Specific Binding of Cancer Cells Using a MicroChamber Array Functionalized with Antibodies X. Zheng1, S.L. Cheung1, L. Wang2, J. Schroeder3, R. Heimark4, J.C. Baygents2, R.Guzman2 & Y. Zohar1 November 19, 2009 1Aerospace
& Mechanical Engineering, 2Chemical & Environmental Engineering 3Molecular and Cellular Biology, 4Department of Surgery
University of Arizona, Tucson, AZ, USA THE UNIVERSITY OF ARIZONA
OUTLINE • Introduction – Motivation, Background & Objective
• Experimental Arrangements – Device fabrication – Surface coating
• Experimental Results – – – – –
Concentration effect Incubation time effect Ambient temperature effect Surface coating effect Cell type effect
• Conclusions THE UNIVERSITY OF ARIZONA
MOTIVATION Microchip for sorting CTCs
• Challenge: Sort 1 CTC in 1-10 billion blood cells
• Approach: Single step from whole blood
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BACKGROUND Cadherins can be used as a highly selective tool to specifically capture target cells in tissue or blood.
Cell
Find the proper lock to a given key ! N-cad E-cad
9 THE UNIVERSITY OF ARIZONA
X
Cell
BACKGROUND Antibody Structure
MD A -M B -2 31
PC 3N
BT -2 0
Western Blotting
Binding site Heavy chain Fab region
N-Cad Light chain
E-Cad
Cad-11
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Hinge
HOOC
COOH
Fc region
OBJECTIVE ¾ Conceptual Design
Advantages: 9 9 9 9 9
Liquid/gas interface (surface tension & liquid evaporation) is eliminated Number & type of cells loaded into each chamber can be determined Total sample volume in each chamber is accurately known Number & type of cells captured in each chamber can be determined Chamber array (5x5) provides multiple data points for statistical analysis THE UNIVERSITY OF ARIZONA
EXPERIMENTAL ARRANGEMENTS ¾ Device fabrication
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EXPERIMENTAL ARRANGEMENTS ¾ Surface functionalization 100ug/ml antibody in PBS, 1 hr 2mg/ml BSA in PBS, 1 hr
O=CH N
O=CH N
O=CH N
CH
CH
CH
=
P
=
P
=
P
NH2
NH2
NH2
Si
Si
Si
OH OH OH OH OH OH OH OH OH
50ug/ml protein G in PBS, overnight, 40C 2% glutaraldehyde in PBS, 2 hrs
P
H 2N
O HC
1% APTES in acetone, 30 mins
C H2
H2 C
O C H2
CH
OCH 2CH 3 CH 3CH 2O
Si OCH 2CH 3
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NH 2
EXPERIMENTAL ARRANGEMENTS ¾ Device testing Anti-Mouse IgG (CY3) 15ug/ml, 1hr
P
P
N=CH
N=CH
N=CH
=
CH
CH
CH
=
=
O
Anti-N-Cadherin
P
N
N
N
Si
Si
Si
O
O
O
O
O
O
O
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(IgG1 from mouse)
• O
Uniform intensity → Uniform antibody activity
EXPERIMENTAL RESULTS
Cell concentration • 104/ml - 2.5x105/ml Incubation time • 15 min - 2 hrs
2
PC3N on anti-N-cad
Attached Cells [cells/mm ]
¾ Cell concentration effect on capture rate 800
600
400 120 min 60 min 30 min 15 min
200
0 0.0
0.5
1.0
1.5
5
2.0
3
2.5
Concentration [10 cells/cm ]
•Density increases linearly with concentration (within the test concentration range; i.e. every cell contact the functionalized surface) THE UNIVERSITY OF ARIZONA
3.0
EXPERIMENTAL RESULTS
Cell concentration • 104/ml - 2.5x105/ml Incubation time • 15 min - 2 hrs
2
PC3N on anti-N-cad
Attached Cells [cells/mm ]
¾ Incubation time effect on capture rate 1200
5 2.5105cells/ml 2.5x10 cells/ml
5 105cells/ml 10 cells/ml
1000
4 5104cells/ml 5x10 cells/ml
4 104cells/ml 10 cells/ml
800 600
Asymptotic density level
400 200 0 0
20
Time constant
40 60 80 100 Incubation time [min]
120
• Captured cell density increased exponentially with incubation time • Asymptotic density level increased almost linearly with concentration • Time constant increases with cell concentration THE UNIVERSITY OF ARIZONA
EXPERIMENTAL RESULTS ¾ Ambient temperature effect on capture rate PC3N on anti-N-cad
Temperature • 4oC - 37oC Utilize NaN3 treatment to prevent possible receptor internalization at 37oC
Attached Cells [%]
Cell concentration • 105/ml
100% 37 °C NaN3 treated 37 °C 25 °C 4 °C
80% 60% 40% 20% 0% 0
• 4oC, capture no cells up to 60 minutes • 25oC & 37oC, 90% after 15 minutes THE UNIVERSITY OF ARIZONA
10
20 30 40 Incubation time [min]
50
60
EXPERIMENTAL RESULTS ¾ Surface coating effect on PC3N capture rate
anti-N-cad
Mouse-IgG
SiO2
Captured PC3N cells, from 5x105 cells/ml suspensions, on various coated surfaces.
Attached Cells [%]
100% 80% 60% 40% 20% 0%
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Anti-N-Cadherin Mouse IgG SiO2
EXPERIMENTAL RESULTS ¾ Cell type effect on cell capture rate
PC3N
BT20
MDA-MB-231 PC3N BT20 MDA-MB-231
Different cell types (5x105/ml) captured on anti-N-cad surfaces
Attached Cells [%]
100% 80% 60% 40% 20% 0%
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1
EXPERIMENTAL RESULTS ¾ Cell type effects N-cad-expressing MDA-MB-231 cells; Green color marks N-cad molecules.
MDA-MB-231
Ncdh+MDA-MB-231
Ncdh+MDA-MB-231 MDA-MB-231
Capture of MDA-MB-231 and Ncdh+MDA-MB-231 cells (105/ml) on anti-Ncadherin surfaces.
Attached Cells [%]
80%
60%
40%
20%
0%
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1
CONCLUSIONS ¾ Microchambers have been fabricated in Si wafers and functionalized with bio-active layers. ¾ Capture of target cells from suspensions increases: • linearly with concentration (dilute suspension). • exponentially with incubation time.
¾ Cell receptor – surface ligand binding affinity is highest at a temperature range of 25-37°C. ¾ The receptor – antibody binding is highly specific; changing cell-type or immobilized-antibody results in very low binding affinity.
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ACKNOWLEDGEMENTS ¾ This work is supported by a BCRP grant, BC061859, and an Arizona Biomedical Research Commission grant 06-080. ¾ The coding of human N-cadherin cDNA was a gift from Dr. Brian McCray (The University of Arizona). ¾ The Si-DRIE was carried out at the Arizona State University, courtesy of Prof. Jun Chae.
Thank you THE UNIVERSITY OF ARIZONA
EXPERIMENTAL RESULTS ¾ Surface coating effect on BT20 capture rate
anti-E-cad
anti-N-cad
Mse-IgG
Captured BT20 cells, from 5x105c cells/ml suspensions, on various coated surfaces.
Attached Cells [%]
100% 80% 60% 40% 20% 0%
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SiO2 Anti-E-Cadherin Anti-N-Cadherin Mouse IgG SiO2