Measurement of Carbonaceous Particles in Biological Samples

• US 20090061473A1
• Filed: 08/29/2007
• Published: 03/05/2009
• Est. Priority Date: 08/29/2007
• Status: Abandoned Application

• Current Assignee: United States Environmental Protection Agency
• Original Assignee: United States Environmental Protection Agency
• Inventors: Hays, Michael D., Saxena, Rajiv Krishna, Gilmour, M. Ian
• Application Number:
  US11/846,587
• Publication Number:
  US 20090061473A1
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/84 involving inorganic compounds or pH

Abstract

Disclosed is a method of quantitative estimation of carbonaceous particles in biological samples such as biological cells and tissues.

Claims

1. A method of determining carbonaceous particle concentration in biological cells, comprising the steps of:

Counting the biological cells;

separating elemental carbon, in the biological cells, in the form of washed pellets; and

analyzing the elemental carbon in the pellets.

2. A method according to claim 1, wherein the step of counting biological cells comprises counting the biological cells using a hemocytometer.

3. A method according to claim 1, wherein the step of separating elemental carbon from the biological cells comprises:

solubilizing the biological cells in a solvent; and

isolating, by high speed centrifugation, residual carbonaceous particles in the form of pellets.

4. A method according to claim 3 wherein the step of solubilizing the biological cells in a solvent is preceded by trypsinizing the biological cells to facilitate isolation of biological cells containing carbonaceous particles.

5. A method according to claim 3 wherein the step of solubilizing the biological cells in a solvent comprises dissolving the biological cells in an alkaline solubilizer.

6. A method according to claim 3 wherein the step of solubilizing the biological cells in a solvent comprises dissolving the biological cells in sodium dodecyl sulfate.

7. A method according to claim 3 wherein the step of solubilizing the biological cells in a solvent comprises dissolving the biological cells in a solvent of normal saline, silica, and sodium dodecyl sulfate.

8. A method according to claim 3 wherein the step of isolating residual carbonaceous particles in the form of pellets is followed by the steps comprising:

washing the pellets to remove impurities from the pellets; and

drying the washed pellets.

9. A method according to claim 8, wherein the step of washing the pellets comprises:

washing the pellets by suspending the pellets in normal saline to form a mixture;

vortexing the mixture; and

centrifuging the mixture to isolate residual carbonaceous particles in the form of pellets.

10. A method according to claim 8, wherein the step of drying the washed pellets comprises transferring sufficient amount of pellets to a quartz filter such that the total mass of the elemental carbon particles in the pellets on the filter is between about 1 μ

g and 100 μ

g.

11. A method according to claim 1, wherein the step of analyzing the elemental carbon comprises measuring the concentration of elemental carbon in the washed pellets using a carbon analyzer.

12. A method of determining carbonaceous particle concentration in biological tissue, comprising the steps of:

weighing a sample of biological tissue;

separating elemental carbon in the form of washed pellets from the biological tissue; and

analyzing the elemental carbon in the pellets.

13. A method according to claim 12, wherein the step of separating elemental carbon from the biological tissue comprises:

homogenizing the cellular component of the biological tissue to form a slurry;

solubilizing the biological tissue in a solvent; and

isolating, by high speed centrifugation, residual carbonaceous particles in the form of pellets.

14. A method according to claim 13, further comprising, prior to the step of homogenizing the cellular component of the biological tissue to form a slurry, dividing the biological tissue into small pieces.

15. A method according to claim 13 wherein the step of solubilizing the biological tissue in a solvent comprises dissolving the biological tissue in an alkaline solubilizer.

16. A method according to claim 13 wherein the step of solubilizing the biological tissue comprises dissolving the biological tissue in sodium dodecyl sulfate.

17. A method according to claim 13 wherein the step of solubilizing the biological tissue comprises dissolving the biological tissue in a solvent of normal saline, silica, and sodium dodecyl sulfate.

18. A method according to claim 13 wherein the step of isolating residual carbonaceous particles in the form of pellets is followed by the steps comprising:

washing the pellets to remove impurities from the pellets; and

drying the washed pellets.

19. A method according to claim 18, wherein the step of washing the pellets comprises:

washing the pellets by suspending the pellets in normal saline to form a mixture;

vortexing the mixture; and

centrifuging the mixture to isolate residual carbonaceous particles in the form of pellets.

20. A method according to claim 18, wherein the step of drying the washed pellets comprises transferring sufficient amount of pellets to a quartz filter such that the total mass of the elemental carbon particles in the pellets on the filter is between about 1 μ

g and 100 μ

g.

21. A method according to claim 12, wherein the step of analyzing the elemental carbon comprises measuring the concentration of elemental carbon in the washed pellets using a carbon analyzer.

22. A method of determining the amount of carbonaceous particles in biological sample comprising the steps of:

weighing the biological sample;

solubilizing the biological sample in a solvent to separate carbonaceous particles including elemental carbon from the biological sample; and

isolating, by high speed centrifugation, residual carbonaceous particles in the form of pellets.

washing the pellets to remove impurities;

drying the pellets; and

analyzing the amounts of organic and elemental carbon in the pellets using a carbon analyzer.

23. A method according to claim 22, wherein the biological sample is selected from a group consisting of lung tissues, lung epithelial cells, and macrophages.

24. A method according to claim 22, wherein the biological sample comprises carbonaceous particles selected from a group consisting of Diesel Exhaust Particles, Ultra-fine Carbon Black, Nano-diamonds, and Carbon-nanotubes.