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    • 专利摘要:
    Method of obtaining an array of planar microparticles with surface molecular multiplexing, array obtained and its use. The invention relates to the controlled manufacture of an array of planar microparticles with multiplexing of molecules on their surface, whose mission is to function as molecular sensors and/or actuators. The present invention proposes a matrix (array) of microparticles on the surface of which are printed all the molecular components necessary to provide them with functionality. It is possible to manufacture this product thanks to the design of a process in which the different molecular elements are multiplexed on the surface of each particle while they are supported on a substrate thanks to the engraving of a foot below them. These microparticles can be mechanically released from the support where they are manufactured by a mechanical method of controlled rupture, which is a non-chemically aggressive method and therefore does not affect the previously printed molecules on its surface. The array and the particles it contains have great versatility in both chemical and/or biological applications. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2555790A1
    申请号:ES201430864
    申请日:2014-06-05
    公开日:2016-01-08
    发明作者:Jaume Esteve Tintó；Jose Antonio Plaza Plaza；Marta Duch Llobera；Núria TORRAS ANDRÉS；Maria Luisa PÉREZ GARCÍA；Juan Pablo AGUSIL ANTONOFF
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；Universitat Autonoma de Barcelona UAB；Universitat de Barcelona UB；
    IPC主号:
    专利说明:

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    DESCRIPTION
    Method of obtaining an array of planar macroparticles with surface molecular multiplexing, obtained array and its use.
    SECTOR AND OBJECT OF THE INVENTION
    The present invention is directed to the application of new techniques for manufacturing microparticle arrays in the wide field of engineering. Due to the nature of the array obtained as well as the microparticles it contains and that can be individualized by mechanical methods, the area of application of this invention is very wide, encompassing both the chemical, cell biology, medicine and medical sectors. pharmacy.
    STATE OF THE TECHNIQUE
    In the field of engineering and materials science, any entity, micro or nanometric scale, equipped with mass and which can be obtained naturally or artificially from physical-chemical procedures is understood as particle. Currently, the different existing techniques for obtaining micro and nanoparticles can be classified into two large groups, depending on whether they are manufactured directly individually, or if they are obtained in the form of an ordered matrix or array.
    All techniques encompassed in the first of the groups listed (individualized manufacturing of particles) are based on two different types of approximations, called top-down approach and bottom-up approach. The first of these is based on obtaining particulate material from a bulk or block of material or larger structures, through progressive size reductions (Dorian A. Canelas, Kevin P. Herlihy and Joseph M. DeSimone. Wiley Inter. Rev. Nanomed. Nanobiotechnol. (2009), 1, 4, 391-404). The ascending approach, on the other hand, consists of supramolecular chemical synthesis, which uses the chemical information contained in the individual components (atoms or molecules) to achieve their spontaneous grouping into larger complex particles, through self-assembly processes (self -assembly; Wei Wang, Baohua Gu, Liyuan Liang and William Hamilton. J. Phys. Chem. B (2003), 107, 3400-3404). In recent years, scientific interest has increased for the development of new materials and polymer-based compounds. Through the use of chemical synthesis techniques, techniques as diverse as flow-focusing or pulverization or microemulsion have been developed which, combined with the microfluldica, are also being used for the manufacture of particles, both simple and compound (KP Yuet, DK Hwang, R. Haghgooie and PS Doyle, Langmuir (2010), 26, 6, 4281-4287).
    All these self-assembling techniques have in common the direct obtaining of large quantities of identical particles, individually and at a low cost. The main drawback presented by these methods of manufacturing particles is that for their functionalization strictly chemical methods must be applied, thus being able to obtain a unique (identical) and total functionalization in all of them, or a combination of two or more functionalizations through the use of chemical substances as long as they do not affect each other; It is a difficult and highly complex process due to the multiple incompatibilities that this entails (neither versatility nor discretization). The particles obtained by the methods described above are often used in the field of pharmacy and biomedicine as drug transport systems or drug delivery systems (Tasciotti E., Liu XW, Bhavane R., Plant K., Leonard AD, Price BK , Cheng MMC, Decuzzi P., Tour JM, Robertson F.
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    and Ferrari M. Nature Nanotechnol. (2008), 3, 3, 151-157) and as carriers or nanocarriers (D. Peer, JM Karp, S. Hong, OC Farokhzad, R. Margalit and R. Langer. Nat. Nanotechnol. (2007), 2, 751-760); although they are also widely used, in the case of magnetic particles, such as magnetic separators or magnetic separation bioprocesses, by magneto- and electromagnetophoresis processes, as! As for the investigation of new materials and compounds.
    On the other hand, there is the set of techniques for manufacturing arrays of particles, based on manufacturing processes of micro- and nanoelectronics. These techniques, both those based on conventional photolithography processes (M.-H. Wu and GM Whitesides. Appl. Phys. Lett., (2001), 78, 16, 2273-2275) and those based on processes known as soft -lithography or soft lithograph (YN Xia and GM Whitesides. Angew. Chem., Int. Ed. (1998), 37, 551-557), such as micromodeling or micromolding, microcontact printing or microcontact printing (MCP), among others, they also allow the simultaneous production of thousands of units (batch processing concept), in a controlled way and at a low cost, but with a high adaptability for the use of materials as diverse as metals and polymers, including a large number of biomaterials, and the possibility of making combinations between them. These processes give the designed particles a great versatility, both in shape and size, being able to obtain, simultaneously, different types of particles. Although in some cases the manufacturing process of this type of particles is more complex than those mentioned above, these techniques allow the obtaining of particles with multiple surfaces, opening the range to a wide variety of possible applications. Thanks to their orderly and controlled location on the surface of the substrate where they are manufactured, these particles allow different types of functionalization to be applied to the same particle, located within it. Currently, existing techniques for particle separation, obtained by lithographic processes, from the manufacturing substrate, a concept known as particle liberation or individualization, require the use of methods based on the use of chemical agents that can be aggressive for multiplexed molecules. on its surface, as is the case with the surface technique (E. Fernandez-Rosas, R. Gomez, E. Ibanez, L. Barrios, M. Duch, J. Esteve, C. Nogues and JA Plaza. Small (2009 ), 5, 21, 2433-2439), where a sacrificial layer, located between the particle and the substrate, is chemically attacked.
    Multiplexed chips (understood as those chips that gather in a single substrate - particle- several types of channels capable of providing and receiving different information, through each of the functionalizations printed on its surface) composed of an ordered array of molecular elements and with dimensions in the order of centimeters, such as DNA chips or DNA chips, have been widely used in fields such as medicine and biology for identification, quantification and determination of the functioning of certain SF Kingsmore molecules. Nat. Rev. Drug. Discov (2006), 5, 310-320). For cases in which the analysis of small volumes of samples is required, the current technological solution is the production of particle suspensions in which each element is comprised of a subpopulation of particles that differ from the other groups by having different anisotropic attributes. (shape, dimensions, color, etc.). The lack of multiplexing in the same particle of those that contain these suspensions, characteristic that if they offer multiplexed chips, constitutes an important limitation when analyzing small volumes (for example, the inside of a cell).
    The present invention includes a new proposal, based on the manufacture of an ordered array of planar microparticles with molecular multiplexing on its surface, of variable geometry and dimensions depending on its final application, obtained from
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    manufacturing techniques of microelectronics. The surface functionalization can be carried out in a controlled and orderly manner with a great variety of molecules simultaneously, such as proteins and DNA. The microparticles are molded and prepared on a substrate and are supported on it thanks to a clamping foot that is etched under them, and can be separated (released) by a mechanical method of controlled rupture and in the absence of chemical release techniques, thanks to the formation of this new structural element called "foot" of the microparticle in the array.
    This foot, similar to a column or pillar, acts as a fastener for said microparticles that make up the array or matrix to the substrate during the molding and molecular multiplexing processes on its surface, and acts as a mechanical stress concentrator element, thus becoming the weakest point of the whole set. This allows, in the case of wanting to release the particles, the application of directed mechanical stresses to break the foot in a controlled manner, without the microparticles or the multiplexed molecules on their surface being damaged because chemical release substances are not used that may affect the structure or function of these molecules. That is, in addition to being able to obtain an array of surface functionalized microparticles, unlike other known methods it is possible to individualize the array microparticles by a chemically non-aggressive method, that is, it does not require the use of chemical agents that can damage the integrity of the microparticle or affecting the molecular functionalization performed previously. Once released, these microparticles, like the array, are capable of acting as sensors or actuators of different activities, both chemical and biological that occur in the environment in which they are found, such as certain chemical reactions or variations of physical parameters such as temperature and pH, among others.
    BRIEF DESCRIPTION OF THE INVENTION
    The invention described here refers to an array of planar microparticles with multiplexed molecules on their surface prepared from an original material deposited or grown on a substrate that serves as support, whose mission is to function as a detector, sensor and / or Molecular actuator in a sample medium that can be both chemical and biological. Specifically, the array is manufactured with microelectronics technology, and the functionalized microparticles it contains are characterized by having dimensions that can be in the range of 1 pm to 100 pm as required in its final application, being possible to prepare them on the substrate in large quantities, with well-defined shapes and dimensions. Due to its size (micrometric) and its surface functionalization, the array of microparticles obtainable from the aforementioned method has great versatility in terms of specific applications, and can be used in both chemical and biological media, always with scientific objectives -technicians.
    The foot that is engraved under the original material of the microparticles (corresponding to the upper part of the support) exerts the function of a structural element, individually joining each microparticle to the substrate during the processes of molecular manufacturing and multiplexing and allowing the localization of the microparticles in an orderly manner in the substrate. In addition, thanks to the formation of this new structural element, the microparticles can be separated from the substrate and individually controlled in a controlled way by applying mechanical stresses, such as transverse mechanical stresses, a chemically non-aggressive method and therefore respecting the integrity of multiplexed molecules on its surface. Thus, all previous functionalization carried out is preserved without being altered, allowing the release of the microparticles after their functionalization.
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    The great industrial advantage of this proposed method lies in the possibility of serially producing functionalized surface microparticles on a substrate, specifically on a foot that supports and allows the use of parallel stamping methods for the molecular functionalization of all microparticles simultaneously, for example in the same way. In addition, in a preferred case of the invention, the foot formed under the source material that gives rise to the microparticles during the manufacturing process allows their release or individualization to be achieved by mechanical separation of the foot by breaking it. Due to its own design and geometry, similar to a column or pillar that supports the micro-grid, this foot becomes the most fragile area of the structure, acting as a mechanical stress concentrator and securing the fracture zone if desired the release of the microparticles for individual use.
    Ideally, the foot should have a variable section (not constant), that is, with two different zones in such a way that one of them is narrower, preferably with the narrowest part of the section in the center. In this way, the foot offers the necessary resistance to support the microparricula during the functionalization process, and at the same time it presents a narrower area where the mechanical stresses are mostly concentrated in the case of wanting its controlled release. In a particular case, the foot may also have a constant section as long as it is smaller than the section of the microparticle itself, preferably less than or equal to 50% of the size of the microparticle section.
    Due to its design and geometry, both the foot and the array microparticles can be formed of the same material, although it is preferable that the foot is made of fragile and non-ductile materials, which further facilitates its controlled breakage.
    Therefore, the first object of the present invention is the method of obtaining an array of planar micrometric surface functionalized, a method comprising the following steps:
    a) preparing a layer of a material of origin of the micro-particles (also called the structuring layer) on a substrate that serves as support, typically a silicon wafer although it may be any other equally suitable for this purpose; said layer of material originating from the micro particles can be of a typical microelectronic material, such as polycrystalline silicon, silicon oxide, silicon nitride, gold, platinum, aluminum, etc .;
    b) Shaping the microparticles in the structuring layer previously prepared on the support by means of customary techniques of microelectronics based on lithography, by which the geometry and lateral dimensions are defined, and engraving techniques with which the thickness is defined after preparing the layer;
    c) the key point of the method is the formation of a foot in the upper part of the substrate that is located under the previously molded microparticles, so that each foot supports a microparticle. The formation of the foot of each microparticle is done by engraving said upper part of the substrate, said engraving being able to be carried out by usual microelectronics techniques. In this way, the feet of the microparriculas are prepared sufficiently stable from the mechanical point of view to support them during the subsequent stage of molecular functionalization on their surface, but also fragile enough to also allow their rupture in a contracted manner, when applied Directed mechanical forces, which allow the foot to rupture and release the microparticle of the
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    array Therefore it is recommended that the foot is made of a fragile material such as silicon; Y
    d) functionalize the surface of the microparticles that are supported by the feet by at least one molecular component, preferably by a method that allows a large number of microparticles to be parallelized, by way of example: soft lithography techniques such as microcontact printing (microcontact printing, MCP), nanolithography by dip-pen (dip-pen nanolithography, DPN), lithograph by polymer-pen (polymer-pen lithography, PPL) or lithograph by nano-printing (nano-imprint lithography, NIL).
    Basically, the substrate that acts as a support for the manufacture of the microparticles is covered with a layer that defines the original material thereof, which will be molded later, and that can be selected within the group of materials composed of: silicon and its derivatives (silicon oxide or nitride, polycrystalline silicon), gold, platinum, aluminum, copper, nickel, cobalt or chromium; metal oxides; and silicates or silicides of compatible metals such as tantalum, iron, or aluminum. This layer is structured or molded on the substrate to define the desired shape of the microparticles, and then the foot that is located beneath them is structured or defined.
    The foot exerts a double function. On the one hand, it holds the microparticle attached to the substrate during the entire manufacturing process of the same! as during the subsequent functionalization steps, ensuring its position at all times. On the other hand, being the weakest and most fragile part of the entire structure, it acts as a stress concentrator element allowing its rupture in the event that it is desired to separate or release the matrix microparticles.
    After preparing or shaping the foot of the microparticles by partial etching (that is, only partially or not the entire section constantly, because a column or pillar shape is preferable), the surface of said microparticles is functionalized with the different elements molecular molecules that are selected (such as organic compounds, polymer chains, proteins, DNA, etc.). This action is carried out in parallel, although since the substrate contains several microparticles on its surface, the parallel functionalization can be repeated in series to provide the particles with more than one functionalization, or it is possible to repeat the impression of the same substance several times. . Thus, thanks to the functionalization, the multiplexing of the microparticles is achieved, in such a way that on the surface of each of them a unique molecular element printed more than once or more of a different molecular element is located, unlike the planar chips of molecular matrices, with dimensions in the order of centimeters, which do not allow the analysis of small volumes such as for example a cell of an ex vivo and in vitro sample, and unlike suspensions of subpopulations of micro-nanoparticles known where each subpopulation has a unique molecular element but does not allow multiplexed analysis in the same microparticle. The realization of an array of molecularly multifunctionalized microparticles in the order of microns as is the case of the present invention also allows multiplexed molecular analysis in small volumes.
    The array of microparticles, after functionalization, can be stored dry.
    A second object of the present invention relates to the array of surface functionalized planar microparticles obtainable from the method described above, as well as from the microparticles themselves that can be separated from the matrix by a method of controlled mechanical breaking of the foot. Preferably, a suspension of these microparticles can be prepared.
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    The array of the microparticles, as well as the functionalized microparticles themselves and released from the support, and the suspension that can be prepared with the microparticles can act thanks to their properties as detectors, sensors and / or molecular actuators. Or put another way, the invention also covers as a third object the use of these products as sensors, actuators or detectors of physical, chemical and biological parameters simultaneously or separately in a medium or sample.
    The present invention is based on the relative observation of the array of the microparticles, which are manufactured on the support with micrometric dimensions (lateral physical dimensions preferably between 1 pm and 100 pm, and preferably with a thickness between 20 nm and 5 pm) and properly molecularly functionalized by certain features traced selectively and controlled on its surface.
    DESCRIPTION OF THE FIGURES
    Figure 1: Representation of two possible configurations of microparticles in the array obtained by the described method, one in the form of a parallelepiped of width A, length L and thickness E, and the other with circular or disk shape, of diameter D and thickness E , according to the invention, where (1) represents the standing material, (2) the microparticle and the substrate (4). 1.A shows the views in perspective and 1.B shows the view of a section.
    Figure 2: Representation of a possible microparticle configuration on the support in the array, where its upper surface has been covered, in a localized way, with different molecular elements, designed for its functionalization (3). 2.A shows a view of a section and 2.B shows a perspective view.
    Figure 3: Scheme of a preferred process of fabrication of the microparticle array based on microelectronic technology, photolithographic processes and layer attacks, according to Example 1, where after obtaining the microparticle matrix these are released and prepared in suspension. Straight section showing the manufacturing process of a microparticle, with a substrate mainly silicon wafer (4) that acts as a support, on which there is a layer (5), typically of polycrystalline silicon, silicon oxide, silicon nitride , gold, platinum, aluminum, chrome, which constituted the original material that gave rise to the microparticles (2) (Fig. 3.A). To define the microparticles, a layer of photoresin (6) (Fig. 3.B) was deposited on said layer of primary material of the microparticles, which was then partially removed from certain areas (7) forming a structured photoresin layer (8) containing the geometry and lateral dimensions of the microparticles to be manufactured (Fig. 3.C). Said microparticles were formed (structured) by means of an attack on the layer of their primary material (5) in the uncovered areas (7) where the photoresin layer (6) had previously been removed, thus forming the body of the / s microparticle / s (2), (Fig. 3.D). Subsequently, an etching of the silicon substrate (4) was made immediately below the microparticles (2) to form the foot (1) (Fig. 3.E). The remaining photoresin (8) was removed from the top of the microparticles (2). Subsequently, the molecular functionalization of the microparticles was carried out with more than one molecular element (3) (Fig. 3.F). Finally, the functionalized microparticles (2) were released from the substrate (4) by controlled rupture, and were collected in an aqueous medium, in this example, demineralized water previously filtered, obtaining the suspension of the microparticles (9).
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    As stated in the previous section, the foot is manufactured by engraving molding the upper part of the substrate, which is in direct contact with the lower part of the layer of material originating from the microparticles. In a particular embodiment of the invention, the substrate is formed of a single material, being more preferably a silicon wafer, although it may be another type of substrate suitable for mechanical support for the manufacture of microparticles, such as borosilicate glass (commonly known by the trade name of Pyrex or Duran) or sodocalcium silicate glass, better known for its English term soda-lime, among others. However, the invention is not limited only to these support materials, since anyone who knows what types of materials are suitable and can perform the intended function; Basically, they are all those that meet the following conditions:
    - be resistant to thermal deposition, evaporation and layer growth processes;
    - be stable at room temperature;
    - be resistant to certain chemical agents (liquid or gas phase compounds) to allow the structuring / engraving / processing in general of the layers in them without the substrate itself being affected (although sometimes, it is necessary to protect them as chemical agents are usually very aggressive); Y
    - allow your own structuring / engraving (partial or total). This would be the case where the foot of the microparticles is manufactured from the same substrate.
    In short, the substrate must primarily exercise the function of support, and therefore must be sufficiently rigid to support the structures and must maintain its integrity against processing them without breaking. And also, in this preferred embodiment, it must also allow the formation of the foot in its upper part, which is in contact with the material originating from the microparticles. In a particular case of the invention, the support or substrate may be of the same material that is used for the original layer of the microparticles. For example, microparticles can be manufactured on a substrate that is a silicon wafer with a silicon foot (ie, etched on the same substrate) where the particles have been molded into a polysilicon layer; this allows the possible subsequent realization of thermal doping processes to provide the microparticles with charges.
    In another particular embodiment of the invention, alternative to the previous one, the substrate is formed by at least two materials, in such a way 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 up In this way, the foot can be molded by engraving of 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 are going to be engraved, this can be the same material as that used for the manufacture of the microparticles themselves or a different material, preferably more fragile and less ductile than the structuring layer to ensure so! its correct behavior in the face of subsequent rupture efforts, such as polycrystalline silicon. For example, it is possible to use a silicon substrate (wafer) only as a support for the layer that will give rise to the foot and the microparticles, without intervening at all in the manufacture of the devices defined therein. Silicon is very advisable, although not the only one, because it is the material par excellence in microelectronics, being compatible with most processes and resistant to temperature changes and chemical agents.
    Similarly, the preparation in step a) of the structuring layer, or what is the same the layer that constitutes the material that gives rise to the microparticles, can be carried
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    carried out by deposition or by growth on the substrate itself. The materials from which the structuring layer can be made can be selected from the group consisting of: silicon and its derivatives (silicon oxide or nitride, 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 currently. The method selected for this will be determined by the choice of the materials to be used.
    To guarantee a good mechanical behavior of the entire structure (microparticle plus foot), it is preferable that the material chosen for the structuring layer and the material of the substrate to be engraved in the form of a foot, be it the substrate of a material or which contains a second material in its upper part, have a relationship between its breaking limits equal to or greater than one (Lrupt_part / Lrupt_pie> 1). In this way, it is guaranteed not only the subjection of the particles during their functionalization, but also that the foot is more fragile than the microparticle and therefore, is more vulnerable to rupture against the application of mechanical stresses in case want the controlled release of said microparticles from the substrate (facilitates their release). However, if the final application requires it, the structuring layer and the second material that contains the substrate in its upper part can be made of the same material, since its own design and geometry allow it.
    The method of manufacturing microparticles based on microelectronic technology allows the definition of their dimensions through the use of photolithographic techniques, preferably techniques commonly used in the field of microelectronics. The use of photolithographic techniques allows the formation of microparticles in specific shapes and dimensions, chosen under a technical criterion, preferably identical to each other, although this technique also allows the manufacture of groups of microparticles identical to each other but different from other groups in the same array. Thus, the micrometric particles of the array obtained by the described method may preferably have dimensions ranging from 1 pm to 100 pm, including both limits in the microparticle plane. Also preferably, the microparticles can have a thickness between 20 nm and 5 pm, including both limits. The microparticles can have varied geometries, such as, for example, paralleleplpedo or circular shape, without these forms being limiting of the invention.
    The definition of the shape of the foot under the microparticle can be carried out by any engraving technique that allows the partial elimination, just below each microparticle, of the material that forms said foot, either the only material that consists of the substrate or the second material that said substrate may contain in its upper part. In this way, it is possible to provide said element with a preferred column or pillar type, with a section that has two different parts, one narrower than the other to force the concentration of mechanical stresses, or with a uniform section throughout its entire extension and at the same time smaller than that of the microparticle itself, preferably less than or equal to 50% of the size of its section. The technique used for the formation of the feet should preferably be a physical attack (reactive dry attack), or a chemical attack (wet), which presents a lateral attack, depending on the material or materials present in the structures (both that it forms the microparticle as the foot) and which in turn will allow obtaining a constant or variable section foot as agreed by the required application.
    In turn, the microparticles can contain one or more kinds of molecules organized in monolayers in localized areas, which allow them to have several utilities
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    Simultaneously, and in turn, make certain measurements or observations of one or several parameters and / or activities inside the environment in which they are located. More specifically, said chemical functionalization can 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, prothels, nucleotides and nucleic acids. Molecules can be deposited on the upper face of the microparticles preferably by techniques of the field of printing of micro and nanometric scale molecules such as microcontact printing, dip-pen nanolithography (dip-pen nanolithography) or the polymer-pen lithography (lithograph by polymer pen).
    As explained above, it may be possible to release the microparticles from the array obtained by the method described. In this particular case of the invention, after step d) of functionalization of the surface of the microparticles, the described method further comprises:
    e) proceed to the controlled breaking of the feet that support the microparticles by means of the application of directed mechanical loads, to separate them from the substrate (individualize them). These loads can be applied by various techniques, such as: foot scraping; application of an adhesive substance on the already functionalized surface of the microparticles and subsequent removal thereof, with subsequent dissolution of the adhesive in media that do not affect the molecular functionalizations; cryofracture, etc.
    In this way, by means of the application of directed mechanical stresses it is possible to break the feet in a controlled way, for example with a clean cut, to release the microparticles of the substrate of the array without them breaking or damaging, preserving intact the functionalization previously applied to the same because it is a completely physical method, not chemically aggressive.
    Preferably, the mechanical breaking of the feet for the individualization of the microparticles they support can be carried out by means of a directed cut, applying a controlled lateral force sufficient to break the foot. Said cutting can be carried out with a micro-tool designed and suitable for this purpose, which comprises a flat-pointed and sharp spatula of micrometric dimensions. In another preferred embodiment, the mechanical rupture can be performed by cryofracture, with the freezing of the entire array structure (the substrate with functionalized microparticles and their respective feet), which comprises: wetting the substrate with a solution, such as a solution phosphate buffered saline (PBS) with a content of 0.05% Tween 20 solution (PBS-T); immerse the entire structure in liquid nitrogen until the solution freezes; wet again in the same way and freeze again with liquid nitrogen; to finally exert a force or lever movement with a clamp or similar element until the foot breaks. The frozen solution containing the microparticles is allowed to melt at room temperature for release. In another different embodiment, an adhesive substance can be deposited on the chemically functionalized microparticles, such as a layer of a polymeric matrix such as Fluoromount®, in liquid phase to allow its entry even below the microparticles, which after polymerization, partially hardens. This substance is a water-based biological mounting medium very commonly used for covering tissues that contain fluorescent markers, for later inspection in optical microscopes such as confocal and fluorescence microscopes, even in transmission and scanning electron microscopes (SEM and TEM) . At that time, said layer of the polymeric matrix can be manually separated from the substrate by carrying the microparticles within it and
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    breaking the feet when separating them, after which this hardened layer can be dissolved in some medium that does not affect the chemical functionalization, for example in aqueous medium, to eliminate it from the surface of the microparticles.
    Also, in a more preferred embodiment of the previous case, the method also includes:
    f) collect the functionalized and separated (or individualized) microparticles in a suspension medium, said medium being able to be any that does not affect the chemical functionalizations.
    Thus, the microparticles, once separated from the substrate by mechanical means, can be kept for storage in a suspension in an aqueous medium that can be acidic, neutral or basic interchangeably, as required by the type of functionalization performed.
    Thanks to the manufacturing method, it is possible to obtain an array of planar microparticles with molecular surface multiplexing. Likewise, in the particular case of the method in which the structure's feet are mechanically broken, the obtaining of these same functionalized, but individualized microparticles is achieved. In another more preferred case, a suspension of these microparticles is achieved, as discussed above. Any of these products, array, microparticle / s and suspension of the microparticles can be used to analyze, for example, "chemical parameters", which are all those measurable chemical quantities such as pH or redox potential (reduction-oxidation ). Even more, it can be used for the simultaneous measurement of several "biological parameters", thus referring to any magnitude that evidences the presence of certain biological compounds, or their action within the medium in which the microparticles are found. These parameters are such as the concentration of ions in solution, the activity of a certain enzyme, the presence of proteins and / or ligands, including the study of DNA, among others. These parameters in a sample medium can be measured through the signal emitted by one or more of the array microparticles, by one or more microparticles released and individualized or by one or more of the individualized and suspended microparticles that are added to the sample medium. The sample medium in which the array, individualized microparticles or suspension thereof can be used as a sensor, actuator or the like, can be any chemical or biological medium, for example an in vitro sample of cells. In fact, a single cell can be a suitable sample medium in which to measure certain parameters thanks to the functionalization of the microparticles, so in this case a microparticle can be separated from the array to be introduced into the cell.
    It is necessary to comment here that if the array, the individualized microparticles or the suspension of microparticles were used as actuators, they can also be used in the most preferred case for the vehiculization of substances, such as drugs or certain reagents. Thus, in some of the examples of use of the array or its microparticles once individualized by the methods described above, it should be noted that they can be used in the field of pharmacy and biomedicine as drug transport systems or drug delivery systems.
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    Example 1: Obtaining an array of planar micropartlcuias, each functionalized with three different proteins and obtained by the method proposed in the present invention, and release of the functionalized microparticles to manufacture a suspension
    The objective of this example is to demonstrate the possibility of manufacturing an array of planar micropartlcuias, with dimensions 3 pm x 3 pm x 1 pm functionalized with three different types of molecules. In this particular case the method of placing the molecules on the planar surface is based on the lithography technique by polymer-pen, polymer-pen lithography. Through this technique three different proteins have been printed.
    A- Fabrication of the microparticles.
    To produce the microparticles, a monocrystalline silicon wafer with crystallographic orientation (100) of a diameter of 100 mm and a thickness of 525 pm was taken. On it a thermal silicon oxide was thermally grown at 1100 ° C. This grown material was used for the structuring or subsequent molding of the microparticles. Then, and as explained in the paragraphs of the Detailed Description, the photolithographic process was carried out, that is, the definition of the structures of the microparticles. For this, 1.2 pm of positive photoresist (HiPR 6512) was deposited on the wafer. Using as a mask a glass grid on which the geometry of the microparticles was defined in chromium, the resin was irradiated with monochromatic light (wavelength 435 nm). For the specific case of this Example of the Invention, square geometric shapes were arranged on the plane, 3 pm sideways and 3 pm apart from each other. After irradiating the photoresist for 5 to 8.5 s, it was partially removed in an ODP 462 developer solution. So that only resin remained on the areas of the silicon oxide layer that the microparticles later defined. Subsequently, the remaining resin was annealed at 200 ° C for 30 min to increase its resistance against subsequent attack. The following process consisted in the realization of a vertical attack on the entire surface, in order to etch the silicon oxide layer in those places that were not protected by the resin. For this, a dry reactive ion (reactive ion etching) equipment was used using a mixture of C2H6 and CHF3. This attack ended upon reaching the silicon wafer. After this process step, the microparticles were already well defined but still attached to the silicon wafer. In the next stage of the manufacturing process an isotropic attack of the silicon wafer was carried out, using as a mask the silicon oxide structures together with the remaining resin layer, in a deep etching process equipment using DRIE reactive ions (Deep Reactive Ion Etching). For this, SF6 and C4F8 gases were used. This process attacked laterally 1.3 pm, from all directions, the silicon located below the silicon oxide microparticles for the formation of the feet that held the microparticles together to the silicon wafer during the chemical functionalization process. Finally, the photoresist that was used as a mask was removed until leaving the surface of the microparticles clean of organic compounds, leaving the microparticles ready for molecular functionalization and subsequent rupture to collect and suspend them.
    B- Functionalization of the surface of the microparticles
    As an example of surface functionalization of the microparticles described above, we proceeded with the application of the technique called polymer-pen lithography 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
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    assembly of molecular monolayers on a large surface, characteristic of the microcontact printing or microcontact printing technique, with the precision of individualized printing using the nanolithography technique by dip-pen or Dip-Pen Nanolithography.
    This technique previously required the fabrication of a mold or seal, called a stamp, of soft polymeric material to transfer the molecules to the surface of the sample. In this example of realization, polydimethylsiloxane (PDMS), an organic polymer based polymer in liquid state, whose components (a curing agent and the base elastomer) are mixed in a ratio of 10: 1 by weight and cured at one temperature between 60 ° C and 100 ° C for a time that can range between 45 min and 120 min, depending on the desired hardness. For the manufacture of the mold for the PDMS seal, another silicon wafer was used where a 1 pm layer of silicon oxide is thermally grown at 1100 ° C. On this layer a photolithography process was performed as described above but with an inverted mask used to define the microparticles (where there was no resin left now, and vice versa). As in the previous case, the silicon oxide layer was etched through the existing mask and subsequently the remaining resin was removed. Once in this state, an anisotropic attack was made in KOH, with which inverted pyramids were defined in areas where silicon oxide does not speak. These pyramids allowed the subsequent obtaining of the polymer tips. Thanks to the use of the same mask but inverted it was possible to manufacture a polymer tip for each microparticle. Therefore, in this specific example, a matrix of inverted pyramids with a square base, 3 pm by 3 pm, separated 3 pm, 2.12 pm deep was defined. Once the mold was obtained, a surface treatment was made with the 97% trichloro-1,1,2,2-tetrahydroperfluorooctylsilane fluorosilane, to avoid adhesion of the polymer with the mold. In this state, liquid PDMS was deposited on this mold and after curing, the PDMS seal was removed.
    This seal was used for the transfer of adsorbed molecules to the tip of the pyramids on the surfaces of the microparticles. To put the molecules on the PDMS mold, the so-called inks were used. These inks are solutions that can contain any type of substance to be printed; from organic molecules, such as fluorescent or fluorophore markers, as well as biomolecules such as DNA strands, proteins, etc., depending on their subsequent application. In the case of this embodiment, three different types of inks were used: i) the WGA lectin (Wheat germ agglutinin) conjugated with the Streptavidin Texas Red® fluorescent marker (SAV-TR) in red; ii) the BSA (Bovine serum albumin) conjugate with the Neutravidin OregonGreen® fluorescent marker (NAV-OG) in green; iii) the Goat Anti-Rabbit IgG antibody conjugated with the AMCA marker (7-amino-4-methyl-3- coumarinyl acetic acid) in blue, respectively. As a process control and for visualization of the results obtained, a fluorescence microscope was used.
    C- Mechanical liberation of previously functionalized microparticles by controlled mechanical fracture
    To release the printed microparticles of the silicon wafer, a drop of Fluoromount® mounting medium was deposited on the wafer, forming a layer that homogeneously covered the wafer microparticles. The medium was allowed to polymerize at room temperature for 1 h, creating a solid layer that envelops the microparticles. This layer was mechanically separated from the wafer, taking with it the microparticles that have been broken by the feet. This method prevented the deterioration of previously printed molecules because the medium was chemically inert.
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    The polymerized and separated layer of the wafer with the separated microparticles could be stored in this state, for later use in suspension. To obtain the suspended microparticles, the separated layer was dissolved in an aqueous medium, such as demineralized water or buffered solutions.
    Example 2: Molecular recognition of proteins: demonstration of the use of the microparticle suspension with molecular multiplexing prepared in Example 1 as a sensor and / or actuator.
    To demonstrate that the functionalized molecules on the surface of the microparticles remain active (they maintain their integrity and functionality and are therefore able to react with different elements of the medium) after being immobilized and once the microparticles have been released from the substrate of the Array by controlled breaking of the feet, an antibody affinity assay was carried out. For this test, the Goat anti-WGA IgG was chosen as the primary antibody and as a secondary antibody, the anti-Goat IgG (H + L), conjugated with the fluorescent marker AMCA (7-amino-4-methyl-3-coumarinilacetic acid) in blue. These antibodies were orderly incorporated (first the primary antibody and then the secondary one) in an aqueous medium, following the standard procedures of these tests, where the suspension of microparticles was previously incorporated, giving rise to the recognition of the proteins by the primary antibody and the consequent union of both molecules (primary and secondary antibodies) to said proteins.
    As a result, the expected changes in fluorescence emissions of previously printed proteins in the microparticles were perceived due to the correct combination of fluorescent marker emissions present in both proteins and antibodies; perfectly visible changes by means of a conventional fluorescence microscope and that demonstrate that the molecular recognition centers of the proteins remain functional. These changes were the following:
    a) the WGA lectin conjugated with SAV-TR that initially emits a fluorescence signal in red, becomes magenta,
    b) the BSA protein conjugated to NAV-OG that initially emits a green fluorescence signal, becomes cyan, and
    c) the Goat Anti-Rabbit IgG antibody conjugated to AMCA that initially emits fluorescence signal in blue, continued to emit in said color.
    As a control of the functionality of the multiplexed system, said immunoassay was performed successfully with the manufactured array, that is, between steps B and C of Example 1 of realization (after the manufacture and functionalization of the microparticles and before their release from substrate), to demonstrate that the process of multiplexing of molecules using the lithograph technique by polymer-pen followed by the system of mechanical release of the microparticles did not affect the correct activity of the molecules.
    权利要求:
    Claims (24)
    [1]
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    1. A method of obtaining an array of surface-functional planar microparticles, comprising the following steps:
    a) preparing a structuring layer of a material originating from the microparticles on a substrate that acts as a support;
    b) molding the microparticles in the structuring layer by means of a microelectronic lithograph technique that shapes the geometry and the lateral dimensions, and an engraving technique with which the thickness of the microparticles is defined;
    c) forming a foot in the upper part of the substrate that is below the structuring layer to support each microparticle, by means of engraving techniques; Y
    d) Chemically functionalize the surface of the microparticles that are held on the substrate by the feet, by one or more molecular components.
    [2]
    2. The method of the preceding claim, wherein the substrate is formed of a single material, said material being a silicon wafer.
    [3]
    3. The method of claim 1, wherein the substrate is formed of two materials, containing a second layer-shaped material that is located in the upper part of the substrate, below the microparticle structuring layer.
    [4]
    4. The method according to any one of the preceding claims, wherein the microparticle structuring layer 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 silicates, iron and aluminum; and silicide selected from the group consisting of tantalum silicide, iron silicide and aluminum silicide.
    [5]
    5. The method according to any one of claims 1 to 4, wherein the material originating from the structuring layer and the upper part of the substrate where the feet are engraved have a relationship between their breaking limits equal to or greater than 1.
    [6]
    6. The method according to any of the preceding claims, wherein the preparation of the structuring layer in step a) is carried out by deposition or by growth of said structuring layer on the substrate, by a microelectronics technique selected from the group consisting of thermal growth, chemical deposition in the vapor phase, cathode pulverization and evaporation.
    [7]
    7. The method according to any of the preceding claims, wherein the formation of the foot of each microparticle in step c) is carried out by partially etching the upper part of the substrate that is under the microparticles by means of a microelectronic technique, with a variable section of the foot of two different parts where one part is narrower than the other, or with a constant section of the foot that is smaller than the microparticle section.
    [8]
    8. The method according to the preceding claim, wherein the foot has a constant section less than or equal to 50% of the section of the microparticles.
    [9]
    9. The method according to any of claims 7 or 8, wherein the partial engraving of the foot is performed by physical lateral attack or chemical side attack.
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    [10]
    10. The method according to any of the preceding claims, wherein the molding of the microparticles in step b) is carried out by photolithographic techniques.
    [11]
    11. The method according to any of the preceding claims, wherein all the microparticles are molded with the same shape and size.
    [12]
    12. The method according to any one of claims 1 to 10, wherein the microparticles are molded into two or more groups of different shape and size.
    [13]
    13. The method according to one of the preceding claims, wherein the functionalization of step d) is carried out by means of a printing technique selected from the group consisting of microcontact printing, nanolithography by dip-pen and lithography technique by polymer-pen
    [14]
    14. The method according to any of the preceding claims, wherein the molecular component is a molecule with chemical and / or biological activity, selected from the group consisting of organic compounds, polymers, peptides, proteins, nucleotides, nucleic acids and any combination thereof. same.
    [15]
    15. The method according to one of the preceding claims, wherein the surface of each microparticle is functionalized in step d) with more than one different molecular element, or with a single molecular element more than once.
    [16]
    16. The method according to any of the preceding claims, further comprising:
    e) break the feet that support the microparticles by applying mechanical breaking loads, to separate said microparticles from the substrate and individualize them.
    [17]
    17. The method according to the preceding claim, wherein the controlled mechanical breaking load of the foot of each microparticle is applied by a technique selected from the group consisting of scraping, cutting, cryofracture and application of an adhesive material on the already functionalized surface of the microparticles and their subsequent removal, with dissolution of the adhesive in media that do not affect the molecular functionalization of the microparticle.
    [18]
    18. The method according to one of claims 16 or 17, further comprising:
    f) collect the individualized microparticles in a suspension medium.
    [19]
    19. The method according to the preceding claim, wherein the suspension medium is an aqueous medium.
    [20]
    20. An array of planar microparticles with molecular surface multiplexing obtainable from the method defined in one of claims 1 to 15.
    [21]
    21. The array according to the preceding claim, wherein the microparticles have a plane size between 1 pm and 100 pm and a thickness between 20 nm and 5 pm.
    [22]
    22. A planar microparticle with individual surface molecular multiplexing and released by the method defined in one of claims 16 or 17.
    [23]
    23. A suspension of microparticles with surface molecular multiplexing obtainable by the method defined in one of claims 18 or 19.
    [24]
    24. Sensor, detector and / or molecular actuator device of physical, chemical and / or biological parameters in a sample medium, characterized in that it comprises an element
    selected from the group consisting of: an array as defined in claims 20 or 21, one or more microparticles as defined in claim 22 and a suspension of microparticles as defined in claim 23.
    10 25. Use of an array as defined in claims 20 or 21, one or more
    microparticles as defined in claim 22 or a suspension of microparticles as defined in claim 23 as a sensor, detector, and / or molecular actuator element in a device.
    15 26. Use according to the preceding claim, for the vehiculization of drugs or agents
    reagents
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    同族专利:
    公开号 | 公开日
    EP3153855A1|2017-04-12|
    ES2555790B1|2016-10-13|
    EP3153855B1|2019-09-04|
    US20210181189A1|2021-06-17|
    US20170184576A1|2017-06-29|
    DK3153855T3|2019-12-09|
    WO2015185782A1|2015-12-10|
    EP3153855A4|2017-05-24|
    ES2758182T3|2020-05-04|
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    US15/315,837| US20170184576A1|2014-06-05|2015-06-03|Method for Producing an Array of Planar Microparticles with Surface Molecular Multiplexing, Resulting Array and Use Thereof|
    DK15803414T| DK3153855T3|2014-06-05|2015-06-03|Process for the preparation of planar microparticles with surface molecular multiplexing, the microparticles and their use|
    ES15803414T| ES2758182T3|2014-06-05|2015-06-03|Method for obtaining planar microparticles with surface molecular multiplexing, the microparticles and their use|
    EP15803414.0A| EP3153855B1|2014-06-05|2015-06-03|Method for producing planar microparticles with surface molecular multiplexing, the microparticles and use thereof|
    PCT/ES2015/070439| WO2015185782A1|2014-06-05|2015-06-03|Method for producing an array of planar microparticles with surface molecular multiplexing, resulting array and use thereof|
    US17/181,958| US20210181189A1|2014-06-05|2021-02-22|Method for producing an array of planar microparticles with surface molecular multiplexing, resulting array and use thereof|
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