# US 10,865,436 B2 · Microfluidic apparatus, method, and applications

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- Number: US 10,865,436 B2
- Document ID: US10865436B2
- Status: Granted
- Kind: B2
- Application number: 14/478,012
- Filed: 2014-09-05
- Published: 2014-12-25
- Granted: 2020-12-15
- Earliest claimed: 2011-11-17
- Assignees: Rheonix, Inc.
- Applicants: Rheonix, Inc.
- Inventors: Peng Zhou
- Web page: https://patentgrep.com/patent/US10865436B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces}
- C07H 1/08: from natural products
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- B01L 2200/0631: Purification arrangements, e.g. solid phase extraction [SPE]
- 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
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A microfluidic apparatus, method, and associated applications utilize and apply to a formalin-fixed paraffin-embedded (FFPE) tissue sample and performing a liquid-liquid extraction to remove the paraffin from the tissue sample prior to a nucleic acid purification step. A microfluidic device includes a dedicated liquid-liquid extraction process vessel, a nucleic acid purification process component, and a nucleic acid amplification reactor. A liquid-liquid extraction and nucleic acid purification kit includes a microfluidic device capable of performing both a liquid-liquid extraction process and a nucleic acid purification process, including a dedicated liquid-liquid extraction process vessel, an immiscible liquid or a precursor phase thereof disposed in the vessel, a nucleic acid purification process component, a nucleic acid amplification reactor fluidically, and a supply of reagents suitable to enable the liquid-liquid extraction process and the nucleic acid purification process.

## Claims

### Claim 1

A microfluidic device having a bottom surface extending along a first plane and a top surface extending along a second plane, comprising:
at least one liquid-liquid extraction process vessel extending orthogonally to the first plane, wherein at least a portion of the at least one liquid-liquid extraction process vessel extends below the first plane;
a separate lumen structure in the form of a tube permanently and fixedly disposed, and protruding into, each of the at least one liquid-liquid extraction process vessel, wherein said lumen structure is disposed at a depth so as to transport only an aqueous phase in a liquid-liquid extraction process comprising the aqueous phase and an immiscible liquid phase;
a nucleic acid purification process component fluidically coupled to the at least one liquid-liquid extraction process vessel via a first microfluidic channel, wherein the first microfluidic channel is directly connected to the lumen structure and extends along a plane parallel to the first plane; and
at least one nucleic acid amplification reactor fluidically coupled to the nucleic acid purification process component via at least a second microfluidic channel that is not continuous with the first microfluidic channel,
wherein the at least one liquid-liquid extraction process vessel, the nucleic acid purification process component, and the at least one nucleic acid amplification reactor are disposed in a single one of the microfluidic device such that a liquid-liquid extraction step performed in the liquid-liquid extraction process vessel comprising removing only the aqueous phase from the liquid-liquid extraction process vessel via the lumen and a nucleic acid purification step performed in the nucleic acid purification process component are both performed in a single one of the microfluidic device.

### Claim 2 (depends on 1)

The microfluidic device of claim 1, consisting of a single microfluidic device.