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Publication Number
US-10081828-B2
Publication Date
2018-09-25
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 concerns a microfluidic apparatus and workflow for processing formalin-fixed paraffin-embedded (FFPE) tissue. A dedicated liquid-liquid extraction vessel performs dewaxing by heating an aqueous solution above the paraffin melting point while an immiscible oil layer remains present as an evaporation barrier and capture medium for melted paraffin.
The liquid-liquid extraction removes paraffin from the tissue sample while the immiscible liquid remains in the vessel, and at least a portion of the paraffin-extracted tissue sample is removed after extraction. The extraction is performed in a single microfluidic device, with a microfluidic device architecture that separates the extraction vessel from a nucleic acid amplification reactor via distinct microfluidic channels.
After dewaxing, the tissue remains in the aqueous phase for subsequent de-crosslinking, cell lysis, and nucleic acid purification, followed by transfer to nucleic acid amplification reactors. A kit is described with integrated extraction/purification components and reagents, including a chaotropic buffer plus a surfactant and a protein digesting enzyme such as Proteinase K.
Claims Coverage
The relevant independent claim covers a microfluidic method for removing paraffin from a paraffin-embedded tissue sample using a liquid-liquid extraction in a single microfluidic device. The claim includes three main inventive themes.
Single microfluidic device liquid-liquid extraction for paraffin removal
Liquid-liquid extraction is performed to remove paraffin from the tissue sample while the extraction is performed in a single microfluidic device.
Heating aqueous solution above paraffin melting point during extraction
The liquid-liquid extraction further comprises raising the temperature of the aqueous solution in the vessel above the melting point of the paraffin embedding the tissue sample.
Immiscible liquid as extraction medium with lower density and higher boiling point
An immiscible liquid having a density less than the density of the aqueous solution and a boiling point greater than the boiling point of the aqueous solution is provided in the vessel, and at least a portion of the liquid-liquid paraffin-extracted tissue sample is removed while the immiscible liquid remains in the vessel.
Across the independent claim, paraffin removal in a single microfluidic device is achieved by liquid-liquid extraction that heats the aqueous solution above the paraffin melting point while an immiscible liquid remains in the vessel, enabling removal of at least a portion of the paraffin-extracted tissue sample.
Stated Advantages
Improved DNA yield and purity compared to traditional xylene extraction (as evidenced by example performance metrics).
Documented Applications
Processing formalin-fixed paraffin-embedded (FFPE) tissue, including dewaxing (paraffin removal), followed by de-crosslinking, cell lysis, nucleic acid purification, and nucleic acid amplification.
Use within a workflow that transfers processed material to nucleic acid amplification reactors via distinct microfluidic channels.
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