Advertisement

Rapid Isolation and Detection of Cell Free Circulating DNA and Other Disease Biomarkers Directly from Whole Blood

  • Rajaram Krishnan
  • Michael J. HellerEmail author
Conference paper

Abstract

The ability to rapidly detect cell free circulating (cfc) DNA biomarkers and drug delivery nanoparticles directly in blood is a major challenge for early disease detection and nanomedicine. We now show that a microarray dielectrophoretic (DEP) device can be used to rapidly isolate and detect high molecular weight (hmw) DNA nanoparticulates and nanoparticles directly from whole blood. At DEP frequencies of 5–10 kHz both fluorescent-stained hmw-DNA and 40 nm fluorescent nanoparticles separate from the blood and become highly concentrated at specific DEP high field regions over the microelectrodes, while blood cells move to the DEP low field regions. The blood cells can then be removed by a simple fluidic wash while the hmw-DNA and nanoparticles remain highly concentrated. The hmw-DNA could be detected at a level of <260 ng/ml, and the nanoparticles at <9.5 × 109 particles/ml, detection levels that are well within the range for viable clinical diagnostics and drug nanoparticle monitoring. Some initial work now indicates the presence of possible cfc-DNA in CLL patient blood samples.

Keywords

Cancer Biomarkers Cell free circulating DNA Diagnostic dielectrophoresis Nanomedicine Nanoparticles Whole blood 

Abbreviations

AC

Alternating current

cfc-DNA

Cell free circulating DNA

DEP

Dielectrophoresis

HC-DEP

High conductance dielectrophoresis

hmw-DNA

High molecular weight DNA

PCR

Polymerase chain reaction

volts pk-pk

Volts peak to peak

Notes

Acknowledgements

We would like to acknowledge David J. Charlot, Roy B. Lefkowitz, Amy L. Hsieh, Jason Steiner, Dr. Dietrich Dehlinger, Robert Littlefield and Jennifer M. Singelyn for their generous help in creating this paper. We would also like to thank Dr. Andrew McCulloch and Dr. Karen Christman and their labs for helping us obtain the whole rat blood, and Dr. Robert Kipps (UCSD Moores Cancer Center) for providing CLL blood samples. This work was supported by the NIH NCI NanoTumor Center Grant (U54-CA119335).

References

  1. Albrecht DR, Underhill GH, Wassermann TB et al (2006) Probing the role of multicellular organization in three-dimensional microenvironments. Nat Methods 3:369–375PubMedCrossRefGoogle Scholar
  2. Asbury CL, Van Den Engh G (1998) Trapping of DNA in nonuniform oscillating electric fields. Biophys J 74:1024–1030PubMedCrossRefGoogle Scholar
  3. Becker FF, Wang XB, Huang Y et al (1995) Separation of human breast cancer cells from blood by differential dielectric affinity. Proc Nat Acad Sci U S A 92:860–864CrossRefGoogle Scholar
  4. Board RE, Knight L, Greystoke A et al (2007) DNA methylation in circulating tumour dna as a biomarker for cancer. Biomarker Insights 2:307–319Google Scholar
  5. Cheng J, Sheldon EL, Wu L et al (1998a) Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips. Nat Biotechnol 16:541–546PubMedCrossRefGoogle Scholar
  6. Cheng J, Sheldon EL, Wu L et al (1998b) Isolation of cultured cervical carcinoma cells mixed with peripheral blood cells on a bioelectronic chip. Anal Chem 70:2321–2326PubMedCrossRefGoogle Scholar
  7. Cui L, Holmes D, Morgan H (2001) The dielectrophoretic levitation and separation of latex beads in microchips. Electrophoresis 22:3893–3901PubMedCrossRefGoogle Scholar
  8. Diehl F, Li M, Dressman D et al (2005) Detection and quantification of mutations in the plasma of patients with colorectal tumors. Proc Natl Acad Sci U S A 102:16368–16373PubMedCrossRefGoogle Scholar
  9. Duncan R. (2003) The dawning era of polymer therapeutics. Nat Rev Drug Discov 2:347–360PubMedCrossRefGoogle Scholar
  10. Editorial (2007) Healthy challenges. Nat Nanotechnol 2:451CrossRefGoogle Scholar
  11. Ermolina I, Milner J, Morgan H (2006) Dielectrophoretic investigation of plant virus particles: cow pea mosaic virus and tobacco mosaic virus. Electrophoresis 27:3939–3948PubMedCrossRefGoogle Scholar
  12. Ferrari M (2005) Cancer nanotechnology: opportunities and challenges. Nat Rev Cancer 5:161–171PubMedCrossRefGoogle Scholar
  13. Gautschi O, Bigosch C, Huegli B et al (2004) Circulating deoxyribonucleic acid as prognostic marker in non-small-cell lung cancer patients undergoing chemotherapy. J Clin Oncol 22:4157–4164PubMedCrossRefGoogle Scholar
  14. Gormally E, Caboux E, Vineis P et al (2007) Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance. Mutat Res-Rev Mutat 635:105–117CrossRefGoogle Scholar
  15. Green NG, Ramos A, Morgan H (2000) Ac electrokinetics: a survey of sub-micrometre particle dynamics. J Phys D: Appl Phys 33:632–641CrossRefGoogle Scholar
  16. Hughes MP (2007) Nanoparticle manipulation by electrostatic forces. In: Goodard WA, Brenner DW, Lyshevski SE, Iafrate GJ (eds) Handbook of nanoscience, engineering, and technology, 2nd edn. CRC Press, FloridaGoogle Scholar
  17. Jen J, Wu L, Sidransky D (2000) An overview on the isolation and analysis of circulating tumor DNA in plasma and serum. Ann NY Acad Sci 906:8–12PubMedCrossRefGoogle Scholar
  18. Krishnan R, Heller MJ (2009) An AC electrokinetic method for enhanced detection of DNA nanoparticles. J Biophotonics 2:253–261PubMedCrossRefGoogle Scholar
  19. Krishnan R, Sullivan BD, Mifflin RL et al (2008) Alternating current electrokinetic separation and detection of DNA nanoparticles in high-conductance solutions. Electrophoresis 29:1765–1774PubMedCrossRefGoogle Scholar
  20. Krishnan R, Dehlinger DA, Gemmen GJ et al (2009) Interaction of nanoparticles at the DEP microelectrode interface under high conductance conditions. Electrochem Comm 11:1661–1666CrossRefGoogle Scholar
  21. Morgan H, Hughes MP, Green NG (1999) Separation of submicron bioparticles by dielectrophoresis. Biophys J 77:516–525PubMedCrossRefGoogle Scholar
  22. Mu L, Feng SS (2003) A novel controlled release formulation for the anticancer drug paclitaxel (Taxol(R)): PLGA nanoparticles containing vitamin E TPGS. J Control Release 86:33–48PubMedCrossRefGoogle Scholar
  23. Nishiyama N (2007) Nanomedicine: nanocarriers shape up for long life. Nat Nanotechnol 2:203–204PubMedCrossRefGoogle Scholar
  24. Nishiyama N, Kataoka K (2006) Current state, achievements, and future prospects of polymeric micelles as nanocarriers for drug and gene delivery. Pharmacol Therapeut 112:630–648CrossRefGoogle Scholar
  25. Ramos A, Morgan H, Green NG et al (1998) Ac electrokinetics: a review of forces in microelectrode structures. J Phys D: Appl Phys 31:2338–2353CrossRefGoogle Scholar
  26. Sozzi G, Conte D, Mariani L et al (2001) Analysis of circulating tumor DNA in plasma at diagnosis and during follow-up of lung cancer patients. Cancer Res 61:4675–4678PubMedGoogle Scholar
  27. Sozzi G, Conte D, Leon M et al (2003) Quantification of free circulating DNA as a diagnostic marker in lung cancer. J Clin Oncol 21:3902–3908PubMedCrossRefGoogle Scholar
  28. Stephens M, Talary MS, Pethig R et al (1996) The dielectrophoresis enrichment of CD34+ cells from peripheral blood stem cell harvests. Bone Marrow Transpl 18:777–782Google Scholar
  29. Stroun M, Anker P, Lyautey J et al (1987) Isolation and characterization of DNA from the plasma of cancer patients. Eur J Cancer Clin Oncol 23:707–712PubMedCrossRefGoogle Scholar
  30. Wu TL, Zhang D, Chia JH et al (2002) Cell-free DNA: measurement in various carcinomas and establishment of normal reference range. Clin Chim Acta 21:77–87CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.Department of Bioengineering, Department of NanoengineeringUCSD Moores Cancer Center, University of California San DiegoLa JollaUSA

Personalised recommendations