# US 11,998,914 B2 · Micro-fluidic devices for assaying biological activity

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- Number: US 11,998,914 B2
- Document ID: US11998914B2
- Status: Granted
- Kind: B2
- Application number: 17/656,244
- Filed: 2022-03-24
- Published: 2022-12-08
- Granted: 2024-06-04
- Earliest claimed: 2013-10-22
- Assignees: Bruker Cellular Analysis, Inc.
- Applicants: Berkeley Lights, Inc.
- Inventors: Kevin T. Chapman; Daniele Malleo; J. Tanner Nevill; Steven W. Short; Mark P. White; M. Jimena Loureiro
- Web page: https://patentgrep.com/patent/US11998914B2

## CPC

- 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}
- B03C 5/005: {Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength}
- B03C 5/026: {using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]}
- G01N 15/1484: {microstructural devices}
- G01N 33/5023: {on expression patterns}
- G01N 33/5047: {Cells of the immune system}
- G01N 33/505: {involving T-cells}
- G01N 33/5052: {involving B-cells}
- G01N 33/54313: {the carrier being characterised by its particulate form}
- G01N 33/6854: {Immunoglobulins}
- B01L 2200/0647: Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L 2200/0668: Trapping microscopic beads
- B01L 2300/0636: Integrated biosensor, microarrays
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/0864: comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
- B01L 2300/087: Multiple sequential chambers
- B01L 2400/0454: radiation pressure, optical tweezers
- B03C 2201/26: for use in medical or biological applications
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B03C: MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS (separating isotopes B01D59/00; combinations of magnetic or electrostatic separation with separation of solids by other means B03B, B07B)
- 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

Biological activity in holding pens in a micro-fluidic device can be assayed by placing in the holding pens capture objects that bind a particular material of interest produced by the biological activity. The biological material of interest that binds to each capture object can then be assessed, either in the micro-fluidic device or after exporting the capture object from the micro-fluidic device. The assessment can be utilized to characterize the biological activity in each holding pen. The biological activity can be production of the biological material of interest. Thus, the biological activity can correspond to or arise from one or more biological cells. Biological cells within a holding pen can be clonal cell colonies. The biological activity of each clonal cell colony can be assayed while maintaining the clonal status of each colony.

## Claims

### Claim 1

A process of assaying biological activity of one or more biological cells in a holding pen of a micro-fluidic device, wherein said biological activity comprises production of a biological material of interest; and
wherein said micro-fluidic device comprises:
a base and a micro-fluidic structure disposed on said base;
wherein said micro-fluidic structure and said base define a flow region and said holding pen, and wherein said holding pen comprises a single opening to said flow region and an unswept region;
said process comprising:
introducing a capture micro-object into said holding pen, wherein said capture object comprises a binding substance that specifically binds to said biological material of interest;
allowing said biological material of interest produced by said one or more biological cells to bind to said capture micro-object; and
assessing whether said biological material of interest is bound to said capture micro-object.

### Claim 2 (depends on 1)

The process of claim 1 further comprising, after allowing said biological material of interest to bind to said capture micro-object but before assessing whether said biological material of interest is bound to said capture micro-object, removing said capture micro-object from said holding pen.

### Claim 3 (depends on 2)

The process of claim 2, wherein removing said capture micro-object comprises moving said capture micro-object to an assay region located within said micro-fluidic device.

### Claim 4 (depends on 2)

The process of claim 2, wherein removing said capture micro-object comprises:
moving said capture micro-object to a channel in said micro-fluidic device; and
exporting said capture micro-object from said micro-fluidic device.

### Claim 5 (depends on 2)

The process of claim 2, wherein said capture micro-object is magnetic, and wherein removing said capture micro-object comprises applying a magnetic field to said micro-fluidic device.

### Claim 6 (depends on 2)

The process of claim 2, further comprising maintaining a correlation between said removed capture micro-object with said holding pen from which said removed capture micro-object was removed, thereby allowing data obtained from said removed capture micro-object to be tracked back to said holding pen.

### Claim 7 (depends on 1)

The process of claim 1, wherein said assessing comprises determining a type or an amount of said biological material of interest that is bound to said capture micro-object.

### Claim 8 (depends on 7)

The process of claim 7, wherein said determining comprises:
binding assay material to said biological material of interest that is bound to said capture micro-object; and
detecting an association between said capture micro-object and radiation originating from said assay material.

### Claim 9 (depends on 1)

The process of claim 1, wherein said biological material of interest is a protein.

### Claim 10 (depends on 9)

The process of claim 9, wherein said protein is an antibody.

### Claim 11 (depends on 1)

The process of claim 1, wherein said assessing is performed while said capture micro-object is in said holding pen.

### Claim 12 (depends on 1)

The process of claim 1, wherein said binding substance of said capture micro-object has a binding affinity for said biological material of interest of at least about 1 μM.

### Claim 13 (depends on 1)

The process of claim 1, wherein said one or more biological cells in said holding pen is a clonal colony of biological cells.

### Claim 14 (depends on 1)

The process of claim 1, wherein said one or more biological cells in said holding pen is a single cell.

### Claim 15 (depends on 1)

The process of claim 1, wherein introducing said capture micro-object comprises introducing a plurality of capture micro-objects, each of which comprises a binding substance that differs from the binding substance of other capture micro-objects in said plurality.

### Claim 16 (depends on 1)

The process of claim 1, wherein said capture micro-object is a y-material capture micro-object comprising y different binding substances, each of which specifically binds to one of n different biological materials of interest.

### Claim 17 (depends on 1)

The process of claim 1, wherein said micro-fluidic device comprises a housing, wherein said housing comprises:
a first electrode;
a second electrode; and
an electrode activation substrate,
wherein said first electrode is part of a first wall of said housing and said second electrode and said electrode activation substrate are part of a second wall of said housing,
wherein said electrode activation substrate has a surface comprising a plurality of DEP electrode regions, and
wherein said surface of said electrode activation substrate is an inner surface of said flow region.

### Claim 18 (depends on 17)

The process of claim 17, wherein removing said capture micro-object comprises:
creating a light trap by projecting a light pattern onto said surface of said electrode activation substrate, wherein said light pattern surrounds and thereby traps said capture micro-object; and
moving said light trap from said holding pen into the flow region of said micro-fluidic device.

### Claim 19 (depends on 17)

The process of claim 17, wherein said DEP electrode regions are light-induced, and wherein removing said capture micro-object comprises:
activating light-induced DEP electrode regions adjacent to said capture micro-object in said holding pen by projecting a light pattern onto said surface of said electrode activation substrate adjacent to said capture micro-object, and
moving said light pattern from said holding pen into said flow region in said micro-fluidic device, whereby the activated DEP electrode regions repel said capture micro-object into said flow region.

### Claim 20 (depends on 17)

The process of claim 17, wherein said electrode activation substrate comprises a photoconductive material or a semiconductor material.

### Claim 21 (depends on 1)

The process of claim 1, wherein said flow region comprises a channel, and wherein said single opening of said holding pen opens into said channel.

### Claim 22 (depends on 1)

The process of claim 1, wherein said microfluidic device comprises a plurality of holding pens.

### Claim 23 (depends on 1)

The process of claim 1, wherein said micro-fluidic structure comprises a multi-component structure that includes a cover.

### Claim 24 (depends on 1)

The process of claim 1, wherein said one or more biological cells are selected from B cell(s), T cell(s), or hybridoma cell(s).

### Claim 25 (depends on 1)

The process of claim 1, wherein said one or more biological cells is/are cancer cell(s).

### Claim 26 (depends on 1)

The process of claim 1, wherein said one or more biological cells is an oocyte.

### Claim 27

A process of assaying biological activity in a micro-fluidic device, wherein said micro-fluidic device comprises: a base and a micro-fluidic structure disposed on said base; wherein said micro-fluidic structure and said base define a flow region and said holding pen, and wherein said holding pen comprises a single opening to said flow region and an unswept region, and said process comprising: culturing one or more biological cells in a holding pen of a micro-fluidic device, wherein said one or more biological cells produce n different biological materials of interest; introducing n different types of capture micro-objects into said holding pen, each said type of capture micro-object comprising a binding substance that specifically binds to one of said n different biological materials of interest; allowing said n different biological materials of interest produced by said one or more biological cells to bind to said n different types of capture micro-objects; and assessing binding between said n different biological materials of interest and said n different types of capture micro-objects.

### Claim 28

A process of assaying biological activity in a micro-fluidic device, wherein said micro-fluidic device comprises: a base and a micro-fluidic structure disposed on said base; wherein said micro-fluidic structure and said base define a flow region and said holding pen, and wherein said holding pen comprises a single opening to said flow region and an unswept region, and said process comprising: culturing one or more biological cells in a holding pen of a micro-fluidic device, wherein said one or more biological cells produce n different biological materials of interest; introducing one or more y-material capture micro-objects into said holding pen, each y-material capture micro-object comprising y different binding substances, each of which specifically binds to one of said n different biological materials of interest; allowing said n different biological materials of interest produced by said one or more biological cells to bind to said y-material capture micro-objects; and assessing binding between said n different biological materials of interest and said y-material capture micro-objects.