Matrix influence on the optical response of composite bacteriorhodopsin films to ammonia

Sensors and Actuators B 133 (2008) 281–290

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Matrix influence on the optical response of composite bacteriorhodopsin films to ammonia S.O. Korposh a , Y.P. Sharkan b , M.Y. Sichka b , D.-H. Yang c , S.-W. Lee c,∗ , J.J. Ramsden a,d,∗∗ a

Cranfield University Kitakyushu Campus, 1-5-4F Hibikino, Kitakyushu 808-0135, Japan Institute of Solid-State Physics & Chemistry, Uzhgorod National University, Voloshina Street 54, 88000 Uzhgorod, Ukraine c Graduate School of Environmental Engineering, University of Kitakyushu, Japan d Department of Materials, Cranfield University, Bedfordshire MK43 0AL, England b

a r t i c l e

i n f o

Article history: Received 10 September 2007 Received in revised form 9 February 2008 Accepted 18 February 2008 Available online 4 March 2008 Keywords: Gelatin PVA Silica sol–gel Photoresponse Photocycle

a b s t r a c t The practical fabrication of thin-film chemical sensors generally requires the responsive element to be embedded in a matrix. The influence of different organic and inorganic matrices (polyvinyl alcohol (PVA), gelatin, and silica) on the static and dynamic optical properties of bacteriorhodopsin (bR)-containing films, including their responses to ammonia, were studied. Morphological and cross-sectional studies of the bR-containing films using atomic force microscopy, scanning electron microscopy, and energy dispersive spectroscopy revealed a uniform distribution of the bR within the films; the silica films were much rougher and more porous than those made with the polymers; gelatin tended to suppress the ammonia response (because of its amine groups); PVA and silica greatly slowed down the photocycle (because of their hydroxyl groups). The silica-based film had the fastest and most sensitive response to ammonia. © 2008 Elsevier B.V. All rights reserved.

1. Introduction For the last few decades biomolecules have attracted considerable research attention as possible components of electronics, optoelectronics and especially photonics devices, because of their densely sophisticated functionality [1]. One of the most important biomolecules in this regard is bacteriorhodopsin (bR), a representative photochromic bioorganic material [2]. It has already attracted attention as an ultrahigh density optical memory [3]. Genuine practical applications of bacteriorhodopsin appear to be feasible due to its ability to remain intact and functional under relatively harsh conditions, such as those in which Halobium salinarum lives [3,4]. Bacteriorhodopsin is naturally found in the outer membrane of the archea H. salinarum, where it constitutes a large fraction of the surface (30–50%). In most of the technological applications, it is however impractical to use the naked molecule: the polypeptide chain is folded nine times across the lipid bilayer membrane; it requires the presence of the lipid in order to maintain its conformation1 . Pure bacteriorhodopsin would presumably col-

∗ Corresponding author. ∗∗ Corresponding author at: Department of Materials, Cranfield University, Bedfordshire MK43 0AL, England. E-mail addresses: [email protected] (S.-W. Lee), j.ramsden@cranfield.ac.uk (J.J. Ramsden). 1 Note that it is possible to maintain bacteriorhodopsin in lipid cubic phases [5]. 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.02.038

lapse into an unspecified, denatured conformation. Fortunately, it is relatively easy to purify bacteriorhodopsin in the form of purple membrane fragments (pmf) [6]. They are rather stable, and indeed the dried form can be kept for decades without apparent deterioration of its properties [7,8]. In sensing and optical memory applications, thin films of bacteriorhodopsin-containing purple membrane fragments are typically required. The fabrication of the thin films is greatly facilitated if the pmf are dispersed in a matrix. The purpose of this paper is to determine the influence of the matrix on relevant properties of bacteriorhodopsin. With this knowledge, it should be possible to select the optimal material for a given application. Bacteriorhodopsin has a characteristic absorptance peak at 570 nm, which is attributed to the retinal chromophore covalently bound to the apoprotein of the bR molecule through a protonated Schiff base linkage. Absorptance of a light quantum by the retinal leads to a well-known sequence of reversible colour changes, from purple to yellow and so forth due to chromophore (retinal) deprotonation and protein conformational changes. The intermediate labelled M410 is the longest lived and the most useful bR state for many photonics applications, because during its creation and decay the largest changes occur in the optical characteristics of the bR molecule compared with those associated with the other intermediates (Fig. 1). Apart from optical memories, another promising application is as a chemical sensor [9,10]. Molecules such as NH3 or Cl2 engen-

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Scheme 3. Fragment of the structure of silica glass prepared by the sol–gel process.

Fig. 1. Absorptance spectra of the bR ground state (purple line, peak at 570 nm) and the M410 intermediate (orange line, peak at 410 nm). The sample was a bR-gelatin film with added triethanolamine (TEA) and dodecyltrimethylammonium bromide (DTMAB) (see Tables 1 and 2), referenced to a bR-free gelatin film. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

amino acids. Gelatin is rich in amine functionality (Scheme 2) [14]. The drawback of these two polymer matrices is their hygroscopicity, which can be problematic when monitoring moist atmospheres. Encapsulation of bR into a transparent, waterinsoluble matrix could be one way to solve this problem, provided a bR-compatible material of adequate optical quality can be found. The sol–gel process is a convenient and versatile method of preparing optically transparent and water-insoluble matrices at moderate temperatures [15]. Ambient processing conditions enable one to create composite optical materials with diverse organic, organometallic or biological molecules embedded within a porous non-crystalline glass matrix [16]. Scheme 3 is representative of the chemical structure of the silica glass prepared by the sol–gel process. Hydroxyl groups are present at the surface, as in PVA. 2. Experimental

Scheme 1. A fragment of the PVA molecule.

der significant changes in the optical spectra of the ground state and the photocycle intermediates. We shall use this phenomenon as a sensitive test of the possible influence of the matrix on the performance of the bR molecule in photonics applications. Matrices made from natural polymers such as gelatin [11] and synthetic polymers such as PVA [12] have already been used for bR film preparation. These matrix materials allow mechanically stable films with good optical quality to be manufactured. We have also recently successfully incorporated bR in an inorganic silica matrix [13]. These matrix materials are not chemically inert, but have functional groups that can in principle interact with the biomolecule—the protein, the chromophore or both—and hence modulate its optical properties in a way comparable to the action of the chemical being sensed. 1.1. Matrix-forming materials PVA is a water-soluble polymer, which forms a thin stable film upon dehydration. The main functional group is hydroxyl (Scheme 1). Gelatin is derived from skin or bones; the principle component is the protein collagen, which in gelatin normally contains 300–4000

2.1. Materials Bacteriorhodopsin was extracted as pmf (bacteriorhodopsin:lipid ratio = 75:25) from H. salinarum strain S9 cultured in our laboratory according to standard procedures [6]. The typical diameter of these fragments is 500 nm. The polymer films were prepared using photographic-grade hide gelatin (Sigma) and PVA (Sigma). Tetraethoxysilane (TEOS) (Fluka) was used as precursor for the preparation of the silica glass. Triethanolamine (TEA) (C6 H15 NO3 ) and dodecyltrimethylammonium bromide (DTMAB) (C28 H21 NBr) were of reagent grade (Sigma) and used without further purification. Film substrates were glass microscope slides (thickness ≈1 mm; roughness ≈0.25 ± 0.1 nm) cut to 5 cm2 squares. 2.2. Preparation of composite polymer films (Table 1) The bR films in gelatin and PVA matrices were prepared using standard procedures [3,17,18]. Briefly, a suspension of pmf (14 mg lyophilized powder/mL) was prepared by soaking for 20 min and then stirring for 6 h in tridistilled water. Solutions of gelatin or PVA were obtained by soaking the powdered polymer in tridistilled water for 20 min at room temperature and then stirring at 60 ◦ C for 40 min to obtain a 6% w/v solution. The film-forming solution, a mixture of bR and gelatin or PVA, was prepared from 0.168 mL of

Scheme 2. A typical fragment of gelatin (the amino acids -Ala-Gly-Pro-Arg-Gly-Glu-4Hyp-Gly-Pro-). 4Hyp denotes 4-hydroxyproline.

S.O. Korposh et al. / Sensors and Actuators B 133 (2008) 281–290 Table 1 Mass ratios of the bR films Sample

Mass ratio(s) (dry films)

bR:gelatin bR:PVA bR:gelatin:TEA bR:gelatin:TEA:DTMAB bR:silica Matrix-free bR

12.5:87.5 12.5:87.5 12.4:73.54:13.8 12.4:73.54:12.8:1.16 5.5:94.5 –

the bR suspension and 0.248 mL of the gelatin or PVA solution to give a final volume with tridistilled water of 0.5 mL. TEA was chosen as a 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 [19]). Since DTMAB also has a photosensitizing effect on bR [19] it can be inferred (by analogy with TEA) that DTMAB is associated with bR [19]. Consequently, DTMAB is useful as a tracer additive for determining the bR distribution in the matrix. The distribution of the bromine, assumed to be primarily associated with the bR, was used in order to distinguish the bR from the gelatin matrix, since both are of organic origin and otherwise would be difficult to distinguish from one another in electron micrographs. Film samples 10–20 ␮m thick were prepared by depositing the suspension onto the substrates by casting, giving a sample with an optical density of about 1 at wavelength  = 570 nm. The thickness can be controlled by deposition of the exact calculated volume of the film-forming solution onto the substrate of known area, followed by drying under ambient conditions. Afterwards, to verify the thickness of the film, its cross-section was analysed using scanning electron microscopy (SEM). The bR-based films were considered to be of good optical quality if they satisfied the criterion A280 /A570 < 2.5 [20]. The films were kept at room temperature. 2.3. Incorporation of bacteriorhodopsin into the silica matrix (Table 1) The silica glass was prepared using a slight modification of the method described by Weetall [21]: 7 mL TEOS, 3.0 mL distilled water and 0.1 mL 0.04 M HCl were mixed together and sonicated for 20 min. The resulting product was diluted with an equal volume of distilled water. 0.5 mL of this solution was mixed with 0.25 mL of a 0.1 mM aqueous sodium borate buffer (pH 9) to form the sol–gel mixture and either 0.1 mL or 0.2 mL bR solution (14 mg/mL) to create film-forming solutions with two different bR concentrations. More bR could not be added to the silica matrix because of excessive light scattering. For the preparation of films with chemical additives, the bR solution was mixed with TEA or DTMAB before mixing with the sol precursor, and afterwards 0.02 mL of the bR-chemical additives mixture was added to 0.12 mL of the sol–gel mixture. 2.4. Preparation of matrix-free bR films (Table 1) Matrix-free bR films were prepared by depositing a solution made up from 14 mg bR (lyophilized powder) in 1 mL water on the substrate, giving a sample with an optical density of about 1 at  = 570 nm. The films were kept at room temperature. 2.5. Structural and chemical characterization The morphology of the surfaces and the film cross-sections (prepared using a diamond cutter) were investigated to deduce

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the distribution of the purple membrane fragments within the films, using a Hitachi S-5200 field emission scanning electron microscope (FE-SEM) equipped with energy dispersive (X-ray) spectroscopy (EDS or EDX). The surface morphology of the silica films was also studied, with a JEOL JSPM-5200 atomic force microscope (AFM) working in non-contact mode using a MicroMash NSC12/Ti-Pt/15 silicon cantilever (curvature tip radius <40 nm, tip length 15–20 ␮m). For the SEM measurements the bR films were deposited onto 2.5 mm × 2 mm glass substrates in order to fit the sample holder of the FE-SEM. Before measurements the films were vacuum-dried for 6 h in order to remove practically all water from the samples, and then coated with a thin (5 nm) platinum film using a Hitachi E-1030 ion sputterer (15 mA, 10 Pa) to allow electrical discharge from the sample surface during its interaction with the electron beam. Porosity measurements of films on the bR-containing films were carried out using a Sorptomatic 1990 (Thermo Scientific) instrument operating on the static volumetric principle, i.e. characterizing porosity via gas adsorption. The specific surface areas of the matrices were determined by the Brunauer–Emmet–Teller (B.E.T.) method [22] and the pore size distribution was determined by the method of nitrogen desorption using the Barret–Joyner–Halenda (B.J.H.) model [23]. 2.6. Optical measurements The approach is described in detail elsewhere [24]; briefly, an Ocean Optics HR2000 spectrophotometer with a charge-coupled device (CCD) detector was used for recording the dynamic changes of the bR films, spectra before and after exposing them to ammonia. A tungsten halogen lamp (Ocean Optics HL-2000) radiating from 360–2000 nm was used as the probe light source for the spectral measurements. Optical fibres with an external diameter of 2 mm (core diameter = 1 mm, cladding thickness = 25 ␮m, and protection cladding thickness = 0.9 mm) were used to connect the sample holder, light source and spectrophotometer. It was verified that the effect of the probe light on the measured spectra (i.e. acting actinically) was negligible. Photo-induced absorptance changes were measured using an Ocean Optics LS-450 probe light source, fitted with light-emitting diodes (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 530 nm, i.e. transmitting only light with wavelength  > 530 nm) or an LED518 emitting at  = 518 nm (irradiances: halogen lamp, 4 mW/cm2 ; LED518 , 0.8 mW/cm2 ; measured using optical power meter PM140, Thorlabs Instrumentation) was used for the photoexcitation of the bR photocycle. Absorptance was measured at 410 and 570 nm, corresponding to the absorptance maxima of the main intermediate state of the bR photocycle (M410 ) and the ground state (bR570 ) (Fig. 1). 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 transmitted by the film. Photo-induced absorptance changes A were normalized according to: A =

A − Aa , A

(1)

where A is the absorptance of the bR film at the probe wavelength ( = 410 or 570 nm) without actinic light (unbleached sample), and Aa is the absorptance of the bR film in the presence of actinic light. Additionally, the lifetime of the M410 state after turning off the actinic illumination was measured. The decay kinetics required a double exponential to be fitted, but in order to characterize the

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Fig. 2. Typical films of bR in a gelatin matrix. (a) Scanning electron micrograph of the surface morphology; (b) scanning electron micrograph of the cross-section.

Fig. 3. Typical films of bR in a PVA matrix. (a) Scanning electron micrograph of the surface morphology; (b) scanning electron micrograph of the cross-section.

Fig. 4. Typical matrix-free bR film. (a) Scanning electron micrograph of the surface morphology; (b) scanning electron micrograph of the cross-section.

decay with a single parameter, we extracted the exact half life of the decay ( 1/2 ) from the fitted curve. Difference absorptance spectra (light minus dark) were obtained by subtraction of the spectrum recorded in the absence of the actinic light (dark) from the spectrum recorded in the presence of the actinic light (light). Normalization of the response to

ammonia was carried out using the following equation, yielding the percentage sensor response (S.R.): S.R. = 100

(A0 − A) , A0

(2)

where A0 is the absorptance of the bR film without ammonia and A

Fig. 5. Typical AFM images of the surface morphologies of films containing bacteriorhodopsin in matrices of: (a) gelatin and (b) PVA.

S.O. Korposh et al. / Sensors and Actuators B 133 (2008) 281–290 Table 2 Surface roughnesses (from the AFM) and thicknesses (from the SEM of the crosssections) of the films Sample

Ra /nm

bR:gelatin bR:PVA bR:gelatin:TEA bR:gelatin:TEA:DTMAB bR:silica Matrix-free bR film Gelatin PVA Silica

3.46 3.10 3.70 3.30 37.30 15.30 0.38 0.33 0.58

Thickness/␮m ± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

20.0 20.0 20.0 20.0 5.0 20.0 20.0 20.0 5.0

± ± ± ± ± ± ± ± ±

5.0 5.0 5.0 5.0 2.5 5.0 5.0 5.0 2.5.0

the absorptance in the presence of ammonia, measured at the same wavelength. 3. Results and discussion 3.1. Structural and chemical characterization 3.1.1. Polymer films The bacteriorhodopsin films with gelatin and PVA matrices have uniform surfaces (Figs. 2a and 3a); cross-sections suggest that the films are somewhat porous (Figs. 2b and 3b). Note that the structural qualities of the matrix-containing films are far superior to those of films cast without a matrix (Fig. 4). The morphologies of the bR films are made with gelatin and PVA matrices are further compared in Fig. 5. The surface roughnesses (Ra ) of gelatin and PVA matrices incorporating bacteriorhodopsin are summarized in Table 2. The uniform distribution of the bR in the gelatin matrix was further shown by measurements of the distribution of the chemical elements using EDS (Fig. 6). Results of the morphological, cross-sectional and energy dispersive studies (Figs. 2–6) of the bR-polymer films showed that there

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are no significant structural differences between the gelatin and PVA film structures; both polymer matrices allow films to be manufactured with uniform surfaces and distributions of the bR within the films. 3.1.2. Silica films The surfaces of the pure silica films (Fig. 7a) are uniform and have a low roughness. Incorporation of bR into silica matrix considerably increases the final film surface roughness (Fig. 7b and Table 2), apparently due to the formation of giant pmf aggregates. The crosssections were grainy, suggesting strongly developed micro- and mesoporosity (Figs. 8–11), which was confirmed by the porosity measurements (Table 3, Section 3.2). 3.2. Porosity Specific surface areas (SSA) and pore size distributions in the polymeric films and the silica are summarized in Table 3. The pore size distributions of all the bR films indicates welldeveloped micro- and mesoporosity. The specific surface area of the bR-containing silica films was 200 times greater than the bRcontaining polymer matrices (and the SSA of the gelatin films was greater than for the PVA). This should result in a faster response time when working as a sensor, which was confirmed by measurements of the optical response of the bR films to the presence of ammonia (Section 3.3.2, Table 5). 3.3. Optical characterization 3.3.1. Matrix effect Incorporation of the bR in the form of pmf into the polymeric matrix of the sensor element does not change the wavelength of the absorptance maximum at 570 nm. The optical absorptance spectra of the bR in the various matrices are compared in Fig. 12. The most significant result to emerge from the comparison (right

Fig. 6. Images of elemental distributions within a bR-containing film (gelatin matrix) formulated with a bromine-containing chemical additive (DTMAB) that is associated with the bR molecules. Data were acquired using an acceleration voltage of 25 kV in raster mode and with a magnification of 3000, from a film surface of 150 ␮m2 . (Panel b) SEM image of the film surface. The scale bars on the bottom of each panel show 10 ␮m.

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Fig. 7. AFM image of the surface morphology of: (a) a pure silica matrix and (b) a silica film incorporating bacteriorhodopsin.

Fig. 8. SEM images of the surface morphology of a film of the silica matrix: (a) scale bar 5 ␮m and (b) scale bar 500 nm.

Fig. 9. SEM images of the cross-section of a silica matrix; (a) scale bar 10 ␮m and (b) scale bar 1 ␮m.

hand plot) is the very high background scattering from the silica matrix. In contrast to the static optical spectra, the matrix has a strong effect on the photo-induced sequence of conformational

changes (photocycle). The difference absorptance spectra (light minus dark) of the films based on bR in different matrices are compared in Fig. 13. Exposure of the bR film to actinic light leads to the well-known photo-induced decrease of the absorptance

Fig. 10. SEM images of the surface morphology of a film of bR in a silica matrix: (a) scale bar 10 ␮m and (b) scale bar 500 nm.

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287

Fig. 11. SEM images of the cross-section of a film of bR in a silica matrix: (a) scale 10 ␮m and (b) scale 1 ␮m.

Fig. 12. Static optical absorptance spectra: (black) line 1, bR:gelatin matrix; (red) line 2, bR:PVA matrix; (orange) line 3, bR:silica matrix; (yellow) line 4, matrix-free bR (see Table 1 for the mass ratios); (a) spectra of the films as measured (see Table 2 for the thicknesses) and (b) absorptances normalized according to the quantity of bR in the film (extinction coefficient at 570 nm ε570 = 63,000 M cm−1 [2]). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Fig. 13. (a) Difference absorptance spectra (light minus dark) of the bR films in different matrices; (black) line 1, bR:gelatin matrix; (red) line 2, bR:PVA matrix; (orange) line 3, bR:silica matrix and (yellow) line 4, matrix-free bR. (b) Spectra in steady state under illumination divided point-by-point by those taken in the dark. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

at the 570 nm, and appearance of a new absorptance maximum at 410 nm. The amplitudes (and decay times—see later) of the M410 intermediate were different with the different matrices. There is already evidence that interaction between the bR amino acid residues and the surrounding matrix groups may chemically modulate the proton transport pathway, which is internally coupled with the photocycle, and consequently modulate the dynamical optical properties of bR-matrix composite films [12]. Moreover, since all of these matrices have different mechanical properties (e.g. the bulk moduli), shape-changing conformational changes [26] during the photocycle will be differently affected by the different matrices. Also, the different matrices interact differently with water [11,12,27]. In PVA, water is strongly attached via numerous hydrogen bonds to the hydroxyl groups of the polyol, and consequently the bR chromophore will be less exposed to water

[11]. Furthermore, different matrices possess different porosities (Table 3), which will also influence the amount of water in the vicinity of the bR molecule, which in turn will influence the photocycle [28,29]. Comparing the four different film types, from the difference absorptance spectra (Fig. 13) and the known fact that dehydration enhances the population of the M410 state under illumination [11] we can infer that the gelatin film contains the largest amount of available water and the PVA film the least. Fig. 14 shows an example of the kinetics of the photoinduced absorptance changes, and Table 4 summarizes the kinetic results for the different matrix materials. Not only is the absorptance change much greater with PVA and silica compared with the gelatin and matrix-free samples, but the lifetimes of the excited state are also significantly extended. The origin of this effect may well be the same as that suggested for the absorptance change enhancement—water scavenging (constrained water

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Table 3 Specific surface areas and pore size distributions Matrixa

Specific surface area (B.E.T.) (m2 g−1 )

Pore specific volume (cm3 g−1 )

Specific surface area (B.J.H.) (m2 g−1 )

Pore classes (B.J.H.) From (nm)

To (nm)

Ab (%)

1.5 3.0 5.0 50

13.9 41.9 8.9 32.0

Gelatin

3

0.003

3.45

0.0 1.5 3.0 10

PVA

2

0.001

2.93

0.0 1.5 3.0 10

1.5 3.0 5.0 50

15.5 44.5 13.9 22.1

Silica

677

0.520

–

0.0 1.5 3.0

1.5 3.0 5.0

17.9 58.9 13.9

Uncertainties were within the ISO limits [25]. a Containing bR (Table 1). b Percentage of pores in each class. Table 4 Photocycle optical parameters of bR-containing films Sample

Aa , b , c

bR:gelatin bR:PVA bR:silica Br

0.5 2.2 2.0 0.4

± ± ± ±

0.1 0.1 0.1 0.1

 1/2 (s)d 9.0 20.0 18.0 6.0

± ± ± ±

0.6 0.4 0.5 0.8

a

Wavelength of the probe light was 410 nm. Percentage of photo-induced absorptance changes, calculated using Eq. (1). c Uncertainties limited by the accuracy of the measurement equipment. d Given uncertainties originated from the fitting of the individual curves to the double exponential decay; uncertainties for consecutive measurements on one sample ≈0.12 (standard deviation derived from 8 measurements); uncertainties for the measurement of different samples ≈0.6 (standard deviation derived from 4 measurements). b

Fig. 14. Time dependence of photo-induced absorptance changes (A) of a bR:PVA film, measured at 25 ◦ C and rH 50%, monitored at 410 nm during and after actinic illumination (LED518 ), as indicated by arrows; normalization was carried out using Eq. (1); the smooth red line shows the double exponential fitting. Table 4 gives the parameters of these curves for all the films investigated. (For interpretation of the reference to colour in this figure legend, the reader is referred to the web version of the article.)

Fig. 15. Absorptance spectra of the bR:silica film measured at 25 ◦ C and rH 50%: (black) line 1, without ammonia and (red) line 2, in 10,000 ppm ammonia. The polymer matrices and matrix-free films demonstrated qualitatively identical responses; quantitative data are summarized in Table 5. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

availability has also been proposed as the reason for slower M state decay kinetics for bR entrapped in a silica xerogel [30]).

3.3.2. Response to ammonia The presence of ammonia leads to a decrease of the absorptance at 570 nm and a small absorptance increase at 410 nm (Fig. 15), in agreement with earlier results [9]. The difference absorptance spectra (ammonia atmosphere minus air) of the various films are compared in Fig. 16. The matrix also affects the optical response to ammonia. Since gelatin contains amine functionalities (Scheme 2), the lower sensitivity to ammonia is in accord with our previously inferred principle of homosuppression of the chemical sensitivity [9], but this obviously cannot apply to the PVA or silica matrices. Fig. 17 shows the sensor response of bR in a silica matrix to increasing ammonia concentrations. From these results the limit of detection (LOD) and sensitivity were estimated (Table 5). These results show that the choice of matrix significantly influences sensor sensitivity. The response time t90 of the bR film in the silica matrix was faster compared to the bR film based on the gelatin matrix and the matrix-free bR film (Table 5). This is most likely a simple consequence of the high porosity of the silica: 200 times greater than that of the polymeric films (Table 3). A higher matrix porosity allows faster diffusion of the ammonia gas into the bR film structure, hence enabling a faster reaction between bR and the analyte.

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Table 5 Response times and limits of detection of bR films to the presence of ammonia (measured at 25 ◦ C and rH 50%) Sample

bR:gelatin bR:PVA bR:silica bR matrix-free

Response timesa

LODb (ppm)

t90

t50

120 min 833 min 10.7 min 28 min

67 min 137 min 113 s 5.8 min

5 10 8 3

Linear range (ppm)

Sensitivity  (%/ppm)

10–200 –c 10–200 10–200

−0.035 −0.020 −0.017 −0.040

± ± ± ±

0.003 0.006 0.002 0.004

a Response times determined as the interval needed for the signal to achieve 90% or 50% of its saturation value when the film was exposed to an ammonia concentration of 1000 ppm. b The limit of detection (LOD) was defined according to LOD = 3/, where  is the standard deviation, and  is the slope (S.R./c) of the calibration curve, where c is ammonia concentration [31]. c For bR:PVA films the response to ammonia (10,000 ppm) was not reproducible, and parameters are evaluated from one measurement, thought to be typical.

Fig. 16. (a) Difference absorptance spectra (10,000 ppm ammonia minus air); (b) ratio of the spectra taken in the ammonia divided point-by-point by those recorded in air; (black) line 1, bR:gelatin; (red) line 2, bR:PVA; (orange) line 3, bR:silica and (yellow) line 4, bR matrix-free film. All samples were measured at 25 ◦ C and rH 50%. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

The effect of the matrix—directly and through its chemical interaction with ambient water, etc.—on the actual response of the active molecule must also be taken into account. The highest gain  was given by the matrix-free film. All the matrices tended to depress , but gelatin did so the least. The introduction of hydroxyl groups (notably by silica and PVA) scavenges water and has the effect of dehydrating in the film [32]. This retards the photocycle. Thus, the fastest one runs in the matrix-free film, the next fastest in gelatin, and finally PVA and silica are the slowest. We note that using the dynamical adsorption properties for sensing purposes enables a much more sensitive response to be obtained then via static absorption. In the latter case, all the matrices are rather similar, except that the silica gives a higher scattering, and hence a higher background and adsorption. Acknowledgements Fig. 17. Sensor response of the bR:silica film to increasing concentrations of ammonia (indicated by the different colours), monitored at 580 nm. The measurements were done by filling the measurement chamber with the ammonia gas of the desired concentration together with nitrogen. Properties of films based on polymer matrices and the matrix-free films gave qualitatively identical responses, and quantitative data are summarized in Table 5. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

We thank O.I. Korposh, I.J. Tcoma and V.V. Yarosh for their expert assistance with the bacteriorhodopsin sample preparation, and A. ´ for calling attention to the important paper by Lazarev and Der Terpugov (ref. [29]). References

4. Conclusions In a design of a sensor, a matrix in which the active molecule is embedded is usually needed for ease of manufacture and to provide good optical quality, and is also very important for film stability. The polymeric matrices gave a higher structural quality, but silica gave much higher porosity. Access of analyte critically depends on the porosity of the matrix. From this viewpoint, silica is the best. The final choice of matrix material depends however on a judicious compromise between conflicting requirements.

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Biographies ´ Serhiy Korposh obtained his master’s degree in physics from Uzhgorod (Ungvar) 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. Mykhaylo Sichka graduated in physics from Uzhgorod National University in 1969, and obtained his doctorate (PhD) in physics from Chernovitski National University in 1982. He is head of the Department of Thin Film Structures within the Institute of Solid State Physics and Chemistry, Uzhgorod National University. His research interests include thin film-based nanocomposites. Do-Hyeon Yang obtained his bachelor’s and master’s degrees in chemistry from Chungbuk National University, Korea (in 1995 and 1998, respectively) and his doctorate from the University of Kitakyushu, Japan (in 2006). His research interests are organic/inorganic thin films and chemical sensing. Seung-Woo Lee obtained his bachelor’s and master’s degrees in chemistry from Chungbuk National University, Korea and his doctorate in chemistry and biochemistry from Kyushu University, Japan (in 1999). He has been a member of the Spatio-Temporal Function Materials Research Group in the Frontier Research System, RIKEN and Associate Professor of the Graduate School of Environmental Engineering of the University of Kitakyushu since 2000. His research interests include molecular imprinting using metal oxide thin films, molecular recognition and chemical sensors. Jeremy Ramsden obtained his bachelor’s and master’s degrees in natural sciences ´ erale ´ from Cambridge University, and his doctorate from the Ecole polytechnique fed 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/nonbio interface, and the use of biomolecules in artificial adaptive systems.