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Photoresponse discrimination of bacteriorhodopsin films to light stimuli of different frequencies
www.elsevier.nl/locate/jphotobiol J. Photochem. Photobiol. B: Biol. 52 (1999) 86–91
Photoresponse discrimination of bacteriorhodopsin films to light stimuli of different frequencies Guangyu Wang b
a,1
, Tao Lu b,2, Kun-Sheng Hu a,*, Long Jiang b
a Institute of Biophysics, Academia Sinica, Beijing 100101, PR China Institute of Photographic Chemistry, Academia Sinica, Beijing 100101, PR China
Received 5 July 1999; accepted 1 September 1999
Abstract The capability of a bacteriorhodopsin-based artificial photosensor to discriminate the adjacent light stimuli from a moving object relies on the intrinsic properties of the differential photoresponse of bacteriorhodopsin films, such as peak value, peak width and degree of distortion of the biphasic spikes, which are related not only to the photoelectric detection system, but also to the pH, temperature, light intensity and the method for depositing films. At higher temperature ()3008C), lower pH (-8.0) or in a deposited thin film rather than a Langmuir–Blodgett film, the relaxation of photoresponse will be accelerated and the sharp photocurrent spike will decrease the pre-excitatory inhibition of the next stimulus and enhance the fusion frequency. The fusion of photoresponse at high stimulus frequencies depends primarily on the response rise time rather than its decay. Under proper conditions, the maximum stimulus frequency corresponding to distinguishable signals in a deposited thin film can be as high as 30–50 Hz, contrasted with 10 Hz in a Langmuir–Blodgett film. q1999 Elsevier Science S.A. All rights reserved. Keywords: Bacteriorhodopsin; Differential photoresponse; Discrimination; Fusion frequency
1. Introduction Bacteriorhodpsin (BR), a retinal-containing protein, possesses the ability to convert light energy to electrochemical energy by pumping protons from the cytoplasmic side to the extracellular side across the membrane under illumination. Under short-circuit measurement conditions, its photoelectric signals elicited from a Langmuir–Blodgett (LB) film and a deposited thin film exhibit a differential photoresponse [1], a fundamental responsivity characteristic of vertebrate photoreceptor cells, by which vertebrates such as frogs can judge whether an object is moving or not. Unlike other biomaterials, the surprising stability, reversibility as well as the unique photochemical and photophysical properties of BR make it the most promising candidate to engineer various technical systems [2–7]. Apart from applications in optical storage and information processing that use * Corresponding author. 1 Present address: MPI f. Biophys. Chem., Am Faßberg 11, D-37077 ¨ Gottingen, Germany. Tel.: q49-551-201 11 59; Fax: q49-551-201 11 68; e-mail: [email protected] 2 Present address: Department of Biology Science, Columbia University, New York, NY 10027, USA.
the photochromic properties of BR[2–4], several motionsensitive artificial retinas based on the differential photoresponse have recently been developed [5–7] and are essentially useful in many aspects, especially in robot vision. Generally, the capability of a motion-sensitive photosensor to discriminate two adjacent light stimuli is determined by the peak value and peak width, especially the rise time of the differential photoresponse. Slow charging and discharging processes will lead to flattening of the response. The serious pre-excitatory inhibition of the next stimulus will finally result in a quick fusion of the response when an increase in the frequency of presentation reaches the so-called fusion frequency. The present study seeks to characterize the rise and decay time scale of a BR film under different measurement conditions. It is demonstrated that pH, temperature and light intensity are of significant influence on both the peak shape and the symmetry of biphasic spikes. Meanwhile, a deposited thin film with less membrane capacitance and resistance presents a sharp photocurrent spike compared with a BR-LB film. These results provide a further understanding of the properties of the differential photoresponse and are helpful in the design of photoelectronic devices.
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2. Materials and methods The purple membrane (PM) was isolated from R1M1 strains of Halobacterium salinarium according to the conventional method [8]. Two kinds of BR films were employed. The preparation of BR-LB films was described previously [9]. The PM spreading solution was prepared by adding 1 ml of PM water stock solution (8.24 mg/ml) to 4 ml of hexane. After sonication three times (15 s each), the mixture was spread on the surface of a 3 mmol/l CaCl2 solution. Films were compressed to an annealing target pressure of 40 dyne/cm2 and then allowed to deposit on the surface of a glass-coated indium–tin-oxide (ITO) electrode to air dry. All BR-LB films consisted of 10 layers of z-type depositions. Concurrently, the deposited thin films were coated on ITO glass by drying diluted PM suspension under ambient atmosphere or under a nitrogen stream to give homogeneous films, the absorbance value of which ranged from 0.2 to 0.25 at 570 nm. These randomly orientated films may be considered as structurally equivalent to LB multilayer films formed on the same kind of electrode [10], which is known to elicit a sufficiently large photoresponse in the method used here [1,11]. An electrochemical liquid junction photocell was built up to measure the photoresponse of the BR film. The cell comprised a BR-LM film-modified working electrode, aqueous electrolyte (0.1 mol/l KCl) and a Pt counter electrode. When irradiated, the photoreceptor yielded a photocurrent, which was recorded with a current amplifier and an oscilloscope (PM 3375 100 MHz). Two current amplifiers were employed: one was a Keithley 427 and the other was home-made. A 300 W halogen lamp was utilized as a light source, which was switched on and off by a shutter. After passing a monochromatic filter, the intensity of green light at 570 nm was about 1 mW/cm2. When strong light was needed, a yellow filter with a wide wavelength range (500 nm) was adopted. Meanwhile, the light intensity was changed by neutral-density filters and calibrated by a commercial radiophotometer. Moreover, the frequency of the light stimulus was varied by means of a controllable light chopper. The temperature was adjusted by means of a home-made regulator and ethylene glycol antifreeze should be used at low temperatures. Finally, the pH was measured with a digital pH meter and adjusted by 0.1 M HCl and KOH.
Fig. 1. The characteristic of differential photocurrent from 10-layer BR-LB films under different light stimulus frequencies (light density is 1 mW/cm2, ls570 nm): (a) the actual features of photocurrent with the rise time, (b) the theoretical features without considering the rise time.
photosensor, which mistakes the intermittent stimuli for a continuous one [12]. In an ideal case, the rise of the rectangular pulse-excited photo e.m.f. is sufficiently fast that the measurement system is a real short circuit. Therefore, the rise time of the photoresponse is negligible. As a result, the fusion of the photoresponse might exhibit only a contraction of spikes rather than a further diminishing to an indistinguishable level at sufficiently high stimulus frequency (Fig. 1(b)). When the rise time of the photoresponse stroke is negligible, the single spike can be fitted by the differential equation described extensively by Hong [13], which is simply expressed as: i(t)sI 0[a exp(yk1 t)qb exp(yk 2 t)]
3. Results and discussion
where a and b are the membrane-related parameters, k1 and k2 are the decay time constants, and I0 is the maximal current initiated by the photo e.m.f. without decay. The multi-exponential decay of photocurrent has been well established theoretically and experimentally [14] and has been observed in our experiment. However, in fact, the rise time of the transient response is by no means negligible. It is determined by many factors, such as the instrumental time constant, the onset time scale of excitation, the proton release and uptake rate, and the parameters of the membrane itself. When the rise time of the photoresponse spike is considered, the relationship between photoresponse and stimulus frequency is given as
3.1. The differential photoresponse of BR film at different stimulus frequencies
t)0 Sf(t) m I(t)T n U Tf(t)q 8 (y1) f(tyn/h) t)n/h
When the light stimulus frequency is gradually increased, the differential photoresponse exhibits a contraction of spikes together with a decline of its magnitude until an indistinguishable level is reached (Fig. 1(a)). The fusion of the photoelectric signals at high stimulus frequency is similar to that of the important responsivity characteristics of a vertebrate
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V
ns1
where f(t)sI 0[1yexp(yk 0 t)][a exp(yk1 t)qb exp(yk 2 t) h is the stimulus frequency, n is the number of stimulations and k0 is the rise time constant. In our experiment, we take k1 and k2 as time constants of the fast and slow decay photores-
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ponse components, respectively, and the ratio a/b reflects the proportions of the two components. Therefore, the photoresponse as a function of stimulus frequency is given theoretically as the line (b) in Fig. 2 (for simplicity a/b is taken as one). It is apparent that when the stimulus rate is faster than the time for the elicited signal to reach its maximum, the following response with opposite polarity diminishes its amplitude. The mutual cancellation of peak values results in a quick decline of the photoresponse until the so-called fusion is reached (Fig. 2). 3.2. The effects of temperature, pH and light intensity A change in temperature and pH influences the lifetime of intermediates (e.g., M, N, O) and thus changes their distribution under photostationary conditions, thereby affecting the intermediate-related photoresponse. Fig. 3 shows several typical photocurrent features at different temperatures. When the electrolyte temperature decreases to as low as y108C, the photoresponse of a BR-LB film is obviously distorted Only a positive stroke is present and exhibits a prolongation not only in the decay time (from 200 to 800 ms) but also in the rise time (from 50 to 100 ms). Under higher resolution,
the negative component that seemed to have disappeared has in fact a low magnitude and an extremely long decay time (5–10 s). After the light is switched off, a relatively longer time is needed (10 s) for the bleached BR to recover. Otherwise, the succeeding light stimulation will result in a drastic drop in the photoresponse magnitude, let alone under highfrequency light stimulation. It is well established that the onset of light is incorporated with the formation of M412, while the steady-state photoresponse corresponds to the light-maintained bleaching of BR. Concurrently, the cancellation of light is related to the recovery of the bleached BR. As the current is directly proportional to the product of charge number and charge-transfer speed, and the light-induced charge translocation directly corresponds to the formation and decay of M412, the slower the rate at which M412 returns to its original state (BR570), the lower the amplitude of the negative photocurrent, the longer the rise time and the lower the photosensitivity to the next excitation. When the experimental rise time of the light shutter is 10 ms, the positive stroke is relatively less influenced than the negative one at lower temperature, and the deceleration of M412 formation (from microseconds to milliseconds) is essentially negligible compared with the retardation of its decay (from milliseconds to seconds). As the temperature ()08C) increases (Fig. 4), two symmetrically distributed spikes exhibit a narrow peak and a typically uni-exponential decay is presented at T)308C. In the meanwhile, the rise of the photoelectric signal is also accelerated (from 100 to 30 ms). On the other hand, the pH in the electrolyte is also an important factor that affects the shape of the response and the
Fig. 2. Photocurrent of BR-LB films as a function of stimulus frequency. The measurement conditions are the same as Fig. 1: (a) experimental result (q); (b) theoretical line with k0s0.1 sy1, k1s0.21 sy1, k2s0.41 sy1, I0s18 nA and without taking k0 into account.
Fig. 3. The behaviour of differential photocurrent of BR-LB films as a function of temperature: (a) at temperatures as low as y108C, the negative stroke almost disappears with a negligible amplitude of photocurrent and a relatively long time scale (10 s); (b) normal presence of photocurrent at room temperature (208C), showing typical biexponential decay time constants k1s0.2 sy1, k2s0.4 sy1; (c) the sharpening photocurrent at higher temperature ()308C) exhibits a uniexponential decay time constant and an acceleration in both rise and decay of the signal, k0s0.05 sy1, k1s0.1 sy1.
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Fig. 4. The temperature dependence of differential photocurrent of BR-LB films: j, positive stroke; q, negative stroke; *, the ratio of positive and negative strokes, which reflects the degree of distortion of the two spikes.
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degree of distortion. High pH has been considered as the simplest way to get the long-lived M412 [15,16]. As discussed above, the slowing down of M412 decay will lead to a distortion of the differential photocurrent, which is presented as the ratio of the two spikes in Fig. 5. When the pH value is increased as high as 9.5, the disparity in the amplitude and the time scales of the two spikes will reach a striking level, as shown in Fig. 5. The effect of a pH decrease is somewhat similar to that resulting from increased temperature. The withdrawal of the slow component at decreased pH will be significantly accelerated at pH-8 until a pure fast component is left at pH-7.5. The distortion of the photoresponse is in no way suitable for the study of intermittent light stimulation as well as for discrimination of adjacent stimuli from a moving object. Such distortion is caused not only by the retardation of M412 decay but also by light excitation of high intensity. It has been proposed by several research groups [17–19] that there are multiple cycles of BR at high pH and high intensity and a long-lived intermediate (N) is involved in such a case. The complicated phenomena in the photocycle of BR will finally lead to an abnormal behaviour of the photoresponse. With the light intensity rising, both positive and negative strokes will increase linearly but with a different ratio; a faster increase is often observed in the positive stroke as shown in Fig. 6. When the light intensity becomes as high as 50 mW/cm2, the amplitude of the positive spike will be doubled compared with that of the negative one, while they have the same values at low light intensity (1 mW/cm2). The disparity of the two spikes at extremely high intensity will result in a distortion in the photocurrent as well as a
Fig. 5. The effect of pH on the differential photocurrent of BR-LB films: j, positive stroke;q, negative stroke; *, the ratio of positive and negative strokes, which reflects the degree of distortion of the two spikes.
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drop in magnitude of the photoresponse from the next stimulation. 3.3. The effects of the membrane and measurement system In photoelectric measurement two types of elements will sometimes make an unwanted contribution to the photoelectric signals. One is the RC charge and discharge characteristics of the membrane system. In a simple equivalent circuit, the membrane can be represented by a capacitor Cm and a resistor Rm. The given relaxation time constant of RC in an aqueous suspension or in gels has been determined to be less than 1 ms in an aqueous medium at a KCl concentration greater than 1 mol/l [20]. However, in the system of PMs absorbed to an air–water interface and transferred onto the surface of conductive glass by using the LB technique, the rims of the PMs are so well sealed that it is difficult for electric signals to discharge through ions surrounding the PMs. In this case, RC)0.1 s [21] and hence the decay of signals may be governed by the relaxation of the membrane. Additionally, the conductive membrane system will slow down not only the relaxation of photoelectric signals but also their rise. The typical rise time of the BR-LB film is about 50–100 ms. Deposited thin films, however, are isotopic and overcome those shortcomings arising from the membrane systems. Therefore, the photoelectric signals from these systems seem to be quite susceptible to frequency limitations of the measuring system and the instrument. Typically, the photoresponse from a deposited thin film reaches a maximum within 20 ms and then returns to zero with a time constant of 50 ms. It should be noted that the deposition of such a solid thin film does not require any special orientation technique. Furthermore, the randomly arrayed PM fragments also manifest a high and rapid differential photoresponse [1,11]. Generally, in order to observe any photoelectric signal from a thin film of BR, it seems to be necessary to orientate the molecules. However, recent reports [22,23] show that the differential photocurrent in fact originates from the change of proton concentration at the electrode/electrolyte interface as a result
Fig. 6. Photocurrent response of BR-LB films for different light intensities. The curves show decreasing amplitude in response to step illumination of the following intensity order: 20, 10, 5, 1 mW/cm2.
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of the proton pumping of BR. This leads to the formation of a transient proton capacitor between the working and counter electrodes. The charging and discharging processes of the proton capacitor yield the differential photoelectric response of BR. Our results also confirm this opinion. As we know, the unidirectionally pumped protons migrate along the PM surface so quickly (within 2 ms) that our measured current (20–200 ms) cannot be the unidirectional proton current. Instead, it is the surface-diffused protons charging/discharging the electrode capacitance surface that induces our differential current. That is why a randomly orientated BR thin film was able to produce a sufficiently large photocurrent response. Under the given rise-time limitation of the light shutter and the given resolution (S/N)2), the maximum fusion frequency can be as high as 50 Hz, contrasted with 10 Hz in a BR-LB film under the given resolution. The second unwanted disturbance is related to the combination of the impedance of the experimental cell and of the bandwidth of the electronic device. The interplay between the sample impedance and the impedance of the detection electronics has been thoroughly investigated [24,25]. In our experiment, current amplifiers with different bandwidths have presented a difference in the time scales of both rise and decay of the signal. With the commercial one (Keithley 427) employed, the rise and decay time scales ranged from 200 to 800 ms, respectively, compared with those of the home-made one (100–400 ms).
4. Conclusions The swift photoresponse under step illumination is significant not only for correct signal recordings but also in the process of discriminating two adjacent stimuli, which is the basis of fabricating a high-speed BR-based photosensor. Under high-frequency stimulation, the maximum discrimination capability of a BR photocell can be defined as the fusion of signals under a given resolution. The fusion is determined primarily by the degree of distortion and the height and half width of the photoresponse spikes. Extreme conditions such as low temperature (-108C), high pH ()9.0) and high light intensity (50 mW/cm2) will result in a striking distortion in two spikes, and thus is no good in the maintenance of the signal amplitude from the next excitation, since a longer time is needed for the long-lived bleached BR to recover. Nevertheless, at temperatures higher than 308C, a higher light intensity (5 mW/cm2-I-50 mW/cm2) or lower pH (-8.0), the acceleration in the build-up of photocurrent attributes directly to the enhancement of the distinguishing capability of the BR photocell. Furthermore, a deposited thin film, other than a BR-LB film, exhibits less disturbance from the RC relaxation characteristic of a membrane and thus is more useful in the construction of highspeed BR-based photosensors.
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5. Abbreviations BR PM LB ITO
bacteriorhodopsin purple membrane Langmuir–Blodgett indium–tin-oxide
Acknowledgements The authors wish to acknowledge the support from the National Science Foundation of China and Grant for Key Program from the Chinese Academy of Sciences (Grant nos. Kj951-A1-501-05 and Kj952-S1-03). References [1] K. Koyama, T. Miyasaka, R. Needleman, J.K. Lanyi, Photoelectrochemical verification of protein-releasing groups in bacteriorhodopsin, Photochem. Photobiol. 68 (1998) 400–406. [2] T. Miyasaka, K. Koyama, Photoelectrochemical behavior of purple membrane Langmuir–Blodgett films at the electrode–electrolyte interface, Chem. Lett., (1991) 1645–1648. [3] R.R. Birge, Protein-based three-dimensional memory, American Scientist 82 (1994) 348–355. [4] R. Thoma, N. Hampp, C. Brauchle, D. Oesterhelt, Bacteriorhodopsin films as spatial light modulators for nonlinear-optical filtering, Opt. Lett. 16 (1991) 651–653. [5] Z. Chen, R. Birge, Protein-based artifical retinas, Trends Biotechnol. 11 (1993) 292–300. [6] T. Miyasaka, K. Koyama, Image sensing and processing by bacteriorhodopsin-based artificial photoreceptor, Appl. Opt. 32 (1993) 6371–6379. [7] K. Fukuzawa, Motion-sensitive position sensor using bacteriorhodopsin, Appl. Opt. 33 (1994) 7489–7495. [8] D. Oesterhelt, W. Stoeckenius, Isolation of the cell membrane of Halobacterium halobium and its fraction in red and purple membrane, Methods Enzymol. 31 (1974) 667–670. [9] T. Miyasaka, K. Koyama, Rectified photocurrents from purple membrane Langmuir–Blodgett films at the electrode–electrolyte interface, Thin Solid Films 210/211 (1992) 146–149. [10] N. Yamaguchi, Y. Jinbo, M. Arai, K. Koyama, Visualization of morphology of purple membrane surfaces by monoclonal antibody techniques, FEBS Lett. 324 (1993) 287–292. [11] K. Koyama, N. Yamaguchi, T. Miyasaka, Antibody-mediated bacteriorhodopsin orientation for molecular device architectures, Science 265 (1994) 762–765. [12] H. Darson, The Physiology of the Eye, Churchill and Livingstone, London, 1972, pp. 221–235. [13] F.T. Hong, Charge transfer across pigmented bilayer lipid membrane and its interfaces, Photochem. Photobiol. 24 (1976) 155–189. [14] S.Y. Liu, T.G. Ebrey, Photocurrent measurements of the purple membrane oriented in a polyacrylamide gel, Biophys. J. 54 (1988) 321–329. [15] P.C. Mowery, R.H. Lozier, Q. Chae, Y.W. Tseng, M. Taylor, W. Stoeckenius, Effects of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin, Biochemistry 18 (1979) 4100– 4107. [16] Y. Kimura, A. Ikegami, W. Stoeckenius, Salt- and pH-dependent changes of the purple membrane absorption spectrum, Photochem. Photobiol. 40 (1984) 641–646. [17] H.C. Bitting, D.J. Jang, M.A. El-Sayed, On the multiple cycles of bacteriorhodopsin at high pH, Photochem. Photobiol. 51 (1990) 593– 598.
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G. Wang et al. / J. Photochem. Photobiol. B: Biol. 52 (1999) 86–91 [18] T. Kouyama, A.N. Kouyama, A. Ikegami, M.A. Mathew, W. Stoeckenius, Bacteriorhodopsin photoreaction: identification of a long-lived intermediate N(P,R350) at high pH and its M-like photoproduct, Biochemistry 27 (1988) 5855–5863. [19] Z. Dancshazy, R. Govindjee, T.G. Ebrey, Independent photocycles of the spectrally distinct forms of bacteriorhodopsin, Proc. Natl. Acad. Sci. USA 85 (1988) 6358–6361. [20] S.Y. Liu, R. Govindjee, T.G. Ebrey, Light-induced current from oriented purple membrane 11. Proton and cation contributions to the photocurrent, Biophys. J. 57 (1990) 951–963. [21] H.W. Trissl, Charge displacements in purple membranes adsorbed to a heptane/water interface, Biochim. Biophys. Acta 723 (1983) 327–331.
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[22] J.P. Wang, S.K. Yoo, L. Song, M.A. El-Sayed, Molecular mechanism of the differential photoelectric response of bacteriorhodopsin, J. Phys. Chem. B 101 (1997) 3420–3423. [23] J.P. Wang, L. Song, S.K. Yoo, M.A. El-Sayed, A comparison of the photoelectric current response resulting from the proton pumping process of bacteriorhodopsin under pulsed and CW laser excitations, J. Phys. Chem. B 101 (1997) 10599–10604. [24] A. Fahr, P. Lauger, E. Bamberg, Photocurrent kinetics of purple membrane sheets bound to the planar bilayer membranes, J. Membr. Biol. 60 (1981) 51–62. [25] L. Keszthelyi, Primary charge motions and light-energy transduction in bacteriorhodopsin, Biophys. Chem. 29 (1988) 127–136.
StyleTag -- Journal: JPB (J. Photochem. Photobiol. B: Biol.)
Article: 7871
Photoresponse discrimination of bacteriorhodopsin films to light stimuli of different frequencies Guangyu Wang b
a,1
, Tao Lu b,2, Kun-Sheng Hu a,*, Long Jiang b
a Institute of Biophysics, Academia Sinica, Beijing 100101, PR China Institute of Photographic Chemistry, Academia Sinica, Beijing 100101, PR China
Received 5 July 1999; accepted 1 September 1999
Abstract The capability of a bacteriorhodopsin-based artificial photosensor to discriminate the adjacent light stimuli from a moving object relies on the intrinsic properties of the differential photoresponse of bacteriorhodopsin films, such as peak value, peak width and degree of distortion of the biphasic spikes, which are related not only to the photoelectric detection system, but also to the pH, temperature, light intensity and the method for depositing films. At higher temperature ()3008C), lower pH (-8.0) or in a deposited thin film rather than a Langmuir–Blodgett film, the relaxation of photoresponse will be accelerated and the sharp photocurrent spike will decrease the pre-excitatory inhibition of the next stimulus and enhance the fusion frequency. The fusion of photoresponse at high stimulus frequencies depends primarily on the response rise time rather than its decay. Under proper conditions, the maximum stimulus frequency corresponding to distinguishable signals in a deposited thin film can be as high as 30–50 Hz, contrasted with 10 Hz in a Langmuir–Blodgett film. q1999 Elsevier Science S.A. All rights reserved. Keywords: Bacteriorhodopsin; Differential photoresponse; Discrimination; Fusion frequency
1. Introduction Bacteriorhodpsin (BR), a retinal-containing protein, possesses the ability to convert light energy to electrochemical energy by pumping protons from the cytoplasmic side to the extracellular side across the membrane under illumination. Under short-circuit measurement conditions, its photoelectric signals elicited from a Langmuir–Blodgett (LB) film and a deposited thin film exhibit a differential photoresponse [1], a fundamental responsivity characteristic of vertebrate photoreceptor cells, by which vertebrates such as frogs can judge whether an object is moving or not. Unlike other biomaterials, the surprising stability, reversibility as well as the unique photochemical and photophysical properties of BR make it the most promising candidate to engineer various technical systems [2–7]. Apart from applications in optical storage and information processing that use * Corresponding author. 1 Present address: MPI f. Biophys. Chem., Am Faßberg 11, D-37077 ¨ Gottingen, Germany. Tel.: q49-551-201 11 59; Fax: q49-551-201 11 68; e-mail: [email protected] 2 Present address: Department of Biology Science, Columbia University, New York, NY 10027, USA.
the photochromic properties of BR[2–4], several motionsensitive artificial retinas based on the differential photoresponse have recently been developed [5–7] and are essentially useful in many aspects, especially in robot vision. Generally, the capability of a motion-sensitive photosensor to discriminate two adjacent light stimuli is determined by the peak value and peak width, especially the rise time of the differential photoresponse. Slow charging and discharging processes will lead to flattening of the response. The serious pre-excitatory inhibition of the next stimulus will finally result in a quick fusion of the response when an increase in the frequency of presentation reaches the so-called fusion frequency. The present study seeks to characterize the rise and decay time scale of a BR film under different measurement conditions. It is demonstrated that pH, temperature and light intensity are of significant influence on both the peak shape and the symmetry of biphasic spikes. Meanwhile, a deposited thin film with less membrane capacitance and resistance presents a sharp photocurrent spike compared with a BR-LB film. These results provide a further understanding of the properties of the differential photoresponse and are helpful in the design of photoelectronic devices.
1011-1344/99/$ - see front matter q1999 Elsevier Science S.A. All rights reserved. PII S 1 0 1 1 - 1 3 4 4 ( 9 9 ) 0 0 1 0 6 - 2
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Article: 7871
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2. Materials and methods The purple membrane (PM) was isolated from R1M1 strains of Halobacterium salinarium according to the conventional method [8]. Two kinds of BR films were employed. The preparation of BR-LB films was described previously [9]. The PM spreading solution was prepared by adding 1 ml of PM water stock solution (8.24 mg/ml) to 4 ml of hexane. After sonication three times (15 s each), the mixture was spread on the surface of a 3 mmol/l CaCl2 solution. Films were compressed to an annealing target pressure of 40 dyne/cm2 and then allowed to deposit on the surface of a glass-coated indium–tin-oxide (ITO) electrode to air dry. All BR-LB films consisted of 10 layers of z-type depositions. Concurrently, the deposited thin films were coated on ITO glass by drying diluted PM suspension under ambient atmosphere or under a nitrogen stream to give homogeneous films, the absorbance value of which ranged from 0.2 to 0.25 at 570 nm. These randomly orientated films may be considered as structurally equivalent to LB multilayer films formed on the same kind of electrode [10], which is known to elicit a sufficiently large photoresponse in the method used here [1,11]. An electrochemical liquid junction photocell was built up to measure the photoresponse of the BR film. The cell comprised a BR-LM film-modified working electrode, aqueous electrolyte (0.1 mol/l KCl) and a Pt counter electrode. When irradiated, the photoreceptor yielded a photocurrent, which was recorded with a current amplifier and an oscilloscope (PM 3375 100 MHz). Two current amplifiers were employed: one was a Keithley 427 and the other was home-made. A 300 W halogen lamp was utilized as a light source, which was switched on and off by a shutter. After passing a monochromatic filter, the intensity of green light at 570 nm was about 1 mW/cm2. When strong light was needed, a yellow filter with a wide wavelength range (500 nm) was adopted. Meanwhile, the light intensity was changed by neutral-density filters and calibrated by a commercial radiophotometer. Moreover, the frequency of the light stimulus was varied by means of a controllable light chopper. The temperature was adjusted by means of a home-made regulator and ethylene glycol antifreeze should be used at low temperatures. Finally, the pH was measured with a digital pH meter and adjusted by 0.1 M HCl and KOH.
Fig. 1. The characteristic of differential photocurrent from 10-layer BR-LB films under different light stimulus frequencies (light density is 1 mW/cm2, ls570 nm): (a) the actual features of photocurrent with the rise time, (b) the theoretical features without considering the rise time.
photosensor, which mistakes the intermittent stimuli for a continuous one [12]. In an ideal case, the rise of the rectangular pulse-excited photo e.m.f. is sufficiently fast that the measurement system is a real short circuit. Therefore, the rise time of the photoresponse is negligible. As a result, the fusion of the photoresponse might exhibit only a contraction of spikes rather than a further diminishing to an indistinguishable level at sufficiently high stimulus frequency (Fig. 1(b)). When the rise time of the photoresponse stroke is negligible, the single spike can be fitted by the differential equation described extensively by Hong [13], which is simply expressed as: i(t)sI 0[a exp(yk1 t)qb exp(yk 2 t)]
3. Results and discussion
where a and b are the membrane-related parameters, k1 and k2 are the decay time constants, and I0 is the maximal current initiated by the photo e.m.f. without decay. The multi-exponential decay of photocurrent has been well established theoretically and experimentally [14] and has been observed in our experiment. However, in fact, the rise time of the transient response is by no means negligible. It is determined by many factors, such as the instrumental time constant, the onset time scale of excitation, the proton release and uptake rate, and the parameters of the membrane itself. When the rise time of the photoresponse spike is considered, the relationship between photoresponse and stimulus frequency is given as
3.1. The differential photoresponse of BR film at different stimulus frequencies
t)0 Sf(t) m I(t)T n U Tf(t)q 8 (y1) f(tyn/h) t)n/h
When the light stimulus frequency is gradually increased, the differential photoresponse exhibits a contraction of spikes together with a decline of its magnitude until an indistinguishable level is reached (Fig. 1(a)). The fusion of the photoelectric signals at high stimulus frequency is similar to that of the important responsivity characteristics of a vertebrate
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V
ns1
where f(t)sI 0[1yexp(yk 0 t)][a exp(yk1 t)qb exp(yk 2 t) h is the stimulus frequency, n is the number of stimulations and k0 is the rise time constant. In our experiment, we take k1 and k2 as time constants of the fast and slow decay photores-
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ponse components, respectively, and the ratio a/b reflects the proportions of the two components. Therefore, the photoresponse as a function of stimulus frequency is given theoretically as the line (b) in Fig. 2 (for simplicity a/b is taken as one). It is apparent that when the stimulus rate is faster than the time for the elicited signal to reach its maximum, the following response with opposite polarity diminishes its amplitude. The mutual cancellation of peak values results in a quick decline of the photoresponse until the so-called fusion is reached (Fig. 2). 3.2. The effects of temperature, pH and light intensity A change in temperature and pH influences the lifetime of intermediates (e.g., M, N, O) and thus changes their distribution under photostationary conditions, thereby affecting the intermediate-related photoresponse. Fig. 3 shows several typical photocurrent features at different temperatures. When the electrolyte temperature decreases to as low as y108C, the photoresponse of a BR-LB film is obviously distorted Only a positive stroke is present and exhibits a prolongation not only in the decay time (from 200 to 800 ms) but also in the rise time (from 50 to 100 ms). Under higher resolution,
the negative component that seemed to have disappeared has in fact a low magnitude and an extremely long decay time (5–10 s). After the light is switched off, a relatively longer time is needed (10 s) for the bleached BR to recover. Otherwise, the succeeding light stimulation will result in a drastic drop in the photoresponse magnitude, let alone under highfrequency light stimulation. It is well established that the onset of light is incorporated with the formation of M412, while the steady-state photoresponse corresponds to the light-maintained bleaching of BR. Concurrently, the cancellation of light is related to the recovery of the bleached BR. As the current is directly proportional to the product of charge number and charge-transfer speed, and the light-induced charge translocation directly corresponds to the formation and decay of M412, the slower the rate at which M412 returns to its original state (BR570), the lower the amplitude of the negative photocurrent, the longer the rise time and the lower the photosensitivity to the next excitation. When the experimental rise time of the light shutter is 10 ms, the positive stroke is relatively less influenced than the negative one at lower temperature, and the deceleration of M412 formation (from microseconds to milliseconds) is essentially negligible compared with the retardation of its decay (from milliseconds to seconds). As the temperature ()08C) increases (Fig. 4), two symmetrically distributed spikes exhibit a narrow peak and a typically uni-exponential decay is presented at T)308C. In the meanwhile, the rise of the photoelectric signal is also accelerated (from 100 to 30 ms). On the other hand, the pH in the electrolyte is also an important factor that affects the shape of the response and the
Fig. 2. Photocurrent of BR-LB films as a function of stimulus frequency. The measurement conditions are the same as Fig. 1: (a) experimental result (q); (b) theoretical line with k0s0.1 sy1, k1s0.21 sy1, k2s0.41 sy1, I0s18 nA and without taking k0 into account.
Fig. 3. The behaviour of differential photocurrent of BR-LB films as a function of temperature: (a) at temperatures as low as y108C, the negative stroke almost disappears with a negligible amplitude of photocurrent and a relatively long time scale (10 s); (b) normal presence of photocurrent at room temperature (208C), showing typical biexponential decay time constants k1s0.2 sy1, k2s0.4 sy1; (c) the sharpening photocurrent at higher temperature ()308C) exhibits a uniexponential decay time constant and an acceleration in both rise and decay of the signal, k0s0.05 sy1, k1s0.1 sy1.
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Fig. 4. The temperature dependence of differential photocurrent of BR-LB films: j, positive stroke; q, negative stroke; *, the ratio of positive and negative strokes, which reflects the degree of distortion of the two spikes.
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degree of distortion. High pH has been considered as the simplest way to get the long-lived M412 [15,16]. As discussed above, the slowing down of M412 decay will lead to a distortion of the differential photocurrent, which is presented as the ratio of the two spikes in Fig. 5. When the pH value is increased as high as 9.5, the disparity in the amplitude and the time scales of the two spikes will reach a striking level, as shown in Fig. 5. The effect of a pH decrease is somewhat similar to that resulting from increased temperature. The withdrawal of the slow component at decreased pH will be significantly accelerated at pH-8 until a pure fast component is left at pH-7.5. The distortion of the photoresponse is in no way suitable for the study of intermittent light stimulation as well as for discrimination of adjacent stimuli from a moving object. Such distortion is caused not only by the retardation of M412 decay but also by light excitation of high intensity. It has been proposed by several research groups [17–19] that there are multiple cycles of BR at high pH and high intensity and a long-lived intermediate (N) is involved in such a case. The complicated phenomena in the photocycle of BR will finally lead to an abnormal behaviour of the photoresponse. With the light intensity rising, both positive and negative strokes will increase linearly but with a different ratio; a faster increase is often observed in the positive stroke as shown in Fig. 6. When the light intensity becomes as high as 50 mW/cm2, the amplitude of the positive spike will be doubled compared with that of the negative one, while they have the same values at low light intensity (1 mW/cm2). The disparity of the two spikes at extremely high intensity will result in a distortion in the photocurrent as well as a
Fig. 5. The effect of pH on the differential photocurrent of BR-LB films: j, positive stroke;q, negative stroke; *, the ratio of positive and negative strokes, which reflects the degree of distortion of the two spikes.
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drop in magnitude of the photoresponse from the next stimulation. 3.3. The effects of the membrane and measurement system In photoelectric measurement two types of elements will sometimes make an unwanted contribution to the photoelectric signals. One is the RC charge and discharge characteristics of the membrane system. In a simple equivalent circuit, the membrane can be represented by a capacitor Cm and a resistor Rm. The given relaxation time constant of RC in an aqueous suspension or in gels has been determined to be less than 1 ms in an aqueous medium at a KCl concentration greater than 1 mol/l [20]. However, in the system of PMs absorbed to an air–water interface and transferred onto the surface of conductive glass by using the LB technique, the rims of the PMs are so well sealed that it is difficult for electric signals to discharge through ions surrounding the PMs. In this case, RC)0.1 s [21] and hence the decay of signals may be governed by the relaxation of the membrane. Additionally, the conductive membrane system will slow down not only the relaxation of photoelectric signals but also their rise. The typical rise time of the BR-LB film is about 50–100 ms. Deposited thin films, however, are isotopic and overcome those shortcomings arising from the membrane systems. Therefore, the photoelectric signals from these systems seem to be quite susceptible to frequency limitations of the measuring system and the instrument. Typically, the photoresponse from a deposited thin film reaches a maximum within 20 ms and then returns to zero with a time constant of 50 ms. It should be noted that the deposition of such a solid thin film does not require any special orientation technique. Furthermore, the randomly arrayed PM fragments also manifest a high and rapid differential photoresponse [1,11]. Generally, in order to observe any photoelectric signal from a thin film of BR, it seems to be necessary to orientate the molecules. However, recent reports [22,23] show that the differential photocurrent in fact originates from the change of proton concentration at the electrode/electrolyte interface as a result
Fig. 6. Photocurrent response of BR-LB films for different light intensities. The curves show decreasing amplitude in response to step illumination of the following intensity order: 20, 10, 5, 1 mW/cm2.
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of the proton pumping of BR. This leads to the formation of a transient proton capacitor between the working and counter electrodes. The charging and discharging processes of the proton capacitor yield the differential photoelectric response of BR. Our results also confirm this opinion. As we know, the unidirectionally pumped protons migrate along the PM surface so quickly (within 2 ms) that our measured current (20–200 ms) cannot be the unidirectional proton current. Instead, it is the surface-diffused protons charging/discharging the electrode capacitance surface that induces our differential current. That is why a randomly orientated BR thin film was able to produce a sufficiently large photocurrent response. Under the given rise-time limitation of the light shutter and the given resolution (S/N)2), the maximum fusion frequency can be as high as 50 Hz, contrasted with 10 Hz in a BR-LB film under the given resolution. The second unwanted disturbance is related to the combination of the impedance of the experimental cell and of the bandwidth of the electronic device. The interplay between the sample impedance and the impedance of the detection electronics has been thoroughly investigated [24,25]. In our experiment, current amplifiers with different bandwidths have presented a difference in the time scales of both rise and decay of the signal. With the commercial one (Keithley 427) employed, the rise and decay time scales ranged from 200 to 800 ms, respectively, compared with those of the home-made one (100–400 ms).
4. Conclusions The swift photoresponse under step illumination is significant not only for correct signal recordings but also in the process of discriminating two adjacent stimuli, which is the basis of fabricating a high-speed BR-based photosensor. Under high-frequency stimulation, the maximum discrimination capability of a BR photocell can be defined as the fusion of signals under a given resolution. The fusion is determined primarily by the degree of distortion and the height and half width of the photoresponse spikes. Extreme conditions such as low temperature (-108C), high pH ()9.0) and high light intensity (50 mW/cm2) will result in a striking distortion in two spikes, and thus is no good in the maintenance of the signal amplitude from the next excitation, since a longer time is needed for the long-lived bleached BR to recover. Nevertheless, at temperatures higher than 308C, a higher light intensity (5 mW/cm2-I-50 mW/cm2) or lower pH (-8.0), the acceleration in the build-up of photocurrent attributes directly to the enhancement of the distinguishing capability of the BR photocell. Furthermore, a deposited thin film, other than a BR-LB film, exhibits less disturbance from the RC relaxation characteristic of a membrane and thus is more useful in the construction of highspeed BR-based photosensors.
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5. Abbreviations BR PM LB ITO
bacteriorhodopsin purple membrane Langmuir–Blodgett indium–tin-oxide
Acknowledgements The authors wish to acknowledge the support from the National Science Foundation of China and Grant for Key Program from the Chinese Academy of Sciences (Grant nos. Kj951-A1-501-05 and Kj952-S1-03). References [1] K. Koyama, T. Miyasaka, R. Needleman, J.K. Lanyi, Photoelectrochemical verification of protein-releasing groups in bacteriorhodopsin, Photochem. Photobiol. 68 (1998) 400–406. [2] T. Miyasaka, K. Koyama, Photoelectrochemical behavior of purple membrane Langmuir–Blodgett films at the electrode–electrolyte interface, Chem. Lett., (1991) 1645–1648. [3] R.R. Birge, Protein-based three-dimensional memory, American Scientist 82 (1994) 348–355. [4] R. Thoma, N. Hampp, C. Brauchle, D. Oesterhelt, Bacteriorhodopsin films as spatial light modulators for nonlinear-optical filtering, Opt. Lett. 16 (1991) 651–653. [5] Z. Chen, R. Birge, Protein-based artifical retinas, Trends Biotechnol. 11 (1993) 292–300. [6] T. Miyasaka, K. Koyama, Image sensing and processing by bacteriorhodopsin-based artificial photoreceptor, Appl. Opt. 32 (1993) 6371–6379. [7] K. Fukuzawa, Motion-sensitive position sensor using bacteriorhodopsin, Appl. Opt. 33 (1994) 7489–7495. [8] D. Oesterhelt, W. Stoeckenius, Isolation of the cell membrane of Halobacterium halobium and its fraction in red and purple membrane, Methods Enzymol. 31 (1974) 667–670. [9] T. Miyasaka, K. Koyama, Rectified photocurrents from purple membrane Langmuir–Blodgett films at the electrode–electrolyte interface, Thin Solid Films 210/211 (1992) 146–149. [10] N. Yamaguchi, Y. Jinbo, M. Arai, K. Koyama, Visualization of morphology of purple membrane surfaces by monoclonal antibody techniques, FEBS Lett. 324 (1993) 287–292. [11] K. Koyama, N. Yamaguchi, T. Miyasaka, Antibody-mediated bacteriorhodopsin orientation for molecular device architectures, Science 265 (1994) 762–765. [12] H. Darson, The Physiology of the Eye, Churchill and Livingstone, London, 1972, pp. 221–235. [13] F.T. Hong, Charge transfer across pigmented bilayer lipid membrane and its interfaces, Photochem. Photobiol. 24 (1976) 155–189. [14] S.Y. Liu, T.G. Ebrey, Photocurrent measurements of the purple membrane oriented in a polyacrylamide gel, Biophys. J. 54 (1988) 321–329. [15] P.C. Mowery, R.H. Lozier, Q. Chae, Y.W. Tseng, M. Taylor, W. Stoeckenius, Effects of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin, Biochemistry 18 (1979) 4100– 4107. [16] Y. Kimura, A. Ikegami, W. Stoeckenius, Salt- and pH-dependent changes of the purple membrane absorption spectrum, Photochem. Photobiol. 40 (1984) 641–646. [17] H.C. Bitting, D.J. Jang, M.A. El-Sayed, On the multiple cycles of bacteriorhodopsin at high pH, Photochem. Photobiol. 51 (1990) 593– 598.
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