Methods and systems for continuous flow cell lysis in a microfluidic device
The present invention relates to methods and systems for cell lysis in a microfluidic device. More specifically, embodiments of the present invention relate to methods and systems for rapid continuous flow mechanical cell lysis. In one embodiment, a microfluidic device includes one or more microfluidic channels, each channel comprising constricted regions and non-constricted regions separating the constricted regions, wherein the constricted regions are configured to disrupt the cellular membranes of cells in fluid flowing through the one or more microfluidic channels.
1. A method for fabricating a microfluidic device using soft-lithography replica molding, comprising:
fabricating a first piece of the microfluidic device in a mold, the first piece including at least one microchannel having features sized to provide for mechanical cell lysis, the features including constricted regions and non-constricted regions separating the constricted regions, the non-constricted regions being shaped to converge at the ends into the constricted regions, wherein fabricating the first piece comprises:
pouring a first layer of a thermoplastic prepolymer onto the mold; and
partially curing the first layer under exposure to UV-light;
fabricating a second piece of the microfluidic device on a glass slide, wherein fabricating the second piece comprises:
coating the glass slide with a layer of the thermoplastic prepolymer; and
partially curing the layer coated on the glass slide under exposure to UV-light;
removing the partially cured first piece from the mold, wherein the partially cured first layer is removed from the mold without loss of the features of the at least one microchannel;
contacting the first piece to the second piece; and
bonding the first piece and the second piece together on the glass slide by curing the contacted first and second pieces under exposure to UV-light.
2. The method of claim 1 , wherein the microfluidic device includes one or more channels comprising features including constricted regions and non-constricted regions separating the constricted regions, wherein the constricted regions are configured to mechanically lyse cells.
3. The method of claim 2 , wherein the one or more microfluidic channels are parallel to each other.
4. The method of claim 2 , wherein the non-constricted regions are arranged in a honeycomb-like pattern.
5. The method of claim 2 , wherein each of the non-constricted regions has a width of 40-100 μm, and each of the constricted regions has a width of 3-10 μm.
6. The method of claim 2 , wherein each of the non-constricted regions has a length of 60-120 μm, and each of the constricted regions has a length of 10-20 μm.
7. The method of claim 2 , wherein there are 1-40 microfluidic channels.
8. The method of claim 2 , wherein each microfluidic channel comprises 3-15 constricted regions.
9. The method of claim 2 , wherein each microfluidic channel comprises a first segment of constricted regions along a fluid flow path having a width of 6-8 μm and a second segment of constricted regions along a fluid flow path having a width of 4-6 μm.
10. The method of claim 2 , wherein each of the constricted regions has a width of 2.5-4.5 μm.
11. The method of claim 2 , wherein each of the microfluidic channels has 4 constricted regions and each constricted region has a width of 3 μm.
12. The method of claim 2 , wherein there are 20 microfluidic channels.
13. The method of claim 2 , wherein there are 40 microfluidic channels.
14. The method of claim 2 , wherein the microfluidic channels are configured to support a flow rate of from about 20 μ/min to about 2000 μL/min.
15. The method of claim 2 , wherein the microfluidic device is configured to support a cell-lysis rate of 85-100%.
16. The method of claim 1 , wherein the thermoplastic prepolymer comprises an off-stoichiometry thiol-ene (OSTE) prepolymer.
17. The method of claim 16 , wherein the OSTE prepolymer has an excess of allyl groups.
18. The method of claim 1 , wherein bonding the first piece and the second piece together on the glass slide further comprises baking the first and second pieces.
19. The method of claim 1 , wherein fabricating the first piece further comprises:
pouring a second layer of the thermoplastic prepolymer onto the mold; and
degassing the second layer under vacuum.
20. The method of claim 1 , wherein fabricating the first piece further comprises removing any air bubbles from the first piece.
21. The method of claim 1 , wherein bonding the first piece and the second piece together on the glass slide further comprises baking the first and second pieces at a temperature close to the glass transition temperature of a thermoplastic polymer made from the thermoplastic prepolymer.
22. The method of claim 1 , wherein fabricating the first piece of the microfluidic device further comprises applying a release agent onto the mold.
23. The method of claim 1 , wherein fabricating the first piece of the microfluidic device further comprises degassing the first layer under vacuum.
24. The method of claim 1 , wherein fabricating the second piece of the microfluidic device further comprises applying a release agent onto the glass.