# US 10,246,700 B2 · Methods and apparatuses for gene purification and imaging

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- Number: US 10,246,700 B2
- Document ID: US10246700B2
- Status: Granted
- Kind: B2
- Application number: 14/948,776
- Filed: 2015-11-23
- Published: 2016-05-26
- Granted: 2019-04-02
- Earliest claimed: 2014-11-24
- Assignees: NanoString Technologies, Inc.
- Applicants: NanoString Technologies, Inc.
- Inventors: Dwayne Dunaway; Rustem Khafizov; Qian Mei; Lucas Dennis; Michael Krouse; Joseph M. Beechem; Isaac Sprague
- Web page: https://patentgrep.com/patent/US10246700B2

## CPC

- C12N 15/1013: {by using magnetic beads}
- B01L 3/502723: {characterised by venting arrangements}
- 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}
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6825: Nucleic acid detection involving sensors
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces}
- B01L 2200/0631: Purification arrangements, e.g. solid phase extraction [SPE]
- B01L 2200/0668: Trapping microscopic beads
- B01L 2200/10: Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
- B01L 2300/0627: Sensor or part of a sensor is integrated
- B01L 2300/0867: Multiple inlets and one sample wells, e.g. mixing, dilution
- B01L 2300/0887: Laminated structure
- B01L 2300/1827: using resistive heater
- B01L 2400/043: magnetic forces
- B01L 2400/0487: fluid pressure, pneumatics
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present disclosure is directed to systems, devices and methods for nucleic acid or protein purification and imaging. A system is provided including a cartridge comprising a sample input area configured to hold a sample, comprising a plurality of hybridized complexes comprising a plurality of target molecules each hybridized with probes and a plurality of non-hybridized probes. The cartridge may also include a first binding chamber configured with first magnetic beads to receive and bind the sample, a first elution channel configured to receive the first magnetic beads and elute the sample from the first magnetic beads, a second binding chamber configured with second magnetic beads to receive and bind the sample, a second elution channel configured to receive the second magnetic beads and elute the sample from the second magnetic beads, and a binding area configured to receive the eluted sample and hold molecules for imaging.

## Claims

### Claim 1

A cartridge configured for purifying a hybridized target molecule sample and imaging a hybridized target molecule, comprising:
a sample input area configured to hold a target molecule sample, the sample comprising:
a plurality of hybridized complexes comprising a plurality of target molecules each hybridized with a first probe and a second probe,
a plurality of non-hybridized first probes, and
a plurality of non-hybridized second probes;
a first binding chamber configured to receive or contain a first affinity matrix and to receive the sample, wherein:
the first affinity matrix is functionalized with first molecules configured to bind with the non-hybridized first probes and hybridized complexes of the sample during a first period of time;
the first binding chamber is additionally configured to receive a first buffer to remove non-hybridized second probes from the sample after the non-hybridized first probes and hybridized complexes of the sample bind with the first affinity matrix;
a first elution channel attached to the first binding chamber and a means for heating, and which is configured to receive the first affinity matrix after the first period of time and configured for heating the first affinity matrix to elute a first eluted sample comprising the plurality of hybridized complexes and plurality of non-hybridized first probes;
a second binding chamber configured to receive or contain a second affinity matrix and to receive the first eluted sample, wherein:
the second affinity matrix is functionalized with second molecules configured to bind with the hybridized complexes during a second period of time;
the second binding chamber is additionally configured to receive a second buffer to remove at least non-hybridized first probes;
a second elution channel attached to the second binding chamber and a means for heating, and which is configured to receive the second affinity matrix after the second period of time and configured for heating the second affinity matrix to elute a second eluted sample comprising the plurality of hybridized complexes; and
a binding area having an active binding surface configured to receive the second eluted sample and bind with the hybridized complexes.

### Claim 2 (depends on 1)

The cartridge of claim 1, wherein the target molecule is a nucleic acid or a protein.

### Claim 3 (depends on 1)

The cartridge of claim 1, wherein the first affinity matrix and the second affinity matrix correspond to a first set of magnetic beads and a second set of magnetic beads, respectively.

### Claim 4 (depends on 1)

The cartridge of claim 1, wherein the active binding surface comprises one of streptavidin, an avidin, and oligonucleotides.

### Claim 5 (depends on 3)

The cartridge of claim 3, wherein the cartridge further comprises:
a plurality of buffer input areas,
a plurality of first binding chambers,
a plurality of waste output areas, and
a plurality of bead pads.

### Claim 6 (depends on 1)

The cartridge of claim 1, wherein the first probes include capture probes.

### Claim 7 (depends on 1)

The cartridge of claim 1, wherein the second probes include reporter probes.

### Claim 8 (depends on 5)

The cartridge of claim 5, wherein the bubble vent configured to separate the sample input and the first binding chamber and to eliminate air bubbles.

### Claim 9 (depends on 3)

The cartridge of claim 3, wherein the first magnetic beads include oligonucleotide-coupled magnetic beads.

### Claim 10 (depends on 9)

The cartridge of claim 9, wherein the oligonucleotide-coupled magnetic beads include F magnetic beads.

### Claim 11 (depends on 3)

The cartridge of claim 3, wherein the second magnetic beads include oligonucleotide-coupled magnetic beads.

### Claim 12 (depends on 11)

The cartridge of claim 11, wherein the oligonucleotide-coupled magnetic beads include G magnetic beads.

### Claim 13 (depends on 1)

The cartridge of claim 1, the cartridge being operatively coupled to:
a plurality of off-card buffer input valves operatively coupled to a fluidic manifold; and
a plurality of waste valves.

### Claim 14 (depends on 1)

The cartridge of claim 1, the cartridge further comprising:
a plurality of on-card buffer input valves operatively coupled to a fluidic manifold.

### Claim 15 (depends on 13)

The cartridge of claim 13, wherein the plurality of off-card buffer input valves are configured to receive the first buffer and the second buffer from the fluidic manifold and provide the buffer to the cartridge.

### Claim 16 (depends on 1)

The cartridge of claim 1, wherein the flow of the second eluted sample onto the binding area is done in small steps that are re-ordered based on pressure profiles.

### Claim 17 (depends on 1)

The cartridge of claim 1, wherein the binding area is further configured to receive a solution formulated to immobilize the second eluted sample on the active binding surface after stretching with flow.

### Claim 18 (depends on 17)

The cartridge of claim 17, wherein the solution includes G-hooks, anti-fade media, and fiducials.

### Claim 19

A cartridge configured for purifying a hybridized target molecule sample and imaging the hybridized target molecule, comprising:
a buffer input area configured to hold a target molecule sample, the sample comprising:
a plurality of hybridized complexes including a plurality of target molecules, and each of reporter probes and capture probes,
a plurality of non-hybridized reporter probes, and
a plurality of non-hybridized capture probes;
a bubble vent configured to separate the sample input and the first binding chamber and to eliminate air bubbles;
a first binding chamber configured to receive or contain F magnetic beads and to receive the sample, wherein:
the F magnetic beads are functionalized with first molecules configured to bind with the non-hybridized reporter probes and hybridized complexes of the sample during a first period of time;
the first binding chamber is additionally configured to receive a first buffer to remove non-hybridized reporter probes from the sample after the non-hybridized reporter probes and hybridized complexes of the sample bind with the F magnetic beads;
a first elution channel attached to the first binding chamber and a means for heating, and which is configured to receive the F magnetic beads after the first period of time and configured for heating the F magnetic beads to elute a first eluted sample comprising the plurality of hybridized complexes and plurality of non-hybridized reporter probes;
a second binding chamber configured to receive or contain G magnetic beads and to receive the first eluted sample, wherein:
the G magnetic beads are functionalized with second molecules configured to bind with the hybridized complexes during a second period of time;
the second binding chamber is additionally configured to receive a second buffer remove at least non-hybridized capture probes;
a second elution channel attached to the second binding chamber and a means for heating, and which is configured to receive the G magnetic beads after the second period of time and configured for heating the G magnetic beads to elute a second eluted sample comprising the plurality of hybridized complexes;
a binding area having an active binding surface configured to receive the second eluted sample and bind with the hybridized complexes;
wherein the cartridge is operatively coupled to:
a plurality of off-card buffer input valves operatively coupled to a fluidic manifold, the plurality of off-card buffer input valves being configured to receive the first buffer and the second buffer from the fluidic manifold and provide the first and buffer to the cartridge; and
a plurality of waste valves configured to collect the first and second buffer from the cartridge.

### Claim 20 (depends on 1)

A system for imaging a plurality of hybridized complexes comprising;
a cartridge according to claim 1;
a cartridge tray operatively coupled to the system and configured to hold the cartridge;
a first heater operatively coupled to the cartridge;
a second heater operatively coupled to the cartridge;
a magnet operatively coupled to the imaging device below the cartridge tray;
a fluidic manifold operatively coupled to the system above the cartridge tray and configured to hold and control the flow of a plurality of buffers;
a plurality of off-card buffer input valves operatively coupled to the fluidic manifold and the cartridge;
a plurality of waste valves operatively coupled to the system above the cartridge tray; and
an imaging reference surface operatively coupled to the imaging device above the cartridge tray.

### Claim 21 (depends on 1)

A method for purifying a hybridized target molecule sample and imaging the hybridized target molecule, comprising:
providing the cartridge of claim 1;
receiving a hybridized sample, the sample comprising a plurality of hybridized complexes comprising target molecules hybridized with first probes and second probes, a plurality of non-hybridized first probes, and a plurality of non-hybridized second probes;
binding the non-hybridized first probes and hybridized complexes of the sample to a first affinity matrix in a first binding chamber during a first period of time;
flowing a first buffer into the first binding chamber to remove non-hybridized second probes from the sample after the non-hybridized first probes and hybridized complexes of the sample bind with the first affinity matrix;
directing the first affinity matrix into a first elution channel;
heating the first affinity matrix to elute a first eluted sample comprising the plurality of hybridized complexes and plurality of non-hybridized first probes;
binding the hybridized complexes of the first eluted sample to a second affinity matrix in a second binding chamber during a second period of time;
flowing a second buffer into the second binding chamber to remove the non-hybridized first probes from the first eluted sample after the hybridized complexes bind with the second affinity matrix;
heating the second affinity matrix to elute a second eluted sample comprising the plurality of hybridized complexes; and
binding the hybridized complexes to an active binding surface for imaging thereof.