# US 12,007,388 B2 · System for single-molecule isolation for cell populations and single cells, and methods and uses thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 12,007,388 B2 - Document ID: US12007388B2 - Status: Granted - Kind: B2 - Application number: 16/999,598 - Filed: 2020-08-21 - Published: 2021-02-25 - Granted: 2024-06-11 - Earliest claimed: 2019-08-22 - Assignees: The Hong Kong University of Science and Technology - Applicants: The Hong Kong University of Science and Technology - Inventors: Pingbo Huang; Qirui Zhao - Web page: https://patentgrep.com/patent/US12007388B2 ## CPC - G01N 33/54333: {Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction} - G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)} - G01N 21/648: {using evanescent coupling or surface plasmon coupling for the excitation of fluorescence} - G01N 27/745: {for detecting magnetic beads used in biochemical assays (concerning the assays G01N33/54326; sensors therefor G01R33/1269; automatic analysers therefor G01N35/0098)} - G01N 33/553: Metal or metal coated - G01N 33/582: {with fluorescent label} - G01N 21/6452: {Individual samples arranged in a regular 2D-array, e.g. multiwell plates} - G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks} - 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) ## Abstract The invention relates to the systems and methods for isolating single cells, capturing selected prey biomolecules within said cells, and detecting said prey biomolecules using a sensitive assay method. Also described herein are functionalized substrates and compositions for enabling the low limit of detection of selected prey biomolecules of selected cells. ## Claims ### Claim 1 A system comprising a glass substrate comprising a plurality of micron-sized features, a plurality of primary antibodies against a plurality of bait molecules, the plurality of bait molecules, a plurality of target prey biomolecules, and a plurality of primary antibodies against said plurality of target prey biomolecules, wherein the plurality of primary antibodies against the plurality of bait molecules coat a plurality of beads. ### Claim 2 (depends on 1) The system of claim 1, wherein a plurality of secondary antibodies against the primary antibodies against the plurality of bait molecules coat the plurality of beads. ### Claim 3 (depends on 1) The system of claim 1, further comprising one or a plurality of types of cells. ### Claim 4 (depends on 3) The system of claim 3, wherein the cells comprise one or a plurality of a type of target prey biomolecule. ### Claim 5 (depends on 3) The system of claim 3, wherein the plurality of cells are located in the plurality of micron-sized features. ### Claim 6 (depends on 3) The system of claim 3, wherein the plurality of types of cells comprises cells having an average diameter of less than the diameter of the mean average round holes of the wells. ### Claim 7 (depends on 6) The system of claim 6, wherein the plurality of types of cells comprises cells having an average diameter of about 30 microns. ### Claim 8 (depends on 3) The system of claim 3, wherein the plurality of target prey biomolecules comprises molecules which are found in the cell membrane of said cells. ### Claim 9 (depends on 3) The system of claim 3, wherein the plurality of target prey biomolecules comprises molecules which are found in the cytoplasm of said cells. ### Claim 10 (depends on 1) The system of claim 1, wherein the plurality of antibody-coated beads are within the plurality of micron-sized features. ### Claim 11 (depends on 1) The system of claim 1, further comprising a cell lysis buffer. ### Claim 12 (depends on 11) The system of claim 11, wherein the cell lysis buffer comprises a non-denaturing detergent. ### Claim 13 (depends on 1) The system of claim 1, wherein the plurality of antibody-coated beads comprises a secondary antibody to the plurality of primary antibodies to a bait biomolecule. ### Claim 14 (depends on 1) The system of claim 1, wherein the plurality of micron-sized features comprises a series of wells having round holes each of which having a mean average diameter between about 15 microns to about 45 microns, a mean average distance between a center of each hole ranging from about 100 microns to about 5000 microns, and a mean average depth of each hole of about 25 microns to about 150 microns. ### Claim 15 (depends on 14) The system of claim 14, wherein the mean average diameter of the series of wells is about 30 microns. ### Claim 16 (depends on 14) The system of claim 14, wherein the mean average distance between the center of each hole is about 150 microns. ### Claim 17 (depends on 14) The system of claim 14, wherein the mean average depth of each hole is about 75 microns. ### Claim 18 (depends on 1) The system of claim 1, wherein the plurality of antibody-coated beads comprise magnetic beads. ### Claim 19 (depends on 18) The system of claim 18, wherein the magnetic bead comprises an iron oxide core. ### Claim 20 (depends on 18) The system of claim 18, further comprising a magnet. ### Claim 21 (depends on 1) The system of claim 1, wherein the plurality of micron-sized features is directly etched into the glass substrate. ### Claim 22 (depends on 1) The system of claim 1, wherein the plurality of micron-sized features is formed from polydimethylsiloxane (PDMS). ### Claim 23 (depends on 22) The system of claim 22, wherein the PDMS is cured PDMS. ### Claim 24 (depends on 1) The system of claim 1, wherein the glass substrate is a glass coverslip having a thickness of 0.085 mm to 0.64 mm. ### Claim 25 (depends on 24) The system of claim 24, wherein the glass substrate is configured to be measureable by TIRF (total internal reflectance spectroscopy). ### Claim 26 (depends on 1) The system of claim 1, wherein the bait molecules and the plurality of target prey biomolecules are independently selected from the group consisting of proteins, DNA, RNA, mRNA, tRNA, cRNA, antibodies, antibody fragments, ScFv's, and peptides. ### Claim 27 (depends on 26) The system of claim 26, wherein the DNA or RNA is modified with biotin.