). After the application of the potential from −0.8 to 0.8 V, an obvious background luminescence was observed in trace a with the peak luminescence at 0.8 V. This observation was similar to our previous result on the aqueous luminol system, which revealed that luminol embedded in the chitosan layer was electrochemically oxidized at the gold layer for the background luminescence as proposed.16 The continuous recording of the luminescence from the electrode in the buffer exhibited the near-constant intensity in 30 min, which was long enough for the following imaging experiment (Figure S1). The phenomenon supported that our PVC/NPOE layer restricted the leakage of luminol from the electrode to the aqueous solution and, then, offered the electrode stability for the cellular analysis. When 0.2 mM hydrogen peroxide was introduced into the solution, more luminescence recorded in trace b confirmed that hydrogen peroxide was reacted with luminol intermediate on the gold layer to produce the additional luminescence. The continuous addition of hydrogen peroxide from 0.2 to 3 mM gave the further increase in the luminescence, as shown in Figure 2B, which exhibited that our microelectrode could reflect the alteration of hydrogen peroxide. The detection limit was poorer than that obtained on our macroscopic ITO electrode (∼50 nM), which might be attributed to small electrode size and the insufficient collision of hydrogen peroxide with the luminol intermediate on the electrode surface.16 A similar electrochemical detection limit on nanoelectrodes was reported supporting the aforementioned explanation.8 To test the feasibility in the visualization of hydrogen peroxide, the electrode was mounted under the microscope, as shown in Figure 3A. Following our previous potential mode for B
DOI: 10.1021/acs.analchem.6b00150 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry
cells or any interspace between the cell membrane and the electrode could induce the membrane impermeant PI into the cells to show red fluorescence. Experimentally, the electrode tip was positioned into the cell for 2 min and, then, withdrawn to leave the cell for the staining of FDA or Hoechst 33342 for 10 min, respectively. Immediately after the electrode was withdrawn, a small hole was observed at the cellular membrane and then disappeared confirming the insertion of the electrode inside the cell and the recovery of the cellular membrane after the electrode removal (Figure S3A−C). The following staining of the cells exhibited green fluorescence at FDA-stained cells and no more blue fluorescence at the Hoechst 33342 stained cell, as shown in Figure S4A−D, which confirmed the cell viability. The success rate to maintain the cellular viability through the electrode insertion was near 50%. Furthermore, to show the tight seal of the cellular membrane around the electrode, the electrode was inserted into the cells and PI was added into the medium for 10 min. After the careful washing of PI away from the cells, no red fluorescence observed at the cells in Figure S4E,F exhibited that no PI diffused into the cells. All these results supported that the insertion of the 2 μm-diameter electrode did not induce the deformation of the cellular membrane. The tight seal between the electrode and the membrane offered the feasibility for the intracellular analysis. For the electrochemical visualization of intracellular hydrogen peroxide at single cells, the cells were physically treated with 10 mM hydrogen peroxide for 5 min so that the intracellular hydrogen peroxide reached a millimolar level. After the cells were washed and reconditioned in PBS, the microelectrode was positioned near the cells and no luminescence was imaged under the potentials. Then, the electrode was inserted into the cells, as shown in Figure 4A, and
Figure 3. Images of the electrode in the presence of aqueous hydrogen peroxide. (A) Bright-field image; (B) the luminescence image with 5 mM hydrogen peroxide; (C) the overlapping image; (D) the sizes of luminescence spot with different concentrations of hydrogen peroxide.
ECL imaging,13 a switching of the potential between 0.8 (2 s) and −0.8 V (0.5 s) was continuously employed on the electrode to induce the luminescence, which was recorded using electron multiply CCD (EM-CCD). The exposure time was set as 1 min. No background luminescence was imaged when the electrode was exposed to PBS indicating that the luminescence from luminol itself was not strong enough for the imaging. After the introduction of 5 mM hydrogen peroxide, a bright spot was observed in Figure 3B. The overlapping of the bright-field and luminescence images in Figure 3C confirmed that the luminescence was generated at the tip of the electrode, which exhibited the electrochemical visualization of hydrogen peroxide. As listed in Figures 3D and S2, the size of luminescence spot was ∼10 μm in the presence of 1 mM hydrogen peroxide and became bigger with more hydrogen peroxide. The luminescence spot was larger than the electrode tip revealing the diffusion of intermediate around the electrode surface. Since the imaging system was not as sensitive as the PMT based system, the detection limit for the imaging of hydrogen peroxide was ∼1 mM. Fortunately, the intracellular hydrogen peroxide after the biological stimulation reached several millimolar, and thus, our electrode was qualified for the visualization of intracellular hydrogen peroxide. To achieve the intracellular analysis, the microelectrode needed to be positioned inside the cell and the cell viability after the electrode insertion was critical. The capillary with a 2 μm tip has been reported to achieve the microinjection in the individual cell with specific care.17,18 In our experiment, three fluorescent dyes, Hoechst 33342, fluorescein diacetate (FDA), and propidium iodide (PI), were used to investigate the cell viability and the membrane sealing around the electrode. In principle, living cells could actively convert the nonfluorescent FDA into fluorescin with green fluorescence as a sign of viability, and the Hoechst 33342 stained cell in the apoptotic process offered more blue fluorescence. Meanwhile, the dead
Figure 4. (A) The bright-field image of the microelectrode in the cell loaded with 10 mM hydrogen peroxide; (B) the luminescence image; (C) the overlapping image; (D) the bright-field image of the microelectrode in the cell stimulated with 800 ng/mL PMA; (E) the luminescence image; (F) the overlapping image.
the potentials were applied on the electrode immediately. The simultaneous recording of the luminescence for 1 min showed the bright spot inside the cells, as shown in Figure 4B. The overlapping of bright-field and luminescence images, as shown in Figure 4C, exhibited that the luminescence spot was positioned at the electrode tip. The control experiment was performed using the electrode contacted with the cell membrane. No luminescence could be visualized suggesting C
DOI: 10.1021/acs.analchem.6b00150 Anal. Chem. XXXX, XXX, XXX−XXX
Letter
Analytical Chemistry Notes
that the electrode outside the cell could not offer the visualization of the released hydrogen peroxide. Therefore, our observation of hydrogen peroxide should be ascribed to a relative high concentration of intracellular hydrogen peroxide. Besides hydrogen peroxide, it was noted that other reactive oxygen species (ROS) and reactive nitrogen species (RNS) were generated inside the cell. The typical concentration of ROS (mainly superoxide) was at the micromolar level and much lower than that of hydrogen peroxide.19 Therefore, the existence of ROS should not induce significant luminescence in our analysis. Meanwhile, in the presence of hydrogen peroxide, NO and nitrite ion inside the cell produced peroxynitrite, the main component of RNS, which had a concentration ranging from a few to a few hundred micromolar in the cell.20 It was reported that peroxynitrite could induce chemiluminescence with luminol without the application of potential.21 To investigate the contribution of RNS in the luminescence, the luminescence of the electrode after the insertion into the cell was recorded in the absence of potential. No luminescence was imaged, confirming that the intracellular peroxynitrite did not generate measurable luminescence in our cell analysis. As a result, the luminescence collected should be mainly attributed to intracellular hydrogen peroxide. To the best of our knowledge, this was the first electrochemical visualization of hydrogen peroxide inside the cells. After the physical elevation of intracellular hydrogen peroxide, the cells were stimulated by 800 ng/mL phorbol myryslate acetate (PMA) which increased the concentration of hydrogen peroxide inside the cells physiologically.22 As shown in Figure 4D−F, the luminescence was observed at the electrode surface, which exhibited that our electrode could reveal the fluctuation of hydrogen peroxide in the biological process. According to the calibration curve in Figure 3D, the intracellular hydrogen peroxide after PMA stimulation reached 5 mM which was similar to our previous result using the electrochemical method.23 In conclusion, a novel comprehensive luminol-Au microelectrode was prepared to induce the electrochemiluminescence with hydrogen peroxide that could be recorded using CCD. By the insertion of microelectrode into the cell, the luminescence imaged revealed the first electrochemical visualization of intracellular hydrogen peroxide. The further development will focus on the optimization of electrode structure to increase the collision possibility between oxygen containing species and luminol species after the oxidation and, thus, improve the detection limit. Also, the oxidases will be introduced on the electrode to image more biomolecules inside the cells electrochemically.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the 973 Program (2013 CB933800) and the National Natural Science Foundation of China (Nos. 21327902, 21135003, and 21575060).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b00150. Experimental section; electrode stability; luminescence images of the electrode; electrode insertion inside the cell; cell viability (PDF)
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DOI: 10.1021/acs.analchem.6b00150 Anal. Chem. XXXX, XXX, XXX−XXX
