DE102013106380A1 - Liquid-based sensor device, useful for detecting the presence of analyte e.g. bacteria cell, comprises substrates, liquid crystal layer, and phospholipid layer, reaction layer, electrodes, and insulating layer and/or alignment layer - Google Patents

Abstract

The liquid-based sensor device comprises a first substrate, a liquid crystal layer disposed on the first substrate, and a phospholipid layer disposed on the liquid crystal layer. The device does not include a detection member or a recognition molecule that is bound directly to a surface of the first substrate. The phospholipid comprises recognition moiety or a recognition molecule such as a receptor, an enzyme, or an antibody, and 10-15 wt.% of cholesterol. The device further comprises reaction layer disposed on the liquid crystal layer, and a first electrode . The liquid-based sensor device comprises a first substrate, a liquid crystal layer disposed on the first substrate, and a phospholipid layer disposed on the liquid crystal layer. The device does not include a detection member or a recognition molecule that is bound directly to a surface of the first substrate. The phospholipid comprises recognition moiety or a recognition molecule such as a receptor, an enzyme, or an antibody, and 10-15 wt.% of cholesterol. The device further comprises a reaction layer disposed on the liquid crystal layer, a first electrode positioned between the first substrate and the liquid crystal layer, an insulating layer and/or an alignment layer or insulation and alignment layers coated the electrode, a second substrate disposed opposite to the first substrate and the liquid crystal layer, an intermediate layer disposed between the first and second substrate, crossed polarizers positioned below and above the liquid crystal layer and in planes of the first and second substrate respectively, a unit to measure an electrical quantity, a microfluidic device in fluid communication with a reaction chamber, and a filter unit for filtering any material from the sample. The reaction layer includes a lipid layer, lipopolysaccharide (LPS) layer and/or a fatty acid layer. A gap is present between the second substrate and the phospholipid layer or between the second substrate and the reaction layer, and acts as the reaction chamber for receiving a sample to be analyzed. The second substrate comprises a second electrode disposed on the second substrate, and an insulating layer on the second electrode. The insulating layer faces the first substrate phospholipid/liquid crystal layer and reaction layer/liquid crystal layer. The first and/or second substrate comprises glass or plastic. The liquid crystal layer has a homeotropic alignment, a homogeneous alignment, or any predefined orientation, and comprises a liquid crystal including thermotropic liquid crystals having positive/negative or no dielectric anisotropy, dual-frequency liquid crystals, discotic and/or lyotropic liquid crystals, a single liquid crystal compound, a mixture of different liquid crystalline compounds and/or a liquid crystal mixture mixed with dopants such as nanoparticles or small organic molecules. The liquid crystal layer is doped with dichroic dye, fluorescence dye or with a quantum dot. The phospholipid and the reaction layers include a single phospholipid compound or a mixture of different phospholipid compounds. The reaction layer comprises a single fatty acid compound or a mixture of different fatty acid compounds. The insulating layer is constructed on the first and/or second electrode. The first electrode and the second electrode include a non-interdigitated electrode, a plane electrode, or an interdigitated electrode. The alignment layer includes a heat/photo-curable polyimide layer providing for a defined pre-tilt angle of the liquid crystal in the liquid crystal layer, a self assembled monolayer, or an obliquely evaporated oxide layer, such as silica providing for a pre-tilt of the liquid crystal in the liquid crystal layer depending on the evaporation angle of the oxide. The first and/or second electrode is a transparent or non-transparent electrode. The crossed polarizers allow to measure changes in the liquid crystal alignment by measuring changes of transmission/reflection of polarized light, and the substrate underneath the liquid crystal layer is a reflective substrate. The measuring unit allows to measure changes in the liquid crystal alignment by measuring changes in such electrical quantity such as capacitance, resistance or current. The reaction chamber has a depth of 900 nm to 30 mu m, includes a circular chamber having a width or diameter of 25-500 mu m, and/or is made of glass, plastic or metal walls such as gold walls.

Description

BACKGROUND

The field of DISCLOSURE relates to the field of liquid crystal based sensor devices.

The present disclosure relates to liquid crystal-based sensor devices for detecting analytes. The present disclosure also relates to liquid crystal based sensor devices for the detection of lipases and / or lipase activities

Furthermore, the present disclosure relates to methods for manufacturing the liquid crystal based sensor devices. Furthermore, the present disclosure relates to methods and uses of the liquid crystal-based sensor devices for detecting analytes, the activity of analytes and for screening compounds that bind analytes and / or modify their activity. Furthermore, the present disclosure relates to methods and uses of liquid crystal-based sensor devices for detecting the presence of lipases and / or lipase activities and for monitoring damage, degradation and / or death of cells or organs in samples. The present disclosure further relates to the use of liquid crystal based sensor devices in toxicity or cytotoxicity assays.

DESCRIPTION OF THE RELATED TECHNIQUE

The description of the "background" herein is for the purpose of a general presentation of the disclosure context. The work of the present inventors, to the extent described in this Background section, as well as aspects of the description that are not otherwise classed as prior art at the time of filing, are neither expressly or implicitly accepted as prior art against the present disclosure.

Liquid crystals are materials that can exhibit the mobility of liquids and the anisotropy of solid crystals. Thermotropic liquid crystals have already been shown to be useful in relaying molecular events at a boundary layer into macroscopic responses that are visible to the naked eye. The long-range orientation and optical anisotropy of LC molecules can transform chemical and biomolecular binding events into amplified optical signals that can be easily observed, even with the naked eye. Boundary layers between thermotropic LCs and immiscible aqueous phases have received considerable attention lately because of the strong coupling that can occur between the placement of the LCs and the organization of molecular arrays that can form at these interfaces. These properties, combined with the optical anisotropy of the LC molecules, make them highly suitable for directly transducing and enhancing the binding of an analyte to a target at a boundary layer in an optical output. Most of the current methods of detecting biological analytes require analytical detectors in a laboratory environment and labeled species such as fluorophores or radioactive isotopes; and therefore, this presents a great potential for providing highly sensitive and inexpensive bioassays that can be performed outside central laboratories without sophisticated instruments.

Self-assembly of surfactants, lipids, and polymers at these interfaces has been shown to be closely related to the order of the LCs, and the presence and organization of these molecules is indicated by changes in the visual appearance of the LCs can be. It has also been reported that more complex interfacial phenomena, such as specific binding events involving proteins, bacteria, viruses, enzyme reactions, hybridization of DNA, and human embryonic stem cell culture, can induce dynamic directional transitions in the LCs at an aqueous LC interface , These studies indicate that the aqueous LC interfaces are a promising class of biomolecular interfaces for displaying interfacial phenomena.

The principles of LC-based detection are based on optical, anchoring and elastic properties resulting from the molecular anisotropies and the unique liquid crystal phase of the LC material. The molecular anisotropy of a liquid crystal sample creates a difference in refractive indices of light parallel or perpendicular to the main molecular orientation, i. H. the LC direction generator. Molecular interactions between an LC and an adjacent interface result in a preferred anchoring angle with respect to the surface normal. Information about the interface, in terms of surface anchoring, is transferred up to 100 μm into the basic quantity as a result of the elastic nature of the LC direction generator field.

It is also reported that in the presence of phospholipases, hydrolysis of the lipid layer occurs at the lipid LC interface, which can then be monitored directly by changes in the optical appearance of the lipid LC layer.

Lipases are hydrolytic enzymes that act on carboxyl ester compounds. These enzymes are widely used in animals, plants, molds and bacteria. The catalytic activities are not limited to hydrolysis; they also catalyze esterification, interesterification and transesterification processes. Therefore, they are widely used in the food, pharmaceutical, chemical and detergent industries. Lipases are also very important in diagnosis settings. Under pathological conditions, they are found, for example, in environments in which they normally do not occur. This suggests that they could be used as markers for processes that destroy cells / organs. A well-known example is the high levels of pancreatic lipases that occur in the blood as a result of pancreatitis. Even when a mammalian cell dies, lipases are released.

Methods are available for the detection of active lipases, which may be referred to as lipase assays. These procedures all depend on the needs of the users and the resources available. Usually, they are time consuming, require large amounts of material / samples, and expensive instruments, such as spectrometers. These assays generally detect the products of the reactions that catalyze lipases, such as the release of fatty acids, fatty acid esters or glycerol, using colorimetric or fluorometric methods or chromatographic analyzes such as HPLC.

SHORT PRESENTATION

• – ein erstes Substrat,
• – eine Flüssigkristallschicht auf dem ersten Substrat,
• – eine Phospholipidschicht auf der Flüssigkristallschicht,

wobei die Vorrichtung keinen Erkennungsteil oder kein Erkennungsmolekül umfasst, das direkt an die Oberfläche des ersten Substrates gebunden ist.The present disclosure provides a liquid crystal-based sensor device capable of detecting the presence of an analyte, such as a prokaryotic cell, a bacterial cell, and / or a prokaryotic agent, such as a toxin, such as a microbial toxin in the order listed includes:

• A first substrate,
• A liquid crystal layer on the first substrate,
• A phospholipid layer on the liquid crystal layer,

wherein the device comprises no recognition part or recognition molecule bound directly to the surface of the first substrate.

• – ein erstes Substrat,
• – eine Flüssigkristallschicht auf dem ersten Substrat,
• – eine Reaktionsschicht auf der Flüssigkristallschicht, wobei die Reaktionsschicht eine Lipidschicht, eine LPS-Schicht und/oder eine Fettsäureschicht ist.

The present disclosure provides a liquid crystal-based sensor device for detecting the presence of lipases or lipase activity, the device comprising in the order listed:

• A first substrate,
• A liquid crystal layer on the first substrate,
• A reaction layer on the liquid crystal layer, wherein the reaction layer is a lipid layer, an LPS layer and / or a fatty acid layer.

• a) zur Verfügung stellen eines ersten Substrats,
• b) Aufbringen einer Flüssigkristallschicht auf das erste Substrat,
• c) Aufbringen einer Phospholipidschicht auf die Flüssigkristallschicht, optional umfassend mindestens ein(en) Erkennungsteil/-molekül, wie beispielsweise einen Rezeptor, und/oder Cholesterol, bzw. Aufbringen einer Reaktionsschicht auf die Flüssigkristallschicht.

The present disclosure provides a method for manufacturing a liquid crystal based sensor device according to the present disclosure, the method comprising the steps of:

• a) providing a first substrate,
• b) applying a liquid crystal layer to the first substrate,
• c) applying a phospholipid layer to the liquid crystal layer, optionally comprising at least one recognition part / molecule, such as a receptor, and / or cholesterol, or applying a reaction layer to the liquid crystal layer.

• a) zur Verfügung stellen einer Flüssigkristall-basierten Sensorvorrichtung gemäß der vorliegenden Offenbarung,
• b) Hinzufügen einer zu analysierenden Probe auf die Phospholipidschicht der Flüssigkristall-basierten Sensorvorrichtung,
• c) Untersuchung auf ein Bindungsereignis, einen zu detektierenden Analyten oder die Aktivität eines Analyten basierend auf Veränderungen der Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht, die von dem Bindungsereignis, dem Analyten oder der Analytenaktivität induziert werden.

The present disclosure provides a method of using the liquid crystal-based sensor device according to the disclosure to detect the presence of an analyte or the activity of an analyte, the method comprising the steps of:

• a) providing a liquid crystal based sensor device according to the present disclosure,
• b) adding a sample to be analyzed to the phospholipid layer of the liquid crystal based sensor device,
• c) Examination for a binding event, an analyte to be detected or the activity of an analyte based on changes in the orientation of the liquid crystals in the liquid crystal layer induced by the binding event, the analyte or the analyte activity.

• a) zur Verfügung stellen einer Flüssigkristall-basierten Sensorvorrichtung gemäß der Offenbarung,
• b) zur Verfügung stellen einer Probe umfassend den Analyten,
• c) zur Verfügung stellen mindestens einer auf Bindung an den Analyten und/oder Modifizierung der Aktivität des Analyten zu screenenden Verbindung.
• d) Hinzufügen von mindestens einer zur screenenden Verbindung zur den Analyten umfassenden Probe,
• e) Hinzufügen der den Analyten sowie die Verbindung umfassenden Probe auf die Phospholipidschicht der Flüssigkristall-basierten Sensorvorrichtung,
• f) Untersuchung auf den Analyten oder die Aktivität des Analyten basierend auf Veränderungen der Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht, die von dem Bindungsereignis, dem Analyten oder der Analytenaktivität induziert werden.

wobei keine Ausrichtungsveränderung anzeigt, dass die zu screenende Verbindung an den Analyten bindet und/oder die Aktivität des Analyten modifiziert, und wobei eine Veränderung der Ausrichtung anzeigt, dass die zu screenende Verbindung den Analyten nicht bindet und/oder die Aktivität des Analyten nicht modifiziert.The present disclosure provides a method of using the liquid crystal based sensor device according to the disclosure to screen for compounds that bind to an analyte and / or modify the activity of an analyte, such as inhibitors of microbial toxins, the method comprising the steps of:

• a) providing a liquid crystal based sensor device according to the disclosure,
• b) providing a sample comprising the analyte,
• c) provide at least one for binding to the analyte and / or modifying the Activity of the analyte to be screened compound.
• d) adding at least one sample to the screening compound to the analyte,
• e) adding the sample comprising the analyte and the compound to the phospholipid layer of the liquid crystal-based sensor device,
• f) Examination for the analyte or the activity of the analyte based on changes in the orientation of the liquid crystals in the liquid crystal layer induced by the binding event, the analyte or the analyte activity.

wherein no orientation change indicates that the compound to be screened binds to the analyte and / or modifies the activity of the analyte, and wherein a change in orientation indicates that the compound to be screened does not bind the analyte and / or modifies the activity of the analyte.

• a) zur Verfügung stellen einer Flüssigkristall-basierten Sensorvorrichtung gemäß der Offenbarung,
• b) Hinzufügen einer zu analysierenden Probe auf die Reaktionsschicht der Flüssigkristall-basierten Sensorvorrichtung,
• c) Untersuchung auf ein Reaktionsereignis basierend auf Veränderungen, die von der/den Lipase(n) oder der Lipasenaktivität(en) an der Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht induziert werden.

The present disclosure provides a method of using the liquid crystal-based sensor device according to the disclosure to detect the presence of lipase (s) or the activity of lipase (s), the method comprising the steps of:

• a) providing a liquid crystal based sensor device according to the disclosure,
• b) adding a sample to be analyzed to the reaction layer of the liquid crystal based sensor device,
• c) assay for a response event based on changes induced by the lipase (s) or lipase activity (s) in the alignment of liquid crystals in the liquid crystal layer.

• a) zur Verfügung stellen einer Flüssigkristall-basierten Sensorvorrichtung gemäß der Offenbarung,
• b) Hinzufügen einer zu analysierenden Probe auf die Reaktionsschicht der Flüssigkristall-basierten Sensorvorrichtung,
• c) Untersuchung auf ein Reaktionsereignis basierend auf Veränderungen der Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht,

wobei eine Änderung der Ausrichtung anzeigt, dass die Zellen und/oder Organe der Probe beschädigt, zersetzt und/oder abgestorben sind.The present disclosure provides a method of using the liquid crystal based sensor device according to the disclosure to monitor for monitoring the damage, degradation, and / or death of cells or organs, the method comprising the steps of:

• a) providing a liquid crystal based sensor device according to the disclosure,
• b) adding a sample to be analyzed to the reaction layer of the liquid crystal based sensor device,
• c) Examination for a reaction event based on changes in the orientation of the liquid crystals in the liquid crystal layer,

wherein a change in orientation indicates that the cells and / or organs of the sample are damaged, degraded and / or dead.

The present disclosure provides the use of a liquid crystal-based sensor device according to the disclosure for detecting the presence of lipase (s) or the activity of lipase (s) in a sample; for monitoring damage, degradation and / or death of cells or organs in a sample, and / or as a lipase screening device in toxicity or cytotoxicity assays.

The foregoing paragraphs have been presented as a general introduction and are not intended to limit the scope of the following claims. The described embodiments, together with further advantages, are best understood in conjunction with the following detailed description, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many related advantages is readily possible as the same becomes better understood by reference to the following detailed description taken in conjunction with the accompanying drawings, wherein:

1A shows a phospholipid, DOPC, with polar head group and non-polar fatty acid chains; 1B shows cholesterol; 1C shows the structure of 5CB, a thermotropic liquid crystal; 1D shows the structure of ganglioside GM1;

2 is a cartoon illustration of phospholipid LC film formation;

3 Figure 12 shows lipid LC layer formation in the presence of various amounts of GM1 embedded in lipid vesicles (which reduces translucency in lipid LC layer formation);

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

5 shows lipid LC layer formation with embedded cholesterol and GM1;

6 Figure 4 shows the detection of CTB in the presence of 10% cholesterol (designated 10 CH in A, B: with cholesterol, C: without cholesterol) (embodiment of Example 1);

7 shows a red dye doped LC sensor on interdigital electrodes (IDEs) (from Example 2);

8th shows a black dye doped LC sensor on interdigital electrodes (IDEs) (from Example 2);

9 Figure 5 shows liquid crystal (5CB) of 100μm diameter "wells" (images taken during the annealing cycle), cooling from 55 ° C (5CB in the liquid phase) to 21 ° C (5CB in the LC phase) (embodiment of Example 2);

10 shows photographs of dye-doped gold lattices with and without DOPC (embodiment of Example 2);

11 shows the definition of reaction times for the embodiment of Example 3;

12 Figure 10 shows the shortening of lipid (DOPC) layer formation and enzyme (PLA1) reaction times when AC20V is applied at different frequencies (embodiment of Example 3).

13A shows trypan blue staining of treated cells compared to untreated cells (embodiment of Example 5A);

13B shows the detection of lipase activity with an LC lipid sensor device (embodiment of Example 5A);

14 shows that treatment with 0.5% Na azide does not inactivate the lipases (embodiment of Example 5B);

15 Figure 4 is a schematic illustration of the toxicity studies performed with 0.5% Na azide and three different cell types (embodiment of Example 5B);

16 Figure 5 shows the lipase effect on LC-based assays (embodiment of example 5B, 2 hours incubation with 0.5% Na azide);

17 Figure 4 shows lipase activity on LC-based assays (embodiment of example 5B, incubation for 24 hours with 0.5% Na azide);

18 shows the construction / set-up of the test plate (A) and the control plate (B) for the acetaminophen treatment (embodiment of Example 5C);

19 Figure 10 shows lipase detection in an LC-based assay after cells were incubated for 30 minutes in 0.05% paracetamol (embodiment of Example 5C);

20 Figure 4 shows control experiments in which cells were not in direct contact with paracetamol-containing media (embodiment of Example 5C).

DETAILED DESCRIPTION OF THE EMBODIMENTS

• – ein erstes Substrat,
• – eine Flüssigkristallschicht auf dem ersten Substrat,
• – eine Phospholipidschicht auf der Flüssigkristallschicht,

wobei die Vorrichtung keinen Erkennungsteil oder kein Erkennungsmolekül umfasst, das direkt an die Oberfläche des ersten Substrates gebunden ist.As discussed above, the present disclosure provides a liquid crystal-based sensor device for detecting the presence of an analyte (referred to herein as an "analyte sensor device"), such as a prokaryotic cell, a bacterial cell, and / or a prokaryotic agent, such as a toxin, such as a microbial toxin, the device comprising in the order listed:

• A first substrate,
• A liquid crystal layer on the first substrate,
• A phospholipid layer on the liquid crystal layer,

wherein the device comprises no recognition part or recognition molecule bound directly to the surface of the first substrate.

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

In the preferred embodiment, the phospholipid layer comprises cholesterol.

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

The phospholipid layer preferably comprises between about 1 to about 20 weight percent cholesterol, preferably about 10 to about 15 weight percent cholesterol.

In one embodiment, the at least one recognition moiety or the at least one recognition molecule included in the phospholipid layer is a receptor, such as ganglioside GM ₁ .

In one embodiment, the phospholipid layer comprises between about 0.1% to about 10% by weight GM ₁ , preferably from about 0.5% to about 5% by weight GM ₁ .

• – ein erstes Substrat,
• – eine Flüssigkristallschicht auf dem ersten Substrat,
• – eine Reaktionsschicht auf der Flüssigkristallschicht, wobei die Reaktionsschicht eine Lipidschicht, eine LPS-Schicht und/oder eine Fettsäureschicht ist.

As discussed above, the present disclosure provides a liquid crystal based sensor device for detecting the presence of lipases or lipase activity (referred to herein as "lipase sensor device"), the device comprising in the order listed:

• A first substrate,
• A liquid crystal layer on the first substrate,
• A reaction layer on the liquid crystal layer, wherein the reaction layer is a lipid layer, an LPS layer and / or a fatty acid layer.

The device preferably comprises no recognition part or recognition molecule bound directly to the surface of the first substrate. When such a recognition member or molecule is used in a device according to the present disclosure, it is preferably included in the reaction layer of the device.

In the following, further embodiments of the above-described sensor devices will be further described.

• – mindestens eine erste Elektrode zwischen dem ersten Substrat und der Flüssigkristallschicht, und
• – eine Isolationsschicht und/oder Ausrichtungsschicht („alignment layer”), oder eine Isolations- und Ausrichtungsschicht, welche die Elektrode beschichtet.

In an embodiment of the sensor devices described above, the sensor device further comprises:

• At least one first electrode between the first substrate and the liquid crystal layer, and
• An insulating layer and / or alignment layer, or an insulating and alignment layer which coats the electrode.

• – ein zweites Substrat, das gegenüber dem ersten Substrat angeordnet ist und die Flüssigkristallschicht und Phospholipidschicht zu Zwischenschichten bzw. die Flüssigkristallschicht und Reaktionsschicht zu Zwischenschichten zwischen dem ersten und zweiten Substrat macht, und wobei es einen Zwischenraum zwischen dem zweiten Substrat und der Phospholipidschicht bzw. zwischen dem zweiten Substrat und der Reaktionsschicht gibt, der als Reaktionskammer zur Aufnahme einer zu analysierenden Probe dient.

In an embodiment of the sensor devices described above, the sensor device further comprises:

• A second substrate disposed opposite to the first substrate and rendering the liquid crystal layer and phospholipid layer into intermediate layers, the liquid crystal layer and reaction layer into intermediate layers between the first and second substrates, and having a gap between the second substrate and the phospholipid layer the second substrate and the reaction layer, which serves as a reaction chamber for receiving a sample to be analyzed.

• – eine zweite Elektrode auf dem zweiten Substrat, und
• – optional, eine Isolationsschicht auf der zweiten Elektrode, wobei die Isolationsschicht der sich auf dem ersten Substrat befindenden Phospholipidschicht/Flüssigkristallschicht bzw. Reaktionsschicht/Flüssigkristallschicht zugewandt ist.

In one embodiment, the second substrate further comprises:

• A second electrode on the second substrate, and
• Optionally, an insulation layer on the second electrode, the insulation layer facing the phospholipid layer / liquid crystal layer or reaction layer / liquid crystal layer located on the first substrate.

• – ein erstes Substrat,
• – eine Flüssigkristallschicht auf dem ersten Substrat,
• – eine Phospholipidschicht auf der Flüssigkristallschicht bzw. eine Reaktionsschicht auf der Flüssigkristallschicht,
• – ein zweites Substrat, das gegenüber dem ersten Substrat angeordnet ist und die Flüssigkristallschicht und Phospholipidschicht zu Zwischenschichten bzw. die Flüssigkristallschicht und Reaktionsschicht zu Zwischenschichten zwischen dem ersten und zweiten Substrat macht, und wobei es einen Zwischenraum zwischen dem zweiten Substrat und der Phospholipidschicht bzw. zwischen dem zweiten Substrat und der Reaktionsschicht gibt, der als Reaktionskammer zur Aufnahme einer zu analysierenden Probe dient,
• – bevorzugt eine zweite Elektrode auf dem zweiten Substrat, oder eine zweite Elektrode auf dem zweiten Substrat und eine Isolationsschicht auf der zweiten Elektrode, wobei die Isolationsschicht der sich auf dem ersten Substrat befindenden Phospholipidschicht/Flüssigkristallschicht bzw. Reaktionsschicht/Flüssigkristallschicht zugewandt ist.

In one embodiment, the sensor device (s) include / include a second electrode, but not a first electrode. In this embodiment, the sensor device may include:

• A first substrate,
• A liquid crystal layer on the first substrate,
• A phospholipid layer on the liquid crystal layer or a reaction layer on the liquid crystal layer,
• A second substrate disposed opposite to the first substrate and rendering the liquid crystal layer and phospholipid layer into intermediate layers, the liquid crystal layer and reaction layer into intermediate layers between the first and second substrates, and having a gap between the second substrate and the phospholipid layer the second substrate and the reaction layer serving as a reaction chamber for receiving a sample to be analyzed,
• Preferably, a second electrode on the second substrate, or a second electrode on the second substrate and an insulating layer on the second electrode, wherein the insulating layer of the located on the first substrate phospholipid layer / liquid crystal layer or reaction layer / liquid crystal layer facing.

In one embodiment of the sensor device described above, the first and / or the second substrate is a solid, preferably glass or plastic. Examples of plastic are cycloolefin polymer (COP), cycloolefin copolymer (COC) and polydimethylsiloxane (PDMS).

In one embodiment of the sensor devices described above, the first and / or the second substrate is a solid, preferably glass or plastic, such as cycloolefin polymer (COP), cycloolefin copolymer (COC) and polydimethylsiloxane (PDMS).

In one embodiment, the liquid crystal layer has a homeotropic alignment, homogeneous alignment, or no predefined orientation.

In an embodiment, the liquid crystal layer comprises a liquid crystal selected from thermotropic liquid crystals positive / negative or no dielectric anisotropy, two-frequency liquid crystals, discotic and / or lyotropic liquid crystals and combinations thereof.

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

In one embodiment of the sensor device described above, the liquid crystal layer is color-doped / dye-doped, preferably with dichroic dye (s), fluorescent dye (s), or doped with quantum dot (s). Examples of liquid crystals are 5CB or MLC6608 liquid crystals. The person skilled in the art is familiar with further liquid crystals.

In one embodiment of the sensor devices described above, the phospholipid layer or reaction layer comprises a single / single phospholipid compound or a mixture of two or more phospholipid compounds.

The phospholipid compounds are preferably selected from
DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)
DPPC (1,2-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-sn-glycero-3-phosphocholine (14: 1, PC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (16: 1, PC),
1,2-diisocyanato-sn-glycero-3-phosphocholine (20: 1, PC),
1,2-Dierucoyl-sn-glycero-3-phosphocholine (22: 1, PC),
1,2-Dinervonoyl-sn-glycero-3-phosphocholine (24: 1, PC),
or combinations thereof.

In the following, further embodiments of the sensor device for detecting the presence of lipases or lipase activity (the lipase sensor device) as described above will be further described.

In one embodiment, the reaction layer comprises a single / single lipopolysaccharide compound or a mixture of two or more different lipopolysaccharides.

The LPS compounds are preferably selected from LPS from E. coli strands, such as
LPS from E. coli 055: B5 (as available, for example, from MP Biomedicals),
LPS from E. coli 0127: B5 (as available, for example, from Sigma-Aldrich, Co.),
LPS from E. coli DH5α (such as isolated from E. coli DH5α bacteria),

In one embodiment, the reaction layer comprises a single / single fatty acid compound or a mixture of two or more different fatty acids.

In one embodiment, the reaction layer is a layer comprising lipid compound (s), LPS compound (s) and / or fatty acid compound (s), ie combinations of, for example,
Lipid compound (s) and LPS compound (s),
Lipid compound (s) and fatty acid compound (s),
LPS compound (s) and fatty acid compound (s),
Lipid compound (s) and LPS compound (s) and fatty acid compound (s).

In the following, further embodiments of the above-described sensor devices will be further described.

In one embodiment of the sensor devices described above, the first electrode and the second electrode, each independently, is a non-interdigitated electrode, a plain eectrode, or an interdigital electrode (IDE).

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

In one embodiment, the alignment layer is composed of a silane, such as octadecyldimethyl (3-trimethosylsilylpropyl) ammonium chloride (DMOAP), or a heat / photocurable polyimide having a certain pretilt angle of the liquid crystal in the liquid crystal layer or a self-locating self-assembling compounds, such as thiols, dithiocarbamates, or an obliquely vaporized oxide layer, such as SiO ₂ , which provides 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 the second electrode is / are transparent or non-transparent, with the electrode (s) preferably being constructed from indium tin oxide (ITO) or fluorine tin oxide (FTO).

In one embodiment, the device further comprises crossed polarizers under and over the liquid crystal layer, preferably on the planes of the first and second substrates, respectively, which allow measurement of the change in liquid crystal orientation by the measurement of transmittance / reflectance of polarized light or wherein the device further comprises at least one polarizer over the liquid crystal layer, and wherein the substrate under the liquid crystal layer is a reflective substrate.

In one embodiment, the device further comprises means for measuring an electrical measure, which means provides for measuring the change in liquid crystal orientation by measuring changes in such electrical measure, for example, capacitance, resistance, or current.

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

In one embodiment, the apparatus further comprises a microfluidic device in fluid communication with the reaction chamber.

In one embodiment, the apparatus further comprises a filter unit for filtering any material from the sample which may be larger than the analyte to be assayed, such as a bacterial cell or a microbial toxin, such that the material does not enter the reaction chamber.

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

As discussed above, the present disclosure provides methods for fabricating the liquid crystal based sensor devices according to the present disclosure.

• a) zur Verfügung stellen eines ersten Substrats,
• b) Aufbringen einer Flüssigkristallschicht auf das ersten Substrat,
• c) Aufbringen einer Phospholipidschicht auf die Flüssigkristallschicht, optional mindestens einen Erkennungsteil/ein Erkennungsmolekül umfassend, wie beispielsweise einen Rezeptor, und/oder Cholesterol, bzw. Aufbringen einer Reaktionsschicht auf die Flüssigkristallschicht.

The manufacturing method of a liquid crystal-based sensor device according to the present disclosure (referred to as "manufacturing method" herein) comprises the steps of:

• a) providing a first substrate,
• b) applying a liquid crystal layer to the first substrate,
• c) applying a phospholipid layer on the liquid crystal layer, optionally at least one recognition part / a recognition molecule comprising, such as a receptor, and / or cholesterol, or applying a reaction layer on the liquid crystal layer.

• a') Aufbringen mindestens einer ersten Elektrode auf dem ersten Substrat, und
• a'') Aufbringen einer Isolationsschicht und/oder Ausrichtungsschicht, oder einer Isolations- und Ausrichtungsschicht, welche die erste Elektrode beschichtet.

In an embodiment, the method further comprises the steps

• a ') applying at least a first electrode on the first substrate, and
• a '') applying an insulating layer and / or alignment layer, or an insulating and alignment layer, which coats the first electrode.

• d) zur Verfügung stellen eines zweiten Substrats, und Aufbringen desselben auf der Phospholipidschicht bzw. der Reaktionsschicht, so dass die Phospholipidschicht bzw. die Reaktionsschicht und die Flüssigkristallschicht sich zwischen dem ersten und dem zweiten Substrat befinden („are sandwiched between”), wobei sich ein Zwischenraum zwischen dem zweiten Substrat und der Phospholipidschicht bzw. der Reaktionsschicht befindet, der als Reaktionskammer zur Aufnahme einer zu analysierenden Probe dient.

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

• d) providing a second substrate, and applying it to the phospholipid layer or the reaction layer so that the phospholipid layer and the reaction layer and the liquid crystal layer are sandwiched between the first and second substrates, respectively is a space between the second substrate and the phospholipid layer or the reaction layer, which serves as a reaction chamber for receiving a sample to be analyzed.

In one embodiment, the second substrate has a second electrode thereon, and thereby step e) is performed so that the second electrode faces the phospholipid layer / liquid crystal layer and the reaction layer / liquid crystal layer, respectively.

In one embodiment, the substrates, electrodes, isolation layer, alignment layer, liquid crystal layer, reaction layer and device are as defined above.

In the following, methods using the analyte sensor device will be described.

• a) zur Verfügung stellen einer Flüssigkristall-basierten Sensorvorrichtung gemäß der vorliegenden Offenbarung (einer Analyten-Sensorvorrichtung),
• b) Hinzufügen einer zu analysierenden Probe auf die Phospholipidschicht der Flüssigkristall-basierten Sensorvorrichtung,
• c) Untersuchung auf ein Bindungsereignis, einen zu detektierenden Analyten oder die Aktivität eines Analyten basierend auf Veränderungen der Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht, die von dem Bindungsereignis, dem Analyten oder der Analytenaktivität induziert werden.

As discussed above, the present disclosure provides a method of using the liquid crystal-based sensor device according to the disclosure (analyte sensor device) to detect the presence of an analyte or the activity of an analyte, the method comprising the steps of:

• a) providing a liquid crystal based sensor device according to the present disclosure (an analyte sensor device),
• b) adding a sample to be analyzed to the phospholipid layer of the liquid crystal based sensor device,
• c) Examination for a binding event, an analyte to be detected or the activity of an analyte based on changes in the orientation of the liquid crystals in the liquid crystal layer induced by the binding event, the analyte or the analyte activity.

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

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

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

In one embodiment, the activity of an analyte is the reaction of the analyte with the phospholipid layer.

• – aufgrund seiner Anheftung („attachment”) oder aufgrund seiner Bindung an das Erkennungsteil oder Erkennungsmolekül (wie beispielsweise einen Rezeptor, ein Enzym oder einen Antikörper, oder
• – aufgrund seiner Reaktion mit dem Erkennungsteil oder dem Erkennungsmolekül (wie beispielsweise einen Rezeptor, Enzym oder Antikörper), oder
• – aufgrund seiner Anheftung („attachment”) oder aufgrund seiner Bindung an die Phospholipidschicht, oder
• – aufgrund seiner Reaktion mit der Phospholipidschicht.

For example, the analyte is capable of altering the alignment of the liquid crystals in the liquid crystal layer

• Due to its attachment or due to its binding to the recognition or recognition molecule (such as a receptor, an enzyme or an antibody, or
• Due to its reaction with the recognition part or the recognition molecule (such as a receptor, enzyme or antibody), or
• Due to its attachment or due to its attachment to the phospholipid layer, or
• Due to its reaction with the phospholipid layer.

The analyte is preferably selected from the group comprising a prokaryotic cell, such as a bacterial cell, and a prokaryotic agent, such as a toxin, such as a microbial toxin, which analyte is capable of controlling the alignment of the liquid crystals at the interface ( "Interface") between the liquid crystal layer and the phospholipid layer.

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

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

Such a second analyte, preferably, binds or responds to the analyte (s) to be detected (indirectly). The second analyte is preferably selected from the components of a medium (such as a nutrient broth) used to grow / cultivate / feed the first analyte, or antibiotics or drugs that act on the first analyte.

• a) zur Verfügung stellen einer Flüssigkristall-basierten Sensorvorrichtung gemäß der vorliegenden Offenbarung (einer Analyten-Sensorvorrichtung),
• b) Hinzufügen einer zu analysierenden Probe auf die Phospholipidschicht der Flüssigkristall-basierten Sensorvorrichtung,
• c) Untersuchung auf ein Bindungsereignis, einen zweiten zu detektierenden Analyten oder die Aktivität eines zweiten Analyten basierend auf Veränderungen der Ausrichtung („change of alignment”) der Flüssigkristalle in der Flüssigkristallschicht, die von dem Bindungsereignis, dem zweiten Analyten oder der Aktivität des zweiten Analyten induziert werden.

In this embodiment, the method is a method of using the liquid crystal-based sensor device according to the disclosure (analyte sensor device) for indirectly detecting the presence of an analyte or the activity of an analyte, and the method comprises the steps of:

• a) providing a liquid crystal based sensor device according to the present disclosure (an analyte sensor device),
• b) adding a sample to be analyzed to the phospholipid layer of the liquid crystal based sensor device,
• c) assay for a binding event, a second analyte to be detected or the activity of a second analyte based on changes in alignment of the liquid crystals in the liquid crystal layer, that of the binding event, the second analyte or the activity of the second analyte be induced.

The binding event is preferably the binding of the second analyte to the recognition part or recognition molecule, such as a receptor included in the phospholipid layer, or the binding of the second analyte to the phospholipid layer.

The activity of the second analyte is preferably the reaction of the second analyte with the recognition moiety or recognition molecule, such as a receptor included in the 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) The liquid crystal layer of the liquid crystal based sensor device according to the present disclosure is observed over a period of time and the alignment change, if any, is measured over a period of time.

In an embodiment with indirect detection of the presence of an analyte, a slower alignment change can be seen, indicating the presence of the analyte.

The term "slowed-down time of the alignment change" as used herein refers to changes in the alignment of the liquid crystals in the (surface) liquid crystal layer of the liquid crystal-based sensor device according to the present disclosure, which are slower in comparison to the alignment changes that occurred before the amount of the active component (the component causing the orientation change, ie, the second analyte) was reduced, e.g. Due to binding to or response to the analyte.

• a) zur Verfügung stellen einer Flüssigkristall-basierten gemäß der vorliegenden Offenbarung (einer Analyten-Sensorvorrichtung),
• b) zur Verfügung stellen einer Probe umfassend den Analyten,
• c) zur Verfügung stellen mindestens einer auf Bindung an den Analyten und/oder Modifizierung der Aktivität des Analyten zu screenenden Verbindung.
• d) Hinzufügen von mindestens einer zur screenenden Verbindung zur den Analyten umfassenden Probe,
• e) Hinzufügen der den Analyten sowie die Verbindung umfassenden Probe auf die Phospholipidschicht der Flüssigkristall-basierten Sensorvorrichtung,
• f) Untersuchung auf den Analyten oder die Aktivität des Analyten basierend auf Veränderungen der Ausrichtung („change of alignment”) der Flüssigkristalle in der Flüssigkristallschicht, die von dem Bindungsereignis, Analyten oder der Analytenaktivität,

wobei keine Ausrichtungsveränderung anzeigt, dass die zu screenende Verbindung an den Analyten bindet und/oder die Aktivität des Analyten modifiziert, und wobei eine Veränderung der Ausrichtung anzeigt, dass die zu screenende Verbindung den Analyten nicht bindet und/oder die Aktivität des Analyten nicht modifiziert.As already stated, the present disclosure provides a method of using the liquid crystal-based sensor device according to the disclosure (analyte sensor device) to screen for compounds that bind to an analyte and / or modify the activity of an analyte, such as inhibitors of microbial toxins , to disposal,
the method comprising the steps of:

• a) providing a liquid crystal based according to the present disclosure (an analyte sensor device),
• 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 at least one sample to the screening compound to the analyte,
• e) adding the sample comprising the analyte and the compound to the phospholipid layer of the liquid crystal-based sensor device,
• f) assaying for the analyte or the activity of the analyte based on changes in alignment of the liquid crystals in the liquid crystal layer resulting from the binding event, analyte or analyte activity,

wherein no orientation change indicates that the compound to be screened binds to the analyte and / or modifies the activity of the analyte, and wherein a change in orientation indicates that the compound to be screened does not bind the analyte and / or modifies the activity of the analyte.

In one embodiment, the orientation or orientation change of the liquid crystals in the (surface) liquid crystal layer of the liquid crystal-based sensor device according to the present disclosure is observed over a period of time and the orientation change, if any, is measured over a period of time.

The term "change of alignment" as used herein in the context of the "screening method" refers to alignment changes of the liquid crystals in the (surface) liquid crystal layer of the liquid crystal-based sensor device according to the present disclosure after adding one Sample comprising the analyte and the compound to be screened (in step e)).

Such an orientation change is visible through a change in (light) transmission or a change in reflection. In a preferred embodiment, such a change is preferably at least 0.1%, more preferably at least 0.5%; even more preferably at least 1% or more change in permeability or reflection.

The term "no orientation change" as used herein in the context of the "screening method" refers to the alignment of the liquid crystals compared to the state prior to adding the sample to the analyte and the compound to be screened (in step e)). remains essentially unchanged, in particular unchanged or without significant change in the (light) permeability or the reflection.

In a preferred embodiment, less than 0.1%, more preferably less than 0.05% or even less change in permeability or reflectance is measured.

In other embodiments, the cut-off value may be higher or slower than the above-mentioned 0.1% depending on e.g. From the sample and its components, media, etc. The cut-off value may also depend on the detector or detection method used, such as the sensitivity of the detector / detection method.

For example, in one embodiment, the efficacy of an antibiotic, drug, or substance may be considered Deactivation or killing bacteria or a toxin. For example, in one case, when a compound such as antibiotic A is not effective in deactivating or killing the bacteria / toxin, it leads to an alignment change of the liquid crystals in the liquid crystal layer. Compound A is thus not effective in deactivating / killing the particular bacterial / toxin. In another case, for example, another compound, such as antibiotic B, is tested which is effective in deactivating or killing the bacterial / toxin; thereby, no alignment change of the liquid crystals in the liquid crystal layer can be observed because the bacteria / toxin is deactivated. Compound B is thus effective in deactivating / killing the particular bacterial / toxin.

The analyte is preferably selected from the group comprising a prokaryotic cell, such as a bacterial cell, and a prokaryotic agent, such as a toxin, such as a microbial toxin, which analyte is capable of aligning the alignment of liquid crystals at the interface the liquid crystal layer and the phospholipid layer to change.

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

Compound (s) that bind an analyte and / or modify the activity of an analyte are preferably screened indirectly, for example, by detecting the presence or activity of a second analyte, which second analyte is capable of controlling the alignment of the liquid crystals in the analyte Change liquid crystal layer.

As described above, the second analyte binds or reacts preferentially to / with the analyte to which the compound (s) to be screened bind, react and / or whose activity they alter.

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

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

In an embodiment of the methods according to the present disclosure, the alignment or orientation change of liquid crystals in the (surface) liquid crystal layer of the liquid crystal-based sensor device according to the present disclosure is observed over a period of time and the orientation change, if any, over a period of time is measured.

The following describes methods using the lipase sensor device.

• a) zur Verfügung stellen einer Flüssigkristall-basierten Sensorvorrichtung gemäß der vorliegenden Offenbarung (eine Lipase-Sensorvorrichtung),
• b) Hinzufügen einer zu analysierenden Probe auf die Reaktionsschicht der Flüssigkristall-basierten Sensorvorrichtung,
• c) Untersuchung auf ein Reaktionsereignis basierend auf Veränderungen, die von der/den Lipase(n) oder der Lipasenaktivität(en) an der Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht induziert werden.

As discussed above, the present disclosure provides a method of using the liquid crystal based sensor device according to the disclosure (lipase sensor device) to detect the presence of lipase (s) or the activity of lipase (s).
the method comprising the steps of:

• a) providing a liquid crystal-based sensor device according to the present disclosure (a lipase sensor device),
• b) adding a sample to be analyzed to the reaction layer of the liquid crystal based sensor device,
• c) assay for a response event based on changes induced by the lipase (s) or lipase activity (s) in the alignment of liquid crystals in the liquid crystal layer.

The sample is preferably a cell culture sample, such as a cell culture supernatant, a cell culture medium, a cell culture solution; or the sample is a body fluid, such as blood, saliva, urine, etc.

In one embodiment, the reaction event is the binding of the lipase (s) to the reaction layer and / or the disruption / disruption of the reaction layer due to lipase (n) activity.

Preferably, the disruption / disruption of the reaction layer due to lipase (n) activity comprises the hydrolysis, esterification, transesterification of the (phospho) lipid (s), LPS and / or the fatty acid (s) of the reaction layer.

• a) zur Verfügung stellen einer Flüssigkristall-basierten Sensorvorrichtung gemäß der vorliegenden Offenbarung (eine Lipase-Sensorvorrichtung),
• b) Hinzufügen einer zu analysierenden Probe auf die Reaktionsschicht der Flüssigkristall-basierten Sensorvorrichtung,
• c) Untersuchung auf ein Reaktionsereignis basierend auf Veränderungen der Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht,

wobei eine Änderung der Ausrichtung anzeigt, dass die Zellen und/oder Organe der Probe beschädigt, zersetzt und/oder abgestorben sind.As discussed above, the present disclosure provides a method of using the liquid crystal based sensor device according to the disclosure (lipase sensor device) to monitor the damage, degradation and / or death of cells or organs.
the method comprising the following steps:

• a) providing a liquid crystal-based sensor device according to the present disclosure (a lipase sensor device),
• b) adding a sample to be analyzed to the reaction layer of the liquid crystal based sensor device,
• c) Examination for a reaction event based on changes in the orientation of the liquid crystals in the liquid crystal layer,

wherein a change in orientation indicates that the cells and / or organs of the sample are damaged, degraded and / or dead.

The sample is preferably a cell culture sample, such as a cell culture supernatant, a cell culture medium, a cell culture solution; or the sample is a body fluid, such as blood, saliva, urine, etc.

In one embodiment, the alignment of the liquid crystals in the liquid crystal layer is observed over a period of time and the orientation change, if any, is measured over a period of time.

In one embodiment, the method comprises repeating steps b) and c) over a period of time. This may damage, decompose and / or kill cells or organs of the sample at different times, i. H. over a period of time, monitored / measured.

In one embodiment, the reaction event is the binding of lipase (s), if present in the sample, to the reaction layer due to damage, decomposition and / or death of cells or organs of the sample, and / or the disruption / disruption of the reaction layer due to Activity (s) of the lipase (s).

Preferably, the disruption / disruption of the reaction layer due to lipase (n) activity comprises the hydrolysis, esterification, transesterification of the (phospho) lipid (s), LPS and / or the fatty acid (s) of the reaction layer.

• a) zur Verfügung stellen einer Flüssigkristall-basierten gemäß der vorliegenden Offenbarung (eine Lipase-Sensorvorrichtung),
• b) zur Verfügung stellen einer Probe umfassend die Lipase(n),
• c) zur Verfügung stellen mindestens einer zu screenenden Verbindung zur Modifizierung der Lipase(n)aktivität,
• d) Hinzufügen von mindestens einer zur screenenden Verbindung zur die Lipase(n) umfassenden Probe,
• e) Hinzufügen der Probe umfassnd die Lipase(n) sowie die zu screenende Verbindung auf die Reaktionsschicht der Flüssigkristall-basierten Sensorvorrichtung,
• f) Untersuchung auf die Lipase(n) oder die Aktivität der Lipase(n) basierend auf Veränderungen an der Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht, die von einem Reaktionsereignis induziert werden,

wobei keine Ausrichtungsveränderung anzeigt, dass die zu screenende Verbindung die Lipase(n)aktivität inhibiert, und eine Ausrichtungsveränderung anzeigrt, dass die zu screenende Verbindung die Lipase(n)aktivität nicht modifiziert, oder die Lipase(n)aktivität verstärkt.The present disclosure provides a method of using the liquid crystal-based sensor device according to the disclosure (lipase sensor device) to screen for compounds that alter the activity of lipase (s), the method comprising the steps of:

• a) providing a liquid crystal-based according to the present disclosure (a lipase sensor device),
• b) providing a sample comprising the lipase (s),
• c) providing at least one compound to be screened for modification of lipase (n) activity,
• d) adding at least one sample comprising the screening compound to the lipase (s),
• e) adding the sample comprising the lipase (s) and the compound to be screened to the reaction layer of the liquid crystal based sensor device,
• f) assaying for the lipase (s) or activity of the lipase (s) based on changes in the alignment of liquid crystals in the liquid crystal layer induced by a reaction event,

wherein no alignment change indicates that the compound to be screened inhibits lipase (n) activity and indicates an orientation change that the compound to be screened does not modify lipase (n) activity or enhances lipase (n) activity.

In one embodiment, the orientation or orientation change of the liquid crystals in the (surface) liquid crystal layer of the liquid crystal-based sensor device according to the present disclosure is observed over a period of time and the orientation change, if any, is measured over a period of time.

The term "change of alignment" as used herein in the context of the "screening method" refers to alignment changes of the liquid crystals in the (surface) liquid crystal layer of the liquid crystal-based sensor device according to the present disclosure after adding one A sample comprising the analyte (ie, lipase (s)) and the candidate compound (in step e)).

Such an orientation change is visible through a change in (light) transmission or a change in reflection. In a preferred embodiment, such a change is preferably at least 0.1%, more preferably at least 0.5%; even more preferably at least 1% or more change in permeability or reflection.

The term "no orientation change" as used herein in the context of the "screening method" refers to the orientation the liquid crystal, which remains essentially unchanged in comparison to the state before the addition of the sample with the analyte (eg lipase (s)) and the compound to be screened (in step e)), in particular unchanged or without significant change in the (light) ) Permeability or reflection.

In a preferred embodiment, less than 0.1%, more preferably less than 0.05% or even less change in permeability or reflectance is measured.

In other embodiments, the cut-off value may be higher or slower than the above-mentioned 0.1% depending on e.g. From the sample and its components, media, etc. The cut-off value may also depend on the detector or detection method used, such as the sensitivity of the detector / detection method.

Hereinafter further embodiments of all methods using an analyte sensor device or lipase sensor device as described above will be further described.

In one embodiment, the examination (in step c) or step f)) is performed by visual examination of the transmittance and / or reflection of polarized light or unpolarized light or by transmission and / or reflection measurements with polarized light or with unpolarized light, and the examination for changes in such a permeability, or the examination (in step c) or step f)) is carried out by measurements of an electrical measure, preferably by measurements of capacitance, resistance, or current, and for example the investigation of changes in such electrical measure.

• d) Überprüfung, dass eine beobachtete Durchlässigkeits- oder Reflektionsveränderung oder eine Veränderung der elektrischen Größe/Maß verursacht wird durch das Bindungsereignis oder die Anwesenheit eines Analyten oder die Aktivität eines Analyten bzw. verursacht wird durch das Reaktionsereignis, bevorzugt an einer Grenzfläche zwischen der Flüssigkristallschicht und der Phospholipidschicht/Reaktionsschicht, und nicht das Ergebnis eines gestörten („disrupted”) Bereichs auf dem Sensor ist, wo keine Flüssigkristalle und keine Phospholipidschicht/Reaktionsschicht vorhanden ist, indem eine Spannung mit Gleich- oder Wechselstrom an die erste und/oder zweite Elektrode angelegt wird und untersucht wird, ob dies zu einer Veränderung der detektierten Durchlässigkeit oder Reflektion von polarisiertem Licht oder der gemessenen elektrischen Größe/Maßes führt.

In an embodiment, the method further comprises the step of:

• d) checking that an observed change in permeability or reflection or a change in electrical size / dimension is caused by the binding event or the presence of an analyte or the activity of an analyte or caused by the reaction event, preferably at an interface between the liquid crystal layer and the phospholipid layer / reaction layer is not the result of a disrupted region on the sensor where there are no liquid crystals and no phospholipid layer / reaction layer by applying a DC or AC voltage to the first and / or second electrodes and whether this leads to a change in the detected transmission or reflection of polarized light or the measured electrical variable / measure.

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

• b') Erhöhung der lokalen Konzentration des Analyten/der Lipase(n), oder Energiezuführen („energizing”) des Analyten/der Lipase(n) an der Grenzfläche zwischen der Phospholipidschicht/Reaktionsschicht und der Probe durch Elektrophorese, Dielektrophorese, Wechselstromelektroosmose (ACEO) oder Joulesche Erwärmung.

In an embodiment, the method further comprises the step of:

• b ') increasing the local concentration of the analyte / lipase (s), or energizing the analyte / lipase (s) at the interface between the phospholipid layer / reaction layer and the sample by electrophoresis, dielectrophoresis, AC electroosmosis (ACEO ) or Joule warming.

• b'') Erhöhung der Reaktionsgeschwindigkeit durch das Anlegen einer Gleich- oder Wechselspannung an die erste und/oder zweite Elektrode, wobei die angelegte Spannung im Bereich von 0,1–25 V eines sinusförmigen/Dreiecks-/Rechtecksignals liegt, und die Frequenz im Bereich von 0 Hz bis 1 GHz, bevorzugt 100 Hz bis 10 MHz, weiter bevorzugt 1 kHz bis 1 MHz liegt.

In an embodiment, the method further comprises the step of:

• b '') Increasing the reaction rate by applying a DC or AC voltage to the first and / or second electrode, wherein the applied voltage is in the range of 0.1-25 V of a sinusoidal / triangular / square wave signal, and the frequency in 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 / reaction event occurs in water or an aqueous solution or in the gas phase, wherein the analyte / lipase (s) is / are in water, an aqueous solution or in the gas phase, or wherein the analyte (s) Lipase (s) to a solid support, such as polymer beads, or immobilized on one of the substrates is / are.

The present disclosure further provides applications of the lipase sensor device.

As discussed above, the present disclosure provides the use of a liquid crystal-based sensor device according to the disclosure (lipase sensor device) for detecting the presence of lipase (s) or the activity of lipase (s) in a sample.

In one embodiment, the sample is a cell culture sample, such as a cell culture supernatant, a cell culture medium, a cell culture solution.

In one embodiment, the sample is a patient sample, such as a body fluid, such as blood, saliva, urine.

As discussed above, the present disclosure provides the use of a liquid crystal sensor device according to the disclosure (lipase sensor device) for monitoring damage, degradation or death of cells or organs in a sample.

In one embodiment, the sample is a cell culture sample, such as a cell culture supernatant, a cell culture medium, a cell culture solution.

In one embodiment, the sample is a patient sample, such as a body fluid, such as blood, saliva, urine.

As discussed above, the present disclosure provides the use of the liquid crystal sensor device according to the disclosure (lipase sensor device) as a lipase screening device in toxicity or cytotoxicity assays.

• (1) Flüssigkristall-basierte Sensorvorrichtung zur Detektion der Anwesenheit eines Analyten, wie beispielsweise einer prokaryotischen Zelle, einer bakteriellen Zelle und/oder eines prokaryotischen Mittels, wie beispielsweise eines Toxins, wie beispielsweise eines mikrobiellen Toxins, wobei die Vorrichtung in der aufgeführten Reihenfolge umfasst:
• – ein erstes Substrat,
• – eine Flüssigkristallschicht auf dem ersten Substrat,
• – eine Phospholipidschicht auf der Flüssigkristallschicht, wobei die Vorrichtung keinen Erkennungsteil oder kein Erkennungsmolekül umfasst, das direkt an die Oberfläche des ersten Substrates gebunden ist.
• (2) Sensorvorrichtung, wie oben in (1) beschrieben, wobei die Phospholipidschicht mindestens einen Erkennungsteil oder ein Erkennungsmolekül, wie beispielsweise einen Rezeptor, ein Enzym oder einen Antikörper, umfasst.
• (3) Sensorvorrichtung, wie oben in (1) oder (2) beschrieben, wobei die Phospholipidschicht Cholesterol umfasst, bevorzugt zwischen 1 und 20 Gew.-%, weiter bevorzugt etwa 10 bis etwa 15 Gew.-% Cholesterol.
• (4) Sensorvorrichtung, wie oben in einem von (1) bis (3) beschrieben, wobei es sich bei dem mindestens einen Erkennungsteil oder Erkennungsmolekül in der Phospholipidschicht um einen Rezeptor handelt, wie beispielsweise Gangliosid GM₁.
• (5) Sensorvorrichtung, wie oben in (7) beschrieben, wobei die Phospholipidschicht zwischen etwa 0,1% bis etwa 10 Gew.-% GM₁, bevorzugt etwa 0,5% bis etwa 5 Gew.-% GM₁ umfasst.
• (6) Flüssigkristall-basierte Sensorvorrichtung zur Detektion der Anwesenheit von Lipasen oder Lipaseaktivität, wobei die Vorrichtung in der aufgeführten Reihenfolge umfasst:
• – ein erstes Substrat,
• – eine Flüssigkristallschicht auf dem ersten Substrat,
• – eine Reaktionsschicht auf der Flüssigkristallschicht, wobei die Reaktionsschicht eine Lipidschicht, eine LPS-Schicht und/oder eine Fettsäureschicht ist.
• (7) Sensorvorrichtung, wie oben in (7) beschrieben, wobei die Vorrichtung keinen Erkennungsteil oder kein Erkennungsmolekül umfasst, das direkt an die Oberfläche des ersten Substrates gebunden ist.
• (8) Sensorvorrichtung, wie oben in einem von (1) bis (7) beschrieben, weiterhin umfassend:
• – mindestens eine erste Elektrode zwischen dem ersten Substrat und der Flüssigkristallschicht, und
• – eine Isolationsschicht und/oder eine Ausrichtungsschicht, oder eine Isolations- und Ausrichtungsschicht, welche die Elektrode beschichtet.
• (9) Sensorvorrichtung, wie oben in einem von (1) bis (8) beschrieben, weiterhin umfassend:
• – ein zweites Substrat, das gegenüber dem ersten Substrat angeordnet ist und die Flüssigkristallschicht und Phospholipidschicht zu Zwischenschichten bzw. die Flüssigkristallschicht und Reaktionsschicht zu Zwischenschichten zwischen dem ersten und zweiten Substrat macht, und wobei es einen Zwischenraum zwischen dem zweiten Substrat und der Phospholipidschicht bzw. zwischen dem zweiten Substrat und der Reaktionsschicht gibt, der als Reaktionskammer zur Aufnahme einer zu analysierenden Probe dient.
• (10) Sensorvorrichtung, wie oben in (9) beschrieben, wobei das zweite Substrat weiterhin umfasst:
• – eine zweite Elektrode auf dem zweiten Substrat, und
• – optional, eine Isolationsschicht auf der zweiten Elektrode, wobei die Isolationsschicht der sich auf dem ersten Substrat befindenden Phospholipidschicht/Flüssigkristallschicht bzw. Reaktionsschicht/Flüssigkristallschicht zugewandt ist.
• (11) Sensorvorrichtung, wie oben in einem von (1) bis (10) beschrieben, wobei das erste und/oder das zweite Substrat einen Feststoff umfasst, bevorzugt Glas oder Kunststoff, wie beispielsweise Cycloolefin-Polymer (COP), Cycloolefin-Copolymer (COC) und Polydimethylsiloxan (PDMS).
• (12) Sensorvorrichtung, wie oben in einem von (1) bis (11) beschrieben, wobei die Flüssigkristallschicht eine homöotrope Ausrichtung, homogene Ausrichtung oder keine vordefinierte Ausrichtung aufweist.
• (13) Sensorvorrichtung, wie oben in einem von (1) bis (12) beschrieben, wobei die Flüssigkristallschicht ein Flüssigkristall umfasst, das ausgewählt ist aus thermotropen Flüssigkristallen mit positiver/negativer oder keiner dielektrischen Anisotropie, Zweifrequenz-Flüssigkristallen, diskotischen und/oder lyotropen Flüssigkristallen und Kombinationen davon.
• (14) Sensorvorrichtung, wie oben in einem von (1) bis (13) beschrieben, wobei die Flüssigkristallschicht eine einzelne Flüssigkristallverbindung oder eine Mischung aus zwei oder mehreren verschiedenen Flüssigkristallverbindungen, oder eine Flüssigkristallmischung gemischt mit Dotanden, wie beispielsweise Nanopartikeln oder kleinen organischen Molekülen, umfasst.
• (15) Sensorvorrichtung, wie oben in (14) beschrieben, wobei die Flüssigkristallschicht farbdotiert ist, bevorzugt mit (einem) dichroitischen Farbstoff(en), Fluoreszenzfarbstoff(en) oder dotiert mit (einem) Quantenpunkt(en).
• (16) Sensorvorrichtung, wie oben in einem von (1) bis (15) beschrieben, wobei die Phospholipidschicht bzw. die Reaktionsschicht eine einzelne Phospholipidverbindung oder eine Mischung aus zwei oder mehreren verschiedenen Phospholipidverbindungen umfasst.
• (17) Sensorvorrichtung, wie oben in (16) beschrieben, wobei die Phopholipidverbindungen ausgewählt sind aus DOPC, (1,2-Dioleoyl-sn-Glycero-3-Phosphocholin), DPPC (1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholin), DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholin), DSPC (1,2-Distearoyl-sn-Glycero-3-Phosphocholin), DLPC (1,2-Dilauroyl-sn-Glycero-3-Phosphocholin), 1,2-Dimyristoleoyl-sn-Glycero-3-Phosphocholin (14:1, PC), 1,2-Dipalmitoleoyl-sn-Glycero-3-Phosphocholin (16:1, PC), 1,2-Dieicosenoyl-sn-Glycero-3-Phosphocholin (20:1, PC), 1,2-Dierucoyl-sn-Glycero-3-Phosphocholin (22:1, PC), 1,2-Dinervonoyl-sn-Glycero-3-Phosphocholin (24:1, PC) oder Kombinationen davon.
• (18) Sensorvorrichtung, wie oben in einem von (6) bis (17) beschrieben, wobei die Reaktionsschicht eine einzelne Lipopolysaccharidverbindung oder eine Mischung aus zwei oder mehreren verschiedenen Lipopolysacchariden umfasst.
• (19) Sensorvorrichtung, wie oben in (18) beschrieben, wobei die LPS-Verbindungen ausgewählt sind aus LPS aus E. coli-Stämmen.
• (20) Sensorvorrichtung, wie oben in einem von (6) bis (17) beschrieben, wobei die Reaktionsschicht eine einzelne Fettsäureverbindung oder eine Mischung aus zwei oder mehreren verschiedenen Fettsäuren umfasst.
• (21) Sensorvorrichtung, wie oben in einem von (6) bis (17) beschrieben, wobei die Reaktionsschicht eine Schicht ist umfassend Lipid-, LPS- und/oder Fettsäureverbindung(en).
• (22) Sensorvorrichtung, wie oben in einem von (8) bis (21) beschrieben, wobei die erste Elektrode und die zweite Elektrode, jeweils unabhängig voneinander, eine nicht-interdigitale Elektrode, eine ebene Elektrode oder eine interdigitale Elektrode (IDE) ist.
• (23) Sensorvorrichtung, wie oben in einem von (8) bis (22) beschrieben, wobei die Isolationsschicht auf der ersten und/oder zweiten Elektrode aus Polyimid aufgebaut ist.
• (24) Sensorvorrichtung, wie oben in einem von (8) bis (23) beschrieben, wobei die Ausrichtungsschicht aufgebaut ist aus einem Silan, wie beispielsweise Oktadecyldimethyl-(3-Trimethoxy-silylpropyl)-Ammoniumchlorid (DMOAP), oder einem hitze/lichtaushärtbaren Polyimid, das einen definierten Vorneigungswinkel des Flüssigkristalls in der Flüssigkristallschicht oder eine selbstanordnende Monoschicht aus selbstanordnenden Verbindungen, wie beispielsweise Thiolen, Dithiocarbamaten, oder einer schräg verdampften Oxidschicht, wie beispielsweise SiO₂, die eine Vorneigung des Flüssigkristalls in der Flüssigkristallschicht je nach dem Verdampfungswinkel des Oxids bereitstellt.
• (25) Sensorvorrichtung, wie oben in einem von (8) bis (24) beschrieben, wobei die erste und/oder die zweite Elektrode transparent oder nicht transparent ist/sind, wobei bevorzugt die Elektrode(n) aus Indium-Zinnoxid (ITO) oder Fluor-Zinnoxid (FTO) aufgebaut ist/sind.
• (26) Sensorvorrichtung, wie oben in einem von (8) bis (25) beschrieben, wobei die Vorrichtung weiterhin gekreuzte Polarisatoren unter und über der Flüssigkristallschicht umfasst, bevorzugt auf den Ebenen des ersten bzw. zweiten Substrats, die eine Messung der Veränderung der Flüssigkristallausrichtung durch die Messung der Durchlässigkeit/Reflektion von polarisiertem Licht erlauben, oder wobei die Vorrichtung weiterhin mindestens einen Polarisator über der Flüssigkristallschicht umfasst, und wobei das unter der Flüssigkristallschicht befindliche Substrat ein reflektierendes Substrat ist.
• (27) Sensorvorrichtung, wie oben in einem von (1) bis (26) beschrieben, wobei die Vorrichtung weiterhin Mittel zur Messung eines elektrischen Maßes umfasst, wobei das/die Mittel eine Messung der Veränderung der Flüssigkristallausrichtung durch die Messung von Veränderungen eines solchen elektrischen Maßes ermöglicht, zum Beispiel Kapazitanz, Widerstand, oder Strom.
• (28) Sensorvorrichtung, wie oben in einem von (8) bis (25) und (27) beschrieben, wobei die Vorrichtung weiterhin nur einen Polarisator umfasst, bevorzugt auf der Ebene entweder des ersten oder des zweiten Substrats, und wobei das entsprechende andere Substrat ohne Polarisator zur Verwendung in Verbindung mit einer Lichtquelle, die polarisiertes Licht zur Verfügung stellt, bestimmt ist, wie zum Beispiel eine Flüssigkristall-Anzeigenvorrichtung.
• (29) Sensorvorrichtung, wie oben in einem von (1) bis (28) beschrieben, weiterhin umfassend eine Mikrofluidvorrichtung in Fluidverbindung mit der Reaktionskammer.
• (30) Sensorvorrichtung, wie oben in einem von (1) bis (29) beschrieben, weiterhin umfassend eine Filtereinheit zum Filtern beliebigen Materials aus der Probe, das größer als der zu untersuchende Analyt sein kann, wie beispielsweise eine Bakterienzelle oder ein mikrobielles Toxin, so dass das Material nicht in die Reaktionskammer eindringt.
• (31) Sensorvorrichtung, wie oben in einem von (1) bis (30) beschrieben, wobei die Reaktionskammer eine Tiefe im Bereich von 800 nm bis 100 µm, bevorzugt von 900 nm bis 30 µm aufweist, und, im Fall einer runden Kammer, eine Breite oder einen Durchmesser im Bereich von 1 µm bis 1 mm, bevorzugt von 25 µm bis 500 µm aufweist, und/oder wobei die Reaktionskammer aus Glas, Kunststoff besteht oder Wände aus Metall hat, wie beispielsweise Goldwände.
• (32) Verfahren zur Herstellung einer Flüssigkristall-basierten Sensorvorrichtung, wie oben in einem von (1) bis (31) beschrieben, wobei das Verfahren die Schritte umfasst:
• d) zur Verfügung stellen eines ersten Substrats,
• e) Aufbringen einer Flüssigkristallschicht auf das erste Substrat,
• f) Aufbringen einer Phospholipidschicht auf die Flüssigkristallschicht, optional umfassend mindestens ein(en) Erkennungsteil/-molekül, wie beispielsweise einen Rezeptor, und/oder Cholesterol, bzw. Aufbringen einer Reaktionsschicht auf die Flüssigkristallschicht.
• (33) Verfahren, wie oben in (32) beschrieben, weiterhin umfassend die Schritte
• a') Aufbringen mindestens einer ersten Elektrode auf dem ersten Substrat, und
• a'') Aufbringen einer Isolationsschicht und/oder Ausrichtungsschicht, oder einer Isolations- und Ausrichtungsschicht, welche die erste Elektrode beschichtet.
• (34) Verfahren, wie oben in (32) oder (33) beschrieben, weiterhin umfassend den Zusatzschritt:
• e) zur Verfügung stellen eines zweiten Substrats, und Aufbringen desselben auf der Phospholipidschicht bzw. der Reaktionsschicht, so dass die Phospholipidschicht bzw. die Reaktionsschicht und die Flüssigkristallschicht sich zwischen dem ersten und dem zweiten Substrat befinden („are sandwiched between”), wobei sich ein Zwischenraum zwischen dem zweiten Substrat und der Phospholipidschicht bzw. der Reaktionsschicht befindet, der als Reaktionskammer zur Aufnahme einer zu analysierenden Probe dient.
• (35) Verfahren, wie oben in einem von (32) bis (34) beschrieben, wobei das zweite Substrat eine zweite darauf befindliche Elektrode auf weist, und wobei Schritt e) so durchgeführt wird, dass die zweite Elektrode der Phospholipidschicht/Flüssigkristallschicht bzw. der Reaktionsschicht/Flüssigkristallschicht zugewandt ist.
• (35) Verfahren, wie oben in einem von (32) bis (35) beschrieben, wobei die Substrate, Elektroden, Isolationsschicht, Ausrichtungsschicht, Flüssigkristallschicht, Reaktionsschicht und Vorrichtung wie oben in einem von (1) bis (31) definiert sind.
• (37) Verfahren zur Verwendung der Flüssigkristall-basierten Sensorvorrichtung, wie oben in einem von (1) bis (3), (8) bis (17) und (22) bis (31) beschrieben, zur Detektion der Anwesenheit eines Analyten oder der Aktivität eines Analyten zur Verfügung, wobei das Verfahren die Schritte umfasst:
• d) zur Verfügung stellen einer Flüssigkristall-basierten Sensorvorrichtung, wie oben in einem von (1) bis (3), (8) bis (17) und (22) bis (31) beschrieben,
• e) Hinzufügen einer zu analysierenden Probe auf die Phospholipidschicht der Flüssigkristall-basierten Sensorvorrichtung,
• f) Untersuchung auf ein Bindungsereignis, einen zu detektierenden Analyten oder die Aktivität eines Analyten basierend auf Veränderungen der Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht, die von dem Bindungsereignis, dem Analyten oder der Analytenaktivität induziert werden.
• (38) Verfahren, wie oben in (37) beschrieben, wobei das Bindungsereignis die Bindung des Analyten an das Erkennungsteil oder das Erkennungsmolekül ist, wie beispielsweise einen Rezeptor, ein Enzym oder einen Antikörper, das in der Phospholipidschicht umfasst ist; oder die Bindung des Analyten an die Phospholipidschicht ist.
• (39) Verfahren, wie oben in (37) oder (38) beschrieben, wobei die Aktivität eines Analyten die Reaktion des Analyten mit dem Erkennungsteil oder Erkennungsmolekül ist, wie beispielsweise einen Rezeptor, Enzym oder Antikörper, der in der Phospholipidschicht umfasst ist; oder die Reaktion des Analyten mit der Phospholipidschicht ist.
• (40) Verfahren, wie oben in einem von (37) bis (39) beschrieben, wobei die Anwesenheit eines Analyten indirekt detektiert wird, wie beispielsweise durch Detektieren der Anwesenheit eines zweiten Analyten, wobei der zweite Analyt in der Lage ist, die Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht zu verändern.
• (41) Verfahren zur Verwendung der Flüssigkristall-basierten Sensorvorrichtung, wie oben in einem von (1) bis (3), (8) bis (17) und (22) bis (31) beschrieben, zum Screening nach Verbindungen, die an einen Analyten binden und/oder die Aktivität eines Analyten modifizieren, wie beispielsweise Inhibitoren mikrobieller Toxine, wobei das Verfahren die Schritte umfasst:
• a) zur Verfügung stellen einer Flüssigkristall-basierten Sensorvorrichtung, wie oben in einem von (1) bis (3), (8) bis (17) und (22) bis (31) beschrieben,
• b) zur Verfügung stellen einer Probe umfassend den Analyten,
• c) zur Verfügung stellen mindestens einer auf Bindung an den Analyten und/oder Modifizierung der Aktivität des Analyten zu screenenden Verbindung.
• d) Hinzufügen von mindestens einer zur screenenden Verbindung zur den Analyten umfassenden Probe,
• e) Hinzufügen der Probe umfassend den Analyten sowie die Verbindung auf die Phospholipidschicht der Flüssigkristall-basierten Sensorvorrichtung,
• f) Untersuchung auf den Analyten oder die Aktivität des Analyten basierend auf Veränderungen der Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht, die von dem Bindungsereignis, dem Analyten oder der Analytenaktivität induziert werden. wobei keine Ausrichtungsveränderung anzeigt, dass die zu screenende Verbindung an den Analyten bindet und/oder die Aktivität des Analyten modifiziert, und wobei eine Veränderung der Ausrichtung anzeigt, dass die zu screenende Verbindung den Analyten nicht bindet und/oder die Aktivität des Analyten nicht modifiziert.
• (42) Verfahren, wie oben in einem von (37) bis (41) beschrieben, wobei der Analyt ausgewählt ist aus der Gruppe umfassend eine prokaryotische Zelle, wie beispielsweise eine Bakterienzelle, und ein prokaryotisches Mittel, wie beispielsweise ein Toxin, wie beispielsweise ein mikrobielles Toxin, wobei der Analyt in der Lage ist, die Ausrichtung der Flüssigkristalle an der Grenzfläche zwischen der Flüssigkristallschicht und der Phospholipidschicht zu verändern.
• (43) Verfahren, wie oben in einem von (37) bis (42) beschrieben, wobei der Analyt ausgewählt ist aus der Gruppe umfassend eine prokaryotische Zelle, wie beispielsweise eine Bakterienzelle, und ein prokaryotisches Mittel, wie beispielsweise ein Toxin, wie beispielsweise ein mikrobielles Toxin, wobei der Analyt in der Lage ist, die Ausrichtung der Flüssigkristalle an der Grenzfläche zwischen der Flüssigkristallschicht und der Phospholipidschicht zu verändern.
• (44) Verfahren, wie oben in einem von (37) bis (43) beschrieben, wobei das Screening auf Verbindungen, die einen Analyten binden und/oder die Aktivität eines Analyten modifizieren, indirekt durchgeführt wird, wie beispielsweise durch Detektieren der Anwesenheit oder Aktivität eines zweiten Analyten, wobei der zweite Analyt in der Lage ist, die Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht zu verändern.
• (45) Verfahren, wie oben in einem von (37) bis (44) beschrieben, wobei die Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht über einen Zeitraum hinweg beobachtet und die Ausrichtungsveränderung, wenn vorhanden, über einen Zeitraum hinweg gemessen wird.
• (46) Verfahren zur Verwendung der Flüssigkristall-basierten Sensorvorrichtung, wie oben in einem von (4) bis (31) beschrieben, zur Detektion der Anwesenheit von Lipase(n) oder der Aktivität von Lipase(n), wobei das Verfahren die Schritte umfasst:
• d) zur Verfügung stellen einer Flüssigkristall-basierten Sensorvorrichtung, wie oben in einem von (4) bis (31) beschrieben,
• e) Hinzufügen einer zu analysierenden Probe auf die Reaktionsschicht der Flüssigkristall-basierten Sensorvorrichtung,
• f) Untersuchung auf ein Reaktionsereignis basierend auf Veränderungen, die von der/den Lipase(n) oder der Lipasenaktivität(en) an der Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht induziert werden.
• (47) Verfahren, wie oben in (46) beschrieben, wobei das Reaktionsereignis die Bindung der Lipase(n) an die Reaktionsschicht und/oder die Störung/Disruption der Reaktionsschicht aufgrund der Lipase(n)aktivität ist.
• (48) Verfahren, wie oben in (47) beschrieben, wobei die Störung/Disruption der Reaktionsschicht aufgrund der Lipase(n)aktivität die Hydrolyse, Veresterung, Trans-Veresterung des (Phospho-)Lipids/der (Phospho-)Lipide, LPS und/oder der Fettsäure(n) der Reaktionsschicht umfasst.
• (49) Verfahren zur Verwendung der Flüssigkristall-basierten Sensorvorrichtung, wie oben in einem von (4) bis (31) beschrieben, zur Überwachung („monitoring”) der Beschädigung, Zersetzung und/oder des Absterbens von Zellen oder Organen überwacht werden, wobei das Verfahren die Schritte umfasst:
• d) zur Verfügung stellen einer Flüssigkristall-basierten Sensorvorrichtung, wie oben in einem von (4) bis (31) beschrieben,
• e) Hinzufügen einer zu analysierenden Probe auf die Reaktionsschicht der Flüssigkristall-basierten Sensorvorrichtung,
• f) Untersuchung auf ein Reaktionsereignis basierend auf Veränderungen der Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht, wobei eine Änderung der Ausrichtung anzeigt, dass die Zellen und/oder Organe der Probe beschädigt, zersetzt und/oder abgestorben sind.
• (50) Verfahren, wie oben in einem von (46) bis (49) beschrieben, wobei die Probe eine Zellkulturprobe ist, wie beispielsweise ein Zellkulturüberstand, ein Zellkulturmedium, eine Zellkulturlösung; oder die Probe eine Körperflüssigkeit ist, wie Blut, Speichel, Urin.
• (51) Verfahren, wie oben in einem von (46) bis (50) beschrieben, wobei die Ausrichtung der Flüssigkristalle in der Flüssigkristallschicht über einen Zeitraum hinweg beobachtet wird und die Ausrichtungsveränderung, wenn vorhanden, über einen Zeitraum hinweg gemessen wird.
• (52) Verfahren, wie oben in einem von (46) bis (51) beschrieben, umfassend die Wiederholung der Schritte b) und c) über einen Zeitraum hinweg.
• (53) Verfahren, wie oben in einem von (46) bis (52) beschrieben, wobei das Reaktionsereignis die Bindung von Lipase(n), wenn anwesend in der Probe aufgrund von Beschädigung, Zersetzung und/oder Absterben von Zellen oder Organen der Probe, an die Reaktionsschicht und/oder die Störung/Disruption der Reaktionsschicht aufgrund der Aktivität(en) der Lipase(n) ist.
• (54) Verfahren, wie oben in (53) beschrieben, wobei die Störung/Disruption der Reaktionsschicht aufgrund der Lipase(n)aktivität die Hydrolyse, Veresterung, Trans-Veresterung des (Phospho-)Lipids/der (Phospho-)Lipide, LPS und/oder der Fettsäure(n) der Reaktionsschicht umfasst.
• (55) Verfahren, wie oben in einem von (37) bis (54) beschrieben, wobei die Untersuchung durch visuelle Prüfung der Durchlässigkeit und/oder Reflektion von polarisiertem Licht oder nicht polarisiertem Licht oder durch Durchlässigkeits- und/oder Reflektionsmessungen mit polarisiertem Licht oder mit nicht polarisiertem Licht erfolgt, und die Untersuchung auf Veränderungen einer solchen Durchlässigkeit, oder die Untersuchung durch Messungen eines elektrischen Maßes, bevorzugt durch Messungen von Kapazität, Widerstand, oder Strom, sowie durch Untersuchung auf Veränderungen eines solchen elektrischen Maßes erfolgt.
• (56) Verfahren, wie oben in einem von (37) bis (55) beschrieben, weiterhin umfassend den Schritt: Überprüfung, dass eine beobachtete Durchlässigkeits- oder Reflektionsveränderung oder eine Veränderung der elektrischen Größe/Maß verursacht wird durch das Bindungsereignis oder die Anwesenheit eines Analyten oder die Aktivität eines Analyten bzw. verursacht wird durch das Reaktionsereignis, bevorzugt an einer Grenzfläche zwischen der Flüssigkristallschicht und der Phospholipidschicht/Reaktionsschicht, und nicht das Ergebnis eines gestörten („disrupted”) Bereichs auf dem Sensor ist, wo keine Flüssigkristalle und keine Phospholipidschicht/Reaktionsschicht vorhanden ist, indem eine Spannung mit Gleich- oder Wechselstrom an die erste und/oder zweite Elektrode angelegt wird und untersucht wird, ob dies zu einer Veränderung der detektierten Durchlässigkeit oder Reflektion von polarisiertem Licht oder der gemessenen elektrischen Größe/Maßes führt.
• (57) Verfahren, wie oben in einem von (37) bis (56) beschrieben, wobei die angelegte Spannung im Bereich von 0,1–25 V eines sinusförmigen/Dreiecks-/Rechtecksignals („sinusoidal/triangular/square signal”) ist, und die Frequenz im Bereich von 0 Hz bis 1 GHz, bevorzugt 100 Hz bis 10 MHz, weiter bevorzugt 1 kHz bis 1 MHz ist.
• (58) Verfahren, wie oben in einem von (37) bis (57) beschrieben, weiterhin umfassend den Schritt:
• b') Erhöhung der lokalen Konzentration des Analyten/der Lipase(n), oder Energiezuführen („energizing”) des Analyten/der Lipase(n) an der Grenzfläche zwischen der Phospholipidschicht/Reaktionsschicht und der Probe durch Elektrophorese, Dielektrophorese, Wechselstromelektroosmose (ACEO) oder Joulesche Erwärmung, und/oder
• b'') Erhöhung der Reaktionsgeschwindigkeit durch das Anlegen einer Gleich- oder Wechselspannung an die erste und/oder zweite Elektrode, wobei die angelegte Spannung im Bereich von 0,1–25 V eines sinusförmigen/Dreiecks-/Rechtecksignals liegt, und die Frequenz im Bereich von 0 Hz bis 1 GHz, bevorzugt 100 Hz bis 10 MHz, weiter bevorzugt 1 kHz bis 1 MHz liegt.
• (59) Verfahren, wie oben in einem von (37) bis (58) beschrieben, wobei das Bindungsereignis/Reaktionsereignis in Wasser oder einer wässrigen Lösung oder in der Gasphase stattfindet, und wobei der Analyt/die Lipase(n) in Wasser, einer wässrigen Lösung oder in der Gasphase vorhanden ist/sind, oder wobei der Analyt/die Lipase(n) an einen festen Träger, wie beispielsweise Polymerperlen, oder an einem der Substrate immobilisiert ist/sind.
• (60) Verwendung einer Flüssigkristall-basierten Sensorvorrichtung, wie oben in einem von (4) bis (31) beschrieben, zur Detektion der Anwesenheit von Lipase(n) oder der Aktivität von Lipase(n) in einer Probe; zur Überwachung von Beschädigung, Zersetzung und/oder des Absterbens von Zellen oder Organen in einer Probe; und/oder als Lipase-Screeningvorrichtung bei Toxizitäts- oder Zytotoxizitätsassays.
• (61) Die Verwendung, wie oben in (60) beschrieben, wobei die Probe eine Zellkulturprobe ist, wie beispielsweise ein Zellkulturüberstand, ein Zellkulturmedium, eine Zellkulturlösung; oder wobei die Probe eine Patientenprobe ist, wie beispielsweise eine Körperflüssigkeit, beispielsweise Blut, Speichel, Urin.

Please note that the present technology can also be configured as described below.

• (1) Liquid crystal-based sensor device for detecting the presence of an analyte, such as a prokaryotic cell, a bacterial cell and / or a prokaryotic agent, such as a toxin, such as a microbial toxin, the device comprising in the order listed:
• A first substrate,
• A liquid crystal layer on the first substrate,
• A phospholipid layer on the liquid crystal layer, the device comprising no recognition part or recognition molecule bound directly to the surface of the first substrate.
• (2) The sensor device as described in (1) above, wherein the phospholipid layer comprises at least one recognition part or recognition molecule such as a receptor, an enzyme or an antibody.
• (3) The sensor device as described in (1) or (2) above, wherein the phospholipid layer comprises cholesterol, preferably between 1 and 20% by weight, more preferably between about 10 and about 15% by weight of cholesterol.
• (4) The sensor device as described in any one of (1) to (3) above, wherein the at least one recognition part or recognition molecule in the phospholipid layer is a receptor such as ganglioside GM ₁ .
• (5) The sensor device as described in (7) above, wherein the phospholipid layer comprises between about 0.1% to about 10% by weight GM ₁ , preferably from about 0.5% to about 5% by weight GM ₁ .
• (6) Liquid crystal-based sensor device for detecting the presence of lipases or lipase activity, the device comprising in the order listed:
• A first substrate,
• A liquid crystal layer on the first substrate,
• A reaction layer on the liquid crystal layer, wherein the reaction layer is a lipid layer, an LPS layer and / or a fatty acid layer.
• (7) The sensor device as described in (7) above, wherein the device does not include a recognition part or a recognition molecule directly bound to the surface of the first substrate.
• (8) The sensor device as described in any one of (1) to (7) above, further comprising:
• At least one first electrode between the first substrate and the liquid crystal layer, and
• An insulating layer and / or an alignment layer, or an insulating and alignment layer, which coats the electrode.
• (9) The sensor device as described in any one of (1) to (8) above, further comprising:
• A second substrate disposed opposite to the first substrate and rendering the liquid crystal layer and phospholipid layer into intermediate layers, the liquid crystal layer and reaction layer into intermediate layers between the first and second substrates, and having a gap between the second substrate and the phospholipid layer the second substrate and the reaction layer, which serves as a reaction chamber for receiving a sample to be analyzed.
• (10) The sensor device as described in (9) above, wherein the second substrate further comprises:
• A second electrode on the second substrate, and
• Optionally, an insulation layer on the second electrode, the insulation layer facing the phospholipid layer / liquid crystal layer or reaction layer / liquid crystal layer located on the first substrate.
• (11) A sensor device as described above in any one of (1) to (10), wherein the first and / or the second substrate comprises a solid, preferably glass or plastic, such as cycloolefin polymer (COP), cycloolefin copolymer ( COC) and polydimethylsiloxane (PDMS).
• (12) The sensor device as described in any of (1) to (11) above, wherein the liquid crystal layer has a homeotropic alignment, homogeneous Has orientation or no predefined orientation.
• (13) The sensor device as described in any one of (1) to (12) above, wherein the liquid crystal layer comprises a liquid crystal selected from thermotropic liquid crystals having positive / negative or no dielectric anisotropy, two-frequency liquid crystals, discotic and / or lyotropic ones Liquid crystals and combinations thereof.
• (14) A sensor device as described in any one of (1) to (13) above, wherein 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. includes.
• (15) The sensor device as described in (14) above, wherein the liquid crystal layer is color-doped, preferably with dichroic dye (s), fluorescent dye (s) or doped with quantum dot (s).
• (16) The sensor device as described in any one of (1) to (15) above, wherein the phospholipid layer or the reaction layer comprises a single phospholipid compound or a mixture of two or more different phospholipid compounds.
• (17) The sensor device as described in (16) above, wherein the phospholipid compounds are selected from DOPC, (1,2-dioleoyl-sn-glycero-3-phosphocholine), DPPC (1,2-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-sn-glycero-3-phosphocholine (14: 1, PC), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (16: 1, PC), 1,2 -Ieicosenoyl-sn-glycero-3-phosphocholine (20: 1, PC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (22: 1, PC), 1,2-dinerononyl-sn-glycero-3 Phosphocholine (24: 1, PC) or combinations thereof.
• (18) The sensor device as described in any one of (6) to (17) above, wherein the reaction layer comprises a single lipopolysaccharide compound or a mixture of two or more different lipopolysaccharides.
• (19) The sensor device as described in (18) above, wherein the LPS compounds are selected from LPS of E. coli strains.
• (20) A sensor device as described in any one of (6) to (17) above, wherein the reaction layer comprises a single fatty acid compound or a mixture of two or more different fatty acids.
• (21) A sensor device as described in any one of (6) to (17) above, wherein the reaction layer is a layer comprising lipid, LPS and / or fatty acid compound (s).
• (22) The sensor device as described in any one of (8) to (21) above, wherein the first electrode and the second electrode, each independently, is a non-interdigital electrode, a planar electrode or an interdigital electrode (IDE).
• (23) A sensor device as described in any of (8) to (22) above, wherein the insulating layer on the first and / or second electrodes is made of polyimide.
• (24) The sensor device as described in any one of (8) to (23) above, wherein the alignment layer is composed of a silane such as octadecyldimethyl- (3-trimethoxy-silylpropyl) ammonium chloride (DMOAP) or a heat-curable type Polyimide having a defined pretilt angle of the liquid crystal in the liquid crystal layer or a self-assembling monolayer of self-assembling compounds such as thiols, dithiocarbamates, or an obliquely evaporated oxide layer such as SiO ₂ , which tends to precoip the liquid crystal in the liquid crystal layer depending on the evaporation angle of the oxide provides.
• (25) A sensor device as described in any of (8) to (24) above, wherein the first and / or the second electrode is transparent or non-transparent, preferably the indium-tin oxide (ITO) electrode (s). or fluorine-tin oxide (FTO) is / are constructed.
• (26) A sensor device as described in any one of (8) to (25) above, wherein the device further comprises crossed polarizers under and over the liquid crystal layer, preferably on the planes of the first and second substrates, respectively, which measure the change in liquid crystal orientation by measuring the transmission / reflection of polarized light, or wherein the device further comprises at least one polarizer over the liquid crystal layer, and wherein the substrate located below the liquid crystal layer is a reflective substrate.
• (27) A sensor device as described in any one of (1) to (26) above, wherein the device further comprises means for measuring an electrical measure, wherein the means comprises measuring the change in liquid crystal orientation by measuring changes in such electric current Dimension allows, for example, capacitance, resistance, or current.
• (28) A sensor device as described in any of (8) to (25) and (27) above, wherein the device further comprises only one polarizer, preferably on the plane of either the first or the second substrate, and wherein the corresponding other substrate without polarizer to Use in conjunction with a light source that provides polarized light is determined, such as a liquid crystal display device.
• (29) The sensor device as described in any of (1) to (28) above, further comprising a microfluidic device in fluid communication with the reaction chamber.
• (30) A sensor device as described in any one of (1) to (29) above, further comprising a filter unit for filtering any material from the sample which may be larger than the analyte to be assayed, such as a bacterial cell or a microbial toxin, so that the material does not penetrate into the reaction chamber.
• (31) A sensor device as described in any of (1) to (30) above, wherein the reaction chamber has a depth in the range of 800 nm to 100 μm, preferably 900 nm to 30 μm, and in the case of a round chamber, a width or a diameter in the range of 1 micron to 1 mm, preferably from 25 microns to 500 microns, and / or wherein the reaction chamber made of glass, plastic or has walls made of metal, such as gold walls.
• (32) A method of manufacturing a liquid crystal-based sensor device as described in any one of (1) to (31) above, the method comprising the steps of:
• d) providing a first substrate,
• e) applying a liquid crystal layer to the first substrate,
• f) applying a phospholipid layer to the liquid crystal layer, optionally comprising at least one recognition part / molecule, such as a receptor, and / or cholesterol, or applying a reaction layer to the liquid crystal layer.
• (33) The method as described in (32) above, further comprising the steps
• a ') applying at least a first electrode on the first substrate, and
• a '') applying an insulating layer and / or alignment layer, or an insulating and alignment layer, which coats the first electrode.
• (34) Method as described in (32) or (33) above, further comprising the additional step:
• e) providing a second substrate, and applying it to the phospholipid layer or the reaction layer such that the phospholipid layer and the reaction layer and the liquid crystal layer are located between the first and second substrates ("are sandwiched between") is a space between the second substrate and the phospholipid layer or the reaction layer, which serves as a reaction chamber for receiving a sample to be analyzed.
• (35) The method as described above in any of (32) to (34), wherein the second substrate has a second electrode thereon, and wherein step e) is performed so that the second electrode of the phospholipid layer / liquid crystal layer or the reaction layer / liquid crystal layer faces.
• (35) The method as described above in any one of (32) to (35), wherein the substrates, electrodes, insulating layer, alignment layer, liquid crystal layer, reaction layer and device are as defined above in any one of (1) to (31).
• (37) A method of using the liquid crystal-based sensor device described above in any one of (1) to (3), (8) to (17) and (22) to (31) for detecting the presence of an analyte or the like Activity of an analyte, the method comprising the steps of:
• d) providing a liquid crystal based sensor device as described in any of (1) to (3), (8) to (17) and (22) to (31) above,
• e) adding a sample to be analyzed to the phospholipid layer of the liquid crystal based sensor device,
• f) Examination for a binding event, an analyte to be detected or the activity of an analyte based on changes in the orientation of the liquid crystals in the liquid crystal layer induced by the binding event, the analyte or the analyte activity.
• (38) The method as described in (37) above, wherein the binding event is the binding of the analyte to the recognition part or the recognition molecule, such as a receptor, an enzyme or an antibody included in the phospholipid layer; or the binding of the analyte to the phospholipid layer.
• (39) The method as described in (37) or (38) above, wherein the activity of an analyte is the reaction of the analyte with the recognition part or recognition molecule, such as a receptor, enzyme or antibody included in the phospholipid layer; or the reaction of the analyte with the phospholipid layer.
• (40) The method as described in any one of (37) to (39) above, wherein the presence of an analyte is indirectly detected, such as by detecting the presence of a second analyte, wherein the second analyte is capable of controlling the orientation of the analyte Liquid crystals in the liquid crystal layer to change.
• (41) A method of using the liquid crystal-based sensor device as described in any one of (1) to (3), (8) to (17) and (22) to (31) for screening for compounds attached to a Bind analytes and / or the Modify the activity of an analyte, such as inhibitors of microbial toxins, the method comprising the steps of:
• a) providing a liquid crystal based sensor device as described in any of (1) to (3), (8) to (17) and (22) to (31) above,
• 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 at least one sample to the screening compound to the analyte,
• e) adding the sample comprising the analyte and the compound to the phospholipid layer of the liquid crystal based sensor device,
• f) Examination for the analyte or the activity of the analyte based on changes in the orientation of the liquid crystals in the liquid crystal layer induced by the binding event, the analyte or the analyte activity. wherein no orientation change indicates that the compound to be screened binds to the analyte and / or modifies the activity of the analyte, and wherein a change in orientation indicates that the compound to be screened does not bind the analyte and / or modifies the activity of the analyte.
• (42) The method as described in any one of (37) to (41) above, wherein the analyte is selected from the group comprising a prokaryotic cell such as a bacterial cell, and a prokaryotic agent such as a toxin such as a microbial toxin, wherein the analyte is capable of altering the alignment of the liquid crystals at the interface between the liquid crystal layer and the phospholipid layer.
• (43) The method as described in any one of (37) to (42) above, wherein the analyte is selected from the group comprising a prokaryotic cell such as a bacterial cell, and a prokaryotic agent such as a toxin such as a microbial toxin, wherein the analyte is capable of altering the alignment of the liquid crystals at the interface between the liquid crystal layer and the phospholipid layer.
• (44) The method as described in any one of (37) to (43) above, wherein the screening for compounds that bind an analyte and / or modify the activity of an analyte is performed indirectly, such as by detecting the presence or activity a second analyte, wherein the second analyte is capable of altering the alignment of the liquid crystals in the liquid crystal layer.
• (45) The method as described in any one of (37) to (44) above, wherein the alignment of liquid crystals in the liquid crystal layer is observed over a period of time, and the orientation change, if any, over a period of time is measured.
• (46) A method of using the liquid crystal-based sensor device as described in any of (4) to (31) above for detecting the presence of lipase (s) or the activity of lipase (s), the method comprising the steps :
• d) providing a liquid crystal based sensor device as described in any one of (4) to (31) above,
• e) adding a sample to be analyzed to the reaction layer of the liquid crystal-based sensor device,
• f) assay for a response event based on changes induced by the lipase (s) or lipase activity (s) in the alignment of the liquid crystals in the liquid crystal layer.
• (47) The method as described in (46) above, wherein the reaction event is the binding of the lipase (s) to the reaction layer and / or the disruption / disruption of the reaction layer due to the lipase (n) activity.
• (48) The method as described in (47) above, wherein the disruption / disruption of the reaction layer due to the lipase (s) activity, the hydrolysis, esterification, trans-esterification of the (phospho) lipid / (phospho) lipids, LPS and / or the fatty acid (s) of the reaction layer.
• (49) A method of using the liquid crystal-based sensor device as described in any one of (4) to (31) above to monitor the damage, decomposition and / or death of cells or organs, wherein the method comprises the steps of:
• d) providing a liquid crystal based sensor device as described in any one of (4) to (31) above,
• e) adding a sample to be analyzed to the reaction layer of the liquid crystal-based sensor device,
• f) Examination for a reaction event based on changes in the orientation of the liquid crystals in the liquid crystal layer, wherein a change in orientation indicates that the cells and / or organs of the sample are damaged, degraded and / or dead.
• (50) The method as described in any one of (46) to (49) above, wherein the sample is a cell culture sample, such as a cell culture supernatant, a cell culture medium, a cell culture solution; or the sample is a body fluid, such as blood, saliva, urine.
• (51) The method as described in any one of (46) to (50) above, wherein the alignment of the liquid crystals in the liquid crystal layer is observed over a period of time, and the orientation change, if any, over a period of time is measured.
• (52) The method as described in any of (46) to (51) above, comprising repeating steps b) and c) over a period of time.
• (53) The method as described in any one of (46) to (52) above, wherein the reaction event involves the binding of lipase (s) when present in the sample due to damage, decomposition and / or death of cells or organs of the sample to the reaction layer and / or the disruption / disruption of the reaction layer due to the activity (s) of the lipase (s).
• (54) The method as described in (53) above, wherein the disruption / disruption of the reaction layer due to the lipase (n) activity, the hydrolysis, esterification, trans-esterification of the (phospho) lipid / the (phospho) lipids, LPS and / or the fatty acid (s) of the reaction layer.
• (55) The method as described in any one of (37) to (54) above, wherein the inspection is by visual inspection of the transmittance and / or reflection of polarized light or unpolarized light or by transmission and / or reflection measurements with polarized light or with non-polarized light, and the examination for changes in such permeability, or the examination by measurements of an electrical measure, preferably by measurements of capacitance, resistance, or current, and by examining for changes in such an electrical measure.
• (56) The method as described in any one of (37) to (55) above, further comprising the step of verifying that an observed change in transmission or reflection or a change in electrical quantity / dimension is caused by the binding event or the presence of a Analyte or the activity of an analyte or is caused by the reaction event, preferably at an interface between the liquid crystal layer and the phospholipid / reaction layer, and not the result of a disrupted region on the sensor, where no liquid crystals and no phospholipid layer / Reaction layer is present by applying a voltage with direct or alternating current to the first and / or second electrode and it is examined whether this leads to a change in the detected transmission or reflection of polarized light or the measured electrical variable / measure.
• (57) The method as described above in any one of (37) to (56), wherein the applied voltage is in the range of 0.1-25 V of a sinusoidal / triangular / square signal , and the frequency is in the range of 0 Hz to 1 GHz, preferably 100 Hz to 10 MHz, more preferably 1 kHz to 1 MHz.
• (58) The method as described above in any one of (37) to (57), further comprising the step of:
• b ') Increasing the local concentration of analyte / lipase (s), or energizing the analyte / lipase (s) at the interface between the phospholipid layer / reaction layer and the sample by electrophoresis, dielectrophoresis, AC electroosmosis (ACEO ) or Joule warming, and / or
• b '') Increasing the reaction rate by applying a DC or AC voltage to the first and / or second electrode, wherein the applied voltage is in the range of 0.1-25 V of a sinusoidal / triangular / square wave signal, and the frequency in Range from 0 Hz to 1 GHz, preferably 100 Hz to 10 MHz, more preferably 1 kHz to 1 MHz.
• (59) The method as described in any one of (37) to (58) above, wherein the binding event / reaction event occurs in water or an aqueous solution or in the gas phase, and wherein the analyte / lipase (s) in water, a aqueous solution or in the gas phase, or wherein the analyte / lipase (s) is / are immobilized on a solid support such as polymer beads or on one of the substrates.
• (60) Use of a liquid crystal-based sensor device as described in any of (4) to (31) above for detecting the presence of lipase (s) or the activity of lipase (s) in a sample; for monitoring damage, degradation and / or death of cells or organs in a sample; and / or as a lipase screening device in toxicity or cytotoxicity assays.
• (61) The use as described in (60) above, wherein the sample is a cell culture sample, such as a cell culture supernatant, a cell culture medium, a cell culture solution; or wherein the sample is a patient sample, such as a body fluid, such as blood, saliva, urine.

The term "analyte" as used herein, particularly in the context of the analyte sensing device, refers to a molecule or cell whose presence (or absence) or activity is to be detected. Examples are prokaryotic agents, such as toxins, such as microbial toxins. An analyte, as used herein, may be also refer to a cell membrane alone or as part of a biological cell, such as a prokaryotic cell, e.g. B. a bacterium.

Examples of microbial toxins include cholera toxin (Vibrio cholerea), heat-sensitive enterotoxin (E. coli), pertussis toxin (Bordetella pertussis), anthrax toxin (Bacillus anthracis), tetanus toxin (Clostridium tetani), streptococcal or staphylococcal toxins.

The term "binding event", particularly in the context of the analyte sensing device, 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. Binding events include, for example, the binding of an analyte to the recognition member or molecule, such as a receptor, that is included in the phospholipid layer.

The term "reaction", particularly in the context of the analyte sensor device, refers to a change in a component (such as the analyte, the phospholipid layer, the second analyte) due to the binding of an analyte to an interaction partner, such as the recognition moiety or recognition molecule, such as receptor / antibody / enzyme, comprises in the phospholipid layer, or with the phospholipid layer, such change being, for example, (partial) hydrolysis of the phospholipid layer, or hydrolysis or chemical modification of the analyte or digestion / digestion of the second analyte by the analyte, etc.

The term "activity", particularly in the context of the analyte sensing device, refers to the ability of a component, such as the analyte, to bind, grow, undergo changes, consume / catalyze, or catalyze, for example, the analyte. B. participate in or inhibit a chemical reaction or growth. The term "activity" also refers to the extent to which a component such as an analyte binds, undergoes changes, consumes / consumes, catalyzes or z. B. participates in or inhibits a chemical reaction or growth.

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

The term "lipase" as used herein, particularly in the context of a lipase sensor device, refers to an enzyme that catalyzes the formation or hydrolysis of lipids. Lipases are hydrolytic enzymes that act on carboxyl ester compounds. Lipases are widely used in animals, plants, molds and bacteria. Their catalytic activities are not limited to hydrolysis but also include esterification, interesterification and transesterification processes. In some cases, the lipases are referred to as "analyte".

The term "reaction event", especially in the context of the lipase sensor device, refers to the molecular interactions of the lipases with the reaction layer of the LC sensor device as well as to the chemical reactions caused by the lipases.

Reaction events are, for example, the binding of the lipase (s) to the reaction layer and / or the disruption / disruption of the reaction layer due to the activity of the lipase (s). The disruption / disruption of the reaction layer due to the lipase (n) activity preferably comprises the hydrolysis, esterification and transesterification of the (phospho) lipid (s) (phospho) lipids, LPS and / or the fatty acid (s) of the reaction layer.

The term "lipid layer" as used herein, particularly in the context of the lipase sensor device, preferably refers to a monolayer of (phospho) lipids.

The term "LPS layer". as used herein, particularly in the context of the lipase sensor device, preferably refers to a monolayer of lipopolysaccharides.

The term "fatty acid layer" as used herein, particularly in the context of the lipase sensor device, preferably refers to a monolayer of fatty acids.

The term "recognition moiety" or "recognition molecule" as used herein refers to a molecule or moiety capable of recognition, ie, capable of specifically binding an analyte, such as a lipase. In accordance with the present disclosure, such recognition portion or recognition molecule is preferably not directly bound to the surface of the first substrate. When such a recognition member or molecule is used in an analyte sensor device according to the present disclosure, it will preferably be included in the phospholipid layer of the device. If such a recognition part or such a recognition molecule is used in a lipase sensor device according to the present disclosure, it will preferably be included in the reaction layer of the device.

It serves the purpose of specifically binding an analyte in the vicinity of the liquid crystal layer, whereupon the analyte induces a change in the orientation of the liquid crystal layer, which in turn can subsequently be detected.

Examples of recognition parts or recognition molecules, in particular in connection with the analyte sensor device, are antibodies, antigens, receptors, transmembrane proteins and enzymes. As an example of a receptor, ganglioside GM _{1 is} mentioned, which binds, for example, cholera toxin.

Examples of recognition parts or recognition molecules, in particular in connection with the lipase sensor device, are antibodies, antigens, receptors, transmembrane proteins and enzymes.

According to the present disclosure, the term "electrode" refers to an electrical line for applying a voltage. An electrode may be "interdigitated", meaning that it has a comb-like shape with two combs facing each other and the respective tines of the combs interlocking. Alternatively, an electrode may not be interdigital (non-intermeshing). An electrode may be transparent or not transparent. For example, a transparent electrode may be formed of indium tin oxide (ITO) or fluorine tin oxide (FTO). A non-transparent electrode may be reflective and may be made of, for example, silver (Ag) or aluminum (Al). The reflective electrodes are ideal when using the liquid crystal based sensor device in combination with a reflection microscope.

The term "insulating layer" as used herein is intended to refer to a layer on the electrode which prevents reaction of molecules from other layers, such as the liquid crystal layer, with the electrode. An insulation layer may be formed of polyimide, for example.

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) but has negative dielectric anisotropy when high AC voltage is applied. As an example of a Merck two-frequency LC called MLA3969, its dielectric anisotropy changes polarity at about 30 kHz. Therefore, the terms "low" and "high" in the context of the AC voltages refer exclusively to the relative positions of the respective voltages to each other. Typically, low can mean 0-100 kHz, and high can mean 10-100 MHz, but the actual values depend on the liquid crystal used.

The term "5CB" refers to 4-cyano-4'-pentyl_biphenyl, a one-component positive dielectric anisotropic liquid crystal commercially available from Aldrich. "MLC6608" refers to the trade name of Merck's Negative LC blend, which is commercially available.

The term "alignment layer" refers to a layer that can induce a specific orientation of a liquid crystal layer when the alignment layer is brought into contact with a liquid crystal layer. For example, an alignment layer may be formed from DMOAP (octadecyldimethyl (3-trimethoxysilylpropyl) ammonium chloride) which induces homeotropic alignment of a liquid crystal layer contacted therewith. Another example of an alignment layer is a polyimide layer.

The term "insulating layer and / or alignment layer" is intended to refer to a scenario in which either or both an insulating layer and a separate alignment layer are present, or where either an insulating layer or an alignment layer is present. In contrast, the term "an isolation and alignment layer" is intended to refer to a scenario where there is only a single layer that has both alignment and isolation properties. In contrast to the aforementioned scenario, the alignment and isolation functions are both integrated into one and the same layer, such an "isolation and alignment layer".

As an example of an alignment layer may be mentioned a polyimide layer, preferably a layer of heat / photocurable polyimide. Such a polyimide layer can provide a certain pretilt angle of the liquid crystal in the liquid crystal layer which is in contact with such a polyimide layer. The change that may be caused by such a polyimide alignment layer may be a change from a homeotropic alignment to a homogeneous alignment, preferably in the range of 90 ° to 0 °, or a change from a homogeneous alignment to a homeotropic alignment.

As used herein, "crossed polarizers" refers to a set of two Polarizers whose polarization axes are oriented perpendicular to each other.

The embodiment using crossed polarizers is one embodiment in which preferably one polarizer is above the liquid crystal layer and another polarizer below the liquid crystal layer. Their corresponding polarization axes are aligned perpendicular to each other. This embodiment makes it possible to measure changes in the liquid crystal orientation by measuring the transmittance 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 below the liquid crystal layer is a reflective substrate, and the embodiment allows the measurement of changes in the liquid crystal orientation by measuring changes in the reflection of polarized light, i. H. Light that is reflected by the reflective substrate.

In yet another embodiment of the liquid crystal based sensor device according to the present disclosure, it comprises only one polarizer, which is preferably over the liquid crystal layer, and the substrate under the liquid crystal layer is a transparent substrate, which can then be used in conjunction with a light source. which provides polarized light.

The term "reaction chamber" refers to the area in which a binding event / reaction event occurs or in which the analyte / lipase (s) or activity of the analyte (s) to be detected / the lipase (s) to be detected is present ,

Typically, in embodiments of the liquid crystal sensor device according to the present disclosure, the reaction chamber is formed by the space above the phospholipid layer / reaction layer and the liquid crystal layer. This area is either open at the top and bordered by suitable margins on the sides to form a closed area where analyte (s) / lipase (s) can be taken up. In other embodiments, this region is formed by the gap between the second substrate and the liquid crystal layer and bordered by edges on the side. This later embodiment is a "closed" embodiment. In the open embodiment, the analyte (s) / lipase (s) may simply be applied to the phospholipid layer / liquid crystal layer (or the reaction layer / liquid crystal layer); in the closed embodiment, the analyte (s) / lipase (s) may be applied to the phospholipid layer / liquid crystal layer (or reaction layer / liquid crystal layer) via dedicated dedicated openings, or may be applied to the phospholipid layer / liquid crystal layer (FIG. or the reaction layer / liquid crystal layer) are applied in the opened state; Thereafter, the device is closed by the lowering of the second substrate from above; or, alternatively, the analyte (s) / lipase (s) may be introduced into the reaction chamber by capillary filling, vacuum filling thereof or by dropping into closed state; In yet another embodiment, the analyte (s) / lipase (s) may also be introduced into the reaction chamber via a suitable pump.

As used herein, the term "polarized light transmission measurement" refers to a transmission measurement performed with a polarized light detector, such as a polarized microscope. The device is usually placed between the crossed polarizers and the light transmitted through them is measured.

The term "electrophoresis" refers to the movement of charged particles / molecules in an electric field. The electric field may be spatially uniform ("uniform") or non-uniform.

As used herein, the term "dielectrophoresis" refers to an effect that induces an attractive or repulsive force on polarizable, uncharged substances through a nonuniform electric field. In this way, this effect can be used to attract / repel bio molecules to an electrode by applying AC voltage to an interdigital electrode (IDE).

The term "AC electroosmosis", "AC electroosmosis" or "ACEO" refers to an effect that induces Joule heating, resulting in a flux movement of free charges in a non-uniform electric field. In this way, any flowing bio-molecules can be mixed in their solution, which shortens the time it takes for free-flowing bio-molecules to impinge on a liquid crystal layer to form a molecular layer (such as a lipid layer).

With respect to the analyte sensor devices according to the present disclosure:
The phospholipid bilayers of cell membranes are normally in a liquid state. They can also be described as lyotropic liquid crystal state or phase. A lyotropic liquid crystal is a material which forms a liquid crystal phase upon addition of a solvent. The term is most commonly used to describe materials consisting of hydrophilic head amphiphilic molecules coupled to a hydrophobic group. Phospholipids (see 1A ) are an example of such amphiphilic compounds which form a liquid crystal phase in the presence of a solvent, in which case the solvent is an aqueous solution. The fluidity of the natural cell membranes is essential for the functioning of membrane-associated systems. For this purpose, cholesterol (see 1B ) is the essential structural component of a mammalian cell membrane and it is necessary to establish the correct membrane permeability and fluidity over the range of physiological temperatures. Cholesterol interacts with the lipid membranes through the interactions between 1) the hydroxyl group of the cholesterol with the polar head group of the phospholipid, 2) hydrocarbon chains, and the cyclic hydrocarbon (steroid) part of the molecule with the nonpolar fatty acid chains of the phospholipid. Through these interactions, the cholesterol enhances the membrane pack. In addition, cholesterol also plays a significant role in intracellular transport, cell signaling events, etc. The fluidity of the phospholipid bilayers of cell membranes enables the movement and spatial reorganization of the membranes, which is essential for many biological processes. The studies presented herein have demonstrated that one can form a phospholipid bilayer such as fluid assemblies with thermotopic liquid crystals (such as the liquid crystals used in display applications) along with phospholipids. A typical structure for such a thermotropic liquid crystal 5CB with polar head group and hydrophobic tail portion is in FIG 1C shown. A general explanatory scheme for phospholipid LC bilayer formation is in 2 shown. The formation of such a bilayer is well documented in the literature. One starts with a quantity of liquid crystal which is homeotropically aligned from one side to a solid substrate which has been pretreated with a special coating material. On the other hand, the upper side of the LC amount in contact with air or water has no homeotropic orientation, and the LC molecules remain substantially parallel to the underlying substrate. When the LC air / water interface is contacted with a lipid vesicle solution (liposomes), a lipid LC bilayer forms almost immediately, converting the parallel alignment of the LC molecules to a homeotropic alignment. This alignment change of liquid crystals can be observed with a polarizing microscope very well by the change in light transmittance.

By very much modeling such a lipid LC layer structure of a natural environment, meaning that the natural cell membranes are readjusted while using a themotropic LC as the signal processor, it is now possible to detect bio events associated with cell membranes. One very important group of events involving the integration of cell membranes is the attack of microbial toxins within the cells. For this purpose, some of the most abundant and structurally similar protein toxins and their bacterial causes are: cholera toxin (V. cholerea), heat-sensitive enterotoxin (E. coli), pertussis toxin (Bordetella pertussis), anthrax toxin (Bacillus anthracis), tetanus toxin ( Clostridium tetani), several streptococcal or staphylococcal toxins and so on. The mode of operation seems to be very similar for these toxins. They usually have discrete subunits or domains: 1) a subunit or domain "A" having a specific enzymatic function and 2) a binding domain, subunit or oligomer "B" that interacts with the cell membrane receptor.

The inventors have surprisingly found that by the generation of a lipid LC layer capable of mimicking a natural cell membrane, an apparatus for efficiently attaching / binding the subunit of the toxin to the LC lipid layer can be provided. The existence of the toxin or the toxic bacteria can thus be detected by simply observing the occurring optical changes (change in light transmittance). This detection is only possible when the toxin is active and this offers a further possibility: With this method / device, the effectiveness of a drug which is intended to inactivate or modify the toxin or the corresponding bacteria can also be detected via this detector technology / detector device become. The present disclosure thus provides a method and apparatus useful: (1) for the detection of toxins and bacteria, (2) for the detection of the activity of toxins and bacteria, and (3) for the screening of drugs. Design and development.

The analyte sensor device according to the present disclosure has the following advantages: (1) It is a very simple device. (2) The detection mechanism works without marking. (3) No complex instrumentation is needed for detection. (4) It can be integrated well together with a microfluidic device. (5) Very fast detection is possible. (6) It has a very good (i.e., sensitive) detection limit. For example, levels as low as 30 pg / mL CTB can be detected (lower concentrations not tested).

With respect to the lipase sensor devices according to the present disclosure: Lipases are hydrolytic enzymes that act on carboxylic ester compounds. These enzymes are widely used in animals, plants, molds and bacteria. The catalytic activities are not limited to hydrolysis; they also catalyze the esterification, interesterification and transesterification processes. Therefore, they are widely used in the food, pharmaceutical, chemical and detergent industries. Lipases are also very important in diagnosis settings. Under pathological conditions, for example, they are found in environments where they do not normally occur, thus being useful markers in cell / organ destructive processes. A well-known example is the high levels of pancreatic lipases that occur in the blood as a result of pancreatitis. Even when a mammalian cell dies, lipases are released.

Phospholipids are basic building blocks of a cell membrane. These lipids are present in the membranes in various structural details, but also share common structural features. These features are the polar headgroup and the non-polar acyl chains. Lipases are responsible for the extracellular degradation and intracellular metabolism of lipids. The different lipases have different selectivity with respect to different lipids depending on the particular chain length, stereoisomers, interfacial properties of the water-lipid interface, etc. Also, the enzyme activity of the lipases can be enhanced or stopped by the use of specific cofactors. In the present disclosure, the inventors tested various lipases from different cells; P19 cells, hepG2 cells and pancreatic cells and the commercially available lipases PLA1, PLA2, PLC and PLD.

The inventors of the present invention have surprisingly found that by providing a sensor device having a lipid LC layer, an LPS LC layer or a fatty acid LC layer, the lipases released from dead cells can even be detected directly testing / analyzing the cell medium or the cell sample. The device according to the present disclosure is based on monitoring the lipase-mediated "disruption" of lipids or LPS (lipopolysaccharides) or fatty acids, including, but not limited to, hydrolysis, esterification, trans-esterification, etc. on the lipid LC layer / LPS. LC layer / fatty acid LC layer of the sensor device. The sensor device according to the present disclosure can be used to monitor not only the activity of the lipases, but also to monitor processes that cause the decomposition, destruction and / or death of cells and organs.

Thus, the present disclosure provides a liquid crystal-based sensor device, a liquid crystal-based lipase assay, a liquid crystal-based toxicity / cytotoxicity test, and a liquid crystal-based analysis tool related to the detection / monitoring of cell / organ destruction / decomposition processes and toxicity / Cytotoxicity studies available.

The present disclosure provides an assay / sensor device that can detect lipases via their activities. The device is based on monitoring lipase activity on a lipid LC layer, such as lipase-mediated lipid hydrolysis or lipase-on lipids, or lipase activity on lipopolysaccharides (LPS) or lipase activity on fatty acids, and can not only Monitoring of lipase activity, but also to monitor processes that cause the decomposition / death of cells, eg. A lipase screening device during toxicity / cytotoxicity assays. The device is also useful in monitoring the presence of lipases, particularly under pathological conditions, when lipases are found in environments in which they are not normally present.

The present disclosure thus provides a method and apparatus useful: (1) for monitoring cell death / organ destruction processes via the alignment change of LC due to lipase release from the dead cells; (2) a toxicity / cytotoxicity assay based on the same working principle; and (3) a lipase assay for the same purpose that can be used in the detergent / detergents industry.

The lipase sensor device according to the present disclosure has the following advantages: (1) It is a very simple device. (2) The detection mechanism works without marking. (3) No complex instrumentation is needed for detection. (4) It can be integrated well together with a microfluidic device. (5) Very fast detection is possible. (6) It is a sensor device in which the lipases released from the dead cells are measured directly by the orientation change of the liquid crystal layer. (7) It allows the direct use of the cell medium for observation.

EXAMPLES

EXAMPLE 1

Used material:

• Lipid vesicles: DOPC-lipid vesicles (1,2-dioleoyl-sn-glycero-3-phosphocholine) or vesicles made from a mixture of DOPC, Gm1 with or without cholesterol.
• Gold Grid: 150 mesh (pitch 165 μm) from Plano. The gold lattice can vary in size, but larger lattices are prone to water disruption and smaller ones take longer to form a lipid layer (LL).
• Note: Instead of gold mesh, other reaction chambers such as glass or plastic wells can be used.
• PBS buffer solution: (Phosphate-buffered saline with a pH of 7.4). The solution is not limited to the PBS solution, as it can be anything, such as water with different pH, TBS (Tris-buffered saline), etc.
• Liquid crystals: 5CB from Aldrich. Liquid crystals are also not limited to 5CB, but may be any molecule capable of showing a liquid crystal phase.
• DMOAP (octadecyldimethyl (3-trimethoxysilylpropyl) ammonium chloride): induces homeotropic alignment of the liquid crystals.
• Cholera toxin B (CTB).

Production of liposomes (unilamellar vesicles)

Liposomes (lipid vesicles) are formed when thin lipid films become hydrated and stacks of liquid crystal bi-layers become fluid and swell. The hydrated lipid films dissolve during agitation / stirring and self-assemble to form large, multilamellar vesicles (LMV) that prevent interaction of the water with the hydrocarbon core of the bilayer at the edges. When these particles have formed, reducing particle size requires energy input in the form of sonic energy (sonication) or mechanical energy (extrusion). The general elements of the procedure include preparation of the lipid for hydration, hydration with agitation, and sizing for a homogeneous vesicle distribution.

In preparing liposomes with a mixed lipid composition, the lipids must first be dissolved and mixed in an organic solvent to ensure homogeneous lipid mixing. For larger volumes, the organic solvent should be removed by rotary evaporation, resulting in 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 having a certain pore size to obtain particles of about the pore size of the filter used. Prior to extrusion through the final pore size, LMV suspensions are disrupted, either by repeated cycles of freezing and thawing, or by pre-filtering the suspension through larger pore sizes (typically 0.2 μm-1.0 μm). This method helps prevent fouling of the membrane and improves the homegenicity of the size distribution in the final suspension.

In a typical procedure, a stock solution of 10 mM DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) in chloroform was prepared. From this solution, various molar concentrations of DOPC (0.01 mM, 0.1 mM, 1 mM) were prepared by dissolving in choloform. The chloroform was then evaporated with nitrogen gas and the solutions dried for 1 hour in a Schlenk apparatus (vacuum line). They were then rehydrated with 5 ml of 0.1 mM PBS (pH 7.0). Small unilamellar vesicles (SUV) were prepared by 3-4 cycles of freezing and heating the solution alternately with a water bath and liquid nitrogen. Small aliquots, 500 μl each, were prepared and stored in the freezer at -20 ° C. If necessary, the solutions were thawed and pressed 21 times with a hand-held extruder through a polycarbonate membrane with pores of 100 nm to produce small unilamellar lipid vesicles. These vesicles were used for lipid layer formation.

Preparation of liposomes from a mixture of phospholipid + GM1 + cholesterol

A stock solution of 10 mM DOPC in chloroform, 1 mg GM1 in 65 μl chloroform: methanol (6: 1) was prepared. Of these solutions, specific concentrations of DOPC + GM1 + cholesterol mixture (90% DOPC [0.01 mM], 0.5% GM1 [0.01 mM], and 9.5% cholesterol [0.01 mM]) or DOPC + GM1 (99.5% DOPC [0.01 mM], 0.5% GM1 [0.01 mM]) by dissolution in chloroform.

The chloroform was then evaporated with nitrogen gas and the solutions dried for 1 hour in a Schlenk apparatus (Schlenk line). They were then rehydrated with 5 ml PBS (pH 7.0). SUVs were prepared by 3-4 cycles of freezing and heating the solution alternately with liquid nitrogen and a water bath. Small aliquots, 500 μl each, were prepared and stored in the freezer at -20 ° C. If necessary, the solutions were thawed and replaced with a hand-held extruder 21 times Polycarbonate membrane with pores of 100 nm pressed to produce small unilamellar lipid vesicles. These vesicles were used for lipid layer formation.

Substrate Preparation: To coat the substrates for the homeotropic alignment of the LC, a DMOAP solution was prepared by mixing 250 ml of octadecyldimethyl (3-trimethoxysilylpropyl) ammonium chloride (60% in methanol, AB 111261) and 150 ml of deionized water (Millipore). produced. Into this solution, the glass substrates were immersed and left for about 5 minutes. After removing the substrates from the solution, remaining drops on the substrate were removed with an air gun. The substrates were then dried in a vacuum oven at 100 ° C for 15 min. Gold bars were then applied to the DMOAP-coated substrate and 2 μl of LC applied to each gold grid. Excess LC was removed by capillary action with a microspray tip. After the gold grid was impregnated with LC, the substrate was placed in an oven at 50 ° C for 10 min. The oven temperature was maintained above the nematic to isotropic temperature of the LC to realize a uniformly aligned LC. The slides with the gold bars were then viewed under the microscope and any gold grid with incorrect alignment of the 5CB was sorted out.

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

Results and discussion

From the molecular structure of GM ₁ (see 1D ) it can be seen that the non-polar hydrocarbon chains of GM1 tend to interact with the nonpolar fatty acid chains of the phospholipid and thus become embedded in the lipid layer. Lipid vesicles (liposomes) with different GM ₁ concentrations were prepared and lipid LC layer formation was observed. For this purpose, 0.5%, 2.5% and 5% (wt%) GM ₁ in 0.01 mM DOPC in PBS buffer were used and the lipid vesicles prepared as described above.

How out 3 As can be seen, all lipid vesicles with different amounts of GM ₁ receptors resulted in lipid LC layer formation. The alignment change in the LC layer is visible through the change in light transmission.

Detection of cholera toxin B (CTB): CTB was applied to lipid LC layers with different amounts of GM ₁ , and it was observed whether a light transmittance change due to the orientation change of the LC layer occurred. A change in the orientation of the LC layer can be observed. As an example, the transmittance change images in the system with 0.5% GM ₁ embedded in the lipid layer are shown in FIG 4 ,

In a natural environment, cholesterol is embedded in the phospholipid bilayer in a healthy mammalian cell membrane. Based on this knowledge, different lipid vesicles were prepared with 0.5% GM ₁ and different concentrations (10, 15 and 20% by weight) of cholesterol and then used to form lipid LC layers with these lipid vesicles on the LC layer , How out 5 As can be seen, lipid LC layers form when 10 and 15% cholesterol are embedded in the lipid vesicles.

Addition of CTB to the lipid LC layers with embedded GM ₁ and cholesterol resulted in very significant changes in light transmission, see 6 ,

Effect of Cholesterol: In order to understand the role of cholesterol in our detection, parallel assays were performed in which the rate of CTB detection was determined in the presence and absence of cholesterol. It can be seen from the pictures (see 6 ) easily deduce that the same amount of CTB can be detected in less than 15 minutes when using a sensor system with cholesterol, while without cholesterol the same effect occurs after about 12 hours. In addition, the show Permeability-time curves in 6A in that the change in LC alignment started within a few minutes in the layers of embedded cholesterol, while no change became apparent after such a short time without the use of cholesterol.

During the tests, it became apparent that CTB loses its activity over time, and in this case, no change in light transmission was observed. However, once a fresh CTB batch is used, the translucency change (due to the LC orientation change) due to the CTB-GM ₁ interaction is detectable again.

EXAMPLE 2: A polarizer / background-illumination-free, color-doped LC-based sensor

This example refers to an LC-based sensor in which dichroic dyes were incorporated into the LC mixture so that no polarizers and backlight are required (polarizer-free and backlight-free, color-doped LC-based sensor).

Used material:

• Target Additive: 1 mM DOPC Lipid (1,2-dioleoyl-sn-glycero-3-phosphocholine): The target molecule can also be mixed with additional dopants / additives such as cholesterol, enzymes, etc., as long as it remains capable of to change the LC alignment at the solution LC interface.
• Gold Grid: 300 mesh (pitch 75 μm) from Plano. The gold lattice can vary in size, but larger lattices are prone to water disruption and smaller ones take longer to form a lipid layer (LL). We can also use Glaswells, an example of glass-filled wells filled with color-doped LC in 9 given is.
• PBS buffer solution: [phosphate buffered saline with a pH of 7.4]. The solution is not limited to the PBS solution, as it can be anything, such as water with different pH, 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 interdigital electrode substrates were purchased from EHC, Japan.
• DMOAP (Octadecyldimethyl (3-trimethoxysilylpropyl) ammonium chloride): Induces homeotropic alignment of the liquid crystals.

Sample preparation:

substrate:

• 1. Treat the substrate with ozone plasma (0.1 mbar O ₂ , 100 W, 1 min) to initiate a reaction with the silane.
• 2. Immerse the substrate in a silane solution for more than 5 minutes.
• 3. Blow off the solution on the substrates with an air gun and dry it in a vacuum oven at 100 ° C for 15 min. Then remove the substrates and allow to cool to room temperature.
• 4. Place the gold grid on the substrates where IDEs are located.
• 5. Apply 2 μl of 5CB to the gold mesh and remove excess LC by capillary filling in a microsyringe tip.
• 6. Place the substrate in an oven at 50 ° C for 10 min. The oven temperature should be kept above the nematic to isotropic temperature of the LC layer to realize a uniform, homeotropically aligned LC layer.

Lipid Solution: Small unilamellar lipid vesicles were prepared by filtering 500 μL of lipid solution (1 mM in PBS) through a filter with pores of 50 nm using a hand-held extruder.

Measurement:

• 1. For measurement with polarizers, calibrate the cross-polarized microscopic nature. 100% transmittance was set when no sample was present and the polarizer and analyzer were parallel to each other. 0% transmittance was set when there was no light source. For a measurement without polarizers, the calibration was set to 100% transmittance when no polarizer and no sample were present.
• 2. Connect the electrical contacts to the sample and place it on the microscope stage.
• 3. Start with taking pictures and measuring the intensity.
• 4. Apply 5 μl lipid solution.
• 5. When a stable lipid layer has formed, apply different AC voltages with a square signal. (The signal may be a sinusoidal / triangular wave)

Results and discussion

Before applying the lipid solution, the LC layer between the cross polarizers appears gray and colored without the polarizers. When applying the lipid solution, the LC layer becomes darker (with polarizers) / lighter (without polarizers). This is due to the induced homeotropic alignment at the solution LC interface due to lipid LC layer formation. Once the transmission change ceases (when the formation of the lipid LC layer is complete), a voltage was applied and the permeability versus time measurements continued.

Before the measurements, the liquid crystal (5CB) was doped with 3% by weight dichroic dye. This means that the sensor component of the system in this example is the dichroic dye-doped LC. 7 and 8th show the experiments carried out with D2 (red azo dye from Merck) and B3 (black-3 dichroic dye mixture from Mitsubishi Chemical). Since the polarizers were not used for these measurements, the permeability during formation of the lipid layer does not decrease. Instead, due to the homeotropic reorientation of surface LC and dye, permeability increases because the dichroic dye absorbs less when its longitudinal axis is parallel to the incident light, ie, becomes more transparent. The initial reduction in permeability is due to the application of the lipid solution and the permeability increases in the course of lipid layer formation. After the rise is saturated, 2, 4, 6 and 8 V (1 kHz) were applied and the sudden reduction in transmittance was recorded. This proves that the color-doped LCs were not interrupted and were still active. Using a higher frequency (eg, MHz) or an improved passivation / isolation layer (eg, thicker PI layer, SU8, etc.) allows higher voltage applications for better contrast.

10 shows photos of the color-doped gold lattice before adding the PBS solution (buffer solution), and after adding the PBS solution with / without lipid (1 mM DOPC). The differences between these gratings with and without DOPC could also be observed without a microscope and without backlighting. Further improvement of the discrimination can be achieved by optimizing the color type / concentration, the LC type and the size and type of the sensor chamber.

It is also possible to use a fluorescent dye or fluorescently labeled LC to obtain a polarizer-free LC sensor.

We disclose here the use of dichroic dyes for an LC-based sensor that does not require polarizers, microscopes or backlighting.

EXAMPLE 3: Acceleration of LC sensor response time by application of an electric field

This example is related to an LC-based sensor as described in Example 2, where the applied voltage is an AC 20V square-wave voltage.

Test conditions:

• LC: 5CB (no dye)
• - Gold Grid: 300 mesh
• Solution: 0.5 mM DOPC. 50% diluted PLA1 (phospholipase A1).
• Substrate:
• IDE (interdigital electrode), spin coated through 100 nm SU8 passivation layer, followed by DMOAP surface treatment.
• • Procedure for the preparation: Annealing (125 ° C, 30 min.) → O ₂ plasma → SU8 (10/25%) coating → O ₂ plasma → DMOAP → annealing (50 ° C, 15 min.)
• - Applied voltage: AC 20V square wave (other waveforms such as triangular / sinusoidal are also possible)

To compare the lipid layer formation and the enzyme reaction times, a method was used with the in 11 used definitions shown.

12 shows how lipid layer formation and enzyme reaction times change at different frequencies. 1 Hz data stands for 0 V (reference) data. The deviation from the data points is still relatively large for the given number of samples, but it can be seen that there is an improvement (reduction) in the response times. The reason for the improvement appears to be either in the dielectrophoresis of the biomolecules, the Joule heating of the 5CG LC, or a combination of both.

EXAMPLE 4

The inventors made liquid crystal lipid layers, as generally already in the earlier patent application EP 11 005 033.3 and described in the above examples. The liquid crystals were homeotropically (vertically) aligned on top of a lower substrate by means of an alignment layer which had been previously deposited on the lower substrate. The alignment layer may be silane, polyimide, SiO ₂ , etc. For the specific examples presented here, a Silane layer (DMOAP, N, N-dimethyl-N-octadecyl-3-aminopropyltrimethoxysilyl chloride) used. Then, lipid vesicles were applied from above in a PBS solution. The lipids also helped the LC molecules to reach a homeotropic orientation from above. The change, namely the formation of LC lipid layers, was observed after a few seconds by the change in light transmission.

Used material:

• Lipid vesicles: Vesicles of DOPC lipid (1,2-dioleoyl-sn-glycero-3-phosphocholine) or lipopolysaccharides (LPS).
• Gold Grid: 150 mesh (pitch 165 μm) from Plano. The gold lattice can vary in size, but larger lattices are prone to water disruption and smaller ones take longer to form a lipid layer (LL).
• Note: Instead of gold mesh, other reaction chambers such as glass or plastic wells can be used.
• PBS buffer solution: (Phosphate-buffered saline with a pH of 7.4). The solution is not limited to the PBS solution, as it can be anything, such as water with different pH, TBS (Tris-buffered saline), etc.
• Liquid crystals: 5CB from Aldrich. Liquid crystals are also not limited to 5CB, but may be any molecule capable of having a liquid crystal phase.
• DMOAP (octadecyldimethyl (3-trimethoxysilylpropyl) ammonium chloride): induces homeotropic alignment of the liquid crystals.

Preparation of liposomes (unilamellar vesicles):

Liposomes (lipid vesicles) are formed when thin lipid films become hydrated and stacks of liquid crystal bilayers become fluid and swell. The hydrated lipid thin films dissolve during agitation and self-assemble into large, multilamellar vesicles (LMV) that prevent interaction of the water with the hydrocarbon core of the bilayer at the edges. When these particles have formed, energy input in the form of sonic energy (sonication) or mechanical energy (extrusion) is required for the particle size reduction. The general elements of the process include preparation of the lipid for hydration, hydration with agitation, and sizing for a homogeneous vesicle distribution.

In preparing liposomes with a mixed lipid composition, the lipids must first be dissolved and mixed in an organic solvent to ensure homogeneous lipid mixing. For larger volumes, the organic solvent should be removed by rotary evaporation, resulting in 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 having a certain pore size to obtain particles of about the pore size of the filter used. Prior to extrusion through the final pore size, LMV suspensions are disrupted, either by repeated cycles of freezing and thawing, or by pre-filtering the suspension through larger pore sizes (typically 0.2 μm-1.0 μm). This method helps prevent fouling of the membrane and improves the homegenicity of the size distribution in the final suspension.

In a typical procedure, a stock solution of 10 mM DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) in chloroform was prepared. From this solution, various molar concentrations of DOPC (0.01 mM, 0.1 mM, 1 mM) were prepared by dissolving in choloform. The chloroform was then evaporated with nitrogen gas and the solutions dried for 1 hour in a Schlenk line (vacuum line). They were then rehydrated with 5 ml of 0.1 mM PBS (pH 7.0). Small unilamellar vesicles (SUV) were prepared by 3-4 cycles of freezing and heating the solution alternately with a water bath and liquid nitrogen. Small aliquots, 500 μl each, were prepared and stored in the freezer at -20 ° C. If necessary, the solutions were thawed and pressed 21 times with a hand-held extruder through a polycarbonate membrane with pores of 100 nm to produce small unilamellar lipid vesicles. These vesicles were used for lipid layer formation.

Substrate Preparation:

To coat the substrates for the homeotropic alignment of the LC, a DMOAP solution was prepared by mixing 250 ml of octadecyldimethyl (3-trimethoxysilylpropyl) ammonium chloride (60% in methanol, AB 111261) and 150 ml of deionized water (Millipore). Into this solution, the glass substrates were immersed and left for about 5 minutes. After removing the substrates from the solution, remaining drops on the substrate were removed with an air gun. The substrates were then dried in a vacuum oven at 100 ° C for 15 min. Gold bars were then applied to the DMOAP-coated substrate and 2 μl of LC applied to each gold grid. Excess LC was removed by capillary action with a microspray tip. After the gold grid was impregnated with LC, the substrate was placed in an oven at 50 ° C for 10 min. The oven temperature was above the nematic to isotropic temperature of the LC layer held to realize a uniformly aligned LC. The slides with the gold bars were then viewed under the microscope and any gold grid with incorrect alignment of the 5CB was sorted out.

Production of LC-LPS coatings:

In another experiment, lipopolysaccharides (LPS) were used instead of the lipid vesicles and liquid crystal LPS layers were prepared. LPS layers aligned themselves like the DOPC on top of the liquid crystals, whereby the LC-LPS layer formation could be followed again by the change of the light transmission. For a typical attempt to form an LC-LPS layer, a solution of 1 mg / mL LPS in water was prepared and from this solution added about 3-5 μL to LC (5CB) resulting in a homeotropic alignment of the 5CB in about 1 Min. Led.

Measurement:

The cross-polarized microsphere brightness was calibrated as follows: 100% transmittance was adjusted when no sample was present and the polarizer and analyzer were parallel to each other. 0% transmittance was set when there was no light source. For the measurement, the polarizer and the analyzer were placed perpendicular to each other, without changing the light intensity. 5 μl of lipid solution or 3-5 μl of LPS was applied to the gold grid at partially aligned 5CB and the change in orientation was then observed and recorded. In the case of lipid, formation on 5CB was completed in a few seconds. In the case of LPS, formation was completed on 5CB in about 1 min. Upon completion of lipid or LPS layering on the LC layer (indicated by a darker image), the gold mesh was washed with PBS to remove excess vesicles or LPS. To this lipid or LPS layer were added lipase samples or cell samples such as cell culture supernatant (treated or untreated to cause cell death / damage as described below) and the change in LC alignment observed and recorded.

EXAMPLE 5

• A) Einfrieren/Auftauen
• B) Na-Azid-Behandlung
• C) Paracetamol-Behandlung.

Sowohl Na-Azid als auch Paracetamol sind als für Zellen toxisch bekannt.The inventors made various kinds of experiments to show that the liquid crystal-based sensor device functions / behaves as claimed. These experiments were designed to kill the cells to release the lipases. The methods chosen for cell killing and for indicating cytotoxicity were

• A) freeze / thaw
• B) Na-azide treatment
• C) paracetamol treatment.

Both Na-azide and paracetamol are known to be toxic to cells.

The inventors used three different types of cells: P-19 cells, HepG2 cells and pancreatic cells. Further details are given below.

EXAMPLE 5A) Freezing / Thawing (Lysis of Cell Membrane)

The following example uses HepG2 cells cultured according to standard cell culture techniques.

• 1. Nach der Trypsinierung werden 5 × 200 μL von Zellproben (300.000 Zellen/mL) gesammelt und in separaten Fläschchen gelagert.
• 2. Diese Proben wurden in flüssigem Stickstoff eingefroren, dann in einem Wasserbad (37°C) aufgetaut, erneut eingefroren, dann wieder im Wasserbad aufgetaut, und zwar 2, 4, 6, 8 und 10 mal.
• 3. Die Proben wurden dann weiterhin für 5 Minuten bei 37°C in einem Ultraschallbad gehalten. Der Ultraschallschritt wurde ausgeführt, um reproduzierbare Ergebnisse für die Lipasendetektion zu erreichen, da durch die Anwendung von Ultraschall (auf tote Zellen) die Lipasen dann vollständig in das Medium freigesetzt werden. Wir haben bereits gezeigt, dass, wenn eine lebende Zellen enthaltende Probe einer Ultraschallbehandlung unterzogen wird, keine Lipasefreisetzung beobachtet wird, was bedeutet, dass die Ultraschallbehandlung keine Zelllyse verursacht.
• 4. 20 μL der Zellprobe wird gesammelt, um sie mit 20 μL Trypanblau zu färben (0,4%), Verhältnis 1:1. Die Färbung mit Typanblau im Verhältnis 1:1 ermöglicht die Identifizierung toter Zellen. Die lebenden Zellen werden durch Trypanblau nicht gefärbt, die toten Zellen werden jedoch dunkel verfärbt (siehe 13A). Dieser Schritt war ein Kontrollschritt, um zu zeigen, dass alle Zellen abgetötet wurden.
• 5. Die verbleibenden Zellproben werden 5 Minuten bei 3.500 U/min zentrifugiert.
• 6. Der Überstand der zentrifugierten Proben wird gesammelt.
• 7. Lipasenaktivität wird dann durch Hinzufügen von 5 μL des Überstands oben auf die LC-Lipidschicht analysiert.

The following procedure is then applied to the freshly cultured and healthy cells to achieve release of lipase as a result of lysis.

• 1. After trypsinization, 5 x 200 μL of cell samples (300,000 cells / mL) are collected and stored in separate vials.
• 2. These samples were frozen in liquid nitrogen, then thawed in a water bath (37 ° C), refrozen, then thawed again in the water bath, 2, 4, 6, 8 and 10 times.
• 3. The samples were then further held for 5 minutes at 37 ° C in an ultrasonic bath. The ultrasound step was performed to achieve reproducible results for lipase detection, since the application of ultrasound (to dead cells) then completely releases the lipases into the medium. We have already shown that when a sample containing living cells is subjected to ultrasound treatment, no lipase release is observed, which means that the ultrasound treatment does not cause cell lysis.
• 4. 20 μL of the cell sample is collected to stain with 20 μL trypan blue (0.4%), 1: 1 ratio. Coloring with Typan Blue in the ratio 1: 1 allows the identification of dead cells. The living cells are not stained by trypan blue, but the dead cells are darkened (see 13A ). This step was a control step to show that all cells were killed.
• 5. Centrifuge the remaining cell samples for 5 minutes at 3500 rpm.
• 6. The supernatant of the centrifuged samples is collected.
• 7. Lipase activity is then analyzed by adding 5 μL of the supernatant on top of the LC-lipid layer.

The pictures in 13B show the detections we made after adding droplets of the supernatants to the LC-based device (LC-Lipid device). The change in the light transmittance / orientation change of the liquid crystal layer occurs due to the lipases interrupting the lipid layer. The alignment change of the LCs was completed after 5 minutes and we were able to successfully detect the detection of active lipases released from the dead cells. To prove this, we performed further control experiments, showing that no detection is possible when the lipases are inactive. We performed these experiments with the commercially available Lipase PLC, in which we inactivated the lipase (1): at a low pH (pH 1 and pH 3); (2) by autoclaving the lipases at 120 ° C for half an hour. The addition of these inactivated enzymes to the LC-based device showed no effect. We also performed control experiments testing the supernatant from the living cell medium. Again, no detection was possible. These experiments proved that the alignment changes observed by us and described above occurred due to the presence of active lipases.

EXAMPLE 5B) Na azide treatment

Na azide is a known toxic compound, and our experiments aimed to kill the cells with Na azide and to detect the toxic effect of Na azide on the lipases released from the cells.

• 1) Wir inkubierten 30 μl Phospholipase A1 (PLA1) mit 30 μl 0,5% Na-Azid-Lösung bei 37°C für 24 Stunden.
• 2) In einem anderen Versuch werden 20 μl einer 30 µM Phospholipase C (PLC) (in PBS-Lösung) mit 20 µl einer 0,5% Na-Azid-Lösung bei 37° für 24 Stunden inkubiert.
• 3) In einem abschließenden Versuch mischten wir 10 µl Phospholipase A1, 10 µl 30 µM Phospholipase C (in Lösung) und 10 µl 100 nM Phospholipase D (PLD) (in Na-Azetat-Puffer) und inkubierten diese Mischung erneut bei 37°C für 24 Stunden mit 30 µl 0,5% Na-Azid (Lipasemischung).

First of all, to make sure that Na-azide does not deactivate the lipases, we performed control experiments in which we incubated various lipases with Na-azide. For these experiments, we used 0.5 mg Na azide dissolved in 100 mL water, referred to herein as 0.5% Na azide. The experiments were as follows:

• 1) We incubated 30 μl of phospholipase A1 (PLA1) with 30 μl of 0.5% Na-azide solution at 37 ° C for 24 hours.
• 2) In another experiment, 20 μl of a 30 μM phospholipase C (PLC) (in PBS solution) is incubated with 20 μl of a 0.5% Na-azide solution at 37 ° for 24 hours.
• 3) In a final experiment, we mixed 10 μl of phospholipase A1, 10 μl of 30 μM phospholipase C (in solution) and 10 μl of 100 nM phospholipase D (PLD) (in Na acetate buffer) and incubate this mixture again at 37 ° C for 24 hours with 30 μl of 0.5% Na azide (lipase mixture).

As seen from the polarized microscope images in 14 can be taken, the Na-azide showed no effect on the inhibition of enzyme activity. The top three pictures in 14 show the normal behavior of active lipases as soon as they come into contact with the LC lipid layers. The second series of images show the same lipase activity, although the lipases were incubated for 24 hours with 0.5% Na azide. These pictures clearly show that the enzymes do not lose their activities through the Na-azide treatment, at least not all enzymes.

Following these experiments, we performed toxicity tests with HepG2 cells, P-19 cells and pancreatic cells, which cells were incubated with 0.5% Na azide for various periods of time. 15 shows the toxicity experiments schematically. On the control plate, we used only cells, but no Na azide.

The cells, both in the control plate and in the Na-azide plate, were incubated for 2, 4 and 24 hours. At the end of each period, 60 μl of solution was taken from each cell well, which was then left for 5 minutes at 37 ° C. in an ultrasonic bath. An aliquot of this solution was used to stain with trypan blue to show the presence of dead cells. The residual solution was centrifuged. 5 μl of supernatant was removed from the centrifuged mixture and added dropwise to the LC-based assay. As seen on the polarized microscope images ( 16 ), cell death was clearly visualized for all cells by means of the active lipases that disrupted the LC lipid layer. ( 16 The cells were incubated with 0.5% Na azide for 2 hours, images were taken 5 and 10 minutes respectively after a droplet supernatant was applied to the LC-based assay.) As expected, the same effect occurred also after the same cells were further incubated for 4 and 24 hours with Na azide. In 17 the images show the effect after 24 hours (the images were taken 10 minutes after applying a droplet supernatant to the LC-based assay).

EXAMPLE 5C) Paracetamol Treatment

• (1) mit verschiedenen Paracetamol-Konzentrationen
• • 0,15 mg Paracetamol in 100 mL Wasser (0,15% Paracetamol)
• • 0,10 mg Paracetamol in 100 mL Wasser (0,10% Paracetamol)
• • 0,05 mg Paracetamol in 100 mL Wasser (0,05% Paracetamol)
• • 0,025 mg Paracetamol in 100 mL Wasser (0,025% Paracetamol)
• (2) unter Verwendung unterschiedlicher Zellen;
• • HepG2-Zellen
• • P19-Zellen
• • Pancreatische Zellen

Very similar experiments were carried out on the Na azide tests described above:

• (1) with different paracetamol concentrations
• 0.15 mg paracetamol in 100 mL water (0.15% paracetamol)
• • 0.10 mg paracetamol in 100 mL water (0.10% paracetamol)
• • 0.05 mg paracetamol in 100 mL water (0.05% paracetamol)
• • 0.025 mg paracetamol in 100 mL water (0.025% paracetamol)
• (2) using different cells;
• HepG2 cells
• • P19 cells
• • Pancreatic cells

As can be seen from the following examples, the high sensitivity of the LC-based assays was again demonstrated in detecting cell death / damage via lipase release.

This in 18A The picture shown generally explains how the incubation of a different number of cells was performed with paracetamol. The yellow solutions seen in the wells on the right side of the plate contain 0.05% paracetamol, 3,000 up to 100,000 cells, and the cell medium. The single well on the left side of the test plate has only 100,000 cells plus cell medium, not paracetamol.

In the 18B The control plate shown contains only wells with 100,000 cells and cell medium, and no paracetamol. Samples from both plates were taken for the experiments and similar experimental procedures were used as for Na-azide. The cells were incubated for 1/2, 2 and 24 hours. At the end of each time period, each cell well was sampled with 60 μL of solution, which was left in an ultrasonic bath for 5 minutes at 37 ° C. The solution was then centrifuged. 5 μl of supernatant was withdrawn and added dropwise to the LC-based assay.

In 19 the pictures in the table show the lipase detection in an LC-based test after the cells were incubated for 30 minutes with 0.05% paracetamol; they show in particular:
Line 1) the LC material (5CB);
Line 2) the lipid layer (DOPC layer) formed on the LC material (5CG-DOPC);
Line 3) the effect of the lipases released after incubation of 3,000 to 100,000 HeopG2 cells with 0.05% paracetamol for only half an hour (lipase detection).

Since we have no paracetamol in the control plate, we do not expect lipase detection from the samples taken from this plate ( 18B ).

19 indeed shows that nothing happens when the supernatant of the incubated cells from the control plate is checked with the LC-based assay. This test was carried out after an incubation period of 24 hours. But like the same 20 can be visualized for the cell supernatants taken from the control wells of the test plate ( 18A , Wells lower right) a lipase effect can be detected. This suggests that although the cells were not incubated directly in a paracetamol-containing medium in these wells, the cells were most likely affected by the diffusion of paracetamol in the atmosphere within that plate. The same diffusion effect was also very clearly visible when we used Na-azide instead of paracetamol.

Therefore, the foregoing discussion discloses and describes merely exemplary embodiments of the present disclosure. As those skilled in the art will appreciate, the present disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics of the disclosure. Accordingly, the disclosure in accordance with the present disclosure is illustrative, but without limiting the scope of the disclosure and other claims. The disclosure, including well-known variants of the teachings herein, in part, defines the scope of the preceding claim terminology such that no inventive subject matter is intended for the public.

The present application claims the benefit of EU Patent Application Nos. EP 12 172 777.0 , filed with the EPO on 20 June 2012, and no. EP 13 161 383.8 , filed with the EPO on March 27, 2013, the entire contents of both applications being incorporated herein by reference.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• EP 11005033 [0219]
• EP 12172777 [0244]
• EP 13161383 [0244]

Claims (31)

Liquid crystal-based sensor device for detecting the presence of an analyte, such as a prokaryotic cell, a bacterial cell and / or a prokaryotic agent, such as a toxin, such as a microbial toxin, the device comprising in the order listed: A first substrate, A liquid crystal layer on the first substrate, A phospholipid layer on the liquid crystal layer, wherein the device comprises no recognition part or recognition molecule bound directly to the surface of the first substrate. A sensor device according to claim 1, wherein the phospholipid layer comprises at least one recognition part or recognition molecule, such as a receptor, an enzyme or an antibody. Sensor device according to claim 1 or 2, wherein the phospholipid layer comprises cholesterol, preferably between 1 and 20 wt .-%, more preferably about 10 to about 15 wt .-% cholesterol. A sensor device according to any one of claims 1-3, wherein the at least one recognition member or recognition molecule in the phospholipid layer is a receptor, such as ganglioside GM ₁ . The sensor device of claim 4, wherein the phospholipid layer comprises between about 0.1% to about 10% by weight GM ₁ , preferably from about 0.5% to about 5% by weight GM ₁ . Liquid crystal-based sensor device for detecting the presence of lipases or lipase activity, the device comprising in the order listed: A first substrate, A liquid crystal layer on the first substrate, A reaction layer on the liquid crystal layer, wherein the reaction layer is a lipid layer, an LPS layer and / or a fatty acid layer. A sensor device according to claim 6, wherein the device comprises no recognition part or recognition molecule bound directly to the surface of the first substrate. Sensor device according to one of claims 1-7, further comprising: At least one first electrode between the first substrate and the liquid crystal layer, and An insulating layer and / or an alignment layer, or an insulating and alignment layer, which coats the electrode. A sensor device according to any one of claims 1-8, further comprising: A second substrate, which is arranged opposite the first substrate and makes the liquid crystal layer and phospholipid layer into intermediate layers or the liquid crystal layer and reaction layer into intermediate layers between the first and second substrate, and wherein there is a gap between the second substrate and the phospholipid layer and between the second substrate and the reaction layer serving as a reaction chamber for receiving a sample to be analyzed. The sensor device of claim 9, wherein the second substrate further comprises: A second electrode on the second substrate, and Optionally, an insulation layer on the second electrode, the insulation layer facing the phospholipid layer / liquid crystal layer or reaction layer / liquid crystal layer located on the first substrate. Sensor device according to one of claims 1-10, wherein the first and / or the second substrate comprises a solid, preferably glass or plastic, such as cycloolefin polymer (COP), cycloolefin copolymer (COC) and polydimethylsiloxane (PDMS). Sensor device according to one of claims 1-11, wherein the liquid crystal layer has a homeotropic alignment, homogeneous alignment or no predefined orientation. A sensor device according to any one of claims 1-12, wherein the liquid crystal layer comprises a liquid crystal selected from thermotropic liquid crystals having positive / negative or no dielectric anisotropy, two-frequency liquid crystals, discotic and / or lyotropic liquid crystals, and combinations thereof. A sensor device according to any one of claims 1-13, wherein 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. Sensor device according to claim 14, wherein the liquid crystal layer is color-doped, preferably with dichroic dye (s), Fluorescent dye (s) or doped with quantum dot (s). A sensor device according to any one of claims 1-15, wherein the phospholipid layer or reaction layer comprises a single phospholipid compound or a mixture of two or more different phospholipid compounds. A sensor device according to claim 16, wherein the phospholipid compounds are selected from DOPC, (1,2-dioleoyl-sn-glycero-3-phosphocholine), DPPC (1,2-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-sn-glycero-3-phosphocholine (14: 1, PC), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (16: 1, PC), 1,2-diisocyanato-sn-glycero-3 Phosphocholine (20: 1, PC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (22: 1, PC), 1,2-dinorononyl-sn-glycero-3-phosphocholine (24: 1, PC ) or combinations thereof. A sensor device according to any one of claims 6-17, wherein the reaction layer comprises a single lipopolysaccharide compound or a mixture of two or more different lipopolysaccharides. The sensor device of claim 18, wherein the LPS compounds are selected from LPS from E. coli strains. A sensor device according to any one of claims 6-17, wherein the reaction layer comprises a single fatty acid compound or a mixture of two or more different fatty acids. A sensor device according to any one of claims 6-17, wherein the reaction layer is a layer comprising lipid, LPS and / or fatty acid compound (s). A sensor device according to any one of claims 8-21, wherein the first electrode and the second electrode, each independently, is a non-interdigital electrode, a planar electrode or an interdigital electrode (IDE). Sensor device according to one of claims 8-22, wherein the insulating layer on the first and / or second electrode is constructed of polyimide. A sensor device according to any one of claims 8-23, wherein the alignment layer is composed of a silane such as octadecyldimethyl- (3-trimethoxysilylpropyl) ammonium chloride (DMOAP) or a heat / photocurable polyimide having a defined pretilt angle of the liquid crystal in the liquid crystal layer or a liquid crystal layer self-assembling monolayer of self-assembling compounds, such as thiols, dithiocarbamates, or an obliquely vaporized oxide layer, such as SiO ₂ , which provides a pre-tilt of the liquid crystal in the liquid crystal layer, depending on the evaporation angle of the oxide. Sensor device according to one of claims 8-24, wherein the first and / or the second electrode is transparent or not transparent, wherein preferably the electrode (s) of indium-tin oxide (ITO) or fluorine-tin oxide (FTO) is constructed / are. A sensor device according to any one of claims 8-25, wherein the device further comprises crossed polarizers under and over the liquid crystal layer, preferably on the planes of the first and second substrates, respectively, which measure the change in liquid crystal orientation by measuring the transmission / reflection of polarized light or wherein the device further comprises at least one polarizer over the liquid crystal layer, and wherein the substrate under the liquid crystal layer is a reflective substrate. A sensor device according to any one of the preceding claims, wherein the device further comprises means for measuring an electrical measure, said means enabling measurement of the change in liquid crystal orientation by measuring changes in such electrical measure, for example capacitance, resistance, or current. A sensor device according to any one of claims 8-25 and 27, wherein the device further comprises only one polarizer, preferably on the plane of either the first or the second substrate, and the corresponding other substrate without polarizer for use in conjunction with a light source which is polarized Light is provided, such as, for example, a liquid crystal display device. The sensor device of any of claims 1-28, further comprising a microfluidic device in fluid communication with the reaction chamber. The sensor device of any one of claims 1-29, further comprising a filter unit for filtering any material from the sample that may be larger than the analyte to be assayed, such as a bacterial cell or a microbial toxin, such that the material does not enter the reaction chamber. Sensor device according to one of claims 1-30, wherein the reaction chamber has a depth in the range of 800 nm to 100 microns, preferably from 900 nm to 30 microns, and, in the case of a round chamber, a width or a diameter in the range of 1 micron to 1 mm, preferably from 25 microns to 500 microns, and / or wherein the reaction chamber made of glass, plastic or has walls made of metal, such as gold walls.

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