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Immobilization of barley protoplasts on a polyelectrolyte modified electrode for measuring the photoelectric behavior of protoplasts
Electrochemistry Communications 4 (2002) 431–435 www.elsevier.com/locate/elecom
Immobilization of barley protoplasts on a polyelectrolyte modified electrode for measuring the photoelectric behavior of protoplasts Yulan Qi, Hongping Zhang, Manming Yan, Zhiyu Jiang
*
Department of Chemistry, Fudan University, Shanghai 200433, China Received 1 March 2002; accepted 18 March 2002
Abstract A novel method to immobilize barley protoplasts on the poly(diallyl dimethyl ammonium chloride) gold/(PDADMAC) electrode was developed for the purpose to measure the photoelectric behavior of barley protoplasts. The electrochemical quartz crystal microbalance (EQCM) results show that the thickness of the adsorbed PDADMAC layer is 2.4 nm. The barley protoplasts are immobilized on the surface of gold/PDADMAC electrode due to the electrostatic adsorption between negatively charged protoplasts and positively charged PDADMAC. The fluorescence image taken by laser scanning confocal microscope shows that the attached barley protoplasts are integrity. For the gold/PDADMAC/barley protoplast electrode an anodic photocurrent was observed under the irradiation of white light (wavelength of 200–800 nm) and its properties are discussed. This novel method may provide a convenient technique for immobilizing cells or other bio-particles on the surface of electrode for studying their electrochemical characters. Ó 2002 Elsevier Science B.V. All rights reserved. Keywords: Barley protoplast; Polyelectrolyte; Modified electrode; Fluorescence image; Photocurrent
1. Introduction The photoelectric behavior of biological materials and particles, such as chlorophyll, photosynthetic pigments, thylakoid and cells, has induced a wide interest for a long time [1–8]. Many experiments in this field have been carried out for a fundamental purpose to investigate the electric charge transferring in photosynthesis or for a practical purpose to get an efficient photoelectric conversion system. However photoelectric behavior of the living protoplasts is rarely studied. In the photoelectrochemical research of living protoplasts, one of the important procedures is to attach protoplasts on the surface of electrode stably. There are same approaches such as Langmuir– Blodgett film technique [7] and anodic stripping
*
Corresponding author. Tel.: +86-21-65642404; Fax: +86-2165641740. E-mail address: [email protected] (Z. Jiang).
method [8] to immobilize biological particles on substrate in the literature. Adsorption of charged particles (such as biological macromolecules DNA, proteins, and dyes) on an oppositely charged surface was a widely used technique to fabricate the self-assembly particles on the substrates in biochemistry and biotechnology [9–13]. But immobilizing cells is a new attempt. It has been known that the membrane of protoplast carries negative charge. In this work, positively charged polyelectrolyte poly(diallyl dimethyl ammonium chloride) (PDADMAC) was chosen as a matrix substance. A novel method was developed to immobilize the barley protoplasts on the surface of PDADMAC modified gold electrode based on electrostatic effect. The electrochemical quartz crystal microbalance (EQCM) method was used to exam the adsorption process of PDADMAC on the electrode. The photoelectric behavior of the barley protoplasts was investigated by measuring the photocurrent under the irradiation of light.
1388-2481/02/$ - see front matter Ó 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 2 4 8 1 ( 0 2 ) 0 0 3 3 8 - 7
432
Y. Qi et al. / Electrochemistry Communications 4 (2002) 431–435
2. Experimental 2.1. Preparation of barley protoplasts modified electrode The procedure for obtaining the barley protoplasts is similar to that described elsewhere [14,15]. A gold plate electrode was prepared by spattering gold on a piece of glass in vacuum. After being washed with plenty of acetone and distilled water successively, the gold plate electrode was immersed into a polyelectrolyte PDADMAC solution (5 g l1 ) for about 60 min to let polyelectrolyte adsorbed on the surface of gold electrode. Then wash it again with plenty of distilled water. After that, the gold/PDADMAC plate electrode was dipped into 5 mol l1 mannitol solution containing barley protoplasts for 15 min. During this process the barley protoplasts was assembled on the surface of PDADMAC layer due to the effect of electrostatic force. The gold/PDADMAC/barley protoplast electrode was washed with distilled water, and then stored in 5 mol l1 mannitol solution for the use in Fluorescence and photocurrent experiments. 2.2. EQCM measurements EQCM measurements were carried out with a CHI400 Time-Resolved EQCM System (CHI, USA). The diameter of gold resonate electrode on EQCM resonator is 5 mm. According to the procedure described previously the gold/PDADMAC and gold/ PDADMAC/barley protoplast resonate electrodes were prepared, respectively. Measuring the frequency change of the EQCM resonator can monitor the adsorption processes taking at the gold electrode. 2.3. Fluorescence images It has been known that chlorophyll can be excited by light and emits peach fluorescence. The gold/PDADMAC/barley protoplast electrode was prepared as described in Section 2.1. The fluorescence images of chloroplasts in barley protoplasts were taken by use of a laser scanning confocal microscopy (LEICA TCS NT) choosing an excitation laser beam with wavelength of 585 nm and an emission pass filter with the wavelength of 660 nm.
Fig. 1. The scheme of photoelectric cell. RE: referent electrode (Ag/ AgCl electrode); CE: counter electrode (platinum electrode); WE: working electrode (gold/PDADMAC/barley protoplast electrode); Electrolyte: 0:5 mol l1 mannitol þ 0:5 mol l1 KCl solution.
lock-in amplifier under the irradiation of a chopped light beam from a 250 W halotungsten lamp. The photocurrents were measured at certain potential or during potential sweep. The measuring signal reflects the variations of the amplitude of photocurent. The electrochemical measurements were carried out using a model ZF-3 potentiostat and an XFD-8 universal programmer. A lock-in amplifier model PAR 5210 with a model PAR 197 chopper at a frequency of 10 Hz was used to measure the amplitudes of photocurrent. The solutions were prepared from analytical reagents and ion free water.
3. Results and discussion 3.1. The adsorption of PDADMAC on gold electrode The assembly of PDADMAC on the surface of gold electrode was monitored by EQCM method, which is an extremely sensitive sensor with the capable of measuring mass changes in the nanogram range. The attachment of a film with mass of Dm on the electrode surface of quartz crystal resonator causes a decrease of resonant frequency DFm . According to Sauerbrey equation [16,17], in vacuum the relationship between Dm and Fm is DFm ¼
2F02 AðlQ qQ Þ1=2
Dm;
ð1Þ
2.4. Photoelectric measurement system The photoelectric measurement system was a threeelectrode cell, consisting of a gold/PDADMAC/barley protoplast working electrode with area of 0:2 cm2 , a platinum counter electrode and an Ag/AgCl reference electrode as shown in Fig. 1. The potentials reported in this paper are related to this reference electrode. The photocurrent measurements were carried out using a
where F0 is the resonant frequency of quartz (F0 ¼ 8:0 106 Hz in this paper), A is the surface area (0:2 cm2 ) of the gold electrode on the quartz crystal, lq is the density of quartz crystal ð2:648 g cm3 Þ and qq is the shear modulus of quartz ð2:947 1011 g cm1 s1 Þ. In liquid, the viscosity gL and density qL of liquid also cause the resonant frequency change DFL of the EQCM as described by equation [18–20]
Y. Qi et al. / Electrochemistry Communications 4 (2002) 431–435 3=2
DFL ¼
F0 ðgL qL Þ1=2 1=2 : plQ qQ
ð2Þ
Thus in liquid, the change of resonant frequency DF measured in experiment is a combination of DFm and DFL , DF ¼ DFm þ DFL . For the case in polyelectrolyte solution, the variation of frequency DFL;P caused by the influence of viscosity and density of polyelectrolyte solution can be calculated from measuring the viscosity g and density q of solution and the resonant frequency change in pure water DFL;H . According to Eq. (2), we can get 1=2 qP qP DFL;P ¼ DFL;H ; ð3Þ gH qH where the subscript P refers to PDADMAC solution and H refers to pure water, respectively. Fig. 2 curves a and b reveal the changes of frequency response after adding distilled water and PDADMAC solution (5 g l1 ) into a EQCM cell, respectively. It can be seen from curve a in Fig. 2 that the value of frequency change DFL;H is 600 Hz after adding water. The viscosity of PDADMAC solution and water were measured using a Ubbelohde Viscometer. The relative viscosity gr was obtained at gr ¼ gP =gH ¼ 5:871. It was measured that qP =qH ¼ 0:9973. Substitution of these values into Eq. (3) yields the contribution of frequency changes caused by the influence of density and viscosity of PDADMAC solution, DFL;P ¼ 1452 Hz. It can be seen from Fig. 2 curve b that after adding PDADMAC solution the frequency decreased gradually, about 9 min later the frequency becomes stable. The total frequency change DF ¼ 1494 Hz. Thus, the decrease of resonant frequency caused by the adsorption of PDADMAC on the gold electrode DFm ¼ DF DFL;P ¼ 42 Hz. It indicates that PDADMAC has been attached on the surface of gold electrode. It is assumed that PDADMAC was adsorbed uniformly on the surface of gold electrode. According to Eq. (1), we can calculate the thickness of adsorbed film
dad ¼
DFm ðlQ qQ Þ 2F02 qad
433
1=2
;
ð4Þ
where qad is the density of pure polyelectrolyte PDADMAC. Normally, the density of polyelectrolytes is about 1:2 g cm3 [21]. Substitution of relevant values into Eq. (4) yields dad ¼ 2:4 nm. This value coincides with what was measured using surface plasmon spectroscopy by Kotov et al. [13]. 3.2. Protoplasts/PDADMAC/Au electrode It has been known that barley protoplast carries negative charge on the surface of membrane [22]. So it can be self-assembled on the surface of positive charged PDADMAC layer. Fig. 3 shows the resonant frequency changes after the immersion of a gold/PDADMAC oscillate electrode into the 0:5 mol l1 mannitol solutions with or without barley protoplasts, respectively. The frequency change induced by 0:5 mol l1 mannitol solution is 632 Hz as shown in Fig. 3(a). Fig. 3 curve b shows the variation of frequency in 0:5 mol l1 mannitol solution containing protoplasts. At first the crystal frequency decreases quickly and 4 min later it becomes much stable. The stable value is around 1946 Hz, which is much lower than that in the solution without protoplasts. This result indicates that barley protoplasts have been successfully adsorbed on the surface of gold/ PDADMAC electrode. It was reported that the adsorption of biologic substances, such as the adsorption of protein on electrode, could be determined by ex situ EQCM technique [23]. But ex situ method is not suitable for study the adsorption of living cells, because the cells will break and die in dry condition. The adsorption of barley protoplasts was confirmed by fluorescent microscopy measurement. A gold/ PDADMAC electrode was fabricated according to the previous procedure. After being immerged in 0:5 mol l1 mannitol solutions containing barley protoplasts for about 15 min, the barley protoplasts could be absorbed on the surface of the gold/PDADMAC
Fig. 2. Frequency–time profiles for gold EQCM electrodes in distilled water (a) and PDADMAC aqueous solution ð5 g l1 Þ (b). The arrows indicate the time at which the solutions were injected into EQCM cell.
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Y. Qi et al. / Electrochemistry Communications 4 (2002) 431–435
Fig. 3. EQCM frequency–time profiles for the PDADMAC preadsorbed gold EQCM electrode immersed into 0:5 mol l1 mannitol solution (a), and 0:5 mol l1 mannitol solution containing barley protoplasts (b). The arrows indicate the time at which the solutions were injected into EQCM cell.
electrode. The electrode was washed using distilled water, and then several drops of 0:5 mol l1 mannitol solution were added on its surface. The selected condition for fluorescent microscopy experiment was similar to that described elsewhere [21]. The wavelength of exciting laser beam was in the range 480–568 nm. Fig. 4 presents the fluorescent image of a barley protoplast being immobilized on the surface of PDADMAC modifying gold electrode. The peach fluorescent light with the wavelength of 665 nm was attributed to the fluorescent emission of chlorophyll in the barley protoplast [22]. It displays that the adsorbed protoplast was integrity with diameter of about 40 lm. This result confirms that barley protoplasts have been successfully immobilized on the surface of gold/PDADMAC electrode. 3.3. Photoresponse of barley protoplasts In the photocurrent experiment the barley protoplasts modified electrode was immersed in containing 0:5 mol l1 mannitol + 0:5 mol l1 KCl solution as shown in Fig. 1. Fig. 5 shows the typical photoelectric
Fig. 4. Fluorescent microphotograph of a barley protoplast adsorbed on a PDADMAC modified gold electrode at 665 nm.
response of gold/PDADMAC/barley protoplast electrode. The light irradiation time was 2 min. The values of photocurrent measured in three repeated measurements were 3.1, 3.1 and 3.0 nA, respectively. Fig. 6 shows the photoelectric responses of gold electrode, gold/PDADMAC electrode and gold/ PDADMAC/barley protoplast electrode in 0:5 mol l1 mannitol þ 0:5 mol l1 KCl solution while the potential was swept at rate of 10 m V s1 , respectively. For gold electrode and gold/PDADMAC electrode almost no photocurrent could be detected as curve a and b, respectively. The barley protoplasts modified electrode revealed considerable anodic photocurrent as curve c. The photocurrent increased with the increasing of potential when the potential was higher than 0.2 V. For compare, we also carried out a photocurrent measurement for a bare gold electrode in the solution of 5 mol l1 monnitol þ 0:5 mol l1 KCl containing protoplasts, but no photocurrent could be observed. These results indicate that the photocurrent can be observed only in the case that the barley protoplasts immobilized on the surface of PDADMAC modified gold electrode. The phenomenon of anodic photocurrent is in agreed with the observation for a chlorophyll modified SiO2 /
Fig. 5. Photocurrent of barley protoplasts measured at an interval of 2 min at a potential of 600 mV.
Y. Qi et al. / Electrochemistry Communications 4 (2002) 431–435
435
technique for immobilizing cells or other bio-particles on electrode to study their electrochemical characters.
Acknowledgements This work was supported by the National Science Foundation of China.
References
Fig. 6. Photoelectric responses of gold electrode (a), gold/PDADMAC electrode (b) and gold/PDADMAC/barley protoplast electrode (c), 0:5 mol l1 monnitol solution, potential sweep rate: 10 mV s1 .
OTE electrode in reference [1]. In our case the situation was more complicated due to the photocurrent was from alive protoplasts. The anodic photocurrent may be attributed to the oxidation of the light exited chlorophyll within chloroplasts of protoplasts. It is possible that electrons or ions may penetrate the polyelectrolyte layer, as it is very thin (2.4 nm). Another possibility may be attributed to the oxidation of some reducing bio-substances produced in the processes of photosystem I (PS I) and photosystem II (PS II).
4. Conclusion A self-assembly system with the electrostatic adsorption of barley protoplasts on the gold/PDADMAC electrode was devised. The adsorption behavior was examined by EQCM, and fluorescence microscope methods. The photoelectric behavior of living barley protoplasts was investigated. It was found an anodic current appeared on the gold/PDADMAC/barley protoplast electrode under the irradiation of white light. This novel method may provide a convenient and quick
[1] T. Miyasaka, T. Watanabe, A. Fujishima, K. Honda, J. Am. Chem. Soc. 100 (1978) 6657. [2] T. Miyasaka, K. Honda, Thin Solid Films 102 (1983) 173. [3] S.I. Ajiki, H. Toyotama, M. Hara, J. Miyake, Bioelectrochem. Bioenerg. 43 (1997) 71. [4] Y.X. Ci, C.Y. Zhang, J. Fen, Bioelectrochem. Bioenerg. 45 (1998) 247. [5] A.A. Bulychev, J.H.A. Dassen, W.J. Vredenberg, V.K. Opanasenko, G.A. Semenova, Bioelectrochem. Bioenerg. 46 (1998) 71. [6] C.Y. Zhang, J. Feng, Y.X. Ci, A.D. Lang, C.H. Huang, Bioelectrochem. Bioenerg. 46 (1998) 145. [7] Y. Yasuda, H. Sugino, H. Tohotama, Y. Hirata, M. Hara, J. Miyake, Bioelectrochem. Bioenerg. 34 (1994) 135. [8] H. Morisaki, M. Sugimoto, H. Shiraishi, Bioelectrochemistry 51 (2000) 21. [9] G. Decher, Science 277 (1997) 1232. [10] Y. Lvov, K. Ariga, I. Ichinose, T. Kunitake, J. Am. Chem. Soc. 117 (1995) 6117. [11] N.A. Kotov, T. Haraszti, L. Turi, G. Zavala, R.E. Geer, I. Dekany, J.H. Fendler, J. Am. Chem. Soc. 119 (1997) 6821. [12] C. Tedeschi, F. Caruso, H. Mohwald, S. Kirstein, J. Am. Chem. Soc. 122 (2000) 5841. [13] N.A. Kotov, I. Dekany, J.H. Fendler, J. Phys. Chem. 99 (1995) 13065. [14] Z.Y. Jiang, H. Berg, Bioelectrochem. Bioenerg. 38 (1995) 383. [15] Z.Y. Jiang, M.Y. Wang, W. Wang, Bioelectrochem. Bioenerg. 48 (1999) 447. [16] G. Sauerbrey, Z. Phys. 155 (1959) 206. [17] G. Sauerbrey, M. Shay, Electrochim. Acta 30 (1985) 1295. [18] K.K. Kanazawa, J.G. Gordon II, Anal. Chem. 57 (1985) 1770. [19] K.K. Kanazawa, J.G. Gordon II, Anal. Chim. Acta 175 (1985) 99. [20] K. Shimazu, I. Yagi, Y. Sato, K. Uosaki, Langmuir 8 (1992) 1385. [21] F. Caruso, K. Niikura, D.N. Furlong, Y. Okahata, Langmuir 13 (1997) 3422. [22] M.Y. Wang, M.M. Yang, Z.Y. Jiang, H. Berg, ElectroMagnetics, accepted. [23] F. Caruso, E. Rodda, D.N. Furlong, Anal. Chem. 69 (1997) 2043.
Immobilization of barley protoplasts on a polyelectrolyte modified electrode for measuring the photoelectric behavior of protoplasts Yulan Qi, Hongping Zhang, Manming Yan, Zhiyu Jiang
*
Department of Chemistry, Fudan University, Shanghai 200433, China Received 1 March 2002; accepted 18 March 2002
Abstract A novel method to immobilize barley protoplasts on the poly(diallyl dimethyl ammonium chloride) gold/(PDADMAC) electrode was developed for the purpose to measure the photoelectric behavior of barley protoplasts. The electrochemical quartz crystal microbalance (EQCM) results show that the thickness of the adsorbed PDADMAC layer is 2.4 nm. The barley protoplasts are immobilized on the surface of gold/PDADMAC electrode due to the electrostatic adsorption between negatively charged protoplasts and positively charged PDADMAC. The fluorescence image taken by laser scanning confocal microscope shows that the attached barley protoplasts are integrity. For the gold/PDADMAC/barley protoplast electrode an anodic photocurrent was observed under the irradiation of white light (wavelength of 200–800 nm) and its properties are discussed. This novel method may provide a convenient technique for immobilizing cells or other bio-particles on the surface of electrode for studying their electrochemical characters. Ó 2002 Elsevier Science B.V. All rights reserved. Keywords: Barley protoplast; Polyelectrolyte; Modified electrode; Fluorescence image; Photocurrent
1. Introduction The photoelectric behavior of biological materials and particles, such as chlorophyll, photosynthetic pigments, thylakoid and cells, has induced a wide interest for a long time [1–8]. Many experiments in this field have been carried out for a fundamental purpose to investigate the electric charge transferring in photosynthesis or for a practical purpose to get an efficient photoelectric conversion system. However photoelectric behavior of the living protoplasts is rarely studied. In the photoelectrochemical research of living protoplasts, one of the important procedures is to attach protoplasts on the surface of electrode stably. There are same approaches such as Langmuir– Blodgett film technique [7] and anodic stripping
*
Corresponding author. Tel.: +86-21-65642404; Fax: +86-2165641740. E-mail address: [email protected] (Z. Jiang).
method [8] to immobilize biological particles on substrate in the literature. Adsorption of charged particles (such as biological macromolecules DNA, proteins, and dyes) on an oppositely charged surface was a widely used technique to fabricate the self-assembly particles on the substrates in biochemistry and biotechnology [9–13]. But immobilizing cells is a new attempt. It has been known that the membrane of protoplast carries negative charge. In this work, positively charged polyelectrolyte poly(diallyl dimethyl ammonium chloride) (PDADMAC) was chosen as a matrix substance. A novel method was developed to immobilize the barley protoplasts on the surface of PDADMAC modified gold electrode based on electrostatic effect. The electrochemical quartz crystal microbalance (EQCM) method was used to exam the adsorption process of PDADMAC on the electrode. The photoelectric behavior of the barley protoplasts was investigated by measuring the photocurrent under the irradiation of light.
1388-2481/02/$ - see front matter Ó 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 2 4 8 1 ( 0 2 ) 0 0 3 3 8 - 7
432
Y. Qi et al. / Electrochemistry Communications 4 (2002) 431–435
2. Experimental 2.1. Preparation of barley protoplasts modified electrode The procedure for obtaining the barley protoplasts is similar to that described elsewhere [14,15]. A gold plate electrode was prepared by spattering gold on a piece of glass in vacuum. After being washed with plenty of acetone and distilled water successively, the gold plate electrode was immersed into a polyelectrolyte PDADMAC solution (5 g l1 ) for about 60 min to let polyelectrolyte adsorbed on the surface of gold electrode. Then wash it again with plenty of distilled water. After that, the gold/PDADMAC plate electrode was dipped into 5 mol l1 mannitol solution containing barley protoplasts for 15 min. During this process the barley protoplasts was assembled on the surface of PDADMAC layer due to the effect of electrostatic force. The gold/PDADMAC/barley protoplast electrode was washed with distilled water, and then stored in 5 mol l1 mannitol solution for the use in Fluorescence and photocurrent experiments. 2.2. EQCM measurements EQCM measurements were carried out with a CHI400 Time-Resolved EQCM System (CHI, USA). The diameter of gold resonate electrode on EQCM resonator is 5 mm. According to the procedure described previously the gold/PDADMAC and gold/ PDADMAC/barley protoplast resonate electrodes were prepared, respectively. Measuring the frequency change of the EQCM resonator can monitor the adsorption processes taking at the gold electrode. 2.3. Fluorescence images It has been known that chlorophyll can be excited by light and emits peach fluorescence. The gold/PDADMAC/barley protoplast electrode was prepared as described in Section 2.1. The fluorescence images of chloroplasts in barley protoplasts were taken by use of a laser scanning confocal microscopy (LEICA TCS NT) choosing an excitation laser beam with wavelength of 585 nm and an emission pass filter with the wavelength of 660 nm.
Fig. 1. The scheme of photoelectric cell. RE: referent electrode (Ag/ AgCl electrode); CE: counter electrode (platinum electrode); WE: working electrode (gold/PDADMAC/barley protoplast electrode); Electrolyte: 0:5 mol l1 mannitol þ 0:5 mol l1 KCl solution.
lock-in amplifier under the irradiation of a chopped light beam from a 250 W halotungsten lamp. The photocurrents were measured at certain potential or during potential sweep. The measuring signal reflects the variations of the amplitude of photocurent. The electrochemical measurements were carried out using a model ZF-3 potentiostat and an XFD-8 universal programmer. A lock-in amplifier model PAR 5210 with a model PAR 197 chopper at a frequency of 10 Hz was used to measure the amplitudes of photocurrent. The solutions were prepared from analytical reagents and ion free water.
3. Results and discussion 3.1. The adsorption of PDADMAC on gold electrode The assembly of PDADMAC on the surface of gold electrode was monitored by EQCM method, which is an extremely sensitive sensor with the capable of measuring mass changes in the nanogram range. The attachment of a film with mass of Dm on the electrode surface of quartz crystal resonator causes a decrease of resonant frequency DFm . According to Sauerbrey equation [16,17], in vacuum the relationship between Dm and Fm is DFm ¼
2F02 AðlQ qQ Þ1=2
Dm;
ð1Þ
2.4. Photoelectric measurement system The photoelectric measurement system was a threeelectrode cell, consisting of a gold/PDADMAC/barley protoplast working electrode with area of 0:2 cm2 , a platinum counter electrode and an Ag/AgCl reference electrode as shown in Fig. 1. The potentials reported in this paper are related to this reference electrode. The photocurrent measurements were carried out using a
where F0 is the resonant frequency of quartz (F0 ¼ 8:0 106 Hz in this paper), A is the surface area (0:2 cm2 ) of the gold electrode on the quartz crystal, lq is the density of quartz crystal ð2:648 g cm3 Þ and qq is the shear modulus of quartz ð2:947 1011 g cm1 s1 Þ. In liquid, the viscosity gL and density qL of liquid also cause the resonant frequency change DFL of the EQCM as described by equation [18–20]
Y. Qi et al. / Electrochemistry Communications 4 (2002) 431–435 3=2
DFL ¼
F0 ðgL qL Þ1=2 1=2 : plQ qQ
ð2Þ
Thus in liquid, the change of resonant frequency DF measured in experiment is a combination of DFm and DFL , DF ¼ DFm þ DFL . For the case in polyelectrolyte solution, the variation of frequency DFL;P caused by the influence of viscosity and density of polyelectrolyte solution can be calculated from measuring the viscosity g and density q of solution and the resonant frequency change in pure water DFL;H . According to Eq. (2), we can get 1=2 qP qP DFL;P ¼ DFL;H ; ð3Þ gH qH where the subscript P refers to PDADMAC solution and H refers to pure water, respectively. Fig. 2 curves a and b reveal the changes of frequency response after adding distilled water and PDADMAC solution (5 g l1 ) into a EQCM cell, respectively. It can be seen from curve a in Fig. 2 that the value of frequency change DFL;H is 600 Hz after adding water. The viscosity of PDADMAC solution and water were measured using a Ubbelohde Viscometer. The relative viscosity gr was obtained at gr ¼ gP =gH ¼ 5:871. It was measured that qP =qH ¼ 0:9973. Substitution of these values into Eq. (3) yields the contribution of frequency changes caused by the influence of density and viscosity of PDADMAC solution, DFL;P ¼ 1452 Hz. It can be seen from Fig. 2 curve b that after adding PDADMAC solution the frequency decreased gradually, about 9 min later the frequency becomes stable. The total frequency change DF ¼ 1494 Hz. Thus, the decrease of resonant frequency caused by the adsorption of PDADMAC on the gold electrode DFm ¼ DF DFL;P ¼ 42 Hz. It indicates that PDADMAC has been attached on the surface of gold electrode. It is assumed that PDADMAC was adsorbed uniformly on the surface of gold electrode. According to Eq. (1), we can calculate the thickness of adsorbed film
dad ¼
DFm ðlQ qQ Þ 2F02 qad
433
1=2
;
ð4Þ
where qad is the density of pure polyelectrolyte PDADMAC. Normally, the density of polyelectrolytes is about 1:2 g cm3 [21]. Substitution of relevant values into Eq. (4) yields dad ¼ 2:4 nm. This value coincides with what was measured using surface plasmon spectroscopy by Kotov et al. [13]. 3.2. Protoplasts/PDADMAC/Au electrode It has been known that barley protoplast carries negative charge on the surface of membrane [22]. So it can be self-assembled on the surface of positive charged PDADMAC layer. Fig. 3 shows the resonant frequency changes after the immersion of a gold/PDADMAC oscillate electrode into the 0:5 mol l1 mannitol solutions with or without barley protoplasts, respectively. The frequency change induced by 0:5 mol l1 mannitol solution is 632 Hz as shown in Fig. 3(a). Fig. 3 curve b shows the variation of frequency in 0:5 mol l1 mannitol solution containing protoplasts. At first the crystal frequency decreases quickly and 4 min later it becomes much stable. The stable value is around 1946 Hz, which is much lower than that in the solution without protoplasts. This result indicates that barley protoplasts have been successfully adsorbed on the surface of gold/ PDADMAC electrode. It was reported that the adsorption of biologic substances, such as the adsorption of protein on electrode, could be determined by ex situ EQCM technique [23]. But ex situ method is not suitable for study the adsorption of living cells, because the cells will break and die in dry condition. The adsorption of barley protoplasts was confirmed by fluorescent microscopy measurement. A gold/ PDADMAC electrode was fabricated according to the previous procedure. After being immerged in 0:5 mol l1 mannitol solutions containing barley protoplasts for about 15 min, the barley protoplasts could be absorbed on the surface of the gold/PDADMAC
Fig. 2. Frequency–time profiles for gold EQCM electrodes in distilled water (a) and PDADMAC aqueous solution ð5 g l1 Þ (b). The arrows indicate the time at which the solutions were injected into EQCM cell.
434
Y. Qi et al. / Electrochemistry Communications 4 (2002) 431–435
Fig. 3. EQCM frequency–time profiles for the PDADMAC preadsorbed gold EQCM electrode immersed into 0:5 mol l1 mannitol solution (a), and 0:5 mol l1 mannitol solution containing barley protoplasts (b). The arrows indicate the time at which the solutions were injected into EQCM cell.
electrode. The electrode was washed using distilled water, and then several drops of 0:5 mol l1 mannitol solution were added on its surface. The selected condition for fluorescent microscopy experiment was similar to that described elsewhere [21]. The wavelength of exciting laser beam was in the range 480–568 nm. Fig. 4 presents the fluorescent image of a barley protoplast being immobilized on the surface of PDADMAC modifying gold electrode. The peach fluorescent light with the wavelength of 665 nm was attributed to the fluorescent emission of chlorophyll in the barley protoplast [22]. It displays that the adsorbed protoplast was integrity with diameter of about 40 lm. This result confirms that barley protoplasts have been successfully immobilized on the surface of gold/PDADMAC electrode. 3.3. Photoresponse of barley protoplasts In the photocurrent experiment the barley protoplasts modified electrode was immersed in containing 0:5 mol l1 mannitol + 0:5 mol l1 KCl solution as shown in Fig. 1. Fig. 5 shows the typical photoelectric
Fig. 4. Fluorescent microphotograph of a barley protoplast adsorbed on a PDADMAC modified gold electrode at 665 nm.
response of gold/PDADMAC/barley protoplast electrode. The light irradiation time was 2 min. The values of photocurrent measured in three repeated measurements were 3.1, 3.1 and 3.0 nA, respectively. Fig. 6 shows the photoelectric responses of gold electrode, gold/PDADMAC electrode and gold/ PDADMAC/barley protoplast electrode in 0:5 mol l1 mannitol þ 0:5 mol l1 KCl solution while the potential was swept at rate of 10 m V s1 , respectively. For gold electrode and gold/PDADMAC electrode almost no photocurrent could be detected as curve a and b, respectively. The barley protoplasts modified electrode revealed considerable anodic photocurrent as curve c. The photocurrent increased with the increasing of potential when the potential was higher than 0.2 V. For compare, we also carried out a photocurrent measurement for a bare gold electrode in the solution of 5 mol l1 monnitol þ 0:5 mol l1 KCl containing protoplasts, but no photocurrent could be observed. These results indicate that the photocurrent can be observed only in the case that the barley protoplasts immobilized on the surface of PDADMAC modified gold electrode. The phenomenon of anodic photocurrent is in agreed with the observation for a chlorophyll modified SiO2 /
Fig. 5. Photocurrent of barley protoplasts measured at an interval of 2 min at a potential of 600 mV.
Y. Qi et al. / Electrochemistry Communications 4 (2002) 431–435
435
technique for immobilizing cells or other bio-particles on electrode to study their electrochemical characters.
Acknowledgements This work was supported by the National Science Foundation of China.
References
Fig. 6. Photoelectric responses of gold electrode (a), gold/PDADMAC electrode (b) and gold/PDADMAC/barley protoplast electrode (c), 0:5 mol l1 monnitol solution, potential sweep rate: 10 mV s1 .
OTE electrode in reference [1]. In our case the situation was more complicated due to the photocurrent was from alive protoplasts. The anodic photocurrent may be attributed to the oxidation of the light exited chlorophyll within chloroplasts of protoplasts. It is possible that electrons or ions may penetrate the polyelectrolyte layer, as it is very thin (2.4 nm). Another possibility may be attributed to the oxidation of some reducing bio-substances produced in the processes of photosystem I (PS I) and photosystem II (PS II).
4. Conclusion A self-assembly system with the electrostatic adsorption of barley protoplasts on the gold/PDADMAC electrode was devised. The adsorption behavior was examined by EQCM, and fluorescence microscope methods. The photoelectric behavior of living barley protoplasts was investigated. It was found an anodic current appeared on the gold/PDADMAC/barley protoplast electrode under the irradiation of white light. This novel method may provide a convenient and quick
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