# US 10,370,653 B2 · Micro-screening apparatus, process, and products > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,370,653 B2 - Document ID: US10370653B2 - Status: Granted - Kind: B2 - Application number: 15/050,142 - Filed: 2016-02-22 - Published: 2016-08-25 - Granted: 2019-08-06 - Earliest claimed: 2015-02-22 - Assignees: The Board of Trustees of the Leland Stanford Junior University - Applicants: The Board of Trustees of the Leland Stanford Junior University - Inventors: Jennifer R. Cochran; Thomas M. Baer; Bob Chen; Spencer Caleb Alford; Arvind Kannan; Sungwon Lim; Ivan Dimov - Web page: https://patentgrep.com/patent/US10370653B2 ## CPC - C12N 15/1079: {Screening libraries by altering the phenotype or phenotypic trait of the host (reporter assays C12N15/1086)} - C40B 30/06: by measuring effects on living organisms, tissues or cells - C40B 30/08: by measuring catalytic activity - G01N 33/502: {for testing non-proliferative effects} - G01N 33/5304: {Reaction vessels, e.g. agglutination plates (for solid-phase systems G01N33/543)} - G01N 33/54366: {Apparatus specially adapted for solid-phase testing} - B01J 2219/00286: Reactor vessels with top and bottom openings - B01J 2219/00317: Microwell devices, i.e. having large numbers of wells - C40B 60/12: for screening libraries - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS ## Abstract Microcavity arrays and methods for quantitative biochemical and biophysical analysis of populations of biological variants. Examples include high-throughput analysis of cells and protein products use a range of fluorescent assays, including binding-affinity measurement and time-resolved enzyme assays. Laser-based extraction of microcavity contents. ## Claims ### Claim 1 A method for screening a library of cells having a plurality of genotypes for a cell having a phenotype of interest for producing a molecule of interest, the method comprising (a) loading a microcavity array with the library of cells; (b) incubating the array under conditions that allow for production of the molecule of interest; (c) imaging the array to identify a cavity comprising cells having the phenotype of interest; and (d) extracting the contents of the cavity comprising cells having the phenotype of interest by directing electromagnetic radiation from a pulsed diode laser that delivers electromagnetic radiation in 2-20 pulses having pulse length of 1-10milliseconds with a pulse separation of 10 to 100 millisecond at a radiation absorbing material associated with the cavity. ### Claim 2 (depends on 1) The method of claim 1, wherein the directing of electromagnetic radiation comprising applying the electromagnetic radiation to the radiation absorbing material to avoid heating a sample liquid in the cavity that is not in contact with the material. ### Claim 3 (depends on 1) The method of claim 1, wherein the cells are expanded in the array. ### Claim 4 (depends on 1) The method of claim 1, wherein the cells are selected from the group consisting of a mammalian cell, a yeast cell, and a bacterial cell. ### Claim 5 (depends on 4) The method of claim 4, wherein the molecule of interest is displayed on the surface of a yeast cell or a bacterial cell. ### Claim 6 (depends on 4) The method of claim 4, wherein the molecule of interest is secreted by a yeast cell or a bacterial cell. ### Claim 7 (depends on 1) The method of claim 1, further comprising culturing the extracted contents of the cavities to produce a second-generation library of cells and repeating steps (a)-(d) with the second generation library. ### Claim 8 (depends on 1) The method of claim 1, further comprising (1) extracting DNA from the cells comprising a gene for the phenotype of interest, (2) amplifying the DNA under conditions to introduce mutations in the gene; (3) creating a second generation library of cells comprising the amplified DNA, and (4) repeating steps (a)-(d) with the second generation library. ### Claim 9 (depends on 1) The method of claim 1, wherein the phenotype of interest comprises a cell producing a binding agent. ### Claim 10 (depends on 9) The method of claim 9, wherein the binding agent is a cell surface binding agent, an antibody, an antibody fragment, a ligand, a small molecule or a receptor. ### Claim 11 (depends on 1) The method of claim 1, wherein the molecule of interest comprises a fluorescent protein that has at least one of an emission intensity of interest and an emission spectra of interest. ### Claim 12 (depends on 11) The method of claim 11, wherein the scanning comprises identifying cavities emitting the at least one of the emission intensity of interest and the emission spectra of interest. ### Claim 13 (depends on 1) The method of claim 1, wherein the phenotype of interest is a protein having an enzymatic activity, a protein having a lack of inhibition of enzyme activity, and a protein having activity in the presence of an inhibitor for the enzyme. ### Claim 14 A method for engineering a property of interest in a fluorescent protein, wherein the property of interest comprises at least one of an emission spectra, an emission intensity, a stokes shift, and an absorption spectra, the method comprising creating a library of cells producing a mutant form of a fluorescent protein, and screening the library for cells producing the mutant form having the property of interest, wherein the screening method comprises: (a) loading a microcavity array with the library of cells; (b) incubating the array under conditions that allow for production of the mutant form; (c) imaging the array to identify a cavity comprising cells producing the mutant form; and (d) extracting the contents of the cavity comprising cells producing the mutant form by directing electromagnetic radiation from a pulsed diode laser that delivers electromagnetic radiation in 2-20 pulses having pulse length of 1-10milliseconds with a pulse separation of 10 to 100 millisecond at a radiation absorbing material associated with the cavity. ### Claim 15 A method for measuring enzyme kinetics for a member of a protein enzyme library produced by a library of cells having a plurality of genotypes for producing mutant forms of a protein enzyme; the method comprising (a) loading a microcavity array with the library of cells; (b) incubating the array in the presence of a substrate for the protein enzyme under conditions that allow for production of the enzyme of interest; (c) imaging the array at selected intervals; (d) measuring difference in enzyme activity for one or more cavities of the array; (e) extracting the contents of a cavity comprising cells having the phenotype of interest by directing electromagnetic radiation from a pulsed diode laser that delivers electromagnetic radiation in 2-20 pulses having pulse length of 1-10milliseconds with a pulse separation of 10 to 100 millisecond at a radiation absorbing material associated with the cavity. ### Claim 16 (depends on 15) The method of claim 15, wherein the library of cells is a library of yeast cells or library of bacterial cells. ### Claim 17 (depends on 16) The method of claim 16, wherein the protein enzymes are displayed on the surface of the cells. ### Claim 18 (depends on 16) The method of claim 16, wherein the protein enzymes are secreted by the cells. ### Claim 19 (depends on 15) The method of claim 15, further comprising adding an inhibitor for the enzyme.