Multistage sample recovery system

Abstract

Multi-stage sample recovery systems are provided that include automated 2-stage and 3-stage sample recovery systems. Such systems enable rapid screening and recovery of samples, including live cell-based samples, from high-throughput screening systems, including systems that utilize large-scale microcapillary arrays. In a particular screening system, each microcapillary includes a solution containing a variant protein, an immobilized target molecule, and a reporter element. The immobilized target molecule can include any molecule of interest, including proteins, nucleic acids, carbohydrates, and other biological molecules. Association of the variant protein with the molecular target is assessed by measuring a signal from the reporter element. The contents of microcapillaries identified in an assay as containing a variant protein of interest can be identified and recovered using the multi-stage systems disclosed herein.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 441,128, filed on December 30, 2016, the entire contents of which are expressly incorporated herein by reference. Background Technology

[0003] The analysis of biological samples, including the identification, characterization, and reengineering of proteins, nucleic acids, carbohydrates, and other important biomolecules, has greatly benefited from increased sample numbers and reduced sample sizes. For example, two-dimensional biomaterial microarrays, such as DNA microarrays, have enabled the development of high-throughput screening methods involving multiple approaches for sample processing and result detection.

[0004] The above methods have benefited in some cases from their combination with optical sensing techniques for identifying samples of interest using fluorescence or other corresponding specific and sensitive labeling methods.

[0005] While this technique provides information about the analysis of a particular sample, such as the presence of a particular biomolecule in solution and the potential amount of said biomolecule or the sequence of a particular nucleic acid or polypeptide, it cannot enable the recovery of biological samples identified by the assay without inactivating or otherwise damaging the sample of interest.

[0006] Therefore, there is a need to continue developing improved micro-screening and analysis methods and systems with high throughput capabilities, and in particular, methods and systems for recovering samples identified in screening and analysis. Summary of the Invention

[0007] This disclosure addresses these or other needs by providing a multi-stage sample recovery system, said multi-stage sample recovery system comprising:

[0008] A selection array level, wherein the selection array level is controllable in two dimensions relative to the microscope objective and is configured to be reversibly correlated with the selection array; and

[0009] A first recovery array stage, wherein the first recovery array stage is controllable in at least one dimension relative to the microscope objective and is configured to be reversibly associated with the recovery array;

[0010] The filtering array level and the first recycling array level are independently controllable.

[0011] In some embodiments, the multi-stage sample recovery system of this disclosure further includes a screening array reversibly associated with the screening array stage and a recovery array reversibly associated with the first recovery array stage.

[0012] In some embodiments, the system further includes an extraction beam generator optically coupled to a micro sample container in the screening array through a hole in the screening array stage.

[0013] In some embodiments, the system further includes a second recovery array stage. Attached Figure Description

[0014] Figure 1A-1C The steps of an exemplary microcapillary screening assay are illustrated schematically. The left-hand illustration in each figure is a cross-sectional view taken from the side of a single microcapillary. The right-hand illustration in each figure is a bottom view of a small portion of the microcapillary array. The shading in each case is intended to illustrate electromagnetic signals such as fluorescence.

[0015] Figure 2A -C shows a small bottom view of the microcapillary array, illustrating hybridoma screening for mammalian cells, using a bright field of view (…). Figure 2A ), LiveGreen Figure 2B ) or fluorescent anti-mouse second antibody ( Figure 2C Imaging of cells.

[0016] Figure 3 Images of microcapillaries containing A431 target cells and hybridoma cells during a 4-hour incubation process are shown.

[0017] Figure 4A and Figure 4B The image shown is a small portion of a microcapillary array, highlighting both expressed and non-expressing yeast cells within mammalian cells, using bright-field perspective (BFS). Figure 4A ) or fluorescent antibody ( Figure 4B Imaging of cells.

[0018] Figure 5A-5G The growth of immortalized human cells in a microcapillary array over a 6-day period was demonstrated.

[0019] Figures 6A-6E These are different views of a microscope system designed to implement the screening methods of this disclosure.

[0020] Figure 7A An exemplary filter array level is shown. Figure 7B An exemplary recycling array level is shown.

[0021] Figure 8 An exemplary positioning of the screening array and the recovery array relative to each other is shown during the recovery of three samples of interest from the screening array facilitated by the sample recovery system of the present invention. Detailed Implementation

[0022] Recently, microcapillary arrays have been used with large numbers of biological samples in methods for high-throughput analysis and protein engineering, such as those known as “microcapillary single-cell analysis and laser extraction” or “μSCALE”. See Chen et al. (2016), *Nature Chem. Biology* 12:76–81; DOI: 10.1038 / NCHEMBIO.1978. This method relies on the spatial isolation of individual cells within the microcapillary array and thus enables repetitive imaging, cell growth, and protein expression of individual samples within each microcapillary of the array. Therefore, this technique enables large-scale, parallel, quantitative biochemical and biophysical measurements of millions of samples within a microcapillary array, for example, in the analysis of millions of protein variants expressed from yeast, bacteria, or other suitable cells distributed throughout the array. Advantageously, this method has allowed for simultaneous time-resolved kinetic analysis of multiple samples, as well as sorting of those cells based on targeted phenotypic features.

[0023] The development of μSCALE methods and devices for quantitative biochemical and biophysical analysis of biological variants has been reported in U.S. Patent Application Publication No. 2016 / 0244749 A1, which is incorporated herein by reference in its entirety. However, extracting the contents of desired microcapillaries according to μSCALE methods requires the inclusion of a radiation-absorbing material in each sample and the introduction of electromagnetic radiation from a pulsed laser into this material, thus increasing the complexity of the extraction method. Furthermore, early methods for screening biological variants in microcavity arrays relied on adding microparticles to the arrayed sample to partially or completely suppress electromagnetic radiation transmission into and out of the sample, thereby minimizing signals emitted from microcavities lacking the desired binding activity. See U.S. Patent Application Publication No. US2014 / 0011690 A1. In some aspects of this disclosure, the screening methods do not rely on these additional sample components or manipulations, thus simplifying and improving the efficiency of the screening technique. The screening method has been described in U.S. Patent Applications No. 62 / 433,210 and No. 15 / 376,588, both filed on December 12, 2016, the disclosures of which are incorporated herein by reference in their entirety.

[0024] In specific applications of these methods, and as will be disclosed in more detail herein, target molecules can be immobilized on surfaces such as particles (e.g., magnetic particles), cell surfaces, or microcapillary walls. The interaction between the variant protein and the target molecule in these methods can then be measured using several approaches, including those utilizing detectable antibodies and measuring detectable signals generated within the target cell. It should be understood that this approach can be used in high-throughput screening to discover protein variants of target molecules bound to target molecules, such as those on cells or other surfaces.

[0025] Filtering methods

[0026] Therefore, in some aspects, this disclosure provides a method for screening variant protein populations, the method comprising the following steps:

[0027] Provided is a microcapillary array comprising multiple microcapillaries, each microcapillary including a variant protein, an immobilized target molecule, and a reporter element, wherein the variant protein associates with the immobilized target molecule with a specific affinity; and

[0028] Measuring signals from at least one reporter element indicating association between at least one variant protein and at least one immobilized target molecule to identify at least one microcapillary of interest.

[0029] In these methods, the microcapillary array preferably comprises a plurality of longitudinal fused capillaries, such as fused silica capillaries, but any other suitable material may also be used in the array. See, for example, PCT International Patent Publications WO2012 / 007537 and WO2014 / 008056, the disclosures of which are incorporated herein by reference in their entirety. Such arrays can be fabricated, for example, by bundling millions or billions of silica capillaries and melting them together through a thermal process, but other suitable fabrication methods may also be employed. The melting process may include, for example, the following steps: i) heating capillary mono-drawn glass drawn under tension into a single-clad fiber; ii) producing capillary multi-drawn mono-capillaries from the mono-drawn glass by bundling, heating, and drawing; iii) producing capillary multi-drawn multi-capillaries from the multi-drawn mono-capillaries by further bundling, heating, and drawing; iv) producing block assemblies of drawn glass from the multi-drawn multi-capillaries by stacking them in briquettes; v) forming block briquettes from the block assemblies by heat and pressure treatment; and vi) forming block-shaped blocks by cutting the block briquettes to a precise length (e.g., 1 mm).

[0030] In some embodiments, the manufacturing method further includes cutting quartz capillaries to form a very high-density array of glass microcapillaries. In some embodiments, the microcapillary array may be cut to a height of approximately 1 mm, but even shorter microcapillary arrays are contemplated, including arrays 10 μm high or even shorter. In some embodiments, even shorter microcapillary arrays are contemplated, including arrays of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, or 10 μm.

[0031] In some embodiments, even longer microcapillary arrays are envisioned, including arrays of 10 mm or even longer. In some embodiments, the height of the array is 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm.

[0032] This process forms a microcapillary array suitable for use in the methods of the present invention, which has a very high density. In an exemplary array, each microcapillary has a diameter of about 5 μm and its open space (i.e., representing the lumen of each microcapillary) is about 66%. In some arrays, the proportion of the array in an open state ranges between about 50% and about 90%, for example, about 60% to 75%, such as the microcapillary array provided by Hamamatsu with an open area of ​​about 67%. In a particular example, a 10 × 10 cm array with microcapillaries of 5 μm in diameter and an open space of about 66% has a total of about 330 million microcapillaries.

[0033] In various embodiments, the inner diameter of each microcapillary in the array ranges from about 1 μm to 500 μm. In some arrays, the inner diameter of each microcapillary ranges from about 1 μm to 300 μm; optionally from about 1 μm to 100 μm; further optionally from about 1 μm to 75 μm; still further optionally from about 1 μm to 50 μm; and still further optionally from about 5 μm to 50 μm.

[0034] In some microcapillary arrays, the open region (OA) of the array occupies 90% of the open region, such that the number of microcapillaries per centimeter of array varies between approximately 4.6 million and over 11 million when the pore size varies between 1 μm and 500 μm. In some microcapillary arrays, the open region occupies 67% of the open region, such that the number of microcapillaries per square centimeter of array varies between approximately 340 and over 800,000 when the pore size varies between 1 μm and 500 μm. In some embodiments, the pore size is 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 250 μm, 350 μm, or 500 μm. In some embodiments, the pore size is between 5 μm and 500 μm. In some embodiments, the pore size is between 10 μm and 450 μm. In some embodiments, the pore size is between 50 μm and 500 μm. In some embodiments, the pore size is between 100 μm and 500 μm. In some embodiments, the pore size is between 250 μm and 500 μm. In some embodiments, the pore size is between 350 μm and 500 μm. In some embodiments, the pore size is between 100 μm and 450 μm. In some embodiments, the pore size is between 250 μm and 450 μm.

[0035] In some embodiments, the number of microcapillaries per square centimeter array is approximately 400; 500; 1000; 2000; 3000; 4000; 5000; 6000; 7000; 8000; 9000; 10000; 20000; 50000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; or 800,000. In some embodiments, the number of microcapillaries per square centimeter array varies between approximately 500 and 800,000. In some embodiments, the number of microcapillaries per square centimeter array varies between approximately 1000 and 700,000. In some embodiments, the number of microcapillaries per square centimeter array varies between about 2,000 and 600,000. In some embodiments, the number of microcapillaries per square centimeter array varies between about 10,000 and 800,000. In some embodiments, the number of microcapillaries per square centimeter array varies between about 10,000 and 700,000. In some embodiments, the number of microcapillaries per square centimeter array varies between about 50,000 and 800,000. In some embodiments, the number of microcapillaries per square centimeter array varies between about 50,000 and 700,000. In some embodiments, the number of microcapillaries per square centimeter array varies between about 100,000 and 700,000. In some embodiments, the number of microcapillaries per square centimeter array varies between about 100,000 and 600,000. In some embodiments, the number of microcapillaries per square centimeter array varies between about 100,000 and 500,000. In some embodiments, the number of microcapillaries per square centimeter array varies between about 500,000 and 800,000.

[0036] In one particular embodiment, a microcapillary array can be fabricated by combining billions of quartz capillaries and then melting them together through a thermal process. Following this, slices (0.5 mm or larger) are cut to form a glass microcapillary array with a very high aspect ratio. Arrays are also commercially available from companies such as Hamamatsu Photonics KK (Japan); Incom, Inc. (Massachusetts); and Photonis Technologies (SAS) (France). In some embodiments, the microcapillaries of the array are sealed at one end with a solid substrate attached to the array.

[0037] The microcapillary array of the screening method of the present invention can include any number of microcapillaries within the array. In some embodiments, the microcapillary array includes at least 100,000, at least 300,000, at least 1,000,000, at least 3,000,000, at least 10,000,000, or even more microcapillaries. Preferably, the number of microcapillaries in the array is selected according to the size of the variant protein library to be screened.

[0038] As described above, each capillary in the microcapillary array used in the screening method of the present invention includes a variant protein, an immobilized target molecule, and a reporter element, wherein the variant protein is one variant protein in a group of variant proteins subjected to the screening method. The group of variant proteins can be any group of proteins that can be suitably distributed within the microcapillary array. Ideally, the group of variant proteins is distributed in the microcapillary array such that each microcapillary includes a small number of different variant proteins, preferably each microcapillary contains only a single different variant protein. Importantly, the immobilized target molecule is combined to select the group of variant proteins such that at least some of the proteins in the group can associate with the immobilized target molecule with a specific affinity, allowing the association to be detected by measuring a signal from the reporter element. In some embodiments, the microcapillary screening method of the present invention allows a screening reaction and / or interaction (including binding interaction) between the variant protein and the target molecule to occur within minutes of adding the component to the microcapillary. In some embodiments, the reaction and / or interaction between the variant protein and the target molecule occurs and / or can be detected within about 1 minute to about 10 minutes. In some embodiments, the reaction and / or interaction between the variant protein and the target molecule occurs and / or is detectable within about 1 hour to about 6 hours. In some embodiments, the reaction and / or interaction between the variant protein and the target molecule occurs and / or is detectable within a period of time during which the cells within the microcapillary are alive and healthy. In some embodiments, the reaction and / or interaction between the variant protein and the target molecule occurs and / or is detectable within a period of time during which the cells within the microcapillary are alive. In some embodiments, the cells may grow after removal from the microcapillary and / or microlumen. In some embodiments, the cells are alive after removal from the microcapillary and / or microlumen. In some embodiments, the reaction and / or interaction between the variant protein and the target molecule occurs within the microcapillary.

[0039] As used herein, the term "protein" refers both to a full-length protein or polypeptide sequence and to a fragment thereof. Such fragments may contain segments that retain functional activities, such as binding activity. The terms "protein" and "polypeptide" are used interchangeably throughout the disclosure and comprise a chain of amino acids covalently linked by peptide bonds, wherein each amino acid in a polypeptide may be referred to as an "amino acid residue." The use of the terms "protein" or "polypeptide" should not be construed as limiting to polypeptides of any particular length, e.g., any particular number of amino acid residues. The subject protein may comprise a protein having non-peptide modifications (including glycosylation, acetylation, phosphorylation, sulfation, etc.) such as post-translational modifications, or other chemical modifications such as alkylation, acetylation, esterification, PEGylation, etc. Additional modifications, such as the inclusion of non-natural amino acids within the polypeptide sequence or the inclusion of non-peptide bonds between amino acid residues, should also be considered within the definition of the terms "protein" or "polypeptide."

[0040] Variant protein groups are preferably groups of proteins with minor variations, such as a group of proteins where each protein has a slightly different amino acid sequence. Therefore, screening assays can identify variant protein sequences with desired properties. Because screening can be performed at such a large scale on a microscopic scale, a large number of variant proteins can be determined in a relatively short time. In some embodiments, the screening process occurs over 4 to 6 hours. In some embodiments, the screening process occurs over 4, 5, or 6 hours. In some embodiments, the screening process requires between 1 and 3 seconds per microcapillary (i.e., lumen, microcapillary, microcavity, pore, and / or micropore). In some embodiments, the screening process requires approximately 1 second per microcapillary (i.e., lumen, microcapillary, microcavity, pore, and / or micropore). In some embodiments, the screening process requires approximately 2 seconds per microcapillary (i.e., lumen, microcapillary, microcavity, pore, and / or micropore). In some embodiments, the screening process requires approximately 3 seconds per microcapillary (i.e., lumen, microcapillary, microcavity, pore, and / or micropore).

[0041] In some embodiments, each microcapillary in the microcapillary array comprises 0 to 5 different variant proteins from the variant protein group. In specific embodiments, each microcapillary in the microcapillary array comprises 0 to 4, 0 to 3, 0 to 2, or even 0 to 1 different variant proteins from the variant protein group. It should be understood that the different variant proteins in the variant protein group have different molecular structures, whether the difference lies in their amino acid sequence or some other chemical modification of the protein.

[0042] It should be understood that each microcapillary will typically include many copies of the same variant protein, depending on the source and expression level of the particular variant protein (see below). In some embodiments, each microcapillary will include thousands, tens of thousands, hundreds of thousands, millions, billions, or even more molecules of the particular variant protein, depending on how the variant protein is delivered to the microcapillary or how it is expressed within the microcapillary.

[0043] Typically used in biological expression systems, such as in vitro (i.e., cell-free) expression systems or in vivo or cellular expression systems, to generate variant protein populations from gene libraries. Exemplary cellular expression systems include, for example, animal systems (e.g., mammalian systems); fungal systems (e.g., yeast systems); bacterial systems; insect systems; or plant systems. In a specific embodiment, the expression system is a mammalian system or a yeast system. Expression systems, whether cellular or cell-free, typically include a library of genetic material encoding variant protein populations. The advantage of cellular expression systems is that cells with the desired phenotype, such as cells expressing specific variant proteins of interest, like variant proteins capable of associating with immobilized target molecules with high affinity, can grow and proliferate, thereby facilitating and simplifying the identification and characterization of the proteins of interest expressed by the cells.

[0044] Gene libraries encoding large variant protein groups are well known in the field of bioengineering. Such libraries are commonly used in systems dependent on directed evolution processes to identify proteins with advantageous properties such as high affinity binding to target molecules, stability, high expression, or specific spectra (e.g., fluorescence) or enzymatic activity. These libraries typically contain gene fusions with sequences from a host expression system, such as protein fragments that guide subcellular localization, where the target fragment directs the expressed variant fusion protein group to a specific location on a cell or viral particle to achieve activity screening of the variant protein group. Large quantities of variant proteins (e.g., 10) can be generated using conventional bioengineering techniques well known in the art. 6 Variations, 10 8 Variations, 10 10 Variations, 10 12 (One or more variants). This library may contain any variant protein described herein, including antibodies, antibody fragments, single-chain variable fragments, or native protein ligands.

[0045] Therefore, in some embodiments, the variant protein is a soluble protein, such as a soluble protein secreted by a cellular expression system. Exemplary soluble variant proteins include antibodies and antibody fragments; alternative protein scaffolds such as disulfide-bonded peptide scaffolds; extracellular domains of cell surface receptor proteins; receptor ligands such as G protein-coupled receptor ligands; other peptide hormones, lectins, etc. Advantageously, in the method of the present invention, variant proteins screened for binding activity do not need to be covalently linked to the cells or viruses expressing them to identify the variant proteins after the screening assay, because the variant protein with the desired binding activity and the cell expressing the variant protein remain colocalized within the same microcapillary throughout the assay. The contents of the desired microcapillary are separated, and then the cell or viral clone responsible for expressing the desired variant protein is proliferated, thereby achieving the identification and characterization of the protein. Unlike screening assays that reveal the variant protein of interest by fusing the protein to molecules on the surface of cell or viral particles, the variant protein identified in the screening method of the present invention does not need to be altered in any way after identification. Therefore, the activity of variant proteins observed during screening is more likely to indicate the actual activity of those proteins in their subsequent applications.

[0046] However, in other embodiments, it may be desirable for the variant protein to be a membrane-associated protein, such as a protein that remains associated with the surface of a cell or viral particle in the expression system. Screening for cell-associated variant proteins may be desirable when the variant protein and its target molecules mediate interactions between two cells within a biological tissue. The ability to screen for cell-associated variant proteins may also be desirable when screening for interactions with conventionally “non-druggable” protein targets such as, for example, G protein-coupled receptors or ion channels.

[0047] In addition to variant proteins, each microcapillary in the microcapillary array of the screening method of the present invention also includes an immobilized target molecule. The immobilized target molecule acts as a potential binding partner for the variant proteins to be screened. Unlike a group of variant proteins in which each microcapillary ideally contains a slightly different sequence, the immobilized target molecule ideally has the same molecular structure in each microcapillary of the array.

[0048] In some embodiments, the target molecule is a target protein or peptide, a target nucleic acid, a target carbohydrate, a target lipid, or a combination of two or more of these target molecules. For example, in some embodiments, the target molecule may be a lipid-modified or glycosylated protein. In some embodiments, the target molecule is anchored to a surface. In more specific embodiments, the target molecule is anchored to the surface of a cell, such as a target cell, the surface of a bead, the surface of a microcapillary wall, or other suitable surface. In other more specific embodiments, the target molecule is a native protein, such as a native protein anchored to the surface of a cell. In still other more specific embodiments, the target molecule is anchored to a surface configured to settle in a microcapillary by gravity.

[0049] As previously mentioned, in the method of this disclosure, the variant protein associates with an immobilized target molecule within a microcapillary with a specific affinity. Importantly, for the variant protein of interest, this affinity should be strong enough that the association can be measured by a signal from a reporter element. This is typically achieved using a dissociation constant (Ki) well known to those skilled in the art. d The binding affinity is assessed, with a lower dissociation constant indicating higher affinity. In some embodiments, the association between the variant protein of interest and the immobilized target molecule exhibits a dissociation constant in the range of millimoles to micromoles. In specific embodiments, the association exhibits a dissociation constant in the range of micromoles to high nanomoles (i.e., 10⁻⁶). -6 M to 10 -8 In more specific embodiments, the association exhibits a dissociation constant ranging from low nanomolar to high picomolar (i.e., 10-1). -8 M to 10 -10 In even more specific embodiments, the dissociation constant exhibited by association is in the picomolar range (i.e., 10 M). -10 M to 10 -12 M) or even lower. In some embodiments, the first cell expresses and secretes a variant protein or polypeptide, and the second cell includes a target such that the first cell binds to the second cell. In some embodiments, the second cell expresses the target. In some embodiments, the second cell is labeled with the target. In some embodiments, the first cell binds to the second cell in a microcapillary. In some embodiments, the first cell binds to the second cell in a microcapillary and / or microcavity.

[0050] In some embodiments, the target molecule is a target protein or peptide, a target nucleic acid, a target carbohydrate, a target lipid, or a combination of two or more of these target molecules. For example, in some embodiments, the target molecule may be a lipid-modified or glycosylated protein. In some embodiments, the target molecule is anchored to a surface. In more specific embodiments, the target molecule is anchored to the surface of a cell, such as a target cell, the surface of a bead, the surface of a microcapillary wall, or other suitable surface. In other more specific embodiments, the target molecule is a native protein, such as a native protein anchored to the surface of a cell. In still other more specific embodiments, the target molecule is anchored to a surface configured to settle in a microcapillary by gravity. In some embodiments, one, two, three, or four or more target molecules are used to identify variants that bind to one, two, three, or four or more target molecules. In some embodiments, the target molecule is individually contained in separate, different microcapillaries. In some embodiments, the target molecule is individually contained in separate, different microcapillaries within a single array. In some embodiments, the target molecule is individually contained in separate, different microcapillaries within one or more arrays. In some embodiments, the target molecule is contained together in a single microcapillary. In some embodiments, the target molecules are contained together in a single microcapillary within a single array. In some embodiments, the variant binds to one, two, three, four or more target molecules that are derivatives or variants of the original target molecule, including chemically modified, post-translational modified, or sequence identity variants (including variants having, for example, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the original nucleic acid or amino acid target sequence).

[0051] In addition to the variant protein and the immobilized target molecule, each microcapillary in the microcapillary array of the screening method of the present invention also includes a reporter element. Importantly, the reporter element provides a measurable signal indicating the association between the variant protein and the immobilized target molecule and is therefore used to identify microcapillaries containing the variant protein of interest.

[0052] In some embodiments, the reporter element is a labeled antibody or other molecule capable of binding to each variant protein in the variant protein population. More specifically, the reporter element is a fluorescently labeled antibody or other binding molecule.

[0053] In some embodiments, the labeled antibody is a labeled first antibody or a labeled second antibody. For the purposes of this disclosure, a first antibody is generally considered to be an antibody that binds directly to the antigen of interest, while a second antibody is generally considered to be an antibody that binds to a constant region on a first antibody for the purpose of labeling the first antibody. Therefore, second antibodies are often labeled with fluorophores or other detectable tags or with enzymes capable of generating detectable signals. They are generally specific to first antibodies from different species. For example, as those skilled in the art will understand, second antibodies against mice, chickens, rabbits, or virtually any first antibody other than those from that animal species can be generated using goats or other animal species. In specific embodiments, the labeled antibody is a fluorescent antibody or an enzyme-linked antibody. In some embodiments, the fluorophore may include, but is not limited to, AlexaFluor 3, AlexaFluor 5, AlexaFluor 350, AlexaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500, AlexaFluor 514, AlexaFluor 532, AlexaFluor 546, AlexaFluor 555, AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, and AlexaFluor 750 (Molecular Probes AlexaFluor dyes, available from Life Technologies, Inc. (USA)). In some embodiments, the fluorophore may comprise, but is not limited to, Cy dyes, including Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7 (available from GE Life Sciences or Lumiprobes). In some embodiments, the fluorophore may comprise, but is not limited to, DyLight 350, DyLight 405, DyLight 488, DyLight 550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 750, and DyLight 800 (available from Thermo Scientific, USA).In some embodiments, the fluorophore may include, but is not limited to, FluoProbes 390, FluoProbes 488, FluoProbes 532, FluoProbes 547H, FluoProbes 594, FluoProbes 647H, FluoProbes 682, FluoProbes 752 and FluoProbes 782, AMCA, DEAC (7-diethylaminocoumarin-3-carboxylic acid); 7-hydroxy-4-methylcoumarin-3; 7-hydroxycoumarin-3; MCA (7-methoxycoumarin-4-acetic acid); 7-methoxycoumarin-3; AMF (4'-(aminomethyl)fluorescein); 5-DTAF (5-(4,6-dichlorotriazinyl)aminofluorescein); 6-DTAF (6-(4,6-dichlorotriazinyl)aminofluorescein); 6- FAM (6-carboxyfluorescein); 5(6)-FAM cadaverine; 5-FAM cadaverine; 5(6)-FAM ethylenediamine; 5-FAM ethylenediamine; 5-FITC (FITC isomer I; fluorescein-5-isothiocyanate); 5-FITC cadaverine; fluorescein-5-maleimide; 5-IAF (5-iodoacetamidofluorescein); 6-JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein); 5-CR 110 (5-Carboxyrhodamine 110); 6-CR 110 (6-Carboxyrhodamine 110); 5-CR6G (5-Carboxyrhodamine 6G); 6-CR6G (6-Carboxyrhodamine 6G); 5(6)-Carboxyrhodamine 6G cadaverine; 5(6)-Carboxyrhodamine 6G ethylenediamine; 5-ROX (5-Carboxy-X-Rhodamine); 6-ROX (6-Carboxy-X-Rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6-TAMRA (6-Carboxytetramethylrhodamine); 5-TAMRA cadaverine; 6-TAMRA cadaverine; 5-TAMRA ethylenediamine; 6-TAMRA ethylenediamine; 5-TMR C6 maleimide; 6-TMR C6 maleimide; TR C2 maleimide; TR cadaverine; 5-TRITC; G isomer (tetramethylrhodamine-5-isothiocyanate); 6-TRITC; R isomer (tetramethylrhodamine-6-isothiocyanate); dansyl cadaverine (5-dimethylaminonaphthalene-l-(N-(5-aminopentyl))sulfonamide); EDANS C2 maleimide; fluorescent amine; NBD; and pyrrole methylene and its derivatives. In some embodiments, the reporter element used may be a donkey anti-goat IgG secondary antibody labeled with AlexaFluor 633.

[0054] In some method implementations, for example in Figure 1A-1CIn the screening method shown, the variant protein mediates the association between the reporter element and the target molecule (in this example, the target molecule on the surface of the target cell). Figure 1B As shown, the variant protein (here named "secreted protein") has sufficient affinity for its target molecule on the target cell to associate with the target cell under microcapillary solution conditions. A reporter element (here named "fluorescent detection antibody") binds to the variant protein, ideally at an epitope that does not affect the variant protein's affinity for the target molecule, such as... Figure 1C As shown.

[0055] As will be understood by those skilled in the art, when soluble reporter elements such as fluorescent antibodies are used in the screening method of the present invention, the signal emitted by any excess reporter elements that remain free in the solution within the microcapillary (i.e., not bound to the variant protein or bound to a variant protein that is not bound to the target molecule) should not be so high as to overwhelm the signal of the reporter elements associated with the target molecule by the variant protein (see, for example, Figure 1C (The unassociated fluorescent detection antibody is shown in the image). However, this background signal can be minimized by limiting the concentration of the labeled antibody or other reporter element in the microcapillary solution. Additionally, when measuring signals from screening methods using fluorescence microscopy, configuring the microscope to image the location of the encircling exposure target molecule with a relatively narrow depth of field (e.g., the bottom of the microcapillary when the target cell has settled there by gravity) can minimize background signal from reporter elements that are not associated with the target molecule.

[0056] In other embodiments, the reporter element is an intracellular reporter element that generates a detectable signal associated with binding events, such as the association of a variant protein with an immobilized target molecule (e.g., a receptor or other target molecules on the surface of a cell). In these embodiments, the reporter element may comprise an entire cellular pathway, such as an intracellular signaling pathway. This pathway should include, or be designed to include, a detectable signal as a downstream readout of the pathway. In contrast, a detectable signal bound to the outer surface of a target cell... Figure 1A-1C The measurements shown in these embodiments indicate that the detectable signal will typically be generated inside the target cell.

[0057] Many intracellular signaling pathways have been developed for use in high-throughput screening assays, particularly in drug discovery screening, and these pathways can be adapted for use in the assays of the present invention. See, for example, Michelini et al. (2010), *Analytical and Bioanalytical Chemistry* 398:227-38. Specifically, any cellular assay in which binding events to target molecules on the surface of a cell result in the generation of a measurable signal, particularly a fluorescent signal, can be used as a reporter element in the assays of the present invention. Preferably, cells can be engineered to express the target molecule of interest on their surface such that binding of a specific variant protein to the target molecule and subsequent activation of intracellular signaling pathways results in a detectable signal from the reporter element, thereby enabling the identification of a microcapillary as a positive hit. Expression of green fluorescent protein (GFP) or any of its variants is commonly used as a readout in such cellular assays and can serve as the reporter element endpoint in the methods of the present invention. The reporter element may also comprise RFP (red fluorescent protein) and YFP (yellow fluorescent protein) and their variants. Alternatively, signal transduction readings can be provided by luciferase or other relevant enzymes that generate bioluminescent signals, as is well known to those skilled in the art. See, for example, Kelkar et al. (2012), *New Insights in Pharmacology* (Curr. Opin. Pharmacol.) 12:592-600. Other known enzyme reporter genes from bacterial and plant systems include β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase (GUS), etc., which can be used in the screening assays of this invention with suitable colorogenic substrates. Transcriptional reporter genes using firefly luciferase and GFP have been widely used to study the function and regulation of transcription factors. These transcriptional reporter genes can also be used in the screening assays of this invention. Exemplary intracellular signal transduction systems are commercially available, such as Cignal from Qiagen. TM Reporter gene assay kits (see, for example, www.sabiosciences.com / reporterassays.php) are available together with luciferase or GFP readings. Such systems can be suitably redesigned for use in the screening methods of this invention.

[0058] It should be understood that variant protein expression systems, particularly variant protein expression systems that are cellular expression systems, can be combined with immobilized target molecules and reporter elements (or suitable components such as cellular components responsible for generating the immobilized target molecules and / or reporter elements) before the expression of the variant protein and / or before the assay mixture is delivered into an array of microcapillaries. Compared to prior art microcapillary screening systems, this approach advantageously allows for timing flexibility and control of interactions between components, where all components for screening assays are typically mixed in a static form and loaded into the microcapillaries. In contrast, the method of the present invention enables some or all of the components to be bound for assay to be generated in situ within the microcapillaries by allowing the growth of cellular components, the expression of genetic components, or both.

[0059] It should also be understood that the concentrations of each component in the screening assay within the microcapillary, including the concentrations of the variant protein, the immobilized target molecule, and the reporter element, can be adjusted as needed during the assay to obtain optimal results. Specifically, it may be desirable to adjust the concentrations of the variant protein and / or the immobilized target molecule to achieve a desired level of association between these components. The level of association will also depend on the specific affinity between these components, where, at a given component concentration, higher affinity results in a higher level of association, and lower affinity results in a lower level of association at a given concentration. Similarly, the concentration of the reporter element can be adjusted to achieve an optimal level of signal output, as will be understood by those skilled in the art. In some embodiments, the reporter element employed comprises a second antibody, comprising a commercially available antibody. In some embodiments, the dilution range is 1:200–1:2000. In some embodiments, the dilution range is 1:300–1:2000. In some embodiments, the dilution range is 1:300–1:1500. In some embodiments, the dilution range is 1:400–1:1500. In some embodiments, the dilution range is 1:500-1:1500. In some embodiments, the dilution range is 1:200-1:1000. In some embodiments, the dilution range is 1:500-1:1000. In some embodiments, the dilution range is 1:1000-1:2000. In some embodiments, the dilution range is 1:1500-1:2000. In some embodiments, the dilution is 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:1500, or 1:2000. In some embodiments, the fluorophore may include, but is not limited to, AlexaFluor 3, AlexaFluor 5, AlexaFluor 350, AlexaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500, AlexaFluor 514, AlexaFluor 532, AlexaFluor 546, AlexaFluor 555, AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, and AlexaFluor 750 (Molecular Probes AlexaFluor dyes, available from Life Technologies, Inc. (USA)).In some embodiments, the fluorophore may comprise, but is not limited to, Cy dyes, including Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7 (available from GE LifeSciences or Lumiprobes). In some embodiments, the fluorophore may comprise, but is not limited to, DyLight 350, DyLight 405, DyLight 488, DyLight 550, DyLight 594, DyLight 633, DyLight 650, DyLight 680, DyLight 750, and DyLight 800 (available from Thermo Scientific, USA).In some embodiments, the fluorophore may include, but is not limited to, FluoProbes 390, FluoProbes 488, FluoProbes 532, FluoProbes 547H, FluoProbes 594, FluoProbes 647H, FluoProbes 682, FluoProbes 752 and FluoProbes 782, AMCA, DEAC (7-diethylaminocoumarin-3-carboxylic acid); 7-hydroxy-4-methylcoumarin-3; 7-hydroxycoumarin-3; MCA (7-methoxycoumarin-4-acetic acid); 7-methoxycoumarin-3; AMF (4'-(aminomethyl)fluorescein); 5-DTAF (5-(4,6-dichlorotriazinyl)aminofluorescein); 6-DTAF (6-(4,6-dichlorotriazinyl)aminofluorescein); 6- FAM (6-carboxyfluorescein); 5(6)-FAM cadaverine; 5-FAM cadaverine; 5(6)-FAM ethylenediamine; 5-FAM ethylenediamine; 5-FITC (FITC isomer I; fluorescein-5-isothiocyanate); 5-FITC cadaverine; fluorescein-5-maleimide; 5-IAF (5-iodoacetamidofluorescein); 6-JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein); 5-CR 110 (5-Carboxyrhodamine 110); 6-CR 110 (6-Carboxyrhodamine 110); 5-CR6G (5-Carboxyrhodamine 6G); 6-CR6G (6-Carboxyrhodamine 6G); 5(6)-Carboxyrhodamine 6G cadaverine; 5(6)-Carboxyrhodamine 6G ethylenediamine; 5-ROX (5-Carboxy-X-Rhodamine); 6-ROX (6-Carboxy-X-Rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6-TAMRA (6-Carboxytetramethylrhodamine); 5-TAMRA cadaverine; 6-TAMRA cadaverine; 5-TAMRA ethylenediamine; 6-TAMRA ethylenediamine; 5-TMR C6 maleimide; 6-TMR C6 maleimide; TR C2 maleimide; TR cadaverine; 5-TRITC; G isomer (tetramethylrhodamine-5-isothiocyanate); 6-TRITC; R isomer (tetramethylrhodamine-6-isothiocyanate); dansyl cadaverine (5-dimethylaminonaphthalene-l-(N-(5-aminopentyl))sulfonamide); EDANS C2 maleimide; fluorescent amine; NBD; and pyrrole methylene and its derivatives. In some embodiments, the reporter element used may be a donkey anti-goat IgG secondary antibody labeled with AlexaFluor633.

[0060] In some embodiments, each microcapillary in the microcapillary array of the screening method of the present invention further comprises one or more agents for enhancing the viability of the cell expression system. Specifically, one or more agents are included to prevent cell damage during a step of separating the contents of the microcapillary of interest, for example, by means of a laser pulse (see below). In preferred embodiments, the agents are methylcellulose (e.g., at 0.001 to 10 wt%); dextran (e.g., at 0.5 to 10 wt%); Pranic F-68 (e.g., at 0.01 to 10 wt%); polyethylene glycol (“PEG”) (e.g., at 0.01 to 10 wt%); polyvinyl alcohol (“PVA”) (e.g., at 0.01 to 10 wt%), etc. Alternatively or additionally, each microcapillary in the microcapillary array of the screening method of the present invention may further comprise a growth additive, such as, for example, 50% conditioned growth medium, 25% standard growth medium, or 25% serum. In some embodiments, the conditioned growth medium is conditioned for 24 hours. In some embodiments, the added reagent is insulin, transferrin, ethanolamine, selenium, insulin-like growth factor, or a combination of these reagents or any of the reagents listed above.

[0061] The screening method of this disclosure preferably includes the additional step of measuring a signal from at least one reporter element indicating the association of at least one variant protein with at least one immobilized target molecule to identify at least one microcapillary of interest. In some embodiments, the measured signal is a fluorescence signal, absorbance signal, bright-field signal, dark-field signal, phase-contrast signal, etc. Therefore, the measurement step can be performed by a suitable detector device, such as a device capable of detecting electromagnetic radiation or any other suitable signal. In a specific embodiment, the measurement step is performed by a microscope, such as a fluorescence microscope or any other microscope configured to detect the signals mentioned above.

[0062] It should be understood that, in preferred embodiments, the microcapillaries used in the screening method of the present invention do not include particles capable of suppressing the transmission of electromagnetic radiation. In other words, the microcapillaries are preferably completely transparent to electromagnetic radiation incident on the microcapillary array, especially electromagnetic radiation incident on the microcapillary array along the longitudinal axis of the microcapillary. In other preferred embodiments, the microcapillaries of the screening method of the present invention do not include magnetic particles or beads. In still other preferred embodiments, the microcapillaries of the screening method of the present invention do not include particles, magnetic particles, or magnetic beads capable of suppressing the transmission of electromagnetic radiation.

[0063] In other preferred embodiments, the microcapillaries used in the screening method of the present invention do not include electromagnetic radiation absorbing materials. However, it should be understood that, for the purposes of this aspect of the invention, the components of the reporting element responsible for generating measurable signals in the screening method, such as fluorophores on fluorescent antibodies, should not be considered as electromagnetic radiation absorbing materials.

[0064] In some embodiments, the screening method of the present invention further includes the step of separating the contents of the microcapillary of interest. In a specific embodiment, the contents of the microcapillary of interest are separated by applying pulses to the microcapillary of interest with a laser. In some embodiments, the laser is a diode laser. In some embodiments, the laser is a nanosecond pulsed laser. In some embodiments, the laser is a picosecond pulsed laser. More specifically, the laser may be a diode laser or a diode-pumped Q-switched Nd:YLF laser. In some embodiments, the laser may be directed to the water-glass interface between the microcapillary wall and the sample contained in the microcapillary. Without intending to be bound by theory, it is believed that exciting a UV laser at this interface can disrupt the meniscus / water surface tension that normally holds the sample in the microcapillary, thereby allowing the sample to fall from the array by gravity. In other embodiments, the contents of the microcapillary of interest are separated by laser-triggered vapor force expansion. In some embodiments, the contents of the microcapillary of interest are separated by disrupting the glass of the microcapillary itself.

[0065] Systems for screening and sample recovery

[0066] According to another aspect of the invention, a system for screening variant protein populations is provided, the system comprising:

[0067] An array of multiple microcapillaries, each microcapillary comprising a variant protein, an immobilized target molecule, and a reporter element, wherein the variant protein associates with the immobilized target molecule with a specific affinity. The components of these screening devices have been described in detail above.

[0068] In some embodiments, the screening system further includes a light source and a detector. In some embodiments, the light source is a Nikon Intensilight Illuminator. In some embodiments, the photodetector is an imaging camera such as a charge-coupled device (CCD) or complementary metal-oxide-semiconductor (CMOS) imaging sensor. In some embodiments, the photodetector is a Hamamatsu ORCA-Flash4.0 CMOS camera. The light source and detector are selected according to the specific reporting element used in the screening system. For example, in the case where the reporting element generates a fluorescence signal, the light source provides excitation light of an appropriate wavelength to excite the fluorescent probe. Similarly, the detector is selected to be sensitive to the wavelength of the light emitted by the fluorescent probe. The light source and detector may be components of a microscope, such as a fluorescence microscope, or they may be separate devices, as will be understood by those skilled in the art. Preferably, the fluorescence microscope is an inverted fluorescence microscope.

[0069] exist Figures 6A-6E The illustration shows an exemplary microscope used for screening variant protein groups and recovering samples of interest from the screening according to the method of the present invention. Figure 6A A perspective view viewed from above the microscope is shown, illustrating the screening array stage 12, the recovery array 14, the recovery array holder 16, the first recovery array stage 18, and the second recovery array stage 20. Figure 6B A front view of the device is shown. Figure 6C The view shown is from the right. Figure 6D The image provides an enlarged view of the right side of the device, which shows in detail the relationship between the screening array level, the recycling array level, and the recycling array in this system. Figure 6E Exploded views of the various components of this particular multi-stage sample recovery system are provided.

[0070] In some respects, this disclosure therefore provides a multi-stage sample recovery system comprising:

[0071] A selection array level, wherein the selection array level is controllable in two dimensions relative to the microscope objective and is configured to be reversibly correlated with the selection array; and

[0072] A first recovery array stage, wherein the first recovery array stage is controllable in at least one dimension relative to the microscope objective and is configured to be reversibly associated with the recovery array;

[0073] The screening array stage and the first recovery array stage are independently controllable. In some embodiments, the screening array stage and the first recovery stage are physically separated from each other. In some embodiments, the screening array stage, which is controllable in two dimensions, is controllable relative to the microscope objective in the horizontal and / or vertical dimensions. In some embodiments, the first recovery array stage, which is controllable in at least one dimension, is controllable relative to the microscope objective in the horizontal and / or vertical dimensions. In some embodiments, the first recovery array stage, which is controllable in at least one dimension, is controllable relative to the microscope objective in the horizontal dimension. In some embodiments, the first recovery array stage, which is controllable in at least one dimension, is controllable relative to the microscope objective in the vertical dimension. In some embodiments, the screening array stage may be positioned closer to the first recovery stage in the vertical dimension. In some embodiments, the screening array stage may be positioned further away from the recovery stage in the vertical dimension. In some embodiments, the screening array stage and the first recovery stage may be moved and / or repositioned during the sample recovery process. In some embodiments, the screening array stage may be moved and / or repositioned during the sample recovery process. In some embodiments, the first recovery stage may be moved and / or repositioned during the sample recovery process. In some embodiments, the screening array stage and the first recovery stage may be moved and / or repositioned in both the horizontal and vertical dimensions during the sample recovery process. In some embodiments, the screening array stage and the first recovery stage may be moved and / or repositioned in the horizontal dimension during the sample recovery process. In some embodiments, the screening array stage and the first recovery stage may be moved and / or repositioned in the vertical dimension during the sample recovery process. In some embodiments, during the sample recovery process, the sample moves from the screening array stage and enters the first recovery stage. In some embodiments, during the sample recovery process, the sample is collected from the screening array stage and enters the first recovery stage. In some embodiments, during the sample recovery process, the sample moves from the screening array stage and enters the first recovery stage, and this movement is facilitated by the movement and / or repositioning of the screening array stage and / or the first recovery stage. In some embodiments, during the sample recovery process, the sample moves from the screening array stage and enters the first recovery stage, and this movement is facilitated by the movement and / or repositioning of the screening array stage. In some embodiments, during the sample recovery process, the sample moves from the screening array stage and enters the first recovery stage, and this movement is facilitated by the movement and / or repositioning of the first recovery stage. In some embodiments, during the sample recovery process, samples are collected from the screening array stage and enter a first recovery stage, and this collection is facilitated by the movement and / or repositioning of the screening array stage and / or the first recovery stage.

[0074] In some embodiments, during the sample recovery process, samples are collected from a screening array stage and enter a first recovery stage, and this collection is facilitated by movement and / or repositioning of the screening array stage. In some embodiments, during the sample recovery process, samples are collected from a screening array stage and enter a first recovery stage, and this collection is facilitated by movement and / or repositioning of the first recovery stage. In some embodiments, the screening array stage may be moved and / or repositioned in a horizontal and / or vertical dimension during the sample recovery process. In some embodiments, the screening array stage may be moved and / or repositioned in a horizontal dimension during the sample recovery process. In some embodiments, the screening array stage may be moved and / or repositioned in a vertical dimension during the sample recovery process. In some embodiments, the first recovery stage may be moved and / or repositioned in a horizontal and / or vertical dimension during the sample recovery process. In some embodiments, the first recovery stage may be moved and / or repositioned in a horizontal dimension during the sample recovery process. In some embodiments, the first recovery stage may be moved and / or repositioned in a vertical dimension during the sample recovery process. In some embodiments, the laser is in a fixed position. In some embodiments, the laser is in a fixed position relative to the microscope objective. In some embodiments, the laser passes through a screening array stage before passing through a first recovery stage. In some embodiments, the laser passes through the screening array stage before passing through the first recovery stage, thereby moving the sample from the screening array stage and into the first recovery stage. In some embodiments, the laser first passes through (e.g., activates and excites) the screening array stage, thereby moving the sample from the screening array stage and into the first recovery stage. In some embodiments, the laser moves the sample from the screening array stage and into the first recovery stage. In some embodiments, the laser passes through a capillary (i.e., cavity, microcapillary, microcavity, pore, and / or micropore) in the screening array stage and enters a capillary (i.e., cavity, microcapillary, microcavity, pore, and / or micropore) in the first recovery stage. In some embodiments, the laser passes through a capillary (i.e., cavity, microcapillary, microcavity, pore, and / or micropore) in the screening array stage and enters a capillary (i.e., cavity, microcapillary, microcavity, pore, and / or micropore) in the first recovery stage, thereby moving the sample from one capillary (i.e., cavity, microcapillary, microcavity, pore, and / or micropore) in the screening array stage and into one capillary (i.e., cavity, microcapillary, microcavity, pore, and / or micropore) in the first recovery stage. In some embodiments, the laser phase remains in a fixed position (i.e., does not move relative to the microscope objective). In some embodiments, while the laser remains in a fixed position (i.e., does not move relative to the microscope objective), the screening array stage and the first recovery stage can be moved and / or repositioned in the horizontal and / or vertical dimensions during the sample recovery process.In some embodiments, while the laser remains in a fixed position (i.e., does not move relative to the microscope objective), the screening array stage and the first recovery stage can be moved and / or repositioned in the horizontal dimension during the sample recovery process. In some embodiments, while the laser remains in a fixed position (i.e., does not move relative to the microscope objective), the screening array stage and the first recovery stage can be moved and / or repositioned in the vertical dimension during the sample recovery process. In some embodiments, while the laser remains in a fixed position (i.e., does not move relative to the microscope objective), the screening array stage can be moved and / or repositioned in the horizontal and / or vertical dimensions during the sample recovery process. In some embodiments, while the laser remains in a fixed position (i.e., does not move relative to the microscope objective), the screening array stage can be moved and / or repositioned in the horizontal dimension during the sample recovery process. In some embodiments, while the laser remains in a fixed position (i.e., does not move relative to the microscope objective), the screening array stage can be moved and / or repositioned in the vertical dimension during the sample recovery process. In some embodiments, while the laser remains in a fixed position (i.e., does not move relative to the microscope objective), the first recovery stage can be moved and / or repositioned in the horizontal and / or vertical dimensions during the sample recovery process. In some embodiments, while the laser remains in a fixed position (i.e., does not move relative to the microscope objective), the first recovery stage can be moved and / or repositioned in the horizontal dimension during the sample recovery process. In some embodiments, while the laser remains in a fixed position (i.e., does not move relative to the microscope objective), the first recovery stage can be moved and / or repositioned in the vertical dimension during the sample recovery process. In some embodiments, the vertical distance between the two stages is approximately 20 mm. In some embodiments, the vertical distance between the screening stage and the recovery stage is approximately 20 mm. In some embodiments, there is a gap between the screening array positioned (e.g., recessed) in the screening stage and the recovery slide positioned (e.g., recessed) in the recovery stage. In some embodiments, the gap between the screening array recessed and / or positioned in the screening stage and the recovery slide recessed and / or positioned in the recovery stage is approximately 1 mm, approximately 2 mm, or approximately 3 mm. In some embodiments, the gap between the screening array recessed and / or positioned in the screening stage and the recovery slide recessed and / or positioned in the recovery stage is approximately 1 mm. In some embodiments, the gap between the screening array recessed and / or positioned in the screening stage and the recovery slide recessed and / or positioned in the recovery stage is approximately 2 mm. In some embodiments, the gap between the screening array recessed and / or positioned in the screening stage and the recovery slide recessed and / or positioned in the recovery stage is approximately 3 mm. In some embodiments, the recovery slide is referred to as the first recovery array.In some embodiments, the gap between the screening array recessed and / or positioned in the screening stage and the first recovery array recessed and / or positioned in the recovery stage is about 1 mm, about 2 mm, or about 3 mm. In some embodiments, the gap between the screening array recessed and / or positioned in the screening stage and the first recovery array recessed and / or positioned in the recovery stage is about 1 mm. In some embodiments, the gap between the screening array recessed and / or positioned in the screening stage and the first recovery array recessed and / or positioned in the recovery stage is about 2 mm. In some embodiments, the gap between the screening array recessed and / or positioned in the screening stage and the first recovery array recessed and / or positioned in the recovery stage is about 3 mm. In some embodiments, the screening array stage and the recovery stage are positioned such that the microscope objective can image both the screening array and the recovery slide. In some embodiments, the screening array stage and the first recovery array stage are positioned such that the microscope objective can image both the screening array and the first recovery array. In some embodiments, the screening array is within the working distance of the objective. In some embodiments, the first recovery array is within the working distance of the objective. In some embodiments, the screening array and the first recovery array are within the working distance of the objective. In some embodiments, the screening array stage is within the working distance of the objective lens. In some embodiments, the first recovery array stage is within the working distance of the objective lens. In some embodiments, the screening array stage and the first recovery array stage are within the working distance of the objective lens. The working distance is generally the distance at which a microscope objective lens can image (i.e., the working distance of the objective lens is the gap or distance between the front lens of the objective lens and the sample). In some embodiments, the working distance of the microscope objective lens is about 1 mm to about 30 mm. In some embodiments, the working distance of the microscope objective lens is about 5 mm to about 25 mm. In some embodiments, the working distance of the microscope objective lens is about 10 mm to about 25 mm. In some embodiments, the working distance of the microscope objective lens is about 10 mm to about 20 mm. In some embodiments, the travel distance of the microscope used with the method of the present invention is about 1 mm to about 30 mm. In some embodiments, the travel distance of the microscope used with the method of the present invention is about 5 mm to about 25 mm. The travel distance is generally the distance that the microscope's Z-axis can travel. In some embodiments, the travel distance of the microscope used with the method of the present invention is about 10 mm to about 25 mm. In some embodiments, the microscope used in conjunction with the method of the present invention travels at a distance of about 10 mm to about 20 mm. In some embodiments, the recovery array is a first recovery array. In some embodiments, the recovery array is a second recovery array. In some embodiments, the microcapillary array is within the working distance. In some embodiments, the microcapillary array is within the working distance of the projection lens or objective lens, such that the microcapillary array can be focused. In some embodiments, the microscope system is capable of focusing both the microcapillary array and the recovery array and / or the recovery slide.In some embodiments, both the screening array and the recovery array and / or the recovery slide are within the objective lens's travel distance. In some embodiments, the screening array is within the objective lens's travel distance. In some embodiments, the recovery array and / or the recovery slide are within the objective lens's travel distance. In some embodiments, both the screening array and the recovery array and / or the recovery slide are within the objective lens's travel distance, such that the screening array and the recovery array and / or the recovery slide can be focused. See the accompanying drawings, provided as exemplary embodiments, which are included. Figures 6A-6E As discussed in the next paragraph and throughout the application.

[0075] As mentioned above, in Figures 6A-6E Various views of an exemplary sample recovery system are provided. Specifically, Figure 6E The relative positioning of the screening array stage 12, the recovery array 14, the recovery array holder 16, the first recovery array stage 18, the second recovery array stage 20, and the microscope objective 22 is shown. (The last sentence appears to be incomplete and possibly refers to a diagram or diagram.) Figures 6B-6D The three angles shown as "laser beam path" and "imaging path" illustrate the optical paths of the extraction beam (in this case, the laser beam) and the screening array image. The screening array stage is preferably configured as an array to accommodate miniature sample containers, specifically within apertures that allow the beam to transmit through the associated array. Figure 7A This level is shown in more detail in the text. Figure 7B An exemplary recycling array level is shown in the figure. At least one recycling array level is preferably connected to a recycling array holder, such as... Figure 6E As shown, this facilitates reversible association between the recovery array and the recovery array stage. Reversible association refers to the ability of the recovery array to be associated with and deassociated with the recovery array stage (e.g., the first recovery stage) before, during, or after the sample recovery process. In some embodiments, reversibility indicates that the recovery array can be placed in and / or removed from the system, in some cases more than once. In some embodiments, the recovery array is reversibly associated with the recovery array stage via spring tension, gravity, magnetism, friction, screws / fasteners, and / or Velcro.

[0076] In a preferred embodiment, the multi-stage sample recovery system further includes a screening array reversibly associated with the screening array level. Reversible association refers to the ability of the screening array to be associated with and deassociated with the screening array level before, during, or after the sample recovery process. In some embodiments, reversibility indicates that the screening array can be placed in and / or removed from the system, in some cases more than once. In some embodiments, the screening array is reversibly associated with the screening array level by spring tension, gravity, magnetism, friction, screws / fasteners, and / or Velcro. As described in more detail above, such a screening array typically comprises multiple microsample containers, preferably multiple microcapillaries, but as those skilled in the art will understand, other screening arrays may be suitably used in the system of the present invention.

[0077] In other preferred embodiments, the multi-stage sample recovery system of the present invention further includes a recovery array reversibly associated with a first recovery array stage. More specifically, the recovery array includes one or more recovery containers. In some embodiments, such as... Figure 6A and 6E The recovery containers shown in recovery array 14 can be configured to prevent cell damage and / or promote cell growth. For example, each recovery container within the recovery array may include one or more reagents for preventing cell damage. In some embodiments, the reagents are methylcellulose (e.g., at 0.001 to 10 wt%); dextran (e.g., at 0.5 to 10 wt%); Pranic F-68 (e.g., at 0.01 to 10 wt%); polyethylene glycol (“PEG”) (e.g., at 0.01 to 10 wt%); polyvinyl alcohol (“PVA”) (e.g., at 0.01 to 10 wt%), etc. Alternatively or additionally, each recovery container may include a growth additive, such as, for example, 50% conditioned growth medium, 25% standard growth medium, 25% serum, or another suitable growth additive. See also U.S. Patent Applications Nos. 62 / 433,210 and 15 / 376,588, both filed December 12, 2016. In some embodiments, the conditioned growth medium is conditioned for 24 hours. In some embodiments, the added reagent is insulin, transferrin, ethanolamine, selenium, insulin-like growth factor, or a combination of these reagents or any of the reagents listed above. Those skilled in the art of cell culture fully understand the configuration of the recovery container used to promote cell growth.

[0078] In some embodiments, the recovery container may be configured to perform amplification reactions such as polymerase chain reaction or reverse transcription polymerase chain reaction, or sequencing reactions such as DNA sequencing reactions. Those skilled in the art will fully understand the configuration of the recovery container used to identify or characterize samples recovered from a screening array using the sample recovery system of the present invention for amplification reactions, sequencing reactions, or any other such analytical reactions.

[0079] In a preferred embodiment, the multi-stage sample recovery system includes both a screening array reversibly associated with a screening array level and a recovery array reversibly associated with a first recovery array level. More specifically, the screening array includes a plurality of miniature sample containers, and the recovery array includes a plurality of recovery containers.

[0080] As previously mentioned, the multi-stage sample recovery system of the present invention typically includes a light source and a photodetector for identifying samples of interest within a screening array. In some cases, such as in monitoring bioluminescence signals, a separate light source may not be necessary, and the system may consist only of a photodetector. In either case, the photodetector is typically configured to optically couple the screening array to the detector through apertures in the screening array stage to monitor optical signals emitted from samples within the screening array. As described above, observing the optical signals from the reporting element within the sample containers of the screening array enables the identification of specific sample containers holding the samples of interest, and the contents of those sample containers can then be recovered by pulses from an extraction beam generator. The photodetector, such as an imaging camera like a charge-coupled device (CCD) or a complementary metal-oxide-semiconductor (CMOS) imaging sensor, is ideally capable of imaging a large number of sample containers from the screening array within a single field of view. In some embodiments, the photodetector is a charge-coupled device (CCD). In some embodiments, the photodetector is a complementary metal-oxide-semiconductor (CMOS) imaging sensor. In some embodiments, the photodetector is a photodiode. When fluorescent markers are used in the reporting element, the photodetector is typically selected based on its sensitivity in the visible range of the electromagnetic spectrum. Fluorescence emission from the screening array is typically guided to the photodetector via the microscope objective and the system's imaging path. As those skilled in the art will understand, commercial microscopes such as the Nikon Eclipse series inverted microscopes can be suitably adapted to the system of this invention.

[0081] In some embodiments, the multi-stage sample recovery system further includes an extraction beam generator optically coupled to a micro-sample container within the screening array through holes in the screening array stage. More specifically, the extraction beam can be a laser beam, such as a beam emitted by a diode laser, a diode-pumped Q-switched laser like a diode-pumped Q-switched Nd:YLF laser, or another suitable laser device. In some embodiments, the laser is a diode laser. In some embodiments, the laser is a nanosecond pulsed laser. In some embodiments, the laser is a picosecond pulsed laser. In cases where the system includes a microcapillary array, the extraction beam can be directed to the water-glass interface between the microcapillary wall and the sample contained within the microcapillary. The use of lasers to separate the contents of specific microcapillaries identified by fluorescence imaging within a microcapillary array has been previously described. See, for example, Chen et al. (2016), Nature Chem. Biology 12:76–81; DOI: 10.1038 / NCHEMBIO.1978 and U.S. Patent Application Publication No. 2016 / 0244749A1.

[0082] In a preferred embodiment, the extraction beam is guided from below the target microsample container. However, it should also be understood that, if desired, the extraction beam may alternatively be guided from above the target microsample container.

[0083] In a specific embodiment, the system further includes a second recovery array stage. In a more specific embodiment, the second recovery array stage is positioned orthogonal to the first recovery array stage. According to these embodiments, samples can be automatically recovered from a screening array into a recovery array having recovery containers arranged in an ordered grid, particularly a grid with x rows and y columns, where x and y can independently be 3, 10, 30, 100, or even more.

[0084] In some embodiments, the screening array stage and one or more recycling array stages may be controlled by one or more electric motors, as will be understood by those skilled in the art.

[0085] In some embodiments, the screening and retrieval arrays of the system of the present invention are configured such that at least one microsample container and at least one retrieval container are positioned within the working distance of the microscope objective. In some embodiments, the working distance (including the vertical distance) is from about 0.1 mm to about 40 mm. In some embodiments, the working distance (including the vertical distance) is from about 1 mm to about 40 mm. In some embodiments, the working distance (including the vertical distance) is from about 2 mm to about 30 mm. In some embodiments, the working distance (including the vertical distance) is from about 1.5 mm to about 30 mm. In some embodiments, the working distance (including the vertical distance) is from about 2.5 mm to about 30 mm. In some embodiments, the working distance (including the vertical distance) is from about 2 mm to about 25 mm. In some embodiments, the working distance (including the vertical distance) is from about 3 mm to about 30 mm. In some embodiments, the working distance (including the vertical distance) is from about 3 mm to about 25 mm. More specifically, the working distance is from about 2.5 mm to about 25 mm. In these embodiments, the system allows for simultaneous imaging of the contents of the microsample container of interest and the associated retrieval container. In more specific embodiments, the working distance of the microscope objective is about 4 mm to about 10 mm, or even about 6 mm to about 8 mm, for example, about 7.4 mm. In some embodiments, the recovery array is a first recovery array. In some embodiments, the recovery array is a second recovery array.

[0086] As previously mentioned, in a preferred embodiment, the screening array of the multi-stage sample recovery system of the present invention includes a plurality of microcapillaries. More specifically, the screening array includes at least 100,000, at least 300,000, at least 1,000,000, at least 3,000,000, at least 10,000,000, or even more microcapillaries. In some embodiments, the array includes at least 100,000, at least 200,000, at least 300,000, at least 400,000, at least 500,000, at least 600,000, at least 700,000, at least 800,000, at least 1,000,000, at least 1,500,000, at least 2,000,000, at least 2,500,000, or at least 3,000,000 or more microcapillaries.

[0087] As previously mentioned, in a preferred embodiment, the recovery array of the multi-stage sample recovery system of the present invention includes one or more recovery containers. Therefore, in such a system, the recovery array may include at least one recovery container, at least three recovery containers, at least ten recovery containers, at least thirty recovery containers, at least one hundred recovery containers, or even more recovery containers.

[0088] In a preferred embodiment, the recycling array of the system of the present invention is positioned below the screening array. In some embodiments, the recycling array and the screening array are separated by at least 25 mm, at least 30 mm, at least 35 mm, at least 40 mm, at least 45 mm, or at least 50 mm or more. In some embodiments, the recycling array and the screening array are separated by at least 30 mm, at least 35 mm, or at least 40 mm. In some embodiments, the recycling array and the screening array are separated by at least 35 mm. In some embodiments, the recycling array is located at least 25 mm, at least 30 mm, at least 35 mm, at least 40 mm, at least 45 mm, or at least 50 mm below the screening array. In some embodiments, the recycling array is located at least 30 mm, at least 35 mm, or at least 40 mm below the screening array. In some embodiments, the recycling array is located at least 35 mm or at least 40 mm below the screening array. In some embodiments, the recycling array is located at least 35 mm below the screening array.

[0089] It will be apparent to those skilled in the art that other suitable modifications and adjustments can be made to the methods and applications described herein without departing from the scope of the invention or any of its embodiments. Having now described the invention in detail, the invention will be more clearly understood by referring to the following examples, which are included herein for illustrative purposes only and are not intended to limit the invention.

[0090] Example

[0091] Example 1. Screening for secreted EGFR-binding proteins

[0092] Figures 1A-1C An exemplary screening method is shown for soluble proteins that can associate with cell surface proteins (e.g., epidermal growth factor receptor (“EGFR”)) that are immobilization target molecules (in this case, immobilization target proteins). Figure 1A (Left image) shows target cells expressing EGFR on their surface. Also shown are “library expression cells” expressing variant protein populations and multiple “fluorescent detection antibodies” in a microcapillary solution. The right image shows a bottom view of the microcapillary array.

[0093] The components of each microcapillary determined based on this screening:

[0094] 1. Cells that secrete the variant protein of interest (“library expression cells”). The variant protein of interest is preferably a group of variant proteins, i.e., a member of the protein library.

[0095] 2. The target protein is anchored on the surface of the “target cell.” In this example, the target protein is a natural cell surface receptor (i.e., EGFR). However, alternatively, the target protein can be anchored on another surface, such as the surface of a bead or the surface of the microcapillary itself.

[0096] 3. Reporting Element

[0097] a. In this example, the reporter element corresponds to a fluorescently labeled antibody (i.e., a "fluorescent detection antibody") that is specific to the secreted protein. The antibody specifically targets the epitope on the secreted protein, but ideally does not interfere with the binding of the secreted protein to the target protein on the target cell.

[0098] b. Alternatively, the reporter element may be an intracellular signaling pathway expressing the target protein. If the secreted variant protein binds to the target protein on the cell surface and activates the intracellular signaling pathway, the binding interaction will produce a fluorescent signal (not shown) within the cell.

[0099] 4. Reaction buffer:

[0100] a. It can be a culture medium for expressing libraries or target cells.

[0101] b. It can be a mammalian imaging solution.

[0102] Method Description

[0103] Step 1: Add all components to the microcapillary (see...) Figure 1A ).

[0104] Step 2: The library-expressing cells that have entered the microcapillaries express specific "secreted proteins". As shown, secreted protein variants that can bind to the target protein are localized on the surface of the target cell (see 1B).

[0105] Step 3: Observe the association of fluorescent detection antibodies with the bound secreted protein variants in specific microcapillaries with the target cells (see...). Figure 1C ).

[0106] Detailed description and sample data:

[0107] To demonstrate this method, a yeast vector library expressing a protein designed to bind to EGFR on human cancer cells was created. Within this library, some yeast variants were able to express the protein, while others were not. Yeast cells, cancer cells, and a fluorescent antibody against the expressed protein were added to microcapillaries. After 18 hours, the microcapillary array was imaged. Further details and results of the screening are provided in Example 3 below.

[0108] Example 2. Hybridoma screening targeting mammalian cells

[0109] General background

[0110] Current methods for screening binding interactions between proteins or other target molecules typically rely on the use of "display" methods, such as phage display, bacterial display, yeast display, mammalian display, or viral display. In display methods, a library of genes encoding protein variants is expressed on the surface of cells or phages. The protein variants are incubated with a soluble version of the target molecule to identify protein variants capable of binding to the target. The library can be screened by panning or by fluorescence-activated cell sorting ("FACS"). This assay has two main limitations: 1) engineered proteins are typically tethered to the display platform; and 2) the presence of a soluble form of the target molecule is usually advantageous. Therefore, it can be difficult to develop reliable assays for variant proteins that bind to multiple target molecules, especially membrane proteins such as G protein-coupled receptors and other such receptors.

[0111] Hybridoma screening for mammalian cells

[0112] To identify antibody variants that specifically bind to a target molecule, hybridomas (which secrete antibody variants) are added to cancer cell lines expressing high levels of EGFR as the target molecule. Then, a labeled antibody specific to the secreted antibody is added.

[0113] Material:

[0114] cell:

[0115] mouse hybridoma

[0116] A431 target cells (human cancer cell line expressing high levels of EGFR)

[0117] Antibody detection:

[0118] Anti-mouse secondary antibody labeled with Alexa488 (fluorophore)

[0119] Culture medium for cell culture:

[0120] DMEM-10% Fetal Bovine Serum

[0121] DMEM-10% Horse Serum

[0122] Cell line growth and preparation.Mouse hybridoma cells were cultured in complete medium (Dulbecco's Modified Eagle's Medium with 10% horse serum). The hybridoma cells were washed twice with PBSA and then resuspended in 600 cells / µL of complete medium. A431 cells were cultured in complete medium (Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum). The A431 cells were washed twice with PBSA and stained with LiveGreen fluorescence. The A431 cells were then resuspended in complete medium containing hybridoma cells at a final concentration of 1800 cells / µL.

[0123] Measurement setup. After mixing the two cell types, the detection antibody was added to the reaction mixture: a 1:100 dilution of the second (anti-mouse Alexa488). This reaction mixture was then loaded into an ethanol-sterilized, corona-treated microcapillary array (40 μm in diameter, 1 mm thick). A 2 mm plate of 1% weight / volume agarose was placed on the array to help prevent evaporation. The samples were imaged under fluorescence and bright-field microscopy every hour.

[0124] Sample data:

[0125] Figure 2A-2C An image showing a small portion of the microcapillary array is displayed, showing all the cells ( Figure 2A Bright field signal), A431 target cells ( Figure 2B LiveGreen signaling or cells labeled with fluorescent anti-mouse secondary antibody ( Figure 2C (Ab-a555 signal). Microcapillaries containing hybridoma cells expressing antibodies specific to EGFR are indicated by two arrows in each image.

[0126] Figure 3 Images of microcapillaries containing A431 target cells and hybridoma cells during a 4-hour incubation process are shown, where antibody binding signals against A431 target cells increased with the production of mouse antibodies specific to EGFR during the measured time period (middle column). LiveGreen staining of A431 target cells decreased during the same time period (right column).

[0127] Example 3. Screening of yeast libraries for mammalian cells

[0128] To determine the optimal secretory yeast plasmid vector, a yeast vector library expressing a scaffold protein designed to bind to the surface of cancer cells was created. This library contained yeast cells with various soluble expression levels of the scaffold protein. Variant expression libraries were screened using the described assays to recover plasmid vectors with high expression of the desired scaffold protein. In this experiment, the secreted scaffold had a c-Myc tag that could be labeled with a fluorescently labeled antibody.

[0129] Material:

[0130] cell:

[0131] Yeast secretion library of scaffold proteins

[0132] A431 cells (a human cancer cell line expressing high levels of EGFR)

[0133] Antibody detection:

[0134] Chicken Anti-c-Myc

[0135] Anti-chicken secondary antibody labeled with Alexa488

[0136] Culture medium for cell culture:

[0137] DMEM-10% FBS

[0138] SD-CAA basic yeast culture medium

[0139] Reaction buffer solution:

[0140] SD-CAA basic yeast culture medium

[0141] method:

[0142] Cell line growth and preparation. Yeast libraries were grown in SD-CAA basal yeast medium (20 g dextrose; 6.7 g Difco yeast nitrogen bases; 5 g Bacto casein amino acids; 5.4 g Na2HPO4; 8.56 g NaH2PO4·H2O; dissolved in deionized H2O to a volume of 1 L). After growth, yeast cells were washed twice with PBSA (phosphate-buffered saline + 1 mg / ml BSA) and then resuspended in SD-CAA at a final concentration of 2,400 cells / µL.

[0143] A431 cells were cultured in complete medium (Dalberg modified Eagle medium with 10% fetal bovine serum). A431 cells were washed twice with PBSA and then suspended in SD-CAA containing yeast cells at a final concentration of 600 cells / µL.

[0144] Measurement setup. After mixing the two cell types, two antibodies were added to the reaction mixture: a 1:250 dilution of unlabeled primary antibody (chicken anti-c-Myc) and a 1:200 dilution of labeled secondary antibody (anti-chicken Alexa488). This reaction mixture was then loaded into an ethanol-sterilized, corona-treated microcapillary array (40 μm in diameter, 1 mm thick). A 2 mm plate containing 1% weight / volume agarose was placed on the array to help prevent evaporation. After 18 hours of growth, the samples were imaged under fluorescence and bright-field microscopy.

[0145] Extraction using microcapillary arrays. The contents of the desired capillary were extracted using a Triton UV laser. The laser ran for 18 ± 2 milliseconds (n = 5 measurements) to deliver a pulse train with a frequency of 2.5 kHz and a total energy of approximately 100 μJ. The microcapillary contents were extracted onto a glass coverslip, which was then placed in yeast growth medium (liquid medium or agar plate) to proliferate the extracted cells.

[0146] Sample data

[0147] Figure 4A and 4B It shows the use of bright field imaging ( Figure 4A ) and fluorescence imaging ( Figure 4B Images of a small portion of a microcapillary array that distinguishes between microcapillaries containing expressed and non-expressing cells.

[0148] Example 4. Growth of cultured human cells in a microcapillary array

[0149] Figure 5A-5G The growth of K562 cells (human immortalized myeloid leukemia cell line) in a microcapillary array in growth medium over a 6-day period is shown. Bright-field images of the same portion of the array are captured every 24 hours. Figure 5A Day 0; Figure 5B Day 1; Figure 5C Day 2; Figure 5D Day 3; Figure 5E Day 4; Figure 5F Day 5; and Figure 5G Day 6. A scale bar of 40 μm is shown in each image.

[0150] Example 5. Hybridoma screening targeting mammalian reporter cells

[0151] To identify antibody variants that activate specific signal transduction pathways, hybridomas secreting different antibody variants are added to a microcapillary array containing reporter cells. For example, the reporter cells can be from Qiagen (see http: / / www.sabiosciences.com / reporter_assay_product / HTML / CCS-013L.html).

[0152] If a protein variant binds to a reporter cell and activates a signaling pathway, the reporter cell expresses a fluorescent protein. The signal fluorescence of the activated cell is observed in a microcapillary containing the desired protein variant, and this signal fluorescence is used to separate the contents of those microcapillaries.

[0153] Example 6. Automated Cell Recovery System (ACRS)

[0154] This example describes a multi-stage sample recovery system already used to recover samples of interest from a large microcapillary array using the screening method described above. The Automated Cell Recovery System (“ACRS”) is configured with three stages (one xy and two linear stages) in two layers, working together to achieve sample recovery from the microcapillary array. The top xy stage holds the microcapillary array and moves it around, allowing the entire array to be imaged by a microscope objective. The two bottom linear stages move the capture surface (e.g., an 18-well slide) to recover the contents of the microcapillaries of interest into separate recovery containers (e.g., new wells on the 18-well slide). The entire configuration fits within the working distance (7.4 mm) of the microscope objective, enabling imaging of both the microcapillary array and the recovery array without removing any components from the microscope.

[0155] Detailed description

[0156] As noted above, ACRS is composed of, for example, Figure 7A The XY order shown and at least one such Figure 7B The X / Y stages are shown. These stages interface with instruments such as the Nikon Ti-E motorized microscope. The X / Y stages hold screening arrays such as microcapillary arrays, and one or more X / Y stages are configured to hold sample recovery arrays such as 18-well glass slides.

[0157] Light from the associated microscope travels through two levels to visualize the contents of each sample in the screening array, such as each microcapillary in the microcapillary array held atop the screening array level. Because the screening array level and the recovery array level are very close together, the objective lens can also image containers associated with the recovery array, such as an 18-well slide.

[0158] These stages operate independently of each other to position the desired micro-sample container (e.g., a microcapillary within a microcapillary array) and the desired capture surface (e.g., a recovery container within a recovery array) at the desired locations relative to the microscope objective. For example, as Figure 8 As shown, if the screening array 10 is found to contain three sample containers of interest, for example, the three sample containers labeled 1, 2, and 3 in the figure, the screening array stage is moved to position the first sample container in a straight line with the optical path of the extraction beam, and the recovery array stage is also moved independently to position the first recovery container of the recovery array 14 in a straight line with the optical path, as shown. Figure 8 As shown in the top left image.

[0159] After the first sample of interest has been transferred to the first recovery container, the screening array stage is moved in the X and Y directions to position the second sample of interest in a straight line with the extraction beam, and the recovery array stage is moved independently to position the second recovery container in a straight line with the beam, as follows. Figure 8 As shown in the upper right figure. After the second sample of interest has been transferred to the second recovery container, the process is repeated as needed by moving the screening array stage in the X and Y directions to position the third sample of interest in a straight line with the extraction beam. The recovery stage is moved independently to position the third recovery container in a straight line with the beam, as shown in the figure above. Figure 8 As shown in the bottom diagram, the sample is transferred to a third recovery container via an extraction bundle.

[0160] In this example, because the first, second, and third recycling containers are positioned in a straight line, only a single recycling array level is needed in the system. If the user wants to use the additional two rows of recycling containers in the illustrated recycling array, the recycling array level can be manually moved, for example, to align the second row of recycling containers with the extraction bundle. However, preferably, the system further includes a second recycling array level positioned orthogonal to the first recycling array level, for example, as shown in the example below. Figures 6A-6E As shown in the system, the second linear stage automatically moves the recovery array in a direction orthogonal to the direction of the first recovery array stage, and thus enables the recovery of additional samples of interest into subsequent rows of the recovery array.

[0161] While specific examples have been provided, the above description is illustrative and not restrictive. Any one or more features described in the foregoing embodiments may be combined in any way with one or more features of any other embodiments of the invention. Furthermore, many variations of the invention will become apparent to those skilled in the art after reading this specification. Many modifications and variations may be made to the invention without departing from the spirit and scope of this application, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are provided by way of example only, and this application is limited only to the terms of the appended claims and the full scope of their equivalents.

[0162] The examples described above are provided to offer a complete disclosure and description of how to manufacture and use the compositions, systems, and methods of the present invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider their invention. Modifications to the modes used for carrying out the invention described above, which are obvious to those skilled in the art, are intended to fall within the scope of the following claims. All patents and publications referenced in this specification indicate the level of skill required for those skilled in the art to which this invention pertains.

[0163] The use of all headings and section names is for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will recognize the usefulness of appropriately combining various aspects from different headings and sections in accordance with the spirit and scope of the invention described herein.

[0164] All references cited in this document, including all patents, patent publications and other published references, are incorporated herein by reference in their entirety and for all purposes, to the extent that each individual publication or patent or patent application is specifically or individually indicated to be incorporated herein by reference in its entirety for all purposes.

Claims

1. A multi-stage sample recovery system, comprising: A screening array stage, wherein the screening array stage is controllable in two dimensions relative to a microscope objective and is configured to be reversibly associated with a screening array, wherein the working distance of the microscope objective is 2.5 mm to 25 mm, and wherein the screening array is reversibly associated with the screening array stage; A first recovery array stage, wherein the first recovery array stage is controllable in at least one dimension relative to the microscope objective and is configured to be reversibly associated with the recovery array; An extraction beam generator, wherein the extraction beam is guided below the first recovery array stage, and the first recovery array stage is positioned below the screening array stage. The filtering array level and the first recycling array level are independently controllable.

2. The multi-stage sample recovery system according to claim 1, wherein the extraction beam is optically coupled through holes in the screening array stage.

3. The multi-stage sample recovery system according to claim 2, wherein the extraction beam is a laser beam.

4. The multi-stage sample recovery system according to claim 1, wherein the screening array comprises a plurality of micro sample containers.

5. The multi-stage sample recovery system according to claim 1, further comprising a recovery array reversibly associated with the first recovery array stage.

6. The multi-stage sample recovery system according to claim 5, wherein the recovery array comprises a plurality of recovery containers.

7. The multi-stage sample recovery system of claim 5, wherein the screening array comprises a plurality of micro sample containers, and the recovery array comprises a plurality of recovery containers, and wherein the screening array and the recovery array are configured to position at least one micro sample container and at least one recovery container within the working distance of the microscope objective.

8. The multi-stage sample recovery system according to claim 1, wherein the screening array comprises a plurality of microcapillaries.

9. The multi-stage sample recovery system according to claim 1, wherein the screening array comprises at least 100,000 microcapillaries.

10. The multi-stage sample recovery system according to claim 1, wherein the recovery array includes a recovery container.

11. The multi-stage sample recovery system of claim 10, wherein the recovery array comprises at least one recovery container.

12. The multi-stage sample recovery system of claim 10, wherein the recovery container is configured to prevent cell damage or promote cell growth.

13. The multi-stage sample recovery system of claim 10, wherein the recovery container is configured to perform an amplification reaction.

14. The multi-stage sample recovery system according to claim 13, wherein the amplification reaction is a polymerase chain reaction.

15. The multi-stage sample recovery system according to claim 13, wherein the amplification reaction is a reverse transcription polymerase chain reaction.

16. The multi-stage sample recovery system of claim 10, wherein the recovery container is configured to perform a sequencing reaction.

17. The multi-stage sample recovery system according to claim 1, wherein the screening array stage and the first recovery array stage are controllable by an electric motor.

18. The multi-stage sample recovery system according to claim 1, further comprising a second recovery array stage.

19. The multi-stage sample recovery system of claim 18, wherein the second recovery array stage is positioned orthogonal to the first recovery array stage.

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