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Supplementary Materials for - Science Translational …2012/06/29 · measured using the µHD...
Transcript of Supplementary Materials for - Science Translational …2012/06/29 · measured using the µHD...
www.sciencetranslationalmedicine.org/cgi/content/full/4/141/141ra92/DC1
Supplementary Materials for
Ultrasensitive Clinical Enumeration of Rare Cells ex Vivo Using a Micro-Hall Detector
David Issadore, Jaehoon Chung, Huilin Shao, Monty Liong, Arezou A. Ghazani, Cesar
M. Castro, Ralph Weissleder,* Hakho Lee*
*To whom correspondence should be addressed. E-mail: [email protected] (R.W.); [email protected] (H.L.)
Published 4 July 2012, Sci. Transl. Med. 4, 141ra92 (2012)
DOI: 10.1126/scitranslmed.3003747
The PDF file includes:
Methods Fig. S1. Fabrication of the µHD. Fig. S2. Design of the flow-focusing microstructures. Fig. S3. Electrical setup and measurement of µHall sensors. Fig. S4. Computer-simulated Hall voltage. Fig. S5. Sensor array design. Fig. S6. Validation of sensor array with magnetic beads. Fig. S7. Effect of MNP incubation time.
SUPPLEMENTARY METHODS
Flow focusing structure
Finite element simulation was performed to determine the optimal structure for coplanar
sheath flow and chevron patterns. The developed system was tested using a sheath flow
stained with a fluorescent dye (fluorescein isothiocyanate, FITC) and a sample flow
containing 2-µm fluorescent beads (Fluospheres F8826, Invitrogen). The ratio of the flow
rate between the sheath flow and the sample streams was controlled using gravitational
flow, with negative pressure provided by a syringe pump on the output (BS8000,
Braintree Scientific). Typical total flow rates were 0.1 to 1 ml/h.
Measurement setup for µHD
For cytometry applications, the voltages from the µHall sensors were amplified, digitized,
and processed using custom-built electronics. The Hall sensors were AC-coupled to the
pre-amplifier through a high pass filter (ƒ3dB = 500 Hz). The Hall sensors had the output
impedance of 100 Ω, a bandwidth of 150 MHz and low input-referred noise (1.3 nV/√Hz).
A typical peak Hall voltage caused by a passing magnetic object is ~1 mV with a
duration of 20 µs. This signal is amplified by a two-stage amplifier with a total gain of 900
(30 × 30) and a bandwidth of 200 kHz. High-speed, low input-impedance, bipolar,
differential amplifiers were used for both stages of amplification (THS4131, Texas
Instruments). These amplifiers were impedance-matched to the Hall sensors. The
amplified signal (~1 V) was then fed to a commercial analog-to-digital converter (PCI
6133, National Instruments) for digitization.
Cell culture
The following human cell lines were cultured cultured in Dulbecco’s modified essential
medium (DMEM, Cellgro), supplemented with 10% fetal bovine serum (Cellgro), 1%
penicillin, and 1% streptomycin (Cellgro): MDA-MB-453, MDA-MB- 468, and A431 cells
were purchased from American Type Culture Collection; SkMG3 cells were provided by
T. Chan (Memorial Sloan-Kettering Cancer Center). All cell lines were maintained at
37°C in a humidified atmosphere containing 5% CO 2. At confluence, cells were washed,
trypsinized, and resuspended in culture media.
Tetrazine (TZ) modification of MNPs and trans-cyclo octene (TCO) modification of
antibodies
Fluorescein-conjugated, amine-terminated, cross-linked iron oxides (magneto-
fluorescent CLIOs) were prepared as described previously (9, 25). These CLIOs
nanoparticles have a core size of 7 nm and a hydrodynamic diameter of 30 nm.
Modification with 2,5-dioxopyrrolidin-1-yl 5-(4-(1,2,4,5-tetrazin-3-yl)benzylamino)-5-
oxopentanoate (TZ-NHS) create CLIO-TZ (25). Briefly, excess TZ-NHS was reacted with
amino-CLIO in phosphate-buffered saline (PBS) containing 0.1 M sodium bicarbonate,
for 3 hours at room temperature. TZ-CLIO was purified using Sephadex G-50 columns
(GE Healthcare). The following monoclonal antibodies were modified with TCO:
Herceptin (anti-HER2/neu), cetuximab (anti-EGFR), anti-EpCAM (R&D Systems), and
anti-Mucin-1 (Fitzgerald Industries).
Antibodies were modified with (E)-cyclooct-4-enyl 2,5-dioxopyrrolidin-1-yl
carbonate (TCO-NHS) as reported previously (25). Briefly, purified antibody was reacted
with TCO-NHS in 10% dimethylformamide for 3 hours at room temperature. TCO-
conjugated antibodies were subsequently buffer-exchanged into PBS and their
concentrations determined by absorbance measurements.
In vitro cell targeting and measurement
Cancer cells were trypsinized and labelled with TCO-conjugated antibodies (10 µg/ml) in
PBS with 0.5% bovine serum albumin (BSA, Sigma) for 45 minutes at 4°C. Following
washing and centrifugation, cells were labeled with FITC-conjugated CLIO-TZ at room
temperature for 30 minutes. After washing twice by centrifugation, FITC fluorescence
was assessed using a LSRII flow cytometer (Becton Dickinson). Mean fluorescence
intensity was determined using FlowJo software, and biomarker expression levels were
normalized with isotype control antibodies. A corresponding magnetic signal was
measured using the µHD sensor.
Cell labeling for multiplexed measurement
Mn-doped ferrite particles (MnFe2O4) of different diameters (10, 12, and 16 nm) were
synthesized as previously reported (21, 30) and coated with BSA. MDA-MB-468 cells
were labeled for HER2/neu, EGFR, and EpCAM. To enable simultaneous targeting of
these three markers, three different bioorthogonal labeling methods were used. For
HER2/neu labeling, anti-HER2 antibody (herceptin) was modified with TCO, and
MnFe2O4 MNPs (12 nm) were modified with TZ. For EGFR labeling, cells were labeled
with biotinylated anti-EGFR (cetuximab) antibody and conjugated with MnFe2O4 MNPs
(10 nm) via a streptavidin linker. The EpCAM-labeling was performed using anti-EpCAM
antibody (R&D Systems) modified with cyclodextrin and 16 nm MnFe2O4 with
adamantine (31). Cells were labeled using the same method as described above. Briefly,
cells were incubated with a mixture of modified antibodies simultaneously for 45 minutes
at 4°C. Following washing and centrifugation, cells were labeled with a cocktail of
different MnFe2O4 MNPs for 30 minutes at room temperature, before being subjected to
µHD measurements.
In vitro drug treatment
Human A431 cells were seeded overnight and then treated with either geldanamycin
(17AAG, Selleck; 500 or 1000 nM) or vehicle (0.1% DMSO in culture medium) for two
days. Cells were trypsinized and targeted with TCO-conjugated EGFR antibody, before
being coupled with magneto-fluorescent CLIO. Flow cytometry and µHD measurements
were performed in addition to Western blotting and fluorescence microscopy to
investigate the reduced expression of EGFR. For Western blotting, cell lysates were
collected from geldanamycin-treated cells using radioimmunoprecipitation buffer
supplemented with protease inhibitor (Thermo Scientific). Protein lysates were resolved
by SDS-PAGE, and EGFR expression was detected using immunostaining and
chemiluminescence. For fluorescence imaging, cells were incubated with FITC-EGFR
antibody (10 µg/ml) and washed three times with PBS before fixation in 2%
paraformaldehyde and permeabilization in 0.2% Triton X100 for nuclear staining
(TOPRO3, Molecular Probes). Images were taken with a fluorescence microscope
(Eclipse 80i, Nikon).
Mouse tumor model
All animal procedures were performed according to guidelines issued by the Committee
of Animal Care of Massachusetts General Hospital. Cultured A431 cells (1 ×106) were
implanted into immunodeficient nu/nu mice (n = 12). Tumors were allowed to grow for
two weeks before mice were randomized into two groups: a control group and a
treatment group. For the treatment group, 50 mg/kg of geldanamycin was administered
intraperitoneally on a daily basis for 6 days. In the control group, animals were
administered with vehicle (90% saline, 10% DMSO, 0.05% Tween 20). Tumor volumes
in both control and treated animals were measured following 1, 2, 4, and 6 days of
continuous treatment. Fine needle aspirate samples were collected on these days.
Samples were magnetically labeled for EGFR detection via the same protocol used for in
vitro cell targeting.
SUPPLEMENTARY FIGURES
Figure S1. Fabrication of the micro-Hall detector (µHD). (A) A schematic of the stepwise fabrication of the µHD. The sensors were built on a PHEMT GaAs wafer using standard semiconductor processing. The PDMS-based microfluidic channels were fabricated using standard two-layer soft lithography followed by polymer molding. The GaAs chip and the PDMS microfluidics were permanently bonded together. (B) A photograph of the µHD. The GaAs chip is on the bottom and the PDMS microfluidics are on top.
Figure S2 . Design of the flow-focusing microstructures. (A) Finite element simulation of sample flow following flow-focusing via patterned chevrons. (Left) A cross-section of the microfluidic channel after lateral flow-focusing showing the sample being focused towards the center of the channel (sample flow in red; sheath flow in blue). (Right) A cross-section of the channel showing the sample being pushed vertically towards the bottom of the channel. (B) Experimental verification of the flow-focusing microstructures for various sheath flow (green) to sample flow (red) ratios. As the sheath flow rate increased relative to the sample flow rate, the width of sample stream decreased.
A
Signal bandwidth
Frequency (Hz)
Noi
se (
dB µ
V)
B (G)
R (Ω
)
C
1
2
.
.
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3
ADC
PC
PA
PA
A
A
I
PA
PA
A
A
I
PA
PA
A
A
I
4
7
8
Howland voltage/current converter
Amplifiers
G = 30 x 30
HPFf3dB = 500 Hz
B
103 106
0
20
40
Figure S3. Electrical setup and measurement of µHall sensors. (A) The magnetic field sensitivity of the Hall sensors was measured using a known magnetic field. (B) The signal bandwidth, marked with dotted lines, and input-referred noise were measured with a spectrum analyzer. (C) The electronic scheme to readout the µHall sensors. Howland voltage-to-current converters were used to bias the Hall sensors with a current from –10 to 10 mA. The Hall sensors were AC coupled to the preamplifier through a high-pass filter with a pass frequency (ƒ3dB) of 500 Hz. The preamplifier and amplifier had a gain (G) of 30 × 30. The conditioned signal was digitized and sent to a computer.
Figure S4. Computer-simulated Hall voltage. (A and B) With the magnetic field either out-of-plane (A) or in-plane (B), the signal from a bead passing over a Hall sensor was simulated.
Figure S5. Sensor array design. (A) The use of sensor arrays enables more magnetic flux to be detected from the magnetic object, while keeping the filling factor high for optimal signal-to-noise performance. (B) Computer-simulated Hall voltage (VH) as a function of height d above an 8 × 8 µHall array.
Figure S6. Validation of sensor array with magnetic beads. (A) Raw data from all eight Hall sensors following the passing of a magnetic bead (diameter, 8 µm). (B) A histogram of the cubic root of the Hall voltage ⟨VH⟩
1/3 for 3 µm (blue) and 8 µm (red) diameter beads. (Inset) Flow cytometry data using the same beads.
Figure S7. Effect of MNP incubation time. Human tumor cells (MDA-MB-453) were labeled with magnetofluorescent nanoparticles for EpCAM expression. Signal was plotted as a function of incubation time. Data are normalized against the signal at incubation time of 25 min.