Dimethylacrylamide (DMAA) Hydrogel for Expansion Microscopy (ExM)
원문보기
IPC분류정보
국가/구분
United States(US) Patent
공개
국제특허분류(IPC7판)
A61L-027/16
G01N-001/31
C08F-220/56
C08L-033/26
A61L-027/56
출원번호
17144551
(2021-01-08)
공개번호
20210196856
(2021-07-01)
발명자
/ 주소
Boyden, Edward Stuart
Obidin, Nikita
Gao, Ruixuan
Gao, Linyi
출원인 / 주소
Boyden, Edward Stuart
인용정보
피인용 횟수 :
0인용 특허 :
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초록▼
The invention provides a method for preparing an expanded cell or tissue sample suitable for microscopic analysis. Expanding the sample can be achieved by binding, e.g., anchoring, key biomolecules to a DMAA-TF polymer network and swelling, or expanding, the polymer network, thereby moving the biomo
The invention provides a method for preparing an expanded cell or tissue sample suitable for microscopic analysis. Expanding the sample can be achieved by binding, e.g., anchoring, key biomolecules to a DMAA-TF polymer network and swelling, or expanding, the polymer network, thereby moving the biomolecules apart as further described herein. As the biomolecules are anchored to the polymer network isotropic expansion of the polymer network retains the spatial orientation of the biomolecules resulting in an expanded, or enlarged, sample.
대표청구항▼
1. A method for preparing an expandable tissue sample, the method comprising the steps of: (a) incubating the sample with a composition comprising acrylamide, dimethylacrylamide, and sodium acrylate linear monomers at a concentration of about 20 about 50 wt % of which sodium acrylate comprises about
1. A method for preparing an expandable tissue sample, the method comprising the steps of: (a) incubating the sample with a composition comprising acrylamide, dimethylacrylamide, and sodium acrylate linear monomers at a concentration of about 20 about 50 wt % of which sodium acrylate comprises about 10 to about 25 mol %, about 0.1 to about 1.0 mol % polymerization initiator, about 0.005 to about 0.02 wt % polymerization inhibitor and about 0.001 to about 0.01 wt % polymerization accelerator; and(b) polymerizing the composition within the sample to form a polymer, wherein the polymer is embedded throughout the sample. 2. The method according to claim 1, wherein prior to incubating the sample with the composition, the sample is treated with a chemical anchoring reagent. 3. The method according to claim 2, wherein the chemical anchoring reagent is acryloyl-X. 4. The method according to claim 2, wherein polymerizing results in covalent anchoring of biomolecules within the sample to the polymer to form a sample-polymer complex. 5. The method according to claim 4, wherein the biomolecules within the sample are anchored to the polymer during and/or after said polymerization. 6. The method according to claim 1, wherein the concentration of linear monomers in the composition is about 30% to about 40%. 7. The method according to claim 6, wherein the concentration of linear monomers is about 35%. 8. The method according to claim 6, wherein the concentration of linear monomers is about 33%. 9. The method according to claim 1, wherein the sodium acrylate is about 15 to about 20 mol % of the linear monomer concentration. 10. The method according to claim 9, wherein the sodium acrylate is about 15 mol % of the linear monomer concentration. 11. The method according to claim 9, wherein the sodium acrylate is about 20 mol % of the linear monomer concentration. 12. The method according to claim 1, wherein the composition comprises about 0.4 mol % polymerization initiator. 13. The method according to claim 12, wherein the polymerization initiator is potassium persulfate (KPS). 14. The method according to claim 1, wherein the composition comprises about 0.005 wt % polymerization accelerator. 15. The method according to claim 14, wherein the polymerization accelerator is TEMED. 16. The method according to claim 1, wherein the composition comprises about 0.01 wt % polymerization inhibitor. 17. The method according to claim 16, wherein the polymerization inhibitor is 4-Hydroxy-TEMPO. 18. The method according to claim 1, wherein prior to performing the permeating step, the sample is treated with a detergent. 19. The method according to claim 1, further comprising the step of adding an aqueous solvent or liquid to cause the sample-polymer complex to swell, thereby physically expanding the complex. 20. The method according to claim 19, wherein the sample-polymer complex expands isotropically. 21. The method according to claim 19, wherein the aqueous solvent or liquid is water. 22. The method according to claim 19, wherein prior to the step of adding the aqueous solvent or liquid to swell the sample-polymer complex, the complex is subjected to digestion. 23. The method according to claim 22, wherein the digestion comprises incubating the complex with a composition comprising a non-specific protease. 24. The method according to claim 23, wherein the composition comprises a non-specific protease in a buffer comprising a metal ion chelator, a non-ionic surfactant, and a monovalent salt. 25. The method according to claim 24, wherein the composition comprises about 1 to about 100 U/ml of a non-specific protease in a buffer having a pH between about 4 and about 12, the buffer comprising about 5 mM to about 100 mM of a metal ion chelator, about 0.1% to about 1.0% of a nonionic surfactant, and about 0.05 M to about 1.0 M monovalent salt, for about 0.5 to about 3 hours at about 50° C. to about 70° C. 26. The method according to claim 19, further comprising the step of producing a high-resolution image of the sample by viewing the enlarged sample under a microscope. 27. The method according to claim 19, further comprising the step of optically imaging the enlarged sample by viewing the sample under a microscope. 28. A method for preparing an expandable cell sample, the method comprising the steps of: (a) incubating the sample with a composition comprising acrylamide, dimethylacrylamide, and sodium acrylate linear monomers at a concentration of about 20 to about 50 wt % of which sodium acrylate comprises about 10 to about 25 mol %, about 0.1 to about 1.0 mol % polymerization initiator, and about 0.001 to about 0.01 wt % polymerization accelerator; and(b) polymerizing the composition within the sample to form a polymer, wherein the polymer is embedded throughout the sample. 29-52. (canceled)
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