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: rweissleder@mgh.harvard.edu (R.W.); hlee@mgh.harvard.edu (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)

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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.