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Taste sensing with cellophane phosphate membrane
Analytica Chimica Acta 554 (2005) 105–112
Taste sensing with cellophane phosphate membrane Sarmishtha Majumdar, Basudam Adhikari ∗ Materials Science Centre, Indian Institute of Technology, Kharagpur 721302, India Received 7 March 2005; received in revised form 5 August 2005; accepted 8 August 2005
Abstract There are reports of taste sensor fabrication by incorporation of lipids in a PVC membrane. Such lipid-based sensors were used to evaluate the senses of tastes such as sourness, saltiness, sweetness, bitterness and umami by measuring the membrane potential using Ag/AgCl electrodes. We have measured the senses of above five basic tastes with a functionalized polymer membrane without using any lipid. Commercial cellophane membrane was reacted with POCl3 for conversion to cellophane phosphate. FT-IR spectroscopy and XRD analysis were done to get an idea about the structure and the morphology of the membranes. The sensor characteristics like temporal stability, response stability, response to different taste substances, and reproducibility of sensing performance were studied using both cellophane and cellophane phosphate membranes. The sensor devices prepared with these membranes showed distinct response patterns in terms of membrane potentials for different taste substances. Both the membranes showed reproducible response patterns with a temporal stability of 30 min in 1 mM KCl solution and a stable response for 5 min in 1 mM solution of each of the taste substances such as HCl for sourness, NaCl for saltiness, sucrose for sweetness, quinine hydrochloride (Q-HCl) for bitterness and monosodium glutamate (MSG) for umami. The concentration threshold values of these membranes were compared with the human threshold values for the above taste substances. Both the membranes responded to lower threshold concentrations than the human detection limits. The membranes also showed characteristic response patterns for organic and mineral acids. Sensor device prepared with cellophane phosphate membrane has excellent shelf life. © 2005 Elsevier B.V. All rights reserved. Keywords: Taste sensor; Taste substances; Cellophane; Functionalized polymer
1. Introduction Sense of taste occurs as a result of interaction between taste buds of tongue and taste substance. The different lipid molecules in the taste buds of tongue are known to play the key role in sensing tastes of food materials [1,2]. Lipid-based multichannel artificial taste sensors were constructed for mimicking the taste sensing ability of humans [3–7]. In these taste sensors various lipids were immobilized, such as n-decyl alcohol, oleic acid, dioctyl phosphate, etc., in plasticized PVC for sensing of sourness, saltiness, bitterness, sweetness and umami. Hayashi et al. [3] fabricated a taste sensor consisting of multichannel electrodes with transducers composed of lipids immobilized within a plasticized PVC matrix. Such a multichannel lipid membrane device was claimed ∗
Corresponding author. Tel.: +91 3222 283966; fax: +91 3222 255303. E-mail address: [email protected] (B. Adhikari).
0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.08.019
to have the ability of sensing tastes as that done by human beings. This sensor was less sensitive to non-electrolytic substances than electrolytes. Although the sensing function of such membranes is dependent on their hydrophilicity as well as the ionic environment at the vicinity of the membrane surface but such membrane surfaces did not have uniform hydrophilicity since the lipid molecules were dispersed in plasticized PVC. A change in charge density on the membrane surface changes the electric potential of the membrane. The surface electric charge density and the permeability of ions of taste substances into the sensing membrane are altered by the physical adsorption of non-electrolytes, which causes changes of electric potential of the membrane [1,7–9]. By a simple method Hayashi et al. [10] adsorbed a lipid monolayer onto the surface of PVC membrane by hydrophobic effect [11,12]. Toko et al. [13] prepared taste-sensing membranes by mixing lipids with silicone rubber. They made three types of silicone membranes using dioctyl phosphate, trioctyl
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methyl ammonium chloride and a 5:5 molar mixture of dioctyl phosphate and trioctyl methyl ammonium chloride. Riul Jr. et al. [14] constructed an electronic tongue composed of polyaniline oligomers (16-mer) and polypyrrole (Ppy), which were able to distinguish salt, sweet, bitter, acidic solutions, different brands of mineral water, tea and coffee proving that conducting polymers are useful sensing materials. Hayashi et al. [15] examined lipid and ion exchange cellulose as transducer materials of a taste sensor. The membrane was constructed with lipid, protein, and glycocalyx. This transducer could detect all basic tastes, excepting the taste of sweet substances. Ethanol was also detected with the lipid/cellulose membrane. A self-assembled artificial membrane of poly(o-ethoxyaniline) with sulfonated lignin was satisfactorily used in the distinction of four brands of coffee [16]. Chitosan was alternated with sulfonated polystyrene (PSS) to build layer-by-layer (LBL) films that were used as sensing units in an electronic tongue [17]. Using impedance spectroscopy as the principal method of detection, an array using chitosan/PSS LBL film and a bare gold electrode as the sensing unit, was capable of distinguishing the basic tastes (salty, sweet, bitter, and sour) to a concentration below human threshold. It is obvious from the past reports that polymers, being tailorable material, are either used as supporting materials for lipids in taste sensing devices or directly contribute to the sensing role. We attempted, therefore, to utilize the tailorability of polymeric materials after suitable functionalization for direct assessment of the sense of taste. Novelty of this work lies in the use of phosphorylated cellophane membrane to act as taste sensing material without lipid molecules, which were used in majority of taste sensor devices. Commercial cellophane was phosphorylated to generate the active functional groups (>P(O) OH) for sensing the basic tastes, viz., sweetness, sourness, saltiness, bitterness and umami.
2. Materials and methods 2.1. Materials Cellophane was obtained from Kesoram Rayons, Kolkata (India). Phosphorus oxychloride (POCl3 ) was procured from Spectrochem Pvt Ltd., Mumbai (India). Triethylamine [(C2 H5 )3 N] was purchased from Qualigens Fine Chemicals, Mumbai (India). Tetrahydrofuran (THF) was taken from E. Merck (India) Limited.
2.3. Membrane characterization 2.3.1. Phosphorus estimation The cellophane membrane was converted to phosphate ester by phosphorylation. Phosphorus content of the membranes thus prepared was estimated by Heraeus Sch¨oniger Combustion Apparatus, Germany following the method of Sch¨oniger [18]. The film was wrapped in ashless filter paper and mounted onto the platinum holder of the Sch¨oniger combustion apparatus. About 10-ml distilled water was taken in the flask for absorption of the combustion products. Then the flask was filled with oxygen and closed with the platinum holder stopper assembly containing the sample. Through some electrical contact the filter paper along with the sample was ignited for complete combustion in oxygen atmosphere. After combustion the flask was allowed to stand for 15 min for absorbing the gaseous products thus formed out of combustion. The platinum holder was rinsed with distilled water. The absorption solution thus obtained was heated to 80◦ C and titrated with 0.005 M Ce3+ ion solution prepared by reducing ceric ammonium nitrate (CAN) solution with hydroxylamine. Eriochrome Black T was used as an indicator with hexamine as buffer. End point was detected by change of color from blue to red. From the titre value, wt.% P in the sample was calculated using the formula: %phosphorus =
titre × f × 15.49 wt. of sample
2.3.2. FT-IR analysis Structural analysis of cellophane and phosphorylated cellophane membranes was done by FT-IR (ATR) analysis using Thermo Nicolet, NEXUS 870 FT-IR spectrophotometer. 2.3.3. Contact angle measurement In order to judge the hydrophilicity [19,20] of the membrane surface contact angle study was done. Contact angle formed by a drop of distilled water on the membrane surface was measured by Rame goniometer (Model 100-00230). 2.3.4. Water absorption study To perform as a taste sensor, the membrane surface should be wetted by the aqueous solution of the substance whose taste is to be assessed. In order to judge the hydrophilicity, the polymer membranes were immersed in water for 24 h at 25 ◦ C and an increase in weight of the membranes was recorded and the result was expressed as %water absorption.
2.2. Membrane preparation Commercial cellophane film (1.4 g) was phosphorylated by refluxing with POCl3 (0.4 ml) in THF (150 ml) at 70 ◦ C for 2 h and 4 h in presence of triethylamine as catalyst. Finally the phosphorylated membrane was washed with distilled water to make it free from POCl3 and THF and dried. The dried membrane was stored in a vacuum desiccator.
2.3.5. Moisture absorption study Moisture absorption behavior of the cellophane membranes was assessed by exposing the membranes to the laboratory environment within a range of 35–55% relative humidity (RH) at 27–30 ◦ C. Weight gain of the membranes was measured at an interval of 24 h up to 7 days.
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Fig. 1. (a) Experimental set-up for measurement; (b) membrane electrode device used in the set-up.
2.3.6. XRD analysis XRD analysis of the cellophane and phosphorylated cellophane films was done with PW 1710 X-ray diffractometer ˚ and Ni-filter between 10◦ and with a Cu-target (λ = 1.5418 A) ◦ 60 (2θ). 2.4. Sensor set-up Fig. 1a shows the experimental set-up for the measurement of tastes of five basic taste substances, for example, NaCl for saltiness, HCl for sourness, quinine-HCl for bitterness, sucrose for sweetness and monosodium glutamate (MSG) for umami. As shown in Fig. 1b the membrane electrode device was fabricated by mounting cellophane and phosphorylated cellophane membranes over a circular cavity on a perspex block. The cavity was filled with a 100 mM KCl solution [3,10] through a narrow hole and a Ag/AgCl electrode was inserted into the cavity. The reference electrode device was constructed with a Ag/AgCl electrode enclosed in a glass tube filled with 100 mM KCl and 1% agar [3,10]. The two electrode terminals were connected to a digital multimeter for measuring the potential across the polymer membrane. The membrane electrode was preconditioned in a 1 mM KCl solution for 30 min. The effect of change in concentration of taste solutions on the potential was measured. Taste substances were dissolved in a 1 mM KCl solution [10]. All experiments were carried out at room temperature (25 ◦ C). 2.4.1. Temporal stability In order to judge the time required for obtaining stable response of electric potential (temporal stability) across the cellophane and phosphorylated cellophane membranes, the membrane electrode device was dipped in 1 mM KCl solution and the potential was measured using a Ag/AgCl reference electrode immediately after dipping at an interval of 1 min. The measurement was continued till stable response was obtained [3].
2.4.2. Response stability of the membrane The response stability of electric potential in a taste solution was studied after pretreatment of the membrane electrode device in a 1 mM KCl solution for 30 min. Then the response stability of electric potential of the cellophane and phosphorylated cellophane membranes was recorded for each of 1 mM HCl, NaCl, MSG, Q-HCl and sucrose solutions at an interval of 30 s. All these 1 mM taste solutions were prepared in 1 mM KCl. 2.4.3. Changes in response with repetitive use The change in response in terms of electric potential of cellophane and phosphorylated cellophane membranes to each taste substance was studied in three consecutive cycles of use. After each cycle of measurement the membrane device was rinsed with water and kept immersed in a 1 mM KCl solution for 5 min prior to next cycle of measurement.
3. Results and discussion 3.1. Membrane preparation In order to mimic the function of phospholipids in lipidbased taste sensors, the hydroxyl groups of cellulose in 20 m thick cellophane (Cello) membrane were phosphorylated by reacting with POCl3 in THF medium at 70 ◦ C in a reflux condition for 2 h (Cello-P-2) and 4 h (Cello-P-4). It was observed that the phosphorus content in the membrane increased from 0.11% to 0.4% with the increase in duration of phosphorylation from 2 to 4 h. For the measurement of taste sensing in terms of membrane potential, wetting of the membrane surface by the taste solution is required. Therefore, water absorption behavior and contact angle were measured for those membranes. Phosphorylation of cellophane caused very little change in its water absorption. Water
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Table 1 Moisture absorption characteristics of cellophane (Cello) and phosphorylated cellophane (Cello-P) membranes after exposure to 35–55% RH at 27–30 ◦ C for different time periods Polymer membrane
Cello Cello-P-2a Cello-P-4a
Moisture absorption (%) 24 h
48 h
72 h
96 h
120 h
144 h
168 h
2.71 3.50 2.63
3.46 3.66 3.31
2.71 2.81 2.72
1.03 1.62 2.04
1.87 2.30 2.21
1.31 2.15 1.87
2.81 2.04 2.21
a
Cello-P-2 is 2 h phosphorylated cellophane and Cello-P-4 is 4 h phosphorylated cellophane.
absorptions of cellophane and 4 h phosphorylated cellophane were 12.9% and 13.08%, respectively. As shown in Table 1, the moisture absorption of cellophane and phosphorylated cellophane membranes fluctuated with the variation of relative humidity (RH) from 35% to 55% at 27–30 ◦ C up to 7 days. Contact angles of cellophane and 4.0 h phosphorylated cellophane were found to be 73◦ and 78◦ , respectively. Thus water absorption, moisture absorption and contact angle studies showed sufficient hydrophilicity of the membrane for taste sensing in aqueous media.
Fig. 2. ATR spectra of cellophane and phosphorylated cellophanes between 1300 and 650 cm−1 .
3.2. Polymer characterization 3.2.1. FT-IR analysis FT-IR analysis (in ATR mode) of the surface of phosphorylated cellophane films was done to study the change in surface chemical structures of the polymer after phosphorylation (Fig. 2). Cello, Cello-P-2 and Cello-P-4 membranes show a broad peak for O H group between 3360 and 3376 cm−1 . The intensity of this peak decreased in case of phosphorylated cellophanes. Peak at 1045 cm−1 in cellophane for C O stretching and O H in-plane bending vibrations coupled disappeared in phosphorylated cellophanes. This may be due to esterification of O H groups to phosphate ester. A new peak appeared at 1119–1121 cm−1 in both the phosphorylated cellophane membranes for P O stretching of R2 (OH)P O group formed after phosphorylation. These observations support the formation of intermolecular phosphate diester linkage between the anhydro glucose mer units of two polymer molecules after phosphorylation as shown in Scheme 1.
Fig. 3. X-ray diffractogram of cellophane and phosphorylated cellophane membranes.
3.2.2. XRD analysis X ray diffractograms of both Cello and Cello-P-4 membranes are shown in Fig. 3. It is observed that the intensity
Scheme 1.
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Fig. 4. Temporal stability of potential of cellophane and phosphorylated cellophane membranes in 1 mM KCl.
of the peak at 12.55◦ (2θ) in cellophane is decreased after phosphorylation (Cello-P-4) indicating a little decrease in crystallinity after phosphorylation. 3.3. Study of taste sensing property of the film 3.3.1. Temporal stability of the membranes Temporal stability of the potential of cellophane and phosphorylated cellophane membranes in a 1 mM KCl is shown in Fig. 4. The potentials were measured immediately after immersion of the membrane electrode device in a 1 mM KCl solution and the membrane potential was measured at an interval of 1 min. As revealed from Fig. 4 cellophane and Cello-P-4 membranes showed stable response after 30 min. Based on this observation, the membrane electrode device was preconditioned for 30 min in a 1 mM KCl solution before measuring the response of taste solutions.
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Fig. 5. Stability of response potential of cellophane phosphate in 1 mM taste solutions (HCl, NaCl, Q-HCl, sucrose and MSG).
3.3.2. Response stability of the membrane In order to judge the response stability of the membrane in a particular taste solution the membrane electrode device was dipped in a taste solution of 1 mM concentration containing 1 mM KCl, and the potential was measured up to 5 min at an interval of 30 s. Fig. 5 shows the stability of response with time to HCl, NaCl, Q-HCl, MSG and sucrose for Cello-P4 membrane. Phosphorylated cellophane membrane showed better response stability than cellophane. This observation indicates that modified cellophane membrane is suitable for sensing taste of different substances. Since the magnitude of response potential is different for different taste substances such membrane is able to recognize different taste substances. 3.3.3. Responses to taste substances The responses of cellophane and phosphorylated cellophane membranes to five taste substances were studied for a concentration range of 0.001–100 mM solution and the results are shown in Fig. 6a and b. Using cellophane higher
Fig. 6. Responses in terms of membrane potential of (a) cellophane and (b) cellophane phosphate to HCl, NaCl, quinine-HCl (Q-HCl), monosodium glutamate (MSG) and sucrose.
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response potential was obtained for monosodium glutamate. This may be due to the formation of NaOH due to the hydrolysis of monosodium glutamate salt of weak acid and strong base. The increase in concentration of HCl, NaCl and quininehydrochloride also causes increase in electric potential. In case of sucrose there is a negligible change in potential. Threshold value for a particular taste substance was taken as the concentration at which upward or downward change in potential occurs [21]. For cellophane the threshold values for HCl, NaCl, Q-HCl, sucrose and MSG are 0.1, 0.008, 0.01, 0.1 and 0.8 mM, respectively. The membrane threshold concentrations were below human threshold concentrations, which are 0.9, 30, 0.03, 170 and 1.6 mM for HCl, NaCl, QHCl, sucrose [22] and MSG [23], respectively. Little improvement in the slope of the response curves for different taste substances was observed in case of Cello-
P-4 membrane (Fig. 6b) in comparison to that of cellophane (Fig. 6a). The response pattern for cellophane phosphate is similar to that of the lipid membranes prepared with dioctyl phosphate [3,10]. Detection threshold values of Cello-P-4 membrane for HCl, NaCl, Q-HCl, sucrose and MSG are 0.008, 0.01, 0.01, 0.1 and 0.1 mM, respectively, which are much below human threshold concentration. We have been able to improve the detection threshold values of the membrane for HCl, NaCl, Q-HCl, sucrose and MSG in comparison to that of lipid membranes prepared by Hayashi et al. [3], which are 0.009, 0.25, 0.06, 160 and 0.08 mM, respectively. The errors (%), for response potentials, defined by the standard deviations divided by the averaged values of cellophane for HCl, NaCl, MSG, Q-HCl and sucrose are 0.76, 1.25, 0.65, 1.44 and 0.52, respectively and that of Cello-P-4 for HCl, NaCl, MSG, Q-HCl
Fig. 7. Responses in terms of membrane potential of cellophane phosphate to (a) HCl, (b) NaCl, (c) Q-HCl, (d) sucrose and (e) MSG in three cycles with an interval of 5 min.
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Fig. 8. Responses in terms of membrane potential of cellophane phosphate to (a) organic acids and (b) mineral acids.
and sucrose are 9.87, 0.75, 1.35, 0.55 and 1.47, respectively. 3.3.4. Changes in response with repetitive use In order to see the reproducibility of response pattern of the membrane to a particular taste solution, the membrane electrode device was tested for repeatability by three consecutive measurements of response potential for each taste solution in the concentration range of 0.001–100 mM. Fig. 7 shows the reproducibility of response of Cello-P4 membrane to five taste substances. It was found that
the phosphorylated cellophane membrane showed almost identical response patterns on repeated measurements though there was a little drift of about 1 mV in each cycle. Cellophane membrane also showed good stability in response. In comparison to the lipid membranes, which are a physical mixture of lipid and polymer, the prepared cellophane phosphate membrane contains the phosphate diester group chemically bonded to the polymer. 3.3.5. Mechanism of response The responses in terms of membrane potentials occur due to some chemical interactions of the hydrophilic groups of the membrane with the ions in the taste solutions. In case of cellophane the increase in potential with the increase in concentrations of HCl, NaCl, Q-HCl may happen due to some ionic association between the cations (H+ , Na+ and Q H+ ) of taste solution and the OH groups of cellophane membrane surface. In case of cellophane phosphate, the phosphate groups >P(O) OH also dissociate in taste solution generating >P(O) O− ions leading to a change
in surface charge density, as occurs in colloidal systems [24,25]. This causes an accumulation of cations, which lead to increase in membrane potential. As quinine is a hydrophobic molecule, the quinine ion, i.e., Q H+ has a tendency to become associated with the hydrophobic part of the membrane. Monosodium glutamate hydrolyses in water forming sodium hydroxide and glutamic acid. In case of cellophane phosphate membrane MSG shows negligible change in potential at lower concentration. This may be due to accelerated dissociation of phosphate group in presence of glutamate ion [26]:
The second dissociation constant (4.9 × 10−5 ) of carboxyl group of MSG is smaller than the first dissociation constant (7.52 × 10−3 ) of phosphoric acid, i.e., of >P(O)O− H+ . This increases the negative charge on the membrane surface hence reduces the effect of Na+ ion on membrane potential. 3.3.6. Response to organic acids and mineral acids Cellophane phosphate membrane showed characteristic response to organic acids like acetic acid, citric acid, formic acid, oxalic acid and lactic acid as shown in Fig. 8a. Responses of cellophane phosphate to mineral acids like hydrochloric, sulphuric and nitric acids are shown in Fig. 8b. The variation in slopes of the curves is due to their differences in dissociation constants. Detailed study with mineral acids will appear in a future communication. 4. Conclusions The taste sensing property of cellophane and phosphorylated cellophane thus prepared was evaluated by studying
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the change in membrane potential with concentration of taste substances. The membranes showed a characteristic curve pattern with each of the taste substances. Phosphorylated cellophane showed improved taste sensor property. The threshold values of membrane response for different taste substances were below human threshold values. The cellophane phosphate membrane showed good stability of response in 1 mM taste solutions. Repeatability in response was quite good with only a small drift in potential (1–2 mV). The cellophane phosphate membrane also showed characteristic response patterns for organic acids and mineral acids.
References [1] K. Kurihara, K. Yoshii, M. Kashiwayanagi, Comp. Biochem. Physiol. 85A (1986) 1. [2] M. Miyake, N. Kamo, K. Kurihara, Y. Kobatake, Biochim. Biophys. Acta 436 (1976) 856. [3] K. Hayashi, M. Yamanaka, K. Toko, K. Yamafugi, Sens. Actuators B: Chem. 2 (1990) 205. [4] K. Toko, K. Hyashi, M. Yamanaka, K. Yamafuji, Ninth Sens. Symp. Tech. Digest., 1990, p. 193. [5] Y. Kikkawa, K. Toko, T. Matsuno, K. Yamafuji, Jpn. J. Appl. Phys. 32 (1993) 5731. [6] K.N. Mikhelson, Sens. Actuators B: Chem 18–19 (1994) 31. [7] S. Iiyama, K. Toko, K. Hayashi, K. Yamafuji, Agric. Biol. Chem. 53 (1989) 675. [8] S. Iiyama, K. Toko, K. Yamafuji, Agric. Biol. Chem. 50 (1986) 2709. [9] K. Hayashi, Y. Matsuki, K. Toko, T. Murata, Ke. Yamafuji, Ka. Yamafuji, Sens. Mater. 1 (1989) 321.
[10] K. Hayashi, K. Toko, M. Yamanaka, H. Yoshihara, K. Yamafuji, H. Ikezaki, R. Toukubo, K. Sato, Sens. Actuators B: Chem. 23 (1995) 55. [11] C. Tanford, The Hydrophobic Effect, Wiley, New York, 1980. [12] D.J. Shaw, Introduction to Colloid and Surface Chemistry, Butterwoth, London, 1980. [13] K. Tanaka, M. Iwakura, T. Onodera, T. Adachi, K. Toko, Chem. Sens. 17 (Suppl. B) (2001) 412. [14] A. Riul Jr, A.M. Gallardo Soto, S.V. Mello, S. Bone, D.M. Taylor, L.H.C. Mattoso, Synth. Met. 132 (2003) 109. [15] K. Hayashi, Y. Wakita, K. Moriyama, Kagoshima Daigaku Kogakubu Kenkyu Hokoku 37 (1995) 27. [16] A. Firmino Jr., R. Antonio, E.C. Venancio, F. Fonseca, L.H.C Mattoso, in: C., Mattoso, L., Henrique, L., Alcides, F., Elisabete (Eds.), Natural Polymers and Composites IV, Proceedings from the International Symposium on Natural Polymers and Composites, 4th, Sao Pedro, Brazil, September 1–4, 2002, p. 55. [17] D.S. dos Santos Jr., A. Riul Jr., R.R. Malmegrim, F.J. Fonseca, O.N. Oliviera Jr., L.H.C. Mattoso, Macro. Biosci. 3 (10) (2003) 591. [18] W. Sch¨oniger, Mikrochim. Acta (1955) 123. [19] C.C. Wamser, M.I. Gilbert, Langmuir 8 (1992) 1608. [20] D. Hosseinpour, N. Mohammadi, S. Moradian, Polym. Testing 22 (2003) 727. [21] K. Toko, Biomimetic Sensor Technology, Cambridge University Press, Cambridge, 2000, p. 120. [22] C. Pfaffmann, in: J. Field (Ed.), The Sense of Taste Handbook of Physiology, Section 1, Neurophysiology, vol. 1, American Physiological Society, Washington, DC, 1959, p. 507. [23] S. Yamaguchi, J. Food. Sci. 32 (1967) 473. [24] H. Tr¨auble, M. Teubner, P. Wooley, H. Eibl, Biophys. Chem. 4 (1976) 319. [25] H. Ohshima, T. Mitsui, J. Colloid Interface Sci. 63 (1978) 525. [26] S. Iiyama, H. Kuga, S. Ezaki, K. Hayashi, K. Toko, Sens. Actuators B 91 (2003) 191.
Taste sensing with cellophane phosphate membrane Sarmishtha Majumdar, Basudam Adhikari ∗ Materials Science Centre, Indian Institute of Technology, Kharagpur 721302, India Received 7 March 2005; received in revised form 5 August 2005; accepted 8 August 2005
Abstract There are reports of taste sensor fabrication by incorporation of lipids in a PVC membrane. Such lipid-based sensors were used to evaluate the senses of tastes such as sourness, saltiness, sweetness, bitterness and umami by measuring the membrane potential using Ag/AgCl electrodes. We have measured the senses of above five basic tastes with a functionalized polymer membrane without using any lipid. Commercial cellophane membrane was reacted with POCl3 for conversion to cellophane phosphate. FT-IR spectroscopy and XRD analysis were done to get an idea about the structure and the morphology of the membranes. The sensor characteristics like temporal stability, response stability, response to different taste substances, and reproducibility of sensing performance were studied using both cellophane and cellophane phosphate membranes. The sensor devices prepared with these membranes showed distinct response patterns in terms of membrane potentials for different taste substances. Both the membranes showed reproducible response patterns with a temporal stability of 30 min in 1 mM KCl solution and a stable response for 5 min in 1 mM solution of each of the taste substances such as HCl for sourness, NaCl for saltiness, sucrose for sweetness, quinine hydrochloride (Q-HCl) for bitterness and monosodium glutamate (MSG) for umami. The concentration threshold values of these membranes were compared with the human threshold values for the above taste substances. Both the membranes responded to lower threshold concentrations than the human detection limits. The membranes also showed characteristic response patterns for organic and mineral acids. Sensor device prepared with cellophane phosphate membrane has excellent shelf life. © 2005 Elsevier B.V. All rights reserved. Keywords: Taste sensor; Taste substances; Cellophane; Functionalized polymer
1. Introduction Sense of taste occurs as a result of interaction between taste buds of tongue and taste substance. The different lipid molecules in the taste buds of tongue are known to play the key role in sensing tastes of food materials [1,2]. Lipid-based multichannel artificial taste sensors were constructed for mimicking the taste sensing ability of humans [3–7]. In these taste sensors various lipids were immobilized, such as n-decyl alcohol, oleic acid, dioctyl phosphate, etc., in plasticized PVC for sensing of sourness, saltiness, bitterness, sweetness and umami. Hayashi et al. [3] fabricated a taste sensor consisting of multichannel electrodes with transducers composed of lipids immobilized within a plasticized PVC matrix. Such a multichannel lipid membrane device was claimed ∗
Corresponding author. Tel.: +91 3222 283966; fax: +91 3222 255303. E-mail address: [email protected] (B. Adhikari).
0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.08.019
to have the ability of sensing tastes as that done by human beings. This sensor was less sensitive to non-electrolytic substances than electrolytes. Although the sensing function of such membranes is dependent on their hydrophilicity as well as the ionic environment at the vicinity of the membrane surface but such membrane surfaces did not have uniform hydrophilicity since the lipid molecules were dispersed in plasticized PVC. A change in charge density on the membrane surface changes the electric potential of the membrane. The surface electric charge density and the permeability of ions of taste substances into the sensing membrane are altered by the physical adsorption of non-electrolytes, which causes changes of electric potential of the membrane [1,7–9]. By a simple method Hayashi et al. [10] adsorbed a lipid monolayer onto the surface of PVC membrane by hydrophobic effect [11,12]. Toko et al. [13] prepared taste-sensing membranes by mixing lipids with silicone rubber. They made three types of silicone membranes using dioctyl phosphate, trioctyl
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methyl ammonium chloride and a 5:5 molar mixture of dioctyl phosphate and trioctyl methyl ammonium chloride. Riul Jr. et al. [14] constructed an electronic tongue composed of polyaniline oligomers (16-mer) and polypyrrole (Ppy), which were able to distinguish salt, sweet, bitter, acidic solutions, different brands of mineral water, tea and coffee proving that conducting polymers are useful sensing materials. Hayashi et al. [15] examined lipid and ion exchange cellulose as transducer materials of a taste sensor. The membrane was constructed with lipid, protein, and glycocalyx. This transducer could detect all basic tastes, excepting the taste of sweet substances. Ethanol was also detected with the lipid/cellulose membrane. A self-assembled artificial membrane of poly(o-ethoxyaniline) with sulfonated lignin was satisfactorily used in the distinction of four brands of coffee [16]. Chitosan was alternated with sulfonated polystyrene (PSS) to build layer-by-layer (LBL) films that were used as sensing units in an electronic tongue [17]. Using impedance spectroscopy as the principal method of detection, an array using chitosan/PSS LBL film and a bare gold electrode as the sensing unit, was capable of distinguishing the basic tastes (salty, sweet, bitter, and sour) to a concentration below human threshold. It is obvious from the past reports that polymers, being tailorable material, are either used as supporting materials for lipids in taste sensing devices or directly contribute to the sensing role. We attempted, therefore, to utilize the tailorability of polymeric materials after suitable functionalization for direct assessment of the sense of taste. Novelty of this work lies in the use of phosphorylated cellophane membrane to act as taste sensing material without lipid molecules, which were used in majority of taste sensor devices. Commercial cellophane was phosphorylated to generate the active functional groups (>P(O) OH) for sensing the basic tastes, viz., sweetness, sourness, saltiness, bitterness and umami.
2. Materials and methods 2.1. Materials Cellophane was obtained from Kesoram Rayons, Kolkata (India). Phosphorus oxychloride (POCl3 ) was procured from Spectrochem Pvt Ltd., Mumbai (India). Triethylamine [(C2 H5 )3 N] was purchased from Qualigens Fine Chemicals, Mumbai (India). Tetrahydrofuran (THF) was taken from E. Merck (India) Limited.
2.3. Membrane characterization 2.3.1. Phosphorus estimation The cellophane membrane was converted to phosphate ester by phosphorylation. Phosphorus content of the membranes thus prepared was estimated by Heraeus Sch¨oniger Combustion Apparatus, Germany following the method of Sch¨oniger [18]. The film was wrapped in ashless filter paper and mounted onto the platinum holder of the Sch¨oniger combustion apparatus. About 10-ml distilled water was taken in the flask for absorption of the combustion products. Then the flask was filled with oxygen and closed with the platinum holder stopper assembly containing the sample. Through some electrical contact the filter paper along with the sample was ignited for complete combustion in oxygen atmosphere. After combustion the flask was allowed to stand for 15 min for absorbing the gaseous products thus formed out of combustion. The platinum holder was rinsed with distilled water. The absorption solution thus obtained was heated to 80◦ C and titrated with 0.005 M Ce3+ ion solution prepared by reducing ceric ammonium nitrate (CAN) solution with hydroxylamine. Eriochrome Black T was used as an indicator with hexamine as buffer. End point was detected by change of color from blue to red. From the titre value, wt.% P in the sample was calculated using the formula: %phosphorus =
titre × f × 15.49 wt. of sample
2.3.2. FT-IR analysis Structural analysis of cellophane and phosphorylated cellophane membranes was done by FT-IR (ATR) analysis using Thermo Nicolet, NEXUS 870 FT-IR spectrophotometer. 2.3.3. Contact angle measurement In order to judge the hydrophilicity [19,20] of the membrane surface contact angle study was done. Contact angle formed by a drop of distilled water on the membrane surface was measured by Rame goniometer (Model 100-00230). 2.3.4. Water absorption study To perform as a taste sensor, the membrane surface should be wetted by the aqueous solution of the substance whose taste is to be assessed. In order to judge the hydrophilicity, the polymer membranes were immersed in water for 24 h at 25 ◦ C and an increase in weight of the membranes was recorded and the result was expressed as %water absorption.
2.2. Membrane preparation Commercial cellophane film (1.4 g) was phosphorylated by refluxing with POCl3 (0.4 ml) in THF (150 ml) at 70 ◦ C for 2 h and 4 h in presence of triethylamine as catalyst. Finally the phosphorylated membrane was washed with distilled water to make it free from POCl3 and THF and dried. The dried membrane was stored in a vacuum desiccator.
2.3.5. Moisture absorption study Moisture absorption behavior of the cellophane membranes was assessed by exposing the membranes to the laboratory environment within a range of 35–55% relative humidity (RH) at 27–30 ◦ C. Weight gain of the membranes was measured at an interval of 24 h up to 7 days.
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Fig. 1. (a) Experimental set-up for measurement; (b) membrane electrode device used in the set-up.
2.3.6. XRD analysis XRD analysis of the cellophane and phosphorylated cellophane films was done with PW 1710 X-ray diffractometer ˚ and Ni-filter between 10◦ and with a Cu-target (λ = 1.5418 A) ◦ 60 (2θ). 2.4. Sensor set-up Fig. 1a shows the experimental set-up for the measurement of tastes of five basic taste substances, for example, NaCl for saltiness, HCl for sourness, quinine-HCl for bitterness, sucrose for sweetness and monosodium glutamate (MSG) for umami. As shown in Fig. 1b the membrane electrode device was fabricated by mounting cellophane and phosphorylated cellophane membranes over a circular cavity on a perspex block. The cavity was filled with a 100 mM KCl solution [3,10] through a narrow hole and a Ag/AgCl electrode was inserted into the cavity. The reference electrode device was constructed with a Ag/AgCl electrode enclosed in a glass tube filled with 100 mM KCl and 1% agar [3,10]. The two electrode terminals were connected to a digital multimeter for measuring the potential across the polymer membrane. The membrane electrode was preconditioned in a 1 mM KCl solution for 30 min. The effect of change in concentration of taste solutions on the potential was measured. Taste substances were dissolved in a 1 mM KCl solution [10]. All experiments were carried out at room temperature (25 ◦ C). 2.4.1. Temporal stability In order to judge the time required for obtaining stable response of electric potential (temporal stability) across the cellophane and phosphorylated cellophane membranes, the membrane electrode device was dipped in 1 mM KCl solution and the potential was measured using a Ag/AgCl reference electrode immediately after dipping at an interval of 1 min. The measurement was continued till stable response was obtained [3].
2.4.2. Response stability of the membrane The response stability of electric potential in a taste solution was studied after pretreatment of the membrane electrode device in a 1 mM KCl solution for 30 min. Then the response stability of electric potential of the cellophane and phosphorylated cellophane membranes was recorded for each of 1 mM HCl, NaCl, MSG, Q-HCl and sucrose solutions at an interval of 30 s. All these 1 mM taste solutions were prepared in 1 mM KCl. 2.4.3. Changes in response with repetitive use The change in response in terms of electric potential of cellophane and phosphorylated cellophane membranes to each taste substance was studied in three consecutive cycles of use. After each cycle of measurement the membrane device was rinsed with water and kept immersed in a 1 mM KCl solution for 5 min prior to next cycle of measurement.
3. Results and discussion 3.1. Membrane preparation In order to mimic the function of phospholipids in lipidbased taste sensors, the hydroxyl groups of cellulose in 20 m thick cellophane (Cello) membrane were phosphorylated by reacting with POCl3 in THF medium at 70 ◦ C in a reflux condition for 2 h (Cello-P-2) and 4 h (Cello-P-4). It was observed that the phosphorus content in the membrane increased from 0.11% to 0.4% with the increase in duration of phosphorylation from 2 to 4 h. For the measurement of taste sensing in terms of membrane potential, wetting of the membrane surface by the taste solution is required. Therefore, water absorption behavior and contact angle were measured for those membranes. Phosphorylation of cellophane caused very little change in its water absorption. Water
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Table 1 Moisture absorption characteristics of cellophane (Cello) and phosphorylated cellophane (Cello-P) membranes after exposure to 35–55% RH at 27–30 ◦ C for different time periods Polymer membrane
Cello Cello-P-2a Cello-P-4a
Moisture absorption (%) 24 h
48 h
72 h
96 h
120 h
144 h
168 h
2.71 3.50 2.63
3.46 3.66 3.31
2.71 2.81 2.72
1.03 1.62 2.04
1.87 2.30 2.21
1.31 2.15 1.87
2.81 2.04 2.21
a
Cello-P-2 is 2 h phosphorylated cellophane and Cello-P-4 is 4 h phosphorylated cellophane.
absorptions of cellophane and 4 h phosphorylated cellophane were 12.9% and 13.08%, respectively. As shown in Table 1, the moisture absorption of cellophane and phosphorylated cellophane membranes fluctuated with the variation of relative humidity (RH) from 35% to 55% at 27–30 ◦ C up to 7 days. Contact angles of cellophane and 4.0 h phosphorylated cellophane were found to be 73◦ and 78◦ , respectively. Thus water absorption, moisture absorption and contact angle studies showed sufficient hydrophilicity of the membrane for taste sensing in aqueous media.
Fig. 2. ATR spectra of cellophane and phosphorylated cellophanes between 1300 and 650 cm−1 .
3.2. Polymer characterization 3.2.1. FT-IR analysis FT-IR analysis (in ATR mode) of the surface of phosphorylated cellophane films was done to study the change in surface chemical structures of the polymer after phosphorylation (Fig. 2). Cello, Cello-P-2 and Cello-P-4 membranes show a broad peak for O H group between 3360 and 3376 cm−1 . The intensity of this peak decreased in case of phosphorylated cellophanes. Peak at 1045 cm−1 in cellophane for C O stretching and O H in-plane bending vibrations coupled disappeared in phosphorylated cellophanes. This may be due to esterification of O H groups to phosphate ester. A new peak appeared at 1119–1121 cm−1 in both the phosphorylated cellophane membranes for P O stretching of R2 (OH)P O group formed after phosphorylation. These observations support the formation of intermolecular phosphate diester linkage between the anhydro glucose mer units of two polymer molecules after phosphorylation as shown in Scheme 1.
Fig. 3. X-ray diffractogram of cellophane and phosphorylated cellophane membranes.
3.2.2. XRD analysis X ray diffractograms of both Cello and Cello-P-4 membranes are shown in Fig. 3. It is observed that the intensity
Scheme 1.
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Fig. 4. Temporal stability of potential of cellophane and phosphorylated cellophane membranes in 1 mM KCl.
of the peak at 12.55◦ (2θ) in cellophane is decreased after phosphorylation (Cello-P-4) indicating a little decrease in crystallinity after phosphorylation. 3.3. Study of taste sensing property of the film 3.3.1. Temporal stability of the membranes Temporal stability of the potential of cellophane and phosphorylated cellophane membranes in a 1 mM KCl is shown in Fig. 4. The potentials were measured immediately after immersion of the membrane electrode device in a 1 mM KCl solution and the membrane potential was measured at an interval of 1 min. As revealed from Fig. 4 cellophane and Cello-P-4 membranes showed stable response after 30 min. Based on this observation, the membrane electrode device was preconditioned for 30 min in a 1 mM KCl solution before measuring the response of taste solutions.
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Fig. 5. Stability of response potential of cellophane phosphate in 1 mM taste solutions (HCl, NaCl, Q-HCl, sucrose and MSG).
3.3.2. Response stability of the membrane In order to judge the response stability of the membrane in a particular taste solution the membrane electrode device was dipped in a taste solution of 1 mM concentration containing 1 mM KCl, and the potential was measured up to 5 min at an interval of 30 s. Fig. 5 shows the stability of response with time to HCl, NaCl, Q-HCl, MSG and sucrose for Cello-P4 membrane. Phosphorylated cellophane membrane showed better response stability than cellophane. This observation indicates that modified cellophane membrane is suitable for sensing taste of different substances. Since the magnitude of response potential is different for different taste substances such membrane is able to recognize different taste substances. 3.3.3. Responses to taste substances The responses of cellophane and phosphorylated cellophane membranes to five taste substances were studied for a concentration range of 0.001–100 mM solution and the results are shown in Fig. 6a and b. Using cellophane higher
Fig. 6. Responses in terms of membrane potential of (a) cellophane and (b) cellophane phosphate to HCl, NaCl, quinine-HCl (Q-HCl), monosodium glutamate (MSG) and sucrose.
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response potential was obtained for monosodium glutamate. This may be due to the formation of NaOH due to the hydrolysis of monosodium glutamate salt of weak acid and strong base. The increase in concentration of HCl, NaCl and quininehydrochloride also causes increase in electric potential. In case of sucrose there is a negligible change in potential. Threshold value for a particular taste substance was taken as the concentration at which upward or downward change in potential occurs [21]. For cellophane the threshold values for HCl, NaCl, Q-HCl, sucrose and MSG are 0.1, 0.008, 0.01, 0.1 and 0.8 mM, respectively. The membrane threshold concentrations were below human threshold concentrations, which are 0.9, 30, 0.03, 170 and 1.6 mM for HCl, NaCl, QHCl, sucrose [22] and MSG [23], respectively. Little improvement in the slope of the response curves for different taste substances was observed in case of Cello-
P-4 membrane (Fig. 6b) in comparison to that of cellophane (Fig. 6a). The response pattern for cellophane phosphate is similar to that of the lipid membranes prepared with dioctyl phosphate [3,10]. Detection threshold values of Cello-P-4 membrane for HCl, NaCl, Q-HCl, sucrose and MSG are 0.008, 0.01, 0.01, 0.1 and 0.1 mM, respectively, which are much below human threshold concentration. We have been able to improve the detection threshold values of the membrane for HCl, NaCl, Q-HCl, sucrose and MSG in comparison to that of lipid membranes prepared by Hayashi et al. [3], which are 0.009, 0.25, 0.06, 160 and 0.08 mM, respectively. The errors (%), for response potentials, defined by the standard deviations divided by the averaged values of cellophane for HCl, NaCl, MSG, Q-HCl and sucrose are 0.76, 1.25, 0.65, 1.44 and 0.52, respectively and that of Cello-P-4 for HCl, NaCl, MSG, Q-HCl
Fig. 7. Responses in terms of membrane potential of cellophane phosphate to (a) HCl, (b) NaCl, (c) Q-HCl, (d) sucrose and (e) MSG in three cycles with an interval of 5 min.
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Fig. 8. Responses in terms of membrane potential of cellophane phosphate to (a) organic acids and (b) mineral acids.
and sucrose are 9.87, 0.75, 1.35, 0.55 and 1.47, respectively. 3.3.4. Changes in response with repetitive use In order to see the reproducibility of response pattern of the membrane to a particular taste solution, the membrane electrode device was tested for repeatability by three consecutive measurements of response potential for each taste solution in the concentration range of 0.001–100 mM. Fig. 7 shows the reproducibility of response of Cello-P4 membrane to five taste substances. It was found that
the phosphorylated cellophane membrane showed almost identical response patterns on repeated measurements though there was a little drift of about 1 mV in each cycle. Cellophane membrane also showed good stability in response. In comparison to the lipid membranes, which are a physical mixture of lipid and polymer, the prepared cellophane phosphate membrane contains the phosphate diester group chemically bonded to the polymer. 3.3.5. Mechanism of response The responses in terms of membrane potentials occur due to some chemical interactions of the hydrophilic groups of the membrane with the ions in the taste solutions. In case of cellophane the increase in potential with the increase in concentrations of HCl, NaCl, Q-HCl may happen due to some ionic association between the cations (H+ , Na+ and Q H+ ) of taste solution and the OH groups of cellophane membrane surface. In case of cellophane phosphate, the phosphate groups >P(O) OH also dissociate in taste solution generating >P(O) O− ions leading to a change
in surface charge density, as occurs in colloidal systems [24,25]. This causes an accumulation of cations, which lead to increase in membrane potential. As quinine is a hydrophobic molecule, the quinine ion, i.e., Q H+ has a tendency to become associated with the hydrophobic part of the membrane. Monosodium glutamate hydrolyses in water forming sodium hydroxide and glutamic acid. In case of cellophane phosphate membrane MSG shows negligible change in potential at lower concentration. This may be due to accelerated dissociation of phosphate group in presence of glutamate ion [26]:
The second dissociation constant (4.9 × 10−5 ) of carboxyl group of MSG is smaller than the first dissociation constant (7.52 × 10−3 ) of phosphoric acid, i.e., of >P(O)O− H+ . This increases the negative charge on the membrane surface hence reduces the effect of Na+ ion on membrane potential. 3.3.6. Response to organic acids and mineral acids Cellophane phosphate membrane showed characteristic response to organic acids like acetic acid, citric acid, formic acid, oxalic acid and lactic acid as shown in Fig. 8a. Responses of cellophane phosphate to mineral acids like hydrochloric, sulphuric and nitric acids are shown in Fig. 8b. The variation in slopes of the curves is due to their differences in dissociation constants. Detailed study with mineral acids will appear in a future communication. 4. Conclusions The taste sensing property of cellophane and phosphorylated cellophane thus prepared was evaluated by studying
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the change in membrane potential with concentration of taste substances. The membranes showed a characteristic curve pattern with each of the taste substances. Phosphorylated cellophane showed improved taste sensor property. The threshold values of membrane response for different taste substances were below human threshold values. The cellophane phosphate membrane showed good stability of response in 1 mM taste solutions. Repeatability in response was quite good with only a small drift in potential (1–2 mV). The cellophane phosphate membrane also showed characteristic response patterns for organic acids and mineral acids.
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