Microfluidic apparatus, method, and applications
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.
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 of 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 fluidically coupled to the lumen structure and extends parallel to the first plane; and
wherein the at least one liquid-liquid extraction process vessel, and the nucleic acid purification process component are disposed in a single one of the microfluidic device such that only the aqueous phase of a liquid-liquid extraction step performed in the liquid-liquid extraction process vessel is removed 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.
2. The microfluidic device of claim 1 , wherein the aqueous phase is a mixture of a chaotropic buffer and a surfactant.
3. The microfluidic device of claim 1 , wherein the aqueous phase includes an added protein digesting enzyme.
4. The microfluidic device of claim 1 , wherein the at least one dedicated liquid-liquid process component enables an extraction of paraffin from a paraffin embedded tissue sample.
5. The microfluidic device of claim 1 , wherein the dedicated liquid-liquid extraction vessel includes a volume of material selected from the group consisting of silicon oil, mineral oil, and a solid silicon oil/wax mixture.
6. The microfluidic device of claim 1 , wherein the liquid-liquid extraction vessel interfaces with a heater to control the temperature of the aqueous phase.
7. The microfluidic device of claim 1 , wherein the nucleic acid purification process component is composed of a silica matrix.
8. The microfluidic device of claim 1 , wherein the nucleic acid purification component is composed of silica beads.
9. The microfluidic device of claim 1 , comprising at least one nucleic acid amplification reactor fluidically coupled to the nucleic acid purification process component.
10. The microfluidic device of claim 9 , wherein the nucleic acid amplification reactor interfaces with a heater to control the temperature of the nucleic acid amplification reactor.
11. The microfluidic device of claim 9 , wherein the aqueous phase is a mixture of a chaotropic buffer and a surfactant.
12. The microfluidic device of claim 9 , wherein the aqueous phase includes an added protein digesting enzyme.
13. The microfluidic device of claim 9 , wherein the at least one dedicated liquid-liquid process component enables an extraction of paraffin from a paraffin embedded tissue sample.
14. The microfluidic device of claim 9 , wherein the dedicated liquid-liquid extraction vessel includes a volume of material selected from the group consisting of silicon oil, mineral oil, and a solid silicon oil/wax mixture.
15. The microfluidic device of claim 9 , wherein the liquid-liquid extraction vessel interfaces with a heater to control the temperature of the aqueous phase.
16. The microfluidic device of claim 9 , wherein the nucleic acid purification process component is composed of a silica matrix.
17. The microfluidic device of claim 9 , wherein the nucleic acid purification component is composed of silica beads.