Controlled release system based on the interaction between biological vesicles and porous materials provided with molecular gates

Abstract

The invention relates to a system comprising (i) biological vesicles which comprise a chemical messenger inside and are able to release said chemical messenger in the presence of an analyte and (ii) a porous material which comprises an indicator and / or an active agent inside the pores thereof and which is joined via its surface to a chemical entity acting as a molecular gate, wherein said molecular gate is arranged in a position such that, in the absence / presence of a chemical messenger released by the biological vesicles, it prevents / permits the output of the indicator and / or the active agent. The invention also relates to the use of said system in detection, quantification and / or diagnostic methods, as well as in therapeutic methods.

Description

[0001] DESCRIPTION

[0002] Controlled release system based on the interaction between organic vesicles and porous materials equipped with molecular gates

[0003] FIELD OF INVENTION

[0004] The present invention has applications in the field of medicine and related areas, such as the environmental and food sectors, and more specifically, relates to the design of controlled release systems for chemical substances, such as indicators and / or active agents, in response to stimuli. Depending on the chemical substance(s) contained therein, the system developed in the present invention can be used, for example, in detection, quantification, and / or diagnostic methods, as well as in therapeutic methods.

[0005] BACKGROUND OF THE INVENTION

[0006] In recent decades, various nano and micrometric scale platforms have been designed to release chemical substances in a controlled manner for various applications in fields such as medicine, detection, biotechnology and environmental sciences.

[0007] A major challenge in local drug delivery is that drug retention within the carrier must be strong enough to prevent premature leakage, while simultaneously ensuring the drug is released once the system reaches its target. Therefore, a fundamental requirement in the development of drug delivery systems is the ability to respond to external stimuli, such as changes in pH or temperature, to achieve optimal drug utilization through protection and selective release at the appropriate site of action.

[0008] Initially, organic systems were the most widely used due to their versatility. Among these systems, micelles, organic vesicles (OVs), and polymeric nanoparticles stand out, all capable of destabilizing or breaking down in response to various stimuli. The design of stimuli-sensitive lipid vesicles (liposomes) has received considerable attention, and several liposome formulations have been prepared, for example, that are sensitive to pH, enzymes, small molecules, or external stimuli (light, temperature), based on the permeation or degradation of their lipid membrane. It should be noted, however, that certain drugs cannot be encapsulated in various types of vesicles, such as lipid vesicles, as they are permeable to the membrane [1].

[0009] One of the most attractive concepts in this area today is that of a pore or channel that acts as a gate and can control the transport of mass, for example, into and out of a cell or organelle. Inspired by these microscopic dual-state (open / closed) valves, many researchers have designed hybrid materials that mimic these processes, developing nanometric porous materials that respond to stimuli through the action of “molecular gates” [2]. These materials equipped with molecular gates (called GMs, short for “gated materials”) are distinguished by their homogeneous porosity, high load-carrying capacity, and high specific surface area. They consist of porous supports functionalized with molecular entities capable of modulating the diffusion of encapsulated species from the interior of the pores into an aqueous solution, depending on a predefined stimulus.Thus, only in the presence of the selected stimulus does the gate "open," allowing the release of the species contained within the pores. The general concept of these closed systems was initially designed for drug delivery and other biomedical applications, but in recent years, various systems have emerged for the detection of different species of interest, in which the encapsulated species is an indicator such as a dye or a fluorophore. Because the presence of just a few analyte molecules triggers the release of hundreds of indicator molecules, these systems amplify the chemical signal, resulting in ultrasensitive detection systems.

[0010] Therefore, porous materials equipped with molecular gates can be loaded with an easily detectable molecule, such as a dye or a fluorophore, thus functioning as sensors, and can also be loaded with various therapeutic compounds, such as antibiotics and anticancer drugs, with the aim of releasing the drug only where and when needed [3-5].

[0011] Over the years, the inventors of the present invention have published several fundamental examples of materials acting as molecular gates coated with antibodies [6-10], aptamers [11-13], oligonucleotides [14-16] for the detection of small organic molecules, proteins, mIR-99a-4p (breast cancer biomarker)

[0017] or genomic DNA, as well as for the detection of fungal infections (Candida albicans, Candida auris), bacterial infections (Staphylococcus aureus), or infections caused by the SARS-CoV-2 virus [18-19]. In another example, the enzyme acetylcholinesterase has been used as a molecular gate, which is attached to the silica surface by cyclic phenylboronic acid esters, thereby blocking the encapsulated compound within the pores. In the presence of acetylcholine, the enzyme is activated and catalyzes its decomposition, producing an instantaneous drop in pH that causes the contents to be released rapidly [20-21].

[0012] On the other hand, and with the aim of ensuring that detection systems for various compounds are rapid, viable, and easy to use, a key point for diagnosis is the development of rapid, ultratrace-level tests that can be performed at the point of need (Point of Care or POC). POC assays and tests for use outside a laboratory must allow the measurement of a single specific parameter or multiple analytes using rapid, easy-to-use, and, ideally, portable methods. For this reason, the demand for miniaturized and powerful detection devices inspired the optimization and adaptation of molecular gate-based materials for implementation in various paper membranes [22-23].

[0013] An interesting approach for chemical signal amplification systems is to mimic certain natural processes and functions by preparing (bio)materials capable of communicating with each other. In a biological sense, these (bio)materials mimic "chemical communication" pathways in which a single molecule, such as a pheromone, triggers a cascade of biochemical reactions. Inspired by this concept, the inventors presented a family of nanoparticles designed to communicate hierarchically [24-26]. The system consisted of a first material released by an enzyme that induced the release of a reducing agent capable of opening a second material capped with a bulky group containing a disulfide bond. This second material released a surfactant capable of opening a third material encased in a lipid bilayer.Since then, very few examples have been described in which micro / nanosystems communicate by exchanging chemical messengers through different means of communication

[0027] .

[0014] Because communication between these particles allows the construction of new systems capable of performing coordinated tasks, chemical communication engineering between micro / nanosystems (through the exchange of chemical messengers) has gained much notoriety in the scientific community in recent years

[0028] .

[0015] However, there is a need to find new chemical transport and release systems that improve upon and / or complement existing state-of-the-art methods for use in detection, quantification, and / or diagnosis, as well as in therapeutic methods. In particular, it would be highly desirable to develop alternative systems that allow for the rapid and selective detection and / or quantification of analytes of interest and / or the diagnosis of diseases, without requiring specialized personnel and equipment.

[0016] BRIEF DESCRIPTION OF THE INVENTION

[0017] The present invention proposes chemical communication / interaction between organic vesicles (OVs) and porous materials equipped with molecular gates (GMs) for use in generating a novel system or platform for the controlled release of chemical substances in response to stimuli. Thus, the use of organic vesicles in combination with a porous material (such as nanoparticles or porous plates) based on molecular gates allows for the detection of compounds (analytes) that interact specifically with the membrane of the organic vesicles. Subsequently, through the emission of a chemical messenger from within the organic vesicles, the porous material equipped with molecular gates is activated, resulting in the release of the chemical substance or substances contained within.The main applications of the proposed system include its use in detection, quantification, and / or diagnostic methods (when the porous material is loaded with an indicator) as well as its use in therapeutic methods (when the porous material is loaded with a therapeutic active agent). However, other uses are also envisioned where the chemical substance released by the porous material is, for example, a plant protection agent, etc.

[0018] To the inventors' knowledge, there is no reported example to date that demonstrates the communication and joint action of organic vesicles and porous materials (supports) equipped with molecular gates.

[0019] This type of system allows for a broader range of detectable compounds, as there are certain analytes for which it is difficult to develop a molecular gate in porous materials, but for which organic vesicles that respond can be designed. Furthermore, it offers the possibility of releasing molecules (e.g., therapeutic agents) from porous materials that cannot be encapsulated in organic vesicles because they are permeable to the membrane [1]. Another interesting aspect is the ability to detect analytes at very low concentrations thanks to a signal amplification process resulting from communication between the organic vesicles and the porous material equipped with molecular gates: one analyte molecule induces the release of multiple messenger molecules from the vesicles, which in turn trigger the opening of the molecular gates, inducing the release of multiple molecules of the encapsulated species from the porous material.

[0020] Advantageously, the system of the invention can be used as a sensor for the detection and / or quantification of analytes indicative of a disease and / or for the diagnosis of a disease in point-of-care (POC) devices or assay kits, presenting a number of advantages over the techniques that are usually used (for example, cell culture or DNA assays), such as: greater speed and selectivity for the detection of different analytes of interest; the possibility of carrying out the measurement in situ, without the need to transfer the sample to specialized laboratories; and it allows reaching very low detection limits due to a signal amplification process thanks to the communication between lipid vesicles and molecular gates.

[0021] In one aspect, the present invention relates to a system, hereinafter the “system of the invention”, comprising:

[0022] - Organic vesicles that contain a chemical messenger and are capable of releasing said chemical messenger in the presence of an analyte; and

[0023] - A porous material comprising an indicator and / or an active agent within its pores and attached on its surface to a chemical entity functioning as a molecular gate, wherein said molecular gate is arranged in a position such that: (i) in the absence of a chemical messenger released by organic vesicles, the molecular gate blocks access to the exterior of the pore, preventing the exit of the indicator and / or the active agent; and

[0024] (i) In the presence of a chemical messenger released by organic vesicles, the molecular gate recognizes and interacts with said chemical messenger, and unlocks access to the outside of the pore, allowing the indicator and / or active agent to exit.

[0025] In another aspect, the invention relates to an in vitro assay kit, hereinafter referred to as the “kit of the invention,” comprising the system of the invention, wherein the porous material comprises an indicator. In another aspect, the invention relates to an in vitro use of the system or kit of the invention for the detection and / or quantification of an analyte in a sample.

[0026] In another aspect, the invention relates to an in vitro use of the system or kit of the invention for the diagnosis of a disease in a subject that is caused by or related to an analyte.

[0027] In another aspect, the invention relates to an in vitro method for detecting and / or quantifying an analyte in a sample comprising: a) contacting the system or kit of the invention with the sample, and b) detecting or measuring the indicator in the medium, wherein the presence of the indicator in the medium is indicative of the presence of the analyte in the sample, and / or the amount of the indicator in the medium is proportional to the concentration of analyte in the sample.

[0028] In another aspect, the invention relates to an in vitro method for diagnosing a disease in a subject caused by or related to an analyte comprising: a) contacting the system or kit of the invention with a sample from the subject, and b) detecting the indicator in the medium, wherein the presence of the indicator in the medium is indicative that the subject suffers from a disease.

[0029] In another aspect, the invention relates to a pharmaceutical composition, hereinafter the “pharmaceutical composition of the invention”, comprising the system of the invention and a pharmaceutically acceptable excipient.

[0030] In another aspect, the invention relates to the system of the invention or the pharmaceutical composition of the invention for use in the detection and / or quantification of an analyte indicative of a disease in a subject and / or for the diagnosis of a disease in a subject and / or for the treatment of a disease in a subject.

[0031] All features and / or method steps described in this document (including claims, description and drawings) may be combined in any combination, except for combinations of such features that are mutually exclusive.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

[0033] These and other features and advantages of the invention will become clearer from the following detailed description of preferred embodiments, given only as illustrative and non-limiting examples, with reference to the accompanying figures.

[0034] Figure 1: Schematic of the communication between organic vesicles (VOs) and porous nanoparticles equipped with molecular gates (GMs) in response to an analyte (substance to be detected) that interacts with the organic membrane of the VOs, producing the release of the chemical messenger contained within, which causes a stimulus that induces the release of the chemical substance contained in the GMs.

[0035] Figure 2: Schematic of the strip lateral flow assay (LFA) kit according to an embodiment of the present invention, representing the communication system between organic vesicles (VOs) and porous nanoparticles equipped with molecular gates (GMs). The presence of analyte induces the release of a messenger from the VOs. In the presence of the messenger, the GMs deposited on the strips release an indicator (reporter) that can be easily detected, for example, with a mobile phone.

[0036] Figure 3: In relation to example 1, transmission electron microscopy (TEM) images of mesoporous silica nanoparticles (MSNs). The scale bar represents 100 nm.

[0037] Figure 4: In relation to example 1, N2 adsorption-desorption isotherms for calcined MSNs, where the characteristic adsorption step at P / P0 of 0.3 for MSNs with empty pores is observed, related to the condensation of nitrogen within the pores by capillarity.

[0038] Figure 5: In relation to example 1, powder X-ray diffraction patterns of MSNs before and after surfactant removal by calcination at 550 °C.

[0039] Figure 6: In relation to example 1, kinetics of TNB formation 2 ' (2-nitro-5-thiobenzoic acid dianion) after the addition of MSNs with the enzyme acetylcholinesterase (AChE) anchored on their surface. The error bars correspond to the standard deviation of three independent samples.

[0040] Figure 7: In relation to example 1, confocal images of giant unilamellar vesicles (GUVs) stained with rhodamine-DOPE with 3 different cholesterol molar ratios, loaded with 50 pM pyranine (HPTS) before and after 10 min of incubation with α-hemolysin (20 pg / ml). Scale bars 25pm.

[0041] Figure 8: In relation to example 1, release kinetics of [Ru(bpy)s]Cl2 from MSNs (2 mg mi' 1 ) in aqueous solution (pH 7.5), in the absence (circles) and in the presence of 20 mM acetylcholine (triangles). Absorbance measured at 452 nm. The error bars correspond to the standard deviation of three independent experiments.

[0042] Figure 9: In relation to example 1, A) Collage of photographs recorded with a smartphone showing the [Ru(bpy)s]Cl2 released from MSNs (5 pg) on ​​strips at different ATCh concentrations after solvent LFA for 5 min. B) Quantified intensity gain of photographs of strips analyzed using ImageJ software. The error bars correspond to the standard deviation of three independent experiments.

[0043] Figure 10: In relation to example 1, chemical communication between GUVs and MSNs in solution, showing the release kinetics of [Ru(bpy)s]Cl2 from MSNs (2 mg mi' 1 ) in aqueous solution (pH 7.5), when a suspension of GUVs was treated (triangles) or not (circles) with α-hemolysin (100 pg / ml) for 10 minutes. Absorbance measured at 452 nm. Error bars correspond to the standard deviation of three independent experiments.

[0044] Figure 11: In relation to Example 1, strip-based chemical communication between GUVs and MSNs. A) Collage of photographs recorded with a smartphone showing the release of [Ru(bpy)s]Cl2 from MSNs (5 pg) on ​​strips over different concentrations of α-HL after lateral solvent flow for 5 min. B) Quantified intensity gain of the strip photographs analyzed using ImageJ software. The error bars correspond to the standard deviation of three independent samples.

[0045] DETAILED DESCRIPTION OF THE INVENTION

[0046] System of the invention

[0047] In a first aspect, the present invention relates to a system comprising (i) organic vesicles comprising a chemical messenger inside and capable of releasing said chemical messenger in the presence of an analyte and (ii) a porous material comprising an indicator and / or an active agent inside its pores and attached on its surface to a chemical entity with a molecular gate function, wherein said molecular gate is disposed in such a position that in the absence / presence of a chemical messenger released by the organic vesicles it prevents / allows the exit of the indicator and / or the active agent.

[0048] Organic vesicles are spherical or substantially spherical structures formed by one or more layers (usually bilayers) of amphiphilic molecules surrounding a water space. Amphiphilic molecules contain a polar hydrophilic end and a nonpolar, hydrophobic end. Due to hydrophobic and hydrophilic interactions, these molecules orient themselves and self-assemble, giving rise to various supramolecular organizations, including vesicles.

[0049] Vesicles can be classified according to the nature of their constituents into liposomes (lipid vesicles, composed of phospholipids), niosomes (composed of non-ionic surfactants), polymersomes (polymeric vesicles, composed of polymers), proteinosomes (composed of protein-polymer amphiphiles), cationic vesicles (composed of cationic surfactants), and cationic vesicles (composed of mixtures of cationic and anionic surfactants). In some embodiments, organic vesicles are selected from the group consisting of lipid vesicles (liposomes), niosomes, polymersomes, proteinosomes, cationic vesicles, and cationic vesicles. In some preferred embodiments, the organic vesicles are lipid vesicles.

[0050] Vesicles can also be classified according to the number of (bi)layers of amphiphilic molecules they possess. If the membrane consists of only one layer / bilayer of amphiphilic molecules, the vesicle is called a "unilamellar" vesicle. Conversely, if the vesicle consists of more than one layer / bilayer arranged concentrically and separated by large aqueous spaces, it is called a "multilamellar" vesicle. In some embodiments, organic vesicles are selected from the group consisting of unilamellar and multilamellar vesicles. In some preferred embodiments, the organic vesicles are unilamellar vesicles.

[0051] Different synthetic methodologies allow for adjusting the size of vesicles from the nanoscale to the microscale. Thus, vesicles can also be classified according to their size into small unilamellar vesicles (SUVs, diameter <100 nm), large unilamellar vesicles (LUVs, diameter 100 nm - <1 pm), and giant unilamellar vesicles (GlIVs, diameter 1-100 pm). In some embodiments, organic vesicles are selected from the group consisting of small unilamellar vesicles, large unilamellar vesicles, and giant unilamellar vesicles. In some preferred embodiments, the organic vesicles are giant unilamellar vesicles, with an average diameter in the range of 1 to 100 pm. Advantageously, GlIVs constitute a versatile synthetic platform with a size and bilayer structure similar to those of natural cells.

[0052] In preferred embodiments, the organic vesicles are unilamellar lipid vesicles (liposomes), preferably giant unilamellar lipid vesicles, most preferably with an average diameter in the range of 5 pm to 40 pm.

[0053] As used in the present invention, the size of the organic vesicle refers to the outer diameter of the organic vesicle. The average diameter of the organic vesicle can be determined by a variety of techniques, including dynamic light scattering (DLS), quasi-elastic light scattering (QELS), electron microscopy, and confocal microscopy.

[0054] As previously stated, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Examples of useful lipids include, but are not limited to, phosphatidylcholine (PC) and derivatives, phosphatidylethanolamine (PE) and derivatives, cholesterol, or a mixture thereof, such as distearoylphosphatidylcholine.

[0055] (DSPC) dioleoyl-phosphatidylethanolamine-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-0- monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine

[0056] (SOPE), cholesterol, or a mixture thereof. In some embodiments, the lipid vesicle bilayer(s) comprises a phospholipid and cholesterol, wherein the phospholipid is preferably phosphatidylcholine or a derivative thereof, and more preferably a mixture of DOPC and POPO. In some embodiments, the molar ratio between the phospholipid (e.g., phosphatidylcholine or a derivative thereof such as a mixture of DOPC and POPO) and the cholesterol varies from approximately 85 / 15 to 40 / 60, for example, from approximately 80 / 20, 75 / 25, 70 / 30, 65 / 35, 60 / 40, 55 / 45 to approximately 45 / 55. In preferred embodiments, the molar ratio of phospholipid (e.g., phosphatidylcholine or a derivative thereof such as a mixture of DOPC and POPC) and cholesterol is approximately 50 / 50 (i.e., 1 / 1).In even more preferred embodiments, the lipid vesicle bilayer (or bilayers) comprises DOPC, POPC, and cholesterol in a molar ratio of approximately 25 / 25 / 50.

[0057] Organic vesicles contain a chemical messenger. In some embodiments, the chemical messenger is acetylthiocholine (ACTh), glucose, sucrose, lactose, an ester derivative, cysteamine, cocaine, a reducing agent, a metal cation, or a mixture thereof. In more specific embodiments, the chemical messenger is acetylthiocholine (ACTh).

[0058] Organic vesicles are capable of releasing the chemical messenger contained within them in the presence of an analyte. The term “analyte,” as used in the present invention, means the substance to be detected or measured, such as an atom, ion, molecule, macromolecule, organelle, or cell.The term analyte includes, but is not limited to, molecules such as proteins, glycoproteins, vitamins, antibodies, antigens, hemoglobin, enzymes, metal salts, ions (e.g., hydrogen ions, hydroxyl ions, sulfates, sulfonates, phosphates, nitrates, nitrites, or electrolytes such as sodium, potassium, lithium, or calcium ions), fatty acids, neurotransmitters, hormones, growth factors, cytokines, monokines, lymphokines, lipocains, nutrients, sugars, receptors, nucleic acids, fragments of DNA or RNA, drugs, pharmaceutical agents, or derivatives or metabolites thereof.The term analyte also includes, but is not limited to, structured elements such as macromolecular structures, organelles, and cells, including, but not limited to, cells of ectodermal, mesodermal, and endodermal origin such as stem cells, blood cells, neural cells, immune cells, and gastrointestinal cells. The term analyte also includes, but is not limited to, microorganisms such as bacteria, fungi, viruses, protozoa, and viroids, or characteristic compounds produced by them. Some analytes indicate a possible disease condition.

[0059] In preferred embodiments, the analyte is indicative of a disease in a subject; that is, the analyte is a substance that can cause a disease in a subject or that denotes the existence of a disease in a subject. In more preferred embodiments, the analyte is a pathogenic microorganism, preferably selected from the group consisting of bacteria, fungi, viruses, and protozoa, as well as characteristic substances produced by or associated with them. In more preferred embodiments, the analyte is a toxin produced by a pathogenic microorganism. In more preferred embodiments, the analyte is α-hemolysin, one of the principal toxins produced by the bacterium Staphylococcus aureus.This toxin is released by the bacterium as a monomer, which, upon contact with cell membranes, oligomerizes, forming pores composed of seven subunits

[0029] . The formation of these pores makes the membranes of organic vesicles permeable to molecules with a molecular weight below 2 kDa, such as acetylcholine (ACh). Other possible analytes capable of inducing pores or destabilizing membranes include: sphingomyelinase, phospholipase, listeriolysin O, surfactants, and cholesterol-dependent cytolysins (CDCs).

[0060] In the present invention, the porous material equipped with molecular gates (MGs) comprises at least one pore along its surface with access to the exterior of the porous material and is loaded with an indicator and / or an active agent within its pore or pores, thereby acting as a porous support for said indicator and / or active agent to be released. In particular, this porous material or support is preferably selected from the group consisting of metals, semiconductors, organic polymers, carbons, or oxides (preferably of different inorganic species), or a mixture thereof. In more particular embodiments, the inorganic species is selected from the group consisting of carbon, titanium, zirconium, silicon, aluminum, magnesium, and boron, as well as any combination thereof. In still more particular embodiments, the porous material is silica, alumina (e.g., porous anodic alumina), or a combination thereof.

[0061] The International Union of Pure and Applied Chemistry (IUPAC) classifies pores by their size, measuring this size by the internal diameter, assuming it is cylindrical, or by the distance between the internal and opposite walls of a pore with a different configuration. Thus, following the IUPAC criteria, if the diameter of a pore (or the distance between the internal or opposite walls of a pore) is less than 2 nm, then the pore is called a micropore; if it is between 2 nm and 50 nm, it is called a mesopore (or nanopore); and if it is greater than 50 nm, then it is a macropore. Taking this classification into account, a material is considered to be microporous when the average size of the pores of said material is less than 2 nm, mesoporous (or nanoporous) when the average size of the pores of said material is between 2 nm and 50 nm, and macroporous when the average size of the pores of said material is greater than 50 nm.In preferred embodiments of the system of the invention, the porous material is microporous, mesoporous, or macroporous. Preferably, the porous material of the invention is mesoporous. In a preferred embodiment of the system of the invention, the average pore size is 2 to 6.5 nm or 2 to 3 nm.

[0062] Methods for determining pore size / diameter are widely known in the prior art. Examples of these methods include, but are not limited to, adsorption-desorption experiments, mercury or nitrogen porosimetry, SAS (small-angle scattering), NMR (nuclear magnetic resonance), and STM-AFM (scanning tunneling and atomic torque microscopy).

[0063] Regarding the porous material, another parameter to consider is its porosity. In the present invention, the porous material can have any porosity. However, preferably the porous material has a pore volume of 1 to 1.2 cm³.3 / g. In another preferred embodiment, the porous material comprises a high surface area, preferably from 800 to 1500 m² 2 / g, more preferably from 900 to 1300 m 2 / go from 1000 to 1200 m 2 / g. Even more particularly the porous material comprises a selected surface area from the list consisting of 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450 and 1500 m 2 / g.

[0064] In another, even more preferred embodiment, the porous material comprises a pore volume of 1 to 1.2 cm³ 3 / gy comprises a surface area of ​​1000 to 1200 m 2 / g.

[0065] The average pore size / diameter, pore volume, and surface area are generally determined by BET-BJH calculation of N2 desorption / adsorption isotherm data.

[0066] Porous materials can have different configurations. In specific embodiments, the porous material is a porous plate or porous nanoparticles.

[0067] The term "porous plate" should be interpreted broadly and encompasses any type of porous element in which two of its dimensions are much larger than the third, such as a plate with a substantially rectangular or square shape. In some embodiments, one of the plate's dimensions is less than 2 mm, while the other dimensions are greater than 8 mm.

[0068] In the present invention, a "nanoparticle" is understood to be a particle having dimensions on the nanoscale, such as dimensions between 1000 nm and 40 nm, between 750 nm and 40 nm, between 500 nm and 40 nm, between 250 nm and 40 nm, between 200 nm and 40 nm, and between 150 nm and 40 nm. The nanoparticle of the invention may be spherical, although other shapes are possible, such as, but not limited to, an ellipsoid, a rod, a cone, a cube, a cuboid (e.g., a rectangular box), a pyramid, and irregular shapes. In certain cases, combinations of different nanoparticle shapes may be included. As indicated above, the nanoparticle may have a substantially spherical shape and, therefore, may have dimensions measured as a diameter of the sphere.Techniques or methods for determining the diameter of nanoparticles are known in the prior art, including, but not limited to, dynamic light scattering (DLS). In some preferred embodiments, the nanoparticle has an average diameter of between 1000 nm and 40 nm, between 750 nm and 40 nm, between 500 nm and 40 nm, between 250 nm and 40 nm, between 200 nm and 40 nm, and between 150 nm and 40 nm. The term "average" as used herein is intended to mean the arithmetic mean. In some more preferred embodiments, the nanoparticle of the system of the invention has an average diameter of between 80 nm and 120 nm. In another, even more preferred embodiment, the nanoparticle is substantially spherical and has an average diameter of between 80 nm and 120 nm.

[0069] The porous nanoparticle employed in the present invention may comprise any substrate material having at least one pore along its surface with access to the exterior of the nanoparticle. However, in a preferred embodiment, the porous nanoparticle is selected from the porous materials defined above and, more preferably, is a silica nanoparticle. In the present invention, "porous silica nanoparticle" means a nanoparticle comprising silicon dioxide (silicon(IV) oxide). However, other porous nanoparticles that could also be used in the present invention are porous silicon or carbon nanoparticles.

[0070] In preferred embodiments of the system of the invention, the nanoparticle is microporous, mesoporous, or macroporous. Preferably, the nanoparticle of the invention is mesoporous. More preferably, the nanoparticle is a mesoporous silica nanoparticle (MSN).

[0071] Examples of porous nanoparticle types include, but are not limited to, MCM-41, MCM-48 (three-dimensional cubic), MCM-50 (layer phase), from the SBA (Santa Barbara Amorphous) family of different types SBA-1, SBA-2, SBA-3, SBA-6, SBA-8, SBA-11, SBA-12, SBA-14, SBA-15, SBA-16; from the FSM family, such as FSM-16, HMS, MSU such as MSU-1, MSU-2, MSU-3, MSII-V, or KIT-1. Thus, in another preferred embodiment, the nanoparticle is of a type selected from the list consisting of MCM-41, MCM-48, MCM-50, SBA-1, SBA-2, SBA-3, SBA-6, SBA-8, SBA-11, SBA-12, SBA-14, SBA-15, SBA-16, FSM-16, HMS, MSU-1, MSU-2, MSU-3, MSU-V, and KIT-1.

[0072] Even more preferably, the nanoparticle is a mesoporous silica nanoparticle of the MCM-41 type.

[0073] The term "MCM-41" (Mobil Composition of Matter 41) refers to a mesoporous material belonging to a family of silicate solids. It features an ordered arrangement of cylindrical mesopores with diameters ranging from 2 nm to 6.5 nm, most typically between 2 nm and 3 nm. These independently adjustable mesopores form a unique, one-dimensional pore system with a defined pore distribution and a large pore surface area and volume.

[0074] In some preferred embodiments, the mesoporous silica nanoparticles are of the MCM-41 type with a particle size between 80 nm and 120 nm and a pore diameter between 2 nm and 6.5 nm (more preferably, with a pore diameter between 2 nm and 3 nm). Furthermore, with regard to porosity, the porous nanoparticle preferably comprises a pore volume and a surface area as defined above for the porous material.

[0075] As already mentioned, another characteristic of porous material is that it comprises at least one pore with access to the exterior of the porous material. Preferably, it comprises a plurality of pores with access to the exterior of the porous material.

[0076] As explained throughout the present invention, in the absence of a chemical messenger released by the organic vesicles, access to the pore's exterior is blocked by a chemical entity, preventing the chemical substance contained within the pores from escaping into the surrounding medium. In the presence of a chemical messenger, access to the pore's exterior is unblocked, allowing the chemical substance, such as an indicator and / or an active agent, to escape.

[0077] In the present invention, the term "indicator" refers to any compound or molecule capable of being detected, visualized, and / or quantified. Furthermore, the term "indicator" may be interchanged with "signal," "signal," or "reporter" compound or molecule. Examples of indicators include, but are not limited to, dyes, fluorophores, redox-active substances, substances with plasmon resonance, or biologically active substances such as cytotoxic agents, proteins, small biomolecules, enzymes, or nucleic acid fragments. Thus, in preferred embodiments of the system of the invention, the indicator is selected from a list consisting of dyes, fluorophores, electrochemiluminescent substances, redox-active substances, substances with plasmon resonance, or biologically active substances such as cytotoxic agents, proteins, small biomolecules, enzymes, or nucleic acid fragments.

[0078] In preferred embodiments, the indicator is selected from the group consisting of a colorimetric indicator (dye), a fluorimetric indicator (fluorophore), an electrochemiluminescent indicator or a mixture thereof, even more preferably a fluorimetric indicator, such as [Ru(bpy)s]Cl2, rhodamine, safranin, fluorescein.

[0079] In the present invention, the term "active agent" is used in its broadest sense and includes any substance or mixture of substances that possess at least one desired property. The term "active agent" includes, for example, therapeutic agents and plant protection products.

[0080] In preferred embodiments, the active agent is a therapeutic agent. Preferably, the therapeutic agent is selected from the group consisting of an antibiotic, an antifungal, an antiviral, an antiprotozoal, an anticancer agent, or a mixture thereof.

[0081] The term “antibiotic agent”, as used in the present invention, means any chemical substance that has the ability to inhibit the growth of, or destroy, bacteria and other microorganisms, used primarily in the treatment of infectious diseases. Examples of antibiotic agents include, but are not limited to, penicillin G, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin, piperacillin, imipenem, aztreonam, cephalothin, cefaclor, cefoxitin, cefuroxime, cefonicid, cefmetazole, cefotetanus, cefprozil, loracarbef, cefetamet, cefoperazone, cefotaxime, ceftizoxime, ceftiaxone, ceftazidime, cefepime, cefixime, cefpodoxime, cefsulodin, fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin, cinoxacin, doxycycline, minocycline, tetracycline, amikacin, gentamicin, kanamycin, netilmicin, tobramycin, streptomycin,Azithromycin, clathromycin, erythromycin, erythromycin estolate, erythromycin ethylsuccinate, erythromycin glucoheptonate, erythromycin lactobionate, erythromycin stearate, vancomycin, teicoplanin, chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, mupirocin, metronidazole, cephalexin, roxithromycin, co-amoxicillin / clavulanate, piperacillin and tazobactam combinations and their various salts, acids, bases and other derivatives. Antibacterial antibiotic agents include, but are not limited to, penicillins, cephalosporins, carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides and fluoroquinolones.

[0082] The term “antifungal agent”, as used in the present invention, means any chemical substance that has the ability to inhibit the growth of, or destroy, fungi. Antifungal agents include, but are not limited to, amphotericin B, candicidin, denostatin, filipin, fungicromin, hachimycin, hamicin, lucensomicin, mepartricin, natamycin, nystatin, pecilocyn, perimyn, azaserin, griseofulvin, oligomycins, neomycin, pyrrolnitrine, sicanin, tubercidine, viridine, butenafine, naftifine, terbinafine, bifonazole, butoconazole, chlordantoin, chlormidazole, cloconazole, clotrimazole, econazole, enilconazole, fenticonazole, flutrimazole, isoconazole, ketoconazole, lanoconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, tolcyclate, tolindate, tolnaftate, fluconazole, itraconazole, saperconazole, terconazole, acrisorcin, amorolfine, biphenamine, bromosalicylchloranilide, buclosamide, calcium propionate, chlorphenesin, cyclopirox, cloxiquin,coparaffinate, diametazole, exalamide, flucytosine, haletazole, hexetidine, loflucarban, nifuratel, potassium iodide, propionic acid, pyrithione, salicylanilide, sodium propionate, sulbentione, tenonitrozole, triacetin, ujothione, undecylenic acid and zinc propionate.

[0083] The term “antiviral agent”, as used in the present invention, means any chemical substance that has the ability to inhibit the replication of, or destroy, viruses, used primarily in the treatment of viral diseases. Antiviral agents include, but are not limited to, acyclovir, cidofovir, cytarabine, didesoxyadenosine, didanosine, edoxudine, famciclovir, floxuridine, ganciclovir, idoxuridine, inosine pranobex, lamivudine, MADU, penciclovir, sorivudine, stavudine, trifluridine, valacyclovir, vidarabine, zalcitabine, zidovudine, acemannan, acetylleucine, amantadine, amidinomycin, delavirdine, foscamet, indinavir, interferon a, interferon p, interferon y, ketoxal, lysozyme, methisazone, moroxidine, nevirapine, podophyllotoxin, ribavirin, rimantadine, ritonavir 2, saquinavir, stalymycin, statolon, tromantadine, zidovudine (AZT), and xenazoic acid.

[0084] The term “antiprotozoal agent,” as used in the present invention, means any chemical substance that has the ability to inhibit the growth of, or destroy, protozoa, used primarily in the treatment of protozoal diseases. Examples of antiprotozoal agents, without limitation, include pyrimethamine (Daraprim®), sulfadiazine, and leucovorin.

[0085] The term "anticancer agent," as used in the present invention, means any chemical substance useful in the treatment of cancer. Anticancer agents can be classified by their mechanism of action into, for example, the following groups:

[0086] - antimetabolites / antineoplastics, such as pyrimidine analogues (floxuridine, capecitabine and cytarabine);

[0087] - purine analogues, folate antagonists and related inhibitors;

[0088] - antiproliferative / antimitotic agents, including natural products such as vinca alkaloid (vinblastine, vincristine) and microtubule inhibitors such as taxane (paclitaxel, docetaxel), vinblastine, nocodazole, epothilones, vinorelbine (NAVELBINE®) and epipodophyllotoxins (etoposide, teniposide);

[0089] - DNA-damaging agents such as actinomycin, amsachna, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide (CYTOXAN®), dactinomycin, daunorubicin, doxorubicin, epirubicin, ifosfamide, melphalan, mercurochrome, mitomycin, mitoxantrone, nitrosourea, procarbazine, taxol, taxotere, teniposide, etoposide, and triethylenethioxyphosphoramide;

[0090] - antibiotics, such as dactinomycin (actinomycin D), daunorubicin, doxorubicin, idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;

[0091] - enzymes, such as L-asparaginase which systemically metabolizes L-asparagine and deprives cells that do not have the ability to synthesize their own asparagine;

[0092] - antiplatelet agents;

[0093] - antiproliferative / antimitotic alkylating agents, such as nitrogen mustards cyclophosphamide and analogues (melphalan, chlorambucil, hexamethylmelamine and thiotepa), alkylnitrosoureas (carmustine) and analogues, streptozocin and triazene (dacarbazine);

[0094] - antiproliferative / antimitotic antimetabolites, such as folic acid analogues (methotrexate);

[0095] - platinum coordination complexes (cisplatin, oxaliplatin and carboplatin), procarbazine, hydroxyurea, mitotane and aminoglutethimide;

[0096] - hormones, hormone analogues (estrogen, tamoxifen, goserelin, bicalutamide and nilutamide), and aromatase inhibitors (letrozole and anastrozole); - anticoagulants, such as heparin, synthetic heparin salts and other thrombin inhibitors;

[0097] - fibrinolytic agents, such as tissue plasminogen activator, streptokinase, urokinase, aspirin, dipyridamole, ticlopidine and clopidogrel;

[0098] - anti-immigration agents;

[0099] - antisecretory agents (breveldin);

[0100] - immunosuppressants tacrolimus, sirolimus, azathioprine and mycophenolate;

[0101] - compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor inhibitors and fibroblast growth factor inhibitors);

[0102] - angiotensin receptor blockers, nitric oxide donors;

[0103] - antisense oligonucleotides;

[0104] - antibodies, such as trastuzumab and rituximab;

[0105] - cell cycle inhibitors and differentiation inducers, such as tretinoin;

[0106] - inhibitors, topoisomerase inhibitors (doxorubicin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mitoxantrone, topotecan and irinotecan) and corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone and prednisolone);

[0107] - growth factor signal transduction kinase inhibitors;

[0108] - inducers of dysfunction;

[0109] - toxins, such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, diphtheria toxin, and caspase inhibitors;

[0110] - and chromatin.

[0111] In some embodiments, the anticancer agent is selected from those mentioned above. Examples of anticancer agents also include, but are not limited to: alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodepa, carboquone, meturedepa, and uredepa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimemelomelamine; acetogenins, for example, bullatacin and bullatacinone; a camptothecin, including a synthetic analogue of topotecan; bryostatin, callistatin; CC-1065, including its synthetic analogues adozelesin, carzelesin, and bizelesin; cryptophycins, particularly cryptophycin 1 and cryptophycin 8; dolastatin; duocarmycin, including the synthetic analogues KW-2189 and CBI-TMI; eleutherobine; 5-azacltidine; pancratistatin; a sarcodictylin; spongistatin;nitrogen mustards such as chlorambucil, clomafazine, cyclophosphamide, glufosfamide, evofosfamide, bendamustine, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, fenesterin, prednimustine, trofosfamide and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, foremustine, lomustine, nimustine and ranimustine;antibiotics such as enediin antibiotics (e.g., caliqueamycin, especially caliqueamycin gamma1 and caliqueamycin phil1), dinemycin including dinemycin A, bisphosphonates such as clodronate, a speramicin, neocarzinostatin chromophore and related chromoprotein antibiotic chromophores (enediin), aclainomycins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carninomycin, carzinophylline, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (Adramycin®) (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxycorubicin), epirubicin, esorubicin, danubian, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porphyromycin, puromycin, chelamicin, rhodorubicin, streptonigrine, steptozocin, tubercidine, ubenimez, zinostatin and zorubicin;Antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as demopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as cladribine, pentostatin, fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azaurididine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid replenishers such as frolinic acid; radiotherapeutic agents such as Radium-223; trichothecenes, especially T-2 toxin, verracurin A, roridine A and anguidin; taxoids such as paclitaxel (TAXOL®), abraxane, docetaxel (TAXOTERE®), cabazitaxel, BIND-014, tesetaxel; platinum analogues such as cisplatin and carboplatin, nanoplatinum NC-6004; aceglatone; aldophosphamide glycoside;aminolevulinic acid; enyluracil; amsacrine; hestrabucil; bisanthrene; edatraxate; defofamine; demecolcine; diaziquone; elformtin; eliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; entinan; leucovorin; lonidamine; maitansinoids such as maitansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenammet; pyrarubicin; losoxantrone; fluoropyrimidine; folinic acid; podophyllinic acid; 2-ethylhydrazide; procarbazine; polysaccharide-K (PSK); razoxane; rhizoxine; sizofiran; spirogermanium; tenuazonic acid; trabectedin; triaziquone; 2,2',2"-thchlorotriem¡lamine; urethane; vindesine; dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacitosine; arabinoside ("Ara-C"); cyclophosphamide; thiopbeta chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; vinblastine; platinum; etoposldo (VP-16);daunomycin; aminopterin; xeoloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids such as retinoic acid; capecitabine; NUC-1031; FOLFOX (folinic acid, 5-fluorouracil, oxaliplatin); FOLFIRI (folinic acid, 5-fluorouracil, irinotecan); FOLFOXIRI (folinic acid, 5-fluorouracil, oxaliplatin, irinotecan), FOLFIRINOX (folinic acid, 5-fluorouracil, irinotecan, oxaliplatin) and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[0112] More particularly, non-limiting examples of anticancer agents usable in the context of the present invention include doxorubicin, cisplatin, palbociclib, navitoclax, etc.

[0113] In preferred embodiments, the chemical entity functioning as a molecular gate is the enzyme acetylcholinesterase (AChE). Other examples of molecular gates include, for instance: oligonucleotides, polymers and cyclodextrins, disulfide-linked polyethylene glycol chains (SS-PEG), an aryl boronate derivative complex with p-cyclodextrin (sensitive to H2O2), enzymes, antibodies, and aptamers.

[0114] In particular embodiments, the chemical entity functioning as a molecular gate is attached to the surface of the porous material by a linker or functional groups, which facilitate its attachment through electrostatic, supramolecular, or covalent interactions. In preferred embodiments, the chemical entity functioning as a molecular gate is attached to the surface of the porous material by a covalent bond. More preferably, it is covalently attached via cyclic phenylboronic acid esters. Other possible attachment methods include, for example, amide bonds, ester bonds, urea bonds, and disulfide bridges.

[0115] In a particular embodiment, the invention relates to the system where: the analyte is α-hemolysin; the chemical messenger of the organic vesicles is acetylcholine (AChC); and the chemical entity with molecular gate function is the enzyme acetylcholinesterase (AChE).

[0116] Invention Kit

[0117] The system of the invention can form part of an in vitro test kit. Therefore, in another aspect, the present invention relates to an in vitro test kit comprising the system of the invention, wherein the porous material comprises an indicator.

[0118] In addition to the system of the invention, the kit may comprise other components useful in implementing the present invention, such as buffers, delivery vehicles, material holders, positive and / or negative control components, etc. The kit may also include instructions for performing the object of the invention. In preferred embodiments, the kit of the invention comprises the system of the invention and instructions for use. These instructions may be present in the kit in a variety of forms, one or more of which may be included. One form in which these instructions may be present is as information printed on a suitable medium or substrate, e.g., a sheet or sheets of paper on which the information is printed, on the kit packaging, in a package insert, etc. Another form would be a computer-readable medium, for example, a CD, a USB drive, etc., on which the information has been recorded.Another possible method is a website address that can be used via the internet to access information at a remote location. Any convenient method can be included in the kit.

[0119] The test kit can be implemented in portable, fast and easy-to-use devices, allowing the analysis to be performed remotely (Point of Care devices, PoC).

[0120] In preferred embodiments, the test kit is a lateral flow assay (LFA) kit. In the context of the present invention, an LFA generally refers to an assay in which a sample flows by capillary action through a strip comprising one or more fibrous membranes to enable the detection of at least one analyte or to enable confirmation of the absence of at least one analyte. The LFA may be purely qualitative, providing a yes / no result, or it may be a quantitative assay to determine the total amount or concentration of an analyte. The LFA may be designed so that the presence and intensity of a signal are proportional to the concentration of an analyte. If the analyte is associated with a disease, the LFA may be used for the diagnosis of that disease.

[0121] In some embodiments, the lateral flow test kit comprises a strip formed by at least one fibrous membrane on which the organic vesicles and the porous material of the system of the invention are immobilized. In this configuration, the fibrous membrane preferably comprises: a sample zone for applying the sample to be analyzed; a deposit zone on which the organic vesicles and the porous material of the system of the invention are immobilized; and a detection zone.

[0122] After application, the sample flows by capillary action through the fibrous membrane into the reservoir zone, where the system of the invention is located. In some embodiments, in the reservoir zone, following the direction of capillary flow, the organic vesicles are immobilized first. If the sample contains the analyte of interest, the chemical messenger contained within the organic vesicles is released. Next, the porous material is immobilized, which, through the action of the chemical messenger, releases the indicator. Finally, the indicator is detected and / or quantified in the detection zone.

[0123] In preferred embodiments, the test kit is a lateral flow test kit comprising a strip formed by at least one fibrous membrane on which the porous material of the system of the invention is immobilized, and the organic vesicles of the system of the invention are provided separately for mixing with the sample to be analyzed. In this configuration, the porous material membrane preferably comprises: a sample zone for applying the sample to be analyzed, previously mixed with the organic vesicles of the system of the invention; a deposit zone on which the porous material of the system of the invention is immobilized; and a detection zone.

[0124] In this case, if the sample contains the analyte of interest, the chemical messenger contained within the organic vesicles is released. The chemical messenger flows by capillary action through the fibrous membrane to the storage zone, where the porous material is immobilized. The chemical messenger then releases the indicator. Finally, the indicator is detected and / or quantified in the detection zone.

[0125] The membrane used in the lateral flow test kit of the present invention may be made of a variety of materials through which the sample to be examined can pass or move and which are known to a person skilled in the art. Examples of useful membrane materials include, but are not limited to, paper (cellulose), nitrocellulose, sintered glass ceramic, fiberglass, polyvinylidene fluoride, nylon, filler-modified nylon, polyethersulfone, and Fusion 5™ (borosilicate glass fibers), or a combination thereof.

[0126] Typically, the membrane is mounted or adhered onto a backing surface that acts as a support and facilitates handling of the strip.

[0127] The lateral flow assay kit can be configured to function as a strip that is immersed in (or otherwise comes into contact with) the sample to be tested, or as a rigid plastic cassette provided with a well onto which the sample is applied.

[0128] Uses and methods of in vitro use of the system and kit

[0129] Throughout the preceding paragraphs, the operation of the system and kit of the invention and its usefulness as a biosensor for analyte detection have been demonstrated. Furthermore, the inventors have observed that the system and kit of the invention are not only useful for detecting an analyte in the medium, but that the amount of indicator released into the medium is proportional to the analyte concentration. Therefore, the system and kit of the invention are also useful for analyte quantification. Thus, in another aspect, the present invention relates to the in vitro use of the system or kit of the invention for the detection and / or quantification of an analyte in a sample.

[0130] Furthermore, if the analyte is indicative of a disease, the system and kit of the invention are also useful in diagnosing diseases caused by or related to that analyte. Thus, in another aspect, the present invention relates to the in vitro use of the system or kit of the invention for diagnosing a disease in a subject that is caused by or related to an analyte.

[0131] Analogous to the in vitro uses of the system and kit of the invention described in previous paragraphs, the present invention also contemplates methods aimed at both detecting and / or quantifying an analyte in a sample, as well as those aimed at diagnosing diseases in a subject caused by or related to an analyte.

[0132] Therefore, in another aspect the invention relates to an in vitro method for detecting and / or quantifying an analyte in a sample comprising: a) contacting the system or kit of the invention with the sample, and b) detecting or measuring the indicator in the medium, wherein the presence of the indicator in the medium is indicative of the presence of the analyte in the sample and / or the amount of the indicator in the medium is proportional to the concentration of analyte in the sample.

[0133] Likewise, in another aspect, the present invention relates to an in vitro method for diagnosing a disease in a subject caused by or related to an analyte comprising: a) contacting the system or kit of the invention with a sample from the subject, and b) detecting the indicator in the medium, wherein the presence of the indicator in the medium is indicative that the subject suffers from a disease.

[0134] The term "analyte" has been defined or explained in previous paragraphs, and those definitions and particular embodiments thereof are also applicable to the present inventive aspects. Thus, in one particular embodiment, the analyte is a pathogenic microorganism, preferably selected from the group consisting of bacteria, fungi, viruses, and protozoa, as well as characteristic substances produced by or associated with them. Therefore, the system and kit of the invention, by detecting and quantifying a substance produced by or associated with a pathogenic microorganism, can be used to indirectly detect the presence / absence of the pathogen in a sample and / or quantify it. In preferred embodiments, the analyte is a toxin produced by a pathogenic microorganism. In a more preferred embodiment, the analyte is α-hemolysin, a characteristic toxin of the bacterium Staphylococcus aureus.Advantageously, as illustrated in the examples, the present invention allows for the detection and / or diagnosis of S. aureus in a rapid, simple manner and with a high level of sensitivity and selectivity.

[0135] S. aureus can be present in a wide variety of environments, from soil, water, and air, to utensils and surfaces, and even humans and animals. In the food industry, the presence of S. aureus in food processing areas can lead to food contamination, with the corresponding risk to human and animal health. Therefore, the detection and / or quantification of S. aureus using the system and kit of the invention can be carried out on any sample that is likely to be contaminated by S. aureus.

[0136] In the present invention, a "sample" means a part or small quantity of something that is considered representative of the whole and that is taken or separated from it for study, analysis, or experimentation. In preferred embodiments, such study, analysis, or experimentation refers to the determination of the presence / absence of a pathogen (e.g., S. aureus) by means of detecting a substance produced by it (e.g., α-hemolysin). Examples of samples suitable for the detection method of the invention include, but are not limited to, clinical samples, food samples, and environmental samples. The term "sample" also includes samples that have been manipulated in some way after collection, for example, by treatment with reagents, solubilization, or enrichment of certain components.In a preferred embodiment, the isolated samples are processed to obtain a liquid solution containing the sample components, including the pathogen cells. In a preferred embodiment, the sample is a food sample, a clinical sample, or an environmental sample.

[0137] In the present invention, "food sample" is understood to mean an isolated sample of food, whether solid or liquid, processed or raw.

[0138] In the present invention, "environmental sample" means a sample that comes from the surroundings or the environment and that is likely to be or be contaminated by a pathogen, such as industrial or domestic wastewater, laboratory solutions such as buffer solutions, culture liquids, reaction solutions, washes and the like.

[0139] In the present invention, a "clinical sample" means a sample taken from a subject. The term "clinical sample" encompasses blood samples and other liquid samples of biological origin, solid tissue samples, such as biopsy samples, tissue cultures, or cells derived from them and their progeny, such as cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. Examples of clinical samples suitable for use with the invention include, but are not limited to, cellular tissues, fecal matter, and body fluids such as blood, urine, saliva, serum, pleural fluid, peritoneal fluid, synovial fluid, and cerebrospinal fluid. In a preferred embodiment of the invention, the biological sample is selected from the group consisting of blood, serum, sputum, pleural fluid, peritoneal fluid, synovial fluid, or cerebrospinal fluid.

[0140] In the present invention, the clinical sample is isolated from a subject. The term "subject," as used herein, refers to any animal, preferably a mammal, and includes, but is not limited to, domestic and farm animals, primates, and humans. In a preferred embodiment, the subject is a human being of any sex, age, or race. In another preferred embodiment, the subject is a bovine animal.

[0141] In the present invention, "detection" refers to reporting or identifying an analyte (for example, α-hemolysin) in a sample. In some cases, the detection of the analyte indirectly allows the detection of another substance, as is the case with α-hemolysin, which allows the indirect detection of the presence of the pathogen S. aureus in a sample.

[0142] In the present invention, "quantification" refers to the determination of the amount, number, or concentration, per unit volume, of an analyte (for example, α-hemolysin) in a sample. In some cases, the quantification of the analyte indirectly allows for the quantification of another substance, as is the case with α-hemolysin, which allows for the indirect quantification of the pathogen S. aureus in a sample.

[0143] In the present invention, "diagnosing" means the procedure by which a specific disease, nosological entity, syndrome, or any health-disease condition is identified through the analysis of a series of clinical parameters or symptoms characteristic of said disease, and which distinguish it from other diseases with similar clinical presentations. In preferred embodiments, in the present invention, the disease to be identified is that caused by a pathogenic microorganism, and the clinical parameter is the presence of a characteristic toxin produced by said pathogen in an isolated sample from the subject. In preferred embodiments, in the present invention, the disease to be identified is that caused by S. aureus, and the clinical parameter is the presence of α-hemolysin in an isolated sample from the subject.

[0144] Staphylococcus aureus is a pathogenic, Gram-positive, non-motile, catalase-positive, non-spore-forming bacterium that can survive for extended periods in dry environments. It grows between 7°C and 50°C, with an optimum growth temperature of 35°C. Its ability to produce heat-resistant enterotoxins within a few hours makes it a common cause of food poisoning, as well as a leading cause of various infectious processes, ranging from skin infections to life-threatening systemic diseases. Examples of diseases caused by S. aureus include, but are not limited to, skin and soft tissue infections, intravascular infections, bone infections, joint infections, respiratory infections, other invasive infections such as meningitis or surgical site infections, toxin-mediated diseases, and infections associated with external materials such as catheters and prostheses.However, in a preferred embodiment of the present invention, the disease caused by S. aureus is selected from the group consisting of staphylococcal scalded skin syndrome, food poisoning, staphylococcal toxic shock syndrome, skin abscess, impetigo, folliculitis, carbuncle (furunculosis), cellulitis of the face and neck, hidradenitis suppurativa, mastitis, bacteremia, sepsis, endocarditis, pneumonia, empyema, osteomyelitis, septic arthritis, meningitis, peritonitis, pericarditis, pyomysitis, and disseminated intravascular coagulation syndrome.

[0145] The first step of the detection and / or quantification method of the invention [step a)] comprises bringing the system or kit of the invention into contact with a sample. Similarly, the first step of the diagnostic method of the invention [step a)] comprises bringing the system or kit of the invention into contact with a sample from the subject. As explained in previous paragraphs, this sample may have been pre-treated before being brought into contact with the system or kit. Techniques for handling or preparing samples for analysis of their components, particularly for the detection of microorganisms, are widely known in the prior art. In a preferred embodiment of the detection and / or quantification method and the diagnostic method of the invention, prior to step a), the method comprises mixing the sample with a buffer solution.

[0146] The term "buffer solution" in the present invention refers to solutions at relatively high concentrations of an acid and its conjugate base, i.e., hydrolytically active salts. These solutions have the property of maintaining a stable pH in a solution upon the addition of relatively small amounts of strong acids or bases. In the detection and / or quantification method of the present invention, as well as in the diagnostic method, the buffer solution allows the pH of the medium to be maintained within a narrow range to prevent changes in the solution that could affect the result of the method. In a preferred embodiment, the "buffer solution" is phosphate buffer solution (PBS). In a preferred embodiment, the pH of the medium is from 6.5 to 8.5, preferably from 7 to 8, and even more preferably 7.5.

[0147] In a second step [step b)], the detection and / or quantification method of the invention comprises detecting or measuring the indicator in the medium. Similarly, in a second step [step b)], the diagnostic method of the invention comprises detecting the indicator in the medium. The means and techniques necessary for detecting or quantifying the indicator in the medium are determined by the indicator used, and such means and techniques are known in the prior art to a person skilled in the art who will know which means and techniques to use. Examples of methods or techniques include, but are not limited to, visual observation, fluorescence spectroscopy, or ultraviolet-visible spectroscopy. In a preferred embodiment, detection and quantification are performed by measuring the fluorescence intensity of the medium.

[0148] Once the amount of indicator in the medium has been detected or measured, the system or kit allows us to conclude that

[0149] - the presence of the indicator in the medium is indicative of the presence of analyte in the sample, and / or

[0150] - The amount of indicator in the medium is proportional to the concentration of analyte in the sample.

[0151] In the present invention, the expression "the presence of the indicator in the medium is indicative of the presence of the analyte in the sample" refers to the fact that the organic vesicles have released the chemical messenger, which has been recognized by the molecular gate of the porous material, unblocking its pores and releasing the indicator into the test medium, thus revealing the presence of the analyte in the sample. As indicated, in some cases the presence of the analyte indirectly denotes the presence of another substance (e.g., a pathogenic microorganism).

[0152] In the present invention, the expression "the amount of indicator in the medium is proportional to the analyte concentration in the sample" refers to a proportional relationship between the indicator in the medium and the analyte in the sample; that is, a change in the amount of indicator in the medium corresponds to a change in the analyte concentration in the sample. As indicated, in some cases the analyte concentration indirectly denotes the concentration of another substance (e.g., a pathogenic microorganism).

[0153] In the present invention, the expression "the presence of the indicator in the medium is indicative that the subject suffers from a disease" refers to the fact that the organic vesicles have released the chemical messenger, which has been recognized by the molecular gate of the porous material, unlocking its pores and releasing the indicator into the test medium, thus revealing the presence of analyte in the sample, and by association the disease caused by or related to said analyte.

[0154] Pharmaceutical Composition of the Invention: The system of the invention, together with pharmaceutically acceptable excipients, can form part of a pharmaceutical composition. Therefore, in another aspect, the present invention relates to a pharmaceutical composition comprising the system of the invention and a pharmaceutically acceptable excipient.

[0155] The term "pharmaceutically acceptable excipient" refers to any ingredient that is essentially non-toxic to a subject at the dose and concentration used and that is added to the system of the invention, among other possible applications, to give it form, facilitate its production, preserve it, facilitate its administration, or regulate its activity in the body. Suitable excipients include excipients commonly used in pharmaceutical products, such as microcrystalline cellulose, lactose, starch, magnesium stearate, crosspovidone, povidone, and talc. Likewise, such pharmaceutical excipients may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably used as vehicles, particularly for injectable solutions.

[0156] The excipients required to manufacture the desired pharmaceutical composition will depend, among other factors, on the chosen route of administration. The system of the invention can be administered by any procedure that delivers the system, preferably to the desired tissue. The compositions can be administered, for example, orally, topically, dermally, nasally, intravenously, intramuscularly, intraperitoneally, intracerebromedullary, intracranially, intramedullary, subcutaneously, intra-articularly, intrasynovially, or intrathecally. Other routes of administration are not excluded.

[0157] Examples of pharmaceutical compositions include any solid composition (e.g., tablets, pills, capsules, granules) or liquid composition (e.g., solutions, suspensions, lotions, or emulsions).

[0158] The composition according to the present invention can be produced according to standard procedures known to those skilled in the art, such as those described or referred to in the Spanish and United States Pharmacopoeias and similar reference texts.

[0159] In particular embodiments, the pharmaceutical composition comprises the system of the invention, where the porous material comprises an indicator, with the objective of detecting and / or quantifying an analyte indicative of a disease and / or diagnosing a disease in a subject.

[0160] In particular embodiments, the pharmaceutical composition comprises the system of the invention, where the porous material comprises a therapeutic agent, with the objective of treating a disease in a subject.

[0161] In particular embodiments, the pharmaceutical composition comprises the system of the invention, where the porous material comprises an indicator and therapeutic agent for the purpose of detecting and / or quantifying an analyte indicative of a disease in a subject and / or for the diagnosis of a disease in a subject and / or for the treatment of a disease in a subject.

[0162] The pharmaceutical composition of the invention allows the indicator and / or therapeutic agent to be released only where and when needed. Thus, only if the system of the invention comes into contact with the analyte will the chemical interaction between the organic vesicles and the porous material occur; that is, the chemical messenger will be released from the organic vesicles, thereby opening the molecular gates of the porous material and releasing the indicator and / or therapeutic agent. In healthy subjects, since there is no analyte present, the indicator and / or therapeutic agent is not released.

[0163] Uses and methods of in vivo use of the system and pharmaceutical composition of the invention

[0164] Throughout the preceding paragraphs, the operation of the system and its usefulness as part of a pharmaceutical composition have been demonstrated. Therefore, in another aspect, the present invention relates to the system or pharmaceutical composition of the invention for use in the detection and / or quantification of an analyte indicative of a disease in a subject and / or for the diagnosis of a disease in a subject and / or for the treatment of a disease in a subject. Likewise, in another aspect, the invention relates to the use of the system of the invention for the preparation of a pharmaceutical composition or medicament for the treatment of a disease in a subject.Likewise, in another aspect, the invention relates to a method of treating a subject, preferably a human or a bovine animal, suffering from a disease, or likely to suffer from a disease, comprising administering to the subject requiring such treatment or prophylaxis an effective quantity of the system of the invention. The terms "disease-indicating analyte," "subject," and "disease" have been defined or explained in preceding paragraphs, and those definitions and particular embodiments thereof are applicable to the present inventive aspect. Thus, in one particular embodiment, the analyte is a toxin produced by a pathogenic organism. In more preferred embodiments, the analyte is α-hemolysin, one of the principal toxins produced by the bacterium Staphylococcus aureus, and the disease is a disease caused by S.aureus, preferably selected from the group consisting of: staphylococcal scalded skin syndrome, food poisoning, staphylococcal toxic shock syndrome, skin abscess, impetigo, folliculitis, anthrax (furunculosis), cellulitis of face and neck, hidradenitis suppurativa, mastitis, bacteremia, sepsis, endocarditis, pneumonia, empyema, osteomyelitis, septic arthritis, meningitis, peritonitis, pericarditis, pyomysitis, and disseminated intravascular coagulation syndrome.

[0165] As used herein, the terms "treat," "treating," and "treatment" generally include the eradication, elimination, reversal, relief, modification, or control of disease after its onset.

[0166] As used herein, the terms "prevention," "preventing," "preventive," "prevent," and "prophylaxis" refer to the ability of a therapeutic agent to avoid, minimize, or hinder the occurrence or development of disease before it arises.

[0167] An "effective" or "therapeutically effective" amount of a drug or active therapeutic agent is understood to be a non-toxic but sufficient quantity of the drug or agent to produce the desired effect. In the therapies of the present invention, an "effective amount" of the system of the invention is the amount of that system that is effective in producing the desired effect. The amount that is "effective" will vary from subject to subject, depending on the individual's age and general condition, the particular active agent(s), and other factors. Therefore, it is not always possible to specify an exact "effective amount." However, a person skilled in the art can determine an appropriate "effective" amount in any individual case using routine experimentation.

[0168] As used herein, the term "approximately" or "around" when applied to one or more values ​​of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "around," as used herein, means that a number may vary up to ±20%, preferably within ±10%, and more preferably within ±5%. When "approximately" is used before a range, it applies to the upper and lower limits of the range.

[0169] In fact, experts in the field know that numerical values ​​related to measurements are subject to measurement errors that limit their precision. When terms such as "around" or "approximately" are applied to a particular value (e.g., "around 200 °C" or "approximately 200 °C") or to a range (e.g., "from approximately x to approximately y"), the value or range can be interpreted as being as precise as the method used to measure it. Unless specifically stated otherwise, the general convention in scientific and technical literature can be applied such that the last digit of numerical values ​​preferably indicates the precision of the measurement. Therefore, unless other margins of error are given, the maximum margin can be determined by applying the rounding convention to the last decimal place.For example, a value of 3.5 preferably has a margin of error of 3.45 to 3.54, and a range of 2% to 10% preferably covers a range of 1.5% to 10.4%. Such variations from a specified value are understood by a person skilled in the art and are within the context of the present invention. Furthermore, for the sake of clarity, some of the quantitative expressions given herein are not qualified by the term "approximately." It is understood that, whether or not the term "approximately" is used explicitly, each quantity given herein is intended to refer to the actual given value, and also to the approximation to that given value that would reasonably be deduced based on ordinary skill in the art, including equivalents and approximations due to experimental and / or measurement conditions for that given value.

[0170] Concentrations, quantities, and other numerical data may be expressed or presented herein in an interval format. It is understood that such an interval format is used simply for convenience and brevity and should therefore be interpreted flexibly to include not only the numerical values ​​explicitly listed as the limits of the interval, but also all individual numerical values ​​or subintervals within that interval as if each numerical value and subinterval were explicitly listed. For illustration, a numerical interval of "approximately 1% by weight to approximately 5% by weight" should be interpreted to include not only the explicitly listed values ​​from approximately 1% by weight to approximately 5% by weight, but also individual values ​​and subintervals within the stated interval.Therefore, this numerical interval includes individual values, such as 2, 3, and 4, and subintervals such as 1-3, 2-4, and 3-5, etc. This same principle applies to intervals that list a single numerical value.

[0171] The following examples are provided solely as a further illustration of the invention and should not be interpreted as defining the limits of the invention.

[0172] EXAMPLES

[0173] Example 1. Sensor for the detection of Staphylococcus aureus through the detection of α-hemolysin based on chemical communication between lipid vesicles and nanoparticles equipped with molecular gates

[0174] The invention will then be illustrated by means of tests carried out by the inventors, which demonstrate the preparation and operation of the system of the invention and its effectiveness as a biosensor for the detection of Staphylococcus aureus.

[0175] In particular, a communication system activated by α-hemolysin, a toxin produced by the bacterium S. aureus, is shown. The presence of α-hemolysin is an indicator of bacterial infection. Specifically, α-hemolysin in milk samples has been proposed as a biomarker for bovine mastitis, a herd disease that causes losses of up to $31.4 billion annually.

[0176] The exemplified system comprises giant unilamellar vesicles (GUVs) and mesoporous silica nanoparticles (MSNs) equipped with molecular gates. The micro-sized lipid vesicles (GUVs) were formulated to allow the insertion of α-hemolysin into their membrane, creating pores that permit the flow of small molecules (<2 kDa). The GUVs were loaded with acetylcholine (ATCh), a chemical messenger derived from the neurotransmitter acetylcholine, which is positively charged and impermeable to lipid membranes. The nanoparticles were loaded with a fluorophore indicator ([Ru(bpy)3]Ch) and coated with the enzyme acetylcholinesterase (AChE), attached to the nanoparticle surface via pH-sensitive bonds.

[0177] In the presence of α-hemolysin (analyte) as a "communication initiator," GUVs release ATCh as a chemical messenger for the nanoparticles. In a second step of the communication, the AChE-functionalized nanoparticles recognize the ATCh. The AChE catalyzes the decomposition of ATCh into thiocholine and acetic acid, increasing the pH and inducing the release of the nanoparticle's payload, i.e., the indicator agent.

[0178] This approach is versatile, as it allows for the combination of modified organic vesicles with nanoparticles equipped with custom-designed molecular gates through the rational design of the vesicle composition, molecular gate, and chemical messenger. Advantageously, this system of sensitive vesicles and nanoparticles with interconnected molecular gates can be easily integrated into detection, quantification, and / or diagnostic kits such as strip-based lateral flow assays (LFAs).

[0179] 1. Reagents

[0180] 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lysamine rhodamine B sulfonyl) (RhB-DOPE) were purchased from Avanti Polar Lipids.

[0181] Tetraethyl orthosilicate (TEOS), n-cetyltrimethylammonium bromide (CTABr), sodium hydroxide (NaOH), tris(2,2'-bipyridyl) dichlorouthenium(II) hexahydrate [Ru(bpy)3]Cl2'6H2O, 3-glycocyclooxypropyltrimethoxysilane, 3-aminophenylboronic acid, cholesterol, paraffin oil, glucose, sucrose, bovine serum albumin (BSA), acetylcholinesterase from Electrophorus electricus (type VI-S), acetylcholine chloride, acetylthiocholine iodide, trisodium salt of 8-hydroxypyrene-1,3,6-thosulfonic acid (HPTS), and α-hemolysin from S. aureus were purchased from Sigma Aldrich.

[0182] The camera microscope slides (p-plate, 18 wells, glass bottom, 1.5H) were acquired from Ibidi.

[0183] The GF / C grade glass microfiber filter papers were purchased from Whatman™.

[0184] 2. Synthesis and functionalization of mesoporous silica nanoparticles (MSNs)

[0185] First, 1 g (2.74 mmol) of n-cetyltrimethylammonium bromide (CTABr) was dissolved in 480 mL of deionized water and stirred for 20 min at 400 rpm. Then, the pH was made basitic by adding 3.5 mL of a 2 mol L NaOH solution. -1The temperature was then increased to 80 °C. Once 80 °C was reached, 5 mL (22.4 mmol) of tetraethyl orthosilicate (TEOS) was added dropwise to the solution while stirring. Magnetic stirring was continued for 2 h to obtain a white suspension. The solid was isolated and washed three times with water by centrifugation at 9500 rpm until a neutral pH was reached. The solid was then dried overnight at 70 °C. To obtain the final mesoporous MCM-41 type nanoparticles, the solid was calcined at 550 °C in an oxidizing atmosphere for 5 h to remove the surfactant.

[0186] To functionalize the surface of the MSNs, 100 mg of MSNs were resuspended in 15 mL of acetonitrile. Then, 500 mL of (3-glycoxypropyl) trimethoxysilane (2.26 mmol) were added, and the mixture was stirred for 5.5 h at room temperature. Subsequently, the solid was isolated by centrifugation at 12,000 rpm for 3 min and dried overnight at 37 °C. The solid was then resuspended in 15 mL of toluene, and 154 mg (1.13 mmol) of 3-aminophenylboronic acid was added. The suspension was then stirred overnight. Finally, the solid was washed by centrifugation once with toluene and once with acetonitrile and resuspended in 15 mL of acetonitrile. To load the MSNs with the indicator, 150 mg of [Ru(bpy)3]Cl2·6H2O were added and the suspension was stirred overnight. The solid was then washed twice with toluene and dried at 37 °C overnight.To coat the MSNs with the enzyme acetylcholinesterase, 0.5 mg of enzyme and 5 mg of MSNs were suspended in 500 ml of 50 mM phosphate buffer at pH 7.5. The mixture was stirred overnight at 4 °C to prevent enzyme degradation. Finally, the functionalized MSNs were washed twice, resuspended in 500 ml of phosphate buffer, and stored at -20 °C until use.

[0187] 3. Synthesis of giant unilamellar vesicles (GUVs)

[0188] Stock solutions of DOPC, POPC, and cholesterol were prepared in chloroform at 100 mg / ml, DSPE-PEG was prepared at 10 mg / ml, and RhB-DOPE was prepared at 1 mg / ml. All solutions were stored at -20 °C until use.

[0189] The drop-transfer method was used to synthesize the GUVs. First, DOPC, POPC, and cholesterol were mixed in a 25 / 25 / 50 molar ratio in a glass vial (70 x 10 x 0.8–1.0 mm) to obtain a combined amount of 2 pmol. DSPE-PEG was added at a 1 molar ratio to increase GUV stability and prevent membrane fusion. RhB-DOPE was added at a 0.2 molar ratio when confocal imaging was required. Then, 200 pL of paraffin oil was added to resuspend the lipids in a nonpolar solution. To evaporate any remaining chloroform, the glass vials were incubated in a sand bath for 30 minutes at 80 °C, with vertex shaking once or twice for 5 seconds between each incubation. Different internal phase compositions (Fl, aqueous phase inside the GUVs) were prepared.The Fl for confocal imaging of GLIVs was prepared using 0.2 M sucrose, 1X PBS (pH 7.5), and 50 pM HPTS. Separately, the Fl for chemical communication between GLIVs and MSNs was prepared with 80 mM acetylcholine, 0.2 M sucrose, and 0.02X PBS. Twenty milliliters of pre-cooled Fl solutions were then introduced into 200 milliliters of paraffin-lipid solutions and vortexed for 30 seconds while tilting the glass tubes. The glass tubes were then incubated again on ice for at least 10 minutes to allow the formation of a lipid monolayer. The external phase (EF, aqueous phase outside the GUVs) for confocal imaging was prepared using 0.2 M glucose and 1X PBS. Whereas the FE when the GUVs were prepared for communication was prepared using 0.2 M glucose, 0.02X PBS and 80 mM NaCl to avoid differences in osmotic pressure.Next, 150 µl of FE were introduced into 1.5 ml Eppendorf tubes and pre-cooled on ice. Lipid emulsions were then placed on top of the 150 µl of FE in the Eppendorf tubes and incubated at 4 °C for 5–10 minutes to allow the formation of the interfacial lipid monolayer. The tubes were then centrifuged at 4 °C for 20 minutes at 3300 x g. After centrifugation, the Eppendorf tubes were pierced with a 21-gauge needle at the site of the GUV pellet, leaving the cap open. The Eppendorf cap was then closed to allow collection of the aqueous solution using forceps, while avoiding the collection of the paraffin phase. To remove any non-encapsulated compound, the GUVs were washed 3-4 times by replacing the supernatant with 80 pl of fresh FE and centrifuging at 2000 xg for 2 min.

[0190] 4. Release of an encapsulated fluorophore in GUVs and confocal imaging

[0191] To visualize the charge release of GUVs using confocal imaging, a camera microscope slide was used. First, the wells of the slide were neutralized with 100 pl of 1 mg mi' 1The GUVs were infused with bovine serum albumin (BSA) for 10 minutes and washed once with ultrapure water. Next, the GUVs loaded with 50 pM HPTS were resuspended in 60 µl of FE. Then, 2.5 µl of the GUV suspension containing 100 µl of FE was introduced into the wells. The GUVs were then allowed to settle to the bottom for 10 minutes. Imaging was performed using a Leica TCS DM18 confocal microscope (Leica Microsystems, n.d.). The encapsulated fluorophore (HPTS) was excited at 488 nm, while the membrane marker (RhB-DOPE) was excited at 532 nm. Fluorescence emissions for HPTS and RhB-DOPE were collected at 500–530 nm and 550–620 nm, respectively. Before and after the addition of α-hemolysin (20 pg / min) 1 ) and 10 min of incubation, images were taken in the same plane with an ACS APO 40x / 1 , 15 oil objective (resolution 512 x 512).

[0192] 5. Release of the ATCh payload from the GUVs

[0193] GUVs were prepared with 80 mM encapsulated ATCh. The GUV pellet was then incubated with 30 µl of external phase containing α-hemolysin for 10 minutes. Subsequently, the samples were centrifuged at 2000 x g for 2 minutes to precipitate the GUVs, and the 30 µl of supernatant were transferred to solutions containing MSNs to study their charge release.

[0194] 6. Release of an indicator from MSNs in aqueous medium

[0195] To study the release of the encapsulated indicator in the MSNs, stock solutions were taken at -20 °C (10 mg / ml) 1 ) and were washed 3 times with 0.02 X PBS at pH 7.5. Subsequently, the MSNs were resuspended in at a concentration of 2 mg / ml 1The samples obtained in step 5, resulting from the incubation of the GUVs with and without α-hemolysin, were used. The samples were shaken at 25 °C and 900 rpm on a thermoshaker, and absorbance measurements were taken at 452 nm at different times. Before each measurement, the MSNs were centrifuged at 12,000 rpm for 3 min to precipitate the solid.

[0196] The quantification of the [Ru(bpy)s]Cl2 released was performed by calculating the concentration using the following formula:

[0197] Where C is the concentration (M), A is the absorbance at 452 nm and E is the molar extinction coefficient of [Ru(bpy)s]Cl2 at 452 nm (14.6 mM' 1 cm -1 )

[0198] 7. Lateral flow experiments on test strips

[0199] As a first step in preparing the strips, two sheets of double-sided tape (2.5 cm wide) were adhered to a plastic sheet, one below the other, to provide a covered surface 5 cm wide. The backings were then removed, and a sheet of glass microfiber filter paper was affixed to the double-sided tape. Finally, 5 µl of MSN suspension (1 mg ml) were deposited onto the tape. 1) 1 cm from the edge, with a distance of 0.5 cm between each drop. After drying the MSN spots for 5 min, individual strips were cut to a size of 0.5 x 5 cm. If necessary, the strips could be stored at 4 °C for up to 4 weeks without loss of enzyme and gate functions. For a lateral flow test, the strips were introduced and incubated for 5 min in Eppendorf tubes containing the GLIV pellet and 120 µl of sample. When studying charge release at increasing concentrations of ATCh, the samples consisted of 0.02 X PBS at pH 7.5 and ATCh at different concentrations. When studying charge release at increasing concentrations of α-hemolysin, the samples consisted of the external phase of GUV with different concentrations of α-hemolysin. The tests were performed directly in the Eppendorf tubes where the GLIVs were prepared.Subsequently, the strips were immediately inserted into a custom 3D-printed strip holder. The fluorescence of the released dye was measured using a smartphone, with an LED emitting at 465 nm, a short-pass filter at 500 nm to eliminate residual light from the LED, and a long-pass filter at 550 nm to efficiently collect the fluorescent emission of the [Ru(bpy)s]Cl2 dye released along the membrane. The collected images of the strips were analyzed using ImageJ software, analyzing the integrated red channel fluorescence density of the samples while correcting for background fluorescence.

[0200] 8. Characterization

[0201] Transmission electron microscopy (TEM) images confirmed the formation of MSNs with a mean diameter of 104 ± 20 nm (n=80), porous structure and spherical morphology (Figure 3).

[0202] The N2 adsorption-desorption isotherm showed the characteristic adsorption step at P / P0 of 0.3 for MSN with empty pores, related to nitrogen condensation within the pores by capillarity (Figure 4). The total specific surface area calculated using the BET model was 1118 m². 2 g -1 On the other hand, the average volume and pore size, obtained using the BJH model associated with adsorption within the pores, were 1.01 cm³ 3 g -1 and 3.08 nm respectively.

[0203] The powder X-ray diffraction of the MSNs before calcination (Figure 5) shows the low-angle reflection peaks characteristic of MCM-41 type mesoporous scaffolds. The peaks of the MSNs after calcination were shifted slightly to the right, which is related to the condensation of the silanol groups after calcination.

[0204] Following hydrolysis of ATCh by AChE, Ellman's reagent (DTNB) is able to interact with the thiol group of thiocholine, which cleaves the disulfide bond of DTNB, generating a yellow compound (TNB 2- ) with a maximum absorbance at 412 nm. A calibration curve was prepared with different concentrations of ATCh using an excess of Ellman reagent (DTNB) and allowing time for the AChE to completely hydrolyze the ATCh.

[0205] The immobilization of the enzyme on the surface of the MSNs was confirmed by an enzymatic assay. The formation of TNB 2 The concentration was monitored over time for 3 minutes using a UV-visible spectrophotometer. To prepare the assay, 100 µl of 1x PBS, 3 µl of 10 mM DTNB, and 2 µl of 0.2 M ATCh were placed in a quartz cuvette. Then, 15 µl of 1x PBS (control) or MSNs (0.1 mg / mL) were added. 1) and were mixed by pipetting. Finally, the absorbance was measured at 412 nm for 3 minutes (Figure 6).

[0206] Acetylcholinesterase activity in MSNs was estimated at 2464 ± 62 U g -1 using the following formula:

[0207] Where, A is the slope of the graph (min -1 ), Abiank is the slope of the graph for white (min -1 ), VT is the total volume in the cuvette, £TNB is the molar extinction coefficient of TNB 2 ' at 412 nm (13.7 mM' 1 cm -1 ), I is the optical path in the cuvette (1 cm), VNPS is the volume of nanoparticles added (mL) and CNPS is the concentration of added nanoparticle suspension (g mL' 1 ).

[0208] 8. Results and discussion

[0209] As a first step, the system components were studied individually, starting with the GUVs and their ability to encapsulate and release their cargo in response to α-hemolysin. To assemble the GUVs, the droplet transfer method was used, which involves preparing a water-in-oil solution with the lipids that will make up the lipid membrane in the oil phase and the material to be encapsulated in the water droplets. It has been previously shown that α-hemolysin can bind to and induce cargo release from lipid vesicles composed of phosphatidylcholines (PCs) such as DOPC and POPC. In this example, the role of cholesterol was studied by encapsulating a fluorophore in various populations of GUVs with different molar ratios of cholesterol and observing its release after 10 minutes of incubation with α-hemolysin (Figure 7).After obtaining confocal images, a cholesterol molar ratio of 50% was chosen for the preparation of GUVs, while DOPC and POPC were set at a molar ratio of 25% each.

[0210] Mesoporous silica nanoparticles (MSNs) were selected as the gating material to function as the acceptor nanoparticle, offering high loading capacity, easy functionalization, and high specificity. The MSNs were prepared using the sol-gel template method and characterized using various standard techniques. Powder X-ray diffraction confirmed the low-angle reflection peaks characteristic of MCM-41 type mesoporous scaffolds. Transmission electron microscopy (TEM) images confirmed the formation of MSNs with a mean diameter of 104 ± 20 nm (n=80), a porous structure, and a spherical morphology. To functionalize the [Ru(bpy)s]Cl2-loaded MSNs, they were coated with the enzyme AChE, which was attached to the nanoparticle surface via cyclic phenylboronic acid esters that cleave upon acidification. The total [Ru(bpy)s]Cl2 loaded in the MSNs was determined to be 76.5 pg mg -1MSNs were agitated by shaking the nanoparticles for 1 h at pH 2. The activity of AChE in the MSNs was also studied using the Ellman assay following the transformation of DTNB to TNB 2 'Over time, in the presence and absence of functionalized MSNs, only rapid TNB formation was observed. 2- in the presence of MSNs, with an estimated AChE activity of 2464 ± 62 U per g of MSNs. The amount of AChE bound to the MSNs was also calculated to be 13.7 pg mg -1 from MSNs, comparing the formation of TNB 2The response of the MSNs to ATCh was studied by monitoring the release of [Ru(bpy)s]Cl2 in the presence and absence of 20 mM ATCh for 1 h (Figure 8). The samples were incubated in 0.02X PBS to avoid buffering the pH drop, and absorbance measurements were taken at 452 nm at scheduled times. Charge release was demonstrated after 1 h of incubation with ATCh alone. Subsequently, functionalized MSNs were deposited onto strips (5 pg), and charge release by LFA was studied as a function of the ATCh concentration. After 5 minutes of LFA, photographs of the strips were immediately taken with a smartphone camera under suitable lighting conditions (ISO 100 and exposure time of 1 / 8 s) (Figure 9A).The fluorescence intensity of the strip photographs was analyzed by extracting the integrated fluorescence density from the charge release using ImageJ software to determine the limit of detection (LOD) (Figure 9B). The LOD was calculated to be 0.18 mM ATCh.

[0211] After studying GUVs and MSNs individually, the feasibility of chemical communication was investigated. To this end, different GUV preparations were incubated for 10 min in the presence and absence of α-hemolysin (100 pg / mL). Subsequently, the released contents of the GUVs were transferred to a tube containing MSNs (2 mg / mL). 1The release of [Ru(bpy)s]Cl2 from these samples was measured. As shown in Figure 10, a significant release of this indicator from the MSNs was observed only when the GUV preparations were incubated with α-hemolysin. Motivated by these results, the inventors aimed to apply this chemical communication system to a strip-based LFA to obtain a rapid, portable, and easy-to-use test for the detection of α-hemolysin. The development of these LFA tests would be particularly interesting in the livestock industry for the rapid detection of the presence of S. aureus using a non-invasive test on milk samples. Strips were prepared with functionalized MSNs (5 pg), and after incubating the GUVs with the samples for 10 min, LFAs were performed for 5 min. The LFA tests were carried out directly in the Eppendorf tubes where the ATCh-loaded GUVs were prepared.In this system, the absorption of GUVs on the surface of the strips was also avoided, since the GUVs sink naturally after 10 minutes due to the higher density of the encapsulated internal aqueous phase, and because the bottom of the strip did not touch the GUV pellet. Therefore, LFA strip tests were performed by exposing the GUV pellet to different concentrations of α-hemolysin (from 12.5 to 100 pg / mL). 1 The strips were photographed immediately (ISO 100 and exposure time of 1 / 8 s) (Figure 11A). The release of the indicator on the strips increased with increasing α-hemolysin concentration. The fluorescence intensity of the dye released from the MSNs was analyzed using ImageJ to establish a calibration curve (Figure 11B), from which an LOD of 15.1 pg / mL was calculated. 1 .

[0212] Conclusions

[0213] This example presents the design of a chemical communication system between GUVs and MSNs that responds to the presence of α-hemolysin, as well as its implementation in strip-based LFA assays as a nanosensor for the detection of S. aureus. In the presence of α-hemolysin, GUVs loaded with the neurotransmitter derivative ATCh release their charge, which is detected by AChE-activated MSNs, which then release a fluorescent marker. In the first step, the charge release of GUVs and MSNs was studied individually and confirmed to occur only in the presence of α-hemolysin and ATCh, respectively. Subsequently, the chemical communication between the two components of the system was also confirmed by studying the release of a marker from the MSNs, while in the absence of α-hemolysin, only the typical residual release was observed. For the development of a nanosensor, MSNs were deposited on strips, and LFA assays were performed for different concentrations of α-hemolysin.The functionality of the strips for detecting alpha-hemolysin was demonstrated. Finally, the nanosensor was implemented in milk samples, and the strips' ability to differentiate samples containing alpha-hemolysin was confirmed. The development of these sensors is of particular importance in the livestock industry to avoid the indiscriminate and widespread use of antibiotics when cattle are sick, which leads to antibiotic resistance. By eliminating the need for complex equipment and techniques, this sensor could be used by non-specialized personnel. Furthermore, the entire assay would only take 15 minutes, making the detection of S. aureus much faster than with cell culture or DNA assays.

[0214] List of references

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[0222] [8] Climent, E.; Martinez-Manez, R.; Maquieira, A.; Sancenón, F.; Dolores Marcos, M.; Brun, E. M.; Soto, J.; Amoros, P., Antibody-Capped Mesoporous Nanoscopic Materials: Design of a Probe for the Selective Chromo-Fluorogenic Detection of Finasteride. ChemistryOpen 2012, 1 (6), 251-259.

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[0224]

[0010] Costa, E.; Climent, E.; Gawlitza, K.; Wan, W.; Weller, M. G.; Rurack, K., Optimisation of analytical assay performance of antibody-gated indicator releasing mesoporous silica particles. Journal of Materials Chemistry B 2020, 8 (22), 4950-4961

[0011] Oroval, M.; Climent, E.; Coll, C.; Eritja, R.; Avino, A.; Marcos, M. D.; Sancenon, F.; Martinez-Manez, R.; Amoros, P., An aptamer-gated silica mesoporous material for thrombin detection. Chem. Commun. 2013, 49 (48), 5480-5482.

[0225]

[0012] Climent, E.; Rurack, K., Combining Electrochemiluminescence Detection with Aptamer- Gated Indicator Releasing Mesoporous Nanoparticles Enables ppt Sensitivity for Strip- Based Rapid Tests. Angew. Chem. Int. Ed. 2021, 60 (50), 26287-26297.

[0226]

[0013] ES2767873A1

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[0014] Climent, E.; Martínez-Máñez, R.; Sancenón, F.; Marcos, M. D.; Soto, J.; Maquieira, A.; Amorós, P., Controlled Delivery Using Oligonucleotide-Capped Mesoporous Silica Nanoparticles. Angew. Chem. Int. Ed. 2010, 49 (40), 7281-7283.

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[0015] Climent, E.; Mondragon, L; Martínez-Máñez, R.; Sancenón, F.; Marcos, M. D.; Murguia, J. R.; Amorós, P.; Rurack, K.; Pérez-Payá, E., Selective, Highly Sensitive, and Rapid Detection of Genomic DNA by Using Gated Materials: Mycoplasma Detection. Angew. Chem. Int. Ed. 2013, 52 (34), 8938-8942.

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[0016] WG2012 / 007623A2

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[0017] Garrido-Cano, I.; Pla, L; Santiago-Felipe, S.; Simon, S.; Ortega, B.; Bermejo, B.; Lluch, A.; Cejalvo, JM; Eroles, P.; Martínez-Mánez, R., ACS Sens., 2021, 6, 3, 1022-1029.

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[0022] Buddingh' BC, Elzinga J, van Hest JCM. Intercellular communication between artificial cells by allosteric amplification of a molecular signal. Nat Commun. 2020;11(1):1652.

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[0023] Climent, E.; Groninger, D.; Hecht, M.; Walter, MA; Martínez-Máñez, R.; Weller, M.G.; Sancenón, F.; Amoros, P.; Rurack, K., Selective, Sensitive, and Rapid Analysis with Lateral-Flow Assays Based on Antibody-Gated Dye-Delivery Systems: The Example of Triacetone Triperoxide. Chem. Eur. J. 2013, 19 (13), 4117-4122.

[0024] Garrido E, Climent E, Marcos MD, Sancenón F, Rurack K, Martínez-Máñez R. Dualplex lateral flow assay for simultaneous scopolamine and "cannibal drug" detection based on receptor-gated mesoporous nanoparticles. Nanoscale. 2022; 14(37): 13505-13513.

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[0025] Aznar E, Oroval M, Pascual L, Murguia JR, Martínez-Máñez R, Sancenón F. Gated Materials for On-Command Release of Guest Molecules. Chem Rev. 2016;116(2):561-

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Claims

1. CLAIMS 1. A system comprising: - Organic vesicles that contain a chemical messenger and are capable of releasing said chemical messenger in the presence of an analyte; and - A porous material comprising an indicator and / or an active agent within its pores and bonded on its surface to a chemical entity functioning as a molecular gate, wherein said molecular gate is arranged in a position such that: (i) in the absence of a chemical messenger released by organic vesicles, the molecular gate blocks access to the exterior of the pore, preventing the exit of the indicator and / or the active agent; and (i) In the presence of a chemical messenger released by organic vesicles, the molecular gate recognizes and interacts with said chemical messenger, and unlocks access to the outside of the pore, allowing the indicator and / or active agent to exit.

2. System according to claim 1, wherein the organic vesicles are selected from the group consisting of lipid vesicles, niosomes, polymersomes, proteinosomes, cationic vesicles, and cationic vesicles.

3. System according to any of claims 1 and 2, wherein the organic vesicles are selected from the group consisting of unilaminar vesicles and multilaminar vesicles.

4. System according to any of claims 1 to 3, wherein the organic vesicles are unilaminar lipid vesicles.

5. System according to claim 4, wherein the unilamellar lipid vesicles are formed by a bilayer comprising a phospholipid and cholesterol.

6. System according to claim 4, wherein the unilamellar lipid vesicles are formed by a bilayer comprising phosphatidylcholine or a derivative thereof and cholesterol in a molar ratio ranging from approximately 85 / 15 to 40 / 60.

7. System according to any of claims 1 to 6, wherein the organic vesicle chemical messenger is selected from the group consisting of acetylcholine, glucose, sucrose, lactose, ester derivative, cysteamine, cocaine, reducing agent, metal cation, or a mixture of the same; preferably, acetylcholine.

8. System according to any of claims 1 to 7, wherein the analyte is α-hemolysin, sphingomyelinase, phospholipase, listeriolysin O, surfactant agents, cholesterol-dependent cytolysins (CDCs) or a mixture thereof; preferably α-hemolysin.

9. System according to any of claims 1 to 8, wherein the porous material is selected from the group consisting of metals, semiconductors, organic polymers, carbons or oxides, or a mixture thereof; preferably silica, alumina or a combination thereof.

10. System according to any of claims 1 to 9, wherein the porous material is a porous plate or porous nanoparticles.

11. System according to any of claims 1 to 10, wherein the porous material is mesoporous; preferably mesoporous silica nanoparticles.

12. System according to claim 11, wherein the mesoporous silica nanoparticles are of type MCM-41 with a particle size between 80 nm and 120 nm and a pore diameter between 2 nm and 6.5 nm.

13. System according to any of claims 1 to 12, wherein the indicator is selected from the group consisting of a colohmethic indicator, a fluorimetric indicator, an electrochemiluminescent indicator, or a mixture thereof.

14. System according to any of claims 1 to 13, wherein the active agent is a therapeutic agent, preferably selected from the group consisting of an antibiotic agent, an antifungal agent, an antiviral agent, an antiprotozoal agent, an anticancer agent, or a mixture thereof.

15. System according to any of claims 1 to 14, wherein the chemical entity with molecular gate function is selected from the group consisting of the enzyme acetylcholinesterase, oligonucleotides, polymers and cyclodextrins, disulfide-linked polyethylene glycol chains (SS-PEG), boronate-derived complex with cyclodextrin, enzymes, antibodies, aptamers or a mixture thereof.

16. In vitro test kit comprising a system according to any of claims 1 to 15, wherein the porous material comprises an indicator.

17. Kit according to claim 16, wherein the kit is a lateral flow assay (LFA) kit.

18. In vitro use of a system according to any of claims 1 to 15 or of an assay kit according to any of claims 16 or 17 for the detection and / or quantification of an analyte in a sample.

19. Use according to claim 18 wherein the sample is selected from the group consisting of a food sample, an environmental sample or a clinical sample, preferably the clinical sample is selected from the group consisting of blood, serum, sputum, pleural, peritoneal, synovial or cerebrospinal fluid.

20. Use according to claim 18 or 19 wherein the analyte is a toxin produced by a pathogenic organism, preferably the analyte is α-hemolysin and the pathogen is S. aureus.

21. In vitro use of a system according to any of claims 1 to 15 or of an assay kit according to any of claims 16 or 17 for the diagnosis of a disease in a subject that is caused by or related to an analyte.

22. Use according to claim 21 wherein the analyte is α-hemolysin and the disease is a disease caused by S. aureus, preferably selected from the group consisting of: staphylococcal scalded skin syndrome, food poisoning, staphylococcal toxic shock syndrome, skin abscess, impetigo, folliculitis, anthrax (furunculosis), cellulitis of face and neck, hidradenitis suppurativa, mastitis, bacteremia, sepsis, endocarditis, pneumonia, empyema, osteomyelitis, septic arthritis, meningitis, peritonitis, pericarditis, pyomysitis, and disseminated intravascular coagulation syndrome.

23. Use according to claim 21 or 22, wherein the subject is a human being or a bovine animal.

24. An in vitro method for detecting and / or quantifying an analyte in a sample comprising: a) contacting a system according to any one of claims 1 to 15 or a kit according to any one of claims 16 or 17 with the sample, and b) detecting or measuring the indicator in the medium, wherein the presence of the indicator in the medium is indicative of the presence of the analyte in the sample, and / or the amount of the The concentration of the indicator in the medium is proportional to the concentration of the analyte in the sample.

25. Method according to claim 24 wherein the sample is selected from the group consisting of a food sample, an environmental sample or a clinical sample, preferably the clinical sample is selected from the group consisting of blood, serum, sputum, pleural, peritoneal, synovial or cerebrospinal fluid.

26. Method according to claim 24 or 25 wherein the analyte is a toxin produced by a pathogenic organism, preferably the analyte is α-hemolysin and the pathogen is S. aureus.

27. An in vitro method for diagnosing a disease in a subject caused by or related to an analyte comprising: a) contacting a system according to any one of claims 1 to 16 or a kit according to any one of claims 17 or 18 with a sample from the subject, and b) detecting the indicator in the medium, wherein the presence of the indicator in the medium is indicative that the subject suffers from a disease.

28. Method according to claim 27, wherein the analyte is α-hemolysin and the disease is a disease caused by S. aureus, preferably selected from the group consisting of: staphylococcal scalded skin syndrome, food poisoning, staphylococcal toxic shock syndrome, skin abscess, impetigo, folliculitis, anthrax (furunculosis), cellulitis of face and neck, hidradenitis suppurativa, mastitis, bacteremia, sepsis, endocarditis, pneumonia, empyema, osteomyelitis, septic arthritis, meningitis, peritonitis, pericarditis, pyomysitis, and disseminated intravascular coagulation syndrome.

29. Method according to claim 27 or 28, wherein the sample is a clinical sample selected from the group consisting of blood, serum, sputum, pleural, peritoneal, synovial or cerebrospinal fluid.

30. Method according to any of claims 27 to 29, wherein the subject is a human being or a bovine animal.

31. Pharmaceutical composition comprising a system according to any of claims 1 to 15 and a pharmaceutically acceptable excipient.

32. System according to any one of claims 1 to 15 or pharmaceutical composition according to claim 30 for use in the detection and / or quantification of an analyte indicative of a disease in a subject and / or for the diagnosis of a disease in a subject and / or for the treatment of a disease in a subject.

33. Pharmaceutical system or composition for use according to claim 32 wherein the analyte is α-hemolysin and the disease is a disease caused by S. aureus, preferably selected from the group consisting of: staphylococcal scalded skin syndrome, food poisoning, staphylococcal toxic shock syndrome, skin abscess, impetigo, folliculitis, anthrax (furunculosis), cellulitis of face and neck, hidradenitis suppurativa, mastitis, bacteremia, sepsis, endocarditis, pneumonia, empyema, osteomyelitis, septic arthritis, meningitis, peritonitis, pericarditis, pyomysitis, and disseminated intravascular coagulation syndrome.

Images