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Transcript
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
• “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
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
Cellular Membrane Potential
Na+ ClNormal cell:
-40 to -90 mV
Malignant cell:
-10 to – 30 mV
Na+ ClK+ ClCl-
K+ ClNa+
Resting Membrane Potential estimated from Goldman Eqn:
RT PK [ K  ]i  PNa [ Na  ]i  PCl [Cl  ]o
V 
ln
F
PK [ K  ]o  PNa [ Na  ]o  PCl [Cl  ]i
Can be measured using “distributional” probes:
Ci
 FV / RT
e
Co
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)]
Assay:
Stain cells, flow cytometry detection of rare events
Fluorescence Intensity
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.”
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
Dielectrophoretic Field Cages
(all field cage images taken from literature)
Electric Field in Cage
Dielectrophoretic Field Cages
200 m
Micro-Channel
Single Cell
Trapped in
Cage
Incoming
Cells
Picture courtesy of Evotec OAI
Picture courtesy of Evotec OAI
Picture courtesy of Evotec OAI
Picture courtesy of Evotec OAI
Multispectral Analysis for Rapid Identification of Single Cells
On-line absorbance, fluorescence, and Raman
Medical diagnostics
Biocomplexity
Technology (e.g., semiconductor industry)
Single Nanoparticle Enumerator “SNaPE”
Mirror
Ar+ Laser
Lens (f = 150 mm)
Premonochromator
Longpass Filter
5X Beam Expander
With 50 m Pinhole
500 m Pinhole
Notch Filter
Dichroic Mirror
PMT
100X Objective
10 m I.D. Capillary
Syringe Pump
3D
Stage
PC
Computer
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
Intensity (arb.)
Bacterial Fluorescence
450
400
350
300
250
200
150
100
50
0
270
E. coli
P. aeruginosa
320
370
Wavelength (nm)
420
470
Raman Intensity
Confocal Raman spectra of Cancer Cells: (glass substrate subtracted)
K562
Jurkat
1500
1000
500
1500
1000
Raman Shift (cm-1)
Bioinformatics
Raman
Extinction
Fluorescence
Raman Shift (cm-1)
Scatter
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
Thanks to the Rowlen Group
Michael
Jessica
Michele
Carrie
Matt
Not pictured: Peter and Jenna