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Photoelectric behavior of mammalian cells and its bioanalytical applications
Bioelectrochemistry and Bioenergetics 45 Ž1998. 247–251
Photoelectric behavior of mammalian cells and its bioanalytical applications Yun-Xiang Ci ) , Chun-Yang Zhang, Jun Feng Department of Chemistry, Peking UniÕersity, Beijing 100871, China Received 10 October 1997; revised 4 March 1998; accepted 9 March 1998
Abstract In this paper, the photoelectric behavior of mammalian cells is first reported by using ITO Ža transparent electrode of indium–tin oxide coated borosilicate. technology. Attached mammalian cells can easily grow on the transparent electrode in a culture flask with general culture medium. This integration of the conductor glass with a layer of mammalian cells and photoelectric-current-measuring system forms a cytosensor when the transparent electrode is mounted into the light path. The attached mammalian cells responds with a negative photoelectric current pulse to white light Ž200–800 nm.. The negative characteristic of the photoelectric current shows electron flow from living cells to the conductor glass. We also found that the fluctuations of the photoelectric current were determined by the coordination of some cell functions. The influence of cell viability, pH and light intensity on the photoelectric behavior is studied. We found that the photoelectric current of tumor cells is higher than that of normal cells, and the photoelectric current of mammalian cells is related to the viability of the cells. q 1998 Elsevier Science S.A. All rights reserved. Keywords: Photoelectric behavior; Mammalian cells; ITO
1. Introduction The photoelectric behavior of living cells, traditionally, is thought to exist in plant cells and photosynthetic bacterium. Great advances have been made over the past few years in the research in harvesting light energy of photosynthesis w1–3x, light-driven electron flow w4–8x and coupling ATP to light-driven electron flow of photosynthetic organisms w9–11x. Light-absorbing pigment in the membranes of chloroplast and photosynthetic bacterium transfer the energy of absorbed light to reaction centers where electron flow is initiated. Electron flow occurs through a series of carriers and drive ATP synthesis by the chemiosmotic mechanism. However, photoelectric behavior of mammalian cells is rarely studied. We first find that photoelectric current is generated when mammalian cells are exited by white light. Generally, the mammalian cells are cultured by attaching on a certain slice of conductor glass and analyzed by a voltage analyzer with a Xe light on. Different kinds of mammalian cells show different photoelectric behaviors. In this paper, we used the cells HeLa Žcell line of cervical cancer., the )
Corresponding author. Fax: q86-10-62751-708.
0302-4598r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved. PII S 0 3 0 2 - 4 5 9 8 Ž 9 8 . 0 0 0 9 7 - X
cells SPr20 Žcell line of mouse Myeloma. and the cells PMEF Žprimary mouse embryo fibroblast. to investigate the different photoelectric behavior between tumor cells and the normal cells, and to try to shed light on the mechanism of photoelectric behavior of mammalian cells by investigating the relationship between the photoelectric behavior and cell physiological behavior.
2. Experiments 2.1. Materials 2.1.1. Cell lines The living cells for the experiment were the cells HeLa, PMEF and SPr20 provided by the Monoclone Laboratory of the Biological Department of Peking University. 2.1.2. Buffer solution The buffer solution was PBS ŽNaCl 136.7 mmol ly1 , KCl 2.7 mmol ly1 , Na 2 HPO4 P 12H 2 O 9.7 mmol ly1 , KH 2 PO4 1.5 mmol ly1 ., which was used as the electrolyte in the measuring system and as washing solution of the cells.
248
Y.-X. Ci et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 247–251
2.2. Apparatus and methods 2.2.1. Cell culture Cells were cultured on the conductor glass in a becco’s Modified Eagle Medium ŽDMEM, Sigma. 10% fetal bovine serum ŽSigma. and 0.05 mg mly1 tamicin ŽSigma. at 378C in a humidified atmosphere taining 5% CO 2 .
Dulwith gencon-
2.2.2. The photoelectric measurement system The photoelectric measurement system used for studying the electron transfer induced by light was a three electrode system ŽFig. 1.. The work electrode was made of ITO embedded on the surface of glass. The window for light transfer was 1.3 cm2 . The counter electrode was a platinum electrode and the reference electrode was a saturated calomel electrode. The electrode voltage of reference electrode to the work electrode was 0 voltage through the experiments.
Fig. 2. A typical photoelectric current of HeLa cells. Ž1. The blank photoelectric current of ITO without HeLa cells on. Ž2. The photoelectric current of HeLa Cells. HeLa cells were cultured on ITO conductor glass in a flask with culture medium at 378C in a humidified atmosphere containing 5% CO 2 . When cells spread fully on the ITO Ž10 5 cmy2 ., the ITO was moved from the culture flask and mounted on the light window of the measuring tube. Then, the photoelectric current was measured with the light intensity of 121.4 mW cmy2 .
2.2.3. Apparatus Measurements were performed by using a model 600 voltage analyzer ŽCH Instrument, USA., which was controlled by an external PC under a Windows environment. The light source was a Xe light ŽUSHID, Japan..
measured by the model 600 voltage analyzer with the Xe light on.
2.2.4. Procedures Before the experiment, the photoelectric current of the ITO conductor glass free of cells was measured. At first, the conductor glass was cleaned by sonicated in acetone solution for 15 min. After that, the ITO conductor glass was washed with PBS and the background photoelectric current was measured. With the same procedure, the same ITO conductor glass was used as substrate for cell growth on. After the cells were cultured for a certain time, the ITO conductor glass was moved from the culture solution and washed by PBS. Then, the ITO conductor glass was mounted on the light window of the measuring tube and
3.1. Photoelectric behaÕior of HeLa cells
3. Results
Fig. 2 is a typical photoelectric current of the HeLa cells. After the dark current is discounted, the photoelectric current of the conductor glass free from cells is in the range of y9.6 nA to y14.9 nA. However, the photoelectric current of the conductor glass with HeLa cells on is from y44.1 nA to y71.0 nA after the dark current is discounted. If the blank photoelectric current of the conductor glass free from cells was discounted, the net photoelectric current of HeLa cells was in the range of y34.5 nA to y59.0 nA ŽTable 1.. This result shows that the distinct photoelectric behavior presents in HeLa cells.
Table 1 Photoelectric current of the HeLa cells ITO number
1
2
3
4
5
6
If darkrnA q16.3 y19.7 q12.0 q3.0 q7.7 q1.3 If lightrnA q3.5 y31.7 y1.9 y12.6 y7.2 y12.6 D If rnA y12.8 y12.0 y13.9 y9.6 y14.9 y13.9 Ic darkrnA y112.3 y63.3 y23.1 y61.2 y44.9 y38.4 Ic lightrnA y162.2 y134.3 y78.9 y105.3 y110.3 y98.5 D Ic rnA y49.9 y71.0 y55.8 y44.1 y65.4 y60.1 Ž D Ic y D If .rnA y37.1 y59.0 y41.9 y34.5 y50.5 y46.2 Fig. 1. The schematic diagram of three-electrode system for photoelectric current analysis. RE: Referent electrode Žsaturated calomel electrode.; CE: counter electrode Žplatinum electrode.; WE: ITO electrode with cells attaching on.
Light intensity is 121.4 mW cmy2 . If , Ic : the current of the ITO conductor glass free from cells and with the cells on; D If , D Ic : the difference of light current and dark current for the ITO conductor glass free from cells and with the cells on.
Y.-X. Ci et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 247–251
249
Table 2 Photoelectric current of the cells PMEF ITO number
1
2
3
Ic darkrnA 0.7 y21.2 y0.8 Ic lightrnA y32.8 y53.4 y32.2 D Ic rnA y33.5 y32.0 y33.0 Ž D Ic y D If .rnA y20.7 y20.2 y19.1
4
5
6
y21.2 y9.4 y54.5 y65.2 y39.4 y87.0 y30.0 y30.0 y32.5 y20.4 y15.1 y18.6
Light intensity is 121.4 mW cmy2 .
3.2. Different photoelectric behaÕior in some mammalian cells In order to investigate some different photoelectric behaviors of tumor cells and normal cells, we chose the cells PMEF that was a kind of normal protype cultured cell, and the cells SPr20 that was a kind of half-attaching tumor cell. Though the attaching force of cells SPr20 on the conductor glass is less than that of the both cells PMEM and cells HeLa, the photoelectric current of cells SPr20 is in the range of y23.2 nA to y33.6 nA after deducting the blank photoelectric current of the ITO. However, the photoelectric current of PMEF is in the range of y15.1 to y20.7 nA ŽTable 2.. From the result above, we can conclude that the photoelectric behavior not only exists in mammalian cells but also shows difference for different kinds of cells. 3.3. Photoelectric behaÕior of mammalian cells at different liÕing states As a living cell, the biochemical reactions and physiological activities taking place in it should be considered having a relationship with the photoelectric behavior. We
Fig. 3. Photoelectric current of HeLa cells at its different living states. Cells were transferred into a series of flasks with the same cell number Ž10 4 mly1 . and same culture condition. After 20 h, cells attached and spread on the ITO glass. Then, the ITO glasses with cells on were moved out from flasks at different culture time and the photoelectric currents were measured with the light intensity of 121.4 mW cmy2 .
Fig. 4. Influence of pH upon the photoelectric current of HeLa cells. Cells were cultured in a series of flasks with the same initial number Ž10 4 mly1 . and same culture condition. After 30 h, the ITO glasses with cells on were transferred into another series of flasks with different pH culture medium. Then, the photoelectric currents were measured after 2 h of culture time Žlight intensity: 121.4 mW cmy2 ..
chose HeLa cells to investigate the photoelectric behavior of living cells at different living state. Fig. 3 shows that the photoelectric current of HeLa cells increases with culture time from 24 h to 48 h. Then it decreases with time from 48 h to 56 h, which demonstrates that the photoelectric current varies with the living state of HeLa cells. The pH in cell culture solution can be taken as a general standard to evaluate the cell viability. We observed the photoelectric behavior of HeLa cells at different pH value. Fig. 4 shows the influence of pH on the photoelectric behavior. From Fig. 4, we can see that the photoelectric current decreases when the acid value of the culture solu-
Fig. 5. Influence of light intensity upon the photoelectric current of HeLa cells.
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Y.-X. Ci et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 247–251
tion increases. These results above show the positive relationship between the photoelectric behavior of mammalian cells and the cell viability. 3.4. Photoelectric current at different light intensities We also investigate the influence of light intensity upon the photoelectric current of living cells. Fig. 5 shows that the variance of light intensity results in the variance of the photoelectric current of HeLa cells. When light intensity is from 8.6 mW cmy2 up to 121.4 mW cmy2 , the photoelectric current of HeLa cells increases rapidly, but it changes slowly while the light intensity is from 121.4 mW cmy2 up to 169.2 mW cmy2 , which demonstrates that the photoelectric current has reached its saturated value.
4. Discussion We first found that the photoelectric behavior is present in living mammalian cells. HeLa cells, PMEF cells and SPr20 cells may generate negative photoelectric current when they are illuminated. The negative photoelectric current results from the electron flow from the cell towards the ITO conductor glass, which means that the negative photoelectric current is generated by the cell itself when the cell is illuminated. In cell biology, the study of mammalian cells is more important because of its close relationship with the health of mankind. The photoelectric behavior of mammalian cells provides a new way to describe cell physiology, to evaluate the effectiveness of chemicals, antitumor drugs, and even to show a new idea to reveal the mechanism of effect of light on living cells. One of the physical chemistry characteristics of living cells is the two phases structure in which exist a huge solid–liquid interface. With its intimate relationship to the huge interface, the protein across the membrane plays a role of bridge of which one end attach on the ITO conductor glass and the other end is in the native place where locates the redox center. In the flash of the light on the attached mammalian cells, the bridge functioned as an electron bridge. So the photoelectric current of mammalian cells relates to the conductance characteristic of proteins in living cells. In 1941, Szent-Gyoryi w12,13x first found that protein demonstrate semiconductor characteristics. Protein demonstrates small conductance in the dark w14x. But in the flash of light more than 320 nm, its conductance increases several hundred times w15x. In addition, superconductive and Josephson junction behavior may also be present in living cells w16x. Josephson junction represents a gap junction between two nearby cells. When electrons passes through Josephson junction, the junction gives a non-ohm behavior ŽJosephson d.c. effect. w17x, this is advantageous to the electron transfer from one cell to another. In particular, when tumor cells attach onto the ITO conductor glass with stack growth, the excited electrons reaching the ITO
conductor glass need to pass a number of cells, Josephson junction allow the electrons to be transmitted from one cell to an adjacent cell without mediation of electrolyte. The positive relationship between the photoelectric behavior and cell viability shows that the photoelectric behavior of mammalian cells is not only relative to the special structure of living cells but also relative to the metabolism. The variance of photoelectric current with the living state of HeLa cells can be explained by the ATP synthesis coupling to the electron transfer within the living cells. In the exponentially growing phase cells with sufficient nutrient, its increase metabolism results in increase oxidative phosphorylation and increase electron transfer, therefore photoelectric current of HeLa cells increases with time from 24 h to 48 h. But in the cell of late growing phase with nutrient running out, its decrease metabolism results in decrease oxidative phosphorylation and decrease electron transfer, therefore the photoelectric current of HeLa cells decreases with time from 48 h to 64 h. The result that the photoelectric current decreases when the pH of the culture solution decreases shows the distinct relationship with metabolism. The optimum pH for the living cell growth is 7.4–7.6, the decrease pH destroys the proton gradient across the inner mitochondrial membrane and inhibits the electron transfer. It is worth noticing that the photoelectric current of tumor cells HeLa is higher than that of normal cells PMEF. In tumor cells, the plasma membrane fluidity increases, the surface negative charges increase Žthe net charge on the membranes of cells is negative w18x., and the permeability of membrane also increases, and the metabolism level of tumor cells is higher than that of normal cells w19x. In the addition, tumor cells behave stack growth because of no contact inhibition. All these are advantageous to electrons transferring from the cells to the conductor glass in flash of light.
Acknowledgements This project was supported by the Natural Science Foundation of Peking University and National Natural Science Foundation of China.
References w1x B.R. Green, E. Pichersky, K. Kloppstech, Chlorophyll arb-binding proteins: an extended family, Trends Biochem. Sci. 16 Ž1991. 181–186. w2x R. Huber, A structural basis of light energy and electron transfer in biology, Eur. J. Biochem. 187 Ž1990. 283–305. w3x H. Zuber, Structure of light-harvesting antenna complexes of photosynthetic bacteria cyanobacteria and red algae, Trends Biochem. Sci. 11 Ž1986. 414–419. w4x L.E. Andreasson, T. Vanngard, Electron transport in photosystems I and II, Annu. Rev. Plant Physiol. Plant Mol. Biol. 39 Ž1988. 379–411.
Y.-X. Ci et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 247–251 w5x J. Deisenhofer, H. Michel, Structures of bacterial photosynthetic reaction centers, Annu. Rev. Cell Biol. 7 Ž1991. 1–23. w6x A.N. Glazer, A. Melis, Photochemical reaction centers: structure, organization, and function, Annu. Rev. Plant Physiol. 38 Ž1987. 11–45. w7x J.H. Golbck, Structure and function of photosystem I, Annu. Rev. Plant Physiol. Plant Mol. Biol. 43 Ž1992. 293–324. w8x W. Nitschke, A.W. Rutherford, Photosynthetic reaction centres: variations on a common structural theme, Trends Biochem. Sci. 16 Ž1991. 241–245. w9x W.A. Gramer, W.R. Widger, R.G. Herrmann, A. Trebst, Topography and function of thylakoid membrane proteins, Trends Biochem. Sci. 10 Ž1985. 125–129. w10x A.T. Jagendorf, Acid–base transitions and phosphorylation by chloroplasts, Fed. Proc. 26 Ž1967. 1361–1369. w11x D.C. Vouvan, B.L. Marrs, Molecular mechanisms of photosynthesis, Sci. Am. 256 Ž1987. 42–48. w12x A. Szent-Gyorgi, The study of energy level in biochemistry, Nature 148 Ž1941. 157–159.
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w13x A. Szent-Gyorgi, Towards a new biochemistry, Science 93 Ž1941. 609–611. w14x D.D. Eley, P.P. Parfitt, M.B. Perry, M.J. Perry, D.H. Taysym, The semiconductivity of organic substances, Trans. Faraday Soc. 49 Ž1953. 79–86. w15x M. Ataka, S. Tanaka, Electrical conductivity of single crystals of lysozyme, Biopolymers 19 Ž1980. 669–679. w16x M. Costato, M. Milani, L. Spinoglio, Quantum mechanics: a breakthrough into biological system dynamics, Bioelectrochem. Bioenerg. 41 Ž1996. 27–30. w17x A. Brone, G. Patermo, Physics and Application of the Josephson Effect, Wiley, New York, 1982. w18x G. Ceve, Membrane electrostatics, Biochim. Biophys. Acta 1031 Ž1990. 311–382. w19x Z.L. Tan, R.L. Zhou, Medical Cell Biology, Bei Jing Medical University and Xie He Medical University Press, Bei Jing, 1996.
Photoelectric behavior of mammalian cells and its bioanalytical applications Yun-Xiang Ci ) , Chun-Yang Zhang, Jun Feng Department of Chemistry, Peking UniÕersity, Beijing 100871, China Received 10 October 1997; revised 4 March 1998; accepted 9 March 1998
Abstract In this paper, the photoelectric behavior of mammalian cells is first reported by using ITO Ža transparent electrode of indium–tin oxide coated borosilicate. technology. Attached mammalian cells can easily grow on the transparent electrode in a culture flask with general culture medium. This integration of the conductor glass with a layer of mammalian cells and photoelectric-current-measuring system forms a cytosensor when the transparent electrode is mounted into the light path. The attached mammalian cells responds with a negative photoelectric current pulse to white light Ž200–800 nm.. The negative characteristic of the photoelectric current shows electron flow from living cells to the conductor glass. We also found that the fluctuations of the photoelectric current were determined by the coordination of some cell functions. The influence of cell viability, pH and light intensity on the photoelectric behavior is studied. We found that the photoelectric current of tumor cells is higher than that of normal cells, and the photoelectric current of mammalian cells is related to the viability of the cells. q 1998 Elsevier Science S.A. All rights reserved. Keywords: Photoelectric behavior; Mammalian cells; ITO
1. Introduction The photoelectric behavior of living cells, traditionally, is thought to exist in plant cells and photosynthetic bacterium. Great advances have been made over the past few years in the research in harvesting light energy of photosynthesis w1–3x, light-driven electron flow w4–8x and coupling ATP to light-driven electron flow of photosynthetic organisms w9–11x. Light-absorbing pigment in the membranes of chloroplast and photosynthetic bacterium transfer the energy of absorbed light to reaction centers where electron flow is initiated. Electron flow occurs through a series of carriers and drive ATP synthesis by the chemiosmotic mechanism. However, photoelectric behavior of mammalian cells is rarely studied. We first find that photoelectric current is generated when mammalian cells are exited by white light. Generally, the mammalian cells are cultured by attaching on a certain slice of conductor glass and analyzed by a voltage analyzer with a Xe light on. Different kinds of mammalian cells show different photoelectric behaviors. In this paper, we used the cells HeLa Žcell line of cervical cancer., the )
Corresponding author. Fax: q86-10-62751-708.
0302-4598r98r$19.00 q 1998 Elsevier Science S.A. All rights reserved. PII S 0 3 0 2 - 4 5 9 8 Ž 9 8 . 0 0 0 9 7 - X
cells SPr20 Žcell line of mouse Myeloma. and the cells PMEF Žprimary mouse embryo fibroblast. to investigate the different photoelectric behavior between tumor cells and the normal cells, and to try to shed light on the mechanism of photoelectric behavior of mammalian cells by investigating the relationship between the photoelectric behavior and cell physiological behavior.
2. Experiments 2.1. Materials 2.1.1. Cell lines The living cells for the experiment were the cells HeLa, PMEF and SPr20 provided by the Monoclone Laboratory of the Biological Department of Peking University. 2.1.2. Buffer solution The buffer solution was PBS ŽNaCl 136.7 mmol ly1 , KCl 2.7 mmol ly1 , Na 2 HPO4 P 12H 2 O 9.7 mmol ly1 , KH 2 PO4 1.5 mmol ly1 ., which was used as the electrolyte in the measuring system and as washing solution of the cells.
248
Y.-X. Ci et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 247–251
2.2. Apparatus and methods 2.2.1. Cell culture Cells were cultured on the conductor glass in a becco’s Modified Eagle Medium ŽDMEM, Sigma. 10% fetal bovine serum ŽSigma. and 0.05 mg mly1 tamicin ŽSigma. at 378C in a humidified atmosphere taining 5% CO 2 .
Dulwith gencon-
2.2.2. The photoelectric measurement system The photoelectric measurement system used for studying the electron transfer induced by light was a three electrode system ŽFig. 1.. The work electrode was made of ITO embedded on the surface of glass. The window for light transfer was 1.3 cm2 . The counter electrode was a platinum electrode and the reference electrode was a saturated calomel electrode. The electrode voltage of reference electrode to the work electrode was 0 voltage through the experiments.
Fig. 2. A typical photoelectric current of HeLa cells. Ž1. The blank photoelectric current of ITO without HeLa cells on. Ž2. The photoelectric current of HeLa Cells. HeLa cells were cultured on ITO conductor glass in a flask with culture medium at 378C in a humidified atmosphere containing 5% CO 2 . When cells spread fully on the ITO Ž10 5 cmy2 ., the ITO was moved from the culture flask and mounted on the light window of the measuring tube. Then, the photoelectric current was measured with the light intensity of 121.4 mW cmy2 .
2.2.3. Apparatus Measurements were performed by using a model 600 voltage analyzer ŽCH Instrument, USA., which was controlled by an external PC under a Windows environment. The light source was a Xe light ŽUSHID, Japan..
measured by the model 600 voltage analyzer with the Xe light on.
2.2.4. Procedures Before the experiment, the photoelectric current of the ITO conductor glass free of cells was measured. At first, the conductor glass was cleaned by sonicated in acetone solution for 15 min. After that, the ITO conductor glass was washed with PBS and the background photoelectric current was measured. With the same procedure, the same ITO conductor glass was used as substrate for cell growth on. After the cells were cultured for a certain time, the ITO conductor glass was moved from the culture solution and washed by PBS. Then, the ITO conductor glass was mounted on the light window of the measuring tube and
3.1. Photoelectric behaÕior of HeLa cells
3. Results
Fig. 2 is a typical photoelectric current of the HeLa cells. After the dark current is discounted, the photoelectric current of the conductor glass free from cells is in the range of y9.6 nA to y14.9 nA. However, the photoelectric current of the conductor glass with HeLa cells on is from y44.1 nA to y71.0 nA after the dark current is discounted. If the blank photoelectric current of the conductor glass free from cells was discounted, the net photoelectric current of HeLa cells was in the range of y34.5 nA to y59.0 nA ŽTable 1.. This result shows that the distinct photoelectric behavior presents in HeLa cells.
Table 1 Photoelectric current of the HeLa cells ITO number
1
2
3
4
5
6
If darkrnA q16.3 y19.7 q12.0 q3.0 q7.7 q1.3 If lightrnA q3.5 y31.7 y1.9 y12.6 y7.2 y12.6 D If rnA y12.8 y12.0 y13.9 y9.6 y14.9 y13.9 Ic darkrnA y112.3 y63.3 y23.1 y61.2 y44.9 y38.4 Ic lightrnA y162.2 y134.3 y78.9 y105.3 y110.3 y98.5 D Ic rnA y49.9 y71.0 y55.8 y44.1 y65.4 y60.1 Ž D Ic y D If .rnA y37.1 y59.0 y41.9 y34.5 y50.5 y46.2 Fig. 1. The schematic diagram of three-electrode system for photoelectric current analysis. RE: Referent electrode Žsaturated calomel electrode.; CE: counter electrode Žplatinum electrode.; WE: ITO electrode with cells attaching on.
Light intensity is 121.4 mW cmy2 . If , Ic : the current of the ITO conductor glass free from cells and with the cells on; D If , D Ic : the difference of light current and dark current for the ITO conductor glass free from cells and with the cells on.
Y.-X. Ci et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 247–251
249
Table 2 Photoelectric current of the cells PMEF ITO number
1
2
3
Ic darkrnA 0.7 y21.2 y0.8 Ic lightrnA y32.8 y53.4 y32.2 D Ic rnA y33.5 y32.0 y33.0 Ž D Ic y D If .rnA y20.7 y20.2 y19.1
4
5
6
y21.2 y9.4 y54.5 y65.2 y39.4 y87.0 y30.0 y30.0 y32.5 y20.4 y15.1 y18.6
Light intensity is 121.4 mW cmy2 .
3.2. Different photoelectric behaÕior in some mammalian cells In order to investigate some different photoelectric behaviors of tumor cells and normal cells, we chose the cells PMEF that was a kind of normal protype cultured cell, and the cells SPr20 that was a kind of half-attaching tumor cell. Though the attaching force of cells SPr20 on the conductor glass is less than that of the both cells PMEM and cells HeLa, the photoelectric current of cells SPr20 is in the range of y23.2 nA to y33.6 nA after deducting the blank photoelectric current of the ITO. However, the photoelectric current of PMEF is in the range of y15.1 to y20.7 nA ŽTable 2.. From the result above, we can conclude that the photoelectric behavior not only exists in mammalian cells but also shows difference for different kinds of cells. 3.3. Photoelectric behaÕior of mammalian cells at different liÕing states As a living cell, the biochemical reactions and physiological activities taking place in it should be considered having a relationship with the photoelectric behavior. We
Fig. 3. Photoelectric current of HeLa cells at its different living states. Cells were transferred into a series of flasks with the same cell number Ž10 4 mly1 . and same culture condition. After 20 h, cells attached and spread on the ITO glass. Then, the ITO glasses with cells on were moved out from flasks at different culture time and the photoelectric currents were measured with the light intensity of 121.4 mW cmy2 .
Fig. 4. Influence of pH upon the photoelectric current of HeLa cells. Cells were cultured in a series of flasks with the same initial number Ž10 4 mly1 . and same culture condition. After 30 h, the ITO glasses with cells on were transferred into another series of flasks with different pH culture medium. Then, the photoelectric currents were measured after 2 h of culture time Žlight intensity: 121.4 mW cmy2 ..
chose HeLa cells to investigate the photoelectric behavior of living cells at different living state. Fig. 3 shows that the photoelectric current of HeLa cells increases with culture time from 24 h to 48 h. Then it decreases with time from 48 h to 56 h, which demonstrates that the photoelectric current varies with the living state of HeLa cells. The pH in cell culture solution can be taken as a general standard to evaluate the cell viability. We observed the photoelectric behavior of HeLa cells at different pH value. Fig. 4 shows the influence of pH on the photoelectric behavior. From Fig. 4, we can see that the photoelectric current decreases when the acid value of the culture solu-
Fig. 5. Influence of light intensity upon the photoelectric current of HeLa cells.
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Y.-X. Ci et al.r Bioelectrochemistry and Bioenergetics 45 (1998) 247–251
tion increases. These results above show the positive relationship between the photoelectric behavior of mammalian cells and the cell viability. 3.4. Photoelectric current at different light intensities We also investigate the influence of light intensity upon the photoelectric current of living cells. Fig. 5 shows that the variance of light intensity results in the variance of the photoelectric current of HeLa cells. When light intensity is from 8.6 mW cmy2 up to 121.4 mW cmy2 , the photoelectric current of HeLa cells increases rapidly, but it changes slowly while the light intensity is from 121.4 mW cmy2 up to 169.2 mW cmy2 , which demonstrates that the photoelectric current has reached its saturated value.
4. Discussion We first found that the photoelectric behavior is present in living mammalian cells. HeLa cells, PMEF cells and SPr20 cells may generate negative photoelectric current when they are illuminated. The negative photoelectric current results from the electron flow from the cell towards the ITO conductor glass, which means that the negative photoelectric current is generated by the cell itself when the cell is illuminated. In cell biology, the study of mammalian cells is more important because of its close relationship with the health of mankind. The photoelectric behavior of mammalian cells provides a new way to describe cell physiology, to evaluate the effectiveness of chemicals, antitumor drugs, and even to show a new idea to reveal the mechanism of effect of light on living cells. One of the physical chemistry characteristics of living cells is the two phases structure in which exist a huge solid–liquid interface. With its intimate relationship to the huge interface, the protein across the membrane plays a role of bridge of which one end attach on the ITO conductor glass and the other end is in the native place where locates the redox center. In the flash of the light on the attached mammalian cells, the bridge functioned as an electron bridge. So the photoelectric current of mammalian cells relates to the conductance characteristic of proteins in living cells. In 1941, Szent-Gyoryi w12,13x first found that protein demonstrate semiconductor characteristics. Protein demonstrates small conductance in the dark w14x. But in the flash of light more than 320 nm, its conductance increases several hundred times w15x. In addition, superconductive and Josephson junction behavior may also be present in living cells w16x. Josephson junction represents a gap junction between two nearby cells. When electrons passes through Josephson junction, the junction gives a non-ohm behavior ŽJosephson d.c. effect. w17x, this is advantageous to the electron transfer from one cell to another. In particular, when tumor cells attach onto the ITO conductor glass with stack growth, the excited electrons reaching the ITO
conductor glass need to pass a number of cells, Josephson junction allow the electrons to be transmitted from one cell to an adjacent cell without mediation of electrolyte. The positive relationship between the photoelectric behavior and cell viability shows that the photoelectric behavior of mammalian cells is not only relative to the special structure of living cells but also relative to the metabolism. The variance of photoelectric current with the living state of HeLa cells can be explained by the ATP synthesis coupling to the electron transfer within the living cells. In the exponentially growing phase cells with sufficient nutrient, its increase metabolism results in increase oxidative phosphorylation and increase electron transfer, therefore photoelectric current of HeLa cells increases with time from 24 h to 48 h. But in the cell of late growing phase with nutrient running out, its decrease metabolism results in decrease oxidative phosphorylation and decrease electron transfer, therefore the photoelectric current of HeLa cells decreases with time from 48 h to 64 h. The result that the photoelectric current decreases when the pH of the culture solution decreases shows the distinct relationship with metabolism. The optimum pH for the living cell growth is 7.4–7.6, the decrease pH destroys the proton gradient across the inner mitochondrial membrane and inhibits the electron transfer. It is worth noticing that the photoelectric current of tumor cells HeLa is higher than that of normal cells PMEF. In tumor cells, the plasma membrane fluidity increases, the surface negative charges increase Žthe net charge on the membranes of cells is negative w18x., and the permeability of membrane also increases, and the metabolism level of tumor cells is higher than that of normal cells w19x. In the addition, tumor cells behave stack growth because of no contact inhibition. All these are advantageous to electrons transferring from the cells to the conductor glass in flash of light.
Acknowledgements This project was supported by the Natural Science Foundation of Peking University and National Natural Science Foundation of China.
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