JP2006528352A - Random array of microspheres - Google Patents

Abstract

  Coating the support with a coating composition to form an element comprising a microsphere array on the support, and a receiving layer having a variable modulus of elasticity; receiving the dispersion of the microspheres in the fluid suspension; Coating on; changing the elastic modulus of the receiving layer to allow the microspheres to be partially submerged in the receiving layer; removing the fluid suspension from the receiving layer and the element to withstand wet processing A method for making the element is described, including the step of securing the microspheres in the receiving layer as described.

Description

  The present invention relates generally to biological or sensor microarray technology. In particular, this relates to a microarray coated on a substrate that does not contain any sites designated to attract microspheres prior to coating.

  Since its discovery in early 1990, high-density arrays formed by spatially addressable synthesis of bioactive probes on two-dimensional solid supports have greatly enhanced biological research and development processes and are simple (Science, 251, 767-773, 1991). The key to current microarray technology is the deposition of bioactive agents at a single spot on the microchip in a “spatial addressable” form.

  Current technology has used various approaches to produce microarrays. For example, US Pat. Nos. 5,412,087 and 5,489,678 demonstrate the use of photolithographic processes to make peptide and DNA microarrays. These patents are intended to prepare peptides and DNA microarrays through a continuous cycle that deprotects well-defined spots on a 1 cm × 1 cm chip by photolithography and then floods the entire surface with activated amino acids or DNA bases. The use of a photoprotective group is taught. By repeating this process, it is possible to construct peptide or DNA microarrays using thousands of arbitrarily different peptide or oligonucleotide sequences at different spots on the array. This method is expensive, and an inkjet approach may be used to produce spatially addressable arrays (eg, US Pat. Nos. 6,079,283; 6,083,762; and 6 , 094,966), however, this technique also has the disadvantage of high manufacturing costs in addition to the relatively large spot size of 40-100 μm. Since the number of bioactive probes to be installed on a single chip is usually somewhere between 1000 and 100,000 probes, the space addressing method is essential regardless of how the chip is manufactured. It is very expensive.

  An alternative approach to spatially addressable methods is the concept of using polymer beads that incorporate fluorescent dyes to create biologically multiplexed arrays. U.S. Pat. No. 5,981,180 discloses a combined method of flow cytometry and color coded beads for performing multiplexed biological assays. Microspheres conjugated on their surface with DNA or monoclonal antibody probes were internally stained with various ratios of two completely different fluorescent dyes. Hundreds of “spectral-addressed” microspheres are reacted with a biological sample, and the “liquid array” passes a single microsphere at a time through a flow cytometry cell to decode the sample information Was analyzed by. US Pat. No. 6,023,540 discloses using an optical fiber bundle with a pre-etched microwell at the distal end to assemble a dye-loaded microsphere. A unique bioactive agent is attached to the surface of each spectrally addressed microsphere, and thousands of microspheres carrying different bioactive probes are combined on the pre-etched microfell of the fiber optic bundle. A bead array is formed.

  More recently, a novel photoencoded microsphere approach has been achieved by using different sizes of zinc sulfide capped cadmium selenide nanocrystals incorporated into the microsphere (Nature Biotech, 19, 631- 635 (2001)). Given the narrow bandwidth demonstrated by these nanocrystals, this approach significantly extends the ability of spectral barcodes within the microsphere.

  While the “spectral addressed microspheres” approach offers just one advantage in terms of its simplicity compared to the older “spatial addressable” approach in microarray fabrication, in the art, biology There is still a need to make manufacturing of microarrays less difficult and less expensive.

  US patent application Ser. No. 09 / 942,241 dated Aug. 29, 2001 “Random array of microspheres” includes a fluid coating comprising microspheres dispersed in gelatin as shown in FIGS. 1a and 1b. Various coating methods for coating a support with the composition are taught and illustrate mechanical coating. As shown in FIG. 1C, immediately after coating, the support is passed through a cooling-setting chamber in the coating machine where gelatin undergoes rapid gelation and the microspheres are immobilized. As shown in FIG. 1d, excess fluid is removed by evaporation. While this process then provides enormous manufacturing advantages over existing technology, some improvement is required to maximize its overall potential value for the art. The problem is that during such mechanical coating and rapid gelation, the gelling agent covers the surface of the microspheres as shown in FIG. 1e, so that the analyte (eg DNA) passes through the gel overcoat. It has a tendency to permeate and prevent hybridization with the probe on the surface of the microsphere.

  US patent application Ser. No. 10 / 062,326 dated Jan. 31, 2002, “Method of making a random array of microspheres using an enzyme” impairs integrity of microspheres or DNA probes on their surface The above problem is overcome by enzymatically removing the gelling agent from the surface of the microspheres. The enzyme treated surface maintains its physical integrity throughout the complete DNA hybridization process and the microarray exhibits a very strong hybridization signal. The advantage of US patent application Ser. No. 10 / 062,326 is that enzymatic digestion can be easily controlled to remove the required amount from the gel overcoat. Furthermore, enzymes and proteases are easily available and can be obtained economically. However, an additional process (enzymatic digestion) is required, which has the disadvantage that additional time and costs are involved.

  In US patent application Ser. No. 10 / 092,803 “Random array of microspheres” dated March 7, 2002, on a receiving layer having the ability to undergo a sol-gel transition, as shown in FIGS. 2a and 2b. Describes a process for preparing random bead microarrays by coating a suspension of microspheres containing no gelling agent but containing a crosslinker for the gelling agent. The microspheres are partially buried in the receiving layer as shown in FIG. 2C, where the receiving layer is crosslinked as shown in FIG. 2d. As shown in FIG. 2e, excess fluid from the suspension is removed by evaporation to form a microarray. Although this approach is an improvement over US patent application Ser. No. 09 / 942,241, the gelling agent in the receiving layer may dissolve in the aqueous suspension used to deposit the microspheres. And can be re-deposited on the microspheres when the suspension is spread on the receiving layer, so that the gelling agent on the surface of the microspheres, as shown in FIG. It is not totally successful in preventing deposition. Furthermore, the presence of a cross-linking agent in the suspension can cross-link biological molecules on the surface of the microsphere and render them ineffective as probes.

  There is a need for a method in which a suspension of microspheres can be spread on a receiving layer and the material of the receiving layer does not dissolve in the suspension or medium in which the microspheres are being transported. Furthermore, the receiving layer composition should be such that sufficient microspheres can be embedded in the receiving layer to prevent lateral aggregation when the solvent in the suspension is removed, such as by evaporation. Don't be.

  The present invention relates to a method of making an element, such as a microarray having microspheres, in which a coating composition is coated on a support to form a receiving layer having a modulus that can be altered by crosslinking; a partially crosslinked receiving layer; Enabling partial cross-linking of the receiving layer to achieve a modulus of elasticity that allows partial embedding of the microspheres therein; dispersion of the microspheres in the fluid suspension to receive the partially cross-linked Coating on a layer, wherein each microsphere has a position; allowing the microsphere to be partially submerged in a partially crosslinked receiving layer; Removing the fluid suspension from the crosslinked receiving layer; and so that the microspheres maintain their respective positions during and after the wet process. Form, it provides a method comprising the step of allowing further crosslink the receiving layer which is partially crosslinked.

  In another embodiment of the present invention, a support; a water-insoluble receiving layer on the support, including a receiving layer material; with randomly spaced microspheres fixed and partially embedded in the receiving layer. A microsphere having a surface exposed above the receiving layer and having at least one probe mounted such that each exposed surface interacts with the analyte. An element is disclosed in which the exposed surface of the substrate does not include a receiving layer material.

  The receiving layer and support are characterized by the absence of sites designed to specifically physically or chemically interact with the microspheres. Therefore, the distribution of microspheres is completely random without being predetermined or induced.

  The invention prepares microarrays on a substrate that do not need to be pre-etched in microwells or pre-marked in any way to attract microspheres, as disclosed in the art. In order to utilize the unique coating technology. By using an unmarked substrate or a substrate that does not require any pre-coating pretreatment, the present invention offers enormous manufacturing advantages over existing technologies. The invention discloses a method of randomly distributing addressable mixed microspheres in dispersion on a receiving layer that does not have any wells or sites to attract the microspheres.

  The present invention provides a microarray that is less costly and easier to prepare than previously disclosed because the substrate does not need to be modified and the microspheres remain immobilized on the substrate.

  In addition, the present invention is disclosed in US patent application filed on June 3, 2002, although the microsphere surface is exposed, in contrast to US patent application Ser. No. 09 / 942,241 dated August 29, 2001. Microarrays are provided that do not utilize the additional process steps (enzymatic digestion) disclosed in US 10 / 062,326. The exposed microsphere surface allows easier access of the analyte to the probe attached to the surface of the microsphere. “Analyte” refers to a molecule having a functional group capable of chemically or physically interacting with a specific molecule on a microsphere surface, referred to herein as a “probe”. In the present invention, the analyte is preferably a nucleic acid or a protein.

  One of the main elements of the present invention is the selection of the receiving layer. The receiving layer must have the desired physical properties such that the microspheres are sufficiently submerged in the receiving layer, thus preventing lateral agglomeration. Specific requirements regarding the physical properties of the receiving layer are detailed below.

  The term “sol-gel transition” or “gelation” as used herein refers to the process by which a fluid solution or suspension of particles forms a continuous three-dimensional network that does not exhibit any steady state flow. This can be achieved by polymerizing in the presence of multifunctional monomers in the polymer; by covalent crosslinking of the dissolved polymer with reactive side chains; and between the polymer molecules in solution, for example hydrogen bonding. Can occur by subsequent binding. Polymers such as gelatin exhibit thermal gelation, which is the last type. The gelling or curing process is characterized by a discontinuous increase in viscosity. See P.I. Rose “Theory of Photographic Processes”, 4th edition, edited by T.H. James, p.

  As used herein, “gelling agent” means a substance that can be gelled as described above. Examples of these include materials that can be gelled by heat, such as gelatin, water-soluble cellulose ethers or poly (n-isopropylacrylamide), or substances that can be chemically crosslinked by boric acid compounds such as poly (vinyl alcohol). It is. Other gelling agents include polymers that can be cross-linked by radiation such as ultraviolet radiation, ionizing radiation or electron beam radiation. Examples of gelling agents include acacia, alginic acid, bentonite, carbomer, sodium carboxymethylcellulose, cetostearyl alcohol, colloidal silicon dioxide, ethylcellulose, gelatin, guar gum, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, magnesium aluminum silicate, maltosilicate Dextrin, methylcellulose, polyvinyl alcohol, povidone, propylene glycol alginate, sodium alginate, sodium glycolate starch, starch, tragacanth and xanthan gum are included. Other gelling agents known in the art such as those described in Secundum Artem Vol. 4, No. 5, Lloyd V. Allen can be used as well. A preferred gelling agent is gelatin pretreated with alkali.

  As used herein, the term “random distribution” means a spatial distribution of elements that does not exhibit any preference or bias. Randomness can be measured in terms of consistency with that expected by the Poisson distribution.

  The present invention teaches a method of making a random array of microspheres, also called “beads”, on a substrate that can include a receiving layer. The microspheres are deposited on the receiving layer such that a portion of the surface of each microsphere is exposed above the receiving layer. The distribution or pattern of microspheres on the receiving layer or substrate is completely random, and the microspheres are pre-marked on the receiving layer or substrate as in other methods previously known in the art. Nor is it attracted to or retained by a predetermined site.

  The random array is first coated with a fluid layer containing a gelling agent and a chemical crosslinker for the gelling agent on any suitable surface or support 1 (FIGS. 3a, 4a, 5a), This is achieved by forming the receiving layer 9 (FIGS. 3b, 4b, 5b). The support 1 can be, for example, glass, paper, metal, a polymeric material, a composite material or a combination thereof as long as it can provide a surface on which a receiving layer can be formed. The gelling agent in the receiving layer 9 can be partially crosslinked to form a partially crosslinked receiving layer 10 (FIGS. 3c, 4c, 5c). Next, the fluid suspension 11 of the microsphere 3 is spread on the partially crosslinked receiving layer 10 (FIGS. 3d, 4d, 5d). This partially cross-linked receiving layer 10 is insoluble in the fluid suspension 11. The microsphere 3 settles at least partially in the partially crosslinked receiving layer 10 (FIGS. 3e, 4e, 5e). The degree of settling is related to the elastic modulus of the partially crosslinked receiving layer 10, the interfacial surface energy of the microsphere 3 material and the interfacial surface energy of the fluid suspension 11. The microspheres 3 can settle throughout the partially crosslinked receiving layer 10 or only a portion thereof. One or more microspheres can settle to the same depth in the partially crosslinked receiving layer 10. According to some embodiments, at least a portion of the microsphere 3 can settle to the bottom of the partially cross-linked receiving layer 10 and ride on the support.

  The modulus of the partially crosslinked receiving layer 10 is controlled by the crosslinking density of the partially crosslinked receiving layer 10 defined as the number of moles of crosslinked material per unit volume. Furthermore, the crosslink density is related to one or more of the chemical crosslinker concentration; duration of chemical crosslink; intensity and time of radiation such as UV, ionization or electron beam radiation, and the type of crosslink used. is doing.

  Alternatively, physical gelling can be used instead of chemical crosslinking or radiation-induced crosslinking. Physical gelation is based on the formation of hydrogen bonds in the receiving layer. The crosslink density in physical gelation is the concentration between the concentration of the gelling agent and the temperature of the partially crosslinked receiving layer and the gel point or sol-gel transition temperature of the gelling agent in the partially crosslinked receiving layer. Related to the difference. In this case, the temperature of the fluid suspension at the time of coating is such that the gelling agent in the partially crosslinked receiving layer has a temperature in the partially crosslinked receiving layer to prevent dissolution of the gelling agent in the fluid suspension. The gelling agent is maintained below the sol-gel transition temperature.

  The evaporation of the fluid suspension 11 can be accomplished by blowing air over the fluid suspension 11 and / or heating the fluid suspension 11 to evaporate the fluid (FIGS. 3f, 4f, 5f) and the array. Achieved by leaving 5. After the array 5 is fully manufactured, the cross-linking reaction of the partially cross-linked receiving layer 10 including the cross-linked gelling agent initiated earlier by addition of the cross-linking agent is completed to cross-link the microsphere 3 Can be permanently fixed in place within the receiving layer 12 (FIGS. 3g, 5g). When gelatin is used as the gelling agent, the preferred cross-linking agent can be a compound such as bis (vinylsulfone) methane, glutaraldehyde or succinaldehyde. Alternatively, additional radiation 13 such as UV radiation, ionizing radiation or electron beam irradiation can be used to provide additional crosslinking, as shown in FIG. 4g. The crosslinked receiving layer is insoluble, allowing wet processing of the formed microarray without dissolution or decomposition of the crosslinked receiving layer.

  As shown in FIGS. 3g, 4g and 5g, the microspheres 3 in the array 5 do not have any receiving layer material attached to or covering its exposed surface. This allows the attachment of functionalized chemical or biological groups, probes and analytes to the exposed surface of the microsphere.

  The above-described method of preparing a partially crosslinked receiving layer by physical gelation, chemical crosslinking or radiation is performed during evaporation of a fluid suspension from the partially crosslinked receiving layer to form an array. It is designed to produce a partially cross-linked receiving layer with the ability to receive microspheres with appropriate physical properties so that no side flocculation of the microspheres occurs. Two factors are important in determining whether the microspheres will agglomerate laterally. One is in "Patterned colloidal deposition controlled by electrostatic and capillary forces" in J. Aizenberg, P. Braun, and P. Wiltzius, Physical Review Letters, Vol. 84, No. 13, 2000. Capillary tubes that drive the microspheres toward each other as described. The other is the degree of indentation of the microspheres into the partially crosslinked receiving layer. The capillary force on the microsphere is directly proportional to the interfacial surface energy between the fluid suspension and the microsphere. During the evaporation phase of the fluid suspension, where the combined thickness of the fluid suspension and the partially crosslinked receiving layer is comparable to the size of the microspheres, capillary forces are microscopic in the partially crosslinked receiving layer. It tends to cause side flocculation of the spheres. On the other hand, the surface force between the microsphere and the partially cross-linked receiving layer is “contact with the surface energy of the elastic solid” K. Johnson et al., Proc. R. Soc. Lond., A.324, 1971. In order to force the microspheres into a relatively soft, partially cross-linked receiving layer and partially prevent lateral agglomeration once the fluid suspension is removed by evaporation. It is possible to allow sufficient subsidence of the microspheres in the cross-linked receiving layer.

  From the above discussion, it can be readily seen that the physical properties of the partially cross-linked receiving layer must meet certain conditions to prevent lateral agglomeration. If the partially cross-linked receiving layer is hard, there is very little microsphere subsidence in the partially cross-linked receiving layer and there is a high probability that side flocculation of the microspheres will occur. On the other hand, if the partially cross-linked receiving layer is too soft, it provides little resistance to capillary forces that drive the lateral aggregation of the microspheres. The deformation resistance characteristics of a partially crosslinked receiving layer can be expressed in terms of Young's modulus. It is possible to determine the lower and upper bounds of the Young's modulus of the partially crosslinked receiving layer within which no side flocculation of the microspheres occurs. A method for determining the Young's modulus limit of a partially crosslinked receiving layer is provided in the Examples section of this document.

  The invention is a polymeric microsphere-based random microarray in which each microsphere in the array has a completely different signature that distinguishes the microsphere from other microspheres in the microarray. Such a signature may be based on the color, shape, size, or combination thereof of the microsphere. For color-based signatures, the primary red color is used to create thousands of identifiable microspheres with completely different “color addresses” (unique RGB values, eg R = 0, G = 204, B = 153). Three dyes representing green and blue can be mixed to derive a color. Microspheres are sites on the surface that are “active” (that is, at such sites, physical or chemical interactions can occur between the microsphere and other molecules or compounds). Can be made along with. Such compounds may be organic or inorganic compounds. Examples of molecules or compounds include organic nucleic acids, proteins or fragments thereof, or ionomers including, for example, metal ions and salts. Pre-synthesized oligonucleotides, monoclonal antibodies or any other biological or chemical agent can be attached to the surface of each microsphere. Thus, each microsphere address, such as color, can correspond to a particular probe. These microspheres can be mixed in equal amounts, and a random microarray can be produced, for example, by coating the mixed microspheres in a single layer.

  Coating methods for coating microsphere suspensions are described by Edward Cohen and Edgar B. Gutoff, Chapter 1 of “Modern Coating and Drying Techniques”, Interface Engineering Series, Volume 1, VCH Publishers Inc., NewYork. , NY 1992). Suitable coating methods may include knife coating, blade coating, dip coating, rod coating, air knife coating, gravure coating, forward and reverse roll coating and slot and extrusion coating. Various coating aids as known in the art can be added to assist in coating the microsphere suspension on the substrate. For example, suitable coating aids can include surfactants, diluents or thinners.

  Fluorescently labeled, chemiluminescent labeled or both biological samples can be hybridized to microsphere-based random microarrays. Signals from both non-selectively labeled biological samples and addressable polymeric microspheres by fluorescence, chemiluminescence or both can be analyzed with a charge coupled device after image enlargement through the optical system. it can. The recorded array image can be automatically analyzed by an image processing algorithm to obtain bioactive probe information based on each microsphere's “address”, such as the color code of each microsphere, and this information can be analyzed with fluorescence / Compared to the chemiluminescent image, a specific biological analyte material in the sample can be detected and quantified. It is also possible to apply optical or other electromagnetic means to confirm the signature.

  Microspheres or particles with a substantially curvilinear shape are preferred for ease of preparation, although other shaped particles such as elliptical or cubic particles can be used as well. Suitable methods of preparing particles are known in the art, including emulsion polymerization as described in “Emulsion polymerization” by I. Piirma, Academic Press, New York (1982), etc., or J Colloid Interface Science, Vol. 169, p. 48-59 (1985), including limited coalescence, as described by TH Whitesides and DS Doss. The particular polymer utilized to make the particles or microspheres is a water immiscible synthetic polymer that is colorable. Preferred polymers are any amorphous water immiscible polymer. Examples of polymer types that are useful are polystyrene, poly (methyl methacrylate) or poly (butyl acrylate). Copolymers such as copolymers of styrene and butyl acrylate can be used as well. Polystyrene polymers are convenient for use. The formed microspheres can be colored with insoluble colorants that are pigments or dyes that do not dissolve during the coating or submerging process. Proper dyeing can be oil-soluble in nature. It is preferred that the dye is non-fluorescent once incorporated into the microsphere.

The microspheres are desirably formed to have an average diameter in the range of 1-100 μm, such as 1-50 μm, more preferably 3-30 μm and most preferably 5-20 μm. The concentration of microspheres in the coating is in the range of 1,000 to 1,000,000 per cm ² , more preferably 1000 to 200,000 per cm ² , most preferably 10,000 to 100,000 per cm ² .

  The microsphere can have a chemical or biological functional group attached to the surface of the microsphere to interact with the desired analyte. Methods for adding chemical or biological functional groups are known in the art.

  The attachment of bioactive agents to the surface of chemically functionalized microspheres is described, for example, in Bangs Laboratories, Inc, Technote 205, Rev. 003, 30 MAR02 (Bangs Laboratories, Inc., Fishers IN). In addition, it can be performed according to procedures published in the art. Some commonly used chemical functional groups include, but are not limited to, carboxy, amino, hydroxy, hydrazide, amide, chloromethyl, epoxy, aldehyde, and the like. Examples of bioactive agents include, but are not limited to, oligonucleotides, DNA and DNA fragments, peptide nucleic acids (PNA), peptides, antibodies, enzymes, proteins and bioactive synthetic molecules.

  The following describes how to determine the Young's modulus range of the receiving layer for a given microsphere composition.

  In the following examples, a Monte Carlo simulation as described in “Random number generation and Monte Carlo method (statistics and calculation)” by James E. Gentle, Springer Verlag (1998) was introduced randomly. Implemented to determine the distance between. The results are then used in an analysis to calculate the Young's modulus of the receiving layer that avoids side flocculation of the microspheres.

In order to simulate the random distribution of microspheres as achieved by the invention, 1000 microspheres with a diameter of 10μ were randomly dropped on the entire substrate with an area of 1 cm ² and shown in FIG. As shown, no two microspheres were duplicated. The distribution of the nearest separation distance between the microspheres in FIG. 6 is shown in Table 1 and plotted in FIG. The microspheres are randomly dropped on the substrate 20 times, and the average of all 20 simulations is shown in Table 2. A cumulative average is provided in Table 2 for each nearest separation distance equal to a percentage of the total number of microspheres separated by at least that separation distance.

Table 2 shows that for this example (1000 / cm ² microsphere; 10 μ diameter microsphere), 95% of the microsphere is at least 30 μ apart from its nearest microsphere. This separation (in this case 30μ) is called “L” and is the minimum measured separation between the microspheres for at least 95% of the microspheres.

  Simulations were repeated for multiple case microsphere density and microsphere diameter, and L was determined for each case as described above over 20 iterations for each microsphere diameter / density combination. The results are summarized in Table 3.

The values from Table 3 can be used to determine the modulus requirements for a partially cross-linked receiving layer to fix the microspheres without lateral aggregation. The normal force P and the side force F acting on each microsphere and causing side aggregation are illustrated in FIG. The normal force P holds the microsphere in place by pushing it down into the receiving layer. P is an acceptor layer partially crosslinked with a microsphere described as γ ₁ as taught by KL Johnson et al., In R.Soc., London A324, 301 (1971). Determined by the interfacial surface energy between and the radius R of the microsphere:
P = 6πRγ ₁ (1)

The horizontal force F acting on each microsphere in order to bring about the side aggregation by the lateral movement of the microsphere is the radius R of the sphere, the distance L between the microspheres, and the microsphere and the microsphere indicated as γ2. Due to the interfacial surface energy between fluid suspensions containing PHYS. REV. LTRS. 84, No. 13 (2000). The lateral force F is determined by the following formula.

From equations (1) and (2), for a given given microsphere radius and distance between the microspheres, the surface energy γ ₁ and γ ₂ represent the normal and lateral forces acting on each microsphere. It can be seen that the amount is determined.

  The Young's modulus of the partially crosslinked receiving layer and the interrelationship of forces P and F are fixed in the partially crosslinked receiving layer in a manner sufficient to resist the microspheres from lateral agglomeration. It will be decided whether or not. When the microspheres are coated in a fluid suspension on a partially crosslinked receiving layer, the microspheres sink through the fluid suspension onto the partially crosslinked receiving layer. Depending on the relationship between the normal force P on the microsphere and the Young's modulus of the partially crosslinked receiving layer, the microsphere will at least partially penetrate into the partially crosslinked receiving layer. As the fluid suspension is removed by evaporation and the fluid suspension level falls below the height of the microspheres above the partially cross-linked receiving layer, the capillary force takes effect and pulls the lateral force F. Will wake up. In order to move laterally, the microspheres need to deform or scrape through the partially cross-linked receiving layer. This motion is resisted by the ability to resist deformation of the partially crosslinked receiving layer, which resistance is represented by the Young's modulus of the partially crosslinked receiving layer. The higher the Young's modulus of the material, the higher its deformation resistance, preserving the microspheres in place within the partially crosslinked receiving layer. If the Young's modulus of the partially cross-linked receiving layer is too low, the partially cross-linked receiving layer is excessively fluid and can allow easy movement of the microspheres, thus leading to side aggregation. There is sex. If the Young's modulus of the partially crosslinked receiving layer is too high, the microspheres will not penetrate into the partially crosslinked receiving layer sufficient to resist lateral movement, thus microscopic The spheres can slide along the surface of the partially crosslinked receiving layer without deformation.

Finite element analysis is performed to determine the range of Young's modulus of the receiving layer that will avoid lateral agglomeration. According to conventional finite element analysis techniques, a geometric representation of the microspheres and the receiving layer is created by dividing the microspheres and layers into discrete elements (also called meshes). For a given normal and lateral force, finite element analysis determines whether the microspheres remain stationary or move laterally to form agglomeration for a selected value of Young's modulus. This analysis provides the lower range or lower bound, which is the lowest modulus at which the microspheres will not move, and the upper range or upper bound, which is the highest modulus at which the microspheres will not move. The results can be plotted as a function of elastic modulus versus γ ₁ / γ ₂ ratio, as shown in FIG.

As shown in FIG. 9 (number of microspheres / cm ² = 1000, microsphere diameter = 10 μ, L = 30 μ), partially crosslinked to keep the microspheres in place while preventing their aggregation The desired Young's modulus of the receiving layer is the region between the lower and upper bounds. The result depends on the absolute value of the interfacial surface energy between the receiving layer partially crosslinked with the microsphere, γ ₁ and the interfacial surface energy between the microsphere and the fluid suspension, γ ₂ . The lower surface energy γ ₁ and γ ₂ is derived from the material properties of the microsphere, fluid suspension and partially crosslinked receiving layer and represents the force acting on the microsphere (see formulas 1 and 2). For example, in FIG. 9, if the ratio of γ ₁ to γ ₂ is equal to 2, the elastic modulus of the partially crosslinked receiving layer should be between 1 MPa and 55 MPa. The results for the other cases of microsphere diameter and density from Table 3 are shown in FIGS. In practice, the lower bound on the elastic modulus can be selected as 1 MPa. The upper and lower bounds of the elastic modulus can be optimized using the formulas provided in this document, depending on the number of microspheres per unit area, the microsphere radius, and the separation distance L.

* ミクロスフィアの９６％が、その最近傍ミクロスフィアからＬより大きい距離だけ離隔している。
** ミクロスフィアの９５％がその最近傍ミクロスフィアからＬより大きい距離だけ離隔している。

* 96% of the microspheres are separated from their nearest microsphere by a distance greater than L.
** 95% of the microspheres are separated from their nearest microsphere by a distance greater than L.

  The invention has been described in detail with particular reference to certain embodiments thereof. Changes and modifications can be made within the spirit and scope of the invention.

FIG. 2 is a schematic diagram illustrating one method that has been utilized in the prior art to prepare microsphere microarrays. FIG. 1a shows any suitable support 1 and FIG. 1b shows a fluid layer 2 comprising microspheres (beads) 3, a gelling agent and a chemical cross-linking agent that are spread across the support of FIG. 1a. FIG. 1C shows a fluid layer in which the gelling agent undergoes a sol-gel transition and thus immobilizes the microspheres 3 in the gel 4; FIG. 1d shows by evaporation of excess fluid 2 from the gel layer 4 FIG. 1 e shows a cross-linked fluid layer 6 that permanently secures the microsphere 3 within the microarray and leaves a film 7 of gel on the surface of the microsphere 3. FIG. 2 is a schematic diagram of another prior art process for preparing random microsphere microarrays, where FIG. 2a shows any suitable support 1; FIG. 2b is coated with an uncrosslinked gelling agent 8 FIG. 2C shows the fluid layer 2 supporting the crosslinker for the gelling agent 8 and the probe-supporting microsphere 3 disposed on the support 1 of FIG. 2b; FIG. 2c shows the microsphere 3 of FIG. 2c submerged in an uncrosslinked gelling agent 8. FIG. As can be seen from FIG. 2 c, the layer with the gelling agent 8 undergoes a sol-gel transition to the gel 4 and thus immobilizes the microspheres 3. FIG. 2e shows the evaporation of fluid from the fluid layer 2: FIG. 2f shows that the microsphere 3 still has a gel coating 4 on its surface due to the dissolution of the gelling agent 8 into the fluid layer 2. FIG. The final microarray 5 is shown. Fig. 3 is a schematic view of one embodiment of the present invention, wherein Fig. 3a shows any suitable support 1; Fig. 3b is extended across the support 1 of Fig. 3a to form a receiving layer 9; Figure 3c shows a crosslinkable composition and a chemical crosslinker; Figure 3c is a portion with a modulus adjusted to allow for the indentation of the microspheres into the fluid suspension that is to be spread over FIG. 3d shows a fluid suspension 11 containing microspheres 3 spread across the partially cross-linked receiving layer of FIG. 3c; FIG. 3f shows the evaporation of the fluid suspension 11 to expose the surface of the microsphere 3; FIG. 3g shows the microsphere 3 partially sinking in the partially cross-linked receiving layer 10; , Make microarray 5 robust to wet processing Shows a receiving layer 12 which is further chemically crosslinked. Fig. 4 is a schematic view of another embodiment of the present invention, where Fig. 4a shows any suitable support 1; Fig. 4b extends across the support 1 of Fig. 4a to form a receiving layer 9; FIG. 4c shows ultraviolet (UV) to an elastic modulus sufficient to allow the microspheres to be pressed into the fluid suspension to be spread over. FIG. 4d shows the partially crosslinked receiving layer 10 cross-linked by radiation 13, such as radiation, ionizing radiation or electron beam irradiation; FIG. 4d covers the entire partially crosslinked receiving layer 10 of FIG. 4c. FIG. 4e shows the microsphere 3 partially submerged in the partially cross-linked receiving layer 10; FIG. 4f shows the microsphere 3 containing the spread microsphere 3; Expose the surface of Sphere 3 FIG. 4g is cross-linked by radiation 13, such as UV radiation, ionizing radiation or electron beam irradiation, in order to make the microarray 5 robust to wet processing. A cross-linked receiving layer 12 is shown. Fig. 5 is yet another schematic diagram of the process of the present invention, where Fig. 5a shows any suitable support 1 and Fig. 5b extends across the support of Fig. 5a to form a receiving layer 9; FIG. 5c shows a gel with sufficient modulus to allow indentation by the microspheres 3; FIG. 5c shows a fluid containing a modified gelling agent and a slow-acting chemical crosslinker for the gelling agent; FIG. 5d shows a sol-gel transitional receiving layer 14 in which the agent is gelled; FIG. 5d shows the sol of gelling agent in the receiving layer extended over the entire gelled receiving layer 14 in FIG. 5c. FIG. 5e shows the microsphere 3 partially submerged in the gelled receiving layer 14; FIG. 5f shows the microsphere 3 containing the microsphere 3 at a temperature below the gel transition. To expose the surface of Sphere 3 Shows the evaporation of the fluid suspension 11; Figure 5g illustrates a chemically crosslinked receiving layer 12 to the microarray 5 to those robust to wet processing. No two microspheres also do not overlap, which is a diagram of an area of 1 cm ² with 1000 microspheres. 7 is a plot of the data in Table 1 showing the distribution of the nearest separation distance between the microspheres of FIG. It is the schematic which shows the force on a microsphere. FIG. 6 is a plot showing the upper and lower bounds of the realizable modulus for a 10 μm microsphere where L is 30 μm. FIG. 5 is a plot showing the upper and lower bounds of the achievable modulus for a 5 μm microsphere where L is 30 μm. FIG. 4 is a plot showing the upper and lower bounds of the achievable modulus for a 15 μm microsphere, where L is 20 μm. FIG. 6 is a plot showing the upper and lower bounds of the achievable modulus for a 20 μm microsphere, where L is 20 μm. FIG. 6 is a plot showing the upper and lower bounds of the achievable modulus for a 10 μm microsphere where L is 5 μm. FIG. 6 is a plot showing the upper and lower bounds of the achievable modulus for a 20 μm microsphere, where L is 25 μm.

Explanation of symbols

1 Support 2 Fluid Layer 3 Microsphere (Bead)
4 Gel 5 Microarray 6 Crosslinked fluid layer 7 Gelling agent film 8 Non-crosslinked gelling agent 9 Receiving layer 10 Partially crosslinked receiving layer 11 Fluid suspension 12 Chemically crosslinked receiving layer 13 Radiation 14 Receiving layer subjected to sol-gel transition

Claims (19)

In a method of manufacturing an element comprising a microsphere array on a support,
a) coating the support with a coating composition to form a receiving layer having an elastic modulus changeable by crosslinking;
b) enabling partial cross-linking of the receiving layer to achieve a modulus of elasticity that allows partial embedding of the microspheres into the partially cross-linked receiving layer;
c) coating a dispersion of microspheres in a fluid suspension onto a partially crosslinked receiving layer, each microsphere having one position;
d) allowing the microspheres to be partially submerged in the partially crosslinked receiving layer;
e) removing the fluid suspension from the partially crosslinked receiving layer; and f) partially crosslinked in such a way that the microspheres maintain their respective positions during and after the wet process. Allowing the receiving layer to be further crosslinked;
Comprising a method. The microspheres form an interface between the partially crosslinked receiving layer, the interface having an interfacial surface energy gamma _1, The method of claim 1. The method of claim 1, wherein the microspheres form an interface with a fluid suspension, the interface having an interfacial surface energy γ ₂ .   The method of claim 1, wherein the elastic modulus of the partially crosslinked receiving layer has a lower bound of 1 MPa. The elastic modulus of the partially crosslinked receiving layer is defined by a monotonically increasing function of the ratio of γ ₁ to γ ₂ where γ ₁ is the interfacial surface energy between the microsphere and the receiving layer and γ ₂ is The method of claim 1, wherein the surface energy of the interface between the microsphere and the fluid suspension. a) a support;
b) a water-insoluble receiving layer on the support comprising the receiving layer material;
c) Randomly spaced microspheres fixed and partially buried in the receiving layer, with exposed surfaces above the receiving layer, each exposed surface interacting with the analyte A microsphere having at least one probe mounted in a manner;
An element wherein the exposed surface of the microsphere does not include a receiving layer material.   The element of claim 6 wherein the crosslinked layer contains gelatin.   The element of claim 6, wherein the microsphere comprises polystyrene or poly (methyl methacrylate).   7. Element according to claim 6, wherein the support comprises glass paper, metal or polymer.   The element of claim 6, wherein the microsphere has an average diameter of 1-100 μ.   The element of claim 6, wherein the microsphere has an average diameter of 5-20 μ. The element according to claim 6, wherein the number of microspheres per cm ² in the crosslinked layer is 100 to 1,000,000. The element according to claim 6, wherein the number of microspheres per cm ² in the crosslinked layer is 10,000 to 100,000. The element according to claim 6, wherein the number of microspheres per 1 cm ² in the cross-linked layer is 1000 to 200,000.   The element of claim 6 wherein the microspheres are color coded.   16. The element of claim 15, wherein the color code of each microsphere identifies a probe on the surface of the microsphere.   The element of claim 6, wherein the probe is a protein or nucleic acid. In the element that contains the microsphere array,
a) coating the support with a coating composition to form a receiving layer having an elastic modulus changeable by crosslinking;
b) enabling partial cross-linking of the receiving layer to achieve a modulus of elasticity that allows partial embedding of the microspheres into the partially cross-linked receiving layer;
c) coating a dispersion of microspheres in a fluid suspension onto a partially crosslinked receiving layer, each microsphere having one position;
d) allowing the microspheres to be partially submerged in the partially crosslinked receiving layer;
e) removing the fluid suspension from the partially crosslinked receiving layer; and f) partially crosslinked in such a way that the microspheres maintain their respective positions during and after the wet process. Allowing the receiving layer to be further crosslinked;
An element produced by a process comprising.   19. The element of claim 18, wherein the modulus of the partially crosslinked receiving layer does not allow any lateral movement of the microspheres in the partially crosslinked receiving layer during removal of the fluid suspension. .

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