Supplementary Information for
Tunable acoustophoretic band-pass particle sorter
Jonathan D. Adams1, H. Tom Soh2,3,*
1
Department of Physics, 2Department of Mechanical Engineering, 3Department of Materials,
University of California, Santa Barbara, CA, 93106, USA
*to whom correspondence should be addressed. email: tsoh@engineering.ucsb.edu
Device Fabrication Process and Experimental Setup:
Standard optical lithography was used to pattern SPR 220-7.0 photoresist (Romm and Hass
Chemicals LLC, Hayward, CA) onto a 500 m thick, 100 mm diameter silicon wafer for use as
an etch mask. The fluidic channels were etched in the Si wafer to a depth of 200 m using a
Bosch deep reactive ion etching process (770 SLR, Plasmatherm, St. Petersburg, FL). Fluidic
access holes were drilled via a CNC drill (Flashcut CNC, Deerfield, IL) with a diamond bit
(Triple Ripple, Abrasive Technology, Lewis Center, OH) in both the silicon wafer (for inlets)
and a 500 m thick, 100 mm diameter Borofloat glass wafer (for outlets). The glass wafer was
anodically bonded (SB6, Suss Microtec AG, Garching, Germany) to the silicon wafer to cap the
microchannel. The wafer was then diced to yield final chip dimensions of 18 mm × 74 mm.
Piezotransducers of diameter 20 mm and thickness 1 mm (PZ26, Ferroperm Piezoceramics,
Kvistgaard, Denmark) were centered over each separation stage and glued to the chip using
superglue (Ross Super Glue Gel, Elmer’s Products, Inc., Columbus, OH). The fluidic inlets and
outlets were attached with 5 minute epoxy (Devcon, Danvers, MA).
The device was then mounted on an inverted fluorescence microscope (TE-2000S, Nikon,
Melville, NY) equipped with a CCD camera (CoolSNAP HQ2, Photometrics, Tuscon, AZ) for
monitoring during operation. Sample and buffer were volumetrically pumped into the device via
syringe pumps (PhD 2000, Harvard Apparatus, Holliston, MA). To excite the acoustic
resonances, separate sinusoidal signals were applied to each piezotransducer via a function
generator (AFG320, Tektronix, Beaverton, OR) and custom amplifier based on an LT1210
operational amplifier (Linear Technology, Milpitas, CA), and the applied voltage signals were
monitored and measured by an oscilloscope (54622A, Agilent, Santa Clara, CA). To minimize
piezotransducer heating during operation, a fan (0925-12LMB, JMC/Da-Tech, Austin TX) was
used for cooling.
Analysis of threshold focusing voltage:
Supplementary Figure 1: The threshold voltage is defined as the voltage required for a transfer
fraction i = 0.5. The values were obtained through linear interpolation of the data presented in
Figure 2. In principle, the threshold transfer i = 0.5 would require a constant focusing rate dy/dt
regardless of particle size which implese V 1 r for a fixed focusing condition. We therefore
plotted the threshold voltage as a function of the reciprocal of the particle diameter. Both the first
stage (black squares) and second stage (red circles) are shown. The data for each stage appear to
follow a linear trend, confirming the inverse relationship between voltage and radius for a fixed
focusing condition.
Frequency response measurement:
We measured the frequency response of each stage of the ABPS. Four cases were measured: 1) f1
swept from 1.6 to 2.5 MHz at V1 = 15 V peak-to-peak with stage 2 off (P1 only). 2) f1 swept
from 1.6 to 2.5 MHz at V1 = 15 V peak-to-peak with f2 = 1.892 MHz and V2 = 43 V peak-to-peak
(P1 and P2). 3) f2 swept from 1.6 to 2.5 MHz at V2 = 15 V peak-to-peak with stage 1 off (P2
only). 4) f2 swept from 1.6 to 2.5 MHz at V2 = 15 V peak-to-peak with f1 = 2.105 MHz and V1 =
43 V peak-to-peak (P2 and P1). 5 m diameter fluorescent particles (G0500, Microgenics Corp.,
Fremont, CA) were suspended in buffer at a concentration of ~106 particles/mL and were
continuously pumped into the sample inlet at a throughput of 2 mL/h, with buffer flow at 14
mL/h in each buffer inlet. (The case P2 only was an exception, where the particle mixture was
pumped at 14 mL/h into the first buffer inlet, with buffer at 2 mL/h into the sample inlet and
second buffer inlet, in order to transfer particles to the side of stage 2 without actuation of the
first piezotransducer.) At each frequency, an image was taken, and an intensity line profile along
a constant transverse path across the channel was recorded. The midpoint of the channel was
determined from intensity profiles taken when neither piezotransducer was actuated. The areas of
the intensity profiles were normalized. A measure of the focusing at each frequency was then
determined by calculating the second moment of area for the normalized intensity profile
I y y   z 2dA ,
A
where z is the distance from the midpoint of the channel, and A is the area of the normalized
intensity profile. The values were then normalized for each case to Iyy measurements from
intensity profiles taken when neither piezotransducer was actuated. Thus, an image recorded
when particles are well focused would have a small Iyy compared with an image when particles
were unfocused. The normalized Iyy values as a function of frequency for each of the four cases
listed above were plotted; the results are shown in Supplementary Figure 2.
Supplementary Fig. 2: Measurement of the frequency response for each stage of the ABPS. a)
Sample image of the device under operation, taken at the end of the first stage. An intensity line
profile was measured along the dotted line. b) Normalized intensity profile along the dotted line
shown in (a). The zero pixel position corresponds to the midpoint of the channel. c) Iyy, the
second moment of area, was measured for the intensity profile at each frequency. The results are
shown for varying f1, with (P1 and P2) and without (P1 only) actuation of the second stage. A
slight shift between ‘P1 only’ and ‘P1 and P2’ is observed, however the shape and amplitude of
the frequency response remains similar, suggesting that the second stage has minimal influence
on the operation of the first stage. The dashed line shows the driving frequency for the second
stage in the ‘P1 and P2’ case. d) Iyy versus f2, without (P2 only) and with (P2 and P1) actuation of
the first stage. The dashed line shows the driving frequency for the first stage. Although a
noticeable amplitude change is present between the two cases, the shape of the frequency
response remains similar.
In both Supplementary Figure 2(c) and 2(d), broad regions of focusing are observable, roughly
between 2.0 and 2.4 MHz for the first stage, and 1.8 and 2.2 MHz for the second stage. In
Supplementary Figure 2(c), both case ‘P1 only’ and ‘P1 and P2’ are very similar both in
resonance peak locations and magnitudes. However, in Supplementary Figure 2(d), the locations
of the resonance peaks are similar while a noticeable magnitude shift is present between the ‘P2
only’ case and the ‘P2 and P1’ case. We believe this magnitude shift is present in only the
second stage, because the driving frequency of the first piezotransducer is near a resonance of the
second stage, while the driving frequency of the second piezotransducer is far from any
resonance in the first stage. Increasing the difference between w1 and w2 would lessen this
coupling, however the piezotransducers were designed for resonance at 2 MHz and become less
efficient as the driving frequencies deviate from this value. Nevertheless, this cross-talk was
insufficient to cause transfer of particles out of the Band-pass Outlet and into the High-pass
Outlet without actuation of the stage 2 piezotransducer, thus permitting independent focusing
control in the two stages.
Although the largest focusing responses were found at f1 = 2.3 MHz and f2 = 2.15 MHz, we
found that these frequencies were less practical to use for a number of reasons: significant
heating of the piezotransducers, high-frequency components in the driving signal, and undesired
behavior such as streaming and off-center particle deflection near the ends of the separation
channels. The driving frequencies f1 = 2.105 MHz and f2 = 1.892 MHz were chosen for the best
overall combination of strong focusing and desirable end-of-channel behavior.