Transcript High Efficiency Capture and Enumeration of Low Abundant

Selection and Enumeration
of Low-Abundance Biological
Cells from Complex Matrices
Udara Dharmasiri
Research Seminar
April 19, 2010
1
“Highly Efficient Capture and Enumeration of Low
Abundance Prostate Cancer Cells Using Prostate-Specific
Membrane Antigen Aptamers Immobilized to a Polymeric
Microfluidic Device”
Electrophoresis, 2009, 30: 1-12
“Microsystems for the Capture of Low-Abundance Cells”
Annual Reviews of Analytical Chemistry, 2010, Vol. 3
“Enrichment and Detection of Escherichia coli 0157:H7 Using
An Antibody Modified Microfluidic Chip”
Analytical Chemistry, 2010, 82 (7), 2844–2849
“High-Throughput Isolation and Electrokinetic Manipulation
of Circulating Tumor Cells Using a Polymeric Microfluidic
Device”
Manuscript in Preparation
2
Highly Efficient Capture and Enumeration
of Low Abundance Prostate Cancer Cells
Using Prostate-Specific Membrane Antigen
Aptamers Immobilized to a Polymeric
Microfluidic Device
Electrophoresis, 2009, 30: 1-12
3
Clinical Utility of
Circulating Tumor
Cells (CTCs)
www.metastasis.cauchoiscar.com
•
•
Cancer metastasis by circulating tumor cells (CTCs)
Elucidating the presence and number of CTCs is emerging
as an effective method for
- diagnosis
- prognosis
- prediction of therapeutic benefits
Loeb, S. The oncologist 2008, 13, 299-305
4
Analyzing Low Abundance Material from
Peripheral Blood
•
•
•
Red Blood Cells
– 109/mL
– 3 – 5 µm
– Biconcave discs
White Blood Cells
– 106/mL
– ~15 µm
– Spherical
1 = CTC
2 = Membrane Pore
3 = Leukocyte
Circulating Tumor Cells
– 1 – 10/mL
– 15 – 30 µm
– Spherical
Hayes, D. F. J. et al. 2008, Clinical Cancer Res., 14, 3646-3650
Allard, W. J. et al. 2004, Clinical Cancer Res., 10, 6897-904
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Existing Tools for Analyzing CTCs in
Peripheral Blood
• Magnetic capture using microbeads coated
with recognition
elements
Dynal
– 5 log enrichment
– Only mononucleated
cell fraction
– 1 CTC in 106 MNC
– Enumeration of cells by fluorescence visualization
• Nuclear tracked polycarbonate membranes
–
–
–
–
Separation based on size
Requires whole blood density gradient centrifugation
1 CTC in 1 mL of peripheral blood
Enumeration of cells by fluorescence visualization
Vona et al., 2000, Am. J. Pathology 256: 57
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CTC-chip
●
Made from silica using DRIE and contains microposts
●
Target CTCs interact with antibody (EpCAM) coated microposts
●
CTCs in the peripheral blood are captured and isolated
●
Capture efficiency - ~65%
Purity - ~50%
● Cell enumeration - by cell staining
●
Nagrath, S. Nature 2007, 450, 1235-1239
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High Throughput Microsampling Unit
(HTMSU)
Selectively and specifically isolate breast cancer cells through a
monoclonal antibody mediated process
● Sampling large input (1 ml) of whole blood in short time (<37 min)
● CTC capture efficiency >97% and purity ~100%
● The released CTCs enumerated on-device using conductivity detector
with 100% detection efficiency
●
8
Adams, A. A. JACS 2008, 130, 8633-8641
Prostate Cancer
●
●
●
Prostate cancer develops
in the prostate gland
The most common type of cancer in men in the USA
Diagnosed by Prostate Specific Antigen (PSA) test
- high false positive and negative errors (30%)
www.prostatecancerfoundation.org
●
●
LNCaP (Prostate cancer cell line) - Metastasize into the lymph nodes
LNCaP cell membrane contains Prostate Specific Membrane Antigen
(PSMA)
PSMA - 750 amino acids
- Mw = 110 kDa
- 1 x 106 molecules/cell
LNCaP Cells
Liu, T. Prostate, 2008, 68, 955-964
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Aptamers for LNCaP Cell Capture
Advantages
● Oligonucleotides
● Chemically robust
● Greater surface density
● Generated by in vitro
● Properties can be changed on
selection process- SELEX demand
• End-point attachment to surfaces
PSMA Aptamer
NH2-(CH2)6-(CH2-CH2-O)6- CCAAGACCUGACUUCUAACUAAGUCUACGUUCC
●
Mw = ~ 10 kDa and Kd = ~ 50 nM-1
Expression level/
Molecules cell-1
Molecular
weight / kDa
Recognition
Element
PSMA
1 x 106
100
PSMA aptamer
EpCAM
5 x 105
33
EpCAM antibody
Lupold, S. E. Cancer Res. 2002, 62, 4029-4033
Parrott, A.M Nuc. 2003, Acids Res. 28, 489-497
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Polymer-based High-Throughput Sampling
Unit for Capturing CTCs (PMMA)
1.
2.
3.
4.
5.
150 µm (depth) x 30 µm (width) x 3 cm (length)
Total volume = 180 nL
Number of parallel channels = 51
Processing time for 1 mL input (4 mm/s) = 9.1 min
4-levels of specificity (capture – 30 µm; detection – 50 µm;
immunoaffinity; shear)
6. Integrated reader for enumerating cells
11
Production of Plastic Fluidic Components from
Metal Molding Tools
(2)
• KERN MMP 2252
– Precision of ±1 µm
– Microstructure aspect
ratios ≤ 20:1
– Milling in metals,
ceramics, polymers
(4)
(1)
(3)
(5)
(6)
3)
(1) CNC controller
(2) 40,000 rpm spindle
(3) Automated tool changer
(4) Laser-based measurement
system
(5) Real-time imaging system
(6) X-Y translational stage
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Producing Parts from Metal Molding Tools
►Jenoptik Microtechnik
HEX 02
– Attach mould insert
– Evacuate chamber
– Heat substrate to Tg
(105oC - PMMA)
– Thermal fusion bonding for
channel enclosure
51-channel linear
(2)
(3)
(4)
20 μm linear
(5)
(1)
35 μm linear
50 μm linear
51-channel curvilinear
(1)
(1) CNC controller
(2) Telescopic upper stage
(3) Lower heating platen
(4) Fixed stage
(5) Vacuum system
13
Immobilization of Aptamers to Polymer
Surfaces
8.4 x 1012 molecules/cm2
Dharmasiri, U.R. Electrophoresis 2009, 30, 3289–3300
McCarley et al., J. Am. Chem. Soc. 2005, 127, 842-843
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Monitoring Fluidic Optimization Measurements
Carl Zeiss Axiovert
Computer controlled
● Programmable motorized
stage
● Inverted optical microscope
● Video CCD inspection
● Fluorescence imaging
●
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Cell Capture Efficiency
●
●
●
0.5 ml of 1,000 cells/ml suspension introduced at linear
velocities between 0.1 to 10 mm/s
Post capture rinse with 150 mM PBS at 50 mm/s linear velocity
The number of selected cells onto the PSMA aptamer and
EpCAM antibody HTMSU counted using fluorescence
Capture
microscopy 100
Capture Efficiency (%)
Capture 90
determined by80
Encounter 70
Rate (ko) 60
ko  DNu
Nu  2 Pe

Pe  0.47Va
■
determined by
reaction
Probability (P)
Aptamer
■
Antibody
k in
P
( k in  1  )
50
40
30
k in   Fs e  E / kT
20
10
  8a 3V
0
D
0
2
4
6
8
10
-1
Linear Velocity (mm s )
Chang and Hammer, Biophys. J.,1999, 76, 1280
Dharmasiri, U.R. Electrophoresis 2009, 30, 3289–3300
16
Non-Specific Cell Adsorption
●
●
●
PSMA aptamer immobilized onto the HTMSU
i) MCF-7 - Human breast cancer cell line, does not express PSMA
ii) WBC - White blood cells
iii) RBC - Red blood cells
introduced at 2.5 mm/s linear flow velocity
A post capture rinse performed with PBS buffer at 50 mm/s
linear velocity
The number of cells adsorbed onto the HTMSU counted using
fluorescence microscopy
Trial
MCF-7
WBC
RBC
I
0
0
0
II
0
0
0
III
0
0
0
●
PSMA aptamer does not interact
with MCF-7, WBC and RBC
Dharmasiri, U.R. Electrophoresis 2009, 30, 3289–3300
17
Cell Release from the Capture Surface
0.25% (w/v) trypsin infused into the HTMSU
● Trypsin (23.8 KDa) – glycoprotein that proteolytically cleaves at
arginine and lysine AA residues. pI = 10.5; Optimal activity at pH = 8.0
● The process of typsination was evaluated by microscopy
●
A. Before trypsin infused
B. Exposed to trypsin for 2 min
C. Exposed to trypsin for 6.5 min - cell was
released
D. Exposed to trypsin for 7.5 min - cell was
removed
Cell Removal Efficiency (%)
100
80
60
40
20
0
0
Dharmasiri, U.R. Electrophoresis 2009, 30, 3289–3300
1
2
3
4
Time (min)
5
6
7
18 8
•
Automated Cell Enumeration
S
N ≥ 3
Whole blood
Whole blood + LNCaP
Number of conductance signal registered
One ml of blood containing 20 LNCaP cells seeded into the HTMSU
● The captured cells released using the trypsin prior to on-chip
conductivity enumeration
● Total of 18 cells were counted at a volume flow rate of 5 µl/min
● The conductance response from whole blood without LNCaP obtained
250
200
150
100
2
R = 0.9997
50
0
0
50
100
150
200
250
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Dharmasiri, U.R. Electrophoresis 2009, 30, 3289–3300
Number of spiked LNCaP cells per mL
Conclusions
● LNCaP cell capture efficiency for PSMA aptamer tethered
microfluidic device was 95 +1%
● MCF-7 cells, WBCs and RBCs do not interfere with LNCaP
cell capture and therefore cell separation purity is ~100%
● 0.25% (w/v) trypsin was an effective reagent for releasing
LNCaP cells from the capture surface
● Conductivity enumeration efficiency was ~100% for the
released LNCaP cells
20
Enrichment and Detection of E. coli
O157:H7 from Water Samples Using an
Antibody Modified Microfluidic Chip
Analytical Chemistry, 2010, 82 (7), 2844–2849
21
E. coli O157:H7
• E. coli O157:H7 - gram negative bacterium
rod-shaped
2 μm long and 0.5 μm diameter
2 m
• The pathogenicity of E. coli O157:H7 is associated
with the production of Shiga-like toxins
-bloody diarrhea
-colitis
• In 2008, 2,000 Americans were hospitalized and
~60 died
Coliform Standards (colonies /100 ml)
Drinking Water
1TC*
Swimming Water
200FC*
Boating Water
1000FC
Treated Sewage Effluent
< 200FC
(TC*- Total Coliform , FC*- Fecal Coliform)
Alocilja, E.C. 2003. Biosens. Bioelectron. 18: 841-84
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EPA Approved Method for E. coli Detection
• ß-D glucuronidase production is not present in O157:H7 serotype
• E. coli O157:H7 viable but not culturable
• Presence of interfering agents alter the accuracy of chromogenic
media
15 cfu/100mL
24 cfu/10mL
4 cfu/1mL
CFU/100 mL
# colonies
x100
volume processed
Cell culturing using EPA Method 1603. (LSU-BR University Lake)
Bennett, A.R. 1996. Letters in Applied Microbiology 22: 237-243
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Anti E.Coli O157:H7 Antibody
• Mw = ~150 kDa
• Polyclonal (pAb)
• Kd = ~50 pM-1
• E. coli O157:H7 are
identified by combination
of O and H antigens
• 9×106 molecules of O
antigen/bacteria
Microchip Enrichment
www.kpl.com
(i) pAb can recognize O157 types for intact and non-culturable cells
(ii) Selective cell capture allows cell enrichment and enumeration from
potentially contaminated samples
Fitzmaurice, J. Mol. Cell. Probes 2004, 18, 123-132
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Polymer-Based (PMMA) Sampling Unit for
E. coli O157:H7 Selection
5.5 mm
11 mm
8 sub devices-16 curvilinear channels-9.5 mm long, 15 µm width/80 µm
depth. Surface area (cell selection bed) = 40 mm2 , volume = 250 nL
Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849
25
E. coli O157:H7 Selection and Enumeration
150 mM PBS solution infused at 50 mm/s linear velocity to remove non-specifically
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absorbed cells
Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849
System Operation
Carl Zeiss Axiovert • Inverted optical microscope
•
•
•
Syringe pump
Fluorescence imaging with
high sensitivity CCD
Syringe pump
E. coli cells were stained
- FITC (PKH67)
- Lipophilic membrane linker
15 m
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E. Coli O157:H7 Enumeration via RT-qPCR
10 cfu
40
D
200
252
Slt!
45
Uid A
•
PCR directed to the conserved regions within the genes encoding
for SLT-I (shiga-like toxins) and the uidA gene, encodes for ßglucuronidase in E. coli O157:H7
Mismatch in G residue, as opposed to the T residue found in other
E. coli strains)
Threshold cycle (Ct)
•
348
500
300
35
6 cfu
30
25
20
0
1
2
3
Log (cell density) [cfu]
4
5
Recognition of Escherichia coli O157:H7 by mismatch amplification assaymultiplex PCR (Cebula et al.,1995, 33, 248)
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RT-qPCR
Experiment Amplification
may27dc.txt
may27ap.txt
and Dissociation Curves
20
0.7
slt1
Fluorescence (-Rn(T))
Column3
Fluorescence (dRn)
0.6
0.5
0.4
0.3
0.2
0.1
specific DNA
product (80 oC)
Ct
15
< 75°C correspond
to non-specific DNA
10
5
0
0.0
-0.1
10
15
20
25
30
Cycle #
Real-time qPCR results
for the uidA gene
35
40
45
50
Single blinded sample
(cfu in the sample)
30
90
150
400
800
-5
50
60
70
80
Temperature
(oC)
90
100
Real-time qPCR results for uidA gene
cfu detected in the sample
(RSD %)
39.3 ±0.2
±4 (12%)
Created with P SI-P lot, Sun Jun 14 21:57:0434
2009
37.6 ±0.1
94 ±2 (2.1%)
37.1 ±0.1
130 ±8 (6.2%)
35.2 ±0.3
405 ±5 (1.2%)
33.9 ±0.1
799 ±15 (1.8%)
Ct ±SD
Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849
29
Cell Capture Efficiency
100
80
Data for curvilinear channel,
width- 15 m and depth- 80 m
60
40
20
0
25
50
75
100
Capture Efficiency (%)
•
3 x 103 cells/mL introduced at different volumetric flow rates
Total input volume analyzed = 500 µL
Capture Efficiency (%)
•
80
Data for linear channel,
depth- 80 m
60
40
20
100
10
20
30
40
Channel width (m)
Linear Flow Velocity (mm/s)
Chang/Hammer model for mobile cell interactions
(1) Encounter rate
(2) Probability of the reaction
Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849
Chang, K. C.; Hammer, D. A. Biophys. J. 1999, 76, 1280–1292
30
Specificity of Polyclonal Anti-E. coli
O157:H7 Antibody
•
3x103 cells/mL were introduced at 5 mm/s linear velocity
A micrograph of capture surface
E. coli O157:H7
15 µm
A micrograph of capture surface
E. coli K12
Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849
31
Cell Release from the Capture Surface
•
•
Mixture of chelators infused into channels at 10 mm/s
Captured cells were observed microscopically until removed by
release solution and Stoke’s force
0 min
0 min
4 min
4 min
Avg. stripping time: 3.4 min +0.35 (n=25)
A.
B.
C.
D.
Brightfield micrograph of the captured cell before being infused the releasing solution
Fluorescent micrograph of the captured cell before being infused the releasing solution
Brightfield micrograph of the cell released surface (4 mins)
Fluorescent field micrograph of the cell released surface (4 mins)
Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849
32
Water Sample Evaluation
Water Sample
cfu in the sample
Real-time qPCR
results for slt1 gene
Standard Sample
200 cfu/200 mL (Spiked)
198 11 cfu
Standard Sample
1,000 cfu/100 mL (Spiked)
1045 100 cfu
Baton Rouge University Lake,
LA
15 cfu/100 mL
(EPA Method 1603)
8 cfu/100 mL
(EPA Method 1603)
2.6 x 106 cfu/100 mL
(EPA Method 1603)
Lake Granbury, TX
Water Treatment Plant , LA
122 cfu
51 cfu
9.6 x 105 2000 cfu *
* Max Capacity of the bed: 260 x 106 cells
LSU Lake
Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849
33
Conclusions
•
•
•
•
Recovery of E. coli O157:H7 was ~72%
E. coli O157:H7 was selected and enumerated without
other serotype interferences
The strategy developed offered the ability to monitor
recreational water quality without the need for a cell
culture step
The entire processing steps were implemented in under 5 h
34
Future Work
High-Throughput Isolation and
Electrokinetic Manipulation of
Circulating Tumor Cells Using a
Polymeric Microfluidic Device
35
Objectives
•
Design a microfluidic device for processing 7.5 mL of
blood to select CTCs in a short time period (~30 min)
No. of
Patients
No. of
specimens
Average No. of
CTCs in 7.5 ml
Prostate cancer
123
188
47 (+13)
Breast cancer
422
1316
80 (+14)
Lung cancer
99
168
92 (+12)
Allard, W.J. Clin. Cancer Res. 2004 10: 6897-904
• Electrokinetic collection of selected CTCs for
molecular profiling
36
Cell Selection and Manipulation
37
High-Throughput Microsampling Unit (HTMSU)
•
•
•
•
Selectively and specifically isolate breast and prostate
cancer cells through an affinity agent mediated process
Sampling 1 ml of whole blood in short time (<37 min)
CTC capture efficiency >97% and purity ~100%
The released CTCs enumerated on-device using
conductivity detector with ~100% detection efficiency
Adams, A. A. JACS 2008, 130, 8633-8641
Dharmasiri, U. Electrophoresis 2009, 30, 3289–3300
38
Cell Capture Section of
µHTMSU
Out put
In put
 460 curvilinear channels
 Volume -100 L
 Process 7.5 mL of sample in 30 min
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Electrophoresis
•
•
Electrophoresis (EP): The force on a charged particle
exerted by an electric field
Most mammalian cells are covered with negatively
charged functional groups at neutral pH
F = qE
F - Coulomb force
q- Net charge on the
object
E- The applied electric
field
• In water, the cells move at a velocity given by the balance
of the Coulomb and viscous drag forces, a process
known as EP
Annu. Rev. Biomed. Eng. 2006. 8:425–54
40
•
Electrokinetics
Utilizes the electroosmotic flow (eof) of the solution and
the electrophoretic mobility (ep) of the material being
transported
• The linear velocity (app)
at which the material moves is
governed by;
CTC Type
Electrophoretic Mobility , (m.u.)
Breast cancer
1.19
Blast cell leukemia (large cells)
1.62
Colon cancer
1.47
Lung cancer
1.32
Vassar, P.S. Nature 1963, 4873, 1215-26
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Cell Manipulation Section of the Microfluidic
Unit
In put
Future Directions
•
•
•
CTCs in large volume of patients’ blood (>7.5 mL) will be
selected in short time (<30 min)
Molecular profiling of CTCs
 Detection of point mutations
 Gene expression profiling
Determine biology of CTCs and cells at primary tumor
 Genetic Make up
 Adhesion properties
 Metastatic potential
43
Acknowledgments
Soper Research Group
Prof. Steven A. Soper
Prof. Robin McCarley
Dr. Maggie Witek
Dr. Robert Truax
Ms. Karen
National Science Foundation
Grant NIH # - 1 R33 CA099246-01
State of Louisiana Board of Regents
Texas Sea Grant (NA06OAR4170076)
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THANK YOU
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