New Research Directions in the Rowlen Group

1. New Research Directions in the Rowlen Group • Detection of cancer cells in blood • Isolation of cancer cells using dielectrophoresis • Multispectral analysis of single cells for identification

2. “One out of every three Americans will be diagnosed with cancer” • http://uch.uchsc.edu/uccc/welcome/index.html • Early diagnosis saves lives • A 1 g tumor, generally undetectable, can exfoliate • up to 106 cells per day into blood stream. • However, 109 – 1010 cells/mL in blood • Detect 4 in 109 ? • If malignant cells could be distinguished/detected: • Early diagnosis • Monitor response to treatment • Evaluate minimum residual disease

3. Fact: Most cancer cells have a lower membrane potential than most normal cells Hypothesis: membrane potential can be used as a biomarker for malignant cells in blood

4. Cellular Membrane Potential Na+ Cl- K+ Cl- Na+ Na+ Cl- K+ Cl- Cl- Normal cell: -40 to -90 mV Malignant cell: -10 to – 30 mV

5. Resting Membrane Potential estimated from Goldman Eqn: Can be measured using “distributional” probes:

6. Ideal characteristics of a fluorescent dye sensitive to cytoplasmic membrane potential: 1) response proportional to membrane potential 2) increased fluorescence with decreased polarization 3) photostable, 4) minimal toxicity to cell 5) immune to drug efflux pump Bis (1,3-dibutyl-barbituric acid) trimethine oxonol [DiBAC4(3)]

7. Assay: Stain cells, flow cytometry detection of rare events Cell Count Fluorescence Intensity

8. National Institutes of Health Program Announcement Objective: “… to develop novel technologies for capturing, enriching, and preserving exfoliated abnormal cells in body fluids or effusions and to develop methods for concentrating the enriched cells for biomarker studies.” “… the number of exfoliated tumor cells [in body fluids] is often small compared to the number of non-neoplastic cells. Therefore, the detection of exfoliated abnormal cells by routine cytopathology is often limited because few atypical cells may be present in the specimen.…” “Thus, the development of enrichment methods is a prerequisite for the routine detection of small numbers of exfoliated cells and small amounts of subcellular materials in biological fluids for molecular analysis.”

9. Dielectrophoresis • Force on particle due to interaction between induced dipole and local electric field • If particle more polarisable than medium, positive dielectrophoresis (as shown) • If particle less polarisable than medium, negative dielectrophoresis, • dipole aligns counter to field, repelled by field

10. Dielectrophoretic Field Cages (all field cage images taken from literature)

11. Electric Field in Cage

12. 200 m Dielectrophoretic Field Cages Micro-Channel Single Cell Trapped in Cage Incoming Cells Picture courtesy of Evotec OAI

13. Picture courtesy of Evotec OAI

14. Picture courtesy of Evotec OAI

15. Picture courtesy of Evotec OAI

16. Multispectral Analysis for Rapid Identification of Single Cells On-line absorbance, fluorescence, and Raman Medical diagnostics Biocomplexity Technology (e.g., semiconductor industry)

17. Single Nanoparticle Enumerator “SNaPE” Mirror Ar+ Laser Lens (f = 150 mm) Premonochromator Longpass Filter 5X Beam Expander With 50 mm Pinhole 500 mm Pinhole 3D Stage Notch Filter Dichroic Mirror PMT 100X Objective 10 mm I.D. Capillary Syringe Pump PC Computer

18. Bacterial Morphology SFM image of E. coli SEM image of P. aeruginosa Images adapted from: http://www.emlab.ubc.ca/gallery/elaineImages/elaine_microorganisms1.html

19. Bacterial Fluorescence

20. Confocal Raman spectra of Cancer Cells: (glass substrate subtracted) K562 Jurkat 1500 Raman Intensity 1000 500 1500 1000 Raman Shift (cm-1)

21. Raman Extinction Fluorescence Raman Shift (cm-1) Scatter Wavelength (nm) Wavelength (nm) Wavelength (nm) Bioinformatics

22. Thanks to the Rowlen Group Carrie Jessica Michael Michele Matt Not pictured: Peter and Jenna