Upload
diane-teresa-strickland
View
214
Download
0
Tags:
Embed Size (px)
Citation preview
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”
2
Manuscript in Preparation
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
Loeb, S. The oncologist 2008, 13, 299-305
www.metastasis.cauchoiscar.com
Clinical Utility of Circulating Tumor Cells (CTCs)
• 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
4
Analyzing Low Abundance Material from Peripheral Blood
• Red Blood Cells– 109/mL– 3 – 5 µm– Biconcave discs
Allard, W. J. et al. 2004, Clinical Cancer Res., 10, 6897-904
• White Blood Cells– 106/mL– ~15 µm– Spherical
• Circulating Tumor Cells– 1 – 10/mL– 15 – 30 µm– Spherical
1 = CTC2 = Membrane Pore3 = Leukocyte
Hayes, D. F. J. et al. 2008, Clinical Cancer Res., 14, 3646-3650 5
Existing Tools for Analyzing CTCs in Peripheral Blood
• 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
• Magnetic capture using microbeads coated with recognition elements– 5 log enrichment– Only mononucleated
cell fraction– 1 CTC in 106 MNC– Enumeration of cells by fluorescence visualization
Dynal
Vona et al., 2000, Am. J. Pathology 256: 576
CTC-chip
● Target CTCs interact with antibody (EpCAM) coated microposts
● Purity - ~50%
● CTCs in the peripheral blood are captured and isolated
● Made from silica using DRIE and contains microposts
● Cell enumeration - by cell staining Nagrath, S. Nature 2007, 450, 1235-1239
● Capture efficiency - ~65%
7
● 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
High Throughput Microsampling Unit (HTMSU)
Adams, A. A. JACS 2008, 130, 8633-86418
● Prostate cancer developsin the prostate gland
● The most common type of cancer in men in the USA
● LNCaP (Prostate cancer cell line) - Metastasize into the lymph nodes
● LNCaP cell membrane contains Prostate Specific Membrane Antigen (PSMA)
www.prostatecancerfoundation.org
● Diagnosed by Prostate Specific Antigen (PSA) test - high false positive and negative errors (30%)
Prostate Cancer
PSMA - 750 amino acids - Mw = 110 kDa - 1 x 106 molecules/cell
Liu, T. Prostate, 2008, 68, 955-964
LNCaP Cells9
NH2-(CH2)6-(CH2-CH2-O)6- CCAAGACCUGACUUCUAACUAAGUCUACGUUCC
Aptamers for LNCaP Cell Capture
● Generated by in vitro
selection process- SELEX
● Oligonucleotides Advantages● Chemically robust● Greater surface density ● Properties can be changed on demand• End-point attachment to surfaces
PSMA Aptamer
Expression level/Molecules cell-1
Molecular weight / kDa
Recognition Element
PSMA 1 x 106 100 PSMA aptamer
EpCAM 5 x 105 33 EpCAM antibody
● Mw = ~ 10 kDa and Kd = ~ 50 nM-1
Lupold, S. E. Cancer Res. 2002, 62, 4029-4033
Parrott, A.M Nuc. 2003, Acids Res. 28, 489-497 10
Polymer-based High-Throughput Sampling Unit for Capturing CTCs (PMMA)
1. 150 µm (depth) x 30 µm (width) x 3 cm (length)2. Total volume = 180 nL3. Number of parallel channels = 514. Processing time for 1 mL input (4 mm/s) = 9.1 min5. 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
(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
(1)
3)
• KERN MMP 2252– Precision of ±1 µm– Microstructure aspect
ratios ≤ 20:1– Milling in metals,
ceramics, polymers
(2)
(3)(5)
(6)
(4)
12
Producing Parts from Metal Molding Tools
(1)
(3)
(5)
(4)
(2)
►Jenoptik Microtechnik HEX 02– Attach mould insert– Evacuate chamber– Heat substrate to Tg
(105oC - PMMA)– Thermal fusion bonding for
channel enclosure
(1) CNC controller(2) Telescopic upper stage(3) Lower heating platen(4) Fixed stage(5) Vacuum system51-channel curvilinear
51-channel linear
50 μm linear
20 μm linear
35 μm linear(1)
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, 842-84314
Monitoring Fluidic Optimization Measurements
Carl Zeiss Axiovert
● Computer controlled● Programmable motorized stage● Inverted optical microscope● Video CCD inspection ● Fluorescence imaging
15
16
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 microscopy
0 2 4 6 8 10
0
10
20
30
40
50
60
70
80
90
100
Cap
ture
Effi
cien
cy (%
)
Linear Velocity (mm s-1)
Dharmasiri, U.R. Electrophoresis 2009, 30, 3289–3300Chang and Hammer, Biophys. J.,1999, 76, 1280
Capture determined by
Encounter Rate (ko)
DNuko
PeNu 2
DVaPe 47.0
■ Aptamer
■ Antibody
Capture determined by
reaction Probability (P)
)1(
in
in
k
kP
kTEsin eFk /
Va
38
17
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
● 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
Cell Release from the Capture Surface
A. Before trypsin infused B. Exposed to trypsin for 2 minC. Exposed to trypsin for 6.5 min - cell was
releasedD. Exposed to trypsin for 7.5 min - cell was
removed
180 1 2 3 4 5 6 7 8
0
20
40
60
80
100
Ce
ll R
em
ov
al E
ffic
ien
cy
(%
)
Time (min)Dharmasiri, U.R. Electrophoresis 2009, 30, 3289–3300
Whole blood
Whole blood + LNCaP
SN ≥ 3
19
Automated Cell Enumeration• 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
0 50 100 150 200 2500
50
100
150
200
250
Number of spiked LNCaP cells per mL
Num
ber of
con
duct
ance
sig
nal r
egis
tere
d
R2 = 0.9997
Dharmasiri, U.R. Electrophoresis 2009, 30, 3289–3300
20
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
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 - gram negative bacterium rod-shaped 2 μm long and 0.5 μm diameter
• 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
E. coli O157:H7
Alocilja, E.C. 2003. Biosens. Bioelectron. 18: 841-84
2 m
Coliform Standards (colonies /100 ml)Drinking Water 1TC*
Swimming Water 200FC*Boating Water 1000FC
Treated Sewage Effluent < 200FC
22
(TC*- Total Coliform , FC*- Fecal Coliform)
EPA Approved Method for E. coli Detection
Bennett, A.R. 1996. Letters in Applied Microbiology 22: 237-243
• 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
Cell culturing using EPA Method 1603. (LSU-BR University Lake)
15 cfu/100mL
x100volume processed# colonies
CFU/100 mL24 cfu/10mL 4 cfu/1mL
23
Anti E.Coli O157:H7 Antibody
Microchip Enrichment
• 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
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, 123-13224
Polymer-Based (PMMA) Sampling Unit for E. coli O157:H7 Selection
11 mm
5.5 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
25Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849
E. coli O157:H7 Selection and Enumeration
26150 mM PBS solution infused at 50 mm/s linear velocity to remove non-specifically absorbed cells Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849
System Operation
Syringe pump15 m
27
• Inverted optical microscope• Fluorescence imaging with
high sensitivity CCD• Syringe pump • E. coli cells were stained
- FITC (PKH67)
- Lipophilic membrane linker
Carl Zeiss Axiovert
0 1 2 3 4 520
25
30
35
40
45
200
500252
348300
Slt!
Uid
AD
Thr
esho
ld c
ycle
(C
t)
Log (cell density) [cfu]
10 cfu
6 cfu
• PCR directed to the conserved regions within the genes encoding for SLT-I (shiga-like toxins) and the uidA gene, encodes for ß-glucuronidase in E. coli O157:H7
• Mismatch in G residue, as opposed to the T residue found in other E. coli strains)
Recognition of Escherichia coli O157:H7 by mismatch amplification assay-multiplex PCR (Cebula et al.,1995, 33, 248)
E. Coli O157:H7 Enumeration via RT-qPCR
28
RT-qPCR Experiment Amplification and Dissociation Curves
Created with PSI-Plot, Sun Jun 14 21:57:04 2009
10 15 20 25 30 35 40 45 50
Co
lum
n3
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
may27ap.txt
50 60 70 80 90 100-5
0
5
10
15
20
may27dc.txt
Fluo
resc
ence
(dRn
)
Cycle # Temperature (oC)Fl
uore
scen
ce (-
Rn(T
))
specific DNA product (80 oC)
< 75°C correspond to non-specific DNA
Ct
slt1
Single blinded sample(cfu in the sample)
Real-time qPCR results for uidA gene
Ct ±SDcfu detected in the sample
(RSD %)
30 39.3 ±0.2 34 ±4 (12%)90 37.6 ±0.1 94 ±2 (2.1%)
150 37.1 ±0.1 130 ±8 (6.2%)400 35.2 ±0.3 405 ±5 (1.2%)800 33.9 ±0.1 799 ±15 (1.8%)
Real-time qPCR results for the uidA gene
29Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849
30
Cell Capture Efficiency• 3 x 103 cells/mL introduced at different volumetric flow rates
• Total input volume analyzed = 500 µL
10 20 30 40
20
40
60
80
100
0 25 50 75 100
20
40
60
80
100
Capt
ure
Effici
ency
(%)
Linear Flow Velocity (mm/s)Chang/Hammer model for mobile cell interactions
(1) Encounter rate(2) Probability of the reaction
Data for curvilinear channel, width- 15 m and depth- 80 m
Chang, K. C.; Hammer, D. A. Biophys. J. 1999, 76, 1280–1292
Data for linear channel, depth- 80 m
Channel width (m)Ca
ptur
e Effi
cien
cy (%
)
Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849
Specificity of Polyclonal Anti-E. coli O157:H7 Antibody
15 µm
A micrograph of capture surface E. coli O157:H7
A micrograph of capture surface E. coli K12
• 3x103 cells/mL were introduced at 5 mm/s linear velocity
31Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849
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
A. Brightfield micrograph of the captured cell before being infused the releasing solutionB. Fluorescent micrograph of the captured cell before being infused the releasing solutionC. Brightfield micrograph of the cell released surface (4 mins) D. Fluorescent field micrograph of the cell released surface (4 mins)
0 min 4 min 4 min0 min
Avg. stripping time: 3.4 min +0.35 (n=25)
Dharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–284932
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) 122 cfu
Lake Granbury, TX 8 cfu/100 mL(EPA Method 1603) 51 cfu
Water Treatment Plant , LA 2.6 x 106 cfu/100 mL(EPA Method 1603) 9.6 x 105 2000 cfu *
33
* Max Capacity of the bed: 260 x 106 cells
LSU LakeDharmasiri U. R. Anal. Chem. 2010, 82 (7), 2844–2849
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
High-Throughput Isolation and Electrokinetic Manipulation of
Circulating Tumor Cells Using a Polymeric Microfluidic Device
35
Future Work
Objectives
No. ofPatients
No. ofspecimens
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
• Design a microfluidic device for processing 7.5 mL of blood to select CTCs in a short time period (~30 min)
• Electrokinetic collection of selected CTCs for molecular profiling
36
Cell Selection and Manipulation
37
High-Throughput Microsampling Unit (HTMSU)
Adams, A. A. JACS 2008, 130, 8633-8641Dharmasiri, U. Electrophoresis 2009, 30, 3289–3300
• 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
38
460 curvilinear channels Volume -100 L Process 7.5 mL of sample in 30 min
Cell Capture Section of µHTMSU
Out put In put
39
Electrophoresis
F - Coulomb force q- Net charge on the object E- The applied electric field
F = qE
Annu. Rev. Biomed. Eng. 2006. 8:425–54
• 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
• In water, the cells move at a velocity given by the balance of the Coulomb and viscous drag forces, a process known as EP
40
Electrokinetics
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
• 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;
41
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
• Determine biology of CTCs and cells at primary tumor
43
Detection of point mutations Gene expression profiling
Genetic Make up Adhesion properties Metastatic potential
AcknowledgmentsSoper Research Group
National Science FoundationGrant NIH # - 1 R33 CA099246-01State of Louisiana Board of RegentsTexas Sea Grant (NA06OAR4170076)
Dr. Maggie WitekDr. Robert TruaxMs. Karen
Prof. Steven A. SoperProf. Robin McCarley
44
THANK YOU
45