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Publication Number
US-10865436-B2
Publication Date
2020-12-15
Expiration Date
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.
Core Innovation
The invention relates to a microfluidic device and kit for processing formalin-fixed paraffin-embedded (FFPE) tissue. It performs aqueous-compatible liquid-liquid extraction to dewax/deparaffinize by separating melted paraffin using an immiscible oil or an immiscible oil/wax precursor layer. The approach avoids xylene by using a phase-separation strategy in which only the aqueous phase is transported away for downstream processing.
The microfluidic system integrates, in a single microfluidic device, a liquid-liquid extraction process vessel, a nucleic acid purification process component, and at least one nucleic acid amplification reactor. A dedicated liquid-liquid extraction vessel is fluidically coupled via a microfluidic channel to a lumen structure configured to transport only an aqueous phase from the liquid-liquid extraction process vessel.
The device further provides a nucleic acid amplification pathway that is fluidically coupled to the nucleic acid purification process component via a second microfluidic channel that is not continuous with the first microfluidic channel. The integrated arrangement enables an end-to-end automated nucleic-acid workflow in which both the liquid-liquid extraction step and a nucleic acid purification step are performed in a single microfluidic device.
Claims Coverage
The partial content identifies one independent claim and one dependent claim. The independent claim covers integrated aqueous-only liquid-liquid extraction, nucleic acid purification, and nucleic acid amplification within a single microfluidic device, using a lumen structure disposed to transport only an aqueous phase and separate microfluidic channeling for purification-to-amplification.
Integrated microfluidic device for aqueous-only liquid-liquid extraction and downstream nucleic-acid processing
The microfluidic device disposes a liquid-liquid extraction process vessel, a nucleic acid purification process component, and at least one nucleic acid amplification reactor in a single microfluidic device such that a liquid-liquid extraction step removing only the aqueous phase from the liquid-liquid extraction process vessel and a nucleic acid purification step are both performed in a single microfluidic device.
Liquid-liquid extraction process vessel extending below the device plane
At least one liquid-liquid extraction process vessel extends orthogonally to a first plane, and at least a portion of the liquid-liquid extraction process vessel extends below the first plane within the microfluidic device.
Lumen structure disposed to transport only the aqueous phase
A separate lumen structure in the form of a tube is permanently and fixedly disposed, protruding into each liquid-liquid extraction process vessel, and disposed at a depth so as to transport only an aqueous phase in a liquid-liquid extraction process comprising an aqueous phase and an immiscible liquid phase.
Purification coupling via a first microfluidic channel parallel to the device plane
A nucleic acid purification process component is fluidically coupled to the at least one liquid-liquid extraction process vessel via a first microfluidic channel directly connected to the lumen structure and extending along a plane parallel to the first plane.
Amplification coupling via a second microfluidic channel not continuous with the first
At least one nucleic acid amplification reactor is 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.
Across the independent claim, the main inventive features focus on an integrated microfluidic device that combines an extraction vessel using a lumen to transport only the aqueous phase, fluidic coupling to a nucleic acid purification component, and separate coupling to nucleic acid amplification reactors through a non-continuous microfluidic channel. The dependent claim adds a structural limitation that the system consists of a single microfluidic device.
Stated Advantages
Avoiding xylene by performing dewax/deparaffinize using aqueous-compatible liquid-liquid extraction with an immiscible oil or silicone-oil/wax precursor layer.
Enabling end-to-end automated nucleic-acid processing in a single microfluidic device by integrating liquid-liquid extraction, nucleic acid purification, and nucleic acid amplification reactors.
Improved or comparable DNA purity (A260/A280) and DNA yield compared with traditional xylene extraction, as reported in the partial content.
Documented Applications
Processing formalin-fixed paraffin-embedded (FFPE) tissue samples for dewaxing/deparaffinization and downstream nucleic-acid processing using microfluidic liquid-liquid extraction and nucleic-acid amplification within a single device.
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