# US 2022/0034869 A1 · Multi-stage, multiplexed target isolation and processing from heterogeneous populations

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- Number: US 2022/0034869 A1
- Document ID: US20220034869A1
- Status: Published application
- Kind: A1
- Application number: 17/163,054
- Filed: 2021-01-29
- Published: 2022-02-03
- Earliest claimed: 2016-02-05
- Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology; The General Hospital Corporation
- Applicants: The Broad Institute, Inc.; Massachusetts Institute of Technology; The General Hospital Corporation
- Inventors: Paul Blainey; Dwayne Vickers; Nir Hacohen
- Web page: https://patentgrep.com/patent/US20220034869A1

## CPC

- G01N 33/5044: {involving specific cell types}
- B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces}
- B01L 3/502761: {specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules}
- G01N 33/536: with immune complex formed in liquid phase
- G01N 33/543: with an insoluble carrier for immobilising immunochemicals
- G01N 33/54326: {Magnetic particles}
- G01N 33/58: involving labelled substances (G01N33/53 takes precedence)
- B01L 2300/0861: Configuration of multiple channels and/or chambers in a single devices
- G01N 33/587: {Nanoparticles}
- 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)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

## Abstract

A system and method for isolating target substrates includes a microfluidic chip, comprising a plurality of processing units, each processing unit comprising: an inlet port, a plurality of first chambers connected to the inlet port by a fluid channel, the fluid channel comprising a plurality of valves, a plurality of second chambers, each of the second chambers connected to a respective first chamber by a fluid channel, each fluid channel including a controllable blocking valve, and a plurality of respective outlet ports, each outlet port in fluid communication with a respective one of said second chambers and each outlet port including a blocking valve. A magnet is adjacent the microfluidic chip and is movable relative to the microfluidic chip. A valve control is capable of actuating certain ones of the controllable blocking valves in response to a control signal.

## Claims

### Claim at position 1

1-61. (canceled)

### Claim 62

A substrate-isolation platform, comprising:
a microfluidic chip, comprising a plurality of processing units, each processing unit comprising: an inlet port, a plurality of first chambers connected to the inlet port by a fluid channel, the fluid channel comprising a plurality of valves, a plurality of second chambers, each of the second chambers connected to a respective first chamber by a fluid channel, each fluid channel including a controllable blocking valve, and a plurality of respective outlet ports, each outlet port in fluid communication with a respective one of said second chambers and each outlet port including a blocking valve;
a magnet adjacent the microfluidic chip, wherein relative positioning of the magnet and the microfluidic chip is variable; and
a valve control capable of actuating certain ones of the controllable blocking valves in response to a control signal.

### Claim 63 (depends on 62)

The platform of claim 62, wherein at least one of the first chambers and the second chambers are ring chambers.

### Claim 64 (depends on 62)

The platform of claim 62, wherein the microfluidic chip comprises a volume of about 10 nl to about 10 μl.

### Claim 65 (depends on 62)

The platform of claim 62, wherein the microfluidic chip comprises at least two layers.

### Claim 66 (depends on 65)

The platform of claim 65, wherein at least one layer comprises high thermal conductivity.

### Claim 67 (depends on 65)

The platform of claim 65, wherein at least one layer comprises a quartz layer and/or at least one layer comprises a silica layer.

### Claim 68

(canceled)

### Claim 69 (depends on 62)

The platform of claim 62, wherein the magnet produces a dynamic magnetic field.

### Claim 70 (depends on 62)

The platform of claim 62, wherein the magnet is an electromagnet.

### Claim 71 (depends on 62)

The platform of claim 62, wherein the magnet is a permanent magnet, optionally wherein the magnet comprises a Rare Earth magnet, optionally wherein the magnet comprises a Neodymium magnet.

### Claim at position 12

72-73. (canceled)

### Claim 74 (depends on 62)

The platform of claim 62, wherein each microchip comprises multiple processing units, optionally wherein each microchip comprises 2-1000 processing units, optionally wherein at least a portion of the first chamber or a portion of the second chamber is transparent, optionally wherein the first chambers comprise a plurality of sub-chambers, optionally comprising a sufficient number of said sub-chambers to enable mixing of at least 10 components in pre-defined ratios and/or the second chambers comprise a plurality of sub-chambers, optionally comprising a sufficient number of said sub-chambers to enable mixing of at least 10 components in pre-defined ratios.

### Claim at position 14

75-80. (canceled)

### Claim 81 (depends on 62)

A method of integrated subtype purification and RNA-Seq on the platform of claim 62.

### Claim at position 16

82-97. (canceled)

### Claim 98

A method of integrated subtype purification and RNA-Seq on a platform comprising a microfluidic chip, comprising a plurality of processing units, each processing unit comprising: an inlet port, a plurality of first chambers connected to the inlet port by a fluid channel, the fluid channel comprising a plurality of valves, a plurality of second chambers, each of the second chambers connected to a respective first chamber by a fluid channel, each fluid channel including a controllable blocking valve, and a plurality of respective outlet ports, each outlet port in fluid communication with a respective one of said second chambers and each outlet port including a blocking valve; and a movable magnet adjacent the microfluidic chip, the method comprising:
providing a plurality of first magnetic beads in a first chamber, said first magnetic beads capable of binding a cell-specific marker so as to attach a specific cell-type;
providing cells to the first chamber, the cells including cells of the specific cell-type, such that cells of the specific cell-type bind to the first magnetic beads;
applying a magnetic field to the first chamber and moving the magnetic field in a first predetermined direction to transfer the first magnetic beads to a second chamber, the second chamber in fluid communication with the first chamber;
blocking fluid communication between the second chamber and the first chamber;
washing the first chamber;
unblocking fluid communication between the second chamber and the first chamber;
releasing the cells from the first magnetic beads;
removing the first magnetic beads from the second chamber by applying a magnetic field from the second chamber in a second predetermined direction;
blocking fluid communication between the second chamber and the first chamber;
lysing the cells in the second chamber;
capturing target substrates from the lysed cells using dynamically altered magnetic beads;
applying a magnetic field to the second chamber and moving the magnetic field in a third predetermined direction to transfer the target substrates captured by the dynamically altered magnetic beads to the first chamber;
mixing the dynamically altered magnetic beads and the captured target substrates with mRNA-seq reagents;
cycling through a range of temperatures to create a PCR product; and
applying DNA-binding beads to the PCR product to clean up DNA.

### Claim 99 (depends on 98)

The method of claim 98, wherein the second magnetic beads are oligo (dT) beads.

### Claim 100 (depends on 98)

The method of claim 98, wherein the second magnetic beads are Solid Phase Reversible Immobilization beads.

### Claim 101 (depends on 98)

The method of claim 98, wherein the DNA-binding beads are charge switch silica beads.

### Claim 102 (depends on 98)

The method of claim 98, wherein the DNA-binding beads are solid phase reversible immobilization (SPRI) beads.

### Claim 103 (depends on 98)

The method of claim 98, wherein the DNA-binding beads are dsDNA antibodies.

### Claim 104

A kit comprising an integrated subtype purification and RNA-Seq on a platform comprising a microfluidic chip, comprising a plurality of processing units, each processing unit comprising: an inlet port, a plurality of first chambers connected to the inlet port by a fluid channel, the fluid channel comprising a plurality of valves, a plurality of second chambers, each of the second chambers connected to a respective first chamber by a fluid channel, each fluid channel including a controllable blocking valve, and a plurality of respective outlet ports, each outlet port in fluid communication with a respective one of said second chambers and each outlet port including a blocking valve; and a movable magnet adjacent the microfluidic chip, the method comprising:
providing a plurality of first magnetic beads in a first chamber, said first magnetic beads capable of binding a cell-specific marker so as to attach a specific cell-type;
providing cells to the first chamber, the cells including cells of the specific cell-type, such that cells of the specific cell-type bind to the first magnetic beads;
applying a magnetic field to the first chamber and moving the magnetic field in a first predetermined direction to transfer the first magnetic beads to a second chamber, the second chamber in fluid communication with the first chamber;
blocking fluid communication between the second chamber and the first chamber;
washing the first chamber;
unblocking fluid communication between the second chamber and the first chamber;
releasing the cells from the first magnetic beads;
removing the first magnetic beads from the second chamber by applying a magnetic field from the second chamber in a second predetermined direction;
blocking fluid communication between the second chamber and the first chamber;
lysing the cells in the second chamber;
capturing target substrates from the lysed cells using dynamically altered magnetic beads;
applying a magnetic field to the second chamber and moving the magnetic field in a third predetermined direction to transfer the target substrates captured by the dynamically altered magnetic beads to the first chamber;
mixing the dynamically altered magnetic beads and the captured target substrates with mRNA-seq reagents;
cycling through a range of temperatures to create a PCR product; and
applying DNA-binding beads to the PCR product to clean up DNA.

### Claim 105 (depends on 104)

The kit according to claim 104, further comprising magnetic beads in a first chamber comprises the first magnetic beads conjugated to an antibody wherein the first magnetic beads are capable of binding a cell-specific marker so as to attach a specific cell-type.

### Claim 106 (depends on 105)

The kit according to claim 105, wherein the antibody and cell-specific marker are cleaved, optionally wherein the antibody and cell-specific marker are enzymatically cleaved.

### Claim 107

(canceled)