ES2499790B2 - Method to amplify the detection of targets in an aligned liquid crystal matrix - Google Patents

Abstract

The present invention relates to a method for amplifying the detection of targets in an aligned liquid crystal matrix and to a device for the amplified detection of targets comprising: one or more binding elements capable of forming a complex with a target, liquid crystal, and polarization means configured to visualize the alignment of the liquid crystal. The device is characterized in that it comprises flow means with the ability to put said liquid crystal in dynamic contact with said joining elements. The amplified detection methods are based on the dynamic contact between a flow of liquid crystal and binding elements capable of complexing with the target.

Description

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METHOD FOR AMPLIFYING THE DETECTION OF TARGETS IN AN ALIGNED LIQUID CRYSTAL MATRIX

DESCRIPTION

FIELD OF THE INVENTION

The present invention falls within the field of the detection of microorganisms (bacteria, viruses and other microbes), microparticles, biomolecules or any type of target capable of being detected with a biorecognition molecule, in liquid crystal matrices. Through the use of a new amplification mechanism, a significant increase in the sensitivity of detectors based on liquid crystals is achieved.

STATE OF THE TECHNIQUE

The first mention of the use of liquid crystal based devices as sensors appears in the article: V.K. Gupta, J.J. Skaife, T.B. Dubrovsky, N.L. Abbott, "Optical amplification of ligand-receptor binding using liquid crystals", Science vol. 279 (5359), 1998, and US patent application 2002/0142453 A, in which the misalignment that occurs in a liquid crystal cell is shown by the change in roughness of its alignment surfaces. Distortions caused in the liquid crystal by targets that have specifically bound to a receptor are described. The areas in which there is no target appear dark, while in the areas where targets have been attached, misalignment of the liquid crystal occurs, causing the appearance of bright spots on the dark background. The size of these points is larger than the targets themselves. Since then, materials and methodologies that improve these systems have been sought, giving them greater sensitivity and reliability.

Thus, in the patent application WO 2007/036905 A2 (C. Woolverton, 2007) a liquid crystal sensor is described, in which superparamagnetic microparticles suspended therein are used. The binding of target particles to these microparticles causes a change in the alignment of the liquid crystal, causing the appearance of bright spots on a dark background when the cells between crossed polarizers are observed with the help of a microscope.

In US Pat. No. 6,411,354 B1 (OD Lavrentovich, T. Ishikawa, 2002), detailed ways to align a specific type of liquid crystals (chromatic liotropic liquid crystals), for the manufacture of liquid crystal cells with applications, for example, to biosensors

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The detection of specific targets at the interface between a thermotropic liquid crystal, generally hydrophobic, and an aqueous sample including the target to be detected is also known. The binding of the target to the receptor modifies the surface, altering the alignment of the liquid crystal. This happens to be aligned perpendicularly to the interface to be aligned in parallel, thus signaling the presence of the target when placing cross polarizers. This technology is related to the original publication: J.M. Brake, M.K. Daschner, Y.Y. Luk, and N.L. Abbott, "Biomolecular interactions at phospholipiddecorated surfaces of liquid crystals", Science vol. 302 (2094), 2003, and with the request of the same author US 2010/233729 A1. In some cases, a drop of liquid crystal deposited on a single substrate is used; in others, the liquid crystal is deposited in a plurality of wells that are exposed to the aqueous sample.

These detection techniques promise important advances, although they still require improvements, for example, in their sensitivity, reliability or simplicity of use. The biggest problem with the mentioned methods is that the amplification factor that is obtained is rarely more than ten times the size of the target. This implies that the detection of microparticles requires the use of a microscope or lens system. The use of microscopy devices not only increases the cost of the detection system, it also reduces the inspection area to a few square millimeters. Reducing the inspection area can significantly reduce the sensitivity of the system, since a smaller number of targets bound to the substrate will be found. In addition, the use of a microscope usually includes a microfluidic system that is incompatible with very viscous samples.

BRIEF DESCRIPTION OF THE INVENTION

The inventors have designed an improved device and method for the detection of targets, based on liquid crystals. Unlike others, this procedure is more sensitive, does not require additional instrumentation, and consequently is easier to handle.

It is therefore a first aspect of the present invention a device for the amplified detection of targets comprising: one or more joining means with the ability to form a complex with a target, liquid crystal, and light polarization means configured to visualize the alignment of said liquid crystal, characterized in that it comprises flow means with the ability to put said liquid crystal and said joining means in dynamic contact.

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A second aspect is the method for detecting a target in a sample, characterized in that it comprises (i) contacting the sample with one or more joining means capable of binding to said target, (ii) operating flow means for create a flow of liquid crystal that contacts the joining means, (iii) visualize the alignment of the liquid crystal by means of polarized light, so that, if the target is present in the sample, one or more areas are observed misalignment in the liquid crystal.

The present invention introduces a method of signal amplification several hundred times the size of the target. The invention represents a significant advance, because the instruments and even the area of application of liquid crystal biosensors are modified. A microscopy system is no longer necessary and large areas can be inspected, even with different receptors (aptamers, antibodies, proteins, ...) in the same device.

The method and device of the present invention makes use of the distortions caused in a liquid crystal matrix in flux by the presence of a body that modifies its alignment. The sensor is based on the optical or electrical detection of this misalignment.

In the sensors known so far, the detection and contact between the target / receptor complex and the liquid crystal did not imply any flow, and the detection was based on the distortion exerted on the resting liquid crystal. Upon detection while the liquid crystal is in dynamic contact with the sample, significantly greater amplification occurs than in other methods. Consequently, the device and method of the present invention differs considerably from existing ones, and provides an amplification of the misalignment of the liquid crystal in the matrix far superior to that of the devices known to date. The detection method is based on the observation of a flow to detect interference in the linear flow of an aligned matrix.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a particular embodiment of a device of the invention in which the liquid crystal molecules (4) are aligned in the absence of the target and the sample appears completely dark (6). The substrates A and B have an alignment layer (1), and a functionalization layer for specific targets (2). Between them are spacers (3) of micrometric calibrated size and, at both ends, crossed polarizers, one of them aligned in the direction of flow. Arrow (5) shows the flow direction of the liquid crystal.

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Figure 2 shows the same device of Figure 1, but in which the presence of a target (7) produces a disturbed flow (8), misaligning the molecules of the liquid crystal and producing a signal (10), when observed between cross polarizers.

Figure 3 is analogous to Figure 1, but in this case the original alignment of the liquid crystal is homeotropic (perpendicular).

Figure 4 is analogous to Figure 2, but in this case the original alignment of the liquid crystal is homeotropic (perpendicular).

Figures 5, 6 and 7 are photographs of some results obtained with devices and methods according to the present invention. In all cases, a flow of the Sunset Yellow liotropic liquid crystal is applied and the results between cross polarizers are observed. The targets that have been detected have a size between 2 to 12 micrometers in diameter. There is a very marked misalignment and a disturbed flow behind these particles.

DETAILED DESCRIPTION OF THE INVENTION

Device

In its simplest form, the device of the present invention comprises: one or more joining means with the ability to form a complex with a target, liquid crystal, light polarization means configured to visualize the alignment of the liquid crystal and means of flow with the ability to put said liquid crystal in dynamic contact with said joining means.

The types of samples that can be analyzed are, in general, any that admits the fixation of the target in the binding means. As examples, although not limited only to them, there may be cited: body fluids of humans and animals including milk and derivatives, water courses, irrigation, sewage and sewage, wells and reservoirs, river and maritime waters for detection of pathogenic microbes or microalgae The device can thus be used at different points in the food chain, manufacturing controls of raw materials of biological origin or destination.

In the present invention, target is understood as any substance capable of specifically binding to the binding means (receptors) and thus causing misalignment in the flow of liquid crystal. In a particular embodiment, the target is selected from the group consisting of microparticles, organic molecules or biomolecules, for example, proteins, DNA, RNA or fragments thereof. In another embodiment, the target is selected from the

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group consisting of microorganisms or fragments thereof. As examples of microorganisms, although not limited only to them, there may be mentioned: bacteria, viruses, yeasts, eukaryotic cells and other microbes.

Liquid crystals are materials that are in an intermediate state between solid and liquid. They are ordered like solids, but they can flow like liquids. Many substances reach the liquid crystal state in certain temperature or concentration ranges. The first are called thermotropic liquid crystals and the second liotropic liquid crystals. In the present invention both types can be used.

Thermotropic liquid crystals can be used, for example 5CB (4-cyano-4'-pentylbiphenyl), MBBA (N- (4-methoxybenzylidene) -4-butylaniline), commercial mixtures of Merck thermotropic liquid crystals such as MDA-98, MLC- 6023-000, MLC-6042-000, MLC-6204-100, MLC-6019-100, MLC-13000-000, MLC-6401, MLC-6290-000, MLC-6025-000, MDA-01-130, MLC-6096, MDA-02-3900, MDA-02-4476, MLC-6025, ZLI-3417-000, ZLI-3417-100, ZLI2788-000, ZLI-3700-000, ZLI-1565-H, ZLI- 3449-000, ZLI-3449-100, ZLI-2116-100, ZLI-3261, ZLI-1957/5, ZLI-3786 and ZLI-811. Lyotropic liquid crystals such as the commercial dye Sunset Yellow FCF ((5E) -6-oxo-5 - [(4-sulfonatophenyl) hydrazinyliden] naphthalen-2-sulphonate disodium) or Cromolyn (5- [3- (2) acid may also be used. -carboxy-4-isochromen-5-yl) oxy-2-hydroxypropoxy] -4-oxochromen-2-carboxylic acid), and also other chromonic liotropics or mixtures of all of the above.

The device of the present invention can adopt various configurations, as long as it is possible that the flow of liquid crystal and the joining means dynamically come into contact. In a preferred embodiment, the device is a cell comprising a first substrate with a first outer face and a first inner face, and a second substrate with a second outer face and a second inner face, wherein at least one of the substrates is totally or partially transparent. According to this particular embodiment, the polarization means, preferably crossed polarizers, are placed on the outer faces. Between both inner faces, facing each other, spacers are placed and at least one of them deposits a functionalization layer. Each of the substrates can be composed, for example, of glass, silicon, quartz or polymeric materials.

A particular embodiment of the device of the invention is exemplified in Figures 1 and 2, with homogeneous alignment of the liquid crystal, and in Figures 3 and 4, with homeotropic alignment.

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At least one of the inner faces includes an alignment layer. Said alignment layer is preferably adequately conditioned using organic or inorganic compounds or other methods to induce alignment by physical procedures. As examples, although not limited only to them, one can cite: conditioning with or without surface rubbing, photoalignment, stabilization in 2D or 3D, or any other type of procedure used as standard, with or without the application of external electric or magnetic fields. In each case, the person skilled in the art can use any of the methods described in the literature see for example:

I. H. Bechtold, M. L. Vega, E. A. Oliveira, J. J. Bonvent “Surface Treatments for Lyotropic Liquid Crystals Alignment”, Molecular Crystals and Liquid Crystals, vol. 391 (1), 2003; U. Kaeder and K. Hiltrop. "Alignment of lyotropic nematics by surface action", Trends in Colloid and Interface Science, vol. 84, 250-252, 1991; L. Parry Jones, "Alignment Properties of Liquid Crystals", Handbook of Visual Display Technology, 1387-1402, 2012, as well as the patent mentioned above US 6,411,354 B1. Preferably, binding elements are fixed on at least one of the two inner faces, forming a functionalization layer, which preferably includes target specific receptors based on antibodies, aptamers, DNA, RNA, proteins, functional fragments of the same or any other type of molecule with recognition function. The binding to the inner face of these joining means can be performed by covalent bonding, ionic bonding, van der Waals interactions, chemisorption, fisisorption or combinations thereof. In order to detect a plurality of targets in the same device, two or more joining means can be used with the ability to complex with one or more different specific targets. It is particularly useful that each of said two or more joining elements are fixed in different regions of the substrate. Preferably, the device is configured so that it lacks joining means in one of its regions, which acts as a negative control.

According to this particular embodiment, the cell is assembled once the substrate surfaces are prepared. In the assembly, spacers are used, preferably microspheres of calibrated size, whose function is to ensure a homogeneous thickness over the entire surface of the cell. To seal the cell and keep the substrates together, an adhesive (for example, a UV curing one) is preferably used. The spacers are preferably placed outside the active area of the device to avoid false positives. According to a particular embodiment, the first substrate and the second substrate are separated by a distance between 1 and 1000 µm, preferably between 5 and 600 µm, more preferably between 10 and 500 µm.

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In a particular embodiment, the cell is filled with the problem liquid sample in which it is desired to evaluate the presence of the target. The sample can be pretreated with microparticles capable of binding to the target. Then, the cell is washed with a suitable buffer solution.

In another particular embodiment, the solution in which it is desired to evaluate the presence of the target and the liquid crystal is mixed before being introduced into the cell, so that the liquid crystal and the target (if present) come into dynamic contact with the means of union.

Then, the flow of liquid crystal is activated, which can be done by positive or negative pressure, centrifugal force or capillarity, depending on the structure of the cell and its thickness. It can also be done with a simple fluidic system. Thus, the flow of liquid crystal in the cell begins, which flows with a preferential direction within the device. When no target has been attached to the inner surface of the substrates, the liquid crystal is unaffected and there is no misalignment, as observed by placing the device between crossed polarizers. When the liquid crystal meets a target that is attached to the surface, a disturbed flow occurs behind that target ("downstream"). When the cell is observed between crossed polarizers a significant misalignment is detected while the liquid crystal flows. This misalignment is sufficient to observe the presence of targets with the naked eye or with a lens.

The device of the present invention is preferably marketed ready to introduce the sample and is small in size, allowing the analysis of samples "in situ". The device may include display and recording media, such as cameras

or videos According to a particular embodiment, the amplification is sufficient to be observed with the naked eye or with a small lens, for example, between 2 and 20 magnifications. According to an alternative embodiment, the misalignment in the liquid crystal is observed by one or more of the methods that are selected from the group consisting of a colorimetric method, by a change in conductivity and / or by a change in permeability.

The manufacturing of the device of the present invention can be carried out following methods described in the literature, related or not related to sensors, such as in: S. Kumar,

J. Noh and C.H. Jang, "State of the Art in Liquid Crystals Nanophysiochemical Properties at Surfaces and Interfaces and Frontier for Posterity", Biochip Journal, vol. 3 (3), 181-189, 2009; and A. Hussain, A.S Pina and A.C.A. Roque, "Bio-recognition and detection using liquid crystals", Biosensors and Bioelectronics, vol. 25 (1), 2009.

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Detection Method

The method of the present invention comprises contacting a sample in which it is desired to detect the presence of a target and a device where the target is to be specifically attached. The target is attached to connecting elements.

When the device is filled with liquid crystal in a cell previously prepared to align it in a specific direction, the presence of the target attached to the binding elements causes a disturbance in the alignment of the liquid crystal molecules.

When the liquid crystal is introduced there is a flow of ordered molecules in one direction. When the liquid crystal encounters a target, a disturbed flow "downstream" is created with respect to it. This disturbance produces a misalignment effect that, as the inventors have discovered, is extraordinarily pronounced, and can be seen as a fine line, significantly greater than any amplification effect described in the literature. The effect can be observed while the liquid crystal flows through the cell, but also shortly after the end of the flow (tens of seconds or minutes, depending on the liquid crystal, thickness, temperature and some other factors). The amplification factor depends on the flow rate; typically, amplification factors of several hundred are achieved. The effect allows even the detection of targets with the naked eye. When the flow stops, the liquid crystal molecules begin to realign. According to an embodiment of the invention, the detection is performed at the time of introducing the liquid crystal or immediately after. In another embodiment, the detection takes place up to 20 minutes later or 1 to 5 minutes later, always before realignment of the liquid crystal molecules occurs.

The amplification method may include specific junctions of secondary groups (for example, aptamers, antibodies, affibodys, peptides, molecules with affinity for any component of the target, etc ...) that specifically adhere to the targets. Secondary groups may include: microparticles (for example, paramagnetic particles, carbon nanotubes, sepharose microparticles, gold nanoparticles, etc ...) or catalysts, especially enzymatic (for example, catalase or peroxidases). Secondary groups that include microparticles would cause or amplify the misalignment that occurs around the target after the secondary group joins. Said misalignment or amplification thereof can be caused by: the volume of the particle, the magnetic or electrical properties of the particle, the

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particle agglutination capacity, etc ... Other microparticles, such as gold nanoparticles, could influence the optical properties of the nearby liquid crystal. Secondary groups that include catalysts can act in different ways. On the one hand, they can degrade specific compounds within the medium in which the liquid crystal is found, producing secondary compounds that give rise to the amplification signal. This could be the case, for example, of a catalase linked to a secondary group that would degrade the H2O2 that could be present in the medium producing oxygen bubbles around the bound target. On the other hand, the catalysts can have a degradation activity of the liquid crystal molecules, causing misalignment in that area, which would result in the passage of light in the affected area in a dark cell or, conversely, a dark area in a cell that allows the passage of light. An example could be chloroperoxidase that has been described as an enzyme capable of degrading Sunset Yellow: J. Zhang, M. Feng, Y. Jiang, M. Hu, S. Li, Q. Zhai, “Efficient decolorization / degradation of aqueous azo dyes using buffered H2O2 oxidation catalyzed by a dosage below ppm level of chloroperoxidase ”, Chemical Engineering Journal, vol. 191, 236-242, 2012.

Next, some preferred embodiments of the invention are offered by way of illustration, which should not be construed as limited only to them.

PREFERRED EMBODIMENTS

one.

Two flat substrates are selected. One of them, at least, must be transparent. The material from which the substrates are made can be glass, quartz, or transparent polymers such as e.g. ex. PMMA If the second substrate is also not transparent, it can be made of metal, ceramic, silicon or other inert and rigid materials. Both substrates must be suitable (and conditioned) for the deposition of layers described below.

2.

An alignment layer is deposited on the inner face of at least one of the two substrates. The alignment layer may include organic or inorganic compounds, or methods for inducing alignment by physical procedures: the layer can be conditioned with or without surface rubbing, or by photoalignment, 2D or 3D stabilization or any other type of method used in a manner standard with or without the application of external electric or magnetic fields.

3.

At least one of the two substrates specific target receptors are fixed based on: antibodies, aptamers, DNA, RNA, proteins or functional fragments of the

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themselves, etc. The substrate can be fully functionalized or only in a specific area. The functionalization consists in modifying the surface of the substrate by introducing chemical functional groups that will bind to specific targets.

4. The two substrates are assembled, forming a cell with the inner faces

5 facing each other and with a calibrated distance (from one micrometer to several hundred micrometers) between both substrates.

5. The test sample, which contains the target to be detected in the cell, is introduced. The cells can be filled by positive or negative pressure, centrifugal force or capillarity, depending on the structure of the cell and its thickness.

10 6. The inside of the cell is washed with a suitable buffer solution, using one of the filling methods in section 5.

7.

The liquid crystal is introduced into the cell using any of the flow elements described above.

8.

The cell is placed between crossed polarizers for the detection of targets.

15 Polarizers can also be placed by attaching them to the outer faces of the substrates prior to cell assembly.

9. The cell between crossed polarizers is observed for the detection of the relevant targets.

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Claims (21)

1. A device for the amplified detection of targets comprising: one or more 5 joining means fixed in the device, with the ability to form a complex with a target, means for initiating and introducing a flow of liquid crystal towards the joining means in a preferential direction within the device, and configured light polarization means to visualize the alignment of said liquid crystal. The device according to claim 1, characterized in that the binding means are selected from the group consisting of: antibodies, aptamers, DNA, RNA and proteins, and functional fragments thereof, which contain specific target binding areas. . 3. The device according to any of claims 1 or 2, characterized in that the The target is specifically linked to secondary groups increasing the sensitivity of the detection or the specificity of the target.

Four.

The device according to claim 3, characterized in that said secondary groups are enzymes.

5.

The device according to any of claims 3 or 4, characterized in that

20 said specific joints to secondary groups degrade specific compounds of the liquid crystal.

6.

The device according to any of claims 3 to 5, characterized in that said specific joints to secondary groups are associated with a microparticle.

7.

The device according to any one of claims 1 to 6, characterized in that the

Liquid crystal is selected from the group consisting of thermotropic, liotropic, chromonic liotropic liquid crystals and mixtures thereof. The device according to any one of claims 1 to 7, characterized in that the target is selected from the group consisting of microparticles, proteins, DNA, RNA, microorganisms and fragments thereof. The device according to any one of claims 1 to 8, characterized in that said flow means are selected from the group consisting of direct microfluidic injection, positive pressure, negative pressure, capillarity, centrifugal force and a mixture thereof. The device according to any one of claims 1 to 9, characterized in that it comprises a first flat substrate with a first outer face and a first face 12 5 10 fifteen twenty 25 30 35 inner, and a second substrate with a second outer face and a second inner face where at least one of the substrates is totally or partially transparent.

eleven.

The device according to claim 10, characterized in that, at least one of the inner faces, comprises joining means forming a functionalization layer, and because, at least one of the inner faces, comprises an alignment layer configured to induce the alignment of The molecules of the liquid crystal.

12.

The device according to any of claims 10 or 11, characterized in that the first substrate and the second substrate are separated by a distance between 1 and 1000 µm.

13.

The device according to any one of claims 1 to 12, characterized by comprising two or more different joining means with the ability to form complexes with one or more targets.

14.

The device according to claim 13, characterized in that it comprises two or more connecting elements with the ability to form complexes with two or more different specific targets.

fifteen.

The device according to claim 14, characterized in that each of said two

or more binding means is in a region other than the first substrate.

16.

The device according to any of claims 1 to 15, characterized in that in one of its regions no connecting elements are fixed

17.

The device according to any one of claims 1 to 16, characterized in that it comprises viewing and recording means that are selected from cameras, videos and lenses.

18.

Method for detecting a target in a sample, characterized in that it comprises (i) contacting the sample with one or more joining means capable of binding to said target, (ii) operating flow means to create a crystal flow liquid that contacts the joining means, (iii) visualize the alignment of the liquid crystal by means of polarized light, so that, if the target is present in the sample, one or more areas of misalignment are observed in the crystal liquid.

19.

The method according to claim 18, characterized in that the detection takes place at the same time as the flow of liquid crystal.

twenty.

The method according to claim 18, characterized in that the detection takes place up to 60 minutes after the end of the liquid crystal flow.

twenty-one.

The method according to any of claims 19 or 20, characterized in that after step (i), and before step (ii), it is washed with a buffer solution.

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22

The method according to any of claims 19 to 21, characterized in that, in said sample, the sample is incubated with secondary groups according to claims 3, 4 or 6 before step (i).

2. 3.

The method according to any of claims 19 to 21, characterized in that, in said sample, the sample is incubated with secondary groups according to claims 3, 4 or 6 before step (ii).

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