METHOD FOR PRODUCING AN ARRAY OF PLANAR MICROPARTICLES WITH SURFACE MOLECULAR MULTIPLEXING, RESULTING ARRAY AND USE THEREOF

Abstract

A method for controlled production of an array of planar microparticles with the multiplexing of molecules on the surface thereof, intended to function as molecular sensors and/or actuators and a matrix (array) of microparticles, the surface thereof being printed with all of the molecular components required to provide the surface with functionality. Different molecular elements are multiplexed on the surface of each particle while they are supported on a substrate by means of a structural foot engraved below the particle. These microparticles can be released mechanically from the support on which they are produced using a controlled mechanical rupture method which is not chemically aggressive and therefore does not affect the molecules previously printed on the surface. The array and the particles contained therein offer great versatility in both chemical and/or biological applications.

Claims

1-26. (canceled)

27. A method for producing planar microparticles, the method comprising: a) preparing a structuration layer of a microparticle starting material on a first surface of substrate that serves as a support; b) shaping the structuration layer to form microparticles, the shaping comprising shaping a geometry and lateral dimensions of the microparticles using a microelectronic lithography technique, and defining a thickness of the microparticles using an engraving technique; c) forming feet in an upper portion of the substrate that is under the structuration layer in order to support each microparticle, by means of engraving techniques; d) forming chemically functionalized microparticles by chemically functionalizing a surface of each microparticle supported on the feet using one or more molecular components and a molecule printing technique; and e) individualizing and releasing the planar microparticles from the substrate by breaking each of the feet of the chemically functionalized microparticles by applying a mechanical breaking load.

28. The method of claim 27, wherein each of the feet are under a respective microparticle, and wherein the formation of each of the feet comprises partially engraving the substrate from the first surface toward a second surface that is opposite the first surface using a microelectronic technique, each of the feet having a height and a width that varies through the height, the width decreasing from a first surface of the substrate to a midpoint of the height and increasing from the midpoint toward the second surface of the substrate.

29. The method according to claim 27, wherein the substrate is formed of a single material, said material being a silicon sheet, or by two materials, including a second material in the form of a layer that is located in the upper part of the substrate beneath the microparticle structuration layer.

30. The method according to claim 27, wherein the structuration layer of the microparticle is a material selected from the group consisting of polycrystalline silicon, silicon oxide, nitride selected from the group consisting of silicon, gold, platinum, copper, aluminum, nickel, cobalt, chromium, metal oxides; tantalum, iron and aluminum silicates; and silicide selected from the group consisting of tantalum silicide, iron silicide and aluminum silicide; and wherein the starting material of the structuration layer and the upper portion of the substrate where the feet are engraved have a relationship between their rupture limits greater than or equal to 1.

31. The method according to claim 27, wherein the preparation of the structuration layer in stage a) is carried out by depositing or by growing said structuration layer on top of the substrate by a microelectronics technique selected from the group consisting of thermal growth, chemical vapor deposition, sputtering and evaporation.

32. The method according to claim 27, wherein the partial engraving of the foot is carried out via a lateral physical etching or a lateral chemical etching.

33. The method according to claim 27, wherein the microparticles are shaped in stage b) by means of photo-lithographic techniques.

34. The method according to claim 27, wherein the microparticles are shaped all with the same shape and size or in two or more groups with different shapes and sizes.

35. The method according to claim 27, wherein the molecule-printing technique is selected from the group consisting of microcontact printing, dip-pen nanolithography and polymer-pen lithography technique.

36. The method according to claim 27, wherein the molecular component is a molecule with chemical or biological activity, or both, selected from the group consisting of organic compounds, polymers, peptides, proteins, nucleotides, nucleic acids and any combination thereof.

37. The method according to claim 27, wherein the surface of each microparticle is functionalized in stage d) with more than one different molecular element, or with a single molecular element more than one time.

38. The method according to claim 27, wherein the mechanical breaking load is applied by means of a technique selected from the group consisting of rasping, cutting, cryofracturing.

39. The method according to claim 27, wherein the mechanical breaking load comprises applying an adhesive material on the already functionalized surface of the microparticles and subsequently pulling it off, and dissolving the adhesive in media that does not affect the molecular functionalization of the microparticle.

40. The method according to claim 27, which further comprises: f) gathering the individualized and released microparticles in a suspension medium.

Description

DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1A is a perspective view of FIG. 1B is a cross-sectional view of a representation of two possible configurations of microparticles in the array obtained through the method described, one in the shape of a parallelepiped with a width A, length L and thickness E, and the other in the shape of a circle or a disk, with a diameter D and thickness E, according to the invention, where (1) represents the material that makes the foot, (2) the microparticle and the substrate (4).

[0028] FIG. 2A is a cross-cectional view and FIG. 2B is a perspective view of a representation of a possible microparticle configuration on the support in the array, where its upper surface has been coated, in a localized manner, with different molecular elements designed for its functionalization (3).

[0029] FIGS. 3A-3H show a manufacturing process of the array of microparticles based on microelectronic technology, photo-lithographic processes and layer etching, according to Example 1, wherein after producing the microparticle matrix, these microparticles are released and prepared in suspension. A straight section that shows the manufacturing process of a microparticle, with a substrate that is mainly a silicon sheet (4) that acts as a support, upon which there is a layer (5), typically made up of polycrystalline silicon, silicon oxide, silicon nitride, gold, platinum, aluminum or chromium, which constituted the original material that became the microparticles (2) (FIG. 3A). To define the microparticles, a photo resin layer (6) was deposited on said layer of original material of the microparticles (FIG. 3B), which was then partially eliminated from specific areas (7), forming a structured photo resin layer (8) that contained the geometry and lateral dimensions of the microparticles to be produced (FIG. 3C). Said microparticles were formed (structured) by etching the layer of their starting material (5) in the uncovered areas (7) where the layer of photo resin (6) had previously been removed, thereby forming the body of the microparticle(s) (2) (FIG. 3D). Afterwards the silicon substrate (4) was engraved immediately below the microparticles (2) to form the foot (1) (FIG. 3E). The remaining photoresin (8) was removed in the upper part of the microparticles (2) (FIG. 3F). After this the molecular functionalization of the microparticles was carried out with more than one molecular element (3) (FIG. 3G). Lastly, the functionalized microparticles (2) were released from the substrate (4) by means of controlled rupture, and were gathered in an aqueous medium, in this example, previously filtered de-mineralized water, thus producing the microparticle suspension (9) (FIG. 3H).

[0030] FIG. 4 is a scanning electron microscope image showing an example of producing the array of parallelepiped-shaped silicon oxide microparticles (dimensions of 3 .Math.m × 3 .Math.m × 1 .Math.m) in accordance with Example 1 - Section A, wherein one may easily make out the microparticles arranged in array and anchored to the substrate by means of a foot. Scale bar = 5 .Math.m.

[0031] FIG. 5 is an optical fluorescence image showing an example of surface functionalization of the microparticles by means of the polymer-pen lithography technique, in accordance with Example 1 - Section B. In this case, multiple printings with two different inks are shown, namely WGA lectin conjugated with the fluorophore Streptavidin Texas Red® (red, ink marked with the letter B in the figure and visualized with darker gray shading on a black background) and the protein BSA conjugated with the fluorophore Neutravidin Oregon Green® (green, ink marked with the letter A in the figure and visualized with lighter gray shading) prior to being functionalized with the antibody Goat Anti-Rabbit IgG conjugated with the marker AMCA and a third ink (blue, not shown in the figure). In this case, the printed pattern was dots. Scale bar = 10 .Math.m.

[0032] FIG. 6 is a scanning electron microscope image showing several microparticles following their mechanical release from the substrate by means of controlled mechanical fracture of the feet in accordance with Example 1 - Section C. Scale bar = 5 .Math.m.

DESCRIPTION OF THE INVENTION

[0033] As mentioned in the foregoing section, the foot is produced through molding by engraving the upper part of the substrate, which is in direct contact with the lower part of the layer of microparticle starting material. In a particular embodiment of the invention, the substrate is formed by a single material, most preferably a silicon sheet, although it may be another suitable type of substrate that offers mechanical support for the fabrication of the microparticles, for example borosilicate glass (commonly known by its commercial name, Pyrex or Duran) or soda-lime-silica glass, among others. Nevertheless, the invention is not limited just to these support materials, since any person skilled in the art will know what types of materials are suitable and may fulfill the intended function; basically, this includes all materials that meet the following conditions: [0034] be resistant to the thermal processes of depositing, evaporating and growing layers; [0035] be stable at ambient temperature; [0036] be resistant to certain chemical agents (compounds in liquid or gas phase) in order to enable the structuration/engraving/processing in general of the layers in them without affecting the substrate (even though sometimes they need to be protected, since the chemical agents tend to be quite aggressive); and [0037] having the ability to itself be structured/engraved (wholly or partially). This would be the case in which the foot of the microparticles is produced out of the substrate itself.

[0038] Thus, the substrate must first and foremost act as a support, and therefore must be rigid enough to support the structures, and, while these structures are being processed, it must maintain its integrity without breaking. Furthermore, in this preferred embodiment, it must also allow for the foot to be formed on its upper part, which is in contact with the microparticle starting material. In a particular embodiment of the invention the support or substrate may be made of the same material used for the microparticle starting layer. For example, microparticles can be manufactured on a substrate that is a silicon sheet with a silicon foot (i.e. it is engraved into the substrate itself), wherein the particles have been molded in a polysilicon layer; this enables the possibility of subsequently carrying out thermal doping processes to provide the microparticles with charges.

[0039] In another particular embodiment of the invention that is an alternative to the preceding one, the substrate is formed by at least two materials, such that it contains a second material in its structure that is located in the upper part in the form of a layer, where it has been deposited or grown. In this way, the foot can be molded by engraving this second material contained in the upper part of the substrate. If the substrate contains a second material in the upper part of its structure, where the feet will be engraved, this second material may be the same material used to produce the microparticles themselves, or a different material, preferably one that is more fragile and less ductile than the structuration layer so as to guarantee that it behaves correctly under the subsequent breaking stress, for instance polycrystalline silicon. For example, it is possible to use a silicon substrate (sheet) only as the support of the layer that will be used as a starting material for the foot and for the microparticles, without intervening in any way in the manufacture of the devices defined in it. Silicon, though not the only option, is highly recommendable because it is the microelectronics material par excellence given its compatibility with most processes and resistance to temperature changes and chemical agents.

[0040] Likewise, the preparation in stage a) of the structuration layer, which is the same thing as the layer constituting the material that gives rise to the microparticles, can be carried out by depositing it or by growing it on top of the substrate itself. The materials that the structuration layer can be made of may be selected from the group consisting of: silicon and derivatives thereof (silicon nitride or silicon oxide, polycrystalline silicon), gold, platinum, aluminum, copper, nickel, cobalt, chromium, metal oxides, and silicates or silicides of compatible metals such as tantalum, iron or aluminum. This layer can be deposited or can be grown by any method used in microelectronics: thermal growth, chemical vapor deposition, sputtering, evaporation, or other common methods used today. The method selected for this will be determined by the choice of materials to use.

[0041] To guarantee good mechanical behavior of the entire structure (microparticle plus foot), it is preferable for the material chosen for the structuration layer and the material of the substrate that will be engraved into a foot, whether the substrate is made of one material or contains a second material in its upper part, to have a relationship between its rupture limits greater than or equal to one (L.sub.rupt_part/L.sub.rupt_foot≥1). This not only secures the particles during functionalization, but also ensures that the foot is more fragile than the microparticle and is therefore more vulnerable to rupture when mechanical stress is applied in cases where one wishes to free said microparticles from the substrate in a controlled manner (facilitate their release). Nevertheless, if the final application so requires, the structuration layer and the second material that contains the substrate in its upper part can be manufactured from the same material, since their own design and geometry enable this.

[0042] The method of manufacturing the microparticles based on microelectronic technology makes it possible to define its dimensions preferably by using photo-lithographic techniques, said techniques being commonly used in the field of microelectronics. The use of photo-lithographic techniques makes it possible to form microparticles into specific shapes and dimensions, chosen with technical criteria, preferably being identical to one another, although this technique also allows for the manufacture of groups of microparticles that are identical to one another but different from other groups in the same array. The micrometric particles in the array produced by means of the described method may preferably have dimensions comprised between 1 .Math.m to 100 .Math.m, both limits included, on the plane of the microparticle. Also preferably, the microparticles may have a thickness comprised between 20 nm and 5 .Math.m, both limits included. The microparticles may have varying geometries, for instance a parallelepiped or circular shape, although these shapes shall not limit the invention.

[0043] The shape of the foot under the microparticle can be defined through any engraving technique that allows for partial elimination, just underneath each microparticle, of the material forming said foot, whether it is the only material making up the substrate or the second material that said substrate may contain in its upper part. In this way said element can be given a preferred shape of the column or pillar type, with a cross section having two differentiated portions, one narrower than the other, to force the mechanical stress to concentrate there, or with a cross section that is uniform throughout its length but smaller than that of the microparticle itself, preferably less than or equal to 50% of the size of the latter’s cross section. The technique used to form the feet should preferably be physical etching (dry reactive etching) or chemical etching (wet), with lateral etching, depending on the material or materials present in the structures (in the shape of both the microparticle and the foot) and which in turn makes it possible to produce a foot with a constant or varying cross section, whichever is best for the required application.

[0044] In turn, the microparticles may contain one or more classes of molecules organized into monolayers in localized areas, which allow them to have several simultaneous uses, and in turn to carry out specific measurements or observations of one or several parameters and/or activities inside the medium where they are found. More specifically, said chemical functionalization may comprise several molecules of natural or synthetic origin, with chemical and/or biological activity, which include, but are not restricted to, simple organic compounds, polymers, peptides, proteins, nucleotides and nucleic acids. The molecules can be deposited on the upper face of the microparticles preferably using techniques from the field of micrometric- and nanometric-scale molecule printing, such as microcontact printing, dip-pen nanolithography or polymer-pen lithography.

[0045] As explained previously, it may be possible to release the microparticles from the array produced by means of the method described. In this particular embodiment of the invention, after stage d) for functionalization of the surface of the microparticles, the described method further comprises:

[0046] e) proceeding to break, in a controlled manner, the feet that support the microparticles by applying directed mechanical loads, in order to separate them from the substrate (individualize them). These loads may be applied by means of a variety of techniques, such as rasping the foot; applying an adhesive substance on the already functionalized surface of the microparticles and subsequently pulling it off, then dissolving the adhesive in media that do not affect the molecular functionalization of the microparticle; cryofracture, etc.

[0047] In this way, by applying directed mechanical stress, the feet can be broken in a controlled manner, for example with a clean cut, in order to release the microparticles from the substrate of the array without breaking or damaging them, preserving intact the functionalization that was previously applied to them, since it is a completely physical method that is not chemically aggressive.

[0048] Preferably, the mechanical rupture of the feet to individualize the microparticles that they sustain may be carried out by means of a directed cut, applying a controlled lateral force strong enough to break the foot. Said cut may be done with a micro-tool appropriately designed for this purpose, comprising a sharpened flat-tipped spatula having micrometric dimensions. In another preferred embodiment, the mechanical rupture may be carried out by means of cryofracture, freezing the entire structure of the array (the substrate with functionalized microparticles and their respective feet), which comprises: wetting the substrate with a solution such as a phosphate-buffered saline solution (PBS) with a content of 0.05% of Tween 20 solution (PBS-T); submerging the entire structure in liquid nitrogen until freezing the solution; re-wetting in the same way and re-freezing with liquid nitrogen; then finally applying a force or movement of leverage with a gripper or similar element until breaking the foot. The frozen solution that contains the microparticles is left to melt at ambient temperature in order to release them. In another preferred embodiment, an adhesive substance may be deposited on top of the chemically functionalized microparticles, for instance a layer of a polymer matrix such as Fluoromount®, in liquid phase so that it can enter even underneath the microparticles, which partially hardens after polymerizing. This substance is an aqueous-based biological mounting medium that is commonly used to cover tissues containing fluorescent markers, for subsequent inspection in optical microscopes such as confocal microscopes, and fluorescence microscopes, including scanning and transmission electron microscopes (SEM and TEM). At this point, said layer of the polymer matrix can be manually separated from the substrate, carrying along with it the microparticles and breaking the feet as it separates them, after which this hardened layer can be dissolved in a medium which does not affect the chemical functionalization, for example in an aqueous medium, in order to eliminate it from the surface of the microparticles.

[0049] Likewise, in a more preferred version of the foregoing embodiment, the method further comprises:

[0050] f) gathering the functionalized and separated (or individualized) microparticles in a suspension medium, which may be any medium that does not affect the chemical functionalizations.

[0051] Thus, the microparticles, once they have been separated from the substrate by mechanical means, can be kept for storage in a suspension in an aqueous medium that may be an acid, neutral or base, it matters not which, as required by the type of functionalization carried out.

[0052] Through the fabrication method it is possible to produce an array of planar microparticles with surface molecular multiplexing. Likewise, in the particular embodiment in which the feet of the structure are mechanically broken, these same functionalized microparticles are obtained but individualized. In an more preferred embodiment, a suspension of these microparticles is achieved, according to the aforementioned. Any of those products, array, microparticle(s) and microparticle suspension may be used to analyze, by way of example, “chemical parameters”, which are all the measurable chemical magnitudes, such as the pH or the redox (oxidation/reduction) potential. What is more, it may be used to simultaneously measure several “biological parameters”, thus referring to any magnitude that proves the presence of specific biological compounds, or the action thereof in the medium in which the microparticles are found. Said parameters may be ion concentration in solution, the activity of a specific enzyme, the presence of proteins and/or ligands, even the study of DNA, among others. These parameters in a sample medium may be measured by means of the signal emitted by one or more microparticles of the array, by one or more released and individualized microparticles or by one or more individualized microparticles and in suspension that is added to the sample medium. The sample medium in which the array may be used, the individualized microparticle or microparticles or the suspension thereof may be used as a sensor, actuator or the like, may be any chemical or biological medium, for example, an in vitro cell sample. In fact, a single cell may be a suitable sample medium in which to measure specific parameters due to the functionalization of the microparticles, which means that, in this embodiment, a microparticle may be separated from the array to insert it into the cell.

[0053] It must be noted here that if the array, the individualized microparticles or the microparticle suspension are used as actuators, these may also serve in the more preferred embodiment for substance vehiculation, such as for example, drugs or specific reagents. As such, in some of the examples of the use of the array or the microparticles thereof once individualized through the methods described above, it must be noted that they may be used in the field of pharmaceuticals and biomedicine as drug transport systems or drug delivery systems.

Examples

Example 1: Producing an Array of Planar Microparticles, Each One Functionalized with Three Different Proteins and Produced Through the Method Proposed in the Present Invention, and Release of Functionalized Microparticles to Produce a Suspension

[0054] The aim of this example is to demonstrate the possibility of manufacturing an array of planar microparticles, with dimensions of 3 .Math.m × 3 .Math.m × 1 .Math.m functionalized with three types of different molecules. In this particular embodiment, the method for placing the molecules on the planar surface is based on the polymer-pen lithography technique. Three different proteins have been printed using this technique.

A- Producing the Microparticles

[0055] To produce microparticles, a monocrystalline silicon sheet with crystallographic orientation (100) with a diameter of 100 mm and thickness of 525 .Math.m was taken. A thermal silicon oxide was thermally grown on it at 1100° C. This grown material was used for the subsequent structuration or molding of microparticles. Then, as set forth in the paragraphs of the Detailed Description, the photolithographic process is carried out, that is, the definition of the structures of the microparticles. To do so, 1.2 .Math.m of positive photoresin (HiPR 6512) was deposited on the sheet. Using a glass grid as a mask on which the geometry of the microparticles was defined in chromium, the resin was irradiated with monochromatic light (wavelength 435 nm). For the specific embodiment of this Example of the Invention, square geometric shapes were arranged on the plane, which were 3 .Math.m long and separated from one another by 3 .Math.m. After irradiating the photoresin for 5 to 8.5 s, it was partially removed in a developer solution ODP 462 so that resin only remained in areas of the silicon oxide layer that subsequently defined the microparticles. Then, the remaining resin was annealed at 200° C. for 30 min in order to increase the strength thereof against subsequent etching. The following process consisted of carrying out vertical etching on the entire surface, in order to engrave the silicon oxide layer in the area that was not protected by the resin. To do so, a dry reactive ion etching equipment was used, using a mixture of C.sub.2H.sub.6 and CHF.sub.3. This etching ended when the silicon sheet was reached. After this step of the process, the microparticles were already well defined but still joined to the silicon sheet. In the following stage of the manufacturing process, an isotropic etching of the silicon sheet was carried out, using the silicon oxide structures as a mask, along with the remaining resin layer, in a deep reactive ion etching (DRIE) process. To do so, SF.sub.6 and C.sub.4F.sub.8 gases were used. This process laterally etched 1.3 .Math.m, from all sides, the silicon located below the silicon oxide microparticles for the formation of the feet the held the microparticles joined to the silicon sheet during the chemical functionalization process. Lastly, the photoresin used as a mask was removed until the microparticle surface was clean of organic compounds, leaving the microparticles ready for their molecular functionalization and subsequent rupture to gather and suspend them.

B- Functionalization of the Surface of the Microparticles

[0056] As an example of functionalization of the surface of the microparticles described above, the technique referred to as polymer-pen lithography was applied. This technique (Fengwei Huo, Zijian Zheng, Gengfeng Zheng, Louise R. Giam, Hua Zhang and Chad A. Mirkin, Science (2008) 321, 1658-1660) combines the possibility of printing or assembling molecular monolayers on a large surface, characteristic of the microcontact printing technique, with the accuracy of individualized printing using the dip-pen nanolithography technique.

[0057] This technique previously required the manufacture of a mold or stamp made of soft polymeric material to transfer the molecules to the surface of the sample. In this exemplary embodiment, polydimethylsiloxane (PDMS), an organic polymer-based silicon in liquid state, was used, the components of which (a curing agent and the base elastomer) are mixed in a ratio of 10:1 by weight and are cured at a temperature between 60° C. and 100° C. for a period of time that may vary between 45 min and 120 min, depending on the hardness desired. To manufacture the mold for the PDMS stamp, another silicon sheet was used where a 1 .Math.m layer of silicon oxide is thermally grown at 1100° C. A photo-lithography process, such as the one described above was used, but with an inverted mask compared to the one used to define the microparticles (where before there was resin, now there is not, and vice versa). Similarly to the previous embodiment, the silicon oxide layer was engraved through the existing mask and the remaining resin was subsequently removed. Once in this state, an anisotropic KOH etching was carried out, with which inverted pyramids were defined in the area where there was no silicon oxide. These pyramids enabled the subsequent production of the polymer points. Due to the use of the same mask, but inverted, it is possible to produce a polymer point for each microparticle. Therefore, in this specific example a matrix of square-based inverted pyramids, of 3 .Math.m by 3 .Math.m, separated by 3 .Math.m, with a depth of 2.12 .Math.m. Once the mold has been obtained, a surface treatment was carried out with fluorosilane trichloro-1,1,2,2-tetrahydroperfluorooctylsilane at 97% to prevent the polymer from adhering to the mold. In this state, liquid PDMS was deposited on this mold and after the curing thereof, the PDMS stamp was removed.

[0058] This stamp was used to transfer the absorbed molecules to the point of the pyramids on the surfaces of the microparticles. In order to put the molecules on the PDMS mold, the so-called inks are used. These inks are solutions that may contain any type of substance that one wishes to print; from organic molecules, such as for example fluorescent or fluorophore markers, as well as biomolecules such as single-strand DNA, proteins, etc. depending on the subsequent application thereof. In this exemplary embodiment, three types of different inks were used: i) wheat germ agglutinin (WGA) lectin conjugated with the Streptavidin Texas Red® fluorescent marker (SAV-TR) in red; ii) bovine serum albumin (BSA) protein conjugated with the Neutravidin OregonGreen® fluorescent marker (NAV-OG) in green; iii) Goat Anti-Rabbit antibody IgG conjugated with the AMCA (7-Amino-4-methyl-3-coumarinylacetic acid) in blue, respectively. As a process control and to visualize the results obtained, a fluorescence microscope was used.

C- Mechanical Release of the Microparticles Previously Functionalized by Means of Controlled Mechanical Fracture

[0059] To release the printed microparticles of the silicon sheet, a drop of Fluoromount® mounting medium is deposited on the sheet, forming a layer that homogeneously covered the microparticles of the sheet. The medium was left to polymerize at room temperature for 1 hours, creating a solid layer that covered the microparticles. This layer is mechanically separated from the sheet, taking the microparticles that had been broken at the feet with it. This method prevents the deterioration of the molecules previously printed since the medium was chemically inert.

[0060] The polymerized layer that is separated from the sheet with the separated microparticles was able to be stored in this state, for the subsequent use thereof in suspension. In order to obtain the microparticles in suspension, the separated layer was dissolved in an aqueous medium, such as for example, de-mineralized water or buffer solutions.

Example 2: Molecular Recognition of Proteins: Demonstration of the Use of the Suspension of Microparticles with Molecular Multiplexing Prepared in Example 1 as Sensor and/or Actuator

[0061] In order to demonstrate that the functionalized molecules on the surface of the microparticles continue to be active (they maintain their integrity and functionality and therefore, are able to react with different elements of the medium) after being immobilized and once the microparticles have been released from the array substrate by means of controlled rupture of the feet, an antibody binding assay was carried out. For this assay, Goat anti-WGA IgG was chosen as the primary antibody and anti-Goat IgG (H+L) conjugated with the fluorescent marker AMCA (7-Amino-4-methyl-3-coumarinylacetic acid) in blue was chosen as the secondary antibody. These antibodies were orderly incorporated (in first place the primary antibody and then the secondary antibody) in an aqueous medium, following the standard methods of these assays, wherein the suspension of microparticles had previously been incorporated, giving rise to the recognition of proteins by the primary antibodies and the resulting bonding of both molecules (primary and secondary antibodies) to said proteins.

[0062] As a result, the expected sums were noted in the fluorescence emissions of the proteins previously printed on the microparticles due to the correct addition of emissions of the fluorescent markers present in both the proteins and the antibodies; perfectly visible changes using a conventional fluorescence microscope and that shows that the molecular recognition centers of the proteins continue to function. Said changes were the following: [0063] a) the fluorescence signal in red emitted by the WGA lectin conjugated with SAV-TR was added to the emission in blue of the secondary antibody conjugated with AMCA, creating a representation in magenta, [0064] b) the fluorescence signal in green emitted by the BSA protein conjugated with NAV-OG was added to the emission in blue of the secondary antibody conjugated with AMCA, creating a representation in cyan, [0065] c) the Goat Anti-Rabbit IgG antibody conjugated with AMCA that initially emitted a blue fluorescence signal, continued to emit in said color.

[0066] As a control of the functionality of the multiplexing system, said immunoassay was successfully carried out with the manufactured array, that is, between steps B and C of Example 1 of the embodiment (after the manufacture and functionalization of the microparticles and before their release from the substrate), in order to demonstrate that the molecule multiplexing system by means of the polymer-pen lithography technique followed by the mechanical release system of the microparticles did not affect the correct activity of the molecules.