Liquid crystal based sensor devices for detecting binding events

or analytes at interfaces

The present invention relates to liquid crystal based sensor devices for detecting binding events or analytes. Moreover, the present invention relates to methods of manufacturing liquid crystal based sensor devices for detecting binding events or analytes. Furthermore, the present invention relates to uses of liquid crystal based sensor devices for detecting binding events or analytes as well as uses of liquid crystal based sensor devices for detecting the activity of analytes and for screening compounds binding to analytes and/or modifying their activity.

Liquid crystals are materials that can exhibit the mobility of liquids and the anisotropy of solid crystals. Thermotropic liquid crystals have demonstrated utility in the transduction of molecular events at an interface into macroscopic responses visible with the naked eye. The long-range orientational order and optical anisotropy of LC molecules can transform chemical and biomolecular binding events into amplified optical signals that can be easily observed, even with naked eye. Interfaces between thermotropic LCs and immiscible aqueous phases have received substantial recent attention owing to the strong coupling that can occur between the ordering of the LCs and organization of assemblies of molecules that can be formed at these interfaces. These properties, combined with the optical anisotropy of LC molecules, make them well-suited for the direct transduction and amplification of the binding of an analyte to a target at an interface into an optical output. Most of the current methods for the detection of biological analytes require laboratory-based analytical detectors and labeled species such as fluorophores or radioactive isotopes and therefore, it is of great potential for providing highly sensitive and low-cost bioassays performed away from central laboratories without the need of sophisticated instrumentation.

It has been shown that the self-assembly of surfactants, lipids, and polymers at these interfaces is strongly coupled to the ordering of the LCs, and that the presence and organization of these molecules can be reported through changes in the optical appearance of the LCs. It has also been reported that more complex interfacial phenomena, such as specific binding events involving proteins, bacteria, viruses, enzymatic reactions, hybridization of DNA, and the culture of human embryonic stem cells at aqueous-LC interfaces can trigger dynamic orientational transitions in the LCs. These studies suggest that aqueous-LC interfaces define a promising class of biomolecular interfaces for reporting interfacial phenomena.

The principles of LC-based detection rely on optical, anchoring, and the elastic properties arising from molecular anisotropics and the unique liquid-crystalline phase of the LC material. The molecular anisotropy of a liquid crystalline sample creates a difference in the refractive indices of light parallel and perpendicular to the bulk molecular orientation, i.e., the LC director. Molecular-scale interactions between an LC and a neighboring interface result in a preferred anchoring angle relative to the surface normal. Information about the interface, in the form of surface anchoring, is transmitted as far as 100 μπι into the bulk as a result of the elastic nature of the LC director field.

Current liquid crystal based sensor devices for detecting binding events or analytes require the presence of molecules or molecular groups that specifically interact with the analyte, such as antibodies. It was an object of the present invention to provide for liquid crystal based sensor devices for detecting binding events or analytes as well as the activity of analytes that are simply and easy to use.

Liquid crystal sensors for detecting a binding event or the presence of an analyte or the presence of an assembly of such analyte(s)

The objects of the present invention are solved by a liquid crystal sensor device for detecting a binding event or the presence of an analyte, such as a nucleic acid, protein, lipid, a prokaryotic cell, such as a bacterial cell, a eukaryotic cell, such as a mammalian cell, a cell organelle, a small molecule compound, or a virus, or for detecting an assembly of such analyte (s), said device comprising, in that order: a first substrate,

at least a first electrode on said first substrate,

an insulating layer and/or an alignment layer, or an insulating and alignment layer, coating said electrode,

a liquid crystal layer on said insulating layer or said alignment layer or said insulating and alignment layer,

wherein said device does not comprise a recognition moiety or molecule that is directly attached to the surface of said first substrate. In one embodiment, the sensor device further comprises a second electrode on said first substrate.

In one embodiment, the sensor device further comprises: a second substrate, located opposite to said first substrate and sandwiching said liquid crystal layer between said first and second substrate, and wherein there is a gap between said second substrate and said liquid crystal layer, which gap acts as a reaction chamber for receiving a sample to be analysed.

In one embodiment, said second substrate has the following components: a second electrode on said second substrate,

an insulating layer on said second electrode, wherein said insulating layer faces said liquid crystal layer located on said first substrate.

In one embodiment, the first and/or second substrate is a solid, preferably glass or plastic.

In one embodiment, said first electrode and said second electrode, independently at each occurrence, is a non-interdigitated, plain electrode or an interdigitated electrode (IDE).

In one embodiment, the insulating layer on the first and/or second electrode is made of polyimide.

In one embodiment, the alignment layer is made of a silane such as (octadecyldimethyl(3- trimethoxy-silylpropyl) ammonium chloride) (DMOAP), or a heat/photo-curable polyimide providing for a defined pre-tilt angle of the liquid crystal in the liquid crystal layer or a self assembled monolayer of self assembling compounds, such as thiols, dithiocarbamates, or an obliquely evaporated oxide layer, such as Si0 ₂ providing for a pre-tilt of the liquid crystal in the liquid crystal layer, depending on the evaporation angle of the oxide.

In one embodiment, the liquid crystal layer has a homeotropic alignment, homogeneous alignment, or no pre-defined alignment. In one embodiment, the liquid crystal layer comprises a liquid crystal selected from thermotropic liquid crystals with positive/negative/or no dielectric anisotropy, dual-frequency liquid crystals, discotic and/or lyotropic liquid crystals, and combinations of the foregoing.

In one embodiment, the liquid crystal layer comprises a single liquid crystal compound or a mixture of two or more different liquid crystal compounds, or a liquid crystal mixture mixed with dopants such as nanoparticles.

In one embodiment, the liquid crystal layer comprises 5CB and/or MLC6608 liquid crystals.

In one embodiment, the first and/or second electrode , is/are transparent or non-transparent, wherein, preferably, the electrode (electrodes) is (are) made of indium tin oxide (ITO) or fluorinated tin oxide (FTO).

In one embodiment, the device further comprises crossed polarizers below and above the liquid crystal layer, preferably in the planes of the first and second substrate, respectively, that allow to measure changes in the liquid crystal alignment by measuring changes of transmission/reflection of polarized light, or wherein the device further comprises at least one polariser above the liquid crystal layer and wherein the substrate underneath the liquid crystal layer is a reflective substrate.

In one embodiment, the device further comprises means to measure an electrical quantity which means allows to measure changes in the liquid crystal alignment by measuring changes in such electrical quantity, for example capacitance, resistance, or current.

In one embodiment, the device further comprises one polarizer only, preferably in the plane of either the first or the second substrate, and wherein the respective other substrate without a polarizer is for use in conjunction with a light source providing polarised light, such as a liquid crystal display device.

The objects of the present invention are also solved by a method of manufacturing a liquid crystal sensor device according to the present invention, said method comprising the steps: a) providing a first substrate,

b) depositing on said first substrate at least a first electrode,

c) depositing an insulating layer and/or an alignment layer, or an insulating and alignment layer, coating said first electrode,

d) depositing a liquid crystal layer on said insulating layer or said alignment layer or said insulating and alignment layer.

In one embodiment, the method further comprises the step b') depositing a second electrode on said first substrate.

In one embodiment, the method further comprises the additional step: e) providing a second substrate and putting it on top of said liquid crystal layer, such that said liquid crystal layer is sandwiched between said first and second substrate, wherein there is a gap between said second substrate and said liquid crystal layer, which gap acts as a reaction chamber for receiving a sample to be analysed.

In one embodiment, said second substrate has a second electrode located on it, and wherein step f) is performed such that said second electrode is facing said liquid crystal layer.

In one embodiment, said substrates, electrodes, insulating layer, alignment layer, liquid crystal layer and device are as defined above.

The objects of the present invention are also solved by a method of using the liquid crystal sensor device of the present invention for detecting a binding event or the presence of an analyte or the presence of an assembly of such analyte, said method comprising the steps: a) providing a liquid crystal sensor device according to the present invention,

b) adding a sample to be analyzed onto the liquid crystal layer of said liquid crystal sensor device,

c) examining for a binding event or an analyte or assembly of analyte to be detected, based on changes induced by said binding event, analyte or assembly of analyte, to the alignment of the liquid crystals in said liquid crystal layer. In one embodiment, said binding event is the formation of a layer of an analyte at an interface between said liquid crystal layer and said sample.

In one embodiment, said analyte is selected from the group comprising a lipid, a protein, a nucleic acid, a small-molecule compound, a prokaryotic cell, such as a bacterial cell, and a eukaryotic cell, such as a mammalian cell, a virus, wherein the analyte has the capability to change the liquid crystal alignment at said interface between said liquid crystal layer and said sample.

In one embodiment, said binding event is a formation or presence of a lipid monolayer or lipid multilayer, wherein said lipid monolayer or multilayer is free or forms part of a cell membrane of a prokaryotic or eukaryotic cell or of a cell organelle.

In one embodiment, said examination in step c) occurs by visual inspection of the transmission and/or reflection of polarized light or by transmission and/or reflection measurements with polarized light, and examining for a change in such transmission, or said examination in step c) occurs by measurements of an electrical quantity, preferably by capacitance, resistance or current measurements, and examining for a change in such electrical quantity.

In one embodiment, the method further comprises the step: d) verifying that an observed change in transmission or reflection or in electrical quantity is caused by said binding event or the presence of an analyte or analyte assembly, preferably, by said formation or presence of said layer, at an interface between said liquid crystal layer and said sample, and is not the result of a disrupted area of the sensor with no liquid crystal and no lipid layer being present, by applying a voltage of direct or alternating current to said first and/or second electrode, and investigating whether this results in a change of detected transmission or reflection of polarized light or measured electrical quantity.

In one embodiment, the applied voltage is in the range of 0.1-25 V sinusoidal/triangular/square signal, and the frequency is in the range from 0 Hz to 1 GHz, preferably 50-10000Hz, more preferably 60-1000Hz. In one embodiment, the method further comprises the step:

b') increasing the local concentration of the analyte at the interface between said liquid crystal layer said sample by electrophoresis, dielectrophoresis or Alternating Current Electroosmosis (ACEO).

In one embodiment, the method further comprises the step:

b ) increasing the reaction speed by applying a voltage of direct or alternating current to said first and/or second electrode, wherein the applied voltage is in the range of 0.1-25 V sinusoidal/triangular/square signal, and the frequency is in the range from 0 Hz to 1 GHz, preferably 100 Hz to 10 MHz, more preferably 1 kHz to 1 MHz.

In one embodiment, the binding event takes place in water or an aqueous solution or in the gas phase, and wherein the analyte or assembly of analyte is present in water, an aqueous solution or in the gas phase, or wherein the analyte or assembly of the analyte is immobilised on a solid support, such as polymeric beads, or one of the substrates.

The objects of the present invention are also solved by a method of using the liquid crystal sensor device of the present invention for identifying an analyte or for the stability of a layer formed by an analyte, said method comprising the steps: a) providing a liquid crystal sensor device according to the present invention,

b) adding or filling a sample to be analyzed onto the liquid crystal layer of said liquid crystal sensor device,

c) examining for the formation of a layer of said analyte at an interface between said liquid crystal layer and said sample based on changes induced to the alignment of the liquid crystals, and, if a layer of said analyte has been formed,

d) applying a voltage of alternating current to said first and/or second electrode, wherein said examination in step c) occurs by visual inspection of the transmission and/or reflection of polarized light or by transmission and/or reflection measurements by polarized light and examining for a change in such transmission and/or reflection, or said examination in step c) occurs by measurements of an electrical quantity, preferably by capacitance, resistance or current measurements, and examining for a change in such electrical quantity and identifying said analyte based on said inspection or measurement.

In one embodiment, the method further comprises the steps:

e) removing the voltage,

f) repeating step c),

g) comparing the result of step f) with that of step c), to verify if the alignment of the liquid crystals has returned to the same alignment as before application of the voltage, indicative that the layer of the analyte is still intact.

In one embodiment, the method further comprises the steps:

h) repeating steps d) - g) with increasing voltage until a threshold voltage is reached in step g) at which the alignment of the liquid crystals has not returned to the same alignment as before application of said threshold voltage, indicative of disruption of said layer of the analyte,

i) determining said threshold voltage applied as a measure for the stability of the layer formed from the analyte under the investigation.

In one embodiment, said analyte is selected from the group comprising a lipid, a protein, a nucleic acid, a small-molecule compound, a prokaryotic cell, such as a bacterial cell, and a eukaryotic cell, such as a mammalian cell, a virus, wherein the analyte has the capability to change the liquid crystal alignment at the interface between said liquid crystal layer and said sample.

In one embodiment, said examination in step f) occurs by visual inspection of the transmission and/or reflection of polarized light or by transmission and/or reflection measurements by polarized light and examining for a change in such transmission and/or reflection, or said examination in step f) occurs by measurements of an electrical quantity, preferably by capacitance, resistance or current measurements, and examining for a change in such electrical quantity.

The objects of the present invention are also solved by a method of using the liquid crystal sensor device of the present invention for identifying a biomolecule, said method comprising the steps: performing the method of using the liquid crystal sensor device according to the present invention, j) identifying the analyte based on its stability of the layer formed, as determined in step i).

In the following are provided definitions of terms used in the context of the liquid crystal sensors for detecting a binding event or the presence of an analyte or the presence of an assembly of such analyte(s):

The term "analyte", as used herein in the above context, refers to any molecule the presence (or absence) of which is to be detected. Examples are "biological" molecules such as nucleic acids, proteins and lipids. An analyte, as used herein, may also refer to a cell membrane, alone or as part of a biological cell, such as a eukaryotic cell, e.g. mammalian cell, or a prokaryotic cell, e.g. a bacterium. The term "analyte" may also include viruses or elements thereof. The term "analyte" further includes proteins, alone or as part of larger assemblies, such as protein complexes, toxins, cell organelles, such as ribosomes, mitochondria etc.

The term "binding event" refers to a change in molecular interactions of an analyte with an interaction partner, which may or may not be an analyte. Binding events are, for example, association or dissociation events, e.g. between antibody and antigen, hormone and receptor, protein and receptor, etc., but also the formation of layers of analytes, the assembly of complexes of analytes etc.

In accordance with the present invention, the term "electrode" refers to an electrical lead to apply voltage. An electrode may be "interdigitated", meaning that it has a comb-like shape with two combs lying opposite each other and the respective figures of the combs engaging with each other. Alternatively, an electrode may be a non-interdigitated. An electrode may be transparent or non-transparent. A transparent electrode may, for example, be formed from indium tin oxide (ITO) or from fluorinated tin oxide (FTO). A non-transparent electrode may be reflective and may, for example, be formed from silver (Ag) or aluminium (Al). The reflective electrodes are ideal when one uses the liquid crystal based sensor device in conjunction with a reflective microscope. The term "insulating layer", as used herein, is meant to refer to a layer on the electrode that prevents reactions of molecules from other layers, such as the liquid crystal layer, with the electrode. An insulating layer may for example be formed from polyimide.

As used herein in the above context, the term "dual-frequency liquid crystal" or "dual- frequency LC" refers to a type of liquid crystal that has positive dielectric anisotropy at low frequency alternating current (AC) voltage, but possesses negative dielectric anisotropy when a high AC voltage is applied. As an example of a dual-frequency LC from Merck called MLA3969, its dielectric anisotropy changes its polarity at around 30kHz. Hence, the terms "low" and " high", in the context of AC voltages only refers to the relative positions of the respective voltages with respect to each other. Typically, low can mean 0-100 kHz, high can mean 10-100 MHz, but the concrete values depend on the liquid crystal used.

The term "5CB" refers to 4-cyano 4'-pentyl biphenyl, a single component liquid crystal with positive dielectric anisotropy commercially available from Aldrich. "MLC6608" refers to a trade name of a negative LC mixture commercially available from Merck.

The term "alignment layer" is meant to refer to a layer that can induce a specific alignment of a liquid crystal layer, if the alignment layer is brought in contact with a liquid crystal layer. For example, an alignment layer may be formed of DMOAP (Octadecyldimethyl(3- trimethoxysilylpropyl)ammonium chloride), which induces homeotropic alignment of a liquid crystal layer brought in contact with it. Another example of an alignment layer is a layer of polyimide.

The term "an insulating layer and/or an alignment layer" is meant to refer to a scenario where, either, both an insulating layer as well as a separate alignment layer are present, or where either an insulating layer is present or an alignment layer is present. In contrast thereto, the term "an insulating and alignment layer" is meant to refer to a scenario where only a single layer is present which has both an alignment as well as an insulating quality. As opposed to the earlier mentioned scenario, the alignment function and the insulating function are both incorporated into one and the same layer in such "insulating and alignment layer".

As an example of an alignment layer, a polyimide layer may be mentioned, preferably a layer of a heat/photo-curable polyimide. Such a polyimide layer may provide for a defined pre-tilt angle of the liquid crystal in a liquid crystal layer that is in contact with such polyimide layer. The change that may be caused by such polyimide alignment layer may be a change from homeotropic alignment to homogenous alignment, preferably in the range of from 90° to 0°, or the change may be from a homogenous alignment to a homeotropic alignment.

As used herein in the above context, a "crossed polarizers" refers to a set of two polarizers having their polarization axis oriented perpendicular to each other.

The embodiment, where crossed polarizers are used is an embodiment, where, preferably one polarizer is above the liquid crystal layer and another polarizer is below the liquid crystal layer. Their respective polarization axes are oriented perpendicular to each other. This embodiment allows the measurement of changes in the liquid crystal alignment by measuring changes of transmission or reflection of polarized light. In another embodiment, the device only comprises at least one polarizer above the liquid crystal layer (and no polarizer below the liquid crystal layer). In this embodiment, the substrate underneath the liquid crystal layer is a reflective substrate, and the embodiment allows to measure changes in the liquid crystal alignment by measuring changes of the reflection of polarized light, i.e. light that is reflected at the reflective substrate.

In yet a further embodiment, the liquid crystal sensor based device according to the present invention only comprises one polarizer, which is, preferably, located above the liquid crystal layer, and the substrate below the liquid crystal layer is a transparent substrate which can be then used in conjunction with a light source that provides polarized light.

The term "reaction chamber" is meant to refer to the space where a binding event takes place, or where the analyte or assembly of analytes to be detected is present.

Typically, the reaction chamber in embodiments of the liquid crystal sensor device according to the present invention is formed by the space above the liquid crystal layer. This space is either open towards the top and encompassed by suitable boundaries at the sides so as to form a containment where an analyte can be received. In other embodiments, this space is formed by the gap between the second substrate and the liquid crystal layer and is encompassed by boundaries at the side. This latter embodiment is a "closed" embodiment. In the open embodiment, the analyte can be simply put on top of the liquid crystal layer; in the closed embodiment, the analyte can be put onto the liquid crystal layer through specific openings especially designated for such purpose, or it can be put onto the liquid crystal layer in an open state, and thereafter the device is closed by lowering the second substrate on top; or, alternatively, the analyte can be put into the reaction chamber by capillary filling it, vacuum filling it or drop casting it in the closed state; yet in an alternative embodiment, the analyte may also be filled into the reaction chamber by using an appropriate pump.

The term "transmission measurements with polarized light", as used herein in the above context, refers to a transmission measurement carried out with a detector for polarized light, such as a polarized microscope. The device is typically placed between the crossed polarizers, and the light transmitting through them is measured.

The term "electrophoresis" refers to the movement of charged particles/molecules in a electric field. The electric field can be spatially uniform or not uniform.

As used herein in the above context, the term "dielectrophoresis" refers to an effect that induces attractive or repulsive force exerted on polarisable, non-charged substances, by means of a non uniform electric field. Thus, this effect can be used to attract/repel bio-molecules to an electrode by applying AC voltage to an interdigitated electrode (IDE).

The term "alternating current electroosmosis", "AC electroosmosis", or "ACEO" refers to an effect that induces a Joule heat resulting in fluid motion of free charges in a non uniform electric field. In this way, randomly floating bio-molecules can be mixed within their solution, shortening the time for a free floating bio-molecule to land on an liquid crystal layer to form a molecular layer (such as lipid layer).

The term "recognition moiety" or "recognition molecule" refers to a molecule or molecular group that is capable of recognizing, i.e. specifically binding an analyte. In accordance with the present invention, such recognition moiety or molecule is not directly attached to the surface of the first substrate. If such recognition moiety or molecule is used in a device according to the present invention, it will preferably be located above the liquid crystal layer of the device. It serves the purpose of specifically binding an analyte in the vicinity of the liquid crystal layer, as a result of which, the analyte will induce a change in the alignment of the liquid crystal layer which can then be subsequently detected. The present inventors have surprisingly found that by using a liquid crystal layer as a sensor in combination with the application of an electric field, binding events that take place in the vicinity of or adjacent to or in contact with the liquid crystal layer can be readily detected. This also opens up a possibility to amplify the detections signal by forcing the analytes to the sensing area of the device, i.e. to the vicinity of the liquid crystal layer.

Liquid crystal sensors for detecting the presence of an analyte or the activity of an analyte, said LC sensors comprising a phospholipid layer

The objects of the present invention are solved by a liquid crystal sensor device for detecting the presence of an analyte, such as a prokaryotic cell, such as a bacterial cell, and/or a procaryotic agent, such as a toxin, such as a bacterial toxin, said device comprising, in that order:

a first substrate,

a liquid crystal layer on said first substrate,

a phospholipid layer on said liquid crystal layer,

wherein said device does not comprise a recognition moiety or molecule that is directly attached to the surface of said first substrate.

In a preferred embodiment, the phospholipid layer comprises at least one recognition moiety or molecule, such as a receptor, an enzyme or an antibody.

In a preferred embodiment, the phospholipid layer comprises cholesterol.

In a preferred embodiment, the phospholipid layer comprises at least one recognition moiety or molecule, such as a receptor, and cholesterol.

The at least one recognition moiety or molecule, such as a receptor, and cholesterol are embedded in said phospholipid layer.

The phospholipid layer preferably comprises between about 1 to about 20 % (by weight) of cholesterol, preferably about 10 to about 15 % (by weight) of cholesterol.

In one embodiment, the sensor device further comprises: - at least a first electrode between said first substrate and said liquid crystal layer, and

- an insulating layer and/or an alignment layer, or an insulating and alignment layer, coating said electrode.

In one embodiment, the sensor device further comprises:

a second substrate, located opposite to said first substrate and sandwiching said liquid crystal layer and said phospholipid layer between said first and second substrate, and wherein there is a gap between said second substrate and said phospholipid layer, which gap acts as a reaction chamber for receiving a sample to be analysed.

In one embodiment, said second substrate further comprises:

- a second electrode on said second substrate, and

optionally, an insulating layer on said second electrode, wherein said insulating layer faces said phospholipid layer/liquid crystal layer located on said first substrate.

In one embodiment, the sensor device comprises a second electrode but no first electrode. In this embodiment, the sensor device can comprise:

a first substrate,

a liquid crystal layer on said first substrate,

a phospholipid layer on said liquid crystal layer,

a second substrate, located opposite to said first substrate and sandwiching said liquid crystal layer and said phospholipid layer between said first and second substrate, and wherein there is a gap between said second substrate and said phospholipid layer, which gap acts as a reaction chamber for receiving a sample to be analysed, preferably a second electrode on said second substrate, or a second electrode on said second substrate and an insulating layer on said second electrode, wherein said insulating layer faces said phospholipid layer/liquid crystal layer located on said first substrate.

In one embodiment, the first and/or second substrate is a solid, preferably glass or plastic.

Examples for plastic are cycloolefin polymer (COP), cycloolefin copolymer (COC) and polydimethyl siloxan (PDMS). In one embodiment, the liquid crystal layer has a homeotropic alignment, homogeneous alignment, or no pre-defined alignment.

In one embodiment, the liquid crystal layer comprises a liquid crystal selected from thermotropic liquid crystals with positive/negative/or no dielectric anisotropy, dual-frequency liquid crystals, discotic and/or lyotropic liquid crystals, and combinations of the foregoing.

In one embodiment, the liquid crystal layer comprises a single liquid crystal compound or a mixture of two or more different liquid crystal compounds, or a liquid crystal mixture mixed with dopants, such as nanoparticles or small organic molecules, such as bent-core organic molecules.

In one embodiment, the liquid crystal layer is dye doped, preferably with dichroic dye(s), fluorescent dye(s), or doped with quantum dot(s).

Examples for liquid crystals are 5CB or MLC6608 liquid crystals. Further liquid crystals are known to the skilled artisan.

In one embodiment, the phospholipid layer comprises a single phospholipid compound or a mixture of two or more different phospholipid compounds.

The phospholipid compounds are preferably selected from

DOPC (1 ,2-dioleoyl-sn-glycero-3-phosphocholine),

DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine),

DMPC ( 1 ,2-dimyristoyl-sn-glycero-3 -phosphocholine),

DSPC (1 ,2-distearoyl-sn-glycero-3 -phosphocholine),

DLPC (1 ,2-dilauroyl-sn-glycero-3 -phosphocholine),

1 ,2-dimyristoleoyl-5«-glycero-3 -phosphocholine (14:1, PC),

1 ,2-dipalmitoleoyl-5«-glycero-3 -phosphocholine (16:1, PC),

1,2 -dieicosenoyl-SM-glycero-3 -phosphocholine (20:1, PC),

1 ,2-dierucoyl-srt-glycero-3 -phosphocholine (22:1, PC),

1 ,2-dinervonoyl-j?7-glycero-3 -phosphocholine (24:1, PC),

or combinations thereof. In one embodiment, the at least one recognition moiety or molecule comprised in said phospholipid layer is a receptor, such as ganglioside GMi.

In one embodiment, the phospholipid layer comprises between about 0.1% to about 10% (by weight) of GMj, preferably about 0.5% to about 5% (by weight) of GMi.

In one embodiment, said first electrode and said second electrode, independently at each occurrence, is a non-interdigitated, plain electrode or an interdigitated electrode (IDE).

In one embodiment, the insulating layer on the first and/or second electrode is made of polyimide.

In one embodiment, the alignment layer is made of a silane such as (octadecyldimethyl(3- trimethoxy-silylpropyl) ammonium chloride) (DMOAP), or a heat/photo-curable polyimide providing for a defined pre-tilt angle of the liquid crystal in the liquid crystal layer or a self assembled monolayer of self assembling compounds, such as thiols, dithiocarbamates, or an obliquely evaporated oxide layer, such as Si0 ₂ providing for a pre-tilt of the liquid crystal in the liquid crystal layer, depending on the evaporation angle of the oxide.

In one embodiment, the first and/or second electrode , is/are transparent or non-transparent, wherein, preferably, the electrode (electrodes) is (are) made of indium tin oxide (ITO) or fluorinated tin oxide (FTO).

In one embodiment, the device further comprises crossed polarizers below and above the liquid crystal layer, preferably in the planes of the first and second substrate, respectively, that allow to measure changes in the liquid crystal alignment by measuring changes of transmission/reflection of polarized light, or wherein the device further comprises at least one polariser above the liquid crystal layer and wherein the substrate underneath the liquid crystal layer is a reflective substrate.

In one embodiment, the device further comprises means to measure an electrical quantity which means allows to measure changes in the liquid crystal alignment by measuring changes in such electrical quantity, for example capacitance, resistance, or current. In one embodiment, the device further comprises one polarizer only, preferably in the plane of either the first or the second substrate, and wherein the respective other substrate without a polarizer is for use in conjunction with a light source providing polarised light, such as a liquid crystal display device.

In one embodiment, the device further comprises a microfiuidic device in fluidic connection with the reaction chamber.

In one embodiment, the device further comprises a filtering unit to filter any material from the sample that might be larger in size than the analyte of interest, such as a bacterial cell or a microbial toxin, such that said material does not enter the reaction chamber.

In one embodiment, the reaction chamber has a depth in the range from 800 nm to 100 μπι, preferably from 900 nm to 30 μηι, and has a width or diameter, in case of a round chamber, in the range of 1 μηι to 1 mm, preferably from 25 μπι to 500 μιη, and/or the reaction chamber is of glass, plastic or has metal walls, such as gold walls.

The objects of the present invention are also solved by a method of manufacturing a liquid crystal sensor device according to the present invention, said method comprising the steps: a) providing a first substrate,

b) depositing a liquid crystal layer on said first substrate,

c) depositing a phospholipid layer on said liquid crystal layer, optionally comprising at least one recognition moiety/molecule, such as a receptor, and/or cholesterol.

In one embodiment, the method further comprises the steps

a') depositing on said first substrate at least a first electrode, and

a") depositing an insulating layer and/or an alignment layer, or an insulating and alignment layer, coating said first electrode.

In one embodiment, the method further comprises the additional step:

d) providing a second substrate and putting it on top of said phospholipid layer, such that said phospholipid layer and said liquid crystal layer are sandwiched between said first and second substrate, wherein there is a gap between said second substrate and said phospholipid layer, which gap acts as a reaction chamber for receiving a sample to be analysed.

In one embodiment, said second substrate has a second electrode located on it, and wherein step e) is performed such that said second electrode is facing said phospholipid layer/liquid crystal layer.

In one embodiment, said substrates, electrodes, insulating layer, alignment layer, liquid crystal layer, phospholipid layer and device are as defined above.

The objects of the present invention are also solved by a method of using the liquid crystal sensor device of the present invention for detecting the presence of an analyte or the activity of an analyte, said method comprising the steps: a) providing a liquid crystal sensor device according to the present invention,

b) adding a sample to be analyzed onto the phospholipid layer of said liquid crystal sensor device,

c) examining for a binding event, an analyte to be detected or the activity of an analyte, based on changes induced by said binding event, analyte or activity of analyte, to the alignment of the liquid crystals in said liquid crystal layer.

In one embodiment, said binding event is the binding of the analyte to the recognition moiety or molecule, such as a receptor, enzyme or antibody, comprised in said phospholipid layer.

In one embodiment, said binding event is the binding of the analyte to the phospholipid layer.

In one embodiment, said activity of an analyte is the reaction of the analyte with the recognition moiety or molecule, such as a receptor such as a receptor, enzyme or antibody, comprised in said phospholipid layer.

In one embodiment, said activity of an analyte is the reaction of the analyte with the phospholipid layer. For example, the analyte is capable of changing the alignment of the liquid crystals in said liquid crystal layer

due to its attachment or due to its binding to the recognition moiety or molecule (such as a receptor, enzyme or antibody), or

due to its reaction with the recognition moiety or molecule (such as the receptor, enzyme or antibody), or

due to its attachment or due to its binding to the phospholipid layer, or

due to its reaction with the phospholipid layer.

Said analyte is preferably selected from the group comprising a prokaryotic cell, such as a bacterial cell, and a procaryotic agent, such as a toxin, such as a microbial toxin, wherein the analyte has the capability to change the liquid crystal alignment at said interface between said liquid crystal layer and said phospholipid layer.

In one embodiment, said examination in step c) occurs by visual inspection of the transmission and/or reflection of polarized light or non-polarized light or by transmission and/or reflection measurements with polarized light or with non-polarized light, and examining for a change in such transmission, or said examination in step c) occurs by measurements of an electrical quantity, preferably by capacitance, resistance or current measurements, and examining for a change in such electrical quantity.

In one embodiment, the method further comprises the step:

d) verifying that an observed change in transmission or reflection or in electrical quantity is caused by said binding event or the presence of an analyte or the activity of an analyte, preferably at an interface between said liquid crystal layer and said phospholipid layer, and is not the result of a disrupted area of the sensor with no liquid crystal and no phospholipid layer being present, by applying a voltage of direct or alternating current to said first and/or second electrode, and investigating whether this results in a change of detected transmission or reflection of polarized light or measured electrical quantity.

In one embodiment, the applied voltage is in the range of 0.1-25 V sinusoidal/triangular/square signal, and the frequency is in the range from 0 Hz to 1 GHz, preferably 100 Hz to 10 MHz, more preferably 1 kHz to 1 MHz. In one embodiment, the method further comprises the step:

b') increasing the local concentration of the analyte or energizing the analyte at the interface between said phospholipid layer and said sample by electrophoresis, dielectrophoresis, Alternating Current Electroosmosis (ACEO) or Joule heating.

In one embodiment, the method further comprises the step:

b ) increasing the reaction speed by applying a voltage of direct or alternating current to said first and/or second electrode, wherein the applied voltage is in the range of 0.1-25 V sinusoidal/triangular/square signal, and the frequency is in the range from 0 Hz to 1 GHz, preferably 100 Hz to 10 MHz, more preferably 1 kHz to 1 MHz.

In one embodiment, the binding event takes place in water or an aqueous solution or in the gas phase, and wherein the analyte is present in water, an aqueous solution or in the gas phase, or wherein the analyte is immobilised on a solid support, such as polymeric beads, or one of the substrates.

In one embodiment, the presence of an analyte is detected indirectly.

The presence of an analyte is preferably detected indirectly by detecting the presence of a second analyte, wherein said second analyte is capable of changing the alignment of the liquid crystals in said liquid crystal layer.

Said second analyte is preferably binding or reacting to the analyte to be (indirectly) detected.

Said second analyte is preferably selected from the components of a medium (such as broth medium) used to grow/culture/feed the first analyte, or antibiotics or drugs that are effective on the first analyte.

In this embodiment, the method of the invention is a method of using the liquid crystal sensor device of the present invention for indirectly detecting the presence of an analyte or the activity of an analyte, and said method comprises the steps:

a) providing a liquid crystal sensor device according to the present invention, b) adding a sample to be analyzed onto the phospholipid layer of said liquid crystal sensor device,

c) examining for a binding event, a second analyte to be detected or the activity of a second analyte, based on changes induced by said binding event, the second analyte or activity of the second analyte, to the alignment of the liquid crystals in said liquid crystal layer.

Said binding event is preferably the binding of the second analyte to the recognition moiety or molecule, such as a receptor, comprised in said phospholipid layer or is the binding of the second analyte to the phospholipid layer.

Said activity of the second analyte is preferably the reaction of the second analyte with the recognition moiety or molecule, such as a receptor, comprised in said phospholipid layer or is the reaction of the second analyte with the phospholipid layer.

In one embodiment, the alignment of the liquid crystals in the (surface) liquid crystal layer of the liquid crystal sensor device according to the present invention is monitored over time and the change in alignment, if present, is measured over time.

In one embodiment of indirect detecting the presence of an analyte, a slowed down time of alignment change can be seen, which is indicative of presence of the analyte.

The term "slowed down time of the alignment change", as used herein, refers to changes of the alignment of the liquid crystals in the (surface) liquid crystal layer in the liquid crystal sensor device according to the present invention which are slower compared to the alignment changes that were taking place before the amount of active component (the component that causes the alignment change, i.e. the second analyte) decreased, due to e.g. binding or reacting to the analyte.

The objects of the present invention are also solved by a method of using the liquid crystal sensor device of the present invention for the screening for compounds that bind to an analyte and/or modify the activity of an analyte, such as inhibitors of microbial toxins,

said method comprising the steps: a) providing a liquid crystal sensor device according to the present invention, b) providing a sample comprising the analyte,

c) providing at least one compound to be screened for binding to the analyte and/or modifying the activity of the analyte,

d) adding said at least one compound to be screened to the sample comprising the analyte,

e) adding the sample comprising the analyte and said compound onto the phospholipid layer of said liquid crystal sensor device,

f) examining for said analyte or the activity of said analyte, based on changes induced by said binding event, analyte or activity of analyte, to the alignment of the liquid crystals in said liquid crystal layer,

wherein no change of the alignment is indicative that the compound to be screened binds to the analyte and/or modifies the activity of the analyte,

and wherein a change of the alignment is indicative that the compound to be screened does not bind to the analyte and/or does not modify the activity of the analyte.

In one embodiment, the alignment or the change of alignment of the liquid crystals in said (surface) liquid crystal layer of the liquid crystal sensor device according to the present invention is monitored over time and the change in alignment, if present, is measured over time.

The term "change of the alignment", as used herein in the context of the "screening method", refers to changes of the alignment of the liquid crystals in the (surface) liquid crystal layer in the liquid crystal sensor device according to the present invention upon addition of sample comprising the analyte and candidate compound (in step e)).

Such change of alignment can be seen in a change of (light) transmission or a change of reflection. In a preferred embodiment such change is preferably at least 0.1 %, more preferably at least 0.5 %, more preferably at least 1 % or more change of transmission or reflection.

The term "no change of the alignment", as used herein in the context of the "screening method", refers to the alignment of the liquid crystals remaining essentially the same as before addition of the sample comprising the analyte and the compound to be screened (in step e)), in particular the same or no essential change in (light) transmission or in reflection.

In a preferred embodiment, less than 0.1 %, more preferably less than 0.05 % or less change in transmission or in reflection.

In other embodiments, the cut-off value can be higher or slower than the above 0.1 %, depending e.g. on the sample and its components, media etc.

The cut-off value can also depend on the detector used or the detection method used, such as the sensitivity of said detector/detection method.

For example, in one embodiment, the effectiveness of an antibiotic, a drug or a substance can be tested with respect to deactivating or killing bacteria or a toxin. For example, in case that one compound, such as antibiotic A, is not effective in deactivating or killing the bacteria/toxin, then it leads to an alignment change of the liquid crystals in the liquid crystal layer. Thus, compound A is not effective in deactivating/killing the respective bacteria/toxin. For example, in another case, another compound, such as antibiotic B, is tested which is effective in deactivating or killing the bacteria/toxin, then no alignment change of the liquid crystals in the liquid crystal layer can be seen, because the bacteria/toxin are deactivated. Thus, compound B is effective in deactivating/killing the respective bacteria/toxin.

Said analyte is preferably selected from the group comprising a prokaryotic cell, such as a bacterial cell, and a procaryotic agent, such as a toxin, such as a microbial toxin, wherein the analyte has the capability to change the liquid crystal alignment at said interface between said liquid crystal layer and said phospholipid layer.

In one embodiment, said examination in step f) occurs by visual inspection of the transmission and/or reflection of polarized light or non-polarized or by transmission and/or reflection measurements with polarized light or with non-polarized, and examining for a change in such transmission, or said examination in step f) occurs by measurements of an electrical quantity, preferably by capacitance, resistance or current measurements, and examining for a change in such electrical quantity. In one embodiment, the method further comprises the step:

d') verifying that an observed change in transmission or reflection or in electrical quantity is caused by said presence of said analyte or the activity of said analyte, preferably at an interface between said liquid crystal layer and said phospholipid layer, and is not the result of a disrupted area of the sensor with no liquid crystal and no phospholipid layer being present, by applying a voltage of direct or alternating current to said first and/or second electrode, and investigating whether this results in a change of detected transmission or reflection of polarized light or measured electrical quantity.

In one embodiment, the applied voltage is in the range of 0.1-25 V sinusoidal/triangular/square signal, and the frequency is in the range from 0 Hz to 1 GHz, preferably 100 Hz to 10 MHz, more preferably 1 kHz to 1 MHz.

In one embodiment, the method further comprises the step:

b') increasing the local concentration of the analyte or energizing the analyte at the interface between said phospholipid layer and said sample by electrophoresis, dielectrophoresis, Alternating Current Electroosmosis (ACEO) or Joule heating.

In one embodiment, the method further comprises the step:

b") increasing the reaction speed by applying a voltage of direct or alternating current to said first and/or second electrode, wherein the applied voltage is in the range of 0.1-25 V sinusoidal/triangular/square signal, and the frequency is in the range from 0 Hz to 1 GHz, preferably 100 Hz to 10 MHz, more preferably 1 kHz to 1 MHz.

In one embodiment, the screening for compounds that bind to an analyte and/or modify the activity of an analyte is performed indirectly.

Compound(s) that bind to an analyte and/or modify the activity of an analyte are preferably screened indirectly, such as by detecting the presence or activity of a second analyte, wherein said second analyte is capable of changing the alignment of the liquid crystals in said liquid crystal layer. As described above, said second analyte is preferably binding or reacting to the/with the analyte to which the compound(s) to be screened bind to, recat to and/or modify the activity of.

This embodiment comprises competitive and non-competitive binding of the second analyte and the compound to be screened to the analyte.

As described above, said second analyte is preferably selected from the components of a medium (such as broth medium) used to grow/culture/feed the first analyte, or antibiotics or drugs that are effective on the first analyte.

In one embodiment of the methods of the present invention, the alignment or the change of alignment of the liquid crystals in said (surface) liquid crystal layer of the liquid crystal sensor device according to the present invention is monitored over time and the change in alignment, if present, is measured over time.

In the following are provided definitions of terms used in the context of liquid crystal sensors for detecting the presence of an analyte or the activity of an analyte, said LC sensors comprising a phospholipid layer:

The term "analyte", as used herein in the above context, refers to a molecule or cell the presence (or absence) or activity of which is to be detected. Examples are procaryotic agents, such as toxins, such as microbial toxins. An analyte, as used herein, may also refer to a cell membrane, alone or as part of a biological cell, such as a prokaryotic cell, e.g. a bacterium. As examples for microbial toxins, Cholera toxin (Vibrio cholered), heat-labile enterotoxin (E. coli), Pertussis toxin (Bordetella pertussis), Anthrax toxin {Bacillus anthracis), Tetanus toxin (Clostridium tetani), streptococcal and staphylococcal toxins, are mentioned.

The term "recognition moiety" or "recognition molecule", as used herein in the above context, refers to a molecule or molecular group that is capable of recognizing, i.e. specifically binding an analyte. In accordance with the present invention, such recognition moiety or molecule is not directly attached to the surface of the first substrate. If such recognition moiety or molecule is used in a device according to the present invention, it will preferably be comprised in the phospholipid layer of the device. It serves the purpose of specifically binding an analyte, in the vicinity of the liquid crystal layer, as a result of which, the analyte will induce a change in the alignment of the liquid crystal layer which can then be subsequently detected.

As examples for recognition moieties or recognition molecules antibodies, antigens, receptors, transmembrane proteins, enzymes are mentioned. As an example of a receptor, ganglioside GMi is mentioned, which for example binds cholera toxin.

The term "binding event" refers to a change in molecular interactions of an analyte with an interaction partner, which may or may not be an analyte. Binding events are, for example, association or dissociation events, e.g. between antibody and antigen, hormone and receptor, protein and receptor, etc. Binding events are for example the binding of the analyte to the recognition moiety or molecule, such as a receptor, comprised in the phospholipid layer.

The term "reaction" refers to a change of a component (such as the analyte, the phospholipid layer, the second analyte) which occurs due to the binding of an analyte with an interaction partner, such as with the recognition moiety or molecule, such as a receptor/antibody/enzyme, comprised in the phospholipid layer, or with the phospholipid layer, wherein such change can be, for example, a (partial) hydrolysis of the phospholipid layer, or a hydrolysis or a chemical modification of the analyte, or a digestion of the second analyte by the analyte etc.

The term "activity" refers to the ability of a component, such as the analyte, to bind, to grow, to undergo changes, to consume, to catalyze or participate in or inhibit e.g. chemical reactions or growth etc. The term "activity" also refers to the extent with which a component, such as the analyte, binds, undergoes changes, consumes, catalyzes or participates in or inhibits e.g. chemical reactions or growth.

The term "phospholipid layer", as used herein in the above context, refers preferably to a monolayer of phospholipids.

In accordance with the present invention, the term "electrode" refers to an electrical lead to apply voltage. An electrode may be "interdigitated", meaning that it has a comb-like shape with two combs lying opposite each other and the respective figures of the combs engaging with each other. Alternatively, an electrode may be a non-interdigitated. An electrode may be transparent or non-transparent. A transparent electrode may, for example, be formed from indium tin oxide (ITO) or from fluorinated tin oxide (FTO). A non-transparent electrode may be reflective and may, for example, be formed from silver (Ag) or aluminium (Al). The reflective electrodes are ideal when one uses the liquid crystal based sensor device in conjunction with a reflective microscope.

The term "insulating layer", as used herein in the above context, is meant to refer to a layer on the electrode that prevents reactions of molecules from other layers, such as the liquid crystal layer, with the electrode. An insulating layer may for example be formed from polyimide.

As used herein, the term "dual-frequency liquid crystal" or "dual-frequency LC" refers to a type of liquid crystal that has positive dielectric anisotropy at low frequency alternating current (AC) voltage, but possesses negative dielectric anisotropy when a high AC voltage is applied. As an example of a dual-frequency LC from Merck called MLA3969, its dielectric anisotropy changes its polarity at around 30kHz. Hence, the terms "low" and " high", in the context of AC voltages only refers to the relative positions of the respective voltages with respect to each other. Typically, low can mean 0-100 kHz, high can mean 10-100 MHz, but the concrete values depend on the liquid crystal used.

The term "5CB" refers to 4-cyano 4'-pentyl biphenyl, a single component liquid crystal with positive dielectric anisotropy commercially available from Aldrich. "MLC6608" refers to a trade name of a negative LC mixture commercially available from Merck.

The term "alignment layer" is meant to refer to a layer that can induce a specific alignment of a liquid crystal layer, if the alignment layer is brought in contact with a liquid crystal layer. For example, an alignment layer may be formed of DMO AP (Octadecyldimethyl(3- trimethoxysilylpropyl)ammonium chloride), which induces homeotropic alignment of a liquid crystal layer brought in contact with it. Another example of an alignment layer is a layer of polyimide.

The term "an insulating layer and/or an alignment layer" is meant to refer to a scenario where, either, both an insulating layer as well as a separate alignment layer are present, or where either an insulating layer is present or an alignment layer is present. In contrast thereto, the term "an insulating and alignment layer" is meant to refer to a scenario where only a single layer is present which has both an alignment as well as an insulating quality. As opposed to the earlier mentioned scenario, the alignment function and the insulating function are both incorporated into one and the same layer in such "insulating and alignment layer".

As an example of an alignment layer, a polyimide layer may be mentioned, preferably a layer of a heat/photo-curable polyimide. Such a polyimide layer may provide for a defined pre-tilt angle of the liquid crystal in a liquid crystal layer that is in contact with such polyimide layer. The change that may be caused by such polyimide alignment layer may be a change from homeotropic alignment to homogenous alignment, preferably in the range of from 90° to 0°, or the change may be from a homogenous alignment to a homeotropic alignment.

As used herein in the above context, a "crossed polarizers" refers to a set of two polarizers having their polarization axis oriented perpendicular to each other.

The embodiment, where crossed polarizers are used is an embodiment, where, preferably one polarizer is above the liquid crystal layer and another polarizer is below the liquid crystal layer. Their respective polarization axes are oriented perpendicular to each other. This embodiment allows the measurement of changes in the liquid crystal alignment by measuring changes of transmission or reflection of polarized light. In another embodiment, the device only comprises at least one polarizer above the liquid crystal layer (and no polarizer below the liquid crystal layer). In this embodiment, the substrate underneath the liquid crystal layer is a reflective substrate, and the embodiment allows to measure changes in the liquid crystal alignment by measuring changes of the reflection of polarized light, i.e. light that is reflected at the reflective substrate.

In yet a further embodiment, the liquid crystal sensor based device according to the present invention only comprises one polarizer, which is, preferably, located above the liquid crystal layer, and the substrate below the liquid crystal layer is a transparent substrate which can be then used in conjunction with a light source that provides polarized light.

The term "reaction chamber" is meant to refer to the space where a binding event takes place, or where the analyte or activity of the analyte to be detected is present.

Typically, the reaction chamber in embodiments of the liquid crystal sensor device according to the present invention is formed by the space above the phospholipid layer and liquid crystal layer. This space is either open towards the top and encompassed by suitable boundaries at the sides so as to form a containment where an analyte can be received. In other embodiments, this space is formed by the gap between the second substrate and the liquid crystal layer and is encompassed by boundaries at the side. This latter embodiment is a "closed" embodiment. In the open embodiment, the analyte can be simply put on top of the phospholipid layer/liquid crystal layer; in the closed embodiment, the analyte can be put onto the phospholipid layer/liquid crystal layer through specific openings especially designated for such purpose, or it can be put onto the phospholipid layer/liquid crystal layer in an open state, and thereafter the device is closed by lowering the second substrate on top; or, alternatively, the analyte can be put into the reaction chamber by capillary filling it, vacuum filling it or drop casting it in the closed state; yet in an alternative embodiment, the analyte may also be filled into the reaction chamber by using an appropriate pump.

The term "transmission measurements with polarized light", as used herein in the above context, refers to a transmission measurement carried out with a detector for polarized light, such as a polarized microscope. The device is typically placed between the crossed polarizers, and the light transmitting through them is measured.

The term "electrophoresis" refers to the movement of charged particles/molecules in a electric field. The electric field can be spatially uniform or not uniform.

As used herein in the above context, the term "dielectrophoresis" refers to an effect that induces attractive or repulsive force exerted on polarisable, non-charged substances, by means of a non uniform electric field. Thus, this effect can be used to attract/repel bio-molecules to an electrode by applying AC voltage to an interdigitated electrode (IDE).

The term "alternating current electroosmosis", "AC electroosmosis", or "ACEO" refers to an effect that induces a Joule heat resulting in fluid motion of free charges in a non uniform electric field. In this way, randomly floating bio-molecules can be mixed within their solution, shortening the time for a free floating bio-molecule to land on an liquid crystal layer to form a molecular layer (such as lipid layer).

The phospholipid bilayers of cell membranes usually exist in a fluidic state. They can also be referred to as lyotropic liquid crystal state or phase. A lyotropic liquid crystal is a material that forms a liquid crystal phase upon addition of a solvent. The term is mostly used to describe materials composed of amphophilic molecules with hydrophilic head-group attached to a hydrophobic group. Phospholipids (see Figure 1A) are an example of such amphophilic compounds, forming liquid crystal phase in the presence of a solvent, solvent being an aqueous solution for this case. Fluidity of natural cell membranes is essential for the functioning of membrane associated systems. Towards this end, cholesterol (see Figure 1 B) is the essential structural component of a mammalian cell membrane, and it is required to establish the proper membrane permeability and fluidity over the range of physiological temperatures. Cholesterol interacts with the lipid membranes through the interactions between 1) hydroxyl group of cholesterol with the polar head group of phospholipid 2) hydrocarbon chains and the cyclic hydrocarbon (steroid) part of the molecule with the non-polar fatty acid chains of the phospholipid. Through these interactions cholesterol increases the membrane packing. Additionally, cholesterol also plays a significant role in intracellular transport, cell signalling, etc. events. The fluidity of phospholipid bilayers of cell membranes enables the motion and spatial reorganization of the membranes which is essential for many biological processes. The studies presented herein have shown that one can form phospholipid bilayer like fluidic assemblies using thermotropic liquid crystals (like the liquid crystals used in display applications) together with phospholipids. A typical structure of such a thermotropic liquid crystal, 5CB, with a polar head group and a hydrophobic tail is shown in Figure 1C. A general explanation scheme of a phospholipid-LC bilayer formation is shown in figure 2. Forming such a bilayer is well documented in literature (see, for example, Bai, N. L. Abbott, Langmuir 2011, 27, 5719 - 5738). One starts with a liquid crystal bulk which is aligned homeotropically from one side on a solid substrate that is pre-treated with a specific coating material. On the other hand, the top side of the LC bulk which is in contact with air or water has no homeotropic alignment, the LC molecules stay rather parallel to the bottom substrate. When the LC air/water interface is brought in contact with a solution of lipid vesicles (liposomes) the lipid-LC bilayer forms almost immediately, turning the parallel orientation of LC molecules to homeotropic orientation. This orientation change of liquid crystals can be very well observed at a polarization microscope from the change in light transmittance.

By making such a lipid-LC layer structure close to a natural environment, meaning mimicking the natural cell membranes while employing a thermotropic LC as signal processor, it is now possible to detect bioevents which are associated with cell membranes. One very important group of events that includes the incorporation of cell membranes is the attack of microbial toxins inside the cells. Towards this end, some of the most common and structurally similar protein toxins and their bacterial sources are: Cholera toxin (V. cholerea), heat-labile entero toxin (E.coli), Pertussis toxin (Bordetella pertussis), Anthrax toxin (Bacillus anthracis), Tetanus toxin (Clostridium tetani), several streptococcal and staphylococcal toxins and so on. What seems to be very common between these toxins is their working mechanisms. They mostly have discreet subunits or domains; 1) a subunit or a domain "A" with a specific enzymatic function and 2) a binding domain, subunit or an oligomer "B" that interacts with the cell membrane receptor.

The present inventors have surprisingly found that by generating a lipid-LC layer that can mimic the natural cell membrane, a device for the effective attachment/binding of the subunit of the toxin to the LC-lipid layer can be provided. The existence of the toxin or the toxic bacteria can thereby be detected by simply looking at the optical changes (change in light transmittance) taking place. This detection is possible only if the toxin is active and this offers a further possibility: By this method/device, also the effectiveness of a drug which is supposed to inactivate or modify the toxin or the respective bacteria can also be detected using this sensing technique/device. The present invention, thus, provides a method and a device which is/are useful for: (1) detection of toxins and bacteria, (2) detection of the activity of toxins and bacteria, and (3) screening for drug design and development.

The sensor device of the present invention has the following advantages: (1) It is a very simple device. (2) The sensing mechanism does not require any labelling. (3) No complex instrumentation is necessary for the detection. (4) It can be very well integrated together with a microfluidic device. (5) Very quick detection is possible. (6) It exhibits a very good (i.e. sensitive) detection limit. For example, as low as 30 pg/mL CTB can be detected (wherein no lower concentrations were tested).

In the following, reference is made to the figures, wherein Figure 1 shows a cartoon of the experimental setup in Example 1 ;

Figure 2 is a cartoon showing the experimental setup of Example 1 in cross-section;

Figure 3 shows cross-polarized microscope images of LC before (left) and after (right) the application of the lipid solution (results obtained in Example 1); Figure 4 shows cross-polarized microscope image of LC upon application of AC 10 V (result obtained in Example 1);

Figure 5 shows the transmission change with time when the lipid solution was applied to the LC layer in Example 1 ;

Figure 6 shows the transmission change with time when the lipid solution was applied to a negative-type LC layer formed from MLC6608;

Figure 7 shows the result obtained in Example 2: Polarized Optical Microscope (POM) images of homogeneous and homeotropically aligned LC (left), and homeotropically aligned LC after lipid layer formation upon applying lmM UPC3 (l,2-dioleyl-5«-glycero-3- phosphocholine) solution;

Figure 8 is a side-view of a setup to detect changes in liquid crystal alignment upon formation of a lipid layer by resistance/capacitance/current measurements according to Example 3 or by optical detection of changes of transmission/reflection of polarized light;

Figure 9 is a side-view of an alternative setup (involving a top and bottom electrode) to detect changes in liquid crystal alignment upon formation of a lipid layer by resistance/capacitance/current measurements or by optical detection of changes of transmission/reflection of polarized light;

Figure 10 is a cartoon showing top view of a setup to detect changes in liquid crystal alignment upon formation of a lipid layer by resistance/capacitance/current measurements or by optical detection of changes of transmission/reflection of polarized light based on a sandwich cell arrangement. Before monolayer formation (top) and after monolayer formation (bottom);

Figure 11 shows pictures of Water-LC and Lipid-LC interfaces taken under a polarized microscope. The LC molecule and lipid cartoons illustrate the alignments which are in accordance with the dark / bright images detected at the interfaces. The rods depict LC molecules, and the colour of them illustrates how bright they appear under the cross-polarized microscope; Figure 12 shows a side view of the sample;

Figure 13 shows a side-view of a sensor setup for reduced detection time and increased sensor sensitivity detection based on dielectrophoresis (in this case through repulsive forces);

Figure 14 shows lipid layer formation time improvement by an application of square wave voltage according to Example 5;

Figure 15 shows lipid layer formation according to Example 5 with higher voltage application;

Figure 16 shows a side-view of a sensor setup for reduced detection time and increased sensor sensitivity detection based on AC electroosmosis (ACEO);

Figure 17 shows a side-view of a sensor setup for reduced detection time and increased sensor sensitivity detection based on electrophoresis;

Figure 17 a-c shows a homeotropically aligned liquid crystal in a gold grid observed under a cross-polarized microscope (a), a microscope image (b) of the same arrangement as in (a), when the buffer solution is capillary filled in the cell, and a microscope image (c) of the same arrangement as in (a), when the liquid crystal is homeotropically aligned due to the formation of a lipid layer.

Figure 18 shows as a cartoon the effects of lipid layer formation on liquid crystal alignment.

Figure 19 A shows a phospholipid, DOPC, with polar head group and non-polar fatty acid chains; Figure 19 B shows cholesterol; Figure 19 C shows the structure of 5CB, a thermotropic liquid crystal; Figure 19 D shows the structure of ganglioside GM1;

Figure 20 is a cartoon showing the phospholipid-LC layer formation; Figure 21 shows lipid-LC layer formation in the presence of different amounts of GM1 embedded in lipid vesicles (wherein upon lipid-LC layer formation the light transmission decreases.);

Figure 22 shows the detection of CTB when 0.5% GM1 is embedded in LC-lipid layer;

Figure 23 shows cholesterol and GM1 embedded lipid-LC layer formation;

Figure 24 shows the detection of CTB in the presence of 10% cholesterol (referred to as 10 CH in A; B: with cholesterol; C: without cholesterol) (embodiment of Example 8);

Figure 25 shows red-dye doped LC sensor on interdigitated electrodes (IDEs) (of Example 9);

Figure 26 shows black-dye doped LC sensor on interdigitated electrodes (IDEs) (embodiment of Example 9);

Figure 27 shows liquid crystal (5CB) on glass wells with 100 μηι diameter (pictures taken during annealing cycle, cooling from 55°C (5CB in liquid phase) to 21°C (5CB in LC phase) (embodiment of Example 9);

Figure 28 shows photos of dye doped gold grids with and without DOPC (embodiment of Example 9);

Figure 29 shows the definition of reaction times of the embodiment of Example 10;

Figure 30 shows the shortening of lipid (DOPC) layer formation and enzyme (PLAl) reaction times when AC20V is applied at different frequencies (embodiment of Example 10).

The invention will now be further described by reference to the following examples which are given to illustrate, not to limit the present invention.

Examples In the following, different liquid crystal based sensor device structures according to the invention are described.

Example 1 Materials used

Target additive: 1 mM DOPC lipid (l,2-dioleoyl-sn-glycero-3-phosphocholine): The target molecule can also be mixed together with additional dopant/additive such as cholesterol, enzymes etc, as long as it has a capability to change the LC alignment at the solution-LC interface.

Gold Grid: 150 mesh (pitch 165 μηι) from Piano. The gold grid can be of various sizes, but larger grids are more susceptible to water disruption, and smaller ones require longer time to form lipid layer (LL).

PBS Buffer solution: [Phosphate-buffered saline with pH of 7.4]. The solution is not restricted to the PBS solution, as it can be anything such as water with different pH values, TBS (Tris- buffered saline) etc.

5CB: Single component LC with positive dielectric anisotropy (positive LC) from Aldrich. Polyimide (PI) coated interdigitated electrode substrates were purchased from EHC, Japan. DMO AP (Octadecyldimethyl(3-trimethoxysilylpropyl)ammonium chloride): Induces homeotropic alignment of the liquid crystals.

Sample preparation Substrate

1. Treat PI substrate with ozone plasma (0.1 mbar 0 ₂ , 100 W, 1 min), in order to react with DMOAP. The ozone treatment can be either carried out or skipped in the case of homeotropic PI, but it is necessary for an insulating layer (e.g. normal PI like in this case, or PMMA).

2. Soak the substrate in a solution of DMOAP for more than 5 min. The solution was prepared by mixing 250 ml of Octadecyldimethyl(3-trimethoxysilylpropyl)ammonium chloride (60% in methanol, AB1 1 1261) and 150 ml of deionized water (Millipore).

3. Blow away the solution on the substrates with an air gun, and dry them in an 100 °C vacuum oven for 15 min. Then take out the substrates and allow to cool down to a room temperature.

4. Place the gold grid on the substrates where IDEs are. 5. Apply 2 μΐ of 5CB on the gold grid, and remove the excess LC with capillary filling in a micro syringe tip.

6. Place the substrate in a 50 °C oven for 10 min. The temperature should be above the nematic to isotropic temperature of the LC, in order to realize a uniformly homeotropically aligned LC.

Lipid solution:

Small unilamellar lipid vesicles were prepared by passing 500 μL lipid solutions (1 mM in PBS) through a 50 nm pore filter with the help of a hand held extruder. Further dilution with PBS was carried out to prepare 0.1 mM, 0.05mM, 0.0 ImM and 0.005mM solutions.

Measurement

1. Calibrate the cross-polarized microscope brightness. 100 % transmittance was set where there is no sample and when the polarizer and the analyzer are parallel to each other. 0% transmittance was set when there is no light source.

2. Attach electrical contacts to the sample, and place it on the microscope stage.

3. Start taking photos and measure the intensity.

4. Apply 5 μΐ lipid solution.

5. When a stable lipid layer is formed, apply AC 1, 2, 3, 4, 5, 6, 10 V square signal with 1 kHz frequency, (the signal can be sine/triangular wave, and the frequency is not limited to 1 kHz)

The experimental setup used is shown in Figures 1 and 2. Results and Discussion

Before the application of the lipid solution, the LC layer appears grey, with its measured transmittance of c.a. 12%. Upon application of the lipid solution droplet, the LC layer turns darker, with its transmittance of c.a. 5%, due to the induced homeotropic alignment at the solution-LC interface (Fig. 3). After the LC layer was darkened and stayed stable for a while, voltages were applied. For this particular sample, when 1kHz AC 10 V was applied, a clear transmittance change could be observed (Fig. 4). The lines from Interdigitated electrodes are visible in both figures, but this is because of the deflection of the source light at ITO, and not because of the LC switching.

Figure 5 shows the transmission change measured during the above mentioned observation. The initial homeotropically aligned state is already dark, but the transmission even lowers when lipid layer is formed upon an addition of a lipid solution droplet. The deep dark state brightens a little upon application of 1 kHz AC 1 V, then returns to its original darkness when turned off. The transmittance increases at voltage on-state and the decrease at the off-state becomes more pronounced at higher voltages. For example, when 1 kHz AC 10 V is applied, the on-state transmittance (7%) becomes higher than the 1 V (5%) because more LC is rotated homogeneously, letting more polarized light to pass through the sample. These abrupt change in the transmission can be easily detected electronically or visibly, can be used to distinguish whether the "dark area" is a lipid layer formed LC, or a disrupted area with no LC and no lipid layer. For comparison purpose, a test with a solution without lipid was carried out. As expected, the lipid layer was not formed, hence the transmission stayed approximately at 10 %.

The layer formed by lipid is surprisingly stable to the alignment change of LC layer, as the lipid layer (LL) returns to its original darkness upon the removal of voltage. Apparently, the LC molecules at the LC-solution interface are strong enough to resist the bulk alignment change. The maximum voltage that can be applied without damaging the lipid layer depends on the LC and/or lipid employed. Thus, IDEs can measure the layer stability, and can further be used to identify a kind of target molecule based on its characteristic layer stability.

The spikes (transmittance change by a voltage application) can be easily detected electronically, leading to an automatic distinction of a reliable layer from a disrupted layer, without solely relying on an operator's observation.

The same experiment was repeated using negative type liquid crystals (MLC6608). Figure 6 shows that, even though in this case a mixture of LC with negative dielectric anisotropy was used, the same effect as with 5CB were observed.

Example 2

A similar experimental setup as in Example 1 was followed. However, this time the liquid crystals of the liquid crystal layer were not homeotropically aligned before application of the sample.

The left image of Figure 7 shows a mixture of homogeneously and homeotropically aligned LC grids. The former are the grids appearing white with a typical schlieren texture, and the latter are the grids appearing grey. The IDE is also visible with the homeotropically aligned LC grids. Upon application of a lipid solution (in this case, 1 mM UPC3), lipid layer is formed, making both grid types homeotropically aligned.

This example shows that the LC does not have to be homeotropically aligned. It is better to have it homeotropically aligned because the LC layer is physically much more stable against the lipid solution. However, when the homogeneously aligned LC layer does not break by application of the lipid solution droplet, it is possible to form a lipid layer on such LC alignment too.

Example 3

This example relates to resistance/capacitance/current measurements as a means to detect changes in liquid crystal alignment upon formation of a lipid layer.

As depicted in Figure 8, ID electrodes can be used to measure a resistance or capacitance or current change of the LC when the lipid layer is formed. This is because the conductivity changes with the orientation of LC due to its anisotropical nature. The resistance/capacitance/current change is small because only the surface LC is expected to be reoriented, and the orientation of the bulk remains the same. The small change may be more visible when the thickness of LC layer is reduced.

Alternatively, a cell where two electrodes are parallel to each other can be used, as shown in Figure 9. The monolayer formation is detected by reading the resistance/conductivity from the top and bottom electrodes.

Another setup for capacitance/resistance/current measurements is a LC sandwich cell, where half of the cell is filled with a lipid solution, and another half filled with LC aligned homogeneously (see Fig. 10). The electrodes are on one substrate as is in a case for interdigitated substrate. Despite the antiparallel (homogeneous) alignment layer employed in the cell, LC at the LC-solution will be reoriented along the interface. However, when lipid molecules form a self-assembled molecule layer at the interface, the LC molecules are expected to reorient themselves perpendicular to the interface. This layer formation can be observed under a cross polarized microscope, or even better by measuring the resistance/capacitance/current change that is associated with LC reorientations. As can be seen from the microscope pictures of Figure 1 1, the brightness of the interface line allows to distinguish whether the solution has lipid or not. The location where while LC molecules are, agrees with the position of white interface line.

Example 4

This example relates to the use of negative or dual-frequency liquid crystals.

Fig. 12 shows homeotropical alignment of negative LC upon voltage is applied on IDE. This is because negative LC aligns perpendicular to an electric field (curved arrow in Figure 12) due to its negative dielectric anisotropy.

The same effect is observed when dual-frequency LC at high frequency AC voltage is used. Dual frequency LC is a type of LC that has positive dielectric anisotropy and low frequency AC voltage, but posses negative dielectric anisotropy when a high AC voltage. The advantage of using negative dielectric anisotropy LC is that one may not need a homeotropic alignment layer on top of the IDEs. Additionally, one may be able to make use of dielectricphoresis or AC electroosmosis effect (described below) at low frequency AC voltage, without disturbing the alignment of homeotropically aligned LC.

Example 5

This example relates to the use of dielectrophoresis in order to increase the local concentration of the analyte at the liquid crystal-aqueous solution interface.

Dielectrophoresis is an effect that induces attractive or repulsive force on non-charged substances using alternating electric field. With the experimental setup of Example 1 it is possible to attract analyte molecules by applying AC voltage to ID electrodes. If the voltage orients positive-type LC to prevent formulation of layers (lipid layers in this case), the reorientation can be avoided by using a negative-type or dual-frequency LC.

Alternatively, with a high frequency AC voltage, positive-type LC can be reoriented. If the analytes are of the type that experience repulsive force from the AC voltage, IDEs can be positioned at the opposite side of the LC layer (Fig. 13). By this induction of attractive/repulsive force on bio-molecules , that are swimming randomly, layer formation can be accelerated (i.e. shorter sensing time) or even forced layer formation can be achieved by forcing all swimming analytes to the LC surface (i.e. increase sensor sensitivity).

Figure 14 demonstrates that applying a voltage indeed results in accelerated lipid layer formation. Negative-type LC (MLC6608) was used. In order not to influence the initial homeotropic alignment of the LC, only a very weak voltage of IV was applied. For higher voltage (5V), higher frequency (200 kHz) was employed in order to maintain the initial homeotropic alignment. The LC does not respond to such high frequency square wave. The results shows a shortening of lipid layer formation when 5 V 200 kHz was applied. The measurement will be repeated in order to confirm the effect.

Application of 7 V and 10 V (200 kHz) was tried as shown in Figure 15, but this did not yield lipid layer with sufficient darkness, i.e. the resultant lipid layer had a transmission of approximately 9 % compared with 3 % for the others. However, the lipid layer formation time seems faster.

Example 6

This example relates to the use of AC electroosmosis (ACEO) in order to increase the local concentration of the analyte at the liquid crystal-aqueous solution interface.

ACEO is an effect that induces a fluid motion of free charges in a non uniform electric field. In this way, randomly floating bio-molecules can be mixed within their solution, shortening the time for a free floating bio-molecule to land on an LC layer to form molecular layer (such as a lipid layer). This is shown in Fig. 16, where an IDE is placed opposite to the LC layer. The arrow indicates the fluid flow due to ACEO.

Example 7

This example relates to the use of electrophoresis in order to increase the local concentration of the analyte at the liquid crystal-aqueous solution interface.

Electrophoresis is the movement of charged substances with electric field. Based on this effect, the molecular layer formation can be accelerated by applying DC electric to the bio- molecules if they are charged. The homeotropically aligned LC is not reoriented by the application of the electric field (Fig. 17). The arrow in the figure indicates an example of the force exerted by the two counter electrodes.

Lipid layer formation by electrophoresis

A similar experimental setup as in Example 1 was followed. An empty sandwich cell with 150-mesh gold grid (Figure 17) was constructed as following. Gold grid was placed on a substrate with a plain ITO (not patterned) and a homeotropic alignment layer. A few spots of UV curable polymer were dispensed at the edge of the gold grid, and they were cured under a UV light, so that the gold grid does not move upon immersing it with a buffer solution. As in the previous case, 5CB LC was applied on the gold grid, and heated up to 50 degrees in order to align LC homeotropically. After cooling down the substrate to a room temperature, two stripes of 120 μπι thick polymer sheets were placed next to the gold grid as spacers. A counter substrate with the same plain ITO was gently placed on the spacers, and the two substrates were clipped together. The homeotropic alignment of the LC was confirmed under a cross- polarized microscope as shown in Figure 17a.

A buffer solution of 0.01 mM DOPC lipid (l,2-dioleoyl-sn-glycero-3-phosphocholine) was capillary filled in the empty cell by capillary action. The LC shows Schlieren-like textures now probably because the LC alignment was disturbed by the force exerted by the buffer solution (Figure 17b).

Figure 17b shows a microscope image when the buffer solution was capillary filled in the cell. The Schlieren-like texture remains same for a few minutes, however, upon applying DC voltage to the cell (0V on the top substrate, and +7V to the bottom substrate), a lipid layer formation was observed (Figure 17c). This is because of the negatively charged lipid was attracted by electrophoretic force to the positive electrode, increasing a concentration of lipid molecules near the LC-buffer interface, consequently resulting in a lipid layer formation (i.e. homeotropic alignment of LC).

Figure 17c shows a microscope image of the LC homeotropically aligned due to a lipid layer formation

Example 8 Materials used:

Lipid vesicles: vesicles of DOPC lipid (l,2-dioleoyl-sn-glycero-3-phosphocholine) or vesicles prepared from a mixture of DOPC, GM1, with/without cholesterol.

Gold Grid: 150 mesh (pitch 165 μπι) from Piano. The gold grid can be of various sizes, but larger grids are more susceptible to water disruption, and smaller ones require longer time to form lipid layer (LL).

Note: instead of gold grids also other reaction chambers, such as glass or plastic wells, can be applied.

PBS Buffer solution: (Phosphate buffered saline with pH of 7.4). The solution is not restricted to the PBS solution, as it can be anything such as water with different pH values, TBS (Tris- buffered saline) etc.

Liquid crystals: 5CB from Aldrich. Liquid crystal is also not restricted to 5CB but any molecules that have ability to show liquid crystalline phase.

DMO AP (Octadecyldimethyl(3-trimethoxysilylpropyl)ammonium chloride): induces homeotropic alignment of the liquid crystals.

Cholera toxin B (CTB).

Preparation of Liposomes (unilamellar vesicles)

Liposomes (lipid vesicles) are formed when thin lipid films are hydrated and stacks of liquid crystalline bilayers become fluid and swell. The hydrated lipid sheets detach during agitation and self-close to form large, multilamellar vesicles (LMV) which prevents interaction of water with the hydrocarbon core of the bilayer at the edges. Once these particles have formed, reducing the size of the particle requires energy input in the form of sonic energy (sonication) or mechanical energy (extrusion). The general elements of the procedure involve preparation of the lipid for hydration, hydration with agitation, and sizing to a homogeneous distribution of vesicles.

When preparing liposomes with mixed lipid composition, the lipids must first be dissolved and mixed in an organic solvent to assure a homogeneous mixture of lipids. For larger volumes, the organic solvent should be removed by rotary evaporation yielding a thin lipid film on the sides of a round bottom flask. Lipid extrusion is a technique in which a lipid suspension is forced through a polycarbonate filter with a defined pore size to yield particles having a diameter near the pore size of the filter used. Prior to extrusion through the final pore size, LMV suspensions are disrupted either by several freeze-thaw cycles or by pre-filtering the suspension through a larger pore size (typically 0.2μηι-1.0μπι). This method helps prevent the membranes from fouling and improves the homogeneity of the size distribution of the final suspension.

In a typical procedure, stock solution of lOmM DOPC (l,2-dioleoyl-sn-glycero-3- phosphocholine) in chloroform was prepared. From this solution various molar concentrations of DOPC (O.OlmM, O.lmM, lmM) were prepared by dissolving them in chloroform. Chloroform was then evaporated with nitrogen gas and solutions were dried for 1 hour in a schlenk line (vacuum line). They were then rehydrated with 5 ml of O.lmM PBS (pH 7.0). Small unilamellar vesicles (SUV) were produced by carrying out 3-4 cycles of freezing and heating of the solution with a water bath and liquid nitrogen alternatively. Small aliquots, 500 μΐ each, were prepared and stored in the freezer at -20°C. When needed, the solutions were thawed and pressed through a polycarbonate membrane, with 100 nm pore, for 21 times using hand held extruder to produce small unilamellar lipid vesicles. These vesicles were used for lipid layer formation.

Preparation of Liposomes from mixture of phospholipid + GM1+ cholesterol

Stock solution of lOmM DOPC in chloroform, lmg of GMl in 65 μΐ in chloroform: methanol (6: 1) was prepared. From these solutions specific concentrations of DOPC+GMl+cholesterol mixture (90% DOPC [O.OlmM], 0.5% GMl [O.OlmM] and 9.5% cholesterol [O.OlmM]) or DOPC+GM1 (99.5 % DOPC [O.OlmM], 0.5% GMl [O.OlmM]) were prepared by dissolving them in chloroform.

Chloroform was then evaporated with nitrogen gas and solutions were dried for 1 hour in a schlenk line. They were then rehydrated with 5 ml PBS (pH 7.0). SUVs were produced by carrying out 3-4 cycles of freezing and heating of the solution with a liquid nitrogen and water bath alternatively. Small aliquots, 500 μΐ each, were prepared and stored in the freezer at - 20°C. When needed, the solutions were thawed and pressed through a polycarbonate membrane, with 100 nm pore, for 21 times using hand held extruder to produce small unilamellar lipid vesicles. These vesicles were used for lipid layer formation.

Substrate preparation To coat the substrates for homeotropic alignment of LC, DMO AP solution was prepared by mixing 250 ml of Octadecyldimethyl(3-trimethoxysilylpropyl)ammonium chloride (60% in methanol, ABl 1 1261) and 150 ml of deionized water (Millipore). To this solution, the glass substrates were dipped in and held there for about 5 min. After the substrates were taken out of the solution the remaining droplets on the substrate were removed with an air gun. Following, the substrates were dried in a 100 °C vacuum oven for 15 min. Then, gold grids were placed on the DMOAP coated substrate and 2 μΐ of LC was applied on each gold grid. The excess LC was removed by capillary action using a micro syringe tip. Once after impregnating the gold grid with LC, the substrate was placed in a 50°C oven for 10 min. The oven temperature was kept above the nematic to isotropic temperature of the LC, in order to realize a uniformly aligned LC. The slides with gold grids were then observed under microscope and any gold grid ^' with no proper alignment of 5CB was discarded.

Measurement

The cross-polarized microscope brightness was calibrated as follows: 100 % transmittance was set when there was no sample and when the polarizer and the analyzer were parallel to each other. 0% transmittance was set when there was no light source. For measurement, the polarizer and the analyzer were placed perpendicular to each other without changing the light intensity. 5 μΐ lipid solution was applied on the gold grid with partially aligned 5CB and the change in alignment was observed and recorded. In case of lipid solution with GM1 the lipid layer (LL) formation on 5CB was completed in 1-2 minutes. On the other hand, the LL formation from a solution with DOPC, GM1 and Cholesterol required longer time, about 8-10 minutes to complete. After completion of lipid layer formation on the LC (indicated by darker image), the gold grid was washed with PBS to remove any excess of vesicles (lipid with or without GM1 and/or cholesterol). To this lipid layer, 2-5 μΐ of CTB solution {toxin) in PBS (various concentrations such as μg/L, 10μg/L etc.) was added and the change in LC alignment was observed and recorded. The observations and recordings were carried out initially for 10-15 min continuously, and then after each hour interval.

Results and Discussion

From the molecular structure of GM _\ (see Figure 19 D) it can be seen that the non-polar hydrocarbon chains of GMj are likely to interact with the non-polar fatty acid chains of the phospholipid, and by this way they will be embedded in the lipid layer. Lipid vesicles (liposomes) with different concentrations of GMi were prepared and the lipid-LC layer formation was observed. For this purpose, 0.5%, 2.5% and 5% (weight%) of in 0.01 mM DOPC in PBS buffer were used and the lipid vesicles were prepared, as described above.

As can be followed from figure 21, all the lipid vesicles containing different amounts of GMi receptor resulted in lipid-LC layer formation. The orientation change of the LC layer is visible though the change in light transmittance.

Detection of Cholera Toxin B (CTB)

CTB was added on the lipid-LC layers bearing different amounts of GMi and it was observed whether there is a light transmittance change happening due to orientation change of LC layer. A change in the orientation of LC layer can be observed. As an example, pictures showing the transmission change in the system where 0.5% GMi was embedded in the lipid layer is given in figure 22.

In a natural environment a healthy mammalian cell membrane has cholesterol embedded in the phospholipid bilayer. Based on this knowledge different lipid vesicles with 0.5% GMi and different concentrations (10, 15 and 20 weight %) of cholesterol were prepared and then used to form lipid-LC layers using these lipid vesicles on the LC. As can be followed from figure 23, lipid-LC layers form when 10 and 15 % of cholesterol are embedded in the lipid vesicles.

Addition of CTB on the lipid-LC layers with and cholesterol embedded in resulted in very clear changes in the light transmittance, see figure 24.

Effect of cholesterol

To understand the role of cholesterol in our detection, parallel tests were performed where the speed of CTB detection in the presence and absence of cholesterol were determined. It can easily be seen from the pictures (see figure 24) that the same amount of CTB can be detected in less than 15 minutes using a sensor system with cholesterol, whereas without cholesterol the same effect is achieved in about 12 hours. Additionally, the transmission-time curves in figure 24 A show that LC orientation change had already started after a few minutes in the layers with embedded cholesterol, whereas no change was observable in such a short time when no cholesterol was used. During the tests, it was seen that CTB loses its activity with time and when that happens no change in the light transmittance could be observed. However, once a fresh batch of CTB is used, the light transmittance change (due to LC orientation change) upon CTB-GMi interaction can again be observed.

Example 9: A polarizer/backlight-free dye-doped LC based sensor

This example relates to a LC based sensor, wherein dichroic dyes were incorporated in the LC mixture such that no polarizers and no backlight are required (polarizer-free and backlight- free dye-doped LC based sensor). This example is similar to Example 1 with the following differences:

the LC mixture was doped with dichroic dyes;

measurements were carried out with and without polarizers.

Materials used:

Target additive: 1 mM DOPC lipid (l,2-dioleoyl-sn-glycero-3-phosphocholine): The target molecule can also be mixed together with additional dopant/additive such as cholesterol, enzymes etc, as long as it has a capability to change the LC alignment at the solution-LC interface.

Gold Grid: 300 mesh (pitch 75 μπι) from Piano. The gold grid can be of various sizes, but larger grids are more susceptible to water disruption, and smaller ones require longer time to form lipid layer (LL). We can also use glass wells, an example of glass wells filled with dye doped LC is given at Figure 27.

PBS Buffer solution: [Phosphate-buffered saline with pH of 7.4]. The solution is not restricted to the PBS solution, as it can be anything such as water with different pH values, TBS (Tris- buffered saline) etc.

5CB: Single component LC with positive dielectric anisotropy (positive LC) from Aldrich. Dichroic Dye: D2 (red azo dye from Merck) and B3 (Black-3 dichroic dye mixture from Mitsubishi Chemical) were used.

Polyimide (PI) coated interdigitated electrode substrates were purchased from EHC, Japan. DMOAP (Octadecyldimethyl(3-trimethoxysilylpropyl)ammonium chloride): Induces homeotropic alignment to the liquid crystals.

Sample preparation: Substrate

7. Treat the substrate with ozone plasma (0.1 mbar 0 ₂ , 100 W, 1 min), in order to get it react with the silane.

8. Soak the substrate in a solution of silane for more than 5 min.

9. Blow away the solution on the substrates with an air gun, and dry them in a 100 °C vacuum oven for 15 min. Then take out the substrates and allow to cool to room temperature.

10. Place the gold grid on the substrates where IDEs are.

1 1. Apply 2 μΐ of 5CB on the gold grid, and remove the excess LC with capillary filling in a micro syringe tip.

12. Place the substrate in a 50 °C oven for 10 min. The temperature should be above the nematic to isotropic temperature of the LC, in order to realize a uniform, homeotropically aligned LC.

13.

Lipid solution:

Small unilamellar lipid vesicles were prepared by passing 500 lipid solutions (1 mM in PBS) through a 50 nm pore filter with the help of a hand held extruder.

Measurement

6. For a measurement with polarizers, calibrate the cross-polarized microscope brightness.

100 % transmittance was set where there is no sample and when the polarizer and the analyzer are parallel to each other. 0% transmittance was set when there is no light source.

For a measurement without polarizers, calibration was made that 100% transmittance was set where there is no polarizer and no sample.

7. Attach electrical contacts to the sample, and place it on the microscope stage.

8. Start taking photos and measure the intensity.

9. Apply 5 μΐ lipid solution.

10. When a stable lipid layer is formed, apply various AC voltages with square signal, (the signal can be sine/triangular wave)

Results and Discussion

Before the application of the lipid solution, the LC layer appears grey between cross- polarizers and colored without the polarizers. Upon application of the lipid solution, the LC layer turns darker (with polarizers) / brighter (without polarizers). This is due to the induced homeotropic alignment at the solution-LC interface, due to lipid-LC layer formation. Once the transmittance change stopped (when the lipid-LC layer formation is completed) voltage was applied and the transmittance vs time measurements continued.

Prior to the measurements, the liquid crystal (5CB) was doped with 3 wt% dichroic dye. This means; dichroic dye doped LC is the sensor component of the system in this example. Figure 25 and Figure 26 show the experiments carried out using D2 (red dichroic dye from Merck) and B3 (Black-3 dichroic dye mixture from Mitsubishi Chemicals), respectively. Because the polarizers were not used for these measurements, the transmittance does not decrease as the lipid layer is formed (as the embodiments described e.g. in Examples 1-7). Instead, due to the homeotropic reorientation of the surface LC and dye, the transmittance increases because the dichroic dye absorbs less when its long axis is parallel to the incident light, i.e. becomes more transparent. The initial transmittance decrease is due to the lipid solution application, and it increases as the lipid layer formation proceeds. Once the increase is saturated, 2, 4, 6 & 8V (1kHz) were applied and the sudden decrease of the transmittance were recorded. This proves that the dye-doped LCs were not disrupted and still functioning. Use of higher frequency (e.g. MHz) or using a better passivation/insulating layer (e.g. thicker PI layer, SU8, etc) will allow higher voltage application for improved contrast.

Figure 28 shows photos of dye doped gold grids before adding PBS solution (buffer solution), and after adding PBS solution with/without lipid (ImM DOPC). The differences between those grids with and without DOPC were observable even without a microscope and without a backlight. Further improvement in the distinction can be possible by optimizing dye type/concentration, LC type and sensor chamber size and type.

It is also possible to use a fluorescent dye or fluorescent marked LC to achieve polarizer-free LC sensor.

We disclose here the use of dichroic dyes, for a LC based sensor which does not require polarizers, microscope and backlight.

Example 10: Speeding up the LC sensor reaction time by applying electric field This example relates to a LC based sensor as described herein (such as Example 9), wherein the applied voltage is AC 20 V square wave.

Experimental condition

- LC: 5CB (no dye)

- Gold grid: 300 mesh

- Solution: 0.5mM DOPC. 50% diluted PLA1 (phospholipase Al).

- Substrate:

• IDE (interdigitated electrode) spin coated by lOOnm SU8 passivation layer, followed by the DMOAP surface treatment.

• Preparation procedure: Annealing (125°C, 30min)→ 0 ₂ plasma→ SU8 (10/25%) coating→ 0 ₂ plasma→ DMOAP→ Annealing (50°C, 15 min)

- Applied voltage: AC 20 V square wave (other waveforms such as triangular/sinusoidal are also possible)

In order to compare the lipid layer formation and enzyme reaction times, the same method was used as for measuring LC response times in Example 1. These definitions are shown in Figure 29.

Figure 30 shows how the lipid layer formation and enzyme reaction times change at various frequencies. lHz data represents 0V (reference) data. The deviation of the data points are still relatively large for the given sample number, but it can be seen that there is an improvement (shortening) of the reaction times. The cause of the improvement appears to be either because of dielectrophoresis of the biomolecules, joule heating of the 5CB LC, or combination of the both.

The features of the present invention disclosed in the specification, the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.