Anal. Chem. 2005, 77, 6715-6718
Amperometric Biosensing Systems Based on Motility and Gravitaxis of Flagellate Algae for Aquatic Risk Assessment Isao Shitanda,† Kazutake Takada,† Yasuyuki Sakai,†,‡ and Tetsu Tatsuma*,†
Institute of Industrial Science, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8505, Japan, and Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Electrochemical biosensing systems for toxic substances were developed on the basis of motility and negative gravitaxis of the unicellular flagellate Chlamydomonas reinhardtii. Changes in the flagellar movement of the flagellates in response to three toxic chemicals, toluene, copper(II) sulfate, and nickel(II) chloride, were monitored as changes in the redox currents for a coexisiting redox marker. The gravitaxis-based flagellate biosensing system was more sensitive to toluene than the motility-based system. A thin-layer flagellate biosensor was also developed. In comparison with the conventional algal biosensors monitoring the photosynthetic activity, the gravitaxisbased thin-layer sensor was more sensitive by more than 1 order of magnitude. The rapid and precise evaluation of water toxicity has been an important issue for environmental risk management. However, it is difficult to measure toxicity of individual chemicals contained in water, since a wide variety of chemicals exist in environmental water, and their mixture may exhibit complex toxicity. In these cases, it is more effective to evaluate the overall toxicity of a sample solution to living organisms rather than determination of individual chemicals using traditional biosensors based on enzymes and antibodies. Bioassay has been one of the most useful methods for the comprehensive determination of toxicity in environmental and industrial wastewater. It is also effective in screening and evaluation of chemicals in terms of toxicity. Various bioassays based on algae,1-4 luminescence bacteria,5 plant tissues,6 and animal cells7 have been developed in recent years. In particular, microalgae have been widely used for toxicity tests because of their high sensitivity and reproducibility. For * Corresponding author. E-mail:
[email protected]. Tel.: +81-3-54526336. Fax: +81-3-5452-6338. † Institute of Industrial Science. ‡ Graduate School of Medicine. (1) Shigeoka, T.; Sato, Y.; Takeda, Y.; Yoshida, K.; Yamauchi, F. Environ. Toxicol. Chem. 1988, 7, 847. (2) Ma, J.; Xu, L.; Wang, S.; Zheng, R.; Jin, S.; Huang, S.; Huang, Y. Ecotoxicol. Environ. Saf. 2002, 51, 128. (3) Kasai, F.; Hatakeyama, S. Chemosphere 1993, 27, 899. (4) Pardos, M.; Benninghoff, C.; Thomas, R. L. J. Appl. Phycol. 1998, 10, 145. (5) Tomulka, K. W.; MacGee, D. J.; Lange, J. H. Bull. Environ. Contam. Toxicol. 1993, 51, 538. (6) Saltzman, S.; Heuer, B. A. Pestic. Sci. 1985, 16, 457. (7) Slabbert, J. L.; Steyn, P. L.; Bateman, G. W.; Kflr, R. Water SA 1984, 10, 1. 10.1021/ac050894b CCC: $30.25 Published on Web 09/16/2005
© 2005 American Chemical Society
instance, an algal growth inhibition test is involved in OECD (Organisation for Economic Co-operation and Development) Guidelines for the Testing of Chemicals (OECD TG201). However, the growth inhibition tests take several days in general and require large culture apparatus. Therefore, on-site monitoring of toxicity is difficult with those methods. To reduce the assay time and apparatus size, whole cell biosensors, in which photosynthetic activity of the microalgae is monitored by optical8-13 or electrochemical14-20 methods, have been developed. These biosensors were found to allow rapid detection of herbicides and organic solvents within a few minutes. As mentioned above, responses of the algal cells to toxic chemicals have generally been evaluated on the basis of their growth and photosynthetic activity. In addition to these activities, algal cells are known to show various other physiological activities such as respiration, metabolism, and swimming motility (in the case of flagellate algae). If inhibition for those activities can simultaneously be detected, the type and mechanism of the toxicity could be elucidated. Also, sensitivity for specific chemicals is expected to be enhanced. For example, new bioassays21,22 based on monitoring swimming velocity of flagellate algae have been developed. However, these toxicity tests have drawbacks such as long measurement times, large errors, and low reproducibility, since the motility of a single algal cell is visually monitored. (8) Van der Heerver, J. A.; Grobelaar, J. U. Arch. Environ. Contam. Toxicol. 1998, 35, 281. (9) Merz, D.; Geyer, M.; Moss, D. A.; Ache, H. J. Fresenius J. Anal. Chem. 1996, 354, 299. (10) Frense, D.; Muller, A.; Beckman, D. Sens. Actuator, B 1998, 51, 256. (11) Rodriguez, M., Jr.; Sanders, C. A.; Greenbaum, E. Biosens. Bioelectron. 2002, 17, 843. (12) Naessens, M.; Leclerc, J. C.; Tran-Minh, C. Ecotoxicol. Environ. Saf. 2000, 46, 181. (13) Brack, W.; Frank, H. Ecotoxicol. Environ. Saf. 1998, 40, 34. (14) Pandard, P.; Rawson, D. M. Environ. Toxicol. Water Qual. 1993, 8, 323. (15) Heever, J.; Grobbelaar, J. Water SA 1997, 23, 233. (16) Duvinsky, Z.; Falkowski, P. G.; Post, A. F.; Van Hes, U. M. J. Plankton Res. 1987, 9, 607. (17) Mingazzini, M.; Saenz, M. E.; Albergoni, F. G.; Marre, M. T. Fresenius Environ. Bull. 1997, 6, 308. (18) Naessens, M.; Tran-Minh, C. Anal. Chim. Acta 1998, 364, 153. (19) Campanella, L.; Cubadda, F.; Sammartino, M. P.; Saoncella, A. Water Res. 2000, 35, 69. (20) Shitanda, I.; Takada, K.; Sakai, Y.; Tatsuma, T. Anal. Chim. Acta 2005, 530, 191. (21) Stallwitz, E.; Hader, D. P. J. Photochem. Photobiol. B 1993, 18, 67. (22) Nultsch, W.; Pfau, J.; Dolle, R. Arch. Microbiol. 1986, 144, 393.
Analytical Chemistry, Vol. 77, No. 20, October 15, 2005 6715
In the present study, we focused on a stirring effect arising from algal flagellar movement and developed compact sensing systems for rapid and sensitive toxicity testing. Changes in the collective flagellar movement of flagellate alga, Chlamydomonas reinhardtii, were monitored as changes in the redox currents for a coexisiting redox marker. Furthermore, the alga exhibits negative gravitaxis, which is one of the algal physiological activities to swim against gravity, in conjunction with swimming motility.22-25 These activities are known to be inhibited by exposure to some organic compounds and heavy metal ions. A system that monitors the gravitaxis as well as motility was also developed. The present method allows not only establishment of a qualtitative and quantitative analytical system for water toxicity but multiple evaluation and screening of newly synthesized chemicals including herbicides. EXPERIMENTAL SECTION Incubation of Algae. Unicellular alga C. reinhardtii strain IAM-C9 (Institute of Applied Microbiology Culture Collection) was used throughout. The algae were grown in tris-acetatephosphate (TAP) medium26 (pH 7.5) in 300-mL culture bottles at 25 °C. The culture bottles were aerated through a membrane filter and illuminated by dim fluorescent light periodically (12-h illumination, 12-h dark). Electrochemical Measurements of Algal Flagellar Movement. Suspensions of the algal cells at logarithmic growth phase (4-6 days after passage) or stationary phase (7-9 days after passage) were centrifuged (300g, 3 min). Then, the algal cells (2 × 107 cell mL-1) were resuspended in the TAP medium. A mixture of the suspension and an equal amount of 2 mM potassium ferrocyanide solution (250 µL each) was added to a cup consisting of a microporous polycarbonate filter at the bottom (Cell Culture Insert, Becton Dickinson Labware; pore size, 3 µm). This cup was immersed in a TAP medium containing 1 mM potassium ferrocyanide. A glassy carbon electrode was used as a working electrode and set at the half depth of the solution in the cup (Figure 1a). A coiled plutinum wire and a Ag/AgCl were used as counter and reference electrodes, respectively. The outer solution was agitated by a magnetic stirrer. Anodic current for ferrocyanide oxidation was monitored with a digital potentiostat (ALS-1202). The potential of the working electrode was +0.5 V versus Ag/AgCl. The electrochemical measurements were carried out in a dark box. Toxicity of the solutions was determined from changes in the anodic current upon addition of toxic compounds to the outer solution. Toluene, copper(II) sulfate, and nickel(II) chloride were used as toxic test chemicals. The toxic chemicals used were at least analytical grade. The test solutions of toluene were prepared by using ethanol. Electrochemical Measurements with a Thin-Layer Sensor. An electrochemical setup and configuration of a working electrode used for a thin-layer system are shown in Figure 1b and c, respectively. The electrode was prepared by sputtering of gold on a glass substrate. A silicone ring (500 µm thick) was used as (23) Yoshimura, K.; Matsuo, Y.; Kamiya, R. Plant Cell Physiol. 2003, 44, 1112. (24) Govorunova, E. G.; Altschuler, I. M.; Hader, D. P.; Sineshchekov, O. A. Photochem. Photobiol. 2000, 72, 320. (25) Kam, V.; Moseyko, N.; Nemson, J.; Feldman, L. J. Int. J. Plant Sci. 1999, 160, 1093. (26) Gorman, D. S.; Levine, R. P. Proc. Natl. Acad. Sci. U.S.A. 1965, 54, 1665.
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Figure 1. Schematic illustration of the experimental setup: (a) used for the algal motility- or gravitaxis-based sensing; (b) used with the thin-layer sensing unit; (c) the electrode used for the thin-layer unit.
a spacer between the electrode and a microporous polycarbonate filter (pore size, 3 µm). An algal suspension containing 1 mM potassium ferrocyanide was introduced into the space between the electrode and the filter. The thus assembled biosensor was mounted to an electrochemical cell. Electrochemical measurements were performed as mentioned in the preceding section. RESULTS AND DISCUSSION Motility-Based Sensing of Toxicity. C. reinhardtii has two flagella at the front edge of the cell and swims by beating them like the breast stroke.22-25 The collective flagellar beatings give rise to bioconvection, which agitates the solution in the vicinity of the electrode, as well as in the solution bulk. As the stirring effect increases, the thickness of the diffusion layer decreases on the electrode surface. Under diffusion-controlled conditions, where the rate of the electrode reaction (i.e., oxidation of ferrocyanide as the redox marker) is determined by the diffusion rate of the marker, the oxidation current is anticipated to increase as the diffusion layer thickness decreases. Thus, the velocity of the flagellar movement and the density of the algal cells near the electrode surface were expected to be reflected by the oxidation current. It should be noted that 1 mM ferrocyanide ion is not toxic to the algae. Phototaxis and gravitaxis of C. reinhardtii are reported to decrease as the algae grow.22,27 To measure the algal swimming motility without interferences from the phototaxis and gravitaxis, the algae cells cultured at stationary phase (incubated for 7-9 days), which are relatively inactive in phototaxis and gravitaxis, were used for the motility-based sensing. Figure 2 shows typical current responses of the electrode shown in Figure 1a upon addition of toluene. As can be seen, the (27) Takahashi, T.; Watanabe, M. FEBS 1993, 336, 516.
Figure 2. Current changes of the motility-based flagellate biosensing system in response to toluene at +0.5 V vs Ag/AgCl in a TAP medium containing 1 mM potassium ferrocyanide (pH 7.0) at 20 °C. Schematic illustrations of algae before and after the addition of toluene are shown in the figure.
steady-state current for ferrocyanide oxidation appeared to decrease, as the concentration of toluene increased (1-10 mM). The current reached steady state within 200 s after the addition toluene and did not decrease further upon addition of higher concentrations of toluene (>10 mM). Microscopic observation of the algal cells right after this experiment showed no swimming motility. Two different control experiments were also carried out. The oxidation current remained constant when the same volume of ethanol used to dissolve toluene was added. In the absence of the algal cells, addition of toluene caused no change in the current. On the basis of these results, it can be concluded that the decrease in the oxidation current is ascribed to inhibition of algal motility by toluene (inset of Figure 2), resulting in a decrease in the convection near the electrode surface, which, in turn, gives rise to an increase in the diffusion layer thickness. Here, the sharp but temporal increase in the oxidation current after the addition of toluene (Figure 2) would be ascribed to a temporary increase in the swimming rate due to stimulus from the toxic chemical. This was supported by the fact that further addition of toluene to algal cells that had been inactivated by 10 mM toluene caused no temporal increase in the oxidation current. Here the inhibition ratio (I) of algal swimming motility is defined by
I ) (∆i/∆imax) × 100 (%)
(1)
where ∆i and ∆imax are decrements of the anodic currents 200 s after the injection of the sample solution and after addition of the same toxic chemical of which concentration is sufficiently high to inactivate the algal cells completely (in the case of toluene, 10 mM), respectively. On the basis of this equation, responses of the present bioassay system were evaluated with three toxic chemicals (toluene, copper(II) sulfate, and nickel(II) chloride). Cu2+ and Ni2+ are known to be inhibitory reagents, which bind to the calcium channel of the flagellar surface and block the flow of Ca2+.22 Dependence of I on the concentration is plotted in Figure
Figure 3. Dose-response (inhibition ratio I vs concentration) curves of the immobilization tests after 200-s exposure to the toxic chemicals toluene (b), copper(II) sulfate (9), and nickel(II) chloride (2).
3. All three chemicals suppressed the oxidation current. Reproducibility was obtained with (7% standard error (500 µM Cu2+). On the other hand, the oxidation current appeared to remain essentially constant when the chemicals were added into the electrolyte solution without algae cells. These results indicate that the present bioassay system can test toxicity of the chemicals within several minutes. Meanwhile, if the substrate examined is oxidizable at the applied electrode potential, ferricyanide ion may be used as a redox marker instead of ferrocyanide ion. The marker will be reduced at a sufficiently negative potential, at which the substrate will not be oxidized. In addition, the present methods assume establishment of disposable and rapid evaluation systems for water toxicity. Thus, recycling of algal cells was not examined. Gravitaxis-Based Sensing of Toxicity. In the case of the bioassay system based on the inhibition of motility, 100 µM toluene was detectable, whereas 10 µM toluene was not detectable. To enhance the sensitivity, we developed a bioassay system based on negative gravitaxis of the algae. Here we used cells at logarithmic growth phase (incubated for 4-6 days). In particular, cells around the solution surface, which are more active in the negative gravitaxis, were collected. Figure 4 shows typical current responses of the system shown in Figure 1a upon addition of toluene. It is apparent that the oxidation current appeared to increase as the concentration of toluene increased (10-100 µM). The current was not perturbed when the same amount of ethanol used to dissolve toluene was added. Moreover, no change in the oxidation current was observed upon addition of toluene into a solution without the algal cells. C. reinhardtii that has high physiological activities (e.g., cells at the logarithmic growth phase) is reported to show pronounced negative gravitaxis and move to the solution surface.23 In fact, the negative gravitaxis activity of the algal cells used was visually verified before the addition of toluene. After the injection of a sublethal amount of toluene (e.g., 10-100 µM), the cells at the solution surface moved to the solution bulk, indicating inactivation of the negative gravitaxis. Analytical Chemistry, Vol. 77, No. 20, October 15, 2005
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Figure 4. Current changes of the gravitaxis-based flagellate biosensing system in response to toluene at +0.5 V vs Ag/AgCl in a TAP medium containing 1 mM potassium ferrocyanide (pH 7.0) at 20 °C. Schematic illustrations of algae before and after the addition of toluene are shown in the figure.
From these results, the observed electrochemical responses can be explained in terms of the inactivation, as follows (inset of Figure 4). Before the addition of toluene, most algal cells stay above the electrode surface level, whereas the addition of toluene inhibits the negative gravitaxis, giving rise to an increase in the number of cells in the vicinity of the electrode surface. The resulting bioconvection around the electrode increases the oxidation current of the redox marker. The detection limit of the gravitaxis inhibition test was found to be 10 µM, which is 1 order of magnitude better than that of the motility-based sensing. Fabrication of the Thin-Layer Biosensor. In the case of the gravitaxis inhibition tests mentioned above, it was demonstrated that this bioassay system is able to detect toluene with high sensitivity. Thus, we developed a more sophisticated thin-layer biosensing unit shown in Figure 1b and c to enhance the detection ability. Figure 5a shows a typical current response upon addition of toluene to be 30 µM. As can be seen in the figure, the current increased sharply to the maximum value and then decreased gradually. Before the addition of toluene, most algal cells stayed around the top of the solution (inset of Figure 5a). The addition of toluene triggered the diving of the cells, and the cells spread over the electrode, giving rise to the current increase. Finally, the cells further spread over the thin-layer unit, and the cell concentration around the electrode decreased, resulting in the current decrease. The height of the current peak (∆ip) was plotted against the concentration of toluene in Figure 5b. Reproducibility was obtained with (7% standard error (10 µM toluene). The ∆ip was found to increase with the concentration below 30 µM and slightly decrease above this value. This behavior could be explained in terms of total inactivation of the negative gravitaxis at ∼30 µM and accompanying inhibition of the flagellar motion above this concentration. The lower detection limit of the present thin-layer sensor was ∼3 µΜ. On the other hand, in the case of the conventional algal biosensing systems based on optical13 and electrochemical20 monitoring of photosynthetic activities, the concentration that gives 10% inhibition of the photosynthetic activities (IC10) is 130 (C. reinhardtii) and 100 µM (Chlorella 6718 Analytical Chemistry, Vol. 77, No. 20, October 15, 2005
Figure 5. (a) Current changes of the thin-layer flagellate biosensing system in response to toluene at +0.5 V vs Ag/AgCl in a TAP medium containing 1 mM potassium ferrocyanide (pH 7.0) at 20 °C. Schematic illustrations of algae before and after the addition of toluene are shown in the figure. (b) Dose-response (height of the current peak vs concentration) curve of the thin-layer flagellate biosensor exprosure to toluene.
vulgaris), respectively. Thus, we conclude that the present gravitaxis-based sensing improved the detection ability by more than 1 order of magnitude. CONCLUSIONS Here we developed motility- and gravitaxis-based algal biosensing systems. In particular, the latter was found to be much more sensitive than the conventional algal biosensing systems based on photosynthetic activities. Since the sensitivity and dynamic range depend on the configuration of the electrochemical cell and the electrode, it is possible to optimize the system for each purpose, and each sample. ACKNOWLEDGMENT We are grateful to Professor R. Kamiya for valuable discussion. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (Area 417, Research 14050028 for T.T.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Received for review May 21, 2005. Accepted August 16, 2005. AC050894B
