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Photoresponsive planar bilayer lipid membranes containing azobenzene amphiphilic derivatives
316
Sensors
and
Achcators B, 13-14 (1993) 376-379
Photoresponsive planar bilayer lipid membranes containing azobenzene amphiphilic derivatives Hajime Yamaguchi and Hiroshi Nakanishi Advanced Research L.abomtoiy, Research and Development Center, Toshiba Corpomtion, 1 Komukai-Toshiba-cho, Saiwai-kq Kawasaki 210 (Japan)
Abstract We have prepared photoresponsive bilayer lipid membranes (BLMs) containing azobenzene derivatives (4’octylazobenzene-4-oxybutyric acid; AZ) and reveal their ability to convert light signals to electrical ones. The changes in the structure of a membrane and in its electrical properties under light irradiation are monitored simultaneously by in situ spectroscopic, electrical and microscopic measurements. The BLMs consist of AZ and glyceryl monooleate. The 360 nm irradiation increases the capacitance and the conductance, which are restored to those in dark conditions upon irradiation by 450 nm light. It is clarified, using simultaneous multi-measurement techniques, that these changes induced by light are caused by the reversible changes in the membrane structure initiated by the trans-cis photoisomerization reaction of AZ and that this structural change of the membrane occurs immediately after the isomerization reaction.
Introduction Biological membranes play an important role in many of the physiological and biological activities of cells [l]. If the function of such membranes could be mimicked,
new artificial sensors and devices with biological functions would become possible. It is known that planar bilayer lipid membranes (BLMs) provide optimum conditions for the production of artificial biological membranes [2]. However, in spite of their excellent characteristics, BLMs have been studied only in a limited area due to difficulties in their preparation and evaluation. Of the various functions of biological membranes, the authors focused on light conversion, which is the first step in visual processing. In the study an azobenxene derivative was chosen as an artificial light receptor molecule. Axohenzene is a well-known photochromic compound, in which proceeds a ~ans-cis photoisomerization similar to the retinal one in rhodopsin existing in the photoreceptor cell. Though the regulation of photoisomerization reactions in azobenzene derivatives has been studied in many membrane types [3], there has been hardly any research reported using BLMs. In this study, we not only prepared a BLM containing azobenzene derivatives, but also measured the changes of the characteristics of the membrane during the formative process as well as the response of the membrane to light irradiation. A multi-observation method was employed, in which spectroscopic, electrical and
measurements were combined. Spectroscopic measurements of microcrystalline semiconductors deposited on BLMs have been reported previously [4], but simultaneous spectroscopic, electrical and microscopic measurements of BLMs themselves have never been tried before. microscopic
Experimental 4’-Octylaxobenzene-4-oxybutyric acid (AZ, Dojin Kagaku Co.), glyceryl monooleate (GMO; Tokyo Kasei Kogyo Co.), and n-decane (Aldrich Co.) were used as the photoreceptor, membrane-forming lipid and solvent, respectively. BLMs were formed across a hole, 0.7 mm in diameter,
punched in a 0.05 mm thick Teflon film. The film was sandwiched between two triangular quartz cells. The two cells were electrically separated from each other and were filled with 0.10 M KC1 aqueous solution. The solution made contact with the BLM through a window in the cell. In the case of absorption spectroscopy, a thin 0.01 mm thick metal plate with a 0.3 mm diameter hole, coated with Teflon, was used instead of the Teflon film to avoid the effects of absorption of AZ residue on the film surfaces. Electrical measurements were performed using two Pt/Pt black electrodes immersed in the cells and connected to an LCR meter (4274A, Hewlett-Packard Co.). Operation of the LCR meter and the data processing
0 1993 - Elsevier Sequoia. All rights reserved
311
were performed with a personal computer (PC98OlRA21; NEC Co.). All electrical measurements using the LCR meter were carried out at a frequency of 1 kHz. The a.c. voltage across the BLM was kept at 7 mV (r.m.s.). Absorption measurements were performed by using a multichannel photodetecting system (MCPD-1000; Otsuka Electronics Co.). Light from a 150 W Dz lamp was introduced via an optical fibre and was focused on the hole. Then the ligbt transmitted through the hole was introduced into the photodetecting system through another optical fibre. A 300 W xenon lamp was used as an excitation source for the photoreaction. The light was focused on the BLM through a monochromator and an optical fibre. Besides electrical and spectroscopic measurements, morphological observations were carried out simultaneously through a microscope (BHMJ-MB; Olympus Co.). The BLM was illuminated by a 50 W halogen lamp through an optical fibre using a 550 nm cut-off filter and a water filter. The cut-off filter prevents AZ photoisomerixation in the membrane. The light passed through an objective (ULWD MSPlan 20 x , Olympus Co.) with a working distance of 11 mm, and through a 0.44X projection lens. The image was picked up by a CCD colour camera (EC-20211; ELM0 Co.). The video signal was displayed on a video monitor (FCM1404 Ikegami Tsushinki Co.). All measurements were performed at 25 f2 “C.
Results and discussion The formation of AZ/GM0 BLMs was monitored simultaneously using three different techniques: spectroscopic, electrical and microscopic measurements. In the spectroscopic measurements an absorption characteristic of AZ was observed near 360 nm, indicating the presence of AZ in the membrane. This fact was also supported by the disappearance of the absorption when the BLM suffered electrical breakdown. The spectrum at 355 nm is the same as that of AZ in solution, suggesting that AZ is dispersed in the BLM on the molecular scale [S]. The specific capacitance of the BLM was determined to be 0.42 ~F/cm*. The BLMs prepared under these conditions were stable, and measurements showed an excellent reproducibility. Figure 1 shows the changes in the BLM’s electrical properties with time when irradiated at two ditferent wavelengths, 360 nm and 450 nm. It is clear that the 360 nm irradiation increases the capacitance and the conductance, and that both of the properties are restored to the original level in dark conditions upon irradiation by 450 nm light. Such changes in capacitance and
1.5
0.4
4*m ,/+
,r-+_;---
o,3
w--,_.L’ 31
1.0 ---i i? C ci
2 min
36Onm
..::..TO.2 $ .. ____ 7.’:. _ _ ..,._.,.__ _. _._‘. -.~,‘.,-.‘_‘,.~ ,.‘.,, _. a ‘,’ ‘t -I).,
., ,..;
0.5 _.. ..--_ t
- .I._.. _... -..
Fig. 1. Changes in capacitance and conductance of the AZ/GM0 BLM upon alternating irradiation by 360 and 450 nm light.
conductance dependent on the wavelength are not seen in the BLM formed from GM0 alone. Therefore, the results indicate that the change in electrical properties can be attributed to AZ in the BLM. To investigate the mechanism of this change in electrical properties, simultaneous spectroscopic, electrical and microscopic measurements were carried out. The results are shown in Fig. 2. The changes in membrane capacitance and absorption spectra with irradiation are shown in Fig. 2(a) and Fig. 2(b) and (c), respectively. During the experiments, the bilayer area was cautiously monitored using an optical microscope to ascertain that the BLM area did not change morphologically. Valleys in the spectra near 360 and 450 run are attributed to the excitation light. AZ in the BLM takes a tmns form before the irradiation, showing a nrr* band near 355 run (spectrum 1). As shown in Fig. 2(b), the intensity of the 355 nm band decreased with the 360 nm irradiation, indicating a decrease of the truns form. It is worth noting that the increase in membrane capacitance clearly corresponds to the spectral change. When the change of the spectrum is completed, the membrane capacitance also reaches a constant value. The decrease in the 355 mn absorption band and the increase in capacitance are completely restored by 450 nm irradiation. These results clearly indicate that the trans-& photoisomerixation of AZ can be controlled in the BLM, and that the electrical changes occur with respect to the photoisomerixation reaction of AZ without delay. The mechanism that gives these results can be explained as follows. In the dark, AZ takes a lrans form in the membrane. When 360 mn irradiation occurs, the trun.s-ci.rphotoisomerixation reaction of AZ is triggered in the membrane. The induced change in the AZ structure disorders the arrangement of matrix molecules (GMO) around AZ, leading to a substantial change in the membrane structure. This phenomenon gives rise to the change in capacitance and conductance. The
310
0.02
diately, as soon as photoisomerization of AZ occurs. Furthermore, the conversion between the two isomers is nearly perfect and the photoisomerization is completely reversible (Fig. 2). In other types of membranes containingazobenzene derivatives,such as Langmuir-Blodgett (LB) films [6] or polymer membranes [7], changes in electrical prop erties are rather slow and the changing ratio of azobenzene derivatives is not so high. These differences can be explained by the fact that in the case of the BLM the structural change of azobenzene derivatives seems to be transmitted directly and swiftly to the neighbouring molecules because of the fluid and ultimately thin structure of the membrane.
0.01
Conclusions
0901
0 Time
(4 .
8 :
c B 2
0
I t
250
I I 300
I
I
I
350
400
450
I 500
Wavelength (nm)
@I
0.01
In this paper, we have described the successfulprep aration of BLMs containing azobenzene derivatives. The electricalproperties of the BLM changedreversibly and veryrapidlyupon irradiationbylight.It wasclarified, usingsimultaneousmulti-measurementtechniques,that these changesinducedby lightwere causedby reversible changes in the membrane structure initiated by the photoisomerizationreaction of azobenzene derivatives. The system developed here offers advantages in the dynamic characterization of BLMs in general, since it allowssimultaneousevaluation of the molecular structures and corresponding electrical properties.
8 :
c 0.01 ::
2
Acknowkdgements (
(c)
Wavelength (nm)
This study was performed through Special Coordination Funds for Promoting Science and Technology of the Science and TechnologyAgency of the Japanese Government.
Fig. 2. Changes in capacitance (a) and absorption spectra of the AZ/GM0 BLM upon alternating irradiation by (b) 360 nm and (c) 450 nm light.
References
reason for the conductance change is thought to be that the structural change makes it easier for ions in the solution to get through the membrane. The change in capacitance is thought to derive from a change in dielectric constant or the thickness of the membrane, although the actual mechanism at work cannot be clarified at this stage. It is noteworthy that the changes in electrical properties in the BLM occurred imme-
M. J. Jain and R. C. Wagner, Intmduch fo Biological hfembnznes, Wiley, New York, 1980. H. T. Tien, Bikrycr L.@d hfembranes (BLM). Theory and Pm&e, Marcel Dekker, New York, 1974. G. S. Kumar and D. C. Neckers, Photochemistry of ambenzene-containing polymers, Chem Rev., 89 (1989) 1915-1925. X. K. Zbao, S. Baral, R. Rolandi and J. H. Fendkr, Semiconductor particles in bilayer lipid membranes. Formation, characterization and photoelectrochemistry, J. Am. Gem. SK, 110 (1988) 1012lu24.
319
5 M. Shimomura, R. Ando and T. Kunitake, Orientation and spectral characteristics of the ambenzene chromophore in the ammonium bilayer assembly,Ber Buns-. Php. Chm., 87 (1983) 1134-1143. 6 H. Tachibana, T. Nakamura, M. Matsumoto, H. Komizu, E. Manda, H. Niino, A. Yabe and Y. Kawabata, Photochemical
switchingin conductiveLangmuir-Blodgett fihns,J.Am Chem. sot., 111 (1989) 3ogcL3081. 7 J. Anzai, H. Sasaki,A. Ueno and T. Osa, Poly(vinylchloride)/ azobenzene-liiedbis(l5-crown-5’)memhranes.Photoinduced potential changes across asymmetric membranes, Chun Left, (1984) 124%120%
Sensors
and
Achcators B, 13-14 (1993) 376-379
Photoresponsive planar bilayer lipid membranes containing azobenzene amphiphilic derivatives Hajime Yamaguchi and Hiroshi Nakanishi Advanced Research L.abomtoiy, Research and Development Center, Toshiba Corpomtion, 1 Komukai-Toshiba-cho, Saiwai-kq Kawasaki 210 (Japan)
Abstract We have prepared photoresponsive bilayer lipid membranes (BLMs) containing azobenzene derivatives (4’octylazobenzene-4-oxybutyric acid; AZ) and reveal their ability to convert light signals to electrical ones. The changes in the structure of a membrane and in its electrical properties under light irradiation are monitored simultaneously by in situ spectroscopic, electrical and microscopic measurements. The BLMs consist of AZ and glyceryl monooleate. The 360 nm irradiation increases the capacitance and the conductance, which are restored to those in dark conditions upon irradiation by 450 nm light. It is clarified, using simultaneous multi-measurement techniques, that these changes induced by light are caused by the reversible changes in the membrane structure initiated by the trans-cis photoisomerization reaction of AZ and that this structural change of the membrane occurs immediately after the isomerization reaction.
Introduction Biological membranes play an important role in many of the physiological and biological activities of cells [l]. If the function of such membranes could be mimicked,
new artificial sensors and devices with biological functions would become possible. It is known that planar bilayer lipid membranes (BLMs) provide optimum conditions for the production of artificial biological membranes [2]. However, in spite of their excellent characteristics, BLMs have been studied only in a limited area due to difficulties in their preparation and evaluation. Of the various functions of biological membranes, the authors focused on light conversion, which is the first step in visual processing. In the study an azobenxene derivative was chosen as an artificial light receptor molecule. Axohenzene is a well-known photochromic compound, in which proceeds a ~ans-cis photoisomerization similar to the retinal one in rhodopsin existing in the photoreceptor cell. Though the regulation of photoisomerization reactions in azobenzene derivatives has been studied in many membrane types [3], there has been hardly any research reported using BLMs. In this study, we not only prepared a BLM containing azobenzene derivatives, but also measured the changes of the characteristics of the membrane during the formative process as well as the response of the membrane to light irradiation. A multi-observation method was employed, in which spectroscopic, electrical and
measurements were combined. Spectroscopic measurements of microcrystalline semiconductors deposited on BLMs have been reported previously [4], but simultaneous spectroscopic, electrical and microscopic measurements of BLMs themselves have never been tried before. microscopic
Experimental 4’-Octylaxobenzene-4-oxybutyric acid (AZ, Dojin Kagaku Co.), glyceryl monooleate (GMO; Tokyo Kasei Kogyo Co.), and n-decane (Aldrich Co.) were used as the photoreceptor, membrane-forming lipid and solvent, respectively. BLMs were formed across a hole, 0.7 mm in diameter,
punched in a 0.05 mm thick Teflon film. The film was sandwiched between two triangular quartz cells. The two cells were electrically separated from each other and were filled with 0.10 M KC1 aqueous solution. The solution made contact with the BLM through a window in the cell. In the case of absorption spectroscopy, a thin 0.01 mm thick metal plate with a 0.3 mm diameter hole, coated with Teflon, was used instead of the Teflon film to avoid the effects of absorption of AZ residue on the film surfaces. Electrical measurements were performed using two Pt/Pt black electrodes immersed in the cells and connected to an LCR meter (4274A, Hewlett-Packard Co.). Operation of the LCR meter and the data processing
0 1993 - Elsevier Sequoia. All rights reserved
311
were performed with a personal computer (PC98OlRA21; NEC Co.). All electrical measurements using the LCR meter were carried out at a frequency of 1 kHz. The a.c. voltage across the BLM was kept at 7 mV (r.m.s.). Absorption measurements were performed by using a multichannel photodetecting system (MCPD-1000; Otsuka Electronics Co.). Light from a 150 W Dz lamp was introduced via an optical fibre and was focused on the hole. Then the ligbt transmitted through the hole was introduced into the photodetecting system through another optical fibre. A 300 W xenon lamp was used as an excitation source for the photoreaction. The light was focused on the BLM through a monochromator and an optical fibre. Besides electrical and spectroscopic measurements, morphological observations were carried out simultaneously through a microscope (BHMJ-MB; Olympus Co.). The BLM was illuminated by a 50 W halogen lamp through an optical fibre using a 550 nm cut-off filter and a water filter. The cut-off filter prevents AZ photoisomerixation in the membrane. The light passed through an objective (ULWD MSPlan 20 x , Olympus Co.) with a working distance of 11 mm, and through a 0.44X projection lens. The image was picked up by a CCD colour camera (EC-20211; ELM0 Co.). The video signal was displayed on a video monitor (FCM1404 Ikegami Tsushinki Co.). All measurements were performed at 25 f2 “C.
Results and discussion The formation of AZ/GM0 BLMs was monitored simultaneously using three different techniques: spectroscopic, electrical and microscopic measurements. In the spectroscopic measurements an absorption characteristic of AZ was observed near 360 nm, indicating the presence of AZ in the membrane. This fact was also supported by the disappearance of the absorption when the BLM suffered electrical breakdown. The spectrum at 355 nm is the same as that of AZ in solution, suggesting that AZ is dispersed in the BLM on the molecular scale [S]. The specific capacitance of the BLM was determined to be 0.42 ~F/cm*. The BLMs prepared under these conditions were stable, and measurements showed an excellent reproducibility. Figure 1 shows the changes in the BLM’s electrical properties with time when irradiated at two ditferent wavelengths, 360 nm and 450 nm. It is clear that the 360 nm irradiation increases the capacitance and the conductance, and that both of the properties are restored to the original level in dark conditions upon irradiation by 450 nm light. Such changes in capacitance and
1.5
0.4
4*m ,/+
,r-+_;---
o,3
w--,_.L’ 31
1.0 ---i i? C ci
2 min
36Onm
..::..TO.2 $ .. ____ 7.’:. _ _ ..,._.,.__ _. _._‘. -.~,‘.,-.‘_‘,.~ ,.‘.,, _. a ‘,’ ‘t -I).,
., ,..;
0.5 _.. ..--_ t
- .I._.. _... -..
Fig. 1. Changes in capacitance and conductance of the AZ/GM0 BLM upon alternating irradiation by 360 and 450 nm light.
conductance dependent on the wavelength are not seen in the BLM formed from GM0 alone. Therefore, the results indicate that the change in electrical properties can be attributed to AZ in the BLM. To investigate the mechanism of this change in electrical properties, simultaneous spectroscopic, electrical and microscopic measurements were carried out. The results are shown in Fig. 2. The changes in membrane capacitance and absorption spectra with irradiation are shown in Fig. 2(a) and Fig. 2(b) and (c), respectively. During the experiments, the bilayer area was cautiously monitored using an optical microscope to ascertain that the BLM area did not change morphologically. Valleys in the spectra near 360 and 450 run are attributed to the excitation light. AZ in the BLM takes a tmns form before the irradiation, showing a nrr* band near 355 run (spectrum 1). As shown in Fig. 2(b), the intensity of the 355 nm band decreased with the 360 nm irradiation, indicating a decrease of the truns form. It is worth noting that the increase in membrane capacitance clearly corresponds to the spectral change. When the change of the spectrum is completed, the membrane capacitance also reaches a constant value. The decrease in the 355 mn absorption band and the increase in capacitance are completely restored by 450 nm irradiation. These results clearly indicate that the trans-& photoisomerixation of AZ can be controlled in the BLM, and that the electrical changes occur with respect to the photoisomerixation reaction of AZ without delay. The mechanism that gives these results can be explained as follows. In the dark, AZ takes a lrans form in the membrane. When 360 mn irradiation occurs, the trun.s-ci.rphotoisomerixation reaction of AZ is triggered in the membrane. The induced change in the AZ structure disorders the arrangement of matrix molecules (GMO) around AZ, leading to a substantial change in the membrane structure. This phenomenon gives rise to the change in capacitance and conductance. The
310
0.02
diately, as soon as photoisomerization of AZ occurs. Furthermore, the conversion between the two isomers is nearly perfect and the photoisomerization is completely reversible (Fig. 2). In other types of membranes containingazobenzene derivatives,such as Langmuir-Blodgett (LB) films [6] or polymer membranes [7], changes in electrical prop erties are rather slow and the changing ratio of azobenzene derivatives is not so high. These differences can be explained by the fact that in the case of the BLM the structural change of azobenzene derivatives seems to be transmitted directly and swiftly to the neighbouring molecules because of the fluid and ultimately thin structure of the membrane.
0.01
Conclusions
0901
0 Time
(4 .
8 :
c B 2
0
I t
250
I I 300
I
I
I
350
400
450
I 500
Wavelength (nm)
@I
0.01
In this paper, we have described the successfulprep aration of BLMs containing azobenzene derivatives. The electricalproperties of the BLM changedreversibly and veryrapidlyupon irradiationbylight.It wasclarified, usingsimultaneousmulti-measurementtechniques,that these changesinducedby lightwere causedby reversible changes in the membrane structure initiated by the photoisomerizationreaction of azobenzene derivatives. The system developed here offers advantages in the dynamic characterization of BLMs in general, since it allowssimultaneousevaluation of the molecular structures and corresponding electrical properties.
8 :
c 0.01 ::
2
Acknowkdgements (
(c)
Wavelength (nm)
This study was performed through Special Coordination Funds for Promoting Science and Technology of the Science and TechnologyAgency of the Japanese Government.
Fig. 2. Changes in capacitance (a) and absorption spectra of the AZ/GM0 BLM upon alternating irradiation by (b) 360 nm and (c) 450 nm light.
References
reason for the conductance change is thought to be that the structural change makes it easier for ions in the solution to get through the membrane. The change in capacitance is thought to derive from a change in dielectric constant or the thickness of the membrane, although the actual mechanism at work cannot be clarified at this stage. It is noteworthy that the changes in electrical properties in the BLM occurred imme-
M. J. Jain and R. C. Wagner, Intmduch fo Biological hfembnznes, Wiley, New York, 1980. H. T. Tien, Bikrycr L.@d hfembranes (BLM). Theory and Pm&e, Marcel Dekker, New York, 1974. G. S. Kumar and D. C. Neckers, Photochemistry of ambenzene-containing polymers, Chem Rev., 89 (1989) 1915-1925. X. K. Zbao, S. Baral, R. Rolandi and J. H. Fendkr, Semiconductor particles in bilayer lipid membranes. Formation, characterization and photoelectrochemistry, J. Am. Gem. SK, 110 (1988) 1012lu24.
319
5 M. Shimomura, R. Ando and T. Kunitake, Orientation and spectral characteristics of the ambenzene chromophore in the ammonium bilayer assembly,Ber Buns-. Php. Chm., 87 (1983) 1134-1143. 6 H. Tachibana, T. Nakamura, M. Matsumoto, H. Komizu, E. Manda, H. Niino, A. Yabe and Y. Kawabata, Photochemical
switchingin conductiveLangmuir-Blodgett fihns,J.Am Chem. sot., 111 (1989) 3ogcL3081. 7 J. Anzai, H. Sasaki,A. Ueno and T. Osa, Poly(vinylchloride)/ azobenzene-liiedbis(l5-crown-5’)memhranes.Photoinduced potential changes across asymmetric membranes, Chun Left, (1984) 124%120%