# US 8,772,017 B2 · Integrated nucleic acid assays

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- Number: US 8,772,017 B2
- Document ID: US8772017B2
- Status: Granted
- Kind: B2
- Application number: 13/492,612
- Filed: 2012-06-08
- Published: 2012-12-27
- Granted: 2014-07-08
- Earliest claimed: 2006-03-15
- Assignees: Micronics, Inc.
- Applicants: C. Frederick Battrell; John Gerdes; John R. Williford; Denise Maxine Hoekstra; Wayne L. Breidford; Stephen Mordue; John Clemmens; Melud Nabavi; Mark Kokoris
- Inventors: C. Frederick Battrell; John Gerdes; John R. Williford; Denise Maxine Hoekstra; Wayne L. Breidford; Stephen Mordue; John Clemmens; Melud Nabavi; Mark Kokoris
- Web page: https://patentgrep.com/patent/US8772017B2

## CPC

- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- B01F 31/65: the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column
- B01F 33/30: Micromixers
- 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}
- C12Q 1/686: Polymerase chain reaction [PCR]
- B01L 3/502723: {characterised by venting arrangements}
- B01L 3/50273: {characterised by the means or forces applied to move the fluids}
- B01L 3/502738: {characterised by integrated valves (throttle valves in microfluidic sample containers B01L3/502746)}
- B01L 3/502746: {characterised by the means for controlling flow resistance, e.g. flow controllers, baffles or throttle valves}
- B01L 7/52: {with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples}
- B01L 2200/082: Handling hazardous material
- B01L 2200/10: Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
- B01L 2200/16: Reagents, handling or storing thereof
- B01L 2300/0663: Whole sensors
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/0867: Multiple inlets and one sample wells, e.g. mixing, dilution
- B01L 2300/087: Multiple sequential chambers
- B01L 2300/0887: Laminated structure
- B01L 2400/0481: squeezing of channels or chambers
- B01L 2400/0638: membrane valves, flap valves
- B01L 2400/0655: pinch valves
- 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
- B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06)
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

## Abstract

Integrated microfluidic cartridges for nucleic acid extraction, amplification, and detection from clinical samples are disclosed. The devices are single-entry, sanitary, and disposable. The devices enable simplex or multiplex nucleic acid target detection, as for example: assay panels for multiple infectious agents, or assay panels for cancerous cell types. Methods for use of microfluidic cartridges in a fully automated, pneumatically controlled apparatus are also disclosed.

## Claims

### Claim 1

A microfluidic cartridge for extracting a nucleic acid fraction from a sample, comprising:
a) a loading port for introduction of the sample;
b) a waste chamber;
c) a lysis sub-circuit operably linked to said loading port, wherein said cell lysis sub-circuit comprises: 1) a mixing chamber; 2) a sealed pouch containing lysis reagent; and 3) a pump member configured for pumping said lysis reagent into said mixing chamber, wherein said lysis sub-circuit is configured to generate a lysed sample from the sample in said mixing chamber; and
d) a nucleic acid extraction sub-circuit operably linked to both of said lysis sub-circuit and said waste chamber, wherein said nucleic acid extraction sub-circuit comprises: 1) a solid phase nucleic acid extraction component configured to bind nucleic acids present in said lysed sample; 2) a sealed pouch containing wash reagent; 3) a sealed pouch containing elution reagent; and 4) a pump member configured for pumping said elution reagent over said solid phase nucleic acid extraction component to generate a mixture of extracted nucleic acids;
wherein said microfluidic cartridge is configured to pump said lysis reagent through a valved connection into said mixing chamber of said lysis subcircuit and said microfluidic cartridge is configured to pump said elution reagent through a valved connection to said solid phase nucleic acid extraction component of said nucleic acid extraction subcircuit;
wherein said pump members are configured to be pneumatically driven by applied positive pressure strokes and suction pressure strokes;
wherein said applied positive pressure strokes and suction pressure strokes enable reciprocating flow through said valved connections; and
wherein the pump member for pumping said elution reagent is a pneumatically actuatable, elastomeric diaphragm configured to enable slug elution of said mixture of extracted nucleic acids.

### Claim 2 (depends on 1)

The microfluidic cartridge of claim 1, wherein said applied positive pressure strokes and suction pressure strokes are configured to enable fragmentation of high molecular weight nucleic acid into fragments of lower molecular weight.

### Claim 3 (depends on 1)

The microfluidic cartridge of claim 1, wherein said lysis reagent comprises a chaeotrope-detergent mixture; said wash reagent is a nucleic acid precipitant; and said elution reagent is an aqueous buffer.

### Claim 4 (depends on 1)

The microfluidic cartridge of claim 1, wherein said elution reagent is formulated to rehydrate a mixture containing reagents and enzymes.

### Claim 5 (depends on 1)

The microfluidic cartridge of claim 1, wherein said nucleic acid eluate is extracted for medical or environmental testing.

### Claim 6 (depends on 1)

The microfluidic cartridge of claim 1, wherein said mixing chamber is provided with a suction port for aspirating the liquid sample thereinto, said suction port having a water impermeable, gas permeable membrane.

### Claim 7 (depends on 6)

The microfluidic cartridge of claim 6, wherein the water impermeable, gas permeable membrane is hydrophobic.

### Claim 8 (depends on 6)

The microfluidic cartridge of claim 6, wherein the water impermeable, gas permeable membrane is hydrophilic.

### Claim 9 (depends on 1)

The microfluidic cartridge of claim 1, wherein said waste chamber comprises a sanitary waste collection chamber for capturing waste fluids on the microfluidic cartridge, said waste collection chamber having a fluid inlet and valved fluid connection to said nucleic acid extraction subcircuit, an absorbent pad within said chamber, and an elastomeric or flexible film separating said fluid inlet from an outside vent, said outside vent for venting said waste chamber through said external surface.

### Claim 10 (depends on 9)

The microfluidic cartridge of claim 9, wherein the elastomeric film covers the absorbent pad.