Methods and systems for using encapsulated microbubbles to process biological samples

• US 9,982,290 B2
• Filed: 10/04/2013
• Issued: 05/29/2018
• Est. Priority Date: 10/04/2012
• Status: Active Grant

• Current Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Janzen, William Perry, Pattenden, Samantha Gail, Jayakody, Chatura Nadisha, Streeter, Jason Eric, Dayton, Paul Alexander, Wood, Cameron Champion, Shenoy, Siddharth Kaup
• Primary Examiner(s): Claytor, Renee
• Assistant Examiner(s): FERNANDEZ, SUSAN EMILY
• Application Number:
  US14/432,747
• Publication Number:
  US 20150252355A1
• Time in Patent Office: 1,698 Days
• CPC Class Codes: C12M 23/36 Means for collection or storage of gas; Gas holders
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: G01N 1/38 Diluting, dispersing or mixing samples
• CPC Class Codes: G01N 2001/387 mixing by blowing a gas, bubbling

Abstract

Methods and systems for using encapsulated microbubbles to process biological samples are disclosed. According to one aspect, a method for using encapsulated microbubbles to process a biological sample includes creating a mixture comprising encapsulated microbubbles mixed with a biological sample and adding activation energy to the mixture to cause at least some of the microbubbles to oscillate or burst and thereby process the sample, including effecting cell lysis, shearing DNA, and/or performing tissue dispersion.

Claims

1. A method for using nanodroplets to process a biological sample, the method comprising:

creating a mixture comprising nanodroplets mixed with a biological sample, wherein the nanodroplets each comprise a liquid core which remains metastable in a liquid form until the application of conversion energy and wherein the biological sample comprises DNA or DNA cross-linked to protein extracted from cells; and

adding the conversion energy to the mixture to cause at least some of the nanodroplets to convert to encapsulated microbubbles, wherein the encapsulated microbubbles comprise of at least one of a lipid, a surfactant, an emulsifier, a polymer, and a protein and adding activation energy to the mixture to cause the encapsulated microbubbles to oscillate or burst, and thereby processing the biological sample sufficiently to shear the DNA or DNA cross-linked to the protein in the biological sample and produce DNA fragments having a size distribution narrower than a size distribution of DNA fragments produced through sonication of the biological sample without using metastable nanodroplets.

2. The method of claim 1 wherein a majority of the encapsulated microbubbles have a diameter in the range from 0.1 microns to 10 microns.

3. The method of claim 1 wherein the biological sample comprises cells and wherein processing the sample comprises effecting cell lysis.

4. The method of claim 3 wherein the cells comprise bacteria or yeast cells.

5. The method of claim 1 wherein the biological sample comprises tissue and wherein processing the sample comprises performing tissue dispersion.

6. The method of claim 1 wherein the biological sample comprises at least one of fresh tissue, cryogenically preserved tissue, and fixed and paraffin embedded tissue.

7. The method of claim 1 wherein adding the activation energy to the mixture comprises sonicating the mixture.

8. The method of claim 7 wherein sonicating the mixture includes applying energy having a frequency in the range from 0.01 MHz to 10.0 MHz.

9. The method of claim 1 wherein adding the activation energy to the mixture comprises exposing the mixture to laser light.

10. The method of claim 1 wherein creating the mixture comprises adding a solution of the nanodroplets to the sample and mixing the solution with the sample.

11. The method of claim 10 wherein adding the solution of nanodroplets to the biological sample comprises adding the solution to a plurality of biological samples located in individual wells of a multi-well sample plate, wherein mixing the solution includes mixing the solution with the biological samples in the wells, and wherein adding the activation energy to the mixtures includes adding the energy to each of the mixtures in the wells to perform high throughput processing of a plurality of biological samples.

12. The method of claim 10 wherein adding conversion energy comprises sonicating the mixture.

13. The method of claim 12 wherein sonicating the mixture includes adding energy having a frequency in the range from 0.01 MHz to 10.0 MHz.

14. The method of claim 10 wherein adding the conversion energy comprises exposing the mixture to laser light.

15. The method of claim 10 where a majority of nanodroplets have a diameter in the range from 100 to 750 nanometers.

16. The method of claim 1 wherein the nanodroplets comprise a shell surrounding the liquid core, wherein the liquid core converts to a gas upon the addition of the conversion energy.

17. The method of claim 16 wherein the liquid core comprises a hydrocarbon or a perfluorocarbon.

18. The method of claim 17 wherein the liquid core comprises at least one of isopentane, perfluoropentane, and perfluorohexane.

19. The method of claim 1 wherein creating the mixture comprises adding the nanodroplets to the biological sample prior to or during the addition of the conversion energy.

20. The method of claim 1 wherein the nanodroplets comprise encapsulated nanodroplets.

21. The method of claim 1 wherein the liquid core comprises perfluorobutane or perfluoropropane.