# US 11,896,978 B2 · Assay cartridges and methods of using the same

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- Number: US 11,896,978 B2
- Document ID: US11896978B2
- Status: Granted
- Kind: B2
- Application number: 17/032,524
- Filed: 2020-09-25
- Published: 2021-03-04
- Granted: 2024-02-13
- Earliest claimed: 2011-01-06
- Assignees: Meso Scale Technologies, LLC.
- Applicants: Meso Scale Technologies, LLC.
- Inventors: Eli N. Glezer; Stephen Higgins; Sandor Kovacs; Sudeep Kumar; Kenneth Page; Kristian Roth; George Sigal
- Web page: https://patentgrep.com/patent/US11896978B2

## CPC

- B01L 7/525: {with physical movement of samples between temperature zones}
- B01L 3/5027: {by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip}
- B01L 2200/027: for microfluidic devices
- B01L 2200/0605: Metering of fluids
- B01L 2200/0621: Control of the sequence of chambers filled or emptied
- B01L 2200/0631: Purification arrangements, e.g. solid phase extraction [SPE]
- B01L 2200/10: Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
- B01L 2200/146: Employing pressure sensors
- B01L 2200/16: Reagents, handling or storing thereof
- B01L 2300/0645: Electrodes
- 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 2300/0867: Multiple inlets and one sample wells, e.g. mixing, dilution
- B01L 2300/087: Multiple sequential chambers
- B01L 2300/0883: Serpentine channels
- B01L 2400/0487: fluid pressure, pneumatics
- B01L 2400/0688: surface tension valves, capillary stop, capillary break
- B01L 2400/0694: vents used to stop and induce flow, backpressure valves
- C12Q 1/686: Polymerase chain reaction [PCR]
- 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

Assay cartridges are described that have purification, reaction, and detection zones and other fluidic components which can include sample chambers, waste chambers, conduits, vents, reagent chambers, reconstitution chambers and the like. The assay cartridges are used to conduct multiplexed nucleic acid measurements. Also described are kits including such cartridges, methods of using the same, and a reader configured to analyze an assay conducted using an assay cartridge.

## Claims

### Claim 1

A method of conducting an analysis of a sample in an assay cartridge, said cartridge comprising a fluidic network and a plurality of chambers, wherein said fluidic network comprises:
a primary flow path and one or more fluidic conduits each intersecting said primary flow path and fluidically connecting said primary flow path to a chamber of said plurality of chambers, wherein said fluidic network is configured to meter a volume of fluid in said fluidic network, said method comprising the steps of:
(i) interfacing the fluidic network with a fluidic control manifold configured to drive fluid motion within said fluidic network;
(ii) metering a volume of sample from said chamber to a second chamber of the plurality of chambers;
(iii) metering a volume of lysis buffer from a third chamber of said plurality of chambers to said second chamber;
(iv) lysing said volume of sample;
(v) moving lysate from said second chamber to a purification zone of said primary flow path;
(vi) extracting nucleic acid from said lysate;
(vii) purifying said nucleic acid;
(viii) moving said purified nucleic acid to a PCR reaction zone of said primary flow path;
(ix) contacting said purified nucleic acid mixture with one or more PCR reagents to form a mixture;
(x) shuttling said mixture formed in step (ix) between first and second temperature controlled zones;
(xi) moving said mixture formed in step (x) to a detection zone; and
(xii) measuring a signal from said detection zone.

### Claim 2 (depends on 1)

The method of claim 1, further comprising the steps of, after step (x), repeating steps (ix) and (x) to form an amplified product mixture;
and contacting said amplified product mixture with a detection reagent.

### Claim 3 (depends on 1)

The method of claim 1, wherein the one or more PCR reagents are dried reagents in the form of a dry pill.

### Claim 4 (depends on 3)

The method of claim 3, wherein dry pill is completely immersed in the purified nucleic acid in the PCR reaction zone.

### Claim 5 (depends on 4)

The method of claim 4, wherein the dry pill is dissolved in a mixture formed in step (ix).

### Claim 6

A method of conducting a PCR analysis of a sample in an assay cartridge, said cartridge comprising a fluidic network and a plurality of chambers, wherein said fluidic network comprises:
(a) a primary flow path comprising, from a proximate to a distal end, (i) an inlet, (ii) a purification zone, (iii) a PCR reaction zone including a first reaction temperature controlled zone and a second reaction temperature controlled zone, and (iv) a detection zone, wherein said primary flow path further comprises (vi) one or more air vent ports, and
(b) one or more fluidic conduits each intersecting said primary flow path and fluidically connected to one or more of said plurality of chambers, wherein each of said plurality of chambers are connected to an additional air vent port,
wherein said plurality of chambers include: a sample chamber, a lysis reagent chamber, a lysis chamber, a purification reagent chamber, a plurality of PCR reagent chambers, and one or more waste chambers;
said method comprising the steps of:
(i) interfacing the fluidic network with a fluidic control manifold configured to drive fluid motion within said fluidic network.
(ii) metering a volume of sample from said sample chamber to said lysis chamber;
(iii) metering a volume of lysis buffer from said lysis reagent chamber to said lysis chamber;
(iv) lysing said volume of sample;
(v) moving lysate from said lysis chamber to said purification zone;
(vi) extracting nucleic acid from said lysate;
(vii) purifying said nucleic acid;
(viii) moving said a purified nucleic acid to said PCR reaction zone;
(ix) contacting said purified nucleic acid with one or more PCR reagents to form a mixture;
(x) shuttling said mixture formed in step (ix) between said first and second temperature controlled zones;
(xi) repeating steps (ix) and (x) to form an amplified product mixture;
(xii) contacting said amplified product mixture with a detection reagent;
(xiii) moving said mixture formed in step (xi) to said detection zone; and
(xiv) measuring a signal from said detection zone.

### Claim 7 (depends on 6)

The method of claim 6, wherein said purification zone comprises, from a proximal to a distal end,
(i) a purification multi-conduit fluidic junction including:
(a) a first purification reagent chamber conduit connecting said primary flow path and said purification reagent chamber; and
(b) a second purification reagent chamber conduit connecting said purification reagent chamber and a purification reagent chamber air vent port;
(ii) an integrated purification membrane positioned in said primary flow path of said purification zone; and
(iii) a waste multi-conduit fluidic junction including:
(a) a first waste chamber conduit connecting said primary flow path and said waste chamber; and
(b) a second waste chamber conduit connecting said waste chamber and a waste chamber air vent port; and
said extracting step (vi) comprises:
(xx) capturing lysate on said membrane;
(yy) removing eluent to said first waste chamber; and
(zz) washing said membrane with one or more purification reagents.

### Claim 8 (depends on 6)

The method of claim 6, wherein said purifying step (vii) comprises
(xx) drying a membrane;
(yy) washing said membrane with purification reagent; and
(zz) eluting nucleic acid from said membrane.

### Claim 9 (depends on 6)

The method of claim 6, wherein said cartridge further comprises a plurality of reconstitution chambers each comprising lyophilized PCR reagents; and
contacting step (ix) further comprises reconstituting one or more of said lyophilized PCR reagents.

### Claim 10 (depends on 9)

The method of claim 9, wherein said cartridge further comprises a first reconstitution chamber comprising a first set of PCR reagents selected from reverse transcriptase and dNTPs, and said method further comprises reconstituting said first set of PCR reagents in said contacting step (ix) and shuttling said mixture formed in said contacting step between said first and second temperature controlled zone.

### Claim 11 (depends on 10)

The method of claim 10, wherein said cartridge further comprises a second reconstitution chamber comprising a second set of PCR reagents selected from dNTPs, primers, and polymerase, and said method further comprises reconstituting said second set of PCR reagents in said contacting step (ix) and shuttling said mixture formed in said contacting step between said first and second temperature controlled zone.

### Claim 12 (depends on 11)

The method of claim 11, wherein said cartridge further comprises a third reconstitution chamber comprising a third set of PCR reagents comprising additional primers, and said method further comprises reconstituting said third set of PCR reagents in said contacting step (ix) and shuttling said mixture formed in said contacting step between said first and second reaction temperature controlled zone.

### Claim 13 (depends on 12)

The method of claim 12, wherein said cartridge further comprises a fourth reconstitution chamber comprising a fourth set of PCR reagents selected from EDTA, and salt, and said method further comprises reconstituting said fourth set of PCR reagents in said contacting step (ix) and shuttling said mixture formed in said contacting step between said first and second reaction temperature controlled zone.

### Claim 14 (depends on 13)

The method of claim 13, wherein said cartridge further comprises a fifth reconstitution chamber comprising detection reagents and said contacting step (xi) comprises reconstituting said detection reagents with said amplified product mixture.

### Claim 15 (depends on 6)

The method of claim 6, wherein said fluidic control manifold comprises a pump and a pressure sensor, the method further comprising aligning said fluidic control manifold with one or more air vent ports positioned in said fluidic network.

### Claim 16 (depends on 6)

The method of claim 6, wherein a fluidic conduit of said one or more fluidic conduits comprises a multi-conduit fluidic junction including (i) a first conduit connecting said primary flow path and a chamber of the plurality of chambers, and (ii) a second conduit connecting said chamber to a respective additional air vent port, wherein said fluidic conduit is in communication with an optical fluid sensor at a position distal to said fluidic junction and said metered volume of fluid is defined by a distance between said fluidic junction and said distal position.

### Claim 17 (depends on 16)

The method of claim 16, further comprising detecting a fluid flow by the optical fluid sensor.

### Claim 18 (depends on 17)

The method of claim 17, further comprising operating at least one of the one or more air vent ports and the additional air vent port to achieve the metered volume of fluid.

### Claim 19 (depends on 6)

The method of claim 6, wherein shuttling said mixture formed in step (ix) between said first and second temperature controlled zones further includes shuttling said mixture through a restriction zone.