Particle sorting system and method

Abstract

Systems and methods for particle sorting are described. The array may include a substrate having a first surface and a second surface opposite the first surface. The substrate may include a plurality of pores defining a lumen extending from the first surface to the second surface. The plurality of pores may be configured to receive a sample solution containing a plurality of particles. The array may further include a surface material disposed on or near the first or second surface. The surface material may include a plurality of materials configured to modify the wetting behavior of the sample solution or the plurality of particles at or near the first or second surface such that one of the first or second surface is hydrophilic and the other of the first or second surface is hydrophobic.

Description

[Technical Field]

[0001] cross reference This application claims the benefits of U.S. Provisional Patent Application No. 62 / 985,257, filed on 4 March 2020. This Provisional Application is incorporated herein by reference. [Background technology]

[0002] background Cell therapy is a cornerstone of regenerative medicine and immunotherapy. While many of the non-therapeutic cells used in this treatment are harmless, certain abnormal cell types can cause serious adverse events in patients, even in small populations. Therefore, it can be crucial to purify therapeutic cells from harmful cells before transplanting them into patients. To accelerate the transition of cell-based regenerative medicine technologies to clinical practice, high-throughput, high-purity methods may be needed to isolate rare stem cells and other immune cell types in a clinically applicable, sterile format based on differential expression of surface markers. [Overview of the Initiative] [Means for solving the problem]

[0003] Abstract Embodiments disclosed herein provide systems, methods, and devices for sorting cells. In some cases, these cells may be sorted with the help of a laser (e.g., laser extraction) and / or a micropore array. The micropore array may include a coating that can interact with the laser to assist in the extraction of cells of interest. The coating may be peeled off in some cases, and at the same time, the meniscus of liquid held within the micropore array may be destroyed. Beneficially, the approaches described herein can increase cell viability and extraction efficiency, for example, because the laser is directed to the array surface rather than directly to the liquid holding the particles of interest.

[0004] In some embodiments, the Disclosure provides an array comprising a substrate having a first surface and a second surface opposite to the first surface, wherein the substrate comprises a substrate material and a surface material, the surface material being located on or adjacent to the first or second surface, the substrate comprising a plurality of pores defining a lumen extending from the first surface to the second surface, the substrate characterized in that each of the plurality of pores has a maximum diameter of 500 microns or less, each of the plurality of pores has an aspect ratio of 5 or greater, and the surface material is selected from a material that absorbs more than 10 percent of incident electromagnetic radiation.

[0005] In some embodiments, the Disclosure provides an array comprising a substrate having a first surface and a second surface opposite to the first surface, wherein the substrate comprises a substrate material and a surface material, the surface material being located on or adjacent to the first or second surface, the substrate comprising a plurality of pores extending from the first surface to the second surface, the substrate having a pore density of 100 or more pores per square millimeter, each of the plurality of pores having an aspect ratio greater than 10, and the surface material being selected from a material that absorbs more than 10 percent of incident electromagnetic radiation.

[0006] In a particular embodiment, each pore is approximately 0.008 mm 2 or has a maximum cross-sectional area of ​​less than that. In a particular embodiment, each pore of a plurality of pores has a pore diameter in the range of 5 microns to 100 microns. In a particular embodiment, each pore of a plurality of pores has a pore diameter in the range of 15 microns to 50 microns. In a particular embodiment, each pore has a length selected from the range of about 1 mm to about 500 mm. In a particular embodiment, each pore has a length selected from the range of about 1 mm to about 100 mm. In a particular embodiment, each pore has a length selected from the range of about 0.1 mm to about 10 mm.

[0007] In certain embodiments, the pore density is in the range of 100 to 2500 pores per square millimeter. In certain embodiments, the pore density is in the range of 500 to 1500 pores per square millimeter. In certain embodiments, the surface material is substantially similar to the substrate material. In certain embodiments, the surface material is different from the substrate material. In certain embodiments, the substrate material is glass, and the surface material is not glass. In certain embodiments, the surface material contains a metal. In certain embodiments, the surface material absorbs more than 10 percent of incident electromagnetic radiation at wavelengths selected from 0.4 to 2.5 microns. In certain embodiments, the surface material absorbs more than 50 percent of the incident radiation. In certain embodiments, the surface material absorbs more than 50 percent of incident electromagnetic radiation at wavelengths selected from 0.4 to 1.5 microns.

[0008] In certain embodiments, the aspect ratio is in the range of 5 to 100. In certain embodiments, the aspect ratio is 20 or greater. In certain embodiments, the aspect ratio is 50 or greater. In certain embodiments, the aspect ratio is 100 or greater. In certain embodiments, the surface material covers or partially covers the second surface. In certain embodiments, the surface material covers or partially covers the first surface. In certain embodiments, the surface material does not obstruct access to the lumen of the pore. In certain embodiments, the surface material has an average thickness of about 20 nm to 500 nm. In certain embodiments, the surface material has an average thickness of about 100 nm to 500 nm. In certain embodiments, the surface material is hydrophobic.

[0009] In certain embodiments, the first and second surfaces are substantially parallel. In certain embodiments, the plurality of pores extend from the first surface to the second surface at an angle to the surface normal. In certain embodiments, the angle is greater than 0 to 90 degrees. In certain embodiments, the plurality of pores extend from the first surface to the second surface at a right angle. In certain embodiments, the plurality of pores pass through an indirect path from the first surface to the second surface.

[0010] In some embodiments, the Disclosure provides a system for sorting components of a mixture, the system comprising an array of any embodiment of the Disclosure and a housing having an inner surface configured to receive selected contents released from the array. In a particular embodiment, the inner surface is located below a second surface of a substrate.

[0011] In some embodiments, the Disclosure provides a method for releasing selected contents from pores in an array, the method comprising the step of identifying pores in an array containing selected contents, the array comprising a substrate having a first surface and a second surface opposite to the first surface, the substrate comprising a substrate material and a surface material, the surface material being located on or adjacent to the first or second surface, the substrate comprising a plurality of pores defining a lumen extending from the first surface to the second surface, the substrate (a) each of the plurality of pores having a maximum diameter of 500 microns or less, A step characterized by one or more of the following: (b) each pore of a plurality of pores has an aspect ratio of 5 or more; (c) a pore density of 100 or more pores per square millimeter; and (d) the surface material is selected from a material that absorbs more than 10 percent of the incident electromagnetic radiation; and a step of removing a portion of the surface material from a first or second surface of the array by electromagnetic radiation directed at the surface material in or adjacent to the identified pore, thereby releasing the contents of the identified pore.

[0012] In certain embodiments, the electromagnetic radiation is selected from a wavelength of 0.2 to 2.5 microns, a fluence level sufficient to break the adhesion between the contents and the pore, and a pulse duration in the range of 1 ns to 1 millisecond. In certain embodiments, the step of removing the surface material includes ablation. In certain embodiments, the step of removing the surface material includes mechanical removal. In certain embodiments, the mechanical removal includes chipping. In certain embodiments, the step of removing the surface material includes photothermal removal. In certain embodiments, the step of removing the surface material includes photochemical removal. In certain embodiments, the step of removing the surface material includes photoacoustic removal.

[0013] In a particular embodiment, the selected contents include cells in an aqueous solution. In a particular embodiment, the cells are selected from INKT cells, Tmem, Treg, HSPC, and combinations thereof. In a particular embodiment, each of the multiple pores is approximately 0.008 mm. 2 or having a cross-sectional area less than that. In a particular embodiment, each pore of a plurality of pores has a pore diameter in the range of 5 microns to 100 microns. In a particular embodiment, each pore of a plurality of pores has a pore diameter in the range of 15 microns to 50 microns. In a particular embodiment, each pore has a length selected from the range of about 1 mm to about 500 mm. In a particular embodiment, each pore has a length selected from the range of about 1 mm to about 100 mm. In a particular embodiment, each pore has a length selected from the range of about 0.1 mm to about 10 mm.

[0014] In a particular embodiment, the pore density is in the range of 100 to 2500 pores per square millimeter in one array. In a particular embodiment, the pore density is in the range of 500 to 1500 pores per square millimeter in one array. In a particular embodiment, the array has 1000 pores / mm 2It includes a pore density exceeding 5000 pores / mm². In a particular embodiment, the pore density is 5000 pores / mm². 2 or greater than that. In certain embodiments, the aspect ratio is in the range of 5 to 100. In certain embodiments, the pore has an aspect ratio of 20 or greater. In certain embodiments, the pore has an aspect ratio of 50 or greater. In certain embodiments, the pore has an aspect ratio of 100 or greater. In certain embodiments, the surface material absorbs more than 10 percent at wavelengths selected from about 0.4 microns to about 2.5 microns. In certain embodiments, the surface material absorbs more than 50 percent of the incident radiation. In certain embodiments, the surface material absorbs more than 50 percent of the incident radiation at wavelengths selected from about 0.4 microns to about 2.5 microns.

[0015] In a particular embodiment, the array is characterized by two or more of the following: (a) each pore of the plurality of pores has a maximum diameter of 500 microns or less; (b) each pore of the plurality of pores has an aspect ratio of 5 or more; (c) a pore density of 100 or more pores per square millimeter; and (d) the surface material is selected from a material that absorbs more than 10 percent of the incident electromagnetic radiation. In a particular embodiment, a portion of the surface material is adjacent to the identified pore. In a particular embodiment, a portion of the surface includes the lumen surface of the identified pore. In a particular embodiment, a portion of the surface is removed to a depth of 100 microns or less. In a particular embodiment, a portion of the surface is removed to a depth of 50 microns or less. In a particular embodiment, the method further includes loading a solution containing the selected contents into the array prior to the step of identifying the pores containing the selected contents. In a particular embodiment, the step of identifying the pores containing the selected contents includes analyzing the electromagnetic radiation emitted from the pores of the array. In a particular embodiment, the step of releasing the contents includes releasing the contents at a rate of approximately 5,000 to approximately 100,000,000 pores per second.

[0016] In some embodiments, the disclosure provides beads comprising an infrared absorbing core and a non-infrared absorbing shell, wherein the outer diameter of the non-infrared absorbing shell is equal to or less than about 10 microns.

[0017] In certain embodiments, the non-infrared absorbing shell comprises agarose, dextran, or both. In certain embodiments, the infrared absorbing core comprises an infrared absorbing dye. In certain embodiments, the beads have a diameter equal to or less than about 20 microns.

[0018] In some embodiments, the Disclosure provides a solution comprising a plurality of beads of any embodiment of the Disclosure; and the particles of the present interest. In certain embodiments, the particles of the present interest are cells. In certain embodiments, the ratio of the number of beads to the number of cells is about 1:1 to 10:1.

[0019] In another aspect of the present disclosure, the array includes a substrate having a first surface and a second surface opposite to the first surface, the substrate comprising a plurality of pores defining a lumen extending from the first surface to the second surface, the plurality of pores configured to receive a sample solution containing a plurality of particles, and a surface material provided on or near the first surface or the second surface, the surface material comprising a plurality of materials configured to modify the wetting behavior of the sample solution or the plurality of particles on or near the first surface or the second surface such that one of the first surface or the second surface is hydrophilic and the other of the first surface or the second surface is hydrophobic.

[0020] In some embodiments, the plurality of materials includes a metal layer (e.g., one that has been sputtered, physically sputtered, chemically coated, modified by functional groups (i.e., surface hydrophilic modification, surface hydrophobic modification), etc.). The metal layer can have a thickness in the range of about 50 nm to about 1 mm. The metal layer can include titanium and / or gold. The first portion of the metal layer may be coated with a first chemical coating. The second portion of the metal layer may be coated with a second chemical coating that is different from the first chemical coating. In some embodiments, the first chemical coating may be provided on the vertical sidewalls of a plurality of pores at or near the first surface or the second surface. The first chemical coating can be configured to reduce or eliminate the attachment of particles to the vertical sidewalls of the pores. The second chemical coating can be configured to reduce or prevent the unwanted leakage of the sample solution from the pores. In some embodiments, the second chemical coating is hydrophobic. The second chemical coating may be provided on a portion of the substrate that is at or near the first surface or the second surface. The portion of the substrate may be near the vertical sidewalls of the plurality of pores. In some cases, the portion of the substrate may be substantially orthogonal to the vertical sidewalls of the plurality of pores.

[0021] In some embodiments, the first chemical coating can include methoxy-poly(ethylene-glycol)-thiol. The second chemical coating can include 1H,1H,2H,2H-perfluorodecanethiol.

[0022] In some embodiments, the plurality of materials further includes a chemical coating that is not on a metal layer (e.g., sputtered, physically sputtered, chemically coated, modified by a functional group (i.e., surface hydrophilic modification, surface hydrophobic modification), etc.). The chemical coating may be provided on one or more portions of the substrate or the plurality of pores that do not have a metal layer (e.g., sputtered, physically sputtered, chemically coated, modified by a functional group (i.e., surface hydrophilic modification, surface hydrophobic modification), etc.). In some embodiments, the chemical coating includes methoxy-poly(ethylene-glycol)-silane.

[0023] In some embodiments, the second surface may be configured to receive a sample solution containing a plurality of particles. The first surface may be configured to be destroyed to release one or more particles from one or more pores. In some embodiments, the second surface may be hydrophilic to facilitate absorption of the sample solution containing a plurality of particles into the plurality of pores. The first surface may be hydrophobic to reduce or eliminate undesirable leakage of the sample solution from the pores.

[0024] In some embodiments, the first surface may be configured to be destroyed by directing electromagnetic radiation at one or more portions of the second surface. In some embodiments, each pore of the plurality of pores has a maximum diameter of 500 microns or less. Each pore of the plurality of pores may have an aspect ratio of 5 or more. The surface material may be selected from materials that absorb more than 10% of the incident electromagnetic radiation. The substrate may have a pore density of 100 or more pores per square millimeter.

[0025] In some embodiments, the particle extraction yield of the array is at least 70%. The particle extraction yield of an array having a functionally modified surface layer (i.e., a chemically coated metal layer) (i.e., surface hydrophilic modification, surface hydrophobic modification) may be higher than that of another array without a functionally modified surface layer (i.e., a chemically coated metal layer) (i.e., surface hydrophilic modification, surface hydrophobic modification). For example, the particle extraction yield of an array having a functionally modified surface layer (i.e., a chemically coated metal layer) (i.e., surface hydrophilic modification, surface hydrophobic modification) may be at least 5% higher than that of another array without a functionally modified surface layer (i.e., a chemically coated metal layer) (i.e., surface hydrophilic modification, surface hydrophobic modification). In some cases, the particle extraction yield of an array having a functionally modified surface layer (i.e., a chemically coated metal layer) (i.e., surface hydrophilic modification, surface hydrophobic modification) is at least 20% higher than that of another array without a functionally modified surface layer (i.e., a chemically coated metal layer) (i.e., surface hydrophilic modification, surface hydrophobic modification).

[0026] In some embodiments, multiple particles contain living cells. The live cell extraction yield of an array having a functionally modified surface layer (i.e., a chemically coated metal layer) (i.e., surface hydrophilic modification, surface hydrophobic modification) may be higher than that of another array without a functionally modified surface layer (i.e., a chemically coated metal layer) (i.e., surface hydrophilic modification, surface hydrophobic modification). For example, the live cell extraction yield of an array having a functionally modified surface layer (i.e., a chemically coated metal layer) (i.e., surface hydrophilic modification, surface hydrophobic modification) may be at least 5% higher than that of another array without a functionally modified surface layer (i.e., a chemically coated metal layer) (i.e., surface hydrophilic modification, surface hydrophobic modification). In some cases, the live cell extraction yield of arrays having a functionally modified surface layer (i.e., a chemically coated metal layer) (i.e., surface hydrophilic modification, surface hydrophobic modification) is at least 20% higher than that of other arrays not having a functionally modified surface layer (i.e., a chemically coated metal layer) (i.e., surface hydrophilic modification, surface hydrophobic modification).

[0027] Embedding by reference All publications, patents, and patent applications referenced herein are invoked by reference to the same extent as each individual publication, patent, or patent application is explicitly and individually invoked by reference. [Brief explanation of the drawing]

[0028] Novel features of the present invention are described in detail in the appended claims. The features and advantages of the present invention will be better understood by referring to the following detailed description and accompanying drawings illustrating illustrative embodiments utilizing the principles of the present invention.

[0029] [Figure 1A] Figure 1A is a side cross-sectional view of an array for sorting cells according to several embodiments.

[0030] [Figure 1B] Figure 1B is a top view of an array for sorting particles according to several embodiments.

[0031] [Figure 1C] Figure 1C shows exemplary images of arrays with different cell concentrations according to several embodiments.

[0032] [Figure 2A] Figure 2A is a side cross-sectional view of an exemplary array for sorting particles according to several embodiments.

[0033] [Figure 2B] Figure 2B is an orthogonal view of an exemplary substrate of an exemplary array according to several embodiments.

[0034] [Figure 3A] Figure 3A is an orthogonal view of an exemplary array for sorting particles according to several embodiments.

[0035] [Figure 3B] Figure 3B is an orthogonal view of an exemplary array for sorting particles, which, according to several embodiments, has a laser-removed coating near the pore.

[0036] [Figure 4A] Figure 4A is an orthogonal view of PBMCs stained with an IR energy-absorbing fluorescent dye in an exemplary first array according to several embodiments.

[0037] [Figure 4B] Figure 4B is an orthogonal view of an exemplary first array after PBMC extraction, according to several embodiments.

[0038] [Figure 5A]Figure 5A shows a side cross-sectional view of an array comprising microspheres according to several embodiments.

[0039] [Figure 5B] Figure 5B shows a side cross-sectional view of an array containing microspheres and aqueous sample solutions according to several embodiments.

[0040] [Figure 6A] Figure 6A shows bright-field images of arrays of microspheres and micropores filled with cells according to several embodiments.

[0041] [Figure 6B] Figure 6B shows bright-field images of cell extraction from a single pore according to several embodiments.

[0042] [Figure 6C] Figure 6C shows images of arrays of microspheres and pores filled with a single cell, according to several embodiments.

[0043] [Figure 6D] Figure 6D shows an image of the array after cells have been extracted from a single micropore, according to several embodiments.

[0044] [Figure 7A] Figure 7A shows exemplary bright-field images of extracted cells according to several embodiments.

[0045] [Figure 7B] Figure 7B shows illustrative images of extracted cells according to several embodiments.

[0046] [Figure 8] Figure 8 shows bright-field images of exemplary microspheres containing agarose and dextran according to several embodiments.

[0047] [Figure 9] Figure 9 shows high-magnification infrared images of exemplary microspheres containing agarose and dextran according to several embodiments.

[0048] [Figure 10A] Figure 10A shows a bright-field image of an exemplary microsphere containing agarose and an IR-absorbing dye according to several embodiments.

[0049] [Figure 10B] Figure 10B shows an infrared image of an exemplary microsphere containing agarose and an IR-absorbing dye, according to several embodiments.

[0050] [Figure 11A] Figure 11A shows a flowchart of the coating procedure according to several embodiments.

[0051] [Figure 11B] Figure 11B shows further details of the coating procedure in Figure 11A according to several embodiments.

[0052] [Figure 12A] Figure 12A is a top view of an array for sorting particles according to one of several embodiments.

[0053] [Figure 12B] Figure 12B shows a cross-sectional view of an array having surface modification at the lower end of the array, according to several embodiments.

[0054] [Figure 13A] Figure 13A shows a cross-sectional view of the bottom end of the array before surface modification, according to several embodiments.

[0055] [Figure 13B]Figure 13B shows a cross-sectional view of the bottom edge of an array coated with one layer of pre-treated material according to several embodiments.

[0056] [Figure 13C] Figure 13C shows a cross-sectional view of the bottom end of an array coated with a first material and a second material according to several embodiments.

[0057] [Figure 13D] Figure 13D shows a cross-sectional view of the bottom end of an array comprising two layers of coating material and surface modification, according to several embodiments.

[0058] [Figure 14A] Figure 14A shows a side cross-sectional view of a system comprising an array, housing, and inner surface according to several embodiments.

[0059] [Figure 14B] Figure 14B shows a side cross-sectional view of a system comprising an array, housing, inner surface, and electromagnetic radiation source according to several embodiments.

[0060] [Figure 15A] Figure 15A is an initial orthogonal view of an exemplary system leak test at time 0 according to several embodiments.

[0061] [Figure 15B] Figure 15B is an orthogonal view of the end of a leak test of an exemplary system after 5 hours, according to several embodiments.

[0062] [Figure 16A] Figure 16A shows a side cross-sectional view of an array comprising multiple pores according to several embodiments.

[0063] [Figure 16B]Figure 16B shows a side cross-sectional view of an array in which an aqueous solution is deposited, according to one of several embodiments.

[0064] [Figure 16C] Figure 16C shows a side cross-sectional view of the exemplary array from Figure 1A being inserted into a cartridge, according to several embodiments.

[0065] [Figure 16D] Figure 16D shows images of signal plots for a first cell and a second cell according to several embodiments.

[0066] [Figure 16E] Figure 16E shows a side cross-sectional view of the extraction of a second cell according to one of several embodiments.

[0067] [Figure 16F] Figure 16F shows a side cross-sectional view of the cell harvesting process according to several embodiments.

[0068] [Figure 17] Figure 17 shows exemplary biofluorescence images of cell arrays according to several embodiments.

[0069] [Figure 18] Figure 18 shows an exemplary scatter plot of 500,000 pores of the array shown in Figure 17, according to several embodiments.

[0070] [Figure 19A] Figures 19A to 19C show a comparison of the performance (extraction yield and cell viability) of Au-coated pore plates and Cr-coated pore plates according to several embodiments. [Figure 19B] Figures 19A to 19C show a comparison of the performance (extraction yield and cell viability) of Au-coated pore plates and Cr-coated pore plates according to several embodiments. [Figure 19C] Figures 19A to 19C show a comparison of the performance (extraction yield and cell viability) of Au-coated pore plates and Cr-coated pore plates according to several embodiments.

[0071] [Figure 20] Figure 20 shows contact angle images and measurements of different coatings according to several embodiments. [Modes for carrying out the invention]

[0072] Detailed explanation There is a need to provide a high-speed, sterile cell sorting system. Therefore, systems, devices, and methods for sorting cells from arrays such as micropore arrays by laser extraction are provided herein. Micropore sorting using the systems, devices, and methods herein may be designed to achieve high sorting speeds of approximately 10,000 cells / second or speeds 100 to 1000 times faster than state-of-the-art methods. Furthermore, embodiments described herein may enable such sorting speeds without impairing cell viability or function, while maintaining sterility and operator biosafety, reducing contamination between samples, and eliminating time constraints on flow rate. In particular, the surface material of the micropore array, as well as the systems and methods using it, can enable the release of pore contents in a manner where thermal effects on the pore contents are negligible. The various systems and methods of this disclosure may be combined with or modified with other systems and methods, such as those described in International Patent Application PCT / US2019 / 049221, entitled "ULTRAFAST PARTICLE SORTING," which is incorporated herein in its entirety by reference.

[0073] array Arrays are provided herein. Arrays described herein may be used to sort particles. These particles may be particles of interest, for example, cells that need to be concentrated for therapeutic use. The array may comprise a substrate. The substrate may comprise a first surface, e.g., a top surface, a second surface opposite the first surface, e.g., a bottom surface, and a plurality of pores extending from the first surface to the second surface. These pores may define lumens that may have various shapes as described herein. These pores may be micropores or microchannels.

[0074] In one non-limiting example, a substrate containing multiple pores may be characterized in that each pore has a maximum diameter of 500 microns or less, each pore has an aspect ratio of 10 or greater, and the surface material is selected from a material that absorbs more than 10 percent of the incident electromagnetic radiation. In further or alternative non-limiting examples, a substrate containing multiple pores may be characterized in that it has a pore density of 100 or more pores per square millimeter, each pore has an aspect ratio of 10 or greater, and the surface material is selected from a material that absorbs more than 10 percent of the incident electromagnetic radiation.

[0075] Figure 1A is a vertical side cross-sectional view of an array for sorting particles according to several embodiments. As shown in Figure 1A, the array 100 may comprise a substrate 110 having (a) a first surface 111 and a second surface 112 opposite the first surface 111, and (b) a plurality of pores 113 extending from the first surface 111 to the second surface 112. These plurality of pores may be substantially parallel to each other and may be configured to hold particles together with a liquid. For example, the liquid may be held within the pores by surface tension and, in some cases, may form a meniscus at one or both ends of each pore.

[0076] The base material 110 may include a base material. The base material may be glass, for example, silicate glass, fused silica, fused quartz, etc. The base material may be plastic, for example, PETG, PEEK, etc. In some embodiments, the base material may be metal, for example, aluminum, steel, chromium, titanium, gold, etc.

[0077] The substrate 110 may contain a plurality of pores 113. In some cases, the plurality of pores 113 contain approximately 100,000 to approximately 100 billion pores. In some cases, the plurality of pores 113 contain approximately 1,000 to approximately 1 billion pores. In some cases, the plurality of pores 113 contain approximately 1 million to approximately 100 billion pores.

[0078] The substrate 110 may contain pores of a certain density. The pore density may include the number of pores per square millimeter of one array. The pore density may be measured on a first surface 111 or a second surface 112. If necessary, in some embodiments, the first array 100 has an open array ratio (packing density) of about 66 percent or about 40 percent to about 75 percent. In some cases, the pore density may be in the range of 100 to 2500 pores per square millimeter. In some cases, the pore density may be in the range of 500 to 1500 pores per square millimeter. A method for producing a high pore density may be by fusing tubes such as capillaries. The pore density may be changed by varying the wall thickness and central diameter of the tubes.

[0079] In one non-limiting example, the first array 110 contains 240 million pores 113, each having a width and length of 10 × 10 inches and a diameter of 15 μm.

[0080] Furthermore, the first array 100 has an array height 110a, which is measured as the vertical distance between the first surface 111 and the second surface 112, as shown in Figure 1A. In some embodiments, the array height 110a may be measured as the maximum or minimum vertical distance between the first surface 111 and the second surface 112. In some embodiments, the array height 110a may be measured as the standard height of the pore 113. In some embodiments, the array height 110a may be measured as the maximum or minimum length of the pore 113. Each pore may have a height (or longitudinal length) 113a. This length may be uniform across pores, or it may vary from pore to pore due to distortion or irregularity during the manufacturing process, etc. If necessary, each pore 113 may have a length equal to or less than about 50 mm. In some cases, each pore may have a length selected from about 1 mm to about 500 mm. In some cases, each pore may have a length selected from approximately 1 mm to approximately 100 mm. In some cases, each pore may have a length selected from approximately 1 mm to approximately 10 mm.

[0081] If necessary, multiple pores 113 may be substantially perpendicular to the first surface 111 and the second surface 112. In some embodiments, multiple pores 113 may be substantially parallel to one another. In some embodiments, the first surface opposite the second surface may be a substantially parallel surface. Multiple pores may extend perpendicularly from the first surface to the second surface. Those pores may extend perpendicularly from the first surface to the second surface. Alternatively, multiple pores may extend from the first surface to the second surface at an angle to the surface normal. That angle may be less than 90 degrees from the normal. That angle may be less than 60 degrees, less than 45 degrees, less than 30 degrees or less. That angle may be in the range of 5 to 90 degrees.

[0082] In some embodiments, multiple pores may traverse indirect paths from a first surface to a second surface. In such embodiments, these pores may be intertwined, glued together, or interlocked. These pores may include one or more curved sections such that the path through the pore substantially changes direction relative to a straight path from the first surface to the second surface.

[0083] Figure 1B shows a top view of an array 100 for sorting particles. In some examples, the array 100 has a number of pores 113. Each of these pores may include a cross-section. The cross-section may be circular, elliptical, polyhedral (e.g., square, hexagonal, octagonal, dodecagonal, etc.), or have an irregular shape. The shape may be uniform across pores, or the pores may differ from pore to pore due to distortion or irregularity during the manufacturing process, etc.

[0084] Referring to Figure 1A, the cross-section of each pore 113 may include cross-sectional dimensions 113b. Cross-sectional dimensions may be measured at either of the two surfaces of the array or at an intermediate position. Cross-sectional dimensions may be measured at a single cross-section. Additionally or alternatively, cross-sectional dimensions may be averaged over many locations along the pore. These dimensions may be measured by many methods, including microscopic methods using a reference, interferometric methods, and methods calculated from flow rates. In some examples, each pore in the array may have cross-sectional dimensions in the range of 5 to 100 microns. In some examples, each pore may have cross-sectional dimensions in the range of 15 to 50 microns.

[0085] In some cases, the cross-sectional dimension can be the diameter. The term diameter is intended to encompass the maximum cross-sectional distance from end to end of a pore that is circular, nearly circular, or elliptical. In some examples, each pore in an array may have a pore diameter ranging from 5 microns to 100 microns. In some examples, each pore may have a diameter ranging from 10 microns to 50 microns.

[0086] Each pore 113 may contain a certain cross-sectional area. This cross-sectional area may be measured in a single cross-section. Additionally or alternatively, the cross-sectional area may be averaged over many locations along the pore. The white areas of the pores 113 shown in Figure 1B may define the cross-sectional area at the first surface of the pore. If necessary, each micropore 113 has a cross-sectional area equal to or less than approximately 1 square millimeter. In some cases, each pore of a group of pores is approximately 0.008 mm 2 Or it may have a maximum cross-sectional area less than that.

[0087] Each pore 113 in the array may have a certain aspect ratio. This aspect ratio may be the ratio of the length of the pore to the maximum cross-sectional dimension of the pore. This aspect ratio may be the ratio of the length of the pore to the diameter of the pore. In some cases, the aspect ratio may be in the range of 10 to 100. In some cases, the aspect ratio may be 10 or greater. In some cases, the aspect ratio may be 20 or greater. In some cases, the aspect ratio may be 100 or greater.

[0088] Figure 1C shows exemplary images of arrays with different cell concentrations. Each well may contain one or more particles of interest, such as cells, as shown in the illustrated embodiment. One or more particles may contain one or more cells. The number of cells may be about 1, about 5, about 25, or more. In some examples, the number of cells may be less than about 100 or less than about 1000.

[0089] In some embodiments, the aqueous sample solution can be deposited on the array 100, for example, by spreading the aqueous sample solution on the array 100. In some embodiments, the first surface 111 of the array 100 may be hydrophilic, and the aqueous sample solution can be absorbed into the pores 113. In some embodiments, the first surface 111 of the array 100 may distribute the target particles, such as cells, in the aqueous sample solution into the micropores 113. In some embodiments, the first surface 111 of the array 100 may randomly distribute the target particles in the aqueous sample solution into the micropores 113. In some embodiments, the target particles may move through the pores and sink to the bottom of each micropore 113. If necessary, in some embodiments, the target particles may be retained in each pore 113 by the surface tension of the aqueous sample solution.

[0090] One or more surface portions of a substrate may be coated with a material. The coated material may be configured to break down in response to electromagnetic radiation directed at or near the coated portion of the substrate. Thus, when a particle of interest is identified as being held within a particular microchannel (pore) of the array, electromagnetic radiation may be directed at the coated portion of the substrate to break down the surface material, thereby breaking the meniscus of liquid held within that microchannel and releasing the particle of interest. In certain embodiments, electromagnetic radiation can remove, for example, excavate, a portion of the coated material in or near a pore in the microarray, thereby breaking the meniscus of liquid held within the microchannel of the pore.

[0091] surface material Non-limiting examples of arrays 100 including surface materials, as shown in Figures 2A-217, are provided herein. Referring to Figure 2A, the surface material 120 may include a coating, which may be bonded to a first surface 111 of the substrate 110. In some embodiments, the surface material 120 may include a material different from that of the substrate material. In one example, the coating may include metals such as transition metals (e.g., gold) and metals that can provide adhesion to gold (e.g., chromium, titanium, nickel, or nickel-chromium). In some embodiments, the surface material may include multiple layers. The surface material may include a combination of metal coatings (e.g., Ti-Au). In some embodiments, the surface material may include metalloids or metal oxides. In some embodiments, examples of surface materials include scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, platinum, gold, mercury, niobium, iridium, molybdenum, silver, cadmium, tantalum, tungsten, aluminum, silicon, phosphorus, tin, oxides of any of the aforementioned, or any combination thereof.

[0092] In some embodiments, the surface material 120 may comprise a polymer. In some embodiments, the surface material may comprise any combination of the coating materials described herein. The surface material or coating may be formed to break away from the first surface 111 of the array in response to electromagnetic radiation directed towards a portion or near a portion of the surface material. Thus, when it is identified that a particle of interest is retained within a particular microchannel of the array, electromagnetic radiation can be directed towards the surface to break and / or peel off the coating, thereby breaking the meniscus of liquid retained within the microchannel and releasing the particle(s) of interest.

[0093] Figure 2A is a side cross-sectional view of an exemplary array for sorting particles according to several embodiments. As illustrated in Figure 2A, the array 100 may comprise a substrate 110, which may include a plurality of pores 113. The substrate 110 may include a second surface 112 and a first surface 111 opposite the second surface 112. Optionally, the plurality of pores 113 may extend from the first surface 111 to the second surface 112. In some embodiments, a coating 120 may be bonded to the first surface 111.

[0094] In some embodiments, the array 100 has an open array ratio (packing density) of about 66 percent. In some embodiments, each pore 113 has a cross-sectional area equal to or less than about 1 square millimeter. In some embodiments, each pore 113 has a diameter of about 50 μm to about 150 μm. In some embodiments, each pore 113 has a length equal to or less than about 50 mm. In some embodiments, the plurality of pores 113 are perpendicular to the second surface 112 and the first surface 111. In some embodiments, each of the pores 113 in the plurality of pores 113 may be substantially parallel to one another. In some embodiments, the plurality of pores 113 may contain about 1 million to about 100 billion pores.

[0095] Furthermore, the array 100 has an array height 110a, which is measured as the distance from the second surface 112 to the surface material 120, as shown in Figure 2A. In some embodiments, the array height 110a may be measured as the vertical distance between the first surface 111 and the second surface 112. In some embodiments, the array height 110a may be measured as the maximum or minimum vertical distance between the first surface 111 and the second surface 112. In some embodiments, the array height 110a may be measured as the standard height of the pore 113. In some embodiments, the array height 110a may be measured as the maximum or minimum height of the pore 113.

[0096] Figure 2B is a top view of an exemplary array according to several embodiments. The multiple pores 113 in the array 100 are arranged in an orthogonal pattern, according to Figure 2B. In some embodiments, the pattern includes a linear pattern, a triangular pattern, a hexagonal pattern, an irregular pattern, or any combination thereof. The orthogonal pattern of pores 113 has at least one of a first spacing 113b and a second spacing 113c, the first spacing 113b and the second spacing being measured between the center points of a continuous pore 113. In some embodiments, at least one of the first spacing 113b and the second spacing is measured as the perpendicular distance between opposing points on the surface of a continuous pore 113. In some embodiments, at least one of the first spacing 113b and the second spacing 113c may be about 10 mm to about 40 mm.

[0097] The array described herein may include a coating 120, which may be bonded to one or more surface portions of a substrate. The coating may be configured to break down when exposed to electromagnetic radiation. For example, in response to electromagnetic radiation from a laser directed at a portion of the coating, the coating may chip or peel off. If necessary, the coating may contain a material different from the material of the substrate. For example, the substrate 110 may contain a first material, and the coating 120 may contain a second material different from the first material.

[0098] In some cases, the surface material (coating 120) may cover or partially cover the second surface 112 of the array. In additional or alternative cases, the surface material may cover or partially cover the first surface 111 of the array. In some cases, the surface material may not substantially block access to the pore lumen. However, in some examples, some pore blockage may occur due to variations in coating thickness during manufacturing, etc. The surface material may have an average thickness of approximately 20 nanometers (nm) to 500 nm. The surface material may have an average thickness of approximately 100 nm to 500 nm.

[0099] In some cases, the surface material (coating 120) may be substantially similar to the base material 110. In some cases, the array may be uniform. In some embodiments, a uniform array may not include or need to include a coating. In some embodiments, a uniform array may include a uniform aggregate or alloy material. In one example, the array may include a metalloid, a metal (e.g., chromium, titanium, gold, iron, nickel, copper, platinum, or palladium) (e.g., a metal that can provide adhesion to gold (e.g., chromium, titanium, nickel, or nickel-chromium)), or any combination thereof. In some embodiments, the base material may include glass, plastic, aluminum, steel, stainless steel, or any combination thereof.

[0100] In some cases, the surface material (coating 120) may be substantially different from the base material 110. The base material may be glass, and the surface material may be a material other than glass. In some cases, the surface material (coating 120) may contain a metal. In some cases, the metal may include titanium, gold, chromium, silver, aluminum, or any other metal. In some cases, the surface material may contain a metal oxide, such as magnesium fluoride, calcium fluoride, silicon dioxide, etc. The surface material may contain layers of metal and / or metal oxide to form individually tailored optical properties such as reflection or absorption.

[0101] In some embodiments, the surface material (coating 120) includes a transition metal (e.g., titanium, gold, etc.). In some embodiments, the second material includes a metalloid. In some embodiments, the second material includes a metal oxide. In some embodiments, the second material may include scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, platinum, gold, mercury, niobium, iridium, molybdenum, silver, cadmium, tantalum, tungsten, aluminum, silicon, phosphorus, tin, oxides of any of the aforementioned, or any combination thereof.

[0102] In some embodiments, the surface material (coating 120) is selected from materials that do not adversely affect cell viability. For example, the surface material may be biocompatible. The surface material may be non-toxic. In certain embodiments, the surface material is selected from materials that do not cause cell damage or cell death when in contact with electromagnetic radiation. For example, the products themselves produced by bringing the surface material into contact with electromagnetic radiation must not cause cell damage or cell death. That is, for example, the products produced by ablation of the surface material may be biocompatible and / or non-toxic to cells. In certain embodiments, the effect on cell viability is evaluated by measuring cell viability before and after exposure of cells to the surface material. In certain embodiments, cell viability remains the same or decreases by only less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, or less than 5%. In certain embodiments, cell viability may be evaluated by measuring cell viability before and after bringing the surface material into contact with electromagnetic radiation. For example, cell viability is evaluated before loading cells into the array and after the surface material is exposed to electromagnetic radiation, releasing the cells from the array's pores. In some cases, viability remains the same or decreases by less than 40%, 30%, 20%, 15%, 10%, 5%, or 1% after the surface material is exposed to electromagnetic radiation.

[0103] The array may, in some cases, have individually tailored hydrophobicity. In one example, the second surface 112 may be hydrophilic. If necessary, the second surface 112 does not need to be hydrophilic itself, but may be operably coupled to a hydrophilic coating. In some embodiments, a portion of the coating 120 may be formed to break away from the first surface 111. In some embodiments, a portion of the coating 120 may be formed to break away from the first surface 111 in response to electromagnetic radiation directed at a portion of the coating. In some embodiments, the coating 120 may be hydrophobic.

[0104] The coating 120 may be configured to break down in response to electromagnetic radiation directed at a portion of the surface material. Therefore, when it is identified that a particle of interest is retained within a specific microchannel (pore) of the array, electromagnetic radiation can be directed at the coating to break down and / or peel off the coating 120, thereby breaking down the liquid meniscus retained within the microchannel (pore 113) and releasing the particle of interest. The coating 120 may absorb radiation at wavelengths or wavelength ranges corresponding to the wavelengths emitted by the electromagnetic radiation source.

[0105] Therefore, once it is identified that the target particles are held within a particular pore of the array, electromagnetic radiation can be directed near or in the vicinity of that particular pore to emit the target particles. In some embodiments, the destruction of the surface material includes removing at least a portion of the material of the array, the coating on the array, or both.

[0106] In some embodiments, array failure may be caused by localized heating. Such a mechanism may occur when the pulse duration is longer, the peak power density is lower, and / or the wavelength of the incident radiation is infrared. Localized heating may cause sublimation of the surface material (coating 120) or the array material. In some embodiments, the substrate material and coating 120 have different coefficients of thermal expansion, which may lead to chipping.

[0107] Additionally or alternatively, array failure may be caused by ablation. Such mechanisms may occur when the incident peak power density is higher, the pulse duration is shorter, the radiated power is higher, and / or the incident radiation is visible. Ablation may involve the breaking of local bonds and / or vaporization of the array or substrate material.

[0108] Additionally or alternatively, array destruction can be caused by plasma generation. This mechanism may occur when the pulse duration of the incident radiation is particularly short, when the wavelength of the incident radiation is in resonance with the multiphoton ionization mechanism, and / or when the wavelength of the incident radiation is very short. Pulse durations in the picosecond to femtosecond range can result in plasma generation faster than localized heating leading to optical etching of the substrate or surface material.

[0109] Additionally or alternatively, array failure can be caused by shock wave generation. Such a mechanism may be more likely to occur when the peak power density is higher, when phonons are resonating, and / or when the pulse duration is shorter. The shock can cause physical vibration, chipping, or shaking of the surface material or array material.

[0110] In one example, the surface material (coating 120) absorbs wavelengths in the visible or infrared range. In some embodiments, the surface material may be opaque. The surface material may absorb a band of at least 5 nanometers selected within the visible and infrared ranges. The surface material may absorb more than 10 percent of incident radiation in a band of at least 5 nanometers selected from 0.4 to 2.5 microns. The surface material may absorb more than 10 percent of incident electromagnetic radiation at wavelengths selected from 0.4 to 2.5 microns. In some cases, the surface material may absorb more than 50 percent of incident radiation in a band of at least 5 nanometers. The 5 nanometer band may be selected within the wavelength range of 0.4 to 2.5 microns. The surface material may absorb more than 50 percent of incident electromagnetic radiation at wavelengths selected from 0.4 to 1.5 microns. The surface material can absorb more than 10 percent of incident radiation at wavelengths selected from the harmonics of a doped ytterbium orthovanadate or ytterbium-aluminum-garnet solid-state laser. The surface material can absorb more than 10 percent of incident radiation at 1064 nanometers.

[0111] In one example, the coating 120 of array 100 has an average thickness of about 600 nm. The thickness of the coating 120 can be reduced by an infrared (IR) laser to about 100 nm or less, for example, about 75 nm or less, or about 50 nm or less. The thickness of the coating can be 10 to 1000 nm. In some embodiments, the coating or any identifiable layer may be 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 300 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, or 1000 nm, or about 10 nm, about 20 The thickness is approximately nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 300 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, or 1000 nm, or any range between two of the aforementioned values.In some embodiments, the coating or any identifiable layer is at least 10 nm, at least 20 nm, at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 300 nm, at least 400 nm, at least 450 nm, at least 500 nm, at least 550 nm, at least 600 nm, at least 650 nm, at least 700 nm, at least 750 nm, at least 800 nm, at least 850 nm, at least 900 nm, at least 950 nm, or at least 1000 nm. The material has a thickness of at least about 10 nm, at least about 20 nm, at least about 30 nm, at least about 40 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, at least about 150 nm, at least about 200 nm, at least about 250 nm, at least about 300 nm, at least about 300 nm, at least about 400 nm, at least about 450 nm, at least about 500 nm, at least about 550 nm, at least about 600 nm, at least about 650 nm, at least about 700 nm, at least about 750 nm, at least about 800 nm, at least about 850 nm, at least about 900 nm, at least about 950 nm, or at least about 1000 nm.In some embodiments, the coating or any identifiable layer is at least 10 nm, at least 20 nm, at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 300 nm, at least 400 nm, at least 450 nm, at least 500 nm, at least 550 nm, at least 600 nm, at least 650 nm, at least 700 nm, at least 750 nm, at least 800 nm, at least 850 nm, at least 900 nm, at least 950 nm, or at least 1000 nm. The material has a thickness of at least about 10 nm, at least about 20 nm, at least about 30 nm, at least about 40 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, at least about 150 nm, at least about 200 nm, at least about 250 nm, at least about 300 nm, at least about 300 nm, at least about 400 nm, at least about 450 nm, at least about 500 nm, at least about 550 nm, at least about 600 nm, at least about 650 nm, at least about 700 nm, at least about 750 nm, at least about 800 nm, at least about 850 nm, at least about 900 nm, at least about 950 nm, or at least about 1000 nm. In some embodiments, the coating thickness may be at least 10 nm, at least 20 nm, at least 30 nm, at least 40 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, at least 800 nm, at least 1000 nm, or greater.In some embodiments, the coating thickness can be at most 1000 nm, at most 800 nm, at most 600 nm, at most 500 nm, at most 400 nm, at most 300 nm, at most 200 nm, at most 100 nm, at most 50 nm, at most 40 nm, at most 30 nm, at most 20 nm, at most 10 nm, or less. The layer structure(s) of the coating can be determined using energy-dispersive X-ray spectroscopy (EDS or EDX).

[0112] In some embodiments, the electromagnetic radiation source can be formed to reduce the average thickness of coating 120 by about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm, about 1 nm to about 30 nm, about 1 nm to about 40 nm, about 1 nm to about 60 nm, about 1 nm to about 70 nm, about 1 nm to about 80 nm, about 1 nm to about 90 nm, or about 1 nm to about 100 nm.

[0113] In some embodiments, the electromagnetic radiation source can be formed to excise a portion of the array at an average depth of about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm, about 1 nm to about 30 nm, about 1 nm to about 40 nm, about 1 nm to about 60 nm, about 1 nm to about 70 nm, about 1 nm to about 80 nm, about 1 nm to about 90 nm, or about 1 nm to about 100 nm.

[0114] In some embodiments, the electromagnetic radiation source can be formed to remove a portion of coating 120 or the array, and that portion has a surface area of about 1 μm 2 to about 30 μm 2 , 1 μm 2 to about 20 μm 2 about 1 μm 2 to about 10 μm 2 or about 1 μm 2 to about 5 μm 2 .

[0115] In some embodiments, the electromagnetic radiation source may be formed to excise a portion of the array at average distances of approximately 1 nm to 5 nm, 1 nm to 10 nm, 1 nm to 20 nm, 1 nm to 30 nm, 1 nm to 40 nm, 1 nm to 60 nm, 1 nm to 70 nm, 1 nm to 80 nm, 1 nm to 90 nm, or 1 nm to 100 nm from the outer edge of the micropore.

[0116] Figure 3A shows a top view of an exemplary array for sorting particles containing a coating, according to several embodiments. Figure 3B shows a top view of a non-limiting example of an array for sorting particles containing a coating to be removed by a laser, according to several embodiments. Referring to Figures 3A and 3B, the coating 120 may absorb electromagnetic energy, thereby breaking it from the substrate 110, shattering the fluid meniscus within each pore 113 and expelling the internal cells. Figure 3B shows a piece of coating 120 removed from the substrate 110 by electromagnetic energy. Referring to Figure 3B, the laser may be focused on a single pore or near a single pore, between two adjacent pores, or equidistant from three pores. In some embodiments, by focusing an infrared laser near a single pore, between two adjacent pores, or equidistant from three pores, the fluid meniscus within one, two, or three pores 113 is shattered, expelling the internal cells. In some embodiments, focusing the laser close to a specific pore reduces the likelihood of unintentionally ejecting cells in nearby pores. In some embodiments, at least one of the intensity and duration of the infrared laser can be set to control the ejection of cells in one, two, or three pores.

[0117] In some embodiments, the surface material (coating 120) can be formed by sputtering the material onto the array 100. In some embodiments, the surface material may include one or more metals (e.g., titanium, gold). The thickness of the surface material may be 10 to 1000 nm. In some embodiments, the thickness of the surface material may be at least 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 800 nm, 1000 nm, or greater. In some embodiments, the thickness of the surface material may be at most 1000 nm, 800 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, or less. In some embodiments, the surface material may include a Ti-Au stack. The thickness of the titanium layer may be at least 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 100 nm, 200 nm, or greater. The gold layer may be formed directly on the titanium layer. The thickness of the gold layer may be at least 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, or greater. In some embodiments, the surface material may include a titanium layer, and the gold layer may be optional.

[0118] In some embodiments, sputtering can be performed under vacuum. In some embodiments, the vacuum may be about 0.08 to about 0.02 mbar. In some embodiments, sputtering can be performed under a voltage of about 100V to 3kV. In some embodiments, the voltage may be at least about 100V, 110V, 130V, 150V, 170V, 220V, 280V, 500V, 1000V, 2000V, 3000V, or more. In some embodiments, the voltage may be at most about 3000V, 2000V, 1000V, 500V, 280V, 220V, 170V, 150V, 130V, 110V, 100V, or less. In some embodiments, sputtering can be performed under a current of 0 to 50mA. In some embodiments, the current may be at least about 0.01 mA, 0.1 mA, 1 mA, 5 mA, 10 mA, 20 mA, 30 mA, 40 mA, 50 mA, or greater. In some embodiments, the current may be at most about 50 mA, 40 mA, 30 mA, 20 mA, 10 mA, 1 mA, 0.1 mA, 0.01 mA, or less. If necessary, in some embodiments, the surface material (coating 120) may be sputtered on only one side (111 or 112) of the array or on both sides (111 and 112). For example, in some embodiments, the surface material may be sputtered on a first face (e.g., 111) of the array. In other embodiments, the surface material may be sputtered on a second face (e.g., 112) of the glass array. In some further embodiments, the surface material may be sputtered on both the first face (e.g., 111) and the second face (e.g., 112) of the array.

[0119] In some embodiments, PBMC extraction includes adding a surfactant and receiving medium to a coated array; inserting the assembled array into a cassette with the coated side facing downward toward the receiving medium; dropping the PBMCs onto the array; and allowing the PBMCs to stand in the pore. In some embodiments, the surfactant preserves the integrity of the cell membrane and improves robustness under liquid shear. In some embodiments, the surfactant includes a nonionic surfactant. In some embodiments, the nonionic surfactant includes 0.1 percent Pluoronic® F68. In some embodiments, the receiving medium includes OptiPEAK T cell medium. In some embodiments, the receiving medium further includes streptavidin. In some embodiments, the PBMCs are allowed to stand in the micropore for about 5 minutes.

[0120] In some embodiments, infrared (IR) energy emitted from a laser and absorbed by the surface material coating 120 (e.g., a Ti-Au stack, a Ti layer, or an Au layer) can cause the coating to expand and peel off at the bottom edge of each micropore, thereby extracting PBMCs from each micropore. The separation of the coating at the bottom edge of each micropore breaks the fluid meniscus within it, releasing the PBMCs.

[0121] Figure 4A is a top view of PBMCs stained with an IR energy-absorbing fluorescent dye in a non-limiting example of an array including a surface material coating (e.g., Ti-Au stack, Ti layer, or Au layer) according to some embodiments. Figure 4B is a top view of an exemplary array with a surface material coating after PBMC extraction, according to some embodiments.

[0122] beads In certain embodiments, the pores of the array may include beads that absorb electromagnetic radiation and affect the disruption of the fluid meniscus within the pore. In some cases, the beads may or may not be bonded to the luminal surface of the pore (they may be added to the pore as a liquid mixture). Beads comprising a core and a shell are provided herein. The beads of this disclosure may be referred to as “microspheres”. The core may include an infrared (IR) absorbing core. The shell may include a non-IR absorbing shell. The beads of this disclosure may be associated with the pores of an array, and the beads may absorb electromagnetic radiation. The non-IR absorbing shell may protect the IR absorbing core from damaging effects of IR absorbing radiation on nearby particles, such as cells, by shielding them from the IR absorbing core. The beads may further contain agarose. The non-IR absorbing shell may contain agarose. The beads may further contain dextran. The beads may be stained with an IR absorbing dye. The beads may include diameters equal to or less than about 20 μm, such as about 1 μm to about 20 μm or about 5 μm to about 20 μm. The beads may include an absorbing shell which may be equal to or less than about 10 microns. In some embodiments, the surface material of the array as described herein may include beads comprising an infrared absorbing core and a non-infrared absorbing shell, the outer diameter of which the non-infrared absorbing shell is equal to or less than about 10 microns.

[0123] Figure 5A shows an array 100 in which beads are arranged. In some cases, the beads may be arranged inside the lumen of the pore. In some cases, the beads may be arranged on a first surface 111. In some cases, the beads may be arranged within the lumen of the pore. Figure 5B shows a side cross-sectional view of an aqueous sample solution in the exemplary array of Figure 5A. In some embodiments, depositing the aqueous sample solution 521 onto the array 100 includes spreading the aqueous sample solution 521 onto the array 100. In some embodiments, the hydrophilic first surface 111 of the array 100 draws the aqueous sample solution 521 into the pore 113. In some embodiments, the hydrophilic first surface 111 of the array 100 evenly distributes the first cells 522 and the second cells 523 in the aqueous sample solution 521 into the pore 113. In some embodiments, the hydrophilic first surface 111 of the array 100 randomly distributes the first cells 522 and the second cells 523 in the aqueous sample solution 521 into the pores 113. In some embodiments, the first cells 522 and the second cells 523 sink to the bottom of each pore 113. If necessary, in some embodiments, the first cells 522 and the second cells 523 are retained in each pore 113 by the surface tension of the aqueous sample solution 521.

[0124] Figure 6A shows a bright-field image of an array of micropores filled with microspheres and cells according to several embodiments. As seen in Figure 6A, each of the micropores 601 in the array 600 can be blocked by microbeads and cells within each of the micropores 601. Figure 6B shows a bright-field image of cell extraction from a single micropore according to several embodiments. As seen in Figure 6B, only one micropore 601 in the array 600 is blocked by cells, suggesting that only cells in a single micropore 601 have been removed. Figure 6C shows an array image of micropores filled with microspheres and cells according to several embodiments. As seen in Figure 6C, only one micropore 601 in the array 600 contains cells. Figure 6D shows an image of the array 600 after cell extraction from a single micropore according to several embodiments. As can be seen in Figure 6D, none of the micropores 601 within array 600 contain cells, suggesting that a single cell in a single micropore 601 was removed.

[0125] Figure 7A shows exemplary bright-field images of extracted cells according to several embodiments. Figure 7B shows exemplary images of extracted cells according to several embodiments.

[0126] Examples of beads or microspheres are provided herein by reference to Figures 8, 9, 10A, and 10B. Figure 8 shows a bright-field image of exemplary agarose and dextran microspheres. In some embodiments, the agarose and dextran microspheres 800 are formed to absorb infrared light. In some embodiments, the agarose and dextran microspheres 800 are opaque, black, or both. In some embodiments, the agarose and dextran microspheres 800 comprise polymer-shelled iron oxide microspheres 800. In some embodiments, the agarose and dextran microspheres 800 have a diameter of about 6 μm to about 20 μm.

[0127] Figure 9 shows a high-magnification infrared image of an exemplary agarose and dextran microsphere. As seen in Figure 9, the agarose and dextran microsphere 800 includes an infrared (IR) absorbing core 910 and a non-IR absorbing shell 920. In some embodiments, the IR absorbing core 910 includes an IR absorbing dye. In some embodiments, the IR absorbing dye includes Epolight 1178. In some embodiments, the non-IR absorbing shell 920 includes agarose and dextran.

[0128] The use of IR core-colored particles may be beneficial for efficient cell extraction. Firstly, dyes incorporated into the molecular structure of the agarose core may increase IR absorption more than dye coatings. Furthermore, non-IR-absorbing softshells can act as a buffer layer to protect cells from stress and thermal shock associated with any potential heat absorption, volume expansion, and / or microbubble formation. Both of these may enable increased extraction efficiency (an increase in the number of successful extraction events) and improved cell viability.

[0129] Figure 10A shows a bright-field image of an exemplary agarose and IR dye microsphere. Figure 10B shows an infrared image of an exemplary agarose and IR dye microsphere. As seen in Figure 10B, the agarose and IR dye microsphere 1000 may absorb infrared (IR) light. In some embodiments, the agarose and IR dye microsphere 1000 comprises agarose. In some embodiments, the agarose and IR dye microsphere 1000 comprises an IR-absorbing dye. In some embodiments, the IR-absorbing dye comprises Epolight. In some embodiments, the dye comprises a green fluorescent protein. In some embodiments, the dye comprises a red fluorescent protein. In some embodiments, the dye comprises cyanine dyes, acridine dyes, fluorescent (flourone) dyes, oxazine dyes, rhodomine dyes, coumarin dyes, phenanthridine dyes, BODIPY dyes, ALEXA dyes, perylene dyes, anthracene dyes, naphthalene dyes, etc. In some embodiments, the agarose and IR dye microspheres 1000 have a diameter of about 2 μm to about 16 μm.

[0130] Figures 11A and 11B illustrate exemplary procedures for modifying all or part of one or more surfaces of the substrate 110. In some embodiments, the bottom surface and a portion of the vertical sidewalls of the pore plate may be first covered with a surface coating (step 1111), for example, as shown in Figures 13B and / or 13C. The surface coating may comprise one or more thermally conductive or conductive materials. Step 1111 may comprise metal deposition. The surface coating may comprise one or more metals selected from the group consisting of chromium, titanium, gold, iron, nickel, copper, platinum, and palladium. The surface coating may comprise one or more metal layers (e.g., 1320 and 1322 shown in Figure 13C), each layer independently selected from the group consisting of chromium, titanium, gold, iron, nickel, copper, platinum, palladium, any mixtures thereof, and any alloys thereof. The surface coating may comprise metal layers and an adhesive layer for the underlying metal layers. An adhesion layer may be used to facilitate adhesion to the substrate material. The surface coating may include a layer of gold and a gold adhesion layer beneath the gold (e.g., chromium, titanium, nickel, or nickel-chromium). The surface coating (step 1111) can be performed using any suitable coating method such as sputtering, spin coating, chemical vapor deposition (CVD), physical vapor deposition (PVD), pulsed laser deposition, atomic layer deposition, low-pressure CVD, or any combination thereof. In some embodiments, the surface coating may be the same as coating 120. In some embodiments, the coating process (step 1111) may include metal deposition. In some cases, the metal coating may include one or more metals (e.g., Cr, Ni, Ti, Au). In some embodiments, the metal coating may include a Ti-Au stack. In some embodiments, the metal coating may include a Cr-Au stack. In some embodiments, the metal coating may include a Ni-Au stack.

[0131] Next, the surface of the pore plate can be cleaned and / or activated by physical or chemical means, such as by plasma cleaning, by immersing the plate in a basic solution, or a combination thereof (step 1113). The physical or chemical means (step 1113) can enhance the adhesion of subsequent layers to the coating obtained from step 1111. In some embodiments, the basic solution may contain a predetermined concentration of NaOH. In some embodiments, the predetermined concentration may be about 1 M to 3 M. In some embodiments, the predetermined concentration may be 3 M, 2.5 M, 2 M, 1.5 M, 1 M, or about 3 M, about 2.5 M, about 2 M, about 1.5 M, about 1 M, or any range between any two of the aforementioned values ​​(including values ​​at both ends). In some embodiments, the predetermined concentration may be at least 1 M, 1.5 M, 2 M, 2.5 M, 3 M, or greater. In some embodiments, the predetermined concentration may be up to 3 M, 2.5 M, 2 M, 1.5 M, 1 M, or less. In some embodiments, the pore plate can be immersed in a basic solution for a predetermined period of time. In some embodiments, the predetermined period may be between approximately 15 minutes and 12 hours. In some embodiments, the predetermined period may be up to 12 hours, 10 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 50 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, or less. In some embodiments, the predetermined period may be at least 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, or more.

[0132] Next, the pore plate can be cleaned to remove impurities, such as residual alkaline solutions, by washing it with deionized water (step 1115). The pore plate can then be dried using pressurized air from, for example, a pressurized air gun.

[0133] In some embodiments, the pore plate may be plasma-cleaned with any suitable plasma in the chamber for a predetermined period of time. Suitable plasmas may be argon plasma, compressed air plasma, flame-based plasma, or vacuum plasma. The predetermined period may be 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 110 seconds, 2 minutes, 130 seconds, 140 seconds, 150 seconds, 160 seconds, 170 seconds, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, 6.5 minutes, 7 minutes, 7.5 minutes, 8 minutes, 8.5 minutes, 9 minutes, 9.5 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, or 15 minutes, or approximately 10 seconds, approximately 20 seconds, approximately 30 seconds, approximately 40 seconds, approximately 50 seconds, approximately 1 minute, approximately 70 seconds It can be seconds, approximately 80 seconds, approximately 90 seconds, approximately 100 seconds, approximately 110 seconds, approximately 2 minutes, approximately 130 seconds, approximately 140 seconds, approximately 150 seconds, approximately 160 seconds, approximately 170 seconds, approximately 3 minutes, approximately 3.5 minutes, approximately 4 minutes, 4.5 minutes, approximately 5 minutes, approximately 5.5 minutes, approximately 6 minutes, approximately 6.5 minutes, approximately 7 minutes, approximately 7.5 minutes, approximately 8 minutes, approximately 8.5 minutes, approximately 9 minutes, approximately 9.5 minutes, approximately 10 minutes, approximately 11 minutes, approximately 12 minutes, approximately 13 minutes, approximately 14 minutes, or approximately 15 minutes, or any range between any two of the aforementioned values ​​(including the values ​​at both ends).The specified period is at least 10 seconds, at least 20 seconds, at least 30 seconds, at least 40 seconds, at least 50 seconds, at least 1 minute, at least 70 seconds, at least 80 seconds, at least 90 seconds, at least 100 seconds, at least 110 seconds, at least 2 minutes, at least 130 seconds, at least 140 seconds, at least 150 seconds, at least 160 seconds, at least 170 seconds, at least 3 minutes, at least 3.5 minutes, at least 4 minutes, at least 4.5 minutes, at least 5 minutes, at least 5.5 minutes, at least 6 minutes, at least 6.5 minutes, at least 7 minutes, at least 7.5 minutes, at least 8 minutes, at least 8.5 minutes, at least 9 minutes, at least 9.5 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, at least 13 minutes, at least 14 minutes, or at least 15 minutes, or at least about 10 seconds, at least about It could be 20 seconds, at least about 30 seconds, at least about 40 seconds, at least about 50 seconds, at least about 1 minute, at least about 70 seconds, at least about 80 seconds, at least about 90 seconds, at least about 100 seconds, at least about 110 seconds, at least about 2 minutes, at least about 130 seconds, at least about 140 seconds, at least about 150 seconds, at least about 160 seconds, at least about 170 seconds, at least about 3 minutes, at least about 3.5 minutes, at least about 4 minutes, at least about 4.5 minutes, at least about 5 minutes, at least about 5.5 minutes, at least about 6 minutes, at least about 6.5 minutes, at least about 7 minutes, at least about 7.5 minutes, at least about 8 minutes, at least about 8.5 minutes, at least about 9 minutes, at least about 9.5 minutes, at least about 10 minutes, at least about 11 minutes, at least about 12 minutes, at least about 13 minutes, at least about 14 minutes, or at least about 15 minutes.The specified period is a maximum of 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 110 seconds, 2 minutes, 130 seconds, 140 seconds, 150 seconds, 160 seconds, 170 seconds, 3 minutes, 3.5 minutes, and a maximum 4 minutes, up to 4.5 minutes, up to 5 minutes, up to 5.5 minutes, up to 6 minutes, up to 6.5 minutes, up to 7 minutes, up to 7.5 minutes, up to 8 minutes, up to 8.5 minutes, up to 9 minutes, up to 9.5 minutes, up to 10 minutes, up to 11 minutes, up to 12 minutes, up to 13 minutes, up to 14 minutes, or up to 15 minutes, or up to approximately 10 seconds, up to approximately 20 seconds, Up to approximately 30 seconds, up to approximately 40 seconds, up to approximately 50 seconds, up to approximately 1 minute, up to approximately 70 seconds, up to approximately 80 seconds, up to approximately 90 seconds, up to approximately 100 seconds, up to approximately 110 seconds, up to approximately 2 minutes, up to approximately 130 seconds, up to approximately 140 seconds, up to approximately 150 seconds, up to approximately 160 seconds, up to approximately 170 seconds, up to approximately 3 minutes, up to approximately 3.5 minutes, up to approximately 4 minutes The maximum duration may be approximately 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, 6.5 minutes, 7 minutes, 7.5 minutes, 8 minutes, 8.5 minutes, 9 minutes, 9.5 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, or 15 minutes. The chamber used for plasma purification may be an ultra-high vacuum (UHV) chamber.

[0134] In some embodiments, the pore plate after step 1113 can be washed in one or more passes using one or more washing solutions (step 1115). In each pass, the washing solution can be independently selected from the group consisting of water, alcohol (e.g., methanol, ethanol, isopropanol, butanol), acetonitrile, acetone, toluene, and mixtures thereof. Each pass can be independently continued for a predetermined period of time. The specified periods are 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 110 seconds, 2 minutes, 130 seconds, 140 seconds, 150 seconds, 160 seconds, 170 seconds, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, 6.5 minutes, 7 minutes, 7.5 minutes, 8 minutes, 8.5 minutes, 9 minutes, 9.5 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 1 hour, or approximately 10 seconds, approximately 20 seconds, approximately 30 seconds, approximately 40 seconds, approximately 50 seconds, approximately 1 minute, approximately 70 seconds, approximately 80 seconds It can be a second, approximately 90 seconds, approximately 100 seconds, approximately 110 seconds, approximately 2 minutes, approximately 130 seconds, approximately 140 seconds, approximately 150 seconds, approximately 160 seconds, approximately 170 seconds, approximately 3 minutes, approximately 3.5 minutes, approximately 4 minutes, approximately 4.5 minutes, approximately 5 minutes, approximately 5.5 minutes, approximately 6 minutes, approximately 6.5 minutes, approximately 7 minutes, approximately 7.5 minutes, approximately 8 minutes, approximately 8.5 minutes, approximately 9 minutes, approximately 9.5 minutes, approximately 10 minutes, approximately 11 minutes, approximately 12 minutes, approximately 13 minutes, approximately 14 minutes, approximately 15 minutes, approximately 20 minutes, approximately 25 minutes, approximately 30 minutes, approximately 35 minutes, approximately 40 minutes, approximately 45 minutes, approximately 50 minutes, approximately 55 minutes, or approximately 1 hour, or any range between any two of the aforementioned values ​​(including the values ​​at both ends).The specified period is at least 10 seconds, at least 20 seconds, at least 30 seconds, at least 40 seconds, at least 50 seconds, at least 1 minute, at least 70 seconds, at least 80 seconds, at least 90 seconds, at least 100 seconds, at least 110 seconds, at least 2 minutes, at least 130 seconds, at least 140 seconds, at least 150 seconds, at least 160 seconds, at least 170 seconds, at least 3 minutes, at least 3.5 minutes, at least 4 minutes, at least 4.5 minutes, at least 5 minutes, at least 5.5 minutes, at least 6 minutes, at least 6.5 minutes, at least 7 minutes, at least 7.5 minutes, at least 8 minutes, at least 8.5 minutes, at least 9 minutes, at least 9.5 minutes, at least 10 minutes, at least 11 minutes, at least 12 minutes, at least 13 minutes, at least 14 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, or 1 hour, or at least about 10 seconds, at least about 20 seconds, less Approximately 30 seconds, at least approximately 40 seconds, at least approximately 50 seconds, at least approximately 1 minute, at least approximately 70 seconds, at least approximately 80 seconds, at least approximately 90 seconds, at least approximately 100 seconds, at least approximately 110 seconds, at least approximately 2 minutes, at least approximately 130 seconds, at least approximately 140 seconds, at least approximately 150 seconds, at least approximately 160 seconds, at least approximately 170 seconds, at least approximately 3 minutes, at least approximately 3.5 minutes, at least approximately 4 minutes, at least approximately 4.5 minutes, at least approximately 5 minutes, at least approximately 5.5 minutes, at least approximately 6 minutes, and at least It could be approximately 6.5 minutes, at least approximately 7 minutes, at least approximately 7.5 minutes, at least approximately 8 minutes, at least approximately 8.5 minutes, at least approximately 9 minutes, at least approximately 9.5 minutes, at least approximately 10 minutes, at least approximately 11 minutes, at least approximately 12 minutes, at least approximately 13 minutes, at least approximately 14 minutes, at least approximately 15 minutes, at least approximately 20 minutes, at least approximately 25 minutes, at least approximately 30 minutes, at least approximately 35 minutes, at least approximately 40 minutes, at least approximately 45 minutes, at least approximately 50 minutes, at least approximately 55 minutes, or at least approximately 1 hour.The specified period is a maximum of 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 110 seconds, 2 minutes, 130 seconds, 140 seconds, 150 seconds, 160 seconds, 170 seconds, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, and more. Large: 6.5 minutes, Maximum: 7 minutes, Maximum: 7.5 minutes, Maximum: 8 minutes, Maximum: 8.5 minutes, Maximum: 9 minutes, Maximum: 9.5 minutes, Maximum: 10 minutes, Maximum: 11 minutes, Maximum: 12 minutes, Maximum: 13 minutes, Maximum: 14 minutes, Maximum: 15 minutes, Maximum: 20 minutes, Maximum: 25 minutes, Maximum: 30 minutes, Maximum: 35 minutes, Maximum: 40 minutes, Maximum: 45 minutes, Maximum: 50 minutes, Maximum: 55 minutes, or up to 1 hour, or up to approximately 10 seconds, up to approximately 20 seconds, up to approximately 30 seconds, up to approximately 40 seconds, up to approximately 50 seconds, up to approximately 1 minute, up to approximately 70 seconds, up to approximately 80 seconds, up to approximately 90 seconds, up to approximately 100 seconds, up to approximately 110 seconds, up to approximately 2 minutes, up to approximately 130 seconds, up to approximately 140 seconds, up to approximately 150 seconds, up to approximately 160 seconds, up to approximately 170 seconds, up to approximately 3 minutes, up to approximately 3.5 minutes, up to approximately 4 minutes, up to approximately 4.5 minutes, up to approximately 5 minutes, up to approximately 5.5 minutes, up to approximately 6 minutes, up to approximately 6 It could be 0.5 minutes, up to approximately 7 minutes, up to approximately 7.5 minutes, up to approximately 8 minutes, up to approximately 8.5 minutes, up to approximately 9 minutes, up to approximately 9.5 minutes, up to approximately 10 minutes, up to approximately 11 minutes, up to approximately 12 minutes, up to approximately 13 minutes, up to approximately 14 minutes, up to approximately 15 minutes, up to approximately 20 minutes, up to approximately 25 minutes, up to approximately 30 minutes, up to approximately 35 minutes, up to approximately 40 minutes, up to approximately 45 minutes, up to approximately 50 minutes, up to approximately 55 minutes, or up to approximately 1 hour.

[0135] In some embodiments, a pore plate having an activated and cleaned surface ready for surface functionalization (after step 1115) can then be coated with one or more surface-modifying materials (step 1117). One or more surface-modifying materials may comprise one or more polymers. One or more surface-modifying materials may comprise one or more hydrophilic materials (e.g., one or more hydrophilic oligomers or one or more hydrophilic polymers), one or more hydrophobic materials (e.g., one or more hydrophobic oligomers or one or more hydrophobic polymers), or any combination thereof.

[0136] In some embodiments, a portion of the vertical sidewall of the pore plate (e.g., the sidewall of pore 113) may be functionalized with a hydrophilic oligomer or polymer such as polyethylene glycol (PEG), poly(hydroxyethyl methacrylate) (PHEMA), polyacrylamide (PAM), polyacrylic acid (PAA), polyvinylpyrrolidone (PVP), polysaccharides, or polylactic acid (PLA). The hydrophilic oligomer or polymer may be linear or branched. The hydrophilic oligomer or polymer may contain a first end group. A hydrophilic oligomer or polymer containing a first end group may contain a second end group. One of the first and second end groups may be configured to react with the activated and cleaned surface (after step 1117) or to form a self-assembled film on that surface, while the other of the first and second end groups, if present, may be configured to remain on the hydrophilic oligomer or polymer after surface functionalization. The first or second terminal group configured to remain on the hydrophilic oligomer or polymer after surface functionalization may be an alkoxy (e.g., methoxy, ethoxy), hydroxyl, amine, or ionic hydrophilic group. The first or second terminal group configured to react with the activated and cleaned surface (after step 1117) or to form a self-assembled film on that surface may be selected from silane, thiol, primary amine (-NH2), carboxylic acid (-COOH), aldehyde, vinyl, epoxy, and chloro. The first or second terminal group configured to react with the activated and cleaned surface (after step 1117) or to form a self-assembled film on that surface may be a silane or thiol. After step 1117, the vertical sidewalls of the pore plate may be functionalized with a hydrophilic oligomer or polymer (e.g., poly(ethylene-glycol) (PEG)) end-capped with an alkoxy group (e.g., methoxy). The hydrophilic oligomer or polymer having a first and second terminus may be a functionalized PEG such as silane-PEG-methoxy (PEG-silane) or thiol-PEG-methoxy (PEG-SH).

[0137] In some embodiments, the hydrophilic oligomer or polymer (e.g., PEG-silane or PEG-SH) is 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5250, 5500, 5750, or 6000 Daltons, or It has a molecular weight of approximately 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5250, 5500, 5750, or approximately 6000 daltons, or any range between any two of the aforementioned values. In some embodiments, the hydrophilic oligomer or polymer (e.g., PEG-silane or PEG-SH) is at least 250, at least 500, at least 750, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, at least 3000, at least 3250, at least 3500, at least 3750, at least 4000, at least 4250, at least 4500, at least 4750, at least 5000, at least 5250, at least 5500, at least 5750, or at least Having a molecular weight of 6000 Daltons, or at least about 250, at least about 500, at least about 750, at least about 1000, at least about 1250, at least about 1500, at least about 1750, at least about 2000, at least about 2250, at least about 2500, at least about 2750, at least about 3000, at least about 3250, at least about 3500, at least about 3750, at least about 4000, at least about 4250, at least about 4500, at least about 4750, at least about 5000, at least about 5250, at least about 5500, at least about 5750, or at least about 6000 Daltons.In some embodiments, the hydrophilic oligomer or polymer (e.g., PEG-silane or PEG-SH) is up to 250, up to 500, up to 750, up to 1000, up to 1250, up to 1500, up to 1750, up to 2000, up to 2250, up to 2500, up to 2750, up to 3000, up to 3250, up to 3500, up to 3750, up to 4000, up to 4250, up to 4500, up to 4750, up to 5000, up to 5250, up to 5500, up to 5750, or up to The molecular weight is 6000 Daltons, or up to about 250, up to about 500, up to about 750, up to about 1000, up to about 1250, up to about 1500, up to about 1750, up to about 2000, up to about 2250, up to about 2500, up to about 2750, up to about 3000, up to about 3250, up to about 3500, up to about 3750, up to about 4000, up to about 4250, up to about 4500, up to about 4750, up to about 5000, up to about 5250, up to about 5500, up to about 5750, or up to about 6000 Daltons. In some embodiments, the PEG-silane used in step 1117 may be a solution dissolved in an alcohol, such as ethanol, for example, 0.5 g / 100 mL of PEG-silane. In some embodiments, the functionalized PEG may include methoxy-poly(ethylene-glycol)-thiol (PEG-SH). In some embodiments, PEG-SH may be a solution dissolved in an alcohol, such as anhydrous ethanol, for example, 0.5 g / 100 mL of PEG-SH. In some embodiments, the hydrophilic oligomer or polymer (e.g., functionalized PEG) may be configured to reduce nonspecific binding, i.e., adhesion, of charged particles, such as cells, to different surfaces of the pore plate 113. The silane groups present in PEG-silane may promote selective affinity to the glass surface of the pore plate. The thiol groups present in PEG-SH may promote selective affinity to the pore plate surface coated with a transition metal, such as Ti-Au. In some embodiments, PEG-SH may specifically adhere to the metal coating portion of the substrate 110, such as Ti-Au.In some embodiments, PEG-silane can adhere specifically to the glass portion of a substrate 110 that does not have any metal coating.

[0138] Figure 12A shows a top view of the array 100 according to some embodiments, and Figure 12B shows a cross-sectional view of the array. Figure 12B shows that one or more surface-modifying materials (e.g., one or more hydrophilic materials) may be added to the first and second portions of the substrate 110. In some embodiments, the first portion may be the upper part of the substrate 110, which is the portion closer to the surface 112. In some embodiments, the second portion may be the bottom part of the substrate 110, which is the portion closer to the surface 111. In some embodiments, the first portion of the substrate 110 (e.g., the portion of the vertical sidewall not covered by 1320 / 1322 in Figure 13B) may be coated with a material 1231 that modifies the surface properties of the substrate 110. The surface material 1231 may include hydrophilic materials such as hydrophilic oligomers or polymers (e.g., functionalized PEG) as described above. In some embodiments, the surface material 1231 may be a functionalized PEG, such as PEG-silane (for example, as described above in this specification with respect to Figure 11 or elsewhere herein). In some embodiments, the functionalized PEG may reduce the nonspecific binding of charged particles, such as cells, to the walls of the pore 113, e.g., adhesion. In some embodiments, the silane groups in PEG-silane can be used to modify the surface properties of the glass portion of the substrate 110. In some embodiments, as described in more detail with reference to 13A-13D, the second portion (bottom) of the substrate 110 may be coated with a plurality of materials 1300.

[0139] Figures 13A to 13D illustrate exemplary processes for forming a multilayer coating 1300 (see Figure 13D) on the bottom of a substrate 110 (for example, as described above in this specification with respect to Figure 11, or elsewhere described herein). In some embodiments, the multilayer coating 1300 can improve or alter the surface properties of the substrate 110. Furthermore, in some cases, the multilayer coating 1300 can exfoliate and simultaneously destroy the liquid meniscus held in the micropore array. In some embodiments, the multilayer coating 1300 or at least one or more portions of the coating 1300 can be destroyed using electromagnetic radiation, such as a laser.

[0140] In some embodiments, a first layer 1320 may be formed on the surface of the substrate 110, as shown in Figure 13B. In some embodiments, the first layer may contain a transition metal, such as Au, Ti, or Cr. Optionally, in some embodiments, a second layer 1322 may be formed on the first layer 1320, as shown in Figure 13C. In some embodiments, the second layer may be made of a different material than the first layer. In some embodiments, the second layer may contain a noble metal, such as Au. In some embodiments, the first layer can facilitate the adhesion of at least one or more subsequent layers of coating material to the substrate. In some embodiments, the first layer may be titanium, which can facilitate the adhesion of a second layer of a different coating material, such as gold. In some embodiments, the second layer, such as gold, may be coated with other materials, such as polymers. For example, thiol (-SH) groups have a high affinity for gold. In some embodiments, the second layer, for example, gold, may be suitable for surface functionalization by using, for example, a thiol (-SH) derivative of a functional surface coating material, such as PEG-SH.

[0141] Next, as shown in Figure 13D, a third layer 1332 may be formed covering the vertical sidewall portion of the pore (e.g., extending along the Z-axis). The vertical sidewall portion may include the first layer 1320 and / or the second layer 1322 (e.g., as described above in this specification with respect to Figure 11, or as described elsewhere in this specification). The first and / or second layer 1320 / 1322 may include a Ti layer, a Ti-Au stack, or an Au layer. In some embodiments, the third layer 1332 may include a polymer. In some embodiments, the polymer may include PEG, or a derivative of PEG, such as PEG-thiol. The thiol groups present in PEG-SH may promote selective affinity to the pore plate surface coated with a metal, such as Ti-Au. In some embodiments, functionalized PEG may reduce nonspecific binding, e.g., adhesion, of charged particles, such as cells, to the wall of the pore 113.

[0142] In some embodiments, a fourth layer 1333 may be formed covering the bottom of the substrate on the first layer 1320 and / or the second layer 1322, as shown in Figure 13D. The bottom may be near the vertical sidewall of the pore. The fourth layer 1333 may extend along the Y-axis, as shown in Figure 13D. In some embodiments, the fourth layer 1333 may contain an oligomer or polymer. In some embodiments, the fourth layer may contain a hydrophobic oligomer or polymer, such as a fluorinated or perfluorinated oligomer or polymer. The fluorinated or perfluorinated oligomer or polymer may be formed from monomers selected from the group consisting of fluorinated dioxol, fluorinated dioxolane, fluorinated cyclic polymerizable alkyl ethers, and combinations thereof. The fluorinated or perfluorinated oligomer or polymer may be a perfluoroalkylthiol, such as perfluorohexanethiol, perfluorooctanthiol, or perfluorodecanethiol. Fluorinated or perfluorinated oligomers or polymers may contain 1H,1H,2H,2H-perfluorodecanethiol (PF-SH). The thiol groups present in PF-SH may have a high affinity for the previous coating layer, for example, gold. Hydrophobic oligomers or polymers may be linear or branched. Hydrophobic oligomers or polymers may contain a first end group. Hydrophobic oligomers or polymers containing a first end group may contain a second end group. One of the first and second end groups may be configured to react with the activated and cleaned surface (after step 1117) or to form a self-assembled film (e.g., a monolayer) on that surface, while the other of the first and second end groups, if present, may be configured to remain on the hydrophobic oligomer or polymer after surface functionalization. The first or second terminal group, configured to react with an activated and cleaned surface or to form a self-assembled film on that surface, can be selected from silanes, thiols, primary amines (-NH2), carboxylic acids (-COOH), aldehydes, vinyls, epoxys, and chloros.The first or second terminal group configured to react with the activated and cleaned surface (after step 1117) or to form a self-assembled film on that surface may be a thiol. Hydrophobic oligomers or polymers (e.g., fluorinated or perfluorinated oligomers or polymers) are 250, 480, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5250, 5500, 5750, or 6000 Daltons, or about 250 It may have a molecular weight of approximately 480, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, 5000, 5250, 5500, 5750, or approximately 6000 daltons, or any range between any two of the aforementioned values.Hydrophobic oligomers or polymers (e.g., fluorinated or perfluorinated oligomers or polymers) are present in quantities of at least 250, at least 480, at least 500, at least 750, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2250, at least 2500, at least 2750, at least 3000, at least 3250, at least 3500, at least 3750, at least 4000, at least 4250, at least 4500, at least 4750, at least 5000, at least 5250, at least 5500, at least 5750, or at least 6000. It may have a molecular weight of Daltons, or at least about 250, at least about 480, at least about 500, at least about 750, at least about 1000, at least about 1250, at least about 1500, at least about 1750, at least about 2000, at least about 2250, at least about 2500, at least about 2750, at least about 3000, at least about 3250, at least about 3500, at least about 3750, at least about 4000, at least about 4250, at least about 4500, at least about 4750, at least about 5000, at least about 5250, at least about 5500, at least about 5750, or at least about 6000 Daltons.Hydrophobic oligomers or polymers (e.g., fluorinated or perfluorinated oligomers or polymers) can be up to 250, up to 480, up to 500, up to 750, up to 1000, up to 1250, up to 1500, up to 1750, up to 2000, up to 2250, up to 2500, up to 2750, up to 3000, up to 3250, up to 3500, up to 3750, up to 4000, up to 4250, up to 4500, up to 4750, up to 5000, up to 5250, up to 5500, up to 5750, or up to 6000. The molecular weight may be Daltons, or up to approximately 250, up to approximately 480, up to approximately 500, up to approximately 750, up to approximately 1000, up to approximately 1250, up to approximately 1500, up to approximately 1750, up to approximately 2000, up to approximately 2250, up to approximately 2500, up to approximately 2750, up to approximately 3000, up to approximately 3250, up to approximately 3500, up to approximately 3750, up to approximately 4000, up to approximately 4250, up to approximately 4500, up to approximately 4750, up to approximately 5000, up to approximately 5250, up to approximately 5500, up to approximately 5750, or up to approximately 6000 Daltons. In some embodiments, the hydrophobic surface coating may form a self-assembled monolayer (SAM). In some embodiments, the self-assembled monolayer may reduce wettability. In some embodiments, self-assembled monolayers can reduce surface energy. In some embodiments, hydrophobic surface coatings, e.g., PF-SH, can have a water contact angle of about 120°. In some embodiments, hydrophobic surface coatings, e.g., PF-SH, can have a water contact angle of 90° to 150°. In some embodiments, hydrophobic surface coatings, e.g., PF-SH, can have a water contact angle of 100°, 105°, 110°, or 115°. In some embodiments, the hydrophobic coating can be used as a sealant. In some embodiments, the sealant can prevent leakage from pore terminations on surface 111. In some embodiments, thiol groups can improve the adhesion of the hydrophobic coating, e.g., PF-SH, to the underlying surface, e.g., Au in Ti-Au.

[0143] In some embodiments, the surface material 1320 and / or 1322 (for example, as described above in this specification with respect to Figure 11, or as described elsewhere in this specification) can be applied to the surface of the substrate 110 by metal sputtering. In some embodiments, the thickness of the surface material 1320 and / or 1322 may be between 10 nm and 1000 nm. In some embodiments, the thickness of the surface material 1320 and / or 1322 may be at least 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 800 nm, 1000 nm, or greater. In some embodiments, the thickness of the surface material 1320 and / or 1322 may be up to about 1000 nm, 800 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm, or less.

[0144] In some embodiments, sputtering can be performed under vacuum. In some embodiments, the vacuum may be about 0.08 to about 0.02 mbar. In some embodiments, the vacuum may be up to about 0.01 mbar, 0.02 mbar, 0.03 mbar, 0.04 mbar, 0.05 mbar, 0.06 mbar, 0.07 mbar, 0.08 mbar, 0.09 mbar, 0.1 mbar, or less. In some embodiments, the vacuum may be at least about 0.1 mbar, 0.09 mbar, 0.08 mbar, 0.07 mbar, 0.06 mbar, 0.05 mbar, 0.04 mbar, 0.03 mbar, 0.02 mbar, 0.01 mbar, or more. In some embodiments, sputtering can be performed under a voltage of about 100 V to 3 kV. In some embodiments, the voltage may be at least about 100V, 110V, 130V, 150V, 170V, 220V, 280V, 500V, 1000V, 2000V, 3000V, or more. In some embodiments, the voltage may be at most about 3000V, 2000V, 1000V, 500V, 280V, 220V, 170V, 150V, 130V, 110V, 100V, or less. In some embodiments, sputtering can be performed under a current of 0 to 50mA. In some embodiments, the current may be at least about 0.01mA, 0.1mA, 1mA, 5mA, 10mA, 20mA, 30mA, 40mA, 50mA, or more. In some embodiments, the current may be approximately 50 mA, 40 mA, 30 mA, 20 mA, 10 mA, 1 mA, 0.1 mA, 0.01 mA, or less. In some embodiments, the surface material may be sputtered onto one or both sides of the glass array (e.g., the top and / or bottom of the plate).

[0145] Methods for forming infrared-absorbing beads are also provided herein. In some embodiments, the method includes the steps of washing agarose beads; coloring agarose beads; and forming the core of agarose beads. In some embodiments, the step of washing agarose beads includes suspending the agarose beads in a first solvent and centrifugating the agarose beads and the first solvent. In some embodiments, the first solvent includes an organic solvent, e.g., acetone, or an aqueous solvent, e.g., water, or a combination thereof. In some embodiments, centrifugation may be performed at a speed of about 1,000 rpm to about 4,000 rpm. In some embodiments, centrifugation may be performed at a speed of about 2,000 rpm. In some embodiments, 1 mL of the first solvent may be used for every 50 mg of agarose beads. In some embodiments, the agarose beads include Superdex beads.

[0146] In some embodiments, the step of coloring the agarose beads includes forming a coloring solution, centrifuging the coloring solution, and adding the coloring solution to the agarose beads. The coloring solution may contain Epolin 1178 and a second solvent. In some embodiments, the second solvent may contain acetone, water, deionized water, or any combination thereof. Centrifugation may be performed at a speed of about 2,000 rpm to about 10,000 rpm, for example, about 5,000 rpm. In some embodiments, the step of coloring the agarose beads further includes incubating the agarose beads and the coloring solution. Incubation may be performed for about 15 minutes to about 1 hour, for example, about 30 minutes. In some embodiments, incubation may be performed at room temperature. Incubation may be performed with constant mixing. In some embodiments, the step of coloring the agarose beads further includes centrifuging the agarose beads after incubation at a speed of, for example, about 750 rpm to about 3,000 rpm. In some embodiments, the step of coloring the agarose beads further includes separating the darker colored beads from the lighter colored beads. In some embodiments, the step of coloring the agarose beads further includes suspending the agarose beads in 0.2 percent BSA-PBS.

[0147] In some embodiments, the step of forming the core of the agarose beads includes suspending the agarose beads in a third solvent and centrifugating the agarose beads and the third solvent. In some embodiments, the third solvent includes a 1:1 acetone-water mixture. In some embodiments, centrifugation may be performed at a speed of about 500 rpm to about 2,000 rpm. In some embodiments, centrifugation may be performed for about 10 seconds to about 60 seconds.

[0148] Alternatively, in some embodiments, the step of forming the core of the agarose beads includes incubating the beads in a buffer. In some embodiments, the buffer includes BSA-PBS. In some embodiments, the buffer has a concentration of about 0.2 percent. In some embodiments, the incubation of the beads in the buffer may be carried out at a temperature of about 4°C. In some embodiments, the incubation of the beads in the buffer may be carried out for at least about 5 days. The step of forming the core of the agarose beads may further include changing the buffer daily.

[0149] Solutions containing a plurality of beads and a target particle as described herein are provided herein. In some cases, the target particle is a cell. In some cases, the solution contains a ratio of the number of beads to the number of cells, approximately 1:1 to 10:1. The solution containing the target particle can be inserted into one or more pores of an array as described herein. Examples of solutions are further described with respect to Examples 5 and 6.

[0150] system Another embodiment provided herein is a system for sorting particles. A system for sorting components of a mixture is provided herein. The system may include any embodiment, variation, or example of an array as described herein.

[0151] Figure 14A shows a system comprising an array 100, a housing 1431, and an inner surface 1432. A system for sorting particles may comprise an array 100 comprising a substrate 110 including a first surface 111; a second surface 112 opposite the first surface 111; and a plurality of pores 113 extending from the first surface 111 to the second surface 112, each of which comprises a cross-sectional area equal to or less than about 1 square millimeter and a length equal to or less than about 10 mm, wherein the substrate 110 comprises a first material; and a coating 120 operably bonded to the second surface 112 (wherein the coating 120 comprises a second material different from the first material, and a portion of the coating 120 may be formed to break away from the second surface 112 in response to electromagnetic radiation directed toward that portion of the coating 120); and a fluid in the plurality of pores 113 of the array 100 (wherein the meniscus of the fluid in the plurality of pores 113 is substantially adjacent to the coating 120).

[0152] In some embodiments, the first surface 111 or the second surface 112 may be hydrophilic. In some embodiments, the first surface 111 or the second surface 112 may be bonded to a hydrophilic coating 120. In some embodiments, the coating 120 may be hydrophobic. In some embodiments, the coating 120 may be capable of preventing leakage from the pore for a period equal to or longer than one hour. In some embodiments, the coating 120 covers the first surface 111 or the second surface 112 entirely.

[0153] In some embodiments, the surface coating material may be titanium. In some embodiments, the surface coating material may include silver, gold, aluminum, copper, platinum, nickel, or cobalt. In some embodiments, the base material may be glass. In some embodiments, the cross-sectional area is about 0.03 mm². 2It may be equal to or less than . In some embodiments, the length may be equal to or less than about 1.5 mm. In some embodiments, the coating 120 includes a thickness of equal to or less than about 200 nm. In some embodiments, the substrate 110 is about 0.5 m -1 This includes a surface area-to-volume ratio. In some embodiments, a portion of the coating 120 may be formed to absorb electromagnetic radiation and to peel off from the second surface 112 in response to electromagnetic radiation directed at the portion of the coating 120. In some embodiments, the plurality of micropores 113 are perpendicular to the first surface 111 and the second surface 112. In some embodiments, the plurality of micropores 113 are substantially parallel to each other. In some embodiments, the plurality of micropores 113 range from about 1 million to about 100 billion micropores 113. In some embodiments, the second material is opaque. The second material may be formed to absorb infrared (IR) energy. The substrate 110 and the coating 120 may have different coefficients of thermal expansion.

[0154] If necessary, the system may further comprise a housing 1431 including an inner surface 1432 configured to receive selected contents discharged from the array. The system may comprise any embodiment, variation, or example of the array as described herein, and a housing including an inner surface, the inner surface of which may be located below a second surface of the substrate. The system may further comprise a cell sorter. The array may be mounted on the cell sorter.

[0155] If necessary, the system for sorting particles may be equipped with an electromagnetic radiation source.

[0156] Figure 14B shows a system for sorting particles, comprising an array 100 and an electromagnetic radiation source 1451. The array may be formed to break down on a first or second surface in response to electromagnetic radiation directed at a portion of a first or second surface. In some cases, for example, when the particles of interest are cells, it may be beneficial for the sorting system to be able to emit particles held within a specific compartment of the array without directly directing a laser or other energy source into the compartment holding the particles of interest, in order to help increase cell viability. By focusing the laser energy on the surface of the array rather than inside the pores of the array, the possibility of damage to the pore contents from thermal shock, thermal expansion, microbubble generation, and localized shear stress can be avoided or reduced.

[0157] The source of electromagnetic radiation may include a laser. The laser may be a doped solid-state laser. The laser may be a fiber laser. The laser may be a semiconductor diode laser. The laser may be a gas laser, e.g., a HeNe laser or an excimer laser. The laser may emit electromagnetic radiation within a range of wavelengths. In some embodiments, the electromagnetic radiation may be emitted as visible light and / or infrared light. The electromagnetic radiation may be emitted within a 5 nanometer band and then emitted as visible light or infrared light. The electromagnetic radiation may be emitted as harmonics of a doped solid-state laser (e.g., doped ytterbium orthovanadate or ytterbium aluminum garnet). The electromagnetic radiation may include radiation at 1064 nm.

[0158] The electromagnetic radiation described above may contain incident energy. The incident energy may exceed 0.1 microjoules per pulse. The incident energy may be less than 1 millijoule per pulse. The incident energy may be in the range of 1 picojoule to 1 joule per pulse. The average power output may be less than 10 watts. The average power output may be less than 100 milliwatts. The average power output may exceed 1 microwatt.

[0159] The electromagnetic radiation described above may include a certain incident peak power density. This peak power density may be less than 10 terawatts per square centimeter. Its peak power may be less than 10 gigawatts per square centimeter.

[0160] The electromagnetic radiation described above may include a certain incident spot diameter. This spot diameter may be small enough to irradiate an area adjacent to the pore without significantly irradiating the cell contents. The spot diameter may be adjusted based on the size and spacing of the pores. The spot diameter may be small enough to irradiate the inner wall of the pore lumen without significantly irradiating the pore contents, such as cells inside the lumen. The spot diameter may be less than 10 millimeters (mm), less than 1 mm, less than 100 microns (μm), less than 10 μm, or less than that.

[0161] The electromagnetic radiation described above may include an incident pulse duration. This pulse duration may be greater than approximately 5 femtoseconds. It may be greater than approximately 100 femtoseconds. It may be greater than or equal to approximately 1 nanosecond. It may be less than approximately 1 microsecond.

[0162] An example of an electromagnetic radiation source is one with an output of 0.1 mJ and an output density of 10 8 ~10 9 W / mm 2It includes a 1064 nm ytterbium fiber laser, which produces a 20 μm spot diameter with a pulse duration of 4 ns at 10 to 30 percent of the maximum laser output, delivering 30 to 90 J / cm² to the array. 2 We can provide this.

[0163] The above system may further comprise one or more lenses for focusing an electromagnetic radiation source. These one or more lenses may include a microscope objective lens. The microscope objective lens may be raster-scanned from edge to edge of the array surface to target a specific portion of the array. The system may comprise one or more moving stages capable of controlling the position of the objective lens relative to the array surface.

[0164] The above system may include one or more beam splitters, filters, or interference filters. One or more beam splitters, filters, or interference filters in the system may allow the user to monitor the surface of the array while aligning the electromagnetic radiation source with the surface of the array or directing the electromagnetic radiation source towards the surface of the array. The alignment may be performed with electromagnetic radiation of a lower or equal output than the output that could destroy the array. The above system may include one or more position-sensitive optical detectors (e.g., CCDs) to monitor the alignment of the electromagnetic radiation source.

[0165] The above system may include a second electromagnetic radiation source. The second electromagnetic radiation source may be used for position adjustment. The second electromagnetic radiation source may be used to excite absorbers such as fluorophores. The second electromagnetic radiation source may be coherent or non-coherent. The second electromagnetic radiation source may be broadband or narrowband. The second electromagnetic radiation source may include any characteristics of electromagnetic radiation sources described herein (e.g., force, pulse duration, wavelength, etc.).

[0166] Figures 15A and 15B show an exemplary system 1400 comprising an array and housing. Figure 15A is an initial top view of the leak test at 0 hours. Figure 18B is an initial top view of the leak test of the exemplary array at 5 hours. According to Figures 15A to 15B, the exemplary array 100 in frame 1510 was leak-tested with deionized water for approximately 5 hours. There was no leakage of deionized water through the micropores of the array. In some embodiments, the coating of the exemplary array 100 may be capable of preventing leakage from the pores for a time equal to or greater than approximately 1 hour. In some embodiments, the coating of the exemplary array 100 may be capable of preventing leakage from the pores for a time equal to or greater than approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours.

[0167] method Embodiments, examples, and variations of arrays described herein may be used in methods for releasing particles from the pores of the array. Embodiments, examples, and variations of systems described herein may be used in methods for releasing particles from the pores of the array. A method for releasing particles from the pores of an array is provided herein, comprising the steps of filling the pore, retaining a portion of a solution in the pore, directing electromagnetic radiation at a portion of the array, destroying a portion of the array, and releasing a portion of the solution containing the particles of interest. The pore may be filled with at least a portion of a solution, which may contain the particles of interest. A portion of the solution may be retained in the pore by surface tension. The step of destroying a portion of the array may break the surface tension of the portion of the solution retained in the pore.

[0168] This specification provides a method for releasing selected contents from pores in an array, the method comprising the step of identifying pores in an array containing selected contents, the array comprising a substrate having a first surface and a second surface opposite to the first surface, the substrate comprising a substrate material and a surface material, the surface material being located on or adjacent to the first or second surface, the substrate comprising a plurality of pores defining a lumen extending from the first surface to the second surface, the substrate having (a) each of the plurality of pores having a maximum diameter of 500 microns or less, and (b) a plurality A step characterized in that each pore of the array has an aspect ratio of 10 or more, (c) a pore density of 100 or more pores per square millimeter, and (d) the surface material is selected from a material that absorbs more than 10 percent of the incident electromagnetic radiation, and a step of removing a portion of the surface material from a first or second surface of the array by electromagnetic radiation directed at the surface material in or adjacent to the identified pore, thereby releasing the contents of the identified pore.

[0169] In some examples, the above array may be characterized by two or more of the following: (a) each pore of the plurality of pores has a maximum diameter of 500 microns or less; (b) each pore of the plurality of pores has an aspect ratio of 10 or more; (c) a pore density of 100 or more pores per square millimeter; and (d) the surface material is selected from a material that absorbs more than 10 percent of the incident electromagnetic radiation.

[0170] Figures 16A–16F show side cross-sectional views of an example of a method for sorting cells using the exemplary array of Figure 1A, as described herein. According to Figures 16A–F, an exemplary method 1600 for sorting cells using an exemplary first array 100 includes a step 1610 of providing an array 100 comprising a plurality of pores 113. In some embodiments, operation 1610 may further include a step of covering a portion of the pores 113 closest to a first surface 111 of the array 100 containing microspheres, according to Figure 5A. Operation 1620 of method 1600 may include a step of depositing an aqueous solution 1621 into the array. In some cases, the array may include depositing first cells 1622 and second cells 1623 onto the first array 100, according to Figure 16B. Operation 1630 of method 1600 may include inserting the array 100 into a housing 1631, according to Figure 16C. In some cases, the housing may comprise a cartridge. The housing may have an inner surface 1632. Operation 1640 of method 100 may include the step of capturing a signal plot of selected particles. The selected particles may include a first cell 1622 and a second cell 1623, according to Figure 16D. According to Figure 16E, method 1600 may further include the step 1640 of locating the signal plot of the first cell 1622 within the signal plots of the first and second cells 1623. According to Figure 16F, method 1600 may further include the step 1640 of extracting the second cell 1623 from the array 100; and the step 1650 of recovering the second cell 1623. The step of extracting cells from the array may include the step of disrupting a coating on or near the surface of the array 100. The disruption step may include the step of providing electromagnetic radiation to selected locations on the surface of the array. Figure 16A shows a side section view of an array comprising multiple pores including a coating, provided according to an exemplary method.

[0171] Figure 16B shows a side cross-sectional view of the deposition of an aqueous sample solution in the exemplary array of Figure 1. In some embodiments, the step 1620 of deposition of the aqueous sample solution 1621 onto the array 100 includes the step of spreading the aqueous sample solution 1621 onto the array 100. In some embodiments, a hydrophilic second surface 112 of the array 100 draws the aqueous sample solution 1621 into a pore 113. In some embodiments, the hydrophilic second surface 112 of the array 100 evenly distributes the first cells 1622 and the second cells 1623 in the aqueous sample solution 1621 into the pore 113. In some embodiments, the hydrophilic second surface 112 of the array 100 randomly distributes the first cells 1622 and the second cells 1623 in the aqueous sample solution 1621 into the pore 113. In some embodiments, the first cells 1622 and the second cells 1623 sink to the bottom of each pore 113. If necessary, in some embodiments, the first cells 1622 and the second cells 1623 are retained in each pore 113 by the surface tension of the aqueous sample solution 1621. In some examples, the cells are selected from INKT cells, Tmem, Treg, HSPC, and combinations thereof. The first surface 111 of the array 100 may be hydrophobic. For example, as described elsewhere in this specification, the bottom side of the pore plate can be coated with 1H,1H,2H,2H-perfluorodecanethiol as a hydrophobic layer to prevent leakage of the pore plate. As described elsewhere in this specification, the vertical sidewalls of the pores near the bottom of the pore plate can be coated with methoxy-poly(ethylene-glycol)-thiol to reduce cell adhesion.

[0172] Figure 16C shows a side cross-sectional view of the exemplary array of Figure 1A inserted into a closed cartridge or housing according to several embodiments. According to Figure 16C, the cartridge 1631 includes a humidifying membrane 1633 on top of the array 100 and also includes a collection tray 1632 for collecting the second cells 1623. Optionally, in some embodiments, the cartridge 1631 includes a closed cartridge 1631. Optionally, in some embodiments, the cartridge 1631 includes a humidity-controlled cartridge 1631. Optionally, in some embodiments, the humidifying membrane 1633 reduces evaporation from the pore 113. Optionally, in some embodiments, the collection tray 1632 may be located below the array 100 in the cartridge 1631. Optionally, in some embodiments, the collection tray 1632 includes a transparent collection tray 1632.

[0173] Figure 16D shows images of signal plots for first and second cells according to several embodiments. According to Figure 16D, a signal plot 1641 for second cells may be measured. In some embodiments, a signal plot 1642 for first cells may be measured. In some embodiments, these plots may be captured by quantifying images taken by an automated fluorescence scanning system. The first cells may be fluorescent at a first wavelength, and the second cells may be fluorescent at a second wavelength. In some embodiments, combined images may be measured. Figure 17 shows a non-limiting example of a raw fluorescence image of an array of cells. Figure 18 shows a non-limiting example of a scatter plot of an array of 500,000 micropores represented in Figure 17.

[0174] Figure 16E shows a side cross-sectional view of extracting a second cell according to one embodiment. According to Figure 16E, the second cell 1623 is extracted from the array 100 by exposing the pore 113 containing the second cell 1623 to pulses from the laser 1651, according to the signal plot of the second cell 1623 in Figure 16D. The laser excites the coating 120. In some embodiments, microspheres may be provided within a particular pore 113. If necessary, in some embodiments, the laser 1651 includes a nanosecond laser 1651.

[0175] Figure 16F shows a side cross-sectional view of cell retrieval according to one embodiment. According to Figure 16F, the second cells 1623 extracted from the array 100 by the laser 1651 can be collected in the retrieval tray 1661.

[0176] Another embodiment provided herein is a method for releasing particles from a pore of an array, the method comprising the steps of: filling the pore with at least a portion of a solution (where at least a portion of the solution contains the particles of interest); holding a portion of the solution in the pore by surface tension; directing electromagnetic radiation to a portion of the array; destroying a portion of the array, thereby breaking the surface tension of the portion of the solution held in the pore; and releasing the portion of the solution containing the particles of interest. In some embodiments, the array comprises a substrate and a coating operably bonded to the substrate. In some embodiments, the substrate comprises a first surface, a second surface opposite the first surface, and a pore, the pore extending from the first surface to the second surface. In some embodiments, the first surface is hydrophilic and the coating is hydrophobic. In some embodiments, a portion of the array is the coating of the array. In some embodiments, a portion of the array is the coating of the array adjacent to the pore. In some embodiments, the coating may comprise one or more metals selected from the group consisting of chromium, titanium, gold, iron, nickel, copper, platinum, and palladium. In some embodiments, the coating may comprise one or more metal layers, each independently selected from the group consisting of chromium, titanium, gold, iron, nickel, copper, platinum, palladium, any mixture thereof, and any alloy thereof. In some embodiments, the coating may comprise a metal layer (e.g., gold) and an adhesion layer for the underlying metal layer. The adhesion layer for the gold layer may comprise chromium, titanium, nickel, or nickel-chromium beneath the gold. In some embodiments, the coating comprises a titanium-gold stack or a titanium layer. In some embodiments, the array comprises a plurality of pores. In some embodiments, the method further comprises the step of filling the plurality of pores with a solution. In some embodiments, the method further comprises the step of releasing a solution held in a subset of the plurality of pores, the subset of which holds a solution containing the particles of interest. The method may further comprise the step of analyzing a plurality of fluorescence signatures for each particle.In some embodiments, the method further includes the step of determining a pore that holds a portion of the solution containing the particles of interest, based on its analysis. In some embodiments, the particles are released at a rate of about 5,000 to about 100,000,000 particles of interest per second. In some embodiments, the particles of interest include cells. In some embodiments, the cells are released with a viability equal to or greater than 60 percent. In some embodiments, the method further includes the step of receiving the particles of interest in a housing, the housing comprising an inner surface for receiving the particles of interest. In some embodiments, the inner surface holds a receiving medium. In some embodiments, the receiving medium includes Pluronic® F68.

[0177] In some embodiments, the method further includes the step of removing a portion of the surface material from a first or second surface of the array by electromagnetic radiation directed at the surface material within or adjacent to the identified pore, thereby releasing the contents of the identified pore. In some examples, the portion of the surface material may be adjacent to the identified pore. The portion of the surface may include the luminal surface of the identified pore. The portion of the surface may be removed to a depth of 100 microns or less. The portion of the surface may be removed to a depth of 50 microns or less.

[0178] In some cases, the process involves loading a solution containing the selected contents into an array before identifying the pores containing the selected contents. In some cases, the process of identifying the pores containing the selected contents involves analyzing electromagnetic radiation emitted from the pores in the array. In some cases, the process of releasing the contents involves releasing the contents at a rate of approximately 5,000 to 100,000,000 pores per second.

[0179] The source of electromagnetic radiation may include a laser. The laser may be a doped solid-state laser. The laser may be a fiber laser. The laser may be a semiconductor diode laser. The laser may be a gas laser, e.g., a HeNe laser or an excimer laser. The laser may emit electromagnetic radiation of a range of wavelengths. In some embodiments, the electromagnetic radiation may be emitted in visible light and / or infrared light. The electromagnetic radiation may be emitted within a 5 nanometer band and then emitted as visible light or infrared light. The electromagnetic radiation may be emitted in the harmonics of a doped solid-state laser (e.g., doped ytterbium orthovanadate or ytterbium aluminum garnet). The electromagnetic radiation may include radiation at 1064 nm.

[0180] Electromagnetic radiation can be selected from wavelengths of 0.2 to 2.5 microns, fluence levels sufficient to break the adhesion between the contents and the pore, and pulse durations in the range of 1 ns to 1 millisecond.

[0181] Therefore, once it is identified that the target particle is held within a particular pore of the array, electromagnetic radiation can be directed near or adjacent to that particular pore to emit the target particle. In some embodiments, the destruction of the second surface includes removing at least a portion of the array material, a coating on the array, or both.

[0182] In some embodiments, the process of removing a portion of the surface material may be caused by localized heating. Such a mechanism may occur when the pulse duration is longer, the peak power density is lower, and / or the wavelength of the incident radiation is infrared. Localized heating may cause sublimation of the surface material or array material. In some embodiments, the substrate material and the coating have different coefficients of thermal expansion, which may lead to chipping.

[0183] In some cases, the process of removing a portion of a surface material can be caused by ablation. Such mechanisms may occur when the incident peak power density is higher, the pulse duration is shorter, the radiated power is higher, and / or the incident radiation is visible. Ablation may involve the breaking of local bonds and / or vaporization of the array or substrate material.

[0184] In some cases, the process of removing a portion of a surface material can be induced by plasma generation. This mechanism can occur when the pulse duration of the incident radiation is particularly short, when the wavelength of the incident radiation is in resonance with the multiphoton ionization mechanism, and / or when the wavelength of the incident radiation is very short. Pulse durations in the picosecond to femtosecond range can result in plasma generation faster than local heating leading to optical etching of the substrate or surface material.

[0185] In some cases, the process of removing a portion of the surface material can be caused by shock wave generation. Such mechanisms are more likely to occur when the peak power density is higher, when phonons are resonating, and / or when the pulse duration is shorter. The shock can cause physical vibration, chipping, or shaking of the surface material or array material.

[0186] In some cases, the process of removing a portion of the surface material is photochemical removal, such as photoionization. In some cases, the process of removing a portion of the surface material includes photoacoustic removal, such as optical generation of shock waves.

[0187] Terms and Definitions Unless otherwise defined, all technical terms used herein have the same meanings as those commonly understood by those skilled in the art of the field to which this disclosure pertains.

[0188] When used herein, the singular forms "a," "an," and "the" refer to plural objects unless the context clearly indicates otherwise. Any reference to "or" herein is intended to include "and / or" unless otherwise stated.

[0189] As used herein, the term “about” means an amount that is close to 10 percent, 5 percent, or 1 percent of the stated amount, including any increments within it.

[0190] As used herein, the term "PBMC" refers to peripheral blood mononuclear cells.

[0191] As used herein, the term “right angle” refers to a perpendicular arrangement or relationship. [Examples]

[0192] The following descriptive examples are representative of, and not intended to limit, embodiments of the software applications, systems, and methods described herein.

[0193] Example 1 - Preparation of Ti-Au coated micropore array: A glass micropore array (20 μm pore, 60 percent pore coverage) was sputtered first with 100 nm thick titanium (Ti), followed by 500 nm thick gold (Au) (vacuum: 8 × 10⁻² to 2 × 10⁻² mbar, sputtering voltage: 100 V to 3 kV, current: 0 to 50 mA). Ti / Au was sputtered on one side of the pore plate. Note that Ti / Au can be sputtered on both sides of the pore plate, or on either side, as described elsewhere in this specification.

[0194] Subsequently, the Ti / Au-coated micropore array was immersed in a 2M NaOH solution at room temperature for 20 minutes. Residual NaOH was washed off with deionized water (DI) and ethanol, and the pore plates were blow-dried.

[0195] PEG-silane coating Methoxy-poly(ethylene-glycol)-silane (PEG-silane) was dissolved in alcohol at a concentration of 0.1–5 g / 100 mL. Acetic acid was added to the solution at a volume ratio of 0.1–5 mL / 100 mL. Dry pore plates from the previous step were immersed in the solution and incubated in an oven at 60–80°C for 10–60 minutes. The pore plates were then washed with deionized water (DI) and ethanol, and blow-dried.

[0196] PEG-SH coating 0.5 g / 100 mL of methoxy-poly(ethylene-glycol)-thiol (PEG-SH) was dissolved in anhydrous ethanol (200 proof) using sonication. A dry pore plate from the previous step was immersed in the solution and incubated in a 30°C oven for 1 hour. The pore plate was then rinsed with deionized water (DI) and ethanol, and blow-dried.

[0197] Hydrophobic coating on the Au side of the pore plate 100 μL of PF-SH was added to 5 mL of 95% ethanol. The solution was then uniformly distributed onto an 8 x 8 inch PDMS sheet while evaporating the ethanol. If no liquid was visible on the PDMS sheet, the PDMS sheet (PF-SH side down) was placed over the Au side of the pore plate for 5 minutes. The PDMS sheet was then removed. The pore plate was allowed to stand for 10 minutes.

[0198] The pore plates were immersed in a PEG-SH coating solution and incubated in a 30°C oven for 15 minutes. The pore plates were then rinsed with deionized water (DI) and / or ethanol, and blow-dried using a pressurized air gun.

[0199] Example 2 - Cassette Assembly: The cassette includes (from top to bottom) a glass sealed to the top of the cassette; an aluminum alloy frame for holding the micropore plate; and a receiving glass plate spaced at a constant or variable distance from the micropore plate. Receiving medium (OptiPEAK T Cell Medium, InVitria, Junction City, KS) containing 0.1 percent of a different volume of Pluronic® F68 (Cat. 24040032, ThermoFisher Scientific Inc.) was added to the receiving plate (depending on the size of the cassette). The coated micropore array was assembled in the cassette with the coated side facing downwards (facing the receiving medium). By adding Pluronic® F68 to the receiving medium, the viability of cells extracted from the pore can be significantly increased from 0 percent viability to >75 percent viability.

[0200] Example 3 - Cell sorting using coated micropore arrays: PBMCs with a density of 2 million / mL were dropped onto the top of a micropore array in OptiPEAK T cell medium and allowed to stand for 5 minutes to allow single cells to be trapped at the bottom of the micropore by surface tension. The cassette was then mounted on a cell sorter. Cells could be extracted from the micropores using laser power of 10–100 percent. The Ti-Au coating on the bottom edge of the micropore absorbed the IR laser energy, and the thin layer of Ti-Au was removed. The meniscus was broken, and cells were released from the desired micropores.

[0201] Example 4 - Production of agarose beads with an IR absorption core: This procedure describes the preparation of agarose beads having a transparent shell and an IR-absorbing core.

[0202] Step 1: Suspend 1.50 mg of Superdex beads (Superdex 75 100 / 300 GL, GE Healthcare Life Sciences) in 1 mL of acetone. Centrifuge at 2000 rpm to collect the Superdex beads. Discard the acetone. Prepare a saturated solution of 1 mL of IR absorption dye (Epolight 1178, Epolin, New Jersey, USA) in acetone. Centrifuge at 5000 rpm to remove any undissolved IR dye. Add the IR dye solution to the Superdex beads. Incubate at room temperature for 30 minutes with constant mixing. Centrifuge the mixture at 1500 rpm. Discard the upper liquid. Leave only the dark-colored pellet at the bottom. Without further washing with acetone, suspend the obtained dark-colored pellet in 0.2 percent BSA-PBS. This will yield Superdex beads with uniformly incorporated IR dye.

[0203] Step 2. To remove the dye from the outer part of the beads, rinse the beads with a 1:1 acetone-water mixture by pipetting for less than 15 seconds. Immediately afterward, centrifuge the mixture at 1000 rpm for 30 seconds and discard the upper liquid. This will yield the IR core structure.

[0204] Alternatively, the IR absorption core can also be prepared by incubating the beads obtained from step 1 in 0.2 percent BSA-PBS at 4 degrees for >5 days. Change the buffer once daily. This allows the IR dye to slowly dissolve from the Superdex beads by molecular diffusion alone.

[0205] The effectiveness of IR dye microspheres is shown in Table 1 below. [Table 1]

[0206] The effectiveness of chromium microspheres is shown in Table 2 below. [Table 2]

[0207] Example 5 - Single PBMC survival rate when Pluronic® F68 is used as a culture medium supplement: This procedure describes media supplements to enhance cell viability during cell sorting. For cell loading and collection, cells were suspended and collected in OptiPEAK T Lymphocyte Complete Media (777OPT069) supplemented with 0.1 percent Pluronic® F68 and 1× penicillin / streptomycin. In this example, for exemplary arrays with a micropore size of 20 μm, the percentage viability for each of the three samples was measured as 81 percent, 74 percent, and 65 percent, respectively.

[0208] Example 6 - PBMC extraction: This procedure describes the solution containing the target particles and beads.

[0209] A solution containing human PBMC cells was dropped onto the top of a micropore array. After 10 minutes, a single PBMC was loading into the micropore. Subsequently, a solution containing either control beads (TiO2 beads coated with IR dye), agarose and dextran beads, or agarose and IR dye microspheres was loaded onto the top of the micropore array. After 15–30 minutes, the beads were loaded into the micropore by gravity. The pore array containing the cells and beads was mounted on top of a receiving reservoir containing cell culture medium. An IR pulsed laser was directed to target the bottom of the pore where the beads were loaded, and the cells were extracted into the cell culture medium. After extraction, the cell culture medium containing the extracted cells was collected for viability assay.

[0210] Example 7 - Cell viability: This procedure describes how to measure cell viability.

[0211] Cell viability was measured using a quantitative sandwich ELISA assay (Human IFN-Gamma ELISA Spot Kit, R&D Systems Inc., No. EL285). This assay uses a capture antibody specific to the human cytokine interferon-gamma (IFN-gamma) pre-coated on a PVDF-coated microplate. When the collected cells are pipetted directly into the wells, immobilized antibodies immediately adjacent to the secreting cells bind to the secreted human IFN-gamma. After washing and incubation with a biotinylation detection antibody, alkaline phosphatase conjugated to streptavidin was added. Unbound enzymes were then removed by washing, and the substrate solution was added. A blue precipitate may appear at the cytokine sites, appearing as spots. Each individual spot corresponds to an individual human IFN-gamma secreting cell. These spots were counted. Standard cell samples of serial dilutions with known viable cell counts were plated in the same way as the collected cell samples. A calibration curve was plotted by counting the blue spots in each well. The number of viable cells in the collected samples was determined using a calibration curve.

[0212] Example 8 - Comparison of performance (extraction yield and cell viability) between different coatings: Figures 19A-19C show a comparison of the performance (extraction yield and cell viability) of Au-coated pore plates and Cr-coated pore plates. Au-coated core plates may include surface modifiers described herein with reference to Figures 11A-13D. For example, Au-coated pore plates may include materials described and shown in Figures 12B and 13D. Referring to the Au-coated pore array in Figure 19A, the upper sidewall portions (glass portions) of the pores in the array may be coated with PEG-silane. The lower vertical sidewall portions of the pores in the array may be coated with Au and PEG-thiol. The bottom of the array (near the vertical sidewalls of the pores) may be coated with Au and stamped with perfluorooctanethiol. Referring to the Cr-coated pore array in Figure 19A, the bottom and bottom vertical sidewalls of the pores in the array may be coated with Cr alone, and the upper sidewall portions (glass portions) of the pores in the array may be coated with PEG-silane.

[0213] Au-coated pore plates with different surface PEG modifications can offer improved extraction yield and cell viability compared to Cr-coated pore plates. For example, the extraction yield of an Au-coated pore plate is 73%, while that of a Cr-coated pore array is 66%. Although the viability of cells extracted from Au-coated and Cr-coated pore plates is similar (66% vs. 68%), the number of viable cells obtained using Au-coated pore plates is greater than that obtained using Cr-coated pore plates because Au-coated core plates have a higher extraction yield compared to Cr-coated pore plates.

[0214] Figure 19B shows another example of yield between Au-coated and Cr-coated pore plates. Extraction yield and cell viability, as well as overall viable cell yield, are improved in Ti-Au-PEG coated pore plates. Peripheral blood mononuclear cells (PBMCs) stained with CD4 / 8-APC T cell marker were loaded onto Au-coated and Cr-coated plates and extracted to compare their extraction yield and viability by dye exclusion. Only the PEG-silane coated Cr plate showed higher dye exclusion-based viability compared to the chemically coated Au plate, but the extraction yield could not be improved considering its laser power limitations. The overall yield (i.e., extraction yield and viability) of the Cr-coated plate appeared to be much lower than that of the Au-coated plate. Figure 19C shows extraction yield images from full plates coated with Ti-Au-PEG and hydrophobic coatings (bright dots are cells stained with fluorescent antibodies under fluorescence imaging). As shown in Figure 19C, a comparison of images before and after extraction reveals a high extraction yield, consistent with the quantitative results described above.

[0215] Example 9 - Contact angle images and measurements of different coatings: Figure 20 shows images and contact angle measurements of a bare Au surface, an mPEG-SH coated surface, and a PF-SH coated surface. Images and measurements were obtained on a glass plate and a pore plate. As shown in Figure 20, different surface coatings can be used to modify the wetting behavior for liquids. For example, a hydrophobic coating can be formed on the bottom of a pore plate (e.g., by stamping PF-SH on the bottom of the array) to prevent leakage from the pore and to form a meniscus sufficient to hold liquids and particles within the pore.

[0216] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only. Those skilled in the art will be able to conceive of numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various substitutes for the embodiments of the present invention described herein may be used in the practice of the invention. The present invention provides, for example, the following items: (Item 1) It is an array, A substrate having a first surface and a second surface opposite to the first surface, wherein the substrate includes a plurality of pores defining a lumen extending from the first surface to the second surface, and the plurality of pores are configured to receive a sample solution containing a plurality of particles, A surface material provided on or near the first or second surface, wherein the surface material includes a plurality of materials configured to modify the wetting behavior of the sample solution or the plurality of particles on or near the first or second surface, such that one of the first or second surface is hydrophilic and the other of the first or second surface is hydrophobic. An array that includes this. (Item 2) The array according to item 1, wherein the plurality of materials include surface layers modified with functional groups, and the surface layers modified with functional groups are, if necessary, hydrophobically modified surface layers, hydrophobically modified surface layers, or a combination thereof, or the surface layers modified with functional groups are, if necessary, chemically coated metal layers. (Item 3) The array according to item 2, wherein the surface layer modified with the functional group comprises titanium and / or gold. (Item 4) The array according to item 2, wherein a first portion of the surface layer modified with the functional group is coated with a first chemical coating. (Item 5) The array according to item 4, wherein a second portion of the surface layer modified with the functional group is coated with a second chemical coating different from the first chemical coating. (Item 6) The array according to item 4, wherein the first chemical coating is provided on the vertical sidewalls of the plurality of pores on or near the first or second surface. (Item 7) The array according to item 6, wherein the first chemical coating is configured to reduce or eliminate the adhesion of the particles to the vertical sidewalls of the pores. (Item 8) The array according to item 5, wherein the second chemical coating is configured to reduce or prevent undesirable leakage of the sample solution from the pore. (Item 9) The array described in item 5, wherein the second chemical coating is hydrophobic. (Item 10) The array according to item 5, wherein the second chemical coating is provided on or near the first or second surface of the substrate, and the portion of the substrate is near the vertical side walls of the plurality of pores. (Item 11) The array according to item 10, wherein the portion of the substrate is substantially perpendicular to the vertical side walls of the plurality of pores. (Item 12) The array according to item 4, wherein the first chemical coating comprises methoxy-poly(ethylene-glycol)-thiol. (Item 13) The array according to item 5, wherein the second chemical coating comprises 1H,1H,2H,2H-perfluorodecanethiol. (Item 14) The array according to item 2, wherein the plurality of materials further include a chemical coating not on a surface layer modified with the functional group. (Item 15) The array according to item 14, wherein the chemical coating is provided on the substrate or on one or more of the plurality of pores that do not have a surface layer modified with the functional group. (Item 16) The array according to item 14, wherein the chemical coating comprises methoxy-poly(ethylene-glycol)-silane. (Item 17) The array according to item 1, wherein the second surface is configured to receive the sample solution containing the plurality of particles. (Item 18) The array according to item 2, wherein the first surface is configured to break apart to release one or more of the particles from one or more of the pores. (Item 19) The array according to item 18, wherein the second surface is hydrophilic to facilitate the absorption of the sample solution containing the plurality of particles into the plurality of pores. (Item 20) The array according to item 18, wherein the first surface is hydrophobic, thereby reducing or eliminating undesirable leakage of the sample solution from the pore. (Item 21) The array according to item 18, wherein the first surface is configured to be destroyed by directing electromagnetic radiation to one or more portions of the second surface. (Item 22) The array according to item 1, wherein each of the plurality of pores has a maximum diameter of 500 microns or less. (Item 23) The array according to item 1, wherein each of the plurality of pores has an aspect ratio of 10 or more. (Item 24) The array according to item 1, wherein the surface material is selected from materials that absorb more than 10% of the incident electromagnetic radiation. (Item 25) The array according to item 1, wherein the substrate has a pore density of 100 or more pores per square millimeter. (Item 26) The array according to item 1, wherein the particle extraction yield of the array is at least 70%. (Item 27) The array according to item 2, wherein the particle extraction yield of the array having a surface layer modified with the functional group is higher than that of another array not having a surface layer modified with the functional group. (Item 28) The array according to item 27, wherein the particle extraction yield of the array having a surface layer modified with the functional group is at least 5% higher than that of the other array not having a surface layer modified with the functional group. (Item 29) The array according to item 28, wherein the particle extraction yield of the array having a surface layer modified with the functional group is at least 20% higher than that of the other array not having a surface layer modified with the functional group. (Item 30) The array according to item 2, wherein the plurality of particles contain living cells and the array has a surface layer modified with the functional group has a higher live cell extraction yield than another array that does not have a surface layer modified with the functional group. (Item 31) The array according to item 30, wherein the live cell extraction yield of the array having a surface layer modified with the functional group is at least 5% higher than that of the other array not having a surface layer modified with the functional group. (Item 32) The array according to item 30, wherein the live cell extraction yield of the array having a surface layer modified with the functional group is at least 20% higher than that of the other array not having a surface layer modified with the functional group. (Item 33) The array according to item 1, wherein the surface layer modified with the functional group has a thickness in the range of approximately 50 nm to approximately 1 mm.

Claims

1. It is an array, A substrate having a first surface and a second surface opposite to the first surface, wherein the substrate includes a plurality of pores defining a lumen extending from the first surface to the second surface, and the plurality of pores are configured to receive a sample solution containing a plurality of particles, A surface material provided on or near the first surface or second surface, wherein the surface material includes a plurality of materials configured such that one of the first surface or the second surface is hydrophilic and the other of the first surface or the second surface is hydrophobic. An array comprising, wherein the plurality of materials comprises a functionally modified surface layer containing a transition metal, wherein the transition metal is titanium and / or gold, and the surface layer is configured to break from the first or second surface of the array in response to electromagnetic radiation being directed to a portion of the surface layer or near a portion of the surface layer.

2. The array according to claim 1, wherein the surface layer modified with the functional group is a hydrophilic surface layer, a hydrophobic surface layer, or a combination of a hydrophilic surface layer and a hydrophobic surface layer, or the surface layer modified with the functional group is a chemically coated transition metal layer.

3. The array according to claim 1, wherein the transition metal is titanium.

4. The array according to claim 1, wherein the transition metal is gold.

5. The array according to claim 1, wherein a first portion of the surface layer modified with the functional group is coated with a first chemical coating, and a second portion of the surface layer modified with the functional group is coated with a second chemical coating different from the first chemical coating.

6. The first chemical coating is provided on the vertical sidewalls of the plurality of pores on or near the first or second surface, and is configured to reduce or eliminate the adhesion of the particles to the vertical sidewalls of the pores, and / or The array according to claim 5, wherein the second chemical coating is hydrophobic and configured to reduce or prevent undesirable leakage of the sample solution from the pore.

7. The second chemical coating is provided on a portion of the substrate that is on or near the first or second surface, and / or The portion of the substrate is located near the vertical side walls of the plurality of pores, and / or The array according to claim 6, wherein the portion of the substrate is substantially perpendicular to the vertical side walls of the plurality of pores.

8. The first chemical coating comprises and / or methoxy-poly(ethylene-glycol)-thiol. The array according to claim 5, wherein the second chemical coating comprises 1H,1H,2H,2H-perfluorodecanethiol.

9. The array according to claim 1, wherein the plurality of materials further comprises a chemical coating not located on a surface layer modified with the functional group.

10. The chemical coating is provided on the substrate or on one or more of the plurality of pores that does not have a surface layer modified with the functional group, and / or The array according to claim 9, wherein the chemical coating comprises methoxy-poly(ethylene-glycol)-silane.

11. The array according to claim 1, wherein the second surface is configured to receive the sample solution containing the plurality of particles.

12. The array according to claim 1, wherein the first surface is configured to break apart in order to release one or more of the particles from one or more of the pores.

13. The second surface is hydrophilic to facilitate the absorption of the sample solution containing the plurality of particles into the plurality of pores, and / or The first surface is hydrophobic to reduce or eliminate undesirable leakage of the sample solution from the pore, and / or The array according to claim 12, wherein the first surface is configured to be destroyed by directing electromagnetic radiation to one or more portions of the first surface.

14. Each of the plurality of pores has a maximum diameter of 500 microns or less, and / or The array according to claim 1, wherein each of the plurality of pores has an aspect ratio of 10 or more.

15. The array according to claim 1, wherein the surface material is selected from materials that absorb more than 10% of the incident electromagnetic radiation.

16. The array according to claim 1, wherein the substrate has a pore density of 100 or more pores per square millimeter.

17. The array according to claim 1, wherein the particle extraction yield of the array is at least 70%.

18. The array according to claim 1, wherein the particle extraction yield of the array having a surface layer modified with the functional group is higher than that of another array not having a surface layer modified with the functional group.

19. The array according to claim 18, wherein the particle extraction yield of the array having a surface layer modified with the functional group is at least 5% higher than that of the other array not having a surface layer modified with the functional group.

20. The array according to claim 1, wherein the plurality of particles contain living cells and the array has a surface layer modified with the functional group has a living cell extraction yield that is at least 5% higher than that of another array that does not have a surface layer modified with the functional group.

21. The array according to claim 1, wherein the surface layer modified with the functional group has a thickness in the range of about 50 nm to about 1 mm.

22. A method for releasing selected contents from the pores of an array according to claim 1, A step of identifying the pore of the array having the selected contents, A step of removing a portion of the surface material from the first or second surface of the array by electromagnetic radiation directed at the surface material within or adjacent to the identified pore, thereby releasing the contents of the identified pore. Methods that include...

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