LT6424B - METHOD OF PREPARING IMOBILIZED ON THE SURFACE PHOSPHOLIPID BILAYER MEMBRANE (tBLM) - Google Patents

Abstract

Claimed method of preparing immobilized on the surface phospholipid bilayer membrane (tBLM) using a multi-layered vesicles (MLV). This method enables fast, without the use of complex equipment to produce tBLM characterised by good insulating properties.

Description

The present invention relates to suspended lipid membranes, in particular to a method for obtaining immobilized bis-layer phospholipid membranes.

State of the art

Cell membrane. The cell membrane, the skeleton (skeleton) without which life could exist, encloses the cell's interior with all its contents and creates a barrier that separates the cell's interior from the outside and serves a protective function. At the same time, this protection is capable of making certain selections, allowing the necessary materials to enter the cell without which vital functions can be sustained. Because the cell membrane is surrounded on all sides by water, only water-soluble and low molecular weight substances can easily pass through it. Other necessary materials, such as proteins or carbohydrates, are transported through carriers and channels.

The major component of the cell membrane structure is the lipid, which forms the membrane backbone and has amphiphilic properties, with one part hydrophobic and the other hydrophilic (Figure 1). This property is due to the chemical composition and structure of the lipid, where this molecule can be divided into two parts: the tail (hydrophobic) and the head (hydrophilic). This distinction allows for the formation of certain defined structures in the aqueous medium, one of which is a bilayer lipid membrane (Fig. 1), consisting of two rows of lipids with hydrophobic tails facing inwards and hydrophilic tails facing outwards. The tail is composed of one or two fatty acids, linear hydrocarbon chains of 12 to 18 carbon atoms in length and terminating in a carboxyl group. These circuits may have several double bonds which, like the number of carbon atoms, affect the flow, viscosity, and phase transition temperature (Tm) of the membrane. The latter hydrocarbon chains are linked to glycerol via one or two ester linkages. And the remaining third bond forms a bond to the lipid head, which consists of phosphate and a certain functional group that may be different in different parts of the body. According to functional groups, phospholipids are separated into several main components: phosphotidylcholine, phosphotidylethanolamine, phosphotidylserine and phosphotidylinositol. This diversity of lipids allows for the composition of different lipid membranes required for one or another group of cells. In addition, membranes exhibit asymmetry in which each layer is dominated by a different amount and type of lipid. Such distribution is required to perform certain functions.

Membrane models. The cell membrane is made up of many different macromolecules (lipids, proteins, carbohydrates), each of which may influence the properties of the membrane itself or its interaction with non-membrane derivatives. Simulated simulation systems, artificial bilayer lipid membranes, the composition of which can already be controlled, are used to isolate one or another phenomenon. The first such systems were bilayer lipid liposomes which were evenly distributed in the liquid. But such lipid systems are only suitable for a narrow range of research methods that do not provide information about the composition and structure of the membrane itself. The membrane composition is studied using a set of assay methods that share one common denominator, the flat solid support (QCM, SPR, AFM, NR, EIS), which is required as a reference point. Thus, there is a need to transfer liposome bilayer lipid membranes to the surface. Here, too, the lipid property of amphiphilicity is very useful, which makes it possible to form a bilayer lipid system on a substrate having a hydrophilic surface. And this is how another membrane model is developed - flat lipid bilayer systems, also known as plain lipid membranes (sBLM) (Figure 2), which are identical in structure and composition to liposomes. In addition, this model is distinguished by the fact that the space between the backing and the lower membrane layer is filled with water, which makes this lipid simulation even more like natural systems. And water is just needed for the formation of protein structures. One of the main advantages of this model is stability, which allows time-consuming experiments (NR, AFM). Secondly, in such membranes, lesions can develop locally, allowing the retention of certain structures on the surface that can already be studied, even with the atomic force microscope (AFM). But this system is not just a simple structural element that can be described by simple parameters (membrane thickness, lesion pore size or height, surface lipid content, etc.). It is also an element with certain electrical properties, which is studied by electrochemical impedance spectroscopy (EIS), which allows to evaluate membrane conductivity. The latter method is particularly suitable for assessing the extent of membrane damage, since pores also increase membrane conductivity. However, the use of this method in sBLM model studies is complicated by membrane motility, which prevents the recording of conductance changes within the membrane itself. To extend these capabilities, hybrid membranes (hBLMs) (Figure 2) are used in which the lower lipid layer is replaced by alkantiols or thiolipids, which create a hydrophobic surface and thus form a stable lipid system firmly attached to the surface with certain electrical properties, and determine membrane conductance or capacitance, which may depend on lipid composition. But in such a system, electrical properties reach another threshold where the absence of a membrane layer allows the formation of highly insulating membranes, but also very rigid, which no longer intervenes to form certain structures that would damage the locally lipid surface and allow membrane conductivity changes.

Thus, these two models (sBLM and hBLM) have certain limitations that preclude the application of assay methods capable of recording changes in membrane conductance. And to solve this problem, a third membrane model is currently being developed for suspended lipid membrane (tBLM) (Fig. 3), which by its structure is an intermediate between sBLM and hBLM because it fulfills two basic requirements: increasing membrane isolation while maintaining its lability. This effect is achieved when the membrane is attached to the surface through special compound anchors [US 20030054431] [H. Lang, C. Duschl, H. Vogei, A new class of thiolipids for attachment of lipid bilayers on gold surfaces, Langmuir, 10 (1974) 197-210]. The latter compounds are structurally similar to lipids: they have a hydrophobic moiety consisting of saturated or unsaturated carbon chains, and the phosphate and functional groups on the hydrophilic moiety are replaced by an ethylene oxide (EO) chain to which a sulfur or sulfur-containing group capable of interacting with gold is attached. surface. The carbon chains of this compound form the bottom layer of the membrane, giving the system stability. And the EO fragments in the membrane layer immediately play two roles: first, to reduce the conductivity of the membrane reservoir, and second, to control the height of the membrane from the sensor surface. But filling the sensor surface with these anchor compounds alone makes the entire lipid system similar to hBLM, which reduces the chances of detecting membrane damage. To prevent this, the anchor compound is mixed with the diluent. The latter molecule is usually a chain of various lengths composed of EO moieties but without a hydrophobic moiety. Thus, the diluent allows the amount of anchor compounds on the surface to be controlled.

Application. Artificial membrane models are currently being developed as certain biosensors capable of detecting various proteins that cause certain damage. These proteins interact with the membrane to form pores of certain structures, thereby damaging the membranes (Fig. 4). And tBLM models are best suited for their detection because (1) an intermembrane water reservoir is created between the membrane and the substrate, which is required for protein interference and for the formation of pores of certain structures; 2) the entrapped protein, by forming a pore in the membrane, increases its electrical conductivity, which can be detected by electrochemical impedance.

Preparation. The protocol for preparation of all these models is currently identical and is carried out in two steps: in Step I, the respective self-assembling monolayers are formed from the spirit solutions (sBLM - hydrophilic monolayer, hBLM - hydrophobic monolayer, tBLM - mixed anchor and diluent); ongoing lipid formation [DJ McGilIivray, G. Valincius, D.J. Vanderah, W. Febo-Ayala, J.T. Woodward, F. Heinrich, J.J. Kasianovvicz, M. Losche, Molecular-Scale Structural and Functional Characterization of Sparsely Tethered Bilayer Lipid Membranes, Biointerphases, 2 (2007) 21-33]. Stage I essentially does not require special conditions except that all thiols must be soluble in alcohol. Stage II is performed using bilayer lipid liposomes (SUVs), i.e. an aqueous solution of liposomes is deposited on the self-assembled monolayer. The problem is, this build method is only suitable for hBLM and sBLM models. The preparation of tBLM from these liposomes is already becoming a more difficult task because of 1) slow formation;

2) not form on the rare anchors on which the membrane damage is best detected by EIS; 3) high membrane defectivity, i.e. membrane conductivity too high to be used as a biosensor.

Also, the technology of liposome preparation itself requires specific equipment and time (Fig. 5). Initially, the lipid powder is dissolved in chloroform, which is first evaporated under a slight stream of nitrogen and then stored under vacuum for 12 hours. Following these procedures, the vessel walls form a lipid film which is filled with buffer and subjected to vigorous shaking to form a multilayer liposome (MLV). In the next step, these structures already yield the aforementioned bilayer liposomes, which are prepared in two ways: MLV digestion with a titanium nozzle ultrasonic mixer to bilayer liposomes, which must then be centrifuged from the titanium waste. Second method: MLV solution is pushed through an extruder polycarbonate membrane containing pores of desired size.

Stage II may also be performed by dissolving the lipid solution in an alcoholic lipid solution, i.e., a self-priming monolayer, and washing the entire surface with a large amount of buffer [B. Raguse, V. Braach-Maksvytis, B.A. Cornell, L.G. King, P.D.J. Osman, R.J. Pace, L. Wieczorek, Tethered Lipid Bilayer Membranes: Formation and Ionic Reservoir Characterization, Langmuir, 14 (1998) 6485

659]. This method is called rapid solvent exchange (RSE). The advantage is that it is a fast method that does not require expensive hardware and is capable of forming insulating (low electrical conductivity) membranes. The first disadvantage of this method is that not all lipids are soluble in alcohol, especially when it comes to creating a membrane containing cholesterol because of the limited solubility of cholesterol. Secondly, some measuring devices are technically difficult to use with two solvents.

Refinement of the problem. Thus, there are a few key points that can be made regarding the preparation of a tBLM to be used as a biosensor:

1) Preparation of tBLM must be fast and do not require specific and expensive hardware.

(2) The tBLM system shall have electrical insulating properties to be suitable for use as a biosensor with the following main applications:

(a) membrane interference of proteins and peptides;

(b) Bacterial and toxin damage to biological membranes for investigation and detection;

(c) Detection and assay of membrane interactions with low molecular weight compounds and associated proteins;

(d) Membrane structure studies.

Brief Description of the Invention

Contract notations

CSF - Quartz Crystal Micro-Scales

AFM - Atomic Force Microscope

NR - neutron reflectometry

EIS - Electrochemical Impedance Spectroscopy

PPR - surface plasmon resonance sBLM - flat lipid bilayer systems, also called plain lipid membranes hBLM - hybrid membrane tBLM - suspended lipid membrane

EO - ethylene oxide chain

SUVs are bilayer lipid liposomes

MLV - Multilayer Liposomes

RSE - solvent replacement method

BRIEF DESCRIPTION OF THE DRAWING FIGURES FIG. Lipid and membrane structure.

Fig. Lipid membranes: plain and hybrid.

Fig. Hanging lipid membrane.

Fig. Damage to the membrane.

Fig. Preparation of bilayer liposomes.

Fig. Representative surface plasmon resonance shift curve obtained from tBLM formation by interaction with phospholipid multilayer liposomes.

Fig. Representative CIS-Cole Complex Capacity Parametric Chart of EIS Data (Parameter - Modulation Frequency): Black curve - initial complex capacitance curve recorded from surface formed by method IA, red curve by fusion of molecular anchor surface with multilayer liposomes prepared by method II. B, for solutions and forming for 30 min according to method IIIA.

Fig. Representation of EIS data obtained by Method III.B on a Bode Complex Conductivity (Admitance) Chart.

This application proposes a solution to the aforementioned problem using multilayer liposomes (MLVs) (Figure 5) for the formation of surface immobilized phospholipid bisplane membranes (tBLM). Multilayer liposomes are not newly invented, they are simply found to exist, but have little application. In our case, the novelty and essence of the invention is that they can also be used in the formation of flat membranes, and in particular tBLM.

Advantages of the proposed method:

1) Quick preparation. SUV preparation can take 8-24 hours. MLVs are prepared in 30 minutes.

2) No additional equipment required. For an SUV, you need to have a vacuum chamber, ultrasonic mixer, centrifuge or extruder. MLV preparation does not need all this, just the simplest dispenser available in every laboratory.

3) Liposome size. The size of the liposomes themselves plays an important role in the preparation of flat membranes (sBLM, tBLM) from SUVs. For a successful result, SUV liposomes must have a diameter of no more than 50 nm, otherwise liposomes will adhere to the surface but will not melt. When using MLV, the size of the liposomes can vary over a wide range (500 nm - 1.5 pm) and their size does not influence the formation of the lipid membrane.

4) Amount of anchor compound in tBLM system. MLVs can be used to form suspended lipid membranes on rare anchor compounds on the surface that are sufficiently stable but also contain large amounts of water in the membrane layer. In the case of SUVs, there must be sufficient anchor compound to allow the liposomes to form a lipid layer.

Electrical isolation of the lipid membrane. MLVs provide enough isolating tBLMs that could already be used as biosensors. In addition, the formation itself does not take more than 1 hour. The insulating properties of a membrane formed from an SUV can be achieved only after a few or several hours.

Detailed Description of the Invention

Materials and Methods

Distilled water - dist. H2O (Milli-Q plus, USA);

Sodium chloride - NaCI (FLUKA, Switzerland);

Sodium dihydrophosphate - NaH2PO4-2H2O (FLUKA,

Switzerland);

Ethanol - EtOH (Vilniaus degtinė AB,

Lithuania);

Chloroform - CHCb (Sigma-Aldrich, Germany);

2-mercaptoethanol (beta mercaptoethanol) - βΜΕ (Sigma-Aldrich, Germany); 20-tetradexyloxy-3,6,9,12,15,18,22-heptaoxa-hexatricontano-1-thiol and [(Z 20 (Z-octadeca-9-enyloxy) -3,6,9,12,15,18, 22-heptaoxatetrakont-31-en-1-thiol, (C18 = oleoyl)] - synthesized by Dr. DJVanderah (University of Maryland);

Dioleoylphosphocholine - DOPC (Avanti Polar Lipids, USA);

Cholesterol ((Avanti Polar Lipids, USA);

EIS measurements were performed on a Zennium instrument (Zahner GmbH, Germany);

PPR spectra were recorded on an Autolab Tvvingle spectrometer (Methrom Lab, The Netherlands).

The method of obtaining tBLM of the present invention comprises three steps:

I. Formation of a surface molecular anchor layer;

II. Preparation of multilayer vesicles;

III. Attachment of tBLM by Anchor Surface Fusion of Multilamellar Vesicles.

The formation of the surface molecular anchor is accomplished in a manner known in the art.

MLVs are set up similarly to SUVs, but with some modifications.

Initially, the lipid powder is dissolved in chloroform, which is evaporated under a gentle stream of nitrogen for about 30 minutes (until chloroform is removed). Following this procedure, a lipid film is formed on the vessel walls, which is filled with buffer and, instead of vigorous shaking, slow mixing of the lipid films with the buffer is performed by means of a dispenser to form multilayer liposomes (MLVs) which are already used for forming.

The following examples illustrate the invention without limiting its scope.

I. Obtaining a surface molecular anchor

I. Example A

1. Plate a 25 × 75 × 1 mm glass slide, or other dimensions, in a solution of 50 g / l ammonium persulfate in concentrated sulfuric acid and keep in this solution for 30 minutes. Remove the plate from this solution and wash with a large quantity (greater than 100 ml / plate) of deionised water. The plate surface is dried in a stream of dry nitrogen (99.99% purity).

2. Place the plate in a vacuum magnetron evaporation chamber (used by PVD 75 Kurt J. Lesker Company, USA evaporation system), activate the vacuum pump and achieve a residual pressure of less than 10 ' ⁷ mTorr.

3. After reaching the specified vacuum level, a 1 nm Ti layer is magnetronically vaporized on the plate and a 50 nm Au layer is evaporated on its surface. The following magnetron evaporation parameters can be used: Ti layer - magnetron power 100 W, argon pressure during evaporation -4.5 mTorr, and Au layer power -120 W, argon pressure 4.2 mTorr. Au (999.9) and Ti (999.9) magnetron evaporation metal targets of 50 mm diameter were used for evaporation. The amount of metal evaporated is recorded by piezoelectric quartz microscales.

4. Upon completion of evaporation, the magnetron evaporation chamber is devacuated and the Ti / Au-coated plate is transferred to a molecular anchor formation solution consisting of 95.6% (or higher) ethanol solvent, 0.03 mM 20- (tetradecyloxy) - 3,6,9,12,15,18,22-heptaoxa-hexatriacontan-1-thiol and 0.07 mM 2-mercaptoethanol. The sample is kept in this solution for 4 hours.

5. After completion of molecular anchor layer formation, the sample (glass plate with Ti / Au layers) is removed and washed with copious amounts (at least 50 ml / plate) of pure solvent, 95.6% (or higher) ethanol. After washing, the plate is dried under a stream of dry nitrogen gas.

Example I.B.

1. Perform the procedures as described in Example I.A under A.1 to A.3; however, a circular glass plate of 25 mm (or other diameter) 1 mm (or other thickness) BK-7 grade glass plate shall be used.

2. Upon completion of evaporation, the magnetron evaporation chamber is devacuated and the Ti / Au-coated plate is transferred to a molecular anchor formation solution consisting of 95.6% ethanol (or higher) solvent, 0.03 mM 20- (tetradecyloxy) - 3,6,9,12,15,18,22-heptaoxa-hexatriacontan-1-thiol disulfide and 0.07 mM 2-mercaptoethanol disulfide. The sample is kept for 1 hour in this solution.

3. After completion of the molecular anchor layer formation, the sample (glass plate with Ti / Au layers) is removed and washed with copious amounts (at least 50 ml / plate) of pure ethanol (95.6% or greater purity). After washing, the plate is dried under a stream of dry nitrogen gas.

II. Preparation of multilayer liposomes (MLV)

II. Example A

1. Dioleoylphosphocholine (dry powder) is dissolved in chloroform to a concentration of 10 mM and the resulting solution is used immediately or, if necessary, can be stored without loss of functionality at -20 ° C for up to 6 months. Other solvents may be used in place of chloroform, such as a mixture of chloroform with another organic solvent, such as ethanol, propanol, isopropanol, butanol, pentanol, 2-ethylpropanol, or hexane, or pentane, or heptane, or octane, or nonane, or squalene.

2. From the resulting solution, withdraw 1 ml of the resulting dioleoylphosphocholine solution into a separate glass vial.

3. The chloroform is evaporated with nitrogen gas (blowing gas for about 30 minutes). The end of evaporation can be considered as the moment when no liquid is left in the vessel plus a further 20 minutes by blowing into a vessel which forms a semi-opaque lipid film on the walls.

4. The lipid film is filled with 10 ml of pH 4.4 buffer containing 10 mM NaH ₂ PO ₄ and 100 mM NaCl.

5. Solution J is labeled with a semi-automatic manual dispenser with nozzle and suction-expulsion cycles to slowly solubilize the lipid film formed on the vessel walls. The process (suction-expulsion) is repeated 50 to 200 times until no film remains on the vessel walls and the contents of the vessel become matte and homogeneous.

6. The MLV solution so prepared is used immediately or can be stored at + 5 ° C for up to 2 weeks.

II. Example B

A mixture of dioleoyl phosphocholine and cholesterol (dry powder) in a molecular weight ratio of 60% / 40% dissolved in chloroform (90% by volume) and ethanol (10% by volume) to a total lipid concentration of 10 mM.

2. From the resulting solution, withdraw 1 ml of the resulting mixed solution of dioleoyl phosphocholine and cholesterol in a separate glass vial.

3. Evaporate the chloroform-ethanol solution with nitrogen gas (by bubbling gas for about 30 minutes). The end of evaporation can be considered as the moment when no liquid is left in the vessel plus a further 20 minutes by blowing into a vessel which forms a semi-opaque lipid film on its walls.

4. The lipid films are filled with 10 ml of pH 4.4 buffer containing 10 mM NahkPCU and 100 mM NaCl.

5. Apply a semi-automatic manual dispenser with a nozzle and a dip-cycle to slowly solubilize the lipid film formed on the vessel walls. The process (suction-expulsion) is repeated 50 to 200 times until no film remains on the vessel walls and the contents of the vessel become matte and homogeneous.

6. The reconstituted MLV solution is used immediately or can be stored at + 5 ° C for up to 2 weeks.

All of the procedures described in Methods A and B are carried out at room temperature (20 ± 2 ° C).

III. Formation of tBLM by Fusion of Multilayer Liposomes to Anchor Surface

III. Example A

7. A plate coated with a molecular anchor, according to Production Example I.A, immersed or otherwise contacted with the MLV solution prepared according to the method described in II.A. The plate is kept in contact with the solution for 1000 s. tBLM spontaneously covers a surface with a degree of fill better than 99%. The formation of tBLM can be monitored in real time by surface plasmon resonance spectroscopy (see Figure 6).

8. Replace the MLV solution with a pure, desired pH buffer, such as pH 7.0, in which NaHPCU and NaH ₂ PO4 (total phosphate concentration 10 mM) and 100 mM NaCl are dissolved.

9. The prepared tBLM is used immediately for assays or can be stored at room temperature for 72 hours.

III. Example B

1. A plate coated with a molecular anchor, immersed or otherwise contacted with an MLV solution prepared according to Production Example I.A or I.B., prepared according to the method described in II.B.

The plate is kept in contact with the solution for 3600 s. tBLM spontaneously covers a surface with a degree of filling greater than 99% and a residual defect density of less than 0.5 μητ 2

2. Formation of tBLM and reduction of defect density can be monitored in real time by surface electrochemical impedance spectroscopy (see below).

3. After 3600 s, the MLV solution is replaced with a pure, desired pH buffer, such as pH 7.0, in which NaHPCU and NaH ₂ PO4 (total phosphate concentration 10 mM) and 100 mM NaCl are dissolved.

4. The prepared tBLM is used immediately for assays or can be stored at room temperature for 72 hours.

IV. Observation of lipid formation

IV. Real-time monitoring of tBLM formation by surface plasmon resonance spectroscopy

The experiment is performed by preparing a tBLM sample using a molecular anchor according to method I.B. The multilayer liposome solution is prepared according to method II.A. The plate prepared according to Method I.A is inserted into the holder of the surface plasmon resonance (PPR) spectrometer Autolab Twingle. Apply approximately 30 ml of a multilayer solution of liposomes prepared by method II.A onto the surface of the plate. activate the surface plasmon resonance spectrometer and record the variation of the minimum position of the resonance curve (in degrees, m °). The minimum position can be related to the amount of bound phospholipid (in this case dioleoyl phosphocholine), so that the completeness of membrane formation can be estimated. A representative tBLM formation curve is presented in Fig. 6.

During the test, we observe that the PPR minimum position reaches a value greater than 400 m ° in about 1000 s. Subsequently, the curve changes slightly, reflecting the surface saturation with phospholipid transported from multilayer liposomes. The wash, which corresponds to a “step” performed 1800 s after the start of interaction with the multilayer liposomes, establishes a final PPR minimum of 410 m °. According to our calibration, 1 m ° change in PPR signal corresponds to 0.62 ng / cm ² lipid binding. Thus, the total amount of phospholipid attached to the surface in this example is 410x0.62 = 254.2 ng / cm ² . This amount of lipid, given that the molar mass of the dioleoyl is 786.11 g / mol, corresponds to 3.23 to ^{10 10} mol / cm ² , or 1.95 to ^{10 14} molecules / cm ² . The dioloyl phosphocholine 1 molecule is known to occupy a surface area of 72.5 A ^{2 in the} planar phospholipid membrane. From the total number of molecules transported from the liposomes to the surface from the PPR data, we find that the transferred dioleoylphosphocholine has a surface area of 1.41 cm ² , which corresponds to a full bilayer if we take into account that the anole compound dioleoyl chains occupy 0.60 cm ² surface. area. Thus, the total amount of dioleoylphosphocholine transported from the multilayer liposomes corresponds to a better than 99% surface coating on the phospholipid bilayer.

IV.2 Real-time monitoring of tBLM formation by electrochemical impedance spectroscopy (EIS)

This method records changes in electrical properties during the tBLM formation process. During tBLM formation, a phospholipid containing a hydrophobic molecular moiety is transferred from the liposomes to the anchor-coated surface and becomes a dielectric layer upon formation of the bilayer. The formation of an additional dielectric layer on the surface is accompanied by a decrease in the electrical capacity of the surface. This process can be monitored using the EIS method.

Sample ready for experiment, method IA Also prepared multilayer liposome solution according to method II.B. The sample (plate) was inserted into an EIS spectroscopy peripheral device described by [Budvytyte, M. Mickiewicz, DJ Vanderah, F. Heinrich, and G. Valincius, Modification of Tethered Bilayers by Phospholipid Exchange with Vesicles. Langmuir. 29, (13), 4320-4327, (2013) Initially recorded the EIS spectrum of anchored compounds, which is represented by a black curve in Fig. 7 in the form of a ColeCole- (complex capacitance) diagram. The right semicircular arc in these diagrams, corresponding to the low frequency range of the EIS spectra, shows the approximate value of the surface electrical capacity, which in this case is 8.8 pF / cm ² .

upon introduction of the vesicle solution into the system, the tBLM formation process is initiated according to the method described in III.B. After 3600 s, the second spectrum (Fig. 7, red curve) is recorded, which indicates that the semicircular curve segment has decreased to about 0.700.80 pF / cm ² . This decrease coincides with the expected effect of the electrical capacitance decrease, which can be estimated by the flat capacitor formula:

The following equation, when rearranged, allows the thickness of the resulting phospholipid derivative to be calculated:

where ε is the relative permeability of the dielectric layer, is the vacuum dielectric constant of 8.85-10 ' ⁸ pF / cm, and d- is the thickness of the dielectric layer. The relative dielectric permeability of the dioleoyl layer is 2.9 [DJ McGilIivray, G. Valincius, F. Heinrich, J. W. F. Robertson, DJ Vanderah, W. Febo-Ayala, I. Ignatjev, M. Losche, J. J. Kasianowicz. Structure of Functional Staphylococcus aureus α-Hemolysin Channels in Tethered Bilayer Lipid Membranes. Biophys. J, 96 (4) 1547-1553 (2009)]. Since 40% cholesterol and 60% dioleoyl were used in this example, the value of the dilational constant is 2.3 [R. Budvytyte, G. Valincius, G. Niaura, V. Voiciuk, M. Mickiewicz, H. Chapman, H. Z. Goh, P. Shekhar, F. Heinrich, S. Shenoy, M. Losche, DJ Vanderah, Langmuir, 29 (2013). 8645-8656. R. Budvytyte, M. Mickiewicz, DJ Vanderah, F. Heinrich, and G. Valincius, Modification of Tethered Bilayers by Phospholipid Exchange with Vesicles. Langmuir. 29, (13), 4320-4327, (2013)]. Placing these constants and the experimental capacitance value C = 0.70 pF / cm ² (Fig. 2, curve 2) in equation (2), the resulting bilayer hydrophobic part has a thickness of 2.9 nm. This value is in good agreement with the dioloyl layer thickness of 3.0 nm as determined by neutron reflectometry. This proves that Method III.B produces a phospholipid, mixed composition (dioleoyl phosphocholine and cholesterol).

In order to estimate the density of residual defects in the tBLM membrane, it is appropriate to map the EIS data to a different coordinate. Figure 8 The Bode diagrams are the same as those shown in Fig. 7. EIS data.

We can see that the surface complex conductivity has a high value before the formation of tBLM (curve 1, red). In the middle frequencies, a "step" of conductivity is observed, with values of 2-10 ' ³ S and higher. In the phase graph (Fig. 8, lower window) this curve is almost minimal, it is observed in the middle frequency range (about 500-600 Hz) and is often blurred with the decreasing part of the high frequency (> 1000 Hz) spectrum.

Formed by method III.B tBLM, the Bode curves are transformed. The admittance curve (Fig. 8, top window, curve 2) decreases. The flat part of the step shifts to the low frequency side, and is reduced by about 1000-fold to about 2Ί0 ^'6 S-value levels.

The formation of tBLM according to Method III.B results in an explicit minimum in the phase curve (Fig. 7, lower window, curve 2), the position of which is determined by the residual defect density [Valincius G., Meškauskas T., Ivanauskas F., Langmuir. 2012 Jan 10; 28 (1): 97790.]. Using the mathematical algorithm of EIS spectra interpretation [Valincius G., Meškauskas T., Ivanauskas F., Langmuir. 2012 Jan 10; 28 (1): 977-90.]. we find that the residual defect density in the present example assumes a natural defect radius of 1 nm to be <0.32 pm ' ² . From here we get that part of the surface occupied by residual defects is not more than 2.5Ί0 ^'7.

Claims (7)

Definition of the Invention CLAIMS 1. A method of producing surface immobilized phospholipid bilayer membranes (tBLM), comprising forming an anchor layer, preparing lipid liposomes, and fusing lipid liposomes to the anchor surface, wherein said fusion uses prepared multilayer lipid liposomes. 2. A process according to claim 1, wherein said multilayer lipid liposomes are obtained by dissolving the phospholipid in an organic solvent, separating 1 ml of the resulting solution, evaporating the organic solvent with nitrogen gas, and filling the residue with appropriate buffer to obtain a multilayer lipid. liposome solution in buffer, which can be used immediately for fusion to the anchor surface. 3. A process according to claim 2 wherein said organic solvent is chloroform or a mixture of chloroform with another organic solvent such as ethanol, propanol, isopropanol, butanol, pentanol, 2-ethylpropanol, or hexane or pentane or heptane, or octane or nonane or squalene. 4. A process according to claim 1 wherein the anchor compound 20- (tetradecyloxy) 3,6,9,12,15,18,22-heptaoxa-hexatriacontan-1-thiol or [(Z 20- (Z) is used to form an anchor monolayer -octadeca-9-enyloxy) -3,6,9,12,15,18,22-heptaoxatetracont-31-en-1-thiol, (Cie = oleoyl)] or disulfides of these compounds by mixing the anchor with a 1-mercaptoethanol surface diluent or its disulfide in a ratio of 1: 9 to 9: 1. 5. The method of claim 1, wherein said surface is a glass plate coated with a titanium layer for adhesion to the glass plate and a gold layer. The surface-immobilized phospholipid bis-membrane (tBLM) obtained by the method of claim 1. Use of a membrane according to claim 6 for the manufacture of a biosensor.