LT6992B - Regenerative phospholipid biosensor on silanized oxides surface - Google Patents

Abstract

The membrane sensor described here is made by using silane compounds forming self-assembled monolayer on electrically conductive and non-conductive oxide surfaces. The self-assembled monolayer is mixed with diluents to form a surface-attached bilayer phospholipid membrane with a submembrane water reservoir. Once formed, the sensitive phospholipid layer can be removed and re-formed several times on the same surface.

Description

FIELD OF THE INVENTION

The invention relates to surface-attached lipid membranes, specifically to the construction of a regenerated phospholipid biosensor on electrically conductive and non-conductive oxide surfaces.

STATE OF THE ART

A cell is the smallest structural unit of life. It is surrounded by a plasma membrane that protects and separates the inside of the cell from the outside. One of the most important components is phospholipids. These are amphiphilic substances that have two hydrophobic carbon chains (tails) and a hydrophilic phosphate group (head). Phospholipids form a bilayer structure, where the hydrophilic heads face outwards and the hydrophobic tails face the interior of the bilayer. Different types of proteins are also interspersed in the membrane for the transport of substances from the inside to the outside of the cell and for other vital functions such as respiration. It is precisely because of this property of the membrane - the ability to insert proteins - that models of lipid membranes were created. They are characterized by a simpler composition that can be controlled. Membrane models are applicable as an experimental platform for the study of protein-membrane interactions, including: assessing the function of integral proteins (pore-forming toxins, ion channels) and peripheral proteins (enzymes, electron transporters). Electrochemical methods, together with microscopic and spectral methods, are very effective in studying membrane processes, but in order to apply such methods, membrane models should be formed on solid electrically conductive surfaces (Ragaliauskas T. et ai., 2017).

One of the most convenient methods of forming a membrane on a solid surface is vesicle casting. Vesicles are liposomes with a spherical structure, consisting of a phospholipid bilayer. After the vesicle solution is poured directly onto the test surface, self-assembly of lipids takes place, when phospholipids move from a spherical shape to a solid surface, forming a bilayer flat structure. Since the membrane forming method is based on self-assembly, it does not require any expensive equipment, making it a cheap and accessible method. This method of membrane formation is also convenient in that the composition of the bilayer can be easily controlled by changing the lipid composition in the vesicles.

One of the simplest membrane models is the planar lipid membrane. It is composed of two layers of phospholipids, but unlike the cell, which is spherical in shape, this bilayer is immobilized in a horizontal plane (Castellana E.T. et ai., 2006). A flat lipid membrane is characterized by having a very thin layer of water between the membrane and the solid surface. This layer of water "simulates" the interior of the cell. With such a model system, proteins can be inserted and subjected to various assays to elucidate membrane permeability, defectivity, and other properties. Although this membrane model is characterized by moderate stability, the various defects in the membrane and their abundance prevent the application of electrochemical measurement methods to evaluate the integrity of flat membranes and to use such membranes as electrochemical biosensors to measure the activity of bacterial toxins and other agents that violate membrane integrity. Hybrid membranes (Plant A.L. 1999) are characterized by an incomparably lower amount of defects. In them, the first layer near the surface consists of a self-assembled monolayer (SAM), and the second layer consists of phospholipids. SAMs consist of molecules containing a chain of carbon atoms with a so-called active functional group (head) on one side, which usually consists of a thiol or thalcoxysilane functional group. SAM adheres to the surface by forming strong covalent bonds between the functional group and the surface. The selection of the active SAM functional group depends on the substrate to be used for SAM formation. If a gold surface is used, a SAM containing a thiol group is selected, and if a metal oxide surface is used, a SAM with a thalcoxysilane group is used. One of the main advantages of hybrid membranes is that they exhibit very high stability (compared to planar lipid membranes) and a low amount of small defects. As a result, it is possible to apply various research methods, including electrochemical ones, in dynamic experiments. However, the hybrid membrane also has a drawback - it does not have a thin layer of water between the membrane and the solid surface, which would allow the insertion of integral proteins into it and the use of such bilayers for biosensors that measure the activity of bacterial toxins embedded in the membranes. Therefore, another model of membranes was developed - these are surface-attached membranes (Junghans A. et al., 2010; McGillivray D.J. et al., 2007). The structure of the membranes attached to the surface is special in that they consist of a self-organized monolayer of "anchor" compounds resembling the structure of lipids. These anchors also have an active functional group that forms strong bonds with the base, as in a conventional SAM, but the tail further down consists of a polyethylene glycol (PEG) chain and this compound ends with two carbon chains that resemble the hydrophobic part of lipids. After the formation of such a self-assembled monolayer, during vesicle casting, a thin layer of water is formed in the part of the self-assembled monolayer where PEG is present due to affinity. Incorporating toxins or other proteins requires "dilution" of the anchors on the surface using short-chain, low-molecular-weight SAM compounds that have a hydrophilic group (such as a hydroxy group) on the outside, also known as diluents. Forming a mixed SAM from long anchor compounds and short diluents and flooding the vesicles results in a surface-tethered bilayer phospholipid membrane that has a submembrane water reservoir. Such a system can be applied to studies of integral proteins, detection of pore-forming toxins.

Membranes can be formed on different surfaces. One of the most popular substrates is an atomically smooth gold surface, which is usually prepared by magnetron sputtering. Such a surface can be used with AFM, SPR or N R methods. Formed membranes are characterized by high homogeneity (minimum defects are obtained during membrane formation) and high stability. The thickness of the submembrane layer of water was determined by the neutron reflectometry method (McGillivray D.J. et al., 2007). Therefore, these membranes are successfully applied to studies of pore-forming toxins, and biological sensors are developed for determining the concentration of bacterial toxins self-intercalated in membranes.

Membrane patterns on the vaporized gold surface is a rather widely researched topic and the optimal parameters for membrane formation have been determined, leading to the first steps towards the commercialization of biosensors (Patent No. LT6424B (2017) "Method for obtaining surface-immobilized phospholipid bilayer membranes (tBLM)"). Knowing that gold and the surface preparation process (electron sputtering) are expensive, the aim is to discover alternative substrates that are cheaper and more easily commercially available in order to create a cheap and easy-to-prepare biosensor. We will note that membrane patterns on metallic substrates such as gold are opaque, so optical methods that require light transmission are difficult to apply to such patterns. It is important to note that there are data (Rakovska et al., 2015) that anchoring compounds, gold thiolates, can easily migrate on the gold surface, which leads to the formation of clusters of anchoring compounds on the surface and the deterioration of functional membrane properties, especially when trying to regenerate membranes in contact with water media.

One alternative to gold substrates could be thin film metal oxides. Recently, tin oxide doped with different elements is getting more and more attention. These are optically transparent and electrically conductive semiconductors, which opens up the opportunity to apply the formed systems in photovoltaics. Optical research methods can also be applied to the research of formed membranes.

In order to form a surface-attached membrane on metal oxide surfaces, it is necessary to use a self-assembled monolayer consisting of trialkoxysilanes. During the hydrolysis reaction, strong Si-O-metal covalent bonds are formed (Wasserman S.R. et al., 1989). In order to form a membrane on metal oxide surfaces, which would have a sub-membrane water reservoir and could be applied to studies of pore-forming proteins, it is necessary to discover such compounds for SAM formation, which would have a hydrophilic part above the functional group (forms connections with the surface) and a hydrophobic tail. After the formation of such a SAM, a thin layer of water would form in the hydrophilic part due to hydrogen bonds, while the hydrophobic part would intercalate between the phospholipids and become part of the membrane during vesicle casting.

Thus, in summary, the main disadvantages for the application of surface-immobilized membranes in the development of biosensors could be identified:

1. Gold membrane substrates are opaque, making it more difficult to apply optical research methods. Meanwhile, oxide substrates can be made optically transparent.

2. Without deteriorating their properties, membranes can be formed on gold surfaces only once, i.e. due to the lability and tendency of gold-thiolates to migrate on the surface, membrane regeneration is not possible. Therefore, it is relevant to apply oxide surfaces suitable for multiple membrane formation.

3. Using thiolate chemistry to form anchor compounds, the range of biosensor substrates is limited to metals, such as gold, while silane anchor compounds allow expanding the list of substrates to semiconductors and dielectrics.

4. The commonly used surface is gold, it is an expensive metal, expensive equipment is used for its preparation, so the aim is to apply cheaper electrically conductive and non-conductive oxide and oxidized metal surfaces.

ESSENCE OF THE INVENTION

Metal and non-metal oxide surfaces have been selected for the implementation of this invention. The application of thin film oxide surfaces for membrane formation has advantages compared to the previously used gold film surfaces for membrane formation, which have a minimum thickness of over 50 nm and are therefore not optically transparent. Since thin-layer oxides are optically transparent, the formed phospholipid membranes can be studied by optical research methods and possibly applied to photocurrent generation or solar cell development.

Due to the chemistry of the surface of the oxides, for the formation of the self-assembled monolayer, silane compounds are used with the surface of metal oxides forming a strong Si-O-metal bond, which ensures that the formed self-assembled monolayer is characterized by high stability. For this reason, silanized oxide surfaces can be suitable for multiple formation of phospholipid membranes with the same properties, in contrast to gold film surfaces characterized by surface mobility of gold thiolate molecules, leading to instability of anchor monolayers and concentration of anchors in surface pools and degradation of functional properties of membranes.

In this work, a new procedure for the synthesis of silanes, based on click-reaction chemistry and requiring no expensive equipment, was used for the formation of SAMs. The mixture of long-chain and short-chain silanes prepared during the synthesis can be immediately applied to monolayer formation without additional purification. Phospholipid membranes formed during vesicle casting are stable and can be regenerated.

Metal or non-metal oxides are cheaper than Au surfaces commonly used for the formation of phospholipid membranes, so there are more opportunities for commercial application of phospholipid biosensors for the detection of toxins and other biologically active substances constructed on electrically conductive and non-conductive oxide surfaces.

Advantages of the proposed method:

1. Using silane anchors, phospholipid biosensors can be constructed on inexpensive, as well as commercially available, electrically conductive and non-conductive oxide and oxidized metal surfaces.

2. The proposed biosensor can be regenerated and used several or more times.

3. The proposed biosensor can be constructed on an optically transparent substrate and integrated into optical research devices/methods.

4. The proposed biosensor can be constructed on conductive, semiconducting and dielectric surfaces.

BRIEF DESCRIPTION OF THE DRAWINGS picture. Electrochemical impedance spectra in Cole-Cole coordinates measured at 0 V potential vs Ag/AgCI,Clsot.: black circles - clean FTO glass, white circles - FTO glass functionalized with TOPS:ATS 1:1 SAM mixture in heptane solvent, black triangles - tBLM, composition - DOPC/Chol 6:4.

picture. Electrochemical impedance spectra in Cole-Cole coordinates measured at 0 V potential vs Ag/AgCI,Clsot.: black circles - clean FTO glass, white circles - FTO glass functionalized with TOPS:ATS 1:1 SAM mixture in toluene solvent, black triangles - tBLM, composition - DOPC/Chol 6:4.

picture. Electrochemical impedance spectra in Cole-Cole coordinates measured at 0 V potential vs Ag/AgCI,Clsot.: black circles - clean ITO glass, white circles - ITO glass functionalized with TOPS:ATS 1:1 SAM mixture in heptane solvent, black triangles - tBLM, composition - DOPC/Chol 6:4.

picture. Fluorescence microscope images, A-D - DOPC/Chol 6:4 tBLM, (where 1% cholesterol is labeled with fluorescent dye (Chol-Cy5)) on TOPS:ATS 1:1 SAM-functionalized glass slides, E-F - same membrane on glass slides plates that were not functionalized with SAM. A, B and C - the edge of the membrane.

picture. Electrochemical impedance spectra in Cole - Cole coordinates measured at 0 V potential vs Ag/AgCI,CLsot.: black circles - clean NP wafer, white circles - NP wafer functionalized with TOPS:ATS 1:1 SAM mixture in heptane solvent, black triangles - tBLM, composition - DOPC/Chol 6:4. A - zoomed-in part of the curve.

picture. Electrochemical impedance spectra in Cole - Cole coordinates measured at 0 V potential vs Ag/AgCI,CL _S ot.: black circles - clean evaporated Ti/TiO2 wafer, white circles - Ti/TiO2 wafer functionalized with TOPS:ATS 1:1 SAM mixture in heptane solvent , black triangles - tBLM, composition - DOPC/Chol 6:4. A zoomed-in part of the curve.

picture. Electrochemical impedance spectra in Cole - Cole coordinates measured at 0 V potential vs Ag/AgCI,Clsot.: black circles - clean Si wafer, white circles - Si wafer functionalized with TOPS:ATS 1:1 SAM mixture in heptane solvent, black triangles - tBLM, composition - DOPC/Chol 6:4.

figure. Electrochemical impedance spectra in Cole - Cole coordinates measured at 0 V potential vs Ag/AgCI,Clsot.: black circles - DOPC/Chol 6:4 tBLM formed on FTO that was functionalized with TOPS:ATS 1:1 SAM, white circles - 60 min after 50 nM melittin insertion into the membrane, black triangles 60 min after 100 nM melittin insertion into the membrane. A - zoomed-in picture of part of the curve. Electrochemical impedance spectra in Cole-Cole coordinates measured at 0 V potential vs Ag/AgCI,Clsot.: black circles - DOPC tBLM formed on FTO that was functionalized with TOPS:ATS 1:1 SAM, white circles - after 60 min after 50 nM melittin of membrane insertion, black triangles 60 min after membrane insertion of 100 nM melittin.

picture. Dependence of the change in conductance of DOPC:Chol 6:4 tBLM upon incorporation of different concentrations of melittin into the membrane.

picture. Electrochemical impedance spectra in Cole - Cole coordinates measured at 0 V potential vs Ag/AgCI,Clsot.: black circles - DOPC/Chol 6:4 tBLM formed on FTO that was functionalized with TOPS:ATS 1:1 SAM, white circles - after 60 min after 50 nM alpha-hemolysin insertion into the membrane, black triangles - 60 min after 100 nM alpha-hemolysin insertion into the membrane. A zoomed-in part of the curve.

picture. Electrochemical impedance spectra in Cole-Cole coordinates measured at 0 V potential vs Ag/AgCI,Clsot.: black circles - DOPC tBLM formed on FTO that was functionalized with TOPS:ATS 1:1 SAM, white circles - 60 min after 100 nM alpha -hemolysin insertion into the membrane.

picture. Dependence of the change in conductance of DOPC:Chol 6:4 tBLM upon incorporation of different concentrations of alpha-hemolysin into the membrane.

picture. Electrochemical impedance spectra in Cole - Cole coordinates measured at 0 V potential vs Ag/AgCI,Clsot.: white symbols - membrane formation several times; black symbols - membrane washing.

DETAILED DESCRIPTION OF THE INVENTION

Taking into account the fact that the development of previous phospholipid biosensors used a gold surface that is not optically transparent, formed self-assembled monolayers of alkylthiols and phospholipid membranes on gold surfaces are not stable enough and cannot be regenerated, as well as considering the cost of gold, metals and non-metals were selected for the implementation of this invention oxide surfaces. The first advantage in the use of these materials is that the oxides in the form of thin films have a high transmittance of visible light and UV radiation. This property allows the application of optical research methods in the development of biosensors, which could not be done with gold surfaces. Optical research methods expand the possible areas of research and pave the way for the application of phospholipid membranes on solid surfaces in the development of photocurrent generating systems.

The formation of a phospholipid biosensor requires a self-assembled monolayer characterized by hydrophobicity and capable of adsorbing and breaking down phospholipid vesicles so that a single phospholipid bilayer is formed. A monolayer is formed on the Au surface using alkylthiol compounds, where covalent Au-S bonds are formed. Although the formed Au-S bonds are strong, but due to the lability of the surface gold atoms, the molecules of the monolayer are mobile on the surface. In case of prolonged contact with the aqueous medium (up to several hours), the monolayer molecules spread over the entire surface rarely form islands, which hinders the formation of a solid phospholipid bilayer - a phospholipid biosensor. Meanwhile, for the formation of a self-assembled monolayer on metal oxide surfaces, compounds containing the trichlorosilane (or trihydroxysilane) functional group are used. Due to the surface chemistry of oxides, strong Si-O-metal covalent bonds are formed, due to which self-assembled monolayers are characterized by high stability (Wasserman S.R. et al., 1989). Prolonged contact with aqueous media or other physical external factors has little or no effect on the properties of the monolayer. This provides additional advantages in the use of oxide surfaces for the formation of phospholipid membranes: the membranes not only exhibit high stability over a long period of time (several days), but also the possibility of regenerating the membranes on the same surface without deteriorating the membrane properties.

Also taking into account the cost of preparing gold surfaces, metal and non-metal oxide surfaces are significantly cheaper, which is relevant in order to apply the phospholipid biosensor for the detection of toxins and other membrane proteins and biologically active substances for commercial use.

In this invention, the lack of commercially available trichlorosilane anchors was taken into account in order to adapt metal and non-metal oxides for the formation of phospholipid biosensors. Based on click-reaction chemistry, which does not require expensive equipment, the synthesis of new silanes has been carried out. This methodology is superior to already known synthesis methods in that a mixture of long-chain and short-chain silane is prepared, where during the formation of a self-organized monolayer, the short chain dilutes the amount of long anchors on the surface, forming a sparsely populated self-organized monolayer. This allows for the formation of a surface-attached membrane.

Conventional designations:

EIS - electrochemical impedance spectroscopy

CV - cyclic voltammetry

AFM - atomic force microscope

SPR - surface plasmon resonance

NR - neutron reflectometry

SAM - self-assembled monolayer

PEG - polyethylene glycol

NMR - nuclear magnetic resonance tBLM - surface attached phospholipid bilayer membranes

FTO - fluorine doped tin oxide glass

ITO - indium tin oxide

ATS - allyltrichlorosilane

TOPS - trichloro(3-(octadecylthio)propyl)silane

ODT - Octadecanethiol

DMPA - 2,2-dimethoxy-2-phenylacetophenone

Materials and methods:

Fluorine doped tin oxide glass - FTO, 300 mm χ 300 mm χ 2.2 mm

Indium tin oxide - ITO, 150 mm x 150 mm x 0.7 mm

Stainless steel - NP, 316L alloy according to AISI nomenclature: composition: Fe/Cr 18%/Ni 10%/Mo 3%, 150 mm χ 150 mm χ 0.5 mm

Glass plate - 25 mm x 75 mm

Trichloro(3-(octadecylthio)propyl)silane - TOPS

Deionized water - dejon. H2O)

Sodium chloride - NaCI

Sodium dihydrogen phosphate - NaH2PO4-2H2O

Sodium hydroxide - NaOH

Allyltrichlorosilane - H2C=CHCH2SiCl3

Octadecanethiol - CH3(CH2)i7SH

2,2-dimethoxy-2-phenylacetophenone - C16H16O3

Heptane - C7H16

Toluene - C7H8

Chloroform - CHCh

1,2-dioleoyl-3-phosphoglycerocholine - DOPC

Cholesterol - chol

Melitin - mel, from bee venom

Alpha hemolysin - aHL, from Staphylococcus aureus

Cholesterol derivative with merocyanine dye: 2-(1E,3E,5E)-5-(1-(5carboxypentyl)-3,3-dimethylindolin-2-ylidene)penta-1,3,3-trimethyl-3H-indole bromide Chol -Cy5

UV lamp - 365 nM 5W ¹³ C and ¹ H NMR spectra were recorded with a Bruker Ascend 400 spectrometer (400 MHz frequency for ¹ H NMR and 100 MHz- ¹³ C NMR), using solvent residue (CDCI3) signals.

The chemical shift is given on the δ scale (ppm).

EIS measurements were performed on a potentiostat/galvanostat pAutolab Type III with installed software FRA. The measurements were carried out in a standard three-electrode cell, in which the working electrode is a metal oxide surface (working area 0.32 cm ² ), the reference electrode is Ag/AgCI, Chsot., and the auxiliary electrode is a platinum wire. The phosphate buffer solution used for electrochemical measurements consisted of 0.01 M NaH2PO4 and 0.1 M NaCl, with pH adjusted to 7.1 using NaOH.

Fluorescence microscopy was performed using an Olympus BX61WI fluorescence microscope, a UMPIanFN-W water immersion objective (10X/0.30 NA) and a Qimaging EXi Aqua camera. A Hg lamp and an Olympus U-MWG2 filter were used to determine the surface distribution of Chol-Cy5. Glass slides were mounted on the bottom of Petri dishes and the vesicle solution was completely washed out of the system with clean PBS pH 7.1 before recording the images.

EXAMPLES OF IMPLEMENTATION OF THE INVENTION example

1. The synthesis of the TOPS compound is carried out by a click-reaction mechanism (TuckerSchwartz A.K. etai., 2011). ATS and ODT are mixed in a molar ratio of 2:1 in a clear tube. 2 mol% DMPA initiator is added and stirred on a magnetic stirrer until a homogeneous mixture is obtained. This mixture is irradiated with a 365 nm wavelength 5 W UV lamp for 24 hours. After this synthesis procedure, we have a ready-to-use silanization mixture (TOPS:ATS 1:1) for SAM formation. The TOPS:ATS 1:1 mixture is kept wrapped in aluminum foil to avoid direct sunlight, which may reduce the chemical activity of the synthesized mixture.

2. Before the formation of the membrane, the FTO-coated glass plate was cut into 25 mm × 10 mm size electrodes and a washing procedure was performed. Washing in an ultrasonic bath in 2 different media for 10 min each: (i) 2% Micro 90 solution; (ii) in deionized water. Next, FTO plate 1 vai. is incubated for approx. H2SO4, followed by a deion rinse. H2O and kept in 2-propanol in an ultrasonic bath for 10 min. In the final step, the FTO is held in dejon. H2O approximately 18-20 years. (overnight) and dried under a stream of N2 gas.

3. The prepared sample is further used in the SAM formation - silanization procedure. 20 ml of heptane (or other saturated alkane) is heated to 60°C in a small beaker and 30 pi of the synthesized TOPS:ATS mixture is added. Then the electrode is inserted in a vertical position into the stirred silanization solution for 1 minute. at a temperature of 60°C. After that, the sample is washed with clean heptane, dried in a stream of N2 gas and heated at 100°C for 1 hour.

4. The formation of tBLM is performed using the vesicle casting method (Figure 1) (Patent No. LT6424B (2017)). 10 mM solutions of lipids in chloroform are prepared and stored at -20°C. In another vessel, such amount of solution is taken to prepare a solution with a concentration of 1 mM in the desired volume. Chloroform is evaporated with a stream of nitrogen gas (blow for about 30 min) until a white lipid film remains on the bottom of the container. It is filled with a buffer solution with a pH of 4.4 and a composition of 0.1 M NaH2PO4 and 0.01 M NaCl. An automatic pipette is dipped into the solution, and cycles of insertion and ejection are repeated until the lipid film bounces off the walls, solubilizes, and forms a homogeneous solution.

example

It is carried out in the same way as described in Example 1, only in point 3 the silanization is carried out in an aromatic solvent (eg toluene) (Figure 2), but benzene can also be used.

example

It is carried out in the same way as described in example 1, only in point 2 it is used:

(i) ITO glass (Figure 3), but it is also possible to use a glass plate coated with other oxides: cadmium tin oxide (CdSnO4), zinc oxide (ZnO), aluminum doped zinc oxide (AI:ZnO), antimony tin oxide (SnO2/ Sb2Os), indium antimony oxide (InSbO), gallium zinc oxide (GaZnO), indium zinc oxide (InZnO), indium indium tin oxide (InSbSnO), indium gallium zinc oxide (InGaZnO), bismuth selenite (Bi2SeOs), yttrium barium cuprate, metal oxide perovskites;

(ii) glass plate (Figure 4), but it is also possible to use colored glass, quartz, sapphire, mica;

(iii) stainless steel (Figure 5), but aluminium, titanium, copper, nickel, nickel-titanium alloys, cobalt and cobalt alloys can also be used;

(iv) vaporized Ti/TiO2 plate (Figure 6), but vaporized Zn/ZnO, Zr/ZrO2, Cr/CrxOy plates can also be used;

(v) Si/SiO2 wafer (Figure 7), but Ge/GeO2 can also be used.

example

It is carried out as described in example 1, only the membrane damage of DOPC/Chol 6:4 and DOPC with different concentrations of proteins - pore-forming toxins is observed: (i) melittin (Figures 8 and 9); (ii) with alpha-hemolysin (Figures 11 and 12), the calibration curves of the phospholipid biosensor are created - an example of the dependence of the change in membrane conductance on the concentration of the toxin (Figures 10 and 13)

This is done as described in Example 1, only observing the removal and re-formation of the phospholipid bilayer on the same surface several times (Figure 14).

The presented examples show that by using the click-reaction chemical process, it is possible to synthesize a mixture of silanes suitable for use, into which a metal oxide surface, such as a glass plate covered with an FTO layer or an ITO layer, or pure glass, colored glass, quartz, sapphire plate, mica, or stainless steel, aluminum, titanium, copper, nickel, nickel-titanium alloy, cobalt-cobalt alloy, or evaporated Ti/TiO2 wafer, or Si/SiO2 wafer, or other oxide-coated metal or semiconductor product, such as cadmium tin oxide (CdSnO4), zinc oxide (ZnO), aluminum alloyed zinc oxide (AI:ZnO), antimony tin oxide (SnO2/Sb2Os), indium antimony oxide (InSbO), gallium zinc oxide (GaZnO), indium zinc oxide (InZnO), antimony indium tin oxide (InSbSnO), indium gallium zinc oxide (InGaZnO), bismuth selenite (Bi2SeOs), yttrium barium cuprate, perovskite, an anchor monolayer is obtained, suitable for attachment of surface-immobilized membranes, tBLM, by the multilayer vesicle casting method described in patent no. LT6424B (2017). The composition of tBLMs can be easily varied depending on the pore-forming protein-toxin that interacts with the membrane and inserts into the membrane, and the resulting tBLMs can be easily regenerated without losing the functional properties of tBLMs. The developed phospholipid biosensors are suitable for the determination of toxin concentration using a calibration curve, such as the dependence of the change in membrane conductance on toxin concentration.

Literature

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2. Eicher-Lorka O., Charkovą T., Matijoška A., Kuodis Z., Urbelis G., Penkauskas T., Mickevičius M., Bulovas A., Valincius G., Cholesterol-based tethers and markers for model membranes investigation , Chemistry and Physics of Lipids 195 (2016) 71-86.

3. Junghans A., Koper I., Structural Analysis of Tethered Bilayer Lipid Membranes, Langmuir 26 (2010) 11035-11040.

4. McGillivray D. J., Valincius G., Vanderah D. J., Febo-Ayala W., Woodward J. T, Heinrich F., Kasianowicz J. J., Losche M., Molecular-scale structural and functional characterization of sparsely tethered bilayer lipid membranes, Biointerphases 2 (2007) 21-33.

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6. Ragaliauskas T., Mickevičius M., Rakovska B., Penkauskas T., Vanderah D.J., Heinrich F., Valincius G., Fast formation of low-defect-density tethered bilayers by fusion of multilamellar vesicles. Biochimica et Biophysica Acta 1859 (2017) 669678.

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Claims (9)

Membrane biosensor, which is different in that it includes silane compounds that form an anchored self-assembled monolayer of silane-organomolecules, which forms a continuous phospholipid bilayer. The biosensor according to claim 1, which differs in that organic solvents such as alkanes and/or their mixtures including heptane, aromatic hydrocarbons and/or their mixtures including toluene and benzene are used for the formation of the self-organizing monolayer. The biosensor according to points 1-2 differs in that the surfaces used for forming the membrane are selected from FTO, ITO, cadmium tin oxide (CdSnO4), zinc oxide (ZnO), aluminum alloyed zinc oxide (Al:ZnO), antimony tin oxide (SnO2/ Sb2O5), indium antimony oxide (InSbO), gallium zinc oxide (GaZnO), indium zinc oxide (InZnO), antimony indium tin oxide (InSbSnO), indium gallium zinc oxide (InGaZnO), bismuth selenite (Bi2SeO5), yttrium barium cuprate and metal oxide perovskites. The biosensor according to points 1-2 differs in that the surfaces used for forming the membrane are selected from glass, colored glass, quartz, sapphire, mica. The biosensor according to points 1-2 differs in that the surfaces used for forming the membrane are selected from stainless steel, aluminum, titanium, copper, nickel, nickel-titanium alloys, cobalt and cobalt alloys. A biosensor according to points 1-2, which differs in that the surfaces of thin active metal films are used for the formation of the membrane, obtained by magnetron or thermal evaporation, forming a natural or artificially obtained oxide layer in the air, selected from Ti/TiO2, Zn/ZnO, Zr/ZrO2 and Cr/CrxOy. The biosensor according to points 1-2 differs in that the semiconductor surfaces used for forming the membrane, having an oxide layer, are selected from Si/SiO2 and Ge/GeO2. A biosensor according to claims 1-7, characterized in that the formed sensitive phospholipid layer can be removed and re-formed one or more times on the same surface. Use of the biosensor according to points 1-8 for the identification of an agent of membrane-damaging proteins.