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ORIGINAL ARTICLE
13 (
1
); 423-430
doi:
10.1016/j.arabjc.2017.05.013

Interaction of cholesterol with artificial bilayer lipid membrane system and development of an electrochemical sensor

, , ,
a Department of Electronics and Communication Engineering, PSG College of Technology, Coimbatore, Tamil Nadu 641 004, India
b Department of Biomedical Engineering, PSG College of Technology, Coimbatore, Tamil Nadu 641 004, India
c Department of Chemistry, PSG College of Technology, Coimbatore, Tamil Nadu 641 004, India

⁎Corresponding author. alagappanpsg@gmail.com (M. Alagappan) man@ece.psgtech.ac.in (M. Alagappan)

Received: , Accepted: ,
© The Author(s) 2017
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
1 Department of Chemistry, Sri Vasavi College, Erode, Tamil Nadu 638 316, India.

Abstract

Interaction of Cholesterol with the bilayer arrangement of phospholipid molecules was studied using electrochemical impedance spectroscopy in Sodium Chloride (NaCl) bath solutions. The membrane resistance (Rm) was decreased from 3.35 GΩ in 1.0 M NaCl bath to 0.756 GΩ in 0.01 M NaCl bath. The cholesterol molecules were found to penetrate into Bilayer Lipid Membrane (BLM) and fluidized the BLM phase. Due to fluidization, the membrane resistance was decreased. The fluidization effect of cholesterol was dependent on the concentration of bath solutions. In 1.0 M NaCl bath solution, the membrane was stable up to 200 µM concentration of cholesterol. With the addition of cholesterol in NaCl bath solutions, the membrane capacitance was increased. An impedimetric sensor was developed based on the membrane resistance in the presence of cholesterol at various concentrations. The detection limit of cholesterol by impedimetric sensor was dependent on the concentration of NaCl in the bath.

Keywords

Cholesterol sensor
Electrochemical sensor
Impedimetric sensor
Bilayer lipid membrane
Charge transfer resistance
1

1 Introduction

Phospholipid molecules are amphipathic molecules that possess both hydrophobic and hydrophilic sections in their structure. These molecules form more than 50% of plasma membranes in living organisms. The plasma membranes play an important role in the cell metabolism and control the materials flow in and out of the cells and cell organelles. The composition and type of phospholipid molecules in the plasma membrane varies in different cells. Mammalian cells require cholesterol for normal cell functions, which can be fulfilled by endogenous biosynthesis of sterol or by extracellular supplements (Bhattacharya and Haldar, 2000). Sometimes both mechanisms are used for cholesterol supply (Yeagle, 1985). The presence of cholesterol in the plasma membrane modifies its physical properties (Vilchèze et al., 1996). Many studies on cholesterol membrane interaction using Electron Spin Resonance (ESR) (McConnell and Hubbell, 1971; Delmelle et al., 1980), Raman Spectra (Lippert and Peticolas, 1971; Mendelsohn, 1972) and Fourier Transform Infrared Spectroscopy (FT-IR) (Umemura et al., 1980) led to a conclusion that cholesterol fluidizes and/or induces disorders in the bilayer arrangement of phospholipid molecules in the cell membranes. The Neutron and X-ray Diffraction studies on cholesterol membrane interactions showed that cholesterol in the bilayer arrangement of phospholipid molecules is held by hydrogen bonding between the carbonyl group of esterified acyl groups and –OH group in cholesterol (Huang, 1977; Franks and Lieb,1979; Bhattacharya and Haldar, 2000). Thus, it is well documented that cholesterol plays an important role in modifying fluidity and mechanical properties of bio membranes (Chiu et al., 2002).

Cholesterol induces fluidity of hydrocarbon chains below the phase transition temperature of membrane and decreases the fluidity of the membrane above phase transition temperature (Lewis and McElhaney, 1992; Maulik and Shipley, 1996; Ohvo-Rekila et al., 2002). The largest contribution to cholesterol / phospholipid interactions appears to be from Van der Waals forces and hydrophobic forces (McMullen and McElhaney, 1996; Israelachvili, 2011). Besides these forces the ionic phospholipids such as phosphatidylcholine can interact electrostatically (Yeagle, 1985; Israelachvili, 2011).

Many sensors have been developed to quantify cholesterol in solution using the enzyme Cholesterol Oxidase and most of them are electrochemical sensors. So far no electrochemical sensor is developed without the enzyme and H2O2. Thus, the present work deals with the ability of cholesterol to fluidize experimental membrane system and to develop a sensor to quantify it using electrochemical impedance spectroscopy.

2

2 Materials and methods

Egg lecithin (L-α-Phosphatidylcholine (99%)) and cholesterol (99%) were purchased from Sigma-Aldrich and used as received without further purification. A stock solution of egg lecithin in chloroform (5 mg/mL) was prepared to form the Bilayer Lipid Membrane (BLM). Under nitrogen atmosphere, 100 µL of the egg lecithin stock solution was evaporated in 2 mL Screw-Cap tube. The lipid film was obtained and is dissolved in 200 µL of n-Decane.

Indigenous chamber was constructed by fixing a 1 mm thick Polymethylmethacrylate (PMMA) septum containing a 1.3 mm diameter aperture between the 5 ml cavities drilled into two halves of the PMMA block cut from 88 × 62 × 24 mm block and BLMs were formed using this chamber (Mallaiya et al., 2014).

By the standard procedure, Stirring device, Faraday cage and Ag/AgCl electrodes were fabricated (Jain, 1972; Coster and Smith, 1974; Tien, 1974; Watts et al., 1981; Favero et al., 2002; Han et al., 2002; Römer and Steinem, 2004; Hanke and Schulue, 2012). To avoid the interference from floor vibrations, the vibration isolated platform (MINUSK USA) was used. Using the Potentiostat (PARSTAT 2273, Princeton Applied Research – USA), electrochemical impedance plots were recorded and the data were analyzed using the ZSimpWin 3.21 software. The impedance spectra were recorded by superimposing an AC sinusoidal voltage of amplitude 25 mV in the frequency range 1 M Hz–10 mHz.

Cholesterol stock solution (13.6 mM) was prepared by dissolving 500 mg of cholesterol in 5 mL of hot Triton-X 100 (Sigma-Aldrich) in a 250 mL wide mouth erlenmeyer flask with continuous stirring. To the solution, so prepared 90 mL double distilled water was added with continuous stirring.

The procedure proposed by Tien (1974) was followed to form the BLMs. 2 μL of the lipid dispersion was applied to the aperture in the precondition process, after which the required bathing solutions were added to the chambers. Then, 5 μL of the BLM forming solution was added directly over the aperture. A bilayer of lipid molecules resulted spontaneously with the exclusion of hydrocarbon solvents.

After the cholesterol solution is added, a stabilization period of 30 min was provided for the cholesterol to equilibrate between the lipid bilayer and aqueous phases. In accordance with earlier reports, each experiment was repeated 3 to 5 times to confirm the trend and to avoid errors.

3

3 Results and discussion

The changes in the electrical properties of black lipid membrane due to addition of cholesterol solution in NaCl bath solutions were studied and compared.

3.1

3.1 Electrical characterization of interaction of cholesterol with BLM

The Nyquist plots obtained for bare and cholesterol penetrated BLMs in the frequency range 10 mHz–1 MHz are shown in Figs. 1–3 for 1.0 M, 0.1 M and 0.01 M NaCl bath solutions respectively. The observed curves are double semicircles, with their centers on the real axis. The first semicircle observed at the highest frequencies represents the BLM surface bath solution interface (Mallaiya et al., 2014). Due to excess negative charge on the membrane surface, it attracts oppositely charged ions from the solution and forms this double layer. The second semicircle observed represents the membrane phase formed by the bilayer arrangement of lipid molecules. The diameter of the semicircles decreases with cholesterol concentration in the bath solution. This indicates that the decrease in membrane resistance or increase in membrane conductance with cholesterol concentration is a characteristic property of the membrane to become much leakier to smaller cations such as Na+ and K+ due to fluidization of the membrane in the presence of cholesterol. Since the curves are double semicircle in nature, the lipid bilayers are dielectric layers and have leakage (Coster, 2003; Naumowicz and Figaszewski, 2003).

Nyquist plots of interaction of cholesterol with BLM in 1.0 M NaCl bath solution.
Figure 1 Nyquist plots of interaction of cholesterol with BLM in 1.0 M NaCl bath solution.
Nyquist plots of interaction of cholesterol with BLM in 0.1 M NaCl bath solution.
Figure 2 Nyquist plots of interaction of cholesterol with BLM in 0.1 M NaCl bath solution.
Nyquist plots of interaction of cholesterol with BLM in 0.01 M NaCl bath solution.
Figure 3 Nyquist plots of interaction of cholesterol with BLM in 0.01 M NaCl bath solution.

The impedance spectra obtained are not straightforward in most of the cases and the analysis is carried out by fitting an equivalent circuit (Tien and Ottova, 2001; Mallaiya et al., 2014) assembled by resistors and capacitors representing the dominant components of biomimetic membranes (Guidelli and Becucci, 2012; Mallaiya et al., 2014). The membrane can be considered to have slabs with different dielectric properties. The ionic current rises due to the flow of ions across each slab and the capacitive current rises due to the accumulation of ions at the boundaries between the slabs (Guidelli and Becucci, 2012; Mallaiya et al., 2014). Therefore, each slab of the membrane can be simulated by a parallel combination of a resistor and capacitor (RC mesh). Hence, a commonly accepted -R(RC)(RC)- model shown in Fig. 4 (Han et al., 2002) was employed to obtain the electrochemical impedance parameters where P and H stand for polar groups and hydrocarbon tails of the bilayer phospholipid membranes respectively.

Equivalent circuit of planar BLM.
Figure 4 Equivalent circuit of planar BLM.

The membrane capacitance (Cm) and its thickness are related by the equation

(1)
C m = ε o ε A / d where ‘εo’ is the permittivity of free space (εo = 8.854 × 10−14 F cm−1), ε is the dielectric constant of the lipid bilayer (ε = 2.05) and ‘A’ is the surface area of BLM (Watts et al., 1981).

Therefore, the thickness of BLM formed in 1.0 M, 0.1 M and 0.01 M NaCl bath solutions is calculated and the values are 4.6 nm, 5.1 nm and 5.5 nm respectively. These values are very close to twice the thickness of lecithin monolayer (2.5 nm) (Favero et al., 2002; Römer and Steinem, 2004; Guidelli and Becucci; 2012) .Hence, the formed lipid membrane is considered as bilayer membrane.

The bilayer lipid membrane is electrically represented by an equivalent circuit shown in Fig. 4. To find out the electrical component showing more contribution to the net impedance at a particular frequency domain, the following rule is applied. When the impedance values of two circuit elements differ greatly, the overall impedance measured at a frequency will have more contribution from the circuit element having higher impedance, if they are connected in series. When the circuit elements are connected in parallel, the overall impedance will have more contribution from the circuit element of lower impedance (Guidelli and Becucci, 2012; Mallaiya et al., 2014). In two RC meshes (Fig. 4) connected in series in the accepted model for black lipid membrane, the first RC mesh represents aqueous solution- membrane interface and the second one represents BLM phase. These circuit elements are possessing different impedance values and the overall impedance at any frequency depends on the circuit element possessing higher impedance, in turn the more contribution from that circuit arrived from a circuit element possessing lower impedance by the above stated rule (Mallaiya et al., 2014).

The dispersion of membrane capacitance (Cm) and conductance (Gm) with frequency are related to the dispersion of conductance (G) and capacitance (C) of polar (P) and hydrocarbon (H) regions of the bilayer phase and are as follows (Coster, 2003).

(2)
C m = C H ( G P / 2 ) 2 + ( C P / 2 ) G H 2 + ω 2 C H ( C P / 2 ) ( C P / 2 + C H ) ( G P / 2 + G H ) 2 + ω 2 ( C P / 2 + C H ) 2
(3)
G m = G H ( G P / 2 ) ( G P / 2 + G H ) + ω 2 C H 2 ( G P / 2 ) + ( ( C P / 2 ) 2 G H ) ( G P / 2 + G H ) 2 + ω 2 ( C P / 2 + C H ) 2

In each RC mesh, the impedance of the capacitive element moves toward the resistance of the resistor while frequency decreases and becomes close to that of resistor at low frequency but still less than that of the resistance of the resistor in the RC mesh.

Fig. 5a–c shows the bode plots on the variation of total impedance of the membrane and the phase angle with respect to the frequency of applied AC signal in 1.0 M, 0.1 M and 0.01 M NaCl bath solutions. At the highest range of frequencies, the overall impedance |Z| of the bilayer membrane system is determined by the résistance of the bath solution (Rs) since at highest range of frequencies the resistance of the bath solution is greater than 1/ωCp and 1/ωCH. To further decrease in frequency, 1/ωCH is comparable with RH and the bode plot becomes independent of applied frequency. This shows that the complete control of the total impedance of the bilayer lipid membrane is influenced by RH or membrane resistance Rm. From the Fig. 5a–c, it can be seen that the membrane resistance (Rm) decreases with decrease in NaCl concentration in the bath. BLM surface has both positive and negative charges due to nitrogen bases and phosphate groups respectively. When BLM is formed in NaCl bath, most of the positive charges are neutralized by Cl− ions while the negative charges are partially neutralized by Na+ ions. This is due to a fact that the binding constant of Cl− ions on the BLM surface is larger than that of Na+ ions (Kotyńska and Figaszewski, 2005). The Cl− ions attached to the BLM surface exert an ionic pressure and this ionic pressure decreases with decrease in NaCl concentration in the bath. The phospholipid molecules generally show lateral movement and this fluidizes the BLM system (Mallaiya et al., 2014). The fluidization of the BLM system makes it much more leakier for the flow of smaller ions such as Na+ and K+. Thus, with a decrease in NaCl concentration, membrane resistance (Rm) decreases.

Bode plots of BLM formed in 1.0 M, 0.1 M and 0.01 M NaCl bath solutions respectively.
Figure 5 Bode plots of BLM formed in 1.0 M, 0.1 M and 0.01 M NaCl bath solutions respectively.

The electrochemical impedance parameters derived using proposed equivalent circuit for black lipid membrane in the presence and absence of cholesterol in the NaCl bath solutions are shown in Tables 1–3. It is observed that the capacitance of the polar region (CP) initially remains constant and then rapidly increases when the cholesterol penetration caused a drastic change in the electrical properties of BLM. At higher concentration of cholesterol its fluidization effect will be large and increases the permeability of smaller ions across the BLM phase, before which, the number of charged species cross the solution – membrane interface and show higher capacitance value due to accumulation of ions. The portion of cholesterol into the BLM separates the phospholipid molecules and affects the Van der Waals forces attractive force at the middle or the core of the BLM system. This shows some contribution to fluidization of BLM system and makes it leakier to smaller ions such as Na+ and K+.

Table 1 Electrochemical impedance parameters of BLM in the presence and absence of cholesterol in 1.0 M NaCl bath solution.
S.No Cholesterol concentration (µM) 1.0 M NaCl Standard deviation for Cm Standard deviation for Rm
Cp (µF) Rp (KΩ) Cm (nF) Rm (GΩ)
1 0 95.00 2.14 5.215 3.35 2.88 × 10−3 1.30 × 10−2
2 10 98.00 2.14 5.219 2.87 1.52 × 10−3 1.58 × 10−2
3 20 98.51 2.23 5.223 2.58 2.07 × 10−3 1.30 × 10−2
4 40 98.62 2.27 5.230 2.17 1.14 × 10−3 1.58 × 10−2
5 60 98.65 2.28 5.237 1.77 1.14 × 10−3 1.92 × 10−2
6 80 97.86 2.28 5.243 1.38 2.30 × 10−3 1.92 × 10−2
7 100 98.00 2.28 5.251 0.986 1.92 × 10−3 3.35 × 10−3
8 200 98.00 2.28 5.259 0.0975 8.36 × 10−4 3.76 × 10−4
9 400 98.00 2.28 15.620 0.00135 1.14 × 10−3 3.19 × 10−5
Table 2 Electrochemical impedance parameters of BLM in the presence and absence of cholesterol in 0.1 M NaCl bath solution.
S.No Cholesterol concentration (µM) 0.1 M NaCl Standard deviation for Cm Standard deviation for Rm
Cp (µF) Rp (KΩ) Cm (nF) Rm (GΩ)
1 0 51.26 28.32 4.684 2.08 1.29 × 10−3 1.71 × 10−2
2 10 51.26 28.35 4.691 1.90 9.57 × 10−4 1.71 × 10−2
3 20 51.32 28.37 4.699 1.72 9.57 × 10−4 1.71 × 10−2
4 40 51.28 28.37 4.709 1.36 1.29 × 10−3 2.59 × 10−2
5 60 55.28 18.37 4.718 0.996 1.29 × 10−3 8.77 × 10−3
6 80 57.28 16.72 4.728 0.638 1.41 × 10−3 4.65 × 10−3
7 100 57.28 16.72 4.739 0.281 1.29 × 10−3 3.86 × 10−3
8 200 278.80 1.023 16.42 0.00104 9.57 × 10−4 5.19 × 10−5
9 400 489.00 0.128 19.52 0.00000236 2.16 × 10−3 5.71 × 10−8
Table 3 Electrochemical impedance parameters of BLM in the presence and absence of cholesterol in 0.01 M NaCl bath solution.
S.No Cholesterol concentration (µM) 0.01 M NaCl Standard deviation for Cm Standard deviation for Rm
Cp (µF) Rp (KΩ) Cm (nF) Rm (X 108 Ω)
1 0 27.32 12.32 4.331 7.56 2.75 × 10−3 2.99 × 10−2
2 10 22.32 11.87 4.341 6.29 1.89 × 10−3 3.87 × 10−2
3 20 27.32 10.22 4.352 5.02 1.70 × 10−3 4.96 × 10−2
4 40 29.47 9.520 4.361 2.48 5.77 × 10−4 3.10 × 10−2
5 60 32.22 2.110 8.452 0.0135 1.15 × 10−3 2.38 × 10−4
6 80 98.26 0.732 14.22 0.0000535 1.50 × 10−3 4.43 × 10−7
7 100 142.5 0.235 19.32 0.00000633 8.16 × 10−4 4.11 × 10−8

The variation of capacitance of BLM phase with cholesterol concentration in NaCl bath solutions is shown in Fig. 6. From this figure, it is observed that the capacitance of BLM phase increases gradually with cholesterol concentration and then exhibited a sudden jump. The initial increase in capacitance is due to penetration of cholesterol into the BLM phase, due to which the tightly bound phospholipid molecules are separated and the area of BLM increases.

Variation of membrane capacitance (Cm) with cholesterol concentration in 1.0 M, 0.1 M and 0.01 M NaCl bath solutions.
Figure 6 Variation of membrane capacitance (Cm) with cholesterol concentration in 1.0 M, 0.1 M and 0.01 M NaCl bath solutions.

The total membrane capacitance (Cm), can be equated to the contribution from both the bilayer hydrophobic domain (CH) and the membrane solution interfaces (CP) which can be explained based on the discussions of Movileanu et al. (2000) as follows:

Since two capacitors CP and CH are in series

(4)
1 C m = 1 C H + 2 C P Accordingly
(5)
C m = C H C P C P + 2 C H

The relation between the capacitance of the hydrophobic domain with the thickness and the dielectric coefficient of the phospholipids acyl chain is given as follows:

(6)
C H = ( ε 0 ε H ) d H where ε H and d H are relative permittivity and thickness of the hydrocarbon core of the membrane respectively. When the cholesterol molecules are inserted within the hydrophobic core of the lipid bilayer, the capacitance of the membrane system changes due to change in the capacitance of hydrophobic domain. Therefore, when the cholesterol molecules are inserted into the lipid bilayer, Eq. (5) changed as follows:
(7)
C m CHO = C P ( C H + C CHO ) C P + 2 ( C H + C CHO ) C m CHO can be expressed as a function that also depends on the capacitance of the membrane in the absence of cholesterol, as follows:
(8)
C m CHO = C m + C P C CHO C P + C H × C P + 2 C H C P + 2 C H + 2 C CHO where
(9)
C CHO = ε 0 ε CHO N CHO V CHO NCHO is the average number of cholesterol molecules per unit area of bilayer, VCHO is the molecular volume of cholesterol and εCHO is the dielectric coefficient of cholesterol.

The components (CH and CP) will be higher than the CCHO even at very high concentrations of cholesterol, since the cross-sectional area of phospholipid occupied domain is much higher than the cross-sectional area of the cholesterol occupied domain.

In this condition,

(10)
C P + 2 C H C P + 2 C H + 2 C CHO ≈ 1 and
(11)
C m CHO = C m + C P C CHO C P + C H Therefore, Cm increases with cholesterol concentration. A sudden jump in capacitance is due to replacement of large numbers of phospholipid molecules by cholesterol molecules.

3.2

3.2 Impedimetric sensor for cholesterol

The variation of membrane resistance (Rm) with cholesterol concentration is shown in Fig. 7a–c for 1.0 M, 0.1 M and 0.01 M NaCl bath solutions respectively. From these figures, it is clear that the membrane resistance follows a linear relationship with cholesterol concentration up to 200 µM, 100 µM and 40 µM in 1.0 M, 0.1 M and 0.01 M NaCl bath solutions respectively. The detection concentration range of cholesterol decreases with decrease in NaCl concentration in bath solution. With the increase in NaCl concentration the binding of Cl− ions on the surface of BLM increases. The binding of Cl− ions imparts a tightening effect on either side of BLM and makes it more stable and less permeable to ions. Due to the same effect the thickness of BLM decreases with NaCl concentration in the bath solution, which is attributed to the calculated capacitance values using equation.1. Hence a relatively more stable membrane formed in 1.0 M NaCl bath solution withstand against fluidization effect of cholesterol and shows a linear relationship between membrane resistance and cholesterol concentration.

Variation of membrane resistance (Rm) with cholesterol concentration in 1.0 M, 0.1 M and 0.01 M NaCl bath solutions respectively.
Figure 7 Variation of membrane resistance (Rm) with cholesterol concentration in 1.0 M, 0.1 M and 0.01 M NaCl bath solutions respectively.

4

4 Conclusions

The penetration of cholesterol into the artificial membrane system was explained using electrochemical impedance spectroscopy. Cholesterol partitioned into the BLM phase showed fluidization effect due to which membrane conductance increases. Thickness of BLM decreases with increase in NaCl concentration in the bath solution. An impedimetric sensor was developed for the quantification of cholesterol without using enzymes. The detection range of cholesterol in the bath solution depends on NaCl concentration.

Acknowledgments

The authors thank the University Grants Commission (UGC), New Delhi, for providing the funds to carry out this project under UGC Major Grant No. 42- 907/2013 (SR).

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