Afg3022b

Ultrasound fields were generated as described previously [28 ] and the acoustic exposure set-up is illustrated in Figure A1 (Appendix A). Briefly, a 1-cm diameter unfocused piezoceramic transducer was mounted at the bottom of a plastic exposure tank filled with degassed, deionized water. The transducer was driven by a continuous sinusoidal signal at its fundamental frequency (8.8 MHz) using a function generator (AFG3022B; Tektronix, Beaverton, OR, USA,), attenuator (837; KayPENTAX, Montvale, NJ, USA), and RF power amplifier (2100L; ENI, Rochester, NY, USA). Acoustic fields were characterized using both needle (HNC-0400; Onda, Sunnyvale, CA, USA) and capsule (HGL-0085; Onda) hydrophones. A location in the far field was selected (10.5 cm from transducer) such that the transaxial beam width was 3 mm. Acoustic fields were calibrated in the free field before and after each experiment for both amplitude (peak positive and peak negative pressure, MPa) and spatial peak pulse average intensity (I_SPPA, W/cm²). Values from each calibration were averaged across all experiments and are reported as mean ± SEM in Table 1.

To visualize the acoustophoretic motion of cells, the chip was mounted upside down in an inverted microscope (Eclipse Ti2, Nikon, Tokyo, Japan) equipped with a CMOS camera (Prime 95B, Teledyne Photometrics, Tucson, Arizona). Stained PBMCs and neutrophils were spiked into the whole blood and imaged at the end of the separation channel. To monitor the density-adjusted medium at the channel outlet, we added a fluorescent tracer molecule (Dextran, Cascade Blue, 3000 MW, Thermo Fisher Scientific) under the assumption that iodixanol and dextran molecules diffuse at the same rate (D_iodixanol ≈ 2.5 × 10^–10 m² s⁻¹ and D_dextran ≈ 2.2 × 10^–10 m² s⁻¹). A laser illumination unit (Celesta light engine, Lumencor, OR, USA) was used with a multiband filter set (CELESTA-DA/FI/TR/Cy5/Cy7-A-000, Semrock optical filters, IDEX Health & Science) in three excitation channels with peak wavelengths at 365 nm, 488 nm, and 561 nm. The excitation channels and the camera were activated through external triggering.
When operating the chip outside of the AcouWash system the PZT transducer was driven by a function generator (AFG3022B, Tektronix, Inc., Beaverton, Oregon, USA) to deliver a resonant frequency (1.99 MHz) and different applied voltages as measured over the piezo with an oscilloscope (TDS1002, Tektronix, Inc., Beaverton, Oregon, USA).

The standing wave field was created using piezoceramic transducers glued underneath the pre-focusing channel as well as underneath the main separation channel. Frequencies were set to 4.831 MHz for the pre-focusing channel with 5 V_pp amplitude and 1.956 MHz for the main separation channel. A dual channel function generator (AFG3022B, Tektronix, Beaverton, OR, USA), connected to signal amplifiers (in-house build) was used to drive both transducers while the voltage over each transducer was measured via a two-channel digital oscilloscope (TDS 1002, Tektronix). Temperature regulation was achieved using a Peltier element and a PT100 resistance temperature detector attached to the acoustophoretic system.

We used an AC voltage to draw a memristor hysteresis loop and a function generator (AFG3022B, Tektronix, Beaverton, OR, USA.) to apply the AC voltage and a picoammweter (6487, Keithley) to measure the ion current. AC voltages were used ranging from −5 V to 5 V; frequencies of 10, 1, and 0.1 Hz; and concentrations of 1, 10, and 100 mM. The order of injecting the solutions and connecting platinum (PT) electrodes was the same as in Section 2.2. The hysteresis loop is plotted for five periods with the gray line from −5 V to 5 V, and the bold black line is the average of the five period ion currents.