BIOMIMETIC MEMBRANE: EFFECT OF CONCENTRATION OF PROPANOL ON THE TRANSMEMBRANE POTENTIAL AND IMPEDANCE

Transcription

1 INTRODUCTION: BIOMIMETIC MEMBRANE: EFFECT OF CONCENTRATION OF PROPANOL ON THE TRANSMEMBRANE POTENTIAL AND IMPEDANCE Deng Da Department of Chemical & Environmental Engineering National University of Singapore 10 Kent Ridge Crescent, Singapore Biological membranes are under intense investigations in the present, as every organism communicates with its environment via the membranes enveloping the material contents of the cells. However owing to the complexity of the biological memebranes, it has long been fashinonable to experiment with artificially consitituted membrane models [1]. Nitrocellulose filters, impregnated with fatty acids and their esters, are shown to be suitable for modelling of many physiochemical properties of biological membranes, such as electric resistance, capacitance, water permeability and ionic selectivity. These model membranes have a pronounced cation-anion and even cation-cation selectivity due to the presence of a nitrocellulose matric with fixed cation-exchange groups in the pores impregnated with lipid-like liquids and the interation of water with those groups [2]. Previous research in our lab under A/P N.M. Kocherginsky s supervision found that isopropyl myristate impregnated nitrocellulose filter is a stable biomimetic membrane, and its physiochemical properties, recalculated to the thickness of biological membrane, were found to be of the same order of magnitude as that of bioligcal membrane. It was discovered that this biomimetic membrane placed between two semi-chambers with concentraction gradient of K + forms stable transmemebrane potential of 0.1V, which agrees well with calculated value, based on Nernst equation for passive diffusion across membrane. Studies were carried out to investigate the effect of ionic surfactants, namely a cationic surfactant Cetyl-trimethy ammonium bromide (CTAB), and an anionic surfactant Sodium n-dodecyl benzyl sulfonate (SDBS), non-ionic surfactants Tween 20,Tween 80, Span 20 and 35 on the transmembrane potinetial (TMP). Sharp changes in TMP were observed in response to the addition of both ionic and non-ionic surfactants. These changes in potential were explained in earlier paper [3] to be the result of the complex formation of ethylene oxide (PO) groups of the surfactants with the cations of the aqueous solution, which are then carried to the membrane surface and hence produce the TMP. However studies in our lab under A/P N.M. Kocherginsky s supervision found that K + concentration and different cations used did not affect the surfactant-induced changes in TMP, and this suggests that the explaination by earlier paper [3] is not applicable to this case. A more appealing explaination is that the addition of surfactants modifies biomimetic membrane and makes it asymmetrical in physiochemical properties, thus resulting in the more cation selectivity of the biomimetic membrane on one side than on another. This UROP project aims to study changes of TMP and the sensitivity of the biomimetic membrane upon addition of propanol. It intends to check whether the TMP changes are a general phenomenon that can be induced by nonionic alcohol and find out the possible mechanism that gives the changes of TMP. It was discovered that the biomimetic membrane is sensitive to concentration of propanol in solution and gives a good calibration line between TMP changes and concentration of propanol. It proposes a possible simple technique for design a propanol sensor. Impedance of the biomimetic membrane was measured and changes of the transmembrane resistance and capacity of the membranes were affected by the concentration of propanol althouth the changes were not very significant. MATERIALS AND METHODS: Preparation of Biomimetic Membrane Nitrocellulose ultrafilter (10-2 cm thickness) with pore size of 0.45µm (Millipore filter category no. HAWP04700), and with pore size of 0.22µm (Millipore filter NO. GSWP04700), Isopropyl myristate ester (from Nakalai Tesuque, lot M7K6108, extra pure grade) were used without further purification. The filters were immersed in a bath of Isopropyl myristate ester for five minutes and pressed between folds of filter paper to remove the excess lipid. In the following paper, the biomimetic membrane will mean 0.45µm Nirocellulose ultrafilter impregnated with Isopropyl myristate ester, unless it was specially stated. Experiments Set Up

2 Autolab PGSTA-20 with General Purpose Electrochemical Station (GPES) software for potential measurement and Frequency Response Analyzer (FRA) for impedance measurement, two Teflon cylinder chambers with volume of 12ml each, buffer solution with 5mM KH 2 PO M KCl at ph5 for the reference chamber and 5mM KH 2 PO 4 at ph5 for the measuring chamber to set up a cation gradient, a magnetic follower for stirring in the measuring chamber, propanol (Merck, >98%) without further purification. The biomimetic membrane was placed between the two chambers of the cell and tightened in place. The buffer solution was added into the two chambers and the Ag/AgCl electodes were placed into the two sides of the chamber and connected to the Autolab for measurment. RESULTS AND DISCUSSION: A group of alcohol family chemicals, such as ethanol, propanol, butanol and pentanol were planned to be tested in our experimental set up to test the general phenomenon of alcohol-induced TMP changes of the biomimatic membrane, at temperature 25 0 C. Propanol was used in this project, and other alcohols will be tested in the further studies. The propanol is usually used as a solvent for extraction of natural products such as flavourings, vegetable oils, resins, waxes and gums, and it is also used as a solvent for synthetic polymers, such as polyvinyl butyral, cellulose esters, lacquers, and PVC adhesives 1. Stability of the TMP of the biomimetic membrane by K + gradient The initial TMP was obtained in the K + gradient (measuring chamber: 5mM KH 2 PO 4 and the reference chamber: 5mM KH 2 PO M KCl). The biomimetic membrane has K + /Cl - selectivity[4]. The experimental TMP is very stable and the value is around 0.1V, which agrees well with Nernst equation for passive diffusion across membrane: E = 2.3(RT/zF)log(C 1 /C 2 )=2.3*(8.314*298/1*96500)log(C 1 /C 2 )=0.059*log(0.505/0.005) 0.1V This TMP of the biomimetic membrane is very stable and can last more than 56 at temperature 25 0 C. 2. Effect of different concentratons of propanol on TMP of the biomimetic membrane After the TMP induced by K + gradient reaches the steady state, propanol was injected into the measuring chamber with stiring by a magnetic follower. The sharp 0.1 drop of TMP was observed immediately. After the minimum value it slowly returns back but it never was able to get back exactly to the initial TMP value. This is a general phenomenon for effect of propanol on biomimetic membrane, see the typical plot of potential changes vs time (Fig 1). Figure 1. Typical TMP change upon propanol added vs time The addition of different concentrations of propanol into the measuring chamber induced sharp changes in TMP, which varied with concentration of propanol (Fig 2). The observed phenomenon may be due to the fast absorption of the propanol at the interface leading to asymmetric membranes, followed by its slow diffusion, eventually reaching at a stable concentration distribution. The higher concentration of propanol injected, the greater immediate drop of the TMP was observed. E/V T/s Figure 2. TMP changes upon different concentrations of propanol (M), 1(0.015M), 2(0.03M), 3(0.066M), 4(0.197M), 5(0.45M), 6(0.63M), 7(0.80M), 8(1.33M), 9(2.42M), 10(2.89M). all at temperature 25 o C.

3 The sharp changes of TMP may be due to the fact that increase of the concentration of the propanol induces stronger interaction of the propanol 0.12 with membrane and assists more cation 0.1 transfer across the membrane. It was y = x discovered that the TMP change of the 0.08 biomimetic membrane is proportional to the 0.06 concentration changes of propanol injected 0.04 into the one side of the chamber at range from 0.015M to 3M. It was also observed that the 0.02 TMP drop magnitudes are a linear function of 0 concentration of the, propanol injected from concentration of propanol/m 0.015M to 3M, with slop (Fig 3.). This indicates that the biomimetic membrane electrode Figure 3. Fast drop of TMP vs concentration of propanol could be used as simple propanol sensor to test the concentration of propanol in aqueous solution. 3. Time dependent TMP changes Upon the injection of the varied concentrations of propanol into the measuring side, the TMP changed immediately without any delay, and researched the minimum eventually within few seconds that may be due to unavoidable operation errors. The fast absorption of the propanol on one side of interface has modified the membrane and maken it physiochemically asymmetric as the propanol disturbs the interface and then enters the membrane. After the TMP reaches the minimum, it slowly returns back towards initial TMP but the steady TMP value is always less than the initial value. The time needed for the TMP to get to a new steady state from the minimum value (lag time) is almost the same for each of the experiment or in other words the lag time is independent of the concentration of propanol. The whole process of the TMP changes can be stated in two steps: fast adsorption of propanol on the interface, and slow diffusion of propanol across the membrane towards stable concentration distribution. Symmetric membrane Highly Asymmetric Low Asymmetric Nearly Symmetric This biomimetic membrane is flexible and ready to change itself to keep a dynamic steady state in response to the changes of the environment (here the injection of propanol), which is very similar to the biological membranes that always keeps at dynamic steady state with the surrounding. 4. The effect of pore size on the kinetics of TMP It was observed that the membranes with smaller pore size of 0.22µm induced bigger TMP sharp drop and faster TMP changes at diffusion step, together with higher new stable TMP of the propanolmodified membrane (Fig 4). The maller pore size of the membrane means more interaction surface for propanol and the membrane. The total interface of the membrane with 0.22µm is bigger. For the same concentration of propanol injected, it has more impact on the interface and leads to larger TMP sharp drop. The molecule size of propanol is very small, so its diffusion across membrane is very fast. There are more pores available for propanol to diffusion across the membrane with 0.22µm than with 0.45µm pore size, which leads to the faster diffusion kinetics of the propanol diffusion step. The higher new TMP of the membrane with 0.22µm pore size may be due to that less propanol was remained in the membrane, as it is easier to across the membrane. Also on average propanol diffusion process is faster across the membrane and it has less delay time for propanol to be in the membrane. Thus the propanol has less modification on the biomimetic membrane. So the membranes with 0.22µm pore size are more sensitive than Figure 4. Comparison TMP changes of membranes with 0.22um and 0.45um membranes with 0.45µm in pore size upone the same amount of propano injected under same condition. response to the injection of the same concentration of propanol at same condition. 5. The steady state TMP changed by varied concentration of propanol. deltat E1/V

4 The propanol-modified membranes also have K + /Cl - selectivity and can form TMP in K + gradient. For the same K + gradient, it observed that the higher the concentration of propanol, the lower the TMP of the propanol-modified membranes (Fig 5). The possible mechanism of this phenomenon is that the propanol can reduce the interaction of ionized groups of the membrane with the ions in the electrolyte. It s also observed that at concentration up to 2.5M propanol, the TMP intends to be stable at 0.07V. This indicates the saturation of the propanol effect on the membranes. steady state TMP concentration of propanol/m Figure 5. New steady TMP vs concentration of propanol 6. Membrane Impedance [5] response to propanol Autolab with FRA software were used to measure the impedance of the biomimetic membrane [5]. The biomimetic membrane surface exposed to buffer 5mM KH 2 PO 4 at ph5 is 3.14cm 2, and the thickness is 10-2 cm. The resistance & capacity of the biomimetic membranes are 2.54x10 6 Ω & 7.13x10-11 F respectively (Fig 6), and it was stable for more than 24 hrs. For varied concentrations of propanol, all the impedance results gave depressed semi-cycles. It was observed that the higher the concentration of the propanol injected the lower the membrane resistance, although the resistance changes were not significant (Fig 7). For CPF, the higher the concentration of the propanol Figure 6. Typical Impedance curve of the injected, the higher the CPF value, although the CPF changes were biomimetic membrane not significant as well (Fig 8). The slight decrease in resistance and increase in capacity are due to the diffusion of propanol into the membrane from one side which has modified the interface, and also the internal structures of water channels of the liquid membrane and made the membrane slightly asymmetric. 1.60E-11 Membrane Resistance (ohm) 2.56E E E+06 Membrane capacitance/f 1.40E E E E E concentration of propanol /M 6.00E Concentration of propanol/m Figure 7. Membrane resistance vs concentration of propanol Figure 8. Transmembrane CPF vs concentration of propanol CONCLUSION Nitrocellulose filter impregnated with isopropyl myristate is a good model for biological membrane. It is cation selective and can form stable TMP with K + gradient. Propanol injected into the buffer in measuring chamber induces sharp decrease of TMP and then the TMP slowly returns towards initial TMP. Fast TMP drop is due to the modification of one interface of the biomimetic membrane by

5 propanol injected and it induced an asymmetry at the membrane. The slow TMP increase after that is due to the diffusion of propanol into the membrane, and after the propanol reaches stable concentration distribution the TMP researched a new stable value. The magnitude of the fast drop of TMP upon propanol injection gives a straight line and this could be used as a simple fast propanol sensor in aqueous solution. The impedance results show that at low concentration of propanol (<1M), the membrane is still can be treated as a biomimetic membrane due to its stable physiochemical properties. ACKNOWLEDGEMENTS The author would like to thank A/P N.M. Kocherginsky for his continual guidance and help. The author also would like to thank postgraduate Tan Chin Lee and the lab officer Sandy for their help and encouragement through the project. REFERENCES [1] R.C. Srivastava, Liquid Membrane Phenomena Biological Implications, Indian society for surface science and technology, Indian, 2002 [2] N.M. Kocherginsky, et al, The Modeling of Biological Membrane Properties by Means of Filters Impregnated with Lipid-like Substances, Journal of Membrane Science, 1987, p [3] N.N. Markuzina, et al, Electrode Properties of Film Membranes containing Alkoxylated Alkylphenols as Nonionic Surfactants, Russian Journal of Applied Chemistry, 66, , 1993 [4] N.M. Kocherginsky, et al, Generation of a Membrane Electrical Potential of Ultrafilters Impregnated with Fatty Acids, Russian Journal of Physical Chemistry, 1987, p.61 [5] Buck, R.P Impedance of Thin and Layered Systems: Cells with Even or Odd Numbers of Interfaces., Annals of Biomedical Engineering, 20, ,1991