Photoelectric properties of a detector based on dried bacteriorhodopsin film

Biosensors and Bioelectronics 21 (2006) 1309–1319

Photoelectric properties of a detector based on dried bacteriorhodopsin film Wei Wei Wang a , George K. Knopf a,∗ , Amarjeet S. Bassi b,∗ a b

Department of Mechanical and Materials Engineering, Faculty of Engineering, The University of Western Ontario, London, Ont., Canada N6A 5B9 Department of Chemical and Biochemical Engineering, Faculty of Engineering, The University of Western Ontario, London, Ont., Canada N6A 5B9 Received 8 March 2005; received in revised form 31 May 2005; accepted 10 June 2005 Available online 21 July 2005

Abstract The photoelectric response of a detector using dried bacteriorhodopsin (bR) film as the light sensing material is mathematically modeled and experimentally verified in this paper. The photocycle and proton transfer kinetics of dried bR film differ dramatically from the more commonly studied aqueous bR material because of the dehydration process. The photoelectric response of the dried film is generated by charge displacement and recombination instead of transferring a proton from the cytoplasmic side to the extracellular side of the cell membrane. In this work, the wild-type bR samples are electrophoretically deposited onto an indium tin oxide (ITO) electrode to construct a simple multiple layered photodetector with high sensitivity to small changes in incident illumination. The light absorption characteristics of the thin bR film are mathematically represented using the kinetics of the bR photocycle and the charge displacement theorem. An electrically equivalent RC circuit is used to describe the intrinsic photoelectric properties of the film and external measurement circuitry to analyze the detector’s response characteristics. Simulated studies and experimental results show that the resistance of the dried bR film is in the order of 1011
. When the input impedance of the measurement circuitry is one order of magnitude smaller than the dried film, the detector exhibits a strong differential response to the original time-varying light signal. An analytical solution of the equivalent circuit also reveals that the resistance and capacitance values exhibited by the dried bR film, in the absence of incident light, are almost twice as large as the values obtained while the material is under direct illumination. Experimental observations and a predictive model both support the notion that dried bR film can be used in simple highly sensitive photodetector designs. © 2005 Elsevier B.V. All rights reserved. Keywords: Bacteriorhodopsin; Photodetector; Photocycle kinetics; Dynamic response; Equivalent RC circuits

1. Introduction Bacteriorhodopsin, a retinal protein found in the cell membranes of Halobacterium salinarum, is one of the simplest known biological energy converters. The absorption of light leads to very rapid (450 fs) retinal isomerization, which triggers the separation of positive and negative charges. Subsequent protein conformational changes drive a proton gradient translated from the cytoplasmic to the extracellular side of the ∗

Corresponding authors. Tel.: +1 5196612130, fax: +1 5196613757. E-mail addresses: [email protected] (G.K. Knopf), [email protected] (A.S. Bassi). 0956-5663/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2005.06.003

membrane (Lanyi, 2000). During this process, the protein undergoes a thermally relaxing photocycle that is characterized by several spectrally distinct intermediates. The electrochemical gradient of protons created across the membrane is utilized for adenosine triphosphate (ATP) synthesis (Hampp, 2000). Intensive research on bacteriorhodopsin has been conducted over the past three decades. The result of these investigative studies is an ongoing understanding of its detailed structure and transport function. Recently, bR has received significant interest as a viable material in designing and fabricating hybrid devices in which the thin bR film functions as a “smart material” to accomplish complex signal transduc-

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tion. The photosensitive properties of bR have been exploited by a number of light sensing and imaging devices. A variety of designs have been demonstrated, such as ultrafast optoelectric signal transducers (Groma et al., 1992), bR-based microarray photodetector (Faruqi, 2001), fundamental element in retinal edge detection (Takei et al., 1991), and monolithically integrated bR-GaAs MOSFET (Bhattacharya et al., 2002). The photoelectric signals generated from reconstituted bR films as observed by a variety of independent research groups have proved that photochemical properties of bR can be maintained in its solid phase because of the unusual stability of purple membrane (PM) sheets (V´ar´o and Keszthelyi, 1983; Kononenko et al., 1987; He et al., 1998; Crittenden et al., 2003). However, the photocycle and proton transfer kinetics of dried bR film dramatically differ from those of the more commonly studied wet bR samples due to the influence of dehydration. In the bulk aqueous phase, the bR molecule acts as a light-driven proton pump. The absorption of a photon by the molecule initiates the isomerization of retinal from alltrans to 13-cis conformation, followed by a proton transport across the cell membrane (K¨uhlbrandt, 2000). The proton transport starts with the release of a proton in the L → M transition and ends with a proton uptake in the following M → N transition during the photocycle (Edman et al., 1999) (Fig. 1a). On the other hand, the photocycle of dried bR film stops at the M state before the Schiff-base is reprotonated. All intermediates coupling conformational change are hindered in the dried form; this prevents switching the accessibility of the Schiff-base from the extracellular to the cytoplasmic side and proton transport does not occur (Ganea et al., 1997). After illumination by a nanosecond laser pulse, the photocycle decays slowly in dried bR films and the protein returns to its ground state through several intermediates with considerably distinctive lifetimes, indicating different paths of the proton back to the Schiff-base (V´ar´o and Lanyi, 1991) (Fig. 1b). The accurate characterization of the photoelectric properties of bR films is essential to designing and developing viable light sensitive devices based on the biomaterial. The desired material properties are, however, strongly influenced by the preparative conditions used to fabricate the film. Due to the large number of variables involved in the process, a mathematical model is more efficient to illustrate the interaction between the variables and the behaviour of bR films. A number of models have been developed to explore the photoelectric properties of bR thin films (Hong, 1999; Der and Keszthelyi, 2001; Huang et al., 2003; Xu et al., 2004; Yao et al., 2003). Most of the models arising from these studies were based on the behaviour of wet bR films, where the kinetics exhibits similar attributes to the wild-type bR in liquid suspension. Furthermore, the bR photocycle kinetics is normally obtained by measuring the transient flash-induced absorption changes in the bR sample and then mathematically analyzing the difference spectra to determine the absorption spectra

Fig. 1. Schematics representing basic photocycles of bR in the bulk aqueous phase where the proton transfer starts with the release of a proton during the L → M transition and end with a proton uptake during the M → N transition (Edman et al., 1999) (a) and in dried film where only K, L and M intermediates are involved and no proton transfers across the protein (b).

of the photocycle intermediates and the kinetics of their rise and decay. In many sensor applications, the light signal that is to be detected by the sensor has a longer temporal duration compared to the lifetime of the bR photocycle. This temporal difference inevitably affects the photocycle kinetics due to the energy coupling during the photo-excitation. Therefore, it is unsuitable to directly correlate photoelectric signals with the photocycle kinetics obtained by the transient spectroscopy approach. Mathematical modeling of the whole bR photodetector allows the complex photoresponse and the underlying mechanisms to be thoroughly investigated and understood. The predictive model is necessary to optimize the performance of any practical device that utilizes this light sensitive biomaterial. Towards achieving this goal, the intrinsic and extrinsic response behaviour of bR-based photodetector are modeled on the time-dependent population distribution of the interme-

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diates in the bR photocycle, the charge displacement theorem, physical model of the orientated bR film, and the equivalent circuit that characterizes the coupling of the dried bR film and external measuring instrument. Experimental evidence, supported by MATLAB and Micro-Cap simulations, is used to assess the capabilities of the proposed model.

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tion of the light. A tunable lamp was used as the light source to excite the photodetector.

3. Theoretical model 3.1. Modeling the bR photocycle

2. Materials and methods The purple membrane (PM) fragments of wild-type bR were extracted from H. salinarum strain S14. The PM suspension was first purified in bi-distilled water by centrifugation for three cycles (50-Ti rotor; 22,400 rpm; 30 min per cycle). After each rinsing cycle, the supernatant was carefully poured out and the PM fragments were re-dissolved in bi-distilled water. The deionized PM fragments were resuspended at 15 mg/ml in bi-distilled water. The sample was then softly sonicated for 5 s to remove aggregation. The pH of the suspension was 6.5. The final conductivity of the suspension was controlled to less than 15 ␮s, which indicates very low contamination by ions. The 50 ␮l PM suspension was injected between two ITO electrodes separated by a plastic spacer with thickness of 1 mm. An electric field of 30 V/cm was applied through the two ITO electrodes. After 5 min, the anode electrode with the deposited PM was removed. Due to its negative charge, the cytoplasmic side of the bR membrane was attached to the ITO conductive surface. The water over the PM fragments was carefully removed with a pipette. The oriented PM film with the ITO electrode was placed in a special chamber to maintain the humidity during drying. One night is required to completely dry the PM film. The optical density of the dried bR thin film was 1.5 at wavelength 570 nm. The dried film was covered by a second ITO electrode and a polyester thin film was used as the spacer. The dried sample was sealed to construct a sandwich-structural photoelectric detector. The diameter of the active area of the detector is approximately 5 mm. The thickness of the dried bR film was measured by a Gaertner Ellipsometer and was 30 ␮m. Measurements of the photoelectric response generated from the bR photodetector were performed at the room temperature with a setup as shown in Fig. 2a. The dynamic response of the photodetector was processed by a readout circuit (Fig. 2b) and an electrometer (Keithley 602). The ITO electrode contacting the cytoplasmic side of the bR membrane was set as the ground. The input of the measuring instrument was connected to the ITO electrode facing the extracellular side of the bR membrane. The conditioned signals were fed directly into a 2G sampling/s 100 MHz digital oscilloscope (Agilent 54825A) via a BNC cable. The photodetector together with the processing circuit was placed in a light sealed metal box, which had a hole on the front side and a BNC connector attached to the back side, to void of the electromagnetic interference of the environment. The inside of the metal box was also painted in black to reduce the reflec-

Under illumination, the bR molecules undergo a complex photocycle, which involves several intermediate states with distinctive lifetimes and absorption maxima. The light absorption properties of bR films are intrinsically related to the population distribution of bR molecules at the different intermediates. As well, the charge motion inside the bR molecule coincides with the intermediates. Studies have revealed that the normal function of bR requires the presence of several water molecules in the proton transfer channel. The kinetics of the photocycle is modified as the humidity of the sample changes (Korenstein and Hess, 1977; V´ar´o, 1981; Cao et al., 1991; Groma et al., 2001). When humidity in the bR film is below 90%, the later intermediates are no longer observable, the primary intermediates are accelerated, and less protons transfer across the protein (Ganea et al., 1997). Hence, the photocycle and proton transfer mechanism in the dried bR film are different from the broadly accepted photoreaction models based on the aqueous bR samples. One observed feature of all photoreaction models developed for dehydrated bR samples is that the dried bR has a truncated photocycle. Fig. 3a illustrates a reversible photocycle model described by Ganea et al. (1997), in which the photocycle terminates at intermediate M and returns back to the ground state B through multi-pathways. The extinction coefficients (rate constants, kij ) of the intermediates were estimated by measuring light absorption spectral at five wavelengths. Transient light pulses with a pulse width in the order of sub-nanosecond are used to initiate the primary intermediate in the photocycle. The derived data show that the backward rate constant of K state, kLK is nearly four times as the forward rate constant, kKL , resulting in both the K state and the long-lived state, M, domain the population distribution for a long time. Furthermore, as the initial charge separation happens in the K state and charge recombination corresponds to the M state, it will be difficult to detect the photoelectric signal generated from the bR film during the same period because of the fewer collected net charges. Therefore, the response time of the bR film to the continuous illumination will be very slow, on the order of a second. This is caused by the long lifetime of the branches of the M state. These phenomena are not, however, observed in related measurements. One reason may be that the measured time constants only reflect the thermal relaxation inside the film. When the duration of light excitation is longer than the photocycle’s lifetime, which means both photochemical and thermal conversions are involved, the thermal isomerization equilibrium is modified and, thereby, the photocycle kinetics are changed simultaneously. As a result, the photoreaction of

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Fig. 2. Experimental setup for bR-based photodetector, (a) laboratory-built measurement unit and (b) signal processing circuit in which the output of the bR photodetector goes directly to the non-inverting input of the precision op-amp LMC 6061. The input impedance of LMC 6061 is about 10 Tera
.

bR in the dried film is accelerated. In reality, the actual transition process is much more complex. Studies on aqueous or wet bR samples have indicated that all the intermediates can be photochemically switched back to the ground state by shining light at a wavelength that corresponds to the absorption of the intermediate state (Birge et al., 1999; Hampp, 2000). Unfortunately no relevant data measured on the dried bR sample are currently available in the published literature. To simplify the actual photoreaction while still retaining the key properties of the photocycle, an irreversible sequential model is applied to demonstrate the photoreaction of the dried bR film under continuous illumination, which is described in Fig. 3b.

The rate equations for time-dependent and intensitydependent concentrations of the different intermediates shown in Fig. 3b can be written as dCB (t) = −κBK (t, I)CB (t) + kM1 B CM1 (t) dt

(1)

dCK (t) = κBK (t, I)CB (t) − kKL CK (t) dt

(2)

dCL (t) = kKL CK (t) − kLM CL (t) dt

(3)

dCM (t) = kLM CL (t) − kMM1 CM (t) dt

(4)

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Thus, the normalized concentration, Ci = Ci /C, for each state at the steady state is given by 

CB =



Cj =

1 1 + bκBK (I)

(9)

κBK (I) , kjl (1 + bκBK (I)) (j = K, L, M and M1 ; l = L, M, M1 and B)

(10)

where b=

Fig. 3. Schematic diagrams of the reversible parallel photocycle in dried bR film excited by untralfast laser pulse (Ganea et al., 1997) (a) and the irreversible sequential photocycle in dried bR film under continuous wave illumination (b).

dCM1 (t) = kMM1 CM (t) − kM1 B CM1 (t) dt

(5)

where κBK represents the rate constant of the photochemical reactions from the ground state B to K state due to the incident light. The parameter kij (i = B, K, L, M and M1 ) represents the rate constant of the thermal relaxation from intermediate i to j. The photochemical rate constant κBK can be written as

1 kKL

+

1 kLM

+

1 kMM1

+1

(11)

The transient response of the bR photocycle to a constant light signal can be evaluated by the time-dependent solution of its rate equations. The rate equations, Eqs. (1)–(5) indicate a non-linear system of first order ordinary differential equations (ODE). If the total concentration of bR molecules, C, is defined as the initial concentration of B state, and there is no molecules in the other intermediates, the initial function values of ODE can be given as [ CB CK CL CM CM1 ] = [ C 0 0 0 0 ]. To find the dynamic solution of ODE is to solve an initial value problem (IVP) using a numerical approach. Since all the derivatives are known at the given initial points, it is possible to start with these initial points and step forward using information provided by the governing differential equations.

κBK (t, I) = (1 − η)σB ΦBK F (t, I)

(6a)

3.2. Modeling photoelectrical response of bR films

I(t) I(t)λi = hv hc

(6b)

Unlike most of the bioelectric signals generated by ionic diffusion, the fast photosignals from dried bR film are referred to as displacement photocurrent (Trissl and Montal, 1977). According to the Ramo–Shockley theorem (Ramo, 1939; Shockley, 1938), the motion of a single charge can induce instantaneous current in neighboring electrode. The current may be detected through the external circuit during the time interval that the charge approaches the electrode due to the instantaneous change of electrostatic flux lines, which end on the electrode. If the bR molecules are properly oriented, then the charges are assumed to move in one direction and are synchronized under the illumination. After absorbing a photon, a proton of charge q is driven to move from one point to another inside the protein. The induced current i received by the connecting electrode is given as

F (t, I) =

where η is the refractive index of the substrate; σ B is the absorption cross-section of B state; ΦBK is the quantum yields of photoreaction B → K; F(t, I) is the photon density flux of the incident light, in which v and c represent the frequency of the incident light and the speed of light in vacuum; and h is the Planck’s constant. As a highly dynamic system, the photocycle of bR molecules approaches its steady state in a short time range (depending on the rate constants involved) after the film is illuminated by a constant light source. The steady-state concentrations of different intermediates determine the intensity-dependent photoresponse of the bR film. Under the continuous illumination the left-hand sides of Eqs. (1)–(5) are zeros dCi (t) = 0, dt

(i = B, K, L, M and M1 )

(7)

The total concentration of bR molecules can be written as the sum of concentrations of all the states
C= Ci , (i = B, K, L, M and M1 ) (8) i

i=

qv εD

(12)

where v is the velocity of the proton’s motion, D is the distance of the electrodes, and ε is the dielectric constant. If the distance between two subsequent intermediates i and i + 1 is represented by di and the lifetime of intermediate i is represented by τ i , the charge qph induced on the electrode

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Fig. 4. (a) Physical model of dried bR thin film containing orientated bR molecules with aligned permanent dipoles, where the molecules are surrounded by lipid, (b) Micro-Cap representation of the equivalent RC circuit of the entire bR-based photodetector, and (c) simplified equivalent circuit of (b).

due to a unit charge transfer from the intermediate i to i + 1, can be written as  τi qdi qph = idt = (13) εD 0 Generally, numerous bR molecules N(t) are excited to participate the photoreaction after absorbing photons. The induced charge on intermediate i is the product of qph and the population on the ith intermediate Ni (t). Thus, the total photogenerated charge, Qph , can be written as
q
qph Ni (t) = Ni (t)di , Qph (t) = εD i

i

(i = B, K, L, M and M1 )

(14) 

where the population of each intermediate, Ni (t) = Ci (t)CV , and V is the volume of bR film. A physical model of the dried film containing oriented bR molecules is derived from the comprehensive analysis of experimental data (Fig. 4a). The excited bR molecules function as a perfect photocurrent source, Iph (t) in parallel with a source resistance, Rs The lipids among the bR molecules in the direction of orientation are represented as a series capacitor, Cl and a series resistor, Rl , due to their high dielectric constant. The resistor Rm and capacitor Cm in parallel with the photocurrent source reflect the effect of the non-illuminated bR molecules as well as the lipids vertical to the orientation direction. Fig. 4b illustrates the equivalent RC circuit, which allows the main characteristics of bR photodetector to be understood. The series resistance, Rc ,

comes from the contact between the film surfaces and the electrodes. The origin of capacitor Cj is the junction capacitance of the external circuit when it is connected to the bR film. The load resistance, Ri , is the input impedance of the measuring instruments. If the connection of bR film to the electrode is considered as low-resistance “ohmic contact”, Rc is negligibly small as compared to the membrane resistance Rm . The junction capacitor can also be ignored for analysis purpose. Therefore, it is reasonable to further simplify the equivalent circuit as the one shown in Fig. 4c, where the photocurrent source, Iph (t) in parallel with source resistance, Rs , is replaced by a photovoltage source, Eph (t) in series with source resistance, Rs . Square wave signal processing provides useful information for analyzing unknown current sources. Analytically, a step change in the input light power causes the output photovoltage to exponentially approach its final value. The intrinsic properties of bR film and the external circuit determine the signal’s amplitude, as well as its response and decay times. A step input signal is defined as  Eph (t) =

E0 0

a
(15)

When t approaches a+ from a− , which means the light is ON, the photocurrent from the source starts to charge both the internal and external circuits. Based on capacitive loading analysis, the transient photovoltage measured on the loading

W.W. Wang et al. / Biosensors and Bioelectronics 21 (2006) 1309–1319

resistor, Ri is given as VC (t) =

Cl E0 (1 − e−t/τ1 ) Cl + C m

If the input photovoltage, Eph (t) is defined as (16)

where the time constant is given by τ 1 = Rs Cl Cm /(Cl + Cm ). Similarly, the resistive loading analysis shows the steadystate response of the output voltage VR (t) =

R E0 R + R l + Rs

(17)

The potential difference between the capacitive loading and the resistive loading tends to charge the output amplitude, which is given by   R Cl E0 (1 − e−t/τ2 ) − VRC (t) = R + R l + Rs Cl + C m (18) where R = Rm Ri /(Rm + Ri ) and the time constant τ 2 = RCm . Therefore, the total output can be written as Von (t) = VC (t) + VRC (t)   Cl E0 (1 − e−t/τ1 ) = Cl + C m   R Cl + E0 (1 − e−t/τ2 ) − R + R l + Rs Cl + C m (19) Eq. (19) can be further simplified as following     Cl RE0 − E0 e−t/τ1 Von (t) = R + R l + Rs Cl + C m   R Cl E0 e−t/τ2 − − R + R l + Rs Cl + C m

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(20)

The first item in Eq. (20) represents a DC signal that is a steady-state response to the constant light. When t approaches b+ from b− , which means the light is OFF, the charges stored on the capacitors begin to discharge through both the internal and external circuits, the light-OFF response can be defined by   Cl E0 e−t/τ1 Voff (t) = Cl + C m   R Cl E0 e−t/τ2 + − (21) R + R l + Rs Cl + C m Convolution is normally used as a method to describe the dynamic response of a system to a time-dependent input signal. Here, the measured photovoltage to a step input function is given as   Cl Eph (t) ⊗ e−t/τ1 V (t) = Cl + C m   R Cl Eph (t) ⊗ e−t/τ2 + − R + R l + Rs Cl + C m (22)

Eph (t) =

(Cl + Cm ) Qph (t) C l Cm

(23)

then the integral form of the convolution can be written as  t 1 V (t) = Qph (u)[e(u−t)/τ1 ] du Cm τ1 0   (Cl + Cm ) Cl R − + R + R l + Rs τ2 Cl Cm Cl + C m  t × Qph (u)[e(u−t)/τ2 ] du (24) 0

4. Results and discussion 4.1. Intrinsic response behaviour of the bR-based photodetector Like any photodetection device, there are two important aspects to the photoresponse of a bR-based detector: the intrinsic response associated with light absorption, and the extrinsic response that characterizes the electrical coupling of the bR film to the external circuit. The former reflects the transit behaviour of the photo-induced charges in the biomaterial, and the latter represents the electrical signal measured by conventional instrumentation. In order to understand the relative contributions of these two aspects to the bR response, it is necessary to evaluate each of them. As discussed in Section 3, the proton transit response of the detector is relevant to the kinetics of the bR photocycle and the physical properties of the dried bR film, such as optical density, active area and thickness. The transit time is attributed to the rise and decay times of the intermediates. The concentration distribution of the intermediates to a step light signal is calculated by using MATLAB. Assuming that the light signal reaches its maximum intensity, 2 mW/mm2 , in milliseconds. The absorption cross-section of B state, σ B = 2.4 × 10−16 cm2 , and the quantum yields of photoreaction B → K, ΦBK = 0.64. The simulation result plotted by logarithm time is presented in Fig. 5a. It shows that the normalized concentrations of the primary intermediates K and L reach their peak values almost at the same time, in 10 ␮s. It is known that the K and L states associate with the charge separation inside the bR molecules. The photoresponse induced by the charge separation is negligible since the concentrations of these two states are very low, in the order of 0.01%. While the concentration of M1 state constantly increases, the other intermediates have relatively low concentrations. The steady-state concentrations of all intermediates are reached simultaneously after about 20 ms, which represents the transit response time of the detector to the continuous illumination. The charge recombination step, M1 state is the dominated state with the normalized concentration of 60% under illumination.

W.W. Wang et al. / Biosensors and Bioelectronics 21 (2006) 1309–1319

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4.2. Extrinsic response behaviour of the bR-based photodetector

Fig. 5. (a) The calculated concentrations of the intermediates of the dried bR film to a step light signal (the rising part) with amplitude of 2 mW/mm2 . Rate constants used in the photocycle simulation: kKL = 3.16 × 105 s−1 , kLM = 2.5 × 105 s−1 , kMM1 = 6.54 × 103 s−1 , kM1 B = 62.5 (Ganea et al., 1997), (b) The calculated photovoltage generated from bR molecules to a square wave light signal with amplitude of 2 mW/mm2 and light-on duration of 10 s.

The photoelectric response of the bR molecules can be calculated based on the time-dependent populations of the intermediates. According to Beer–Lambert law, the total concentration of the dried bR film, C, is equal to A/σ B × d = 4.76 × 1018 cm−3 , where A = 1.5 is the optical density of the film, d = 30 ␮m is the film thickness. Therefore, the total population of bR molecules, N = CV= 2.8 × 1015 , where V = 5.89 × 10−4 cm3 is the volume of the film. The intensity of the step light signal is given as  I(t) =

2 mw/mm2 0

1 s ≤ t ≤ 11 s otherwise

(25)

The subsequent photovoltage generated from the bR molecules is plotted in Fig. 5b. On the second time scale, the photo-induced voltage responses to the step light signal instantaneously. The transient characteristics of the photovoltage, which are determined by the kinetic parameters of the bR photocycle do not show in this time regime.

In general, the measured photoelectric signal of the bRbased detector represents an extrinsic response that depends on the sensing material and the interconnection between the detector and the signal processing circuitry. To explore the impact of the external measuring instruments, an electrometer with the adjustable input impedance from 1 M
to 100 Tera
and a readout circuit are both used to measure the photoelectric response of the detector to step light signals. The experimental results show that the measured photoelectric signal starts to exhibit differential characters when the input impedance of the electrometer is set to a value that is lower than 100 G
. It is also found that the photo-induced signal obtained when light is turned ON has a bigger amplitude and a faster decay time compared to the signal measured when light is turned OFF. Theoretical analysis reveals the effects of the input impedance of the measuring instrument on the photoelectric response of the detector. When light is ON, Eq. (19) gives two exponential items whose polarities are determined by the value of the input impedance, Ri . The first item describes the transient photovoltage change that charges the capacitors, Cl and Cm . If assuming Cl is equal to Cm , the output voltage due to the capacitive loading will be equal to E0 /2, which is always positive. The second item indicates the potential difference between the capacitive loading and the resistive loading. When Ri is larger than Rm , the total resistive loading on the measuring instrument is bigger than E0 /2, which causes the second item is positive. Consequently, the membrane capacitor is continuously charged. Oppositely, when Ri is smaller than Rm , the potential on the measuring instrument is smaller than E0 /2, which results the second item is negative. The capacitor Cm is, therefore, discharged through RCm , and the measured photovoltage is a differential signal. Eq. (21) produces the same effect of the input impedance Ri on the polarity of the exponential items when light is OFF. Experimental results are in good agreement with the above analysis. Fig. 6 illustrates photoelectric signals measured by the electrometer with the input impedance of 10 G
(a) and 100 G
(b), respectively. When light is ON, the time constants, τ 1 in both measurements are equal, which is 2 s. Since τ 1 = Rs Cl Cm /(Cl + Cm ), varying the input impedance will not affect τ 1 . However, the time constants, τ 2 changes with the input impedance because of τ 2 = RCm . τ 2 is around 2.5 s for the input impedance of 10 G
and 22 s for 100 G
. Calculation yields the values of the components in the equivalent circuit, Rs = 16 G
, Rm = 650 G
, Rl = 634 G
and Cl ≈ Cm = 63 pF. When the light is OFF, the time constants, τ 1 in both measurements are identical, which is around 4 s. Evidently, it is about twice as long as τ 1 of light-ON response. This indicates that the intrinsic properties of the bR film are affected by illumination. Meanwhile, the time constants, τ 2 measured in both

W.W. Wang et al. / Biosensors and Bioelectronics 21 (2006) 1309–1319

Fig. 6. Influence of low input impedance on bR photodetector responses. Photoelectric signals (noisy curves) to square wave light signals with amplitude of 2 mW/mm2 are measured by electrometer with the input impedance of 10 G
(a) and 100 G
(b). Micro-Cap simulated signals (thin solid curves) are superimposed on measured signals.

cases exhibit the same properties as in the light-ON measurement, except that they are also two times longer. This confirms that the intrinsic resistance and capacitance of the bR film vary to the incident light. The measured time constants are used to compute the remaining parameters, Rs = 23.8 G
, Rm = 650 G
, Rl = 626 G
, Cl = 63 pF and Cm = 126 pF. To verify the calculated parameters, Micro-Cap simulation is applied to the equivalent circuit of Fig. 4c. The calculated results are superimposed on the measured data in Fig. 6. It can be seen that two data sets closely match except that the measured curves have smaller negative peaks. This is additional proof that the internal resistance and capacitance of the bR film increases when the light source is OFF. To investigate the effect of the high input impedance from the measurement instrumentation on the photoresponse generated from the bR photodetector, a signal processing circuit using precision CMOS single micropower operational amplifier is designed. The operational amplifier LMC 6061 was selected because it requires an ultra low input bias current. Moreover, the electrometer with input impedance of

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Fig. 7. Influence of high input impedance on bR photodetector responses. Photoelectric signals (noisy curves) to square wave light signals with amplitude of 2 mW/mm2 are measured by signal processing circuit with input impedance of 10 Tera
(a) and by electrometer with the input impedance of 100 Tera
(b). Micro-Cap simulated signals (thin solid curves) are superimposed on measured signals.

100 Tera
is chosen for the same purpose. Fig. 7 shows that measured photoelectric signals display the different signal profiles from those measured with low input impedances. Due to the high input impedance, the potential difference between the capacitive loading and the resistive loading have a fixed polarity that is always positive. As a result, the membrane capacitor, Cm is continuously charged to its maximum value during the illumination and discharged to the minimum when light is OFF. Micro-Cap simulation is applied to the simplified equivalent circuit using the same experimentally determined parameters as above except for the values of the input impedance. A comparative analysis of measured data and calculated data reveals that the real signals decay much slowly and the baselines do not go back to zero when light is switched OFF. The former is caused by the sudden increase in the resistance and capacitance of the film when the light is turned OFF. The latter effect can be explained as the capacitor Cm , not fully discharging when both resistances of R and Rl have relatively high values.

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In order to achieve improved functionality and performance, eliminating the unwanted interconnection between the detector and the readout circuit is a critical mission for the design of bR photodetector. Like any other biosensor, the capacitance and resistance of the bR film depend directly on the physical parameters of the film, which are fixed at the moment of fabrication. Therefore, it is very important to optimize the relevant parameters to reduce the unwanted electric components during the preparative process. Eqs. (16) and (18) in Section 3.2 shows the extrinsic response time is proportional to the film capacitance, thereby a smaller capacitance is preferred for the high response speed and wide bandwidth. Moreover, Eq. (23) indicates that a smaller film capacitance results in larger amplitudes in the measured photoresponse. Furthermore, the inherent capacitance of the bR film decreases linearly with increasing the thickness of the film. Clearly, to develop a fast and sensitive bR photodetector, a large thickness of the dried bR film is required. This prediction is also supported by the experimental observation reported by Hong (1999, Fig. 27).

5. Conclusions The photoelectric response of an innovative light detector constructed from dried bacteriorhodopsin (bR) film had been characterized both experimentally and analytically. The photocycle of the bR molecules in a dried film involved fewer intermediates than the more commonly studied aqueous film. Under constant illumination the photo-induced voltage reached steady-state values in milliseconds. The light absorption properties were represented by an irreversible sequential photocycle model and the Ramo–Shockley theorem. The electrically equivalent circuit demonstrated the intrinsic electric properties of the film and the influence that the external measurement circuitry had on the photoresponse of the proposed detector. Experimental observations and simulation studies had shown that the photoresponse of the dried bR film was attributed to the intrinsic transit response of the charge displacement and recombination, and the extrinsic response caused by the electrical coupling of the bR film to the measurement circuitry. The input impedance of the measuring instruments had significant influence over the observed photoelectric signal. Decreasing the input impedance induced a differential signal and also lowered the photoresponse amplitude. This was caused by interactions between the measuring instrument and inherent RC network of bR film. Another significant observation was that the resistance and capacitance values of the dried bR film were reduced considerably when illuminated. As a result, the measured photoelectric signal exhibited smaller amplitude and slower decay time when the light was off as compared to when the light was on. The validity of the proposed model is confirmed by the consistent agreement between experimental and simulated results. Prediction with this model is also supported by other published

experimental results. This work tries to illustrate the potential for integrating dried bR film into practical devices and applications, such as movement or edge enhancement detection and high-speed photodetection.

Acknowledgements This work has been supported by research grants to Profs. A.S. Bassi and G.K. Knopf from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation (CFI), and Photonics Research Ontario (PRO).

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