Anal. Chem. 2007, 79, 4237-4240
Microimaging of Algal Bioconvection by Scanning Electrochemical Microscopy Isao Shitanda,† Yutaka Yoshida, and Tetsu Tatsuma*
Institute of Industrial Science, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8505, Japan
Local bioconvection generated by algal flagellar movement was imaged by scanning electrochemical microscopy. As a microelectrode probe vertically approached an individual multicellular flagellate alga, Volvox carteri, an oxidation current of a coexisting redox marker ([Fe(CN)6]4-) increased gradually, due to bioconvective enhancement of mass transport, and eventually decreased because the algal body blocked the diffusion of the marker. Twodimensional imaging of the bioconvection of an individual alga was also possible. The bioconvective enhancement of the current was hindered by a toxic compound that inhibits the flagellar movement. Scanning electrochemical microscopy (SECM) has been shown to be a powerful tool for imaging concentration profiles of a redoxactive species.1-3 Recently, SECM has also been used for bioimaging of cells and organisms in terms of photosynthetic activity,4,5 respiratory activity,6,7 production activity of dopamine,8 osteoclastic activity,9 membrane permeability,10 and redox activity.11,12 In the present study, we applied SECM to imaging of biologically induced convection (i.e., bioconvection). Although fluid flows through porous materials including biomaterials13-15 and magnetrohydrodynamic flows16 have been imaged by SECM, imaging * Corresponding author. E-mail:
[email protected]. † Present address: Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, Japan. (1) Bard, A. J.; Fan, F.-R. F.; Kwak, J.; Lev, O. Anal. Chem. 1989, 61, 132. (2) Bard, A. J.; Li, X.; Zhan, W. Biosens. Bioelectron. 2006, 22, 461. (3) Amemiya, S.; Guo, J.; Xiong, H.; Gross, D. A. Anal. Bioanal. Chem. 2006, 386, 458. (4) Tsionsky, M.; Cardon, Z. G.; Bard, A. J.; Jackson, R. B. Plant Physiol. 1997, 113, 895. (5) Yasukawa, T.; Kaya, T.; Matsue, T. Anal. Chem. 1999, 71, 4637. (6) Torisawa, Y.; Shiku, H.; Kasai, S.; Nishizawa, M.; Matsue, T. Int. J. Cancer 2004, 109, 302. (7) Holt, K. B.; Bard, A. J. Biochemistry 2005, 44, 13214. (8) Liebetrau, J. M.; Miller, H. M.; Baur, J. E.; Takacs, S. A.; Anupunpisit, V.; Garris, P. A.; Wipf, D. O. Anal. Chem. 2003, 75, 563. (9) Berger, C. E. M.; Horrocks, B. R.; Datta, H. K. Electrochim. Acta 1999, 44, 2677. (10) Guo, J.; Amemiya, S. Anal. Chem. 2005, 77, 2147. (11) Liu, B.; Rotenberg, S. A.; Mirkin, M. V. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9855. (12) Cai, C.; Liu, B.; Mirkin, M. V.; Frank, H. A.; Rusling, J. F. Anal. Chem. 2002, 74, 114. (13) Edwards, M. A.; Martin, S.; Whitworth, A. L.; Macpherson, J. V.; Unwin, P. R. Physiol. Meas. 2006, 27, R63. (14) Macpherson, J. V.; Beeston, M. A.; Unwin, P. R. Langmuir 1995, 11, 3959. (15) Macpherson, J. V.; Beeston, M. A.; Unwin, P. R. J. Chem. Soc. Faraday Trans. 1995, 91, 1407. (16) Ragsdale, S. R.; White, H. S. Anal. Chem. 1999, 71, 1923. 10.1021/ac070150t CCC: $37.00 Published on Web 05/02/2007
© 2007 American Chemical Society
Figure 1. Schematic illustration for SECM imaging of bioconvection of V. carteri.
of biologically induced convection has not yet been reported, to the best of our knowledge. Local imaging of biological fluid flow caused by cell motility, such as flagellar, ciliary, peristaltic, and cardiac movements, is important in characterization of their behavior and elucidation of their mechanisms. In addition, monitoring of cell motility could be applied to sensitive bioassay techniques regarding aquatic risk assessment for environmental water and industrial, agricultural, and domestic wastewater. It would also be effective in screening and evaluation of chemicals in terms of toxicity. In view of this, we recently developed compact monitoring systems for algal flagellar movement.17,18 A solution containing flagellate alga, Chlamydomonas reinhardtii, and a redox marker, [Fe(CN)6]4-, is stirred by a (17) Shitanda, I.; Takada, K.; Sakai, Y.; Tatsuma, T. Anal. Chem. 2005, 77, 6715. (18) Shitanda, I.; Tatsuma, T. Anal. Chem. 2006, 78, 349.
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taxis,17 and phototaxis18 of the flagellate. These activities were modified or inhibited by different chemicals. However, microimaging of the bioconvection was not possible with the conventional electrode. Here we employed a microelectrode instead of the macroscopic electrode and Volvox carteri as a flagellate alga. Volvox is a spherical multicellular green alga, whose diameter is 100-500 µm (Figure 1). The alga contains many small biflagellate somatic cells and a few large, nonmotile reproductive cells called gonidia and swims with a characteristic rolling motion.19 Fluid flow generated by the collective flagellar movement of an individual of the alga, which was entrapped on a glass plate, was monitored by SECM in the presence of the redox marker. Since the bioconvective flow enhances mass transport and an oxidation current of the marker, the enhanced current can be an index of the bioconvection.
Figure 2. Typical vertical approach curves for entrapped V. carteri recorded in SVM containing 1 mM K4[Fe(CN)6] (pH 7.5, 25 °C) in the absence (a) or presence (b) of 1 mM diltiazem. Probe potential, +0.5 V vs Ag|AgCl; approach rate, 1.0 µm s-1. Zero distance is determined to be the probe height at which the normalized current ) 0.8.
bioconvection due to collective flagellar movement. A diffusionlimited oxidation current for the redox marker is observed at a conventional “macroscopic” electrode in the solution. The current increases with the convection rate, which is a function of algal population and average intensity of flagellar movement. This system was applied to monitoring of motility,17 negative gravi-
EXPERIMENTAL SECTION Incubation of Algae. Flagellate alga V. carteri strain (wildderived, EVE10) was used throughout. The algae were grown in standard volvox medium (SVM, pH 7.5)20 in a 300-mL culture bottle at 25 °C. The culture bottle was aerated through a membrane filter and illuminated by dim fluorescent light periodically (12-h illumination, 12-h dark). Sample Preparation. Algae were entrapped on a glass plate by using a polyion complex film prepared from poly(L-lysine) (PLL; Aldrich, Mw ) 100 000) as a polycation and poly(styrenesulfonate) (PSS; Aldrich, Mw ) 70 000) as a polyanion. PLL (25 mmol dm-3 monomer unit, 2 µL) and PSS (25 mmol dm-3 monomer unit, 1 µL) solutions were successively cast on a glass surface, followed by additional dropping of the PLL solution (2 µL). The plate was rinsed with ultrapure water and dried for 1 h at room temperature. The incubated algae were cast on the plate to be entrapped on the polyion complex film. SECM Measurements. Figure 1 shows the setup for SECM measurements, which is composed of a SECM equipment (ALS900) with a digital potentiostat (ALS-600) and a piezo-driven XYZ stage, an inverted microscope (CKX41, Olympus), and a CCD camera (VB7010, Keyence). The sample plate with the entrapped algae was set in a glass vessel containing a mixture of the SVM and 2 mM aqueous K4[Fe(CN)6] as the redox marker (250 µL each). A platinum disk microelectrode (diameter, 10 µm), a coiled
Figure 3. Schematic illustrations for the diffusion layer around the microelectrode surface and the bioconvection layer around the V. carteri. (a) The electrode is sufficiently apart from the alga. (b) The diffusion layer is overlapped with the bioconvection layer. (c) The diffusion is restricted by the algal body. 4238 Analytical Chemistry, Vol. 79, No. 11, June 1, 2007
entrapped alga and polarized at +0.5 V versus Ag|AgCl, where the redox marker [Fe(CN)6]4- is oxidized to give diffusion-limited anodic currents. Current changes were monitored during vertical approach of the probe to the alga at 1.0 µm s-1. The anodic current increased gradually and dropped eventually. It should be noted that 1 mM [Fe(CN)6]4- is not toxic to the algae. V. carteri is a spherical multicellular green alga (diameter, 100-500 µm), which contains many small biflagellate somatic cells. Collective flagellar beatings give rise to bioconvection, which agitates the solution around itself. If the probe is sufficiently apart from the alga, the bioconvection has no influence on the anodic current. In this case, a hemispherical diffusion arises around the probe (Figure 3a), and the steady-state current i is given by the following equation,21
i ) 4nFDCa
Figure 4. Typical horizontal scan curves for entrapped V. carteri recorded in SVM containing 1 mM K4[Fe(CN)6] (pH 7.5, 25 °C). Probe potential, +0.5 V vs Ag|AgCl; probe scan rate, 5 µm s-1. The probe position in (a) and (b) was higher than that in (c) by 60 and 30 µm, respectively.
plutinum wire, and a Ag|AgCl were used as working, counter, and reference electrodes, respectively. The working electrode was polarized at +0.5 V versus Ag|AgCl, and an anodic current for oxidation of [Fe(CN)6]4- was monitored. The current was typically 0.5-0.8 nA. RESULTS AND DISCUSSION Vertical Approach Curves. Figure 2a shows a typical vertical approach curve. The probe was set above an individual of the
(1)
where n, F, D, C, and a are the number of electrons involved (n ) 1 in the present case), the Faraday constant, the diffusion coefficient of the redox marker, the bulk concentration of the marker, and the radius of the electrode, respectively. As the probe approaches the alga, the diffusion layer on the electrode eventually comes into contact with the bioconvection layer around the alga (Figure 3b). As a result, the diffusion layer thickness decreases and the mass transport to the electrode surface as well as the anodic current increases. In the present case, however, the enhancement factor of the current was as small as 1.05. Hence, the diffusion likely falls into an intermediate one between the hemispherical and the planar diffusions, probably because the algal bioconvection is relatively weak. When the probe is so close to the algal surface that the algal body blocks the diffusion of the redox marker to the electrode, the current decreases (Figure 3c). This behavior shares mechanisms with so-called negative feedback mode of SECM,22 in which electrolytic currents decrease as a probe approaches an electrically insulating substrate. Effect of an Inhibitor for Flagellar Movement. Figure 2b shows a typical approach curve obtained after addition of 1 mM diltiazem to the solution. Diltiazem is known to bind to calcium channels at the flagellar surface and block the flow of Ca2+ to inhibit the flagellar movement.23 In the course of the approach to the alga, a sharp current decrease was observed, although it was not preceded by a current increase. This result indicates that the algal flagellar movement is responsible for the current increase observed in the absence of diltiazem (Figure 2a). Thus, we conclude that the present system can evaluate changes in the local fluid flow generated by an individual flagellate alga. Additionally, the present method allows detection of a toxic compound that inhibits the flagellar movement and algal motility. Horizontal Scan Curves. Figure 4 shows typical changes in the current when the probe at +0.5 V versus Ag|AgCl was scanned horizontally at 5 µm s-1. The scanning was repeated eight times at different heights; the probe was lowered by 10 µm after each scan. In the first to third scans, the oxidation current slightly (19) (20) (21) (22) (23)
Kirk, D. L. Curr. Biol. 2004, 14, R599. Kirk, M. M.; Kirk, D. L. Cell 1985, 41, 419. Saito, Y. Rev. Polarogr. 1968, 15, 178. Kwak, J.; Bard, A. J. Anal. Chem. 1989, 61, 1799. Nultsch, W.; Pfau, J.; Dolle, R. Arch. Microbiol. 1986, 144, 393.
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as shown in Figure 4b. Finally, in the seventh and eighth scans, the current peak was almost totally eroded and the scan curve was dominated by a large “current hole” (Figure 4c; the eighth scan). The current hole reflects the blocked mass transport to the probe by the algal body, as mentioned above. Two-dimensional mapping of the bioconvection was also possible (Figure 5). In this experiment, the probe was scanned horizontally at 12.5 µm s-1, since the increase in the scan rate did not modify the scan curve significantly. As shown in the figure, the oxidation current increased at around an individual alga.
Figure 5. Typical two-dimensional bioconvection image for entrapped V. carteri recorded in SVM containing 1 mM K4[Fe(CN)6] (pH 7.5, 25 °C). Probe potential. +0.5 V vs Ag|AgCl; probe scan rate, 12.5 µm s-1.
increased when the probe passed above the alga (Figure 4a; the second scan). In the fourth to sixth scans, the peak height gradually increased (Figure 4b; the fifth scan). As mentioned above, the results are explained in terms of the enhanced mass transport due to the bioconvection (Figure 3b). At the same time, however, a “hole” gradually broke into the current peak at its top
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CONCLUSIONS It was proved that bioconvection can be imaged by SECM in the presence of an appropriate redox marker. Since the present system can evaluate an influence of a chemical species on the bioconvection, it would be applied to aquatic risk assessment and screening of newly synthesized chemicals and found biochemical compounds. ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (Area No. 417, Research No. 14050028 for T.T.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Received for review January 25, 2007. Accepted March 30, 2007. AC070150T
