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Response of bacteriorhodopsin thin films to ammonia
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Sensors and Actuators B 129 (2008) 473–480
Response of bacteriorhodopsin thin films to ammonia S.O. Korposh a , Y.P. Sharkan b , J.J. Ramsden a,c,∗ a
Cranfield University Kitakyushu Campus, 1-5-4F Hibikino, Kitakyushu 808-0135, Japan b Institute of Solid-State Physics & Chemistry, Uzhgorod National University, Voloshina St. 54, 88000 Uzhgorod, Ukraine c Department of Materials, Cranfield University, Bedfordshire , United Kingdom
Received 22 April 2007; received in revised form 2 August 2007; accepted 3 August 2007 Available online 19 August 2007
Abstract The optical response of optical fibres end-coated with bacteriorhodopsin-containing films to ammonia vapour was investigated. Both static absorptance changes and changes in the dynamic parameters of the actinic light-induced photocycle were determined. Simple optical adsorption measurements can be used to detect ammonia with a detection limit of 5 ppm and a dynamic range of 10–10,000 ppm, over which the response is logarithmic. By analysing the modulation of the photocycle response by ammonia, the limit of detection can be decreased to 0.9 ppm. The incorporation of chemicals with an amine functionality, such as triethanolamine, in the film suppresses the ammonia response, and is therefore useful for referencing. © 2007 Elsevier B.V. All rights reserved. Keywords: Fibre-optic sensor; Bacteriorhodopsin; Photocycle; Chemical additive
1. Introduction Ammonia is currently regarded as a sensing challenge, for which a suitable sensing device for widespread industrial and domestic application is still sought [1]. Recently, we have proposed bacteriorhodopsin (bR) as a universal material for sensitizing optical sensors [2]. Its complex optical response allows sophisticated and informative data analysis with a great variety of possibilities for extracting highly sensitive and selective responses to the presence of gaseous analytes. Furthermore, the material can be chemically treated to confer a particular profile of sensitivities onto the material [3]. In this work, we investigate the optical response of bacteriorhodopsin thin films to the presence of ammonia vapour. Both static and dynamic measurements have been carried out. In the former, the sensing film is equilibrated with the ammonia and its optical densities at different wavelengths compared with those of the ammonia-free film. Transients leading to equilibrium have also been characterized. Dynamic measurements refer
to following the result of the initiation of the bacteriorhodopsin photocycle [4] by actinic light; the photocycle dynamics are modulated by the presence of ammonia. Their characterization allows a multiparameter response to be associated with a particular ammonia concentration, and results in significantly enhanced sensitivity compared with the static measurements. Optical response measurements were also carried out using end-coated optical fibres, with a view to the development of a complete miniature sensor for ammonia. It was demonstrated that an amine-containing chemical additive suppresses the response of the bacteriorhodopsin film to ammonia. This suggests that a general strategy for enhancing the chemical sensitivity is to choose additives that are chemically complementary to the analyte being detected, and in order to suppress the chemical sensitivity, additives that are chemically similar to the analyte being detected should be chosen. The latter procedure is useful for creating a reference pad. 2. Experimental 2.1. Materials
∗
Corresponding author at: B-70, Cranfield University, Bedfordshire, MK43 0AL, United Kingdom. E-mail address: [email protected] (J.J. Ramsden). 0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2007.08.024
Bacteriorhodopsin was extracted as purple membrane fragments (pmf) according to standard procedures [5] from
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Halobacterium salinarum strain S9 cultured in our laboratory. The polymer film samples based on bacteriorhodopsin were prepared using a photographic grade hide gelatin (Sigma). 2.2. Sample preparation Bacteriorhodopsin films in a gelatin matrix were prepared using standard procedures [6–9]. A suspension of pmf (14 mg lyophilized powder/mL) was prepared by soaking the powder for 20 min and then stirring for 6 h in tridistilled water. A solution of gelatin was obtained by soaking the powder in tridistilled water for 20 min at room temperature and stirring it at 60 ◦ C for 40 min to obtain a 6% (w/v) solution. The film-forming solution, a mixture of bR and gelatin, was prepared from 0.168 mL of the bR suspension and 0.248 mL of the gelatin solution to give a final volume with tridistilled water of 0.5 mL. Triethanolamine (TEA) (Sigma) was used as the sensitizing additive; a 0.4 M aqueous solution was added to the suspension to give a TEA:bR molar ratio of 250:1 (which was previously found to give maximal photosensitivity [10]). Film samples 10–20 m thick were prepared by depositing the suspension onto 5 cm2 planar glass substrates by casting, giving a sample with an optical density (OD) of about 1 at λ = 570 nm, and kept at room temperature. A small number of experiments were carried out using an optical fibre coated with the bR film. In this case 10 L of the film-forming solution was deposited onto the distal end of a 200 m diameter fibre-optic Y-type coupler to produce a film of approximately the same thickness as those cast on the planar glass substrate. 2.3. Optical measurement set-up A specially designed and laboratory-built sensor chamber (Fig. 1) was used for measuring the effects of the presence of
ammonia on the optical properties of the bR films. The sample holder with the bR film was placed inside the chamber and connected to the light source (LS) and spectrophotometer (SF) by means of optical fibres through vacuum feed-through connexions. An Ocean Optics HR-2000 spectrophotometer with a chargecoupled device (CCD) detector was used for measurement of the dynamic changes of the bR film spectra before and after exposing the films to the ammonia. A tungsten halogen lamp (Ocean Optics HL-2000) radiating from 360 nm to 2000 nm was used as the probe LS for the spectral measurements. A single optical fibre with an external diameter of 2 mm (diameter of the core, 1 mm; cladding thickness, 25 m; protection cladding thickness, 0.9 mm) was used to connect the sample holder (SH), LS and SF. Photo-induced absorptance changes were measured using a light emitting diode (LED) (Ocean Optics “LS-450 blue LED”) as the probe light source fitted with LEDs emitting at either 380 or 590 nm. A tungsten halogen lamp (Ocean Optics HL-2000) with a high pass filter (Ocean Optics OF2-OG515, cutoff wavelength λ = 530 nm (i.e. transmitting only light with λ > 530 nm)) was used for the photo-excitation of the bR photocycle. Irradiance was measured using a PM140 (Thorlabs Instrumentation) optical power meter. The absorptance measurements were carried out at 410 and 570 nm, which correspond to the maxima of the main intermediate state of the bR photocycle (M410 ) and the ground state (bR570 ), respectively. Optical density (absorptance) was determined by taking the logarithm of the ratio of the irradiance incident on the film (measured prior to inserting the bR-containing films) to the irradiance measured after the light had passed through the film. Photo-induced absorptance changes A were normalized according to: A =
Aλ − Aλa , Aλ
(1)
Fig. 1. Measurement set-up (see text). The chamber volume was 7 L. OF, optical fibre; SF, spectrophotometer; for which the light source (LS) was a halogen lamp (HL); AL, actinic light source used for the photo-excitation of the bR photocycle; PG, pressure gauge; V, valves. The inset shows the arrangement of the sample holder and optical fibres inside the measurement chamber: OF1, optical fibre connected to halogen lamp; OF2, optical fibre connected to the actinic light source using vacuum feedthroughs (for the bR film illumination); and OF3, optical fibre connected to the spectrophotometer.
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where Aλ is the absorptance of the bR film at the probe wavelength (410 or 570 nm) without actinic light (unbleached sample) and Aλa is the absorptance of the bR film in the presence of actinic light. The decay kinetics of the photo-induced absorptance changes measured while exposed to ammonia atmospheres of different concentrations after turning off the actinic illumination were fitted by a double exponential decay (required since it was clearly not single exponential): t t A(t) = A1 exp − + A2 exp − , (2) τ1 τ2 where A1 and A2 are the relative amplitudes of the decays with time constants τ 1 and τ 2 , respectively. In order to heuristically characterize the decay with a single parameter, we extracted the half-life of the decay (τ 1/2 ) from the fitted curve. This measurement methodology yields two parameters: the amplitude of the photo-induced absorptance changes (A), determined using Eq. (1), and the time constant τ 1/2 of the intermediate M410 . 2.4. Response measurements 2.4.1. Static conditions A gas cylinder containing nitrogen mixed with ammonia at a calibrated concentration c (four cylinders containing 100 ppm, 1000 ppm, 0.1% and 10% ammonia by volume were available) was connected to the gas chamber in order to provide known concentrations of ammonia within the chamber. Pressure gauges were used to monitor the admission of gas into the chamber (Fig. 1). The same (35 cm3 /s) flow rate was always used. For intermediate ammonia concentrations (and also concentrations less than 100 ppm) the mixture at the highest available concentration was admitted to the chamber containing pure nitrogen at a pressure of 1 atm, to give a small pressure increase P. The final ammonia concentration is then cP (for example, 10 ppm is obtained with c = 100 ppm and P = 0.1 atm). It was verified using pure nitrogen that a pressure increase of at least 20% did not affect the optical absorptance of the bR. 2.4.2. Dynamic conditions A 30% (w/w) aqueous ammonia solution was placed in a flask, and humidified air bubbled through it in order to create an atmosphere saturated with ammonia (see Fig. 2), whose
Fig. 2. Scheme of the gas generation system. The measurement chamber had a volume of ca. 500 cm3 ; L1 and L2 , flow rates; and FC, flow controller (Kofloc, RK1000).
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concentration z in the flask headspace was calculated from the difference in flask weight before (W0 ) and after (W1 ) flowing the humidified air through the flask for 10 min at a flow rate of 1 L/min (giving a volume V = 10 L), making allowance for water evaporation. A second air line by-passed the flask, and the two were finally recombined, with the final ammonia concentration (volume fraction) c in the sensor measurement chamber being calculated using c=
L1 , L1 (1 + z) + L2
(3)
where z is the mole fraction of the analyte (ammonia) in the flask headspace (i.e. the ratio of the partial pressure of the solution ps = (W0 – W1 )/V at the given temperature to the atmospheric pressure P, i.e. z = ps /P); and L1 and L2 the flow rates of the air passed through the flask and by-passing it, respectively. Note that the concentration of the ammonia in the headspace remains constant. Water evaporation was negligible even during the 4–5 h period that some experiments lasted. L1 was kept constant at 1 L/min and by varying L2 the different concentrations of analyte were obtained. The baseline of each experiment was recorded by flowing humidified air through the chamber until the absorptance of the bR film reached a steady state. This measurement chamber was used to record the reversibility of the sensor response, using bR-coated optical fibres. 3. Results 3.1. Response to ammonia The absorptance spectra of the bR films were measured with and without ammonia (Fig. 3a). The presence of the ammonia leads to a decrease of the absorptance at 570 nm and a small absorptance increase at 410 nm. As well as the former, a shift of the wavelength of the absorptance maximum from 565 nm to 550 nm was observed. The difference absorptance spectrum (i.e. the spectrum taken without ammonia subtracted from the spectrum taken in the presence of ammonia) is shown in Fig. 3b; it indicates that the largest absorptance changes occur at 580 nm. Normalization was carried out using the following equation, yielding the percentage sensor response (S.R.) to ammonia S.R. = 100
A0 − A , A0
(4)
where A0 is the absorptance of the bR film without ammonia and A is the absorptance in the presence of ammonia, measured at the same wavelength (at or near the trough minimum of the difference spectrum (Fig. 3b)). Response kinetics are shown in Fig. 4. From the results of the ammonia-induced absorptance changes over a range of concentrations (Fig. 4b), a calibration curve was plotted that shows a linear response over the ammonia concentration range of 0–200 ppm when monitored at either 570 nm or 580 nm, see Fig. 5a. The slope diminishes at higher concentrations, but from 1000 ppm to 10,000 ppm the response can be approximated as a straight line (Fig. 5b), but with a much diminished slope com-
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Fig. 3. (a) Absorptance spectra of the bR-gelatin film (mass ratio of the purple membrane fragments to gelatin (bR:gelatin) = 12.5:87.5, calculated as mass present in the dry film) measured at 25 ◦ C and rH 50% without flow, using the procedure in Section 2.4.1; black line, without ammonia; red line, in a 10% ammonia atmosphere. (b) Difference absorptance spectrum (ammonia − air) of the bR-gelatin film. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
pared with the low concentration range. The sensor response over the entire concentration range is nonlinear (Fig. 5c). The limit of detection (LOD) was defined heuristically according to the following equation [11]: LOD =
3σ , m
(1000 s) had to be applied to achieve complete relaxation of the S.R. 3.2. Effect of ammonia on the photocycle parameters of the bR
(5)
where σ is the standard deviation (derived in the present work from 220 blank measurements and found to be equal to 0.058), and m is the slope of the calibration curve (whose values are given in the legend of Fig. 5). According to Eq. (5), the LOD for the bR film was 5 ppm. The reversibility of the ammonia response was investigated by flushing ammonia gas at the desired concentration through the measurement chamber (Section 2.4.2) with a flow rate of 1 L/min, followed by flushing ammonia-free humidified air at a flow rate of 1 L/min until the full relaxation of S.R. was achieved. The response of the sensing element (the bR-coated optical fibre) is shown in Fig. 6. For the lower ammonia concentrations (up to 100 ppm) the response was completely reversible with a relaxation time of 100 s; at higher concentrations (>100 ppm) a longer flushing time
Photo-induced absorptance changes of the bR film in the presence of ammonia are shown in Fig. 7. The probe light and actinic light were brought to the sample via a fibre-optic Y-type coupler (Fig. 1). The amplitude of the photo-induced absorptance changes increased almost sixfold and the half-life of the intermediate M410 state doubled when the bR film was exposed to an ammonia concentration of 10,000 ppm. The presence of ammonia gas at the lowest concentration (10 ppm) already caused noticeable changes in the photocycle parameters of the bR film. The results of the exponential fitting of the decay are summarized in Table 1. 3.3. Response in the presence of chemical additives The ammonia-induced absorptance changes of films incorporating TEA are compared with those of TEA-free films in Fig. 8.
Fig. 4. (a) Temporal behaviour of S.R. The bR film had a mass ratio (bR:gelatin) = 12.5:87.5 measured at 25 ◦ C and rH 50%. Response induced by the presence of 10,000 ppm ammonia (obtained by admitting 0.1 atm of 10% ammonia at a flow rate of 35 cm3 /s into the nitrogen filled 7 L chamber) monitored at: 570 nm (black line); 580 nm (blue line); and 410 nm (red line). t90 is defined as the response time needed for the signal to achieve 90% of its maximum value at 570 nm (about 2000 s in this case). (b) Response of the bR film to increasing the concentration of ammonia from 10 ppm to 10,000 ppm monitored at 580 nm, using the procedure in Section 2.4.1 to fix the ammonia concentration. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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Fig. 5. Calibration curves constructed from the results of ammonia-induced optical absorptance changes in the bR film (Fig. 3b). (a) Ammonia concentration range 0–200 ppm—squares, slope: −0.030 ± 0.003 at 570 nm; circles, slope: −0.035 ± 0.003 at 580 nm; triangles, bR film with added TEA (mass ratio bR:gelatin = 12.5:87.5; bR:gelatin:TEA = 12.4:73.54:13.8; slope: −0.012 ± 0.003, see later). (b) Ammonia concentration range 1000–10,000 ppm—squares, slope: −0.0030 ± 0.0003 at 570 nm; circles, slope: −0.0050 ± 0.0003 at 580 nm. (c) Calibration curve plotted for the entire concentration range on a logarithmic scale.
The results demonstrated that the sensitivity of the bR film to ammonia is diminished by the addition of TEA. The calibration curve for the bR film with added TEA is included in Fig. 5a. The presence of additive also slows down
the response to ammonia (data not shown); for the bR film with added TEA the response time to ammonia was seven times slower than for the additive-free film. The LOD, calculated using Eq. (5), for the bR film with added TEA was 18 ppm,
Fig. 6. Time dependence of the response of the bR film deposited onto the distal end of the fibre-optic Y-type coupler 200 m in diameter (mass ratio bR:gelatin = 12.5:87.5 measured at 25 ◦ C and rH 50%; probe light LED590 emission wavelength 590 nm). Measurements were undertaken in flow mode (Section 2.4.2). Initially the ammonia concentration was increased and then the system was flushed with clean air at a flow rate of 1 L/min until full recovery of the S.R.
Fig. 7. Time dependence of the photo-induced absorptance changes of the bR film (mass ratio bR:gelatin = 12.5:87.5 measured at 25 ◦ C and rH 50%) monitored at 410 nm during and after actinic illumination (halogen light source with a high pass filter (>530 nm), arrows indicates the turning on and off of the actinic light source) in the presence of ammonia gas at different concentrations. Normalization was carried out using Eq. (1); the fitted line (Eq. (2)) is shown for the 20 ppm decay data.
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Table 1 Photocycle parameters obtained from Eq. (1) and via Eq. (2) Sample name
Ammonia concentration (ppm)
Aa,b
bR + gelatin
0 10 20 100 200 1,000 2,000 10,000
1.4 3.6 3.8 4.2 5.2 6.8 7.0 7.2
± ± ± ± ± ± ± ±
τ 1/2 /sc 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
15.0 19.0 20.0 23.0 25.0 27.0 28.0 31.0
± ± ± ± ± ± ± ±
1.4 0.5 0.7 0.2 0.1 0.1 0.1 0.1
a
Percentage of photo-induced absorptance changes, calculated using Eq. (1). Uncertainties reflect the accuracy of the measurement equipment. c Uncertainties reflect the fitting of individual curves to the double exponential decay (see Fig. 7, line fitted (Eq. (2)) for the 20 ppm decay data). Uncertainties for the measurement of different samples ≈0.6 (standard deviation derived from four measurements); and uncertainties for separate measurements of one sample ca. ≈0.12 (standard deviation derived from eight measurements). b
that is, three times higher than for the bR film without chemical additives. Furthermore, ammonia had a negligible influence on the photocycle parameters of the bR film with added TEA (Fig. 9). The calibration curves plotted for bR + gelatin and bR + gelatin + TEA using data from the exponential fitting (Eq. (2)) are compared in Fig. 10. The lifetime constant of the decay of the M410 state of the bR film with added TEA became dependent on the presence of ammonia only when film was exposed to high ammonia concentrations (>2000 ppm). The LODS calculated using Eq. (5) and the limiting linear slopes at the lowest detectable ammonia concentrations were 0.9 ppm and 24 ppm, and 0.2 s ppm−1 and 0.007% ppm−1 for the lifetime and photoinduced absorptance change, respectively.
Fig. 9. Time dependence of the photo-induced absorptance changes of the bR film with added TEA (mass ratio bR:gelatin:TEA = 12.4:73.5:13.8 measured at 25 ◦ C and rH 50%) monitored at 410 nm during and after actinic illumination (halogen light source with a high pass filter (>530 nm), arrows indicates the turning on and off of the actinic light source) in the presence of ammonia at different concentrations.
4. Discussion 4.1. Ammonia effect on the absorptance spectrum The results shown in Figs. 3 and 4a indicate that the absorptance decreases at 570 nm and increases at 410 nm; and the 570 absorptance maximum shifts towards the blue. These effects are similar to those engendered by dehydration of bR [9], which causes the absorptance to decrease at 570 nm and increase at 410 nm, along with a blue shift of the absorptance maximum at 570 nm. Dyukova et al. have proposed that this is due to dissociation of a water molecule specifically bound to the retinal chromophore [9]. Note that ammonia abstracts water to form hydroxyl ions − NH3 + H2 O NH+ 4 + OH
Fig. 8. Ammonia-induced absorptance changes shown as the ratio of the spectrum taken in 10,000 ppm ammonia to the spectrum taken in air: black line (1), bR film without added TEA (dried at rH 50%); red line (2), bR film with added TEA (mass ratio bR:gelatin = 12.5:87.5; bR:gelatin:TEA = 12.4:73.5:13.8; measured at 25 ◦ C and rH 50% without flow (see Section 2.4.1). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
(6)
and may therefore induce dehydration [12]. However, as demonstrated by Hildebrandt et al. [13], the presence of ammonia in a hydrated bR film leads to insignificant changes in the chromophoric structure. Thus, it seems that there is no structural disruption of the bR molecule, implying that ammonia is likely to have a reversible influence on the bR optical parameters, as is indeed corroborated by the results shown in Fig. 6. Note that the effect of pH per se is very much smaller [9] compared with our observation of ammonia effect. The absorptance decrease at 570 nm and increase at 410 nm may also be thought as a shift of the equilibrium between the ground state (bR570 ) and the intermediate (M410 ) state, similar to the action of actinic light, i.e. bR + NH3 bR∗
(7)
where bR* represents a modified form of bR (e.g. M410 ). The suppression of the sensitivity to ammonia by addition of TEA to the bR film (Figs. 5a and 8) would appear to result from
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Fig. 10. Calibration curves were plotted using the data from the double-exponential fitting (Eq. (2)) and represents (a) the lifetime of the M410 state; (b) the photoinduced absorptance changes (Eq. (1)); squares, bR film without added TEA; circles, bR + gelatin + TEA; points are connected merely to guide the eye. Measured in the presence of ammonia gas of different concentrations at room conditions of 25 ◦ C and rH 50%, mass ratio bR:gelatin = 12.5:87.5; bR:gelatin:TEA = 12.4:73.5:13.8 (see Figs. 6–8).
the fact that the NH2 group of the TEA then takes up the rˆole of the proton acceptor in the bR film. A possible mechanism of the TEA effect on bR was previously suggested by Batori-Tartsi and Ludmann [3]: they demonstrated that chemical additives act on the extracellular side of the purple membrane and take the rˆole of proton acceptor, i.e. the electronegative functional groups present in the TEA can capture the proton released on the extracellular side of the membrane during the photocycle [10]. Our results show that the presence of ammonia causes the bR photocycle to change in a similar way as with added TEA: the amplitude of the photo-induced absorptance changes and the lifetime of the bR film without TEA when exposed to ammonia was the same as that of the bR film with added TEA without ammonia exposure (see Fig. 10). Our results (Figs. 3 and 7) imply that ammonia (cf. Eq. (6)) decreases the energy barrier of the transition of the intermediate bR570 state to the M410 state. Even the relatively weak (∼0.1 mW/cm2 ) probe light causes a slight actinic effect on the
bR film when it is exposed to ammonia, i.e. shifts the equilibrium between the bR570 ground state and the intermediate M410 state (Fig. 4a). Moreover, an increase of the probe light irradiance (by about 10 times) caused a threefold increase of the S.R. (Fig. 11). 5. Summarizing conclusions The effect of ammonia on the optical properties of bR films was studied quantitatively. Sensor response of the bR film was linear in the 10–200 ppm range of ammonia concentration. The slope diminishes at higher concentrations, but from 1000 ppm to 10,000 ppm the response can be approximated as linear. Response was completely reversible with a recovery time of 100 s for the lower ammonia concentrations (up to 100 ppm), but at higher concentrations (>100 ppm) a longer flushing time (1000 s) was needed in order to achieve a complete recovery of the sensor response. The LOD for the bR film without added TEA was 5 ppm for ammonia-induced absorptance changes, 0.9 ppm when the time constant of the M410 state decay was measured, and 24 ppm for the photo-induced absorptance changes. The incorporation of chemicals with an amine functionality, such as triethanolamine, in the film suppresses the ammonia response, and is therefore useful for referencing. Acknowledgments We thank O.I. Korposh, I.J. Tcoma and V.V. Yarosh for their expert assistance with the bacteriorhodopsin sample preparation, and Dr S.-W. Lee of the University of Kitakyushu for providing us with some of the instrumentation. References
Fig. 11. Sensor response of the bR film to 500 ppm of ammonia at different light sources irradiances; black line, LED595 (0.4 mW/cm2 ); red line, halogen lamp with high pass filter (passing 530–2000 nm) (4 mW/cm2 ). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
[1] J. Hodgkinson, J. Saffell, J. Luff, J. Shaw, J.J. Ramsden, C. Huggins, R. Bogue, R. Carline, MNT Gas Sensors Roadmap, MNT Gas Sensors Forum, Cranfield University, 2006. [2] J.P. Sharkany, S.O. Korposh, Z.I. Batori-Tarci, I.I. Trikur, J.J. Ramsden, Bacteriorhodopsin-based biochromic films for chemical sensors, Sens. Actuator B 107 (2005) 70–81.
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[3] Z. Batori-Tartsi, K. Ludmann, Bioelectronic applications of photochromic pigments, in: A. D´er, L. Keszthelyi (Eds.), Bioelectronic Applications of Photochromic Pigments (NATO Science Series, Subseries I, Life and Behavioural Sciences), IOS Press, Amsterdam, 2001, pp. 137–148. [4] R.H. Lozier, R.A. Bogomolni, W. Stoeckenius, Bacteriorhodopsin: a lightdriven proton pump in Halobacterium halobium, Biophys. J. 15 (1975) 955–962. [5] D. Oesterhelt, W. Stoeckenius, Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane, Methods Enzymol. 31 (1974) 667–678. [6] Z. B´athori T´arczy, J. S´arkany, I. Bandrovszkaja, J. Sersun, O. Korpos, V. Jaros, Bakteriorodopszin-alapanyag´u fotochrom r´etegek, Tudom´anyos tal´alkoz´o, Budapest, 1992, pp. 110–112. [7] A. Kikineshy, Z. Bathori-Tarczy, Y. Sharkany, Materials and devices for fiber optic sensors, in: W.K. Jones, G. Hars´anyi (Eds.), Multichip Modules with Integrated Sensors (NATO ASI Series, Subseries 3, High Technology), Dordrecht, Kluwer, 1996, pp. 181–184. [8] L. Lensu, M. Frydrych, J. Parkkinen, S. Parkkinen, T. Jaaskelainen, Photoelectric properties of bacteriorhodopsin analogs for color-sensitive optoelectronic devices, Opt. Mater. 27 (2004) 57–62. [9] T. Dyukova, B. Robertson, H. Weetall, Optical and electrical characterization of bacteriorhodopsin films, Biosystems 41 (1997) 91–98. [10] Z. Batori-Tartsi, K. Ludman, G. V´ar´o, The effect of chemical additives on the bacteriorhodopsin photocycle, J. Photochem. Photobiol. B 49 (1999) 192–197. [11] M.E. Swartz, I.S. Krull, Analytical Method Development and Validation, Dekker, New York, 1997.
[12] T.V. Dyukova, E.P. Lukashev, Dehydration effects on D96N bacteriorhodopsin films, Thin Solid Films 283 (1–4) (1996). [13] P. Hildebrandt, M. Stockburger, Role of water in bacteriorhodopsin’s chromophore: a resonance Raman study, Biochemistry 23 (1984) 5539–5548.
Biographies Serhiy Korposh obtained his master’s degree in physics from Uzhgorod (Ungv´ar) National University, Transcarpathia, Ukraine (in 2002), and his doctorate from Cranfield University (in 2007). His research interests are metrology and chemical sensing using optical methods. Yosyp Sharkan obtained his master’s degree in physics and his doctorate (Cand. Sci.) from Lviv (Lemberg) National University, Ukraine. Since 1993 he has been head of the Department of Integrated Optics within the Institute of Solid-State Physics and Chemistry at Uzhgorod National University. From 2003 until 2006 he was a visiting researcher at Cranfield University at Kitakyushu. His research interests include thin film-based nanocomposites. Jeremy Ramsden obtained his bachelor’s and master’s degrees in natural sciences from Cambridge University, and his doctorate from the Ecole polytechnique f´ed´erale de Lausanne (EPFL). He has been a member of the faculty of Natural Philosophy at the University of Basel since 1994, and professor of Nanotechnology at Cranfield University (formerly Cranfield Institute of Technology) since 2002. He is also research director for bionanotechnology at Cranfield University at Kitakyushu. His research interests include the bio/non-biointerface, and the use of biomolecules in artificial adaptive systems.
Sensors and Actuators B 129 (2008) 473–480
Response of bacteriorhodopsin thin films to ammonia S.O. Korposh a , Y.P. Sharkan b , J.J. Ramsden a,c,∗ a
Cranfield University Kitakyushu Campus, 1-5-4F Hibikino, Kitakyushu 808-0135, Japan b Institute of Solid-State Physics & Chemistry, Uzhgorod National University, Voloshina St. 54, 88000 Uzhgorod, Ukraine c Department of Materials, Cranfield University, Bedfordshire , United Kingdom
Received 22 April 2007; received in revised form 2 August 2007; accepted 3 August 2007 Available online 19 August 2007
Abstract The optical response of optical fibres end-coated with bacteriorhodopsin-containing films to ammonia vapour was investigated. Both static absorptance changes and changes in the dynamic parameters of the actinic light-induced photocycle were determined. Simple optical adsorption measurements can be used to detect ammonia with a detection limit of 5 ppm and a dynamic range of 10–10,000 ppm, over which the response is logarithmic. By analysing the modulation of the photocycle response by ammonia, the limit of detection can be decreased to 0.9 ppm. The incorporation of chemicals with an amine functionality, such as triethanolamine, in the film suppresses the ammonia response, and is therefore useful for referencing. © 2007 Elsevier B.V. All rights reserved. Keywords: Fibre-optic sensor; Bacteriorhodopsin; Photocycle; Chemical additive
1. Introduction Ammonia is currently regarded as a sensing challenge, for which a suitable sensing device for widespread industrial and domestic application is still sought [1]. Recently, we have proposed bacteriorhodopsin (bR) as a universal material for sensitizing optical sensors [2]. Its complex optical response allows sophisticated and informative data analysis with a great variety of possibilities for extracting highly sensitive and selective responses to the presence of gaseous analytes. Furthermore, the material can be chemically treated to confer a particular profile of sensitivities onto the material [3]. In this work, we investigate the optical response of bacteriorhodopsin thin films to the presence of ammonia vapour. Both static and dynamic measurements have been carried out. In the former, the sensing film is equilibrated with the ammonia and its optical densities at different wavelengths compared with those of the ammonia-free film. Transients leading to equilibrium have also been characterized. Dynamic measurements refer
to following the result of the initiation of the bacteriorhodopsin photocycle [4] by actinic light; the photocycle dynamics are modulated by the presence of ammonia. Their characterization allows a multiparameter response to be associated with a particular ammonia concentration, and results in significantly enhanced sensitivity compared with the static measurements. Optical response measurements were also carried out using end-coated optical fibres, with a view to the development of a complete miniature sensor for ammonia. It was demonstrated that an amine-containing chemical additive suppresses the response of the bacteriorhodopsin film to ammonia. This suggests that a general strategy for enhancing the chemical sensitivity is to choose additives that are chemically complementary to the analyte being detected, and in order to suppress the chemical sensitivity, additives that are chemically similar to the analyte being detected should be chosen. The latter procedure is useful for creating a reference pad. 2. Experimental 2.1. Materials
∗
Corresponding author at: B-70, Cranfield University, Bedfordshire, MK43 0AL, United Kingdom. E-mail address: [email protected] (J.J. Ramsden). 0925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2007.08.024
Bacteriorhodopsin was extracted as purple membrane fragments (pmf) according to standard procedures [5] from
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Halobacterium salinarum strain S9 cultured in our laboratory. The polymer film samples based on bacteriorhodopsin were prepared using a photographic grade hide gelatin (Sigma). 2.2. Sample preparation Bacteriorhodopsin films in a gelatin matrix were prepared using standard procedures [6–9]. A suspension of pmf (14 mg lyophilized powder/mL) was prepared by soaking the powder for 20 min and then stirring for 6 h in tridistilled water. A solution of gelatin was obtained by soaking the powder in tridistilled water for 20 min at room temperature and stirring it at 60 ◦ C for 40 min to obtain a 6% (w/v) solution. The film-forming solution, a mixture of bR and gelatin, was prepared from 0.168 mL of the bR suspension and 0.248 mL of the gelatin solution to give a final volume with tridistilled water of 0.5 mL. Triethanolamine (TEA) (Sigma) was used as the sensitizing additive; a 0.4 M aqueous solution was added to the suspension to give a TEA:bR molar ratio of 250:1 (which was previously found to give maximal photosensitivity [10]). Film samples 10–20 m thick were prepared by depositing the suspension onto 5 cm2 planar glass substrates by casting, giving a sample with an optical density (OD) of about 1 at λ = 570 nm, and kept at room temperature. A small number of experiments were carried out using an optical fibre coated with the bR film. In this case 10 L of the film-forming solution was deposited onto the distal end of a 200 m diameter fibre-optic Y-type coupler to produce a film of approximately the same thickness as those cast on the planar glass substrate. 2.3. Optical measurement set-up A specially designed and laboratory-built sensor chamber (Fig. 1) was used for measuring the effects of the presence of
ammonia on the optical properties of the bR films. The sample holder with the bR film was placed inside the chamber and connected to the light source (LS) and spectrophotometer (SF) by means of optical fibres through vacuum feed-through connexions. An Ocean Optics HR-2000 spectrophotometer with a chargecoupled device (CCD) detector was used for measurement of the dynamic changes of the bR film spectra before and after exposing the films to the ammonia. A tungsten halogen lamp (Ocean Optics HL-2000) radiating from 360 nm to 2000 nm was used as the probe LS for the spectral measurements. A single optical fibre with an external diameter of 2 mm (diameter of the core, 1 mm; cladding thickness, 25 m; protection cladding thickness, 0.9 mm) was used to connect the sample holder (SH), LS and SF. Photo-induced absorptance changes were measured using a light emitting diode (LED) (Ocean Optics “LS-450 blue LED”) as the probe light source fitted with LEDs emitting at either 380 or 590 nm. A tungsten halogen lamp (Ocean Optics HL-2000) with a high pass filter (Ocean Optics OF2-OG515, cutoff wavelength λ = 530 nm (i.e. transmitting only light with λ > 530 nm)) was used for the photo-excitation of the bR photocycle. Irradiance was measured using a PM140 (Thorlabs Instrumentation) optical power meter. The absorptance measurements were carried out at 410 and 570 nm, which correspond to the maxima of the main intermediate state of the bR photocycle (M410 ) and the ground state (bR570 ), respectively. Optical density (absorptance) was determined by taking the logarithm of the ratio of the irradiance incident on the film (measured prior to inserting the bR-containing films) to the irradiance measured after the light had passed through the film. Photo-induced absorptance changes A were normalized according to: A =
Aλ − Aλa , Aλ
(1)
Fig. 1. Measurement set-up (see text). The chamber volume was 7 L. OF, optical fibre; SF, spectrophotometer; for which the light source (LS) was a halogen lamp (HL); AL, actinic light source used for the photo-excitation of the bR photocycle; PG, pressure gauge; V, valves. The inset shows the arrangement of the sample holder and optical fibres inside the measurement chamber: OF1, optical fibre connected to halogen lamp; OF2, optical fibre connected to the actinic light source using vacuum feedthroughs (for the bR film illumination); and OF3, optical fibre connected to the spectrophotometer.
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where Aλ is the absorptance of the bR film at the probe wavelength (410 or 570 nm) without actinic light (unbleached sample) and Aλa is the absorptance of the bR film in the presence of actinic light. The decay kinetics of the photo-induced absorptance changes measured while exposed to ammonia atmospheres of different concentrations after turning off the actinic illumination were fitted by a double exponential decay (required since it was clearly not single exponential): t t A(t) = A1 exp − + A2 exp − , (2) τ1 τ2 where A1 and A2 are the relative amplitudes of the decays with time constants τ 1 and τ 2 , respectively. In order to heuristically characterize the decay with a single parameter, we extracted the half-life of the decay (τ 1/2 ) from the fitted curve. This measurement methodology yields two parameters: the amplitude of the photo-induced absorptance changes (A), determined using Eq. (1), and the time constant τ 1/2 of the intermediate M410 . 2.4. Response measurements 2.4.1. Static conditions A gas cylinder containing nitrogen mixed with ammonia at a calibrated concentration c (four cylinders containing 100 ppm, 1000 ppm, 0.1% and 10% ammonia by volume were available) was connected to the gas chamber in order to provide known concentrations of ammonia within the chamber. Pressure gauges were used to monitor the admission of gas into the chamber (Fig. 1). The same (35 cm3 /s) flow rate was always used. For intermediate ammonia concentrations (and also concentrations less than 100 ppm) the mixture at the highest available concentration was admitted to the chamber containing pure nitrogen at a pressure of 1 atm, to give a small pressure increase P. The final ammonia concentration is then cP (for example, 10 ppm is obtained with c = 100 ppm and P = 0.1 atm). It was verified using pure nitrogen that a pressure increase of at least 20% did not affect the optical absorptance of the bR. 2.4.2. Dynamic conditions A 30% (w/w) aqueous ammonia solution was placed in a flask, and humidified air bubbled through it in order to create an atmosphere saturated with ammonia (see Fig. 2), whose
Fig. 2. Scheme of the gas generation system. The measurement chamber had a volume of ca. 500 cm3 ; L1 and L2 , flow rates; and FC, flow controller (Kofloc, RK1000).
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concentration z in the flask headspace was calculated from the difference in flask weight before (W0 ) and after (W1 ) flowing the humidified air through the flask for 10 min at a flow rate of 1 L/min (giving a volume V = 10 L), making allowance for water evaporation. A second air line by-passed the flask, and the two were finally recombined, with the final ammonia concentration (volume fraction) c in the sensor measurement chamber being calculated using c=
L1 , L1 (1 + z) + L2
(3)
where z is the mole fraction of the analyte (ammonia) in the flask headspace (i.e. the ratio of the partial pressure of the solution ps = (W0 – W1 )/V at the given temperature to the atmospheric pressure P, i.e. z = ps /P); and L1 and L2 the flow rates of the air passed through the flask and by-passing it, respectively. Note that the concentration of the ammonia in the headspace remains constant. Water evaporation was negligible even during the 4–5 h period that some experiments lasted. L1 was kept constant at 1 L/min and by varying L2 the different concentrations of analyte were obtained. The baseline of each experiment was recorded by flowing humidified air through the chamber until the absorptance of the bR film reached a steady state. This measurement chamber was used to record the reversibility of the sensor response, using bR-coated optical fibres. 3. Results 3.1. Response to ammonia The absorptance spectra of the bR films were measured with and without ammonia (Fig. 3a). The presence of the ammonia leads to a decrease of the absorptance at 570 nm and a small absorptance increase at 410 nm. As well as the former, a shift of the wavelength of the absorptance maximum from 565 nm to 550 nm was observed. The difference absorptance spectrum (i.e. the spectrum taken without ammonia subtracted from the spectrum taken in the presence of ammonia) is shown in Fig. 3b; it indicates that the largest absorptance changes occur at 580 nm. Normalization was carried out using the following equation, yielding the percentage sensor response (S.R.) to ammonia S.R. = 100
A0 − A , A0
(4)
where A0 is the absorptance of the bR film without ammonia and A is the absorptance in the presence of ammonia, measured at the same wavelength (at or near the trough minimum of the difference spectrum (Fig. 3b)). Response kinetics are shown in Fig. 4. From the results of the ammonia-induced absorptance changes over a range of concentrations (Fig. 4b), a calibration curve was plotted that shows a linear response over the ammonia concentration range of 0–200 ppm when monitored at either 570 nm or 580 nm, see Fig. 5a. The slope diminishes at higher concentrations, but from 1000 ppm to 10,000 ppm the response can be approximated as a straight line (Fig. 5b), but with a much diminished slope com-
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Fig. 3. (a) Absorptance spectra of the bR-gelatin film (mass ratio of the purple membrane fragments to gelatin (bR:gelatin) = 12.5:87.5, calculated as mass present in the dry film) measured at 25 ◦ C and rH 50% without flow, using the procedure in Section 2.4.1; black line, without ammonia; red line, in a 10% ammonia atmosphere. (b) Difference absorptance spectrum (ammonia − air) of the bR-gelatin film. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
pared with the low concentration range. The sensor response over the entire concentration range is nonlinear (Fig. 5c). The limit of detection (LOD) was defined heuristically according to the following equation [11]: LOD =
3σ , m
(1000 s) had to be applied to achieve complete relaxation of the S.R. 3.2. Effect of ammonia on the photocycle parameters of the bR
(5)
where σ is the standard deviation (derived in the present work from 220 blank measurements and found to be equal to 0.058), and m is the slope of the calibration curve (whose values are given in the legend of Fig. 5). According to Eq. (5), the LOD for the bR film was 5 ppm. The reversibility of the ammonia response was investigated by flushing ammonia gas at the desired concentration through the measurement chamber (Section 2.4.2) with a flow rate of 1 L/min, followed by flushing ammonia-free humidified air at a flow rate of 1 L/min until the full relaxation of S.R. was achieved. The response of the sensing element (the bR-coated optical fibre) is shown in Fig. 6. For the lower ammonia concentrations (up to 100 ppm) the response was completely reversible with a relaxation time of 100 s; at higher concentrations (>100 ppm) a longer flushing time
Photo-induced absorptance changes of the bR film in the presence of ammonia are shown in Fig. 7. The probe light and actinic light were brought to the sample via a fibre-optic Y-type coupler (Fig. 1). The amplitude of the photo-induced absorptance changes increased almost sixfold and the half-life of the intermediate M410 state doubled when the bR film was exposed to an ammonia concentration of 10,000 ppm. The presence of ammonia gas at the lowest concentration (10 ppm) already caused noticeable changes in the photocycle parameters of the bR film. The results of the exponential fitting of the decay are summarized in Table 1. 3.3. Response in the presence of chemical additives The ammonia-induced absorptance changes of films incorporating TEA are compared with those of TEA-free films in Fig. 8.
Fig. 4. (a) Temporal behaviour of S.R. The bR film had a mass ratio (bR:gelatin) = 12.5:87.5 measured at 25 ◦ C and rH 50%. Response induced by the presence of 10,000 ppm ammonia (obtained by admitting 0.1 atm of 10% ammonia at a flow rate of 35 cm3 /s into the nitrogen filled 7 L chamber) monitored at: 570 nm (black line); 580 nm (blue line); and 410 nm (red line). t90 is defined as the response time needed for the signal to achieve 90% of its maximum value at 570 nm (about 2000 s in this case). (b) Response of the bR film to increasing the concentration of ammonia from 10 ppm to 10,000 ppm monitored at 580 nm, using the procedure in Section 2.4.1 to fix the ammonia concentration. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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Fig. 5. Calibration curves constructed from the results of ammonia-induced optical absorptance changes in the bR film (Fig. 3b). (a) Ammonia concentration range 0–200 ppm—squares, slope: −0.030 ± 0.003 at 570 nm; circles, slope: −0.035 ± 0.003 at 580 nm; triangles, bR film with added TEA (mass ratio bR:gelatin = 12.5:87.5; bR:gelatin:TEA = 12.4:73.54:13.8; slope: −0.012 ± 0.003, see later). (b) Ammonia concentration range 1000–10,000 ppm—squares, slope: −0.0030 ± 0.0003 at 570 nm; circles, slope: −0.0050 ± 0.0003 at 580 nm. (c) Calibration curve plotted for the entire concentration range on a logarithmic scale.
The results demonstrated that the sensitivity of the bR film to ammonia is diminished by the addition of TEA. The calibration curve for the bR film with added TEA is included in Fig. 5a. The presence of additive also slows down
the response to ammonia (data not shown); for the bR film with added TEA the response time to ammonia was seven times slower than for the additive-free film. The LOD, calculated using Eq. (5), for the bR film with added TEA was 18 ppm,
Fig. 6. Time dependence of the response of the bR film deposited onto the distal end of the fibre-optic Y-type coupler 200 m in diameter (mass ratio bR:gelatin = 12.5:87.5 measured at 25 ◦ C and rH 50%; probe light LED590 emission wavelength 590 nm). Measurements were undertaken in flow mode (Section 2.4.2). Initially the ammonia concentration was increased and then the system was flushed with clean air at a flow rate of 1 L/min until full recovery of the S.R.
Fig. 7. Time dependence of the photo-induced absorptance changes of the bR film (mass ratio bR:gelatin = 12.5:87.5 measured at 25 ◦ C and rH 50%) monitored at 410 nm during and after actinic illumination (halogen light source with a high pass filter (>530 nm), arrows indicates the turning on and off of the actinic light source) in the presence of ammonia gas at different concentrations. Normalization was carried out using Eq. (1); the fitted line (Eq. (2)) is shown for the 20 ppm decay data.
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Table 1 Photocycle parameters obtained from Eq. (1) and via Eq. (2) Sample name
Ammonia concentration (ppm)
Aa,b
bR + gelatin
0 10 20 100 200 1,000 2,000 10,000
1.4 3.6 3.8 4.2 5.2 6.8 7.0 7.2
± ± ± ± ± ± ± ±
τ 1/2 /sc 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
15.0 19.0 20.0 23.0 25.0 27.0 28.0 31.0
± ± ± ± ± ± ± ±
1.4 0.5 0.7 0.2 0.1 0.1 0.1 0.1
a
Percentage of photo-induced absorptance changes, calculated using Eq. (1). Uncertainties reflect the accuracy of the measurement equipment. c Uncertainties reflect the fitting of individual curves to the double exponential decay (see Fig. 7, line fitted (Eq. (2)) for the 20 ppm decay data). Uncertainties for the measurement of different samples ≈0.6 (standard deviation derived from four measurements); and uncertainties for separate measurements of one sample ca. ≈0.12 (standard deviation derived from eight measurements). b
that is, three times higher than for the bR film without chemical additives. Furthermore, ammonia had a negligible influence on the photocycle parameters of the bR film with added TEA (Fig. 9). The calibration curves plotted for bR + gelatin and bR + gelatin + TEA using data from the exponential fitting (Eq. (2)) are compared in Fig. 10. The lifetime constant of the decay of the M410 state of the bR film with added TEA became dependent on the presence of ammonia only when film was exposed to high ammonia concentrations (>2000 ppm). The LODS calculated using Eq. (5) and the limiting linear slopes at the lowest detectable ammonia concentrations were 0.9 ppm and 24 ppm, and 0.2 s ppm−1 and 0.007% ppm−1 for the lifetime and photoinduced absorptance change, respectively.
Fig. 9. Time dependence of the photo-induced absorptance changes of the bR film with added TEA (mass ratio bR:gelatin:TEA = 12.4:73.5:13.8 measured at 25 ◦ C and rH 50%) monitored at 410 nm during and after actinic illumination (halogen light source with a high pass filter (>530 nm), arrows indicates the turning on and off of the actinic light source) in the presence of ammonia at different concentrations.
4. Discussion 4.1. Ammonia effect on the absorptance spectrum The results shown in Figs. 3 and 4a indicate that the absorptance decreases at 570 nm and increases at 410 nm; and the 570 absorptance maximum shifts towards the blue. These effects are similar to those engendered by dehydration of bR [9], which causes the absorptance to decrease at 570 nm and increase at 410 nm, along with a blue shift of the absorptance maximum at 570 nm. Dyukova et al. have proposed that this is due to dissociation of a water molecule specifically bound to the retinal chromophore [9]. Note that ammonia abstracts water to form hydroxyl ions − NH3 + H2 O NH+ 4 + OH
Fig. 8. Ammonia-induced absorptance changes shown as the ratio of the spectrum taken in 10,000 ppm ammonia to the spectrum taken in air: black line (1), bR film without added TEA (dried at rH 50%); red line (2), bR film with added TEA (mass ratio bR:gelatin = 12.5:87.5; bR:gelatin:TEA = 12.4:73.5:13.8; measured at 25 ◦ C and rH 50% without flow (see Section 2.4.1). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
(6)
and may therefore induce dehydration [12]. However, as demonstrated by Hildebrandt et al. [13], the presence of ammonia in a hydrated bR film leads to insignificant changes in the chromophoric structure. Thus, it seems that there is no structural disruption of the bR molecule, implying that ammonia is likely to have a reversible influence on the bR optical parameters, as is indeed corroborated by the results shown in Fig. 6. Note that the effect of pH per se is very much smaller [9] compared with our observation of ammonia effect. The absorptance decrease at 570 nm and increase at 410 nm may also be thought as a shift of the equilibrium between the ground state (bR570 ) and the intermediate (M410 ) state, similar to the action of actinic light, i.e. bR + NH3 bR∗
(7)
where bR* represents a modified form of bR (e.g. M410 ). The suppression of the sensitivity to ammonia by addition of TEA to the bR film (Figs. 5a and 8) would appear to result from
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Fig. 10. Calibration curves were plotted using the data from the double-exponential fitting (Eq. (2)) and represents (a) the lifetime of the M410 state; (b) the photoinduced absorptance changes (Eq. (1)); squares, bR film without added TEA; circles, bR + gelatin + TEA; points are connected merely to guide the eye. Measured in the presence of ammonia gas of different concentrations at room conditions of 25 ◦ C and rH 50%, mass ratio bR:gelatin = 12.5:87.5; bR:gelatin:TEA = 12.4:73.5:13.8 (see Figs. 6–8).
the fact that the NH2 group of the TEA then takes up the rˆole of the proton acceptor in the bR film. A possible mechanism of the TEA effect on bR was previously suggested by Batori-Tartsi and Ludmann [3]: they demonstrated that chemical additives act on the extracellular side of the purple membrane and take the rˆole of proton acceptor, i.e. the electronegative functional groups present in the TEA can capture the proton released on the extracellular side of the membrane during the photocycle [10]. Our results show that the presence of ammonia causes the bR photocycle to change in a similar way as with added TEA: the amplitude of the photo-induced absorptance changes and the lifetime of the bR film without TEA when exposed to ammonia was the same as that of the bR film with added TEA without ammonia exposure (see Fig. 10). Our results (Figs. 3 and 7) imply that ammonia (cf. Eq. (6)) decreases the energy barrier of the transition of the intermediate bR570 state to the M410 state. Even the relatively weak (∼0.1 mW/cm2 ) probe light causes a slight actinic effect on the
bR film when it is exposed to ammonia, i.e. shifts the equilibrium between the bR570 ground state and the intermediate M410 state (Fig. 4a). Moreover, an increase of the probe light irradiance (by about 10 times) caused a threefold increase of the S.R. (Fig. 11). 5. Summarizing conclusions The effect of ammonia on the optical properties of bR films was studied quantitatively. Sensor response of the bR film was linear in the 10–200 ppm range of ammonia concentration. The slope diminishes at higher concentrations, but from 1000 ppm to 10,000 ppm the response can be approximated as linear. Response was completely reversible with a recovery time of 100 s for the lower ammonia concentrations (up to 100 ppm), but at higher concentrations (>100 ppm) a longer flushing time (1000 s) was needed in order to achieve a complete recovery of the sensor response. The LOD for the bR film without added TEA was 5 ppm for ammonia-induced absorptance changes, 0.9 ppm when the time constant of the M410 state decay was measured, and 24 ppm for the photo-induced absorptance changes. The incorporation of chemicals with an amine functionality, such as triethanolamine, in the film suppresses the ammonia response, and is therefore useful for referencing. Acknowledgments We thank O.I. Korposh, I.J. Tcoma and V.V. Yarosh for their expert assistance with the bacteriorhodopsin sample preparation, and Dr S.-W. Lee of the University of Kitakyushu for providing us with some of the instrumentation. References
Fig. 11. Sensor response of the bR film to 500 ppm of ammonia at different light sources irradiances; black line, LED595 (0.4 mW/cm2 ); red line, halogen lamp with high pass filter (passing 530–2000 nm) (4 mW/cm2 ). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
[1] J. Hodgkinson, J. Saffell, J. Luff, J. Shaw, J.J. Ramsden, C. Huggins, R. Bogue, R. Carline, MNT Gas Sensors Roadmap, MNT Gas Sensors Forum, Cranfield University, 2006. [2] J.P. Sharkany, S.O. Korposh, Z.I. Batori-Tarci, I.I. Trikur, J.J. Ramsden, Bacteriorhodopsin-based biochromic films for chemical sensors, Sens. Actuator B 107 (2005) 70–81.
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[3] Z. Batori-Tartsi, K. Ludmann, Bioelectronic applications of photochromic pigments, in: A. D´er, L. Keszthelyi (Eds.), Bioelectronic Applications of Photochromic Pigments (NATO Science Series, Subseries I, Life and Behavioural Sciences), IOS Press, Amsterdam, 2001, pp. 137–148. [4] R.H. Lozier, R.A. Bogomolni, W. Stoeckenius, Bacteriorhodopsin: a lightdriven proton pump in Halobacterium halobium, Biophys. J. 15 (1975) 955–962. [5] D. Oesterhelt, W. Stoeckenius, Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane, Methods Enzymol. 31 (1974) 667–678. [6] Z. B´athori T´arczy, J. S´arkany, I. Bandrovszkaja, J. Sersun, O. Korpos, V. Jaros, Bakteriorodopszin-alapanyag´u fotochrom r´etegek, Tudom´anyos tal´alkoz´o, Budapest, 1992, pp. 110–112. [7] A. Kikineshy, Z. Bathori-Tarczy, Y. Sharkany, Materials and devices for fiber optic sensors, in: W.K. Jones, G. Hars´anyi (Eds.), Multichip Modules with Integrated Sensors (NATO ASI Series, Subseries 3, High Technology), Dordrecht, Kluwer, 1996, pp. 181–184. [8] L. Lensu, M. Frydrych, J. Parkkinen, S. Parkkinen, T. Jaaskelainen, Photoelectric properties of bacteriorhodopsin analogs for color-sensitive optoelectronic devices, Opt. Mater. 27 (2004) 57–62. [9] T. Dyukova, B. Robertson, H. Weetall, Optical and electrical characterization of bacteriorhodopsin films, Biosystems 41 (1997) 91–98. [10] Z. Batori-Tartsi, K. Ludman, G. V´ar´o, The effect of chemical additives on the bacteriorhodopsin photocycle, J. Photochem. Photobiol. B 49 (1999) 192–197. [11] M.E. Swartz, I.S. Krull, Analytical Method Development and Validation, Dekker, New York, 1997.
[12] T.V. Dyukova, E.P. Lukashev, Dehydration effects on D96N bacteriorhodopsin films, Thin Solid Films 283 (1–4) (1996). [13] P. Hildebrandt, M. Stockburger, Role of water in bacteriorhodopsin’s chromophore: a resonance Raman study, Biochemistry 23 (1984) 5539–5548.
Biographies Serhiy Korposh obtained his master’s degree in physics from Uzhgorod (Ungv´ar) National University, Transcarpathia, Ukraine (in 2002), and his doctorate from Cranfield University (in 2007). His research interests are metrology and chemical sensing using optical methods. Yosyp Sharkan obtained his master’s degree in physics and his doctorate (Cand. Sci.) from Lviv (Lemberg) National University, Ukraine. Since 1993 he has been head of the Department of Integrated Optics within the Institute of Solid-State Physics and Chemistry at Uzhgorod National University. From 2003 until 2006 he was a visiting researcher at Cranfield University at Kitakyushu. His research interests include thin film-based nanocomposites. Jeremy Ramsden obtained his bachelor’s and master’s degrees in natural sciences from Cambridge University, and his doctorate from the Ecole polytechnique f´ed´erale de Lausanne (EPFL). He has been a member of the faculty of Natural Philosophy at the University of Basel since 1994, and professor of Nanotechnology at Cranfield University (formerly Cranfield Institute of Technology) since 2002. He is also research director for bionanotechnology at Cranfield University at Kitakyushu. His research interests include the bio/non-biointerface, and the use of biomolecules in artificial adaptive systems.