# Fast switching and durable on-chip spark and cavitation bubble cell sorter

The design of the cell sorter is shown in Figs. 1a, b. The microfluidic chip consists of five glass plates, as shown in Fig. 1 C. The three inner plates are 150 μm thick, where the microfluidic channels complete the 3D hydrodynamic focusing of the sample flow27. The third layer is composed of two upstream sheath flow channels for horizontal hydrodynamic focusing, a straight main channel, a waste channel, a collection channel and a pair of electrodes. The main channel is connected to the downstream collection channel and the waste channel via a bifurcation. The fine needle-shaped structure and the arc-shaped parts are respectively positive electrodes (PE) and negative electrodes (NE). The actuation chamber is connected to the main channel by a nozzle close to the bifurcating junction. The second and fourth layers of glass contain microchannels for vertical hydrodynamic focusing. The two outer 1mm glass plates cover the microfluidic chip and provide an observation window and fluid connection ports.

The moving particles are successively illuminated by a rectangular flat-topped 488 nm laser beam, which is shaped by a diffractive optical element (DOE)28, as shown in Figure 1a. If a target particle is detected, a high voltage electric discharge will be applied to the electrodes after a certain delay, generating a dielectric breakdown of the solution between the electrodes. The deposited energy leads to localized heat and initiates a cavitation bubble29. The bubble expands, entraining surrounding liquids and giving a jet stream through the nozzle into the main channel. As a result, the target particle is deflected by the jet stream and flows into the collection channel. Once the maximum volume is reached, the microbubble will quickly shrink and collapse. Then the liquids cover the insulation and the sample returns to the waste channel. The electric discharge is confined within the actuation chamber to prevent the electric field shock from damaging the cells in the main channel (Fig. S1 shows the simulated electric field). The flow rate of the sheath flow is 120 μL/s and that of the sample flow is 0.5 to 2 μL/s. The sheath flow confines the sample stream to a narrow stream with a diameter of 15 μm and a velocity of 5 m/s. This high quality 3D hydrodynamic focus is fundamental for accurate sorting. We applied a buffer flow (40 μL/s) to refresh the liquids between the electrodes and sweep away the electrolyte microbubbles30 which are generated simultaneously with the cavitation bubble. Sheath and buffer fluids are 1× phosphate buffered saline (PBS), which can maintain cell osmolality and reduce threshold voltage for bubble generation31.

Glass plates and metal electrodes are manufactured by laser engraving and integrated by thermocompression32, as shown in Figure 1d. The chip in this work has the same five-layer structure as our previous work32, except for the insertion of three metal electrodes in the third layer (Fig. S2). Manufacturing details are listed in a previous book32, including laser processing, glass cleaning, chip alignment, thermocompression bonding and cooling. A chip is produced in 5 hours and costs no more than 20 USD. The low cost makes the chip disposable to eliminate cross-contamination. Platinum and tungsten are selected as materials for PE and NE, respectively. The deviation between PE and NE is determined by experiments (see Fig. S3 and the explanation). Figure 2 proves that the lifetime of the electrodes is more than 108 spark discharges. After 10seven actions, the electrodes remained intact without degeneration. After 108 actions, the cavitation bubble shrinks due to spark induced erosion on the PE tip. The bubble size can be restored by slightly increasing the discharge energy from 3.0 to 3.5 mJ. In other words, the sorter is still operational after 108 Shares. Stainless steel PE is used in our previous design26 with a lifetime of less than 105as shown in Fig. S4.

### System Setup

The optical detection of the cell sorter is shown in Fig. 3a. A 488 nm (20 mW) laser is shaped by a diffractive optical element28 (DOE) and focused through a lens (F= 40 mm) to form a flat-topped rectangular spot (10 μm × 60 μm). The fluorescence emitted from each particle is collected by the same lens, extracted by two dichroic mirrors (longpass 505 nm and longpass 550 nm) and a bandpass filter (530 nm/43 nm), then detected by using a photomultiplier tube (PMT, R928 + C7427, Hamamatsu, Japan). A sorting command will be given to the high voltage circuit with some delay to trigger a spark cavitation bubble if the PMT signal of the fluorescence exceeds the threshold value (Fig. S5). With the LED flashlight, a high-speed camera (Photron FastCam SA-Z, Photron Inc., Japan) is installed on the opposite side of the chip to capture the sorting process. Three syringe pumps (TS-1B, LongerPump, China) drive sample flow, sheath flow and buffer flow. The high voltage circuit shown in Figure 3b generates individual spark discharges. A 900 V electrical discharge lasting between 2 and 4 µs is applied to the electrodes when a sort command is received.

During the sorting operation, bubbles of cavitation sparks are produced repeatedly with varying time intervals. Since spark discharges increase the conductivity of aqueous solution, although we use buffer flow to refresh the cavitation region, the successive cavitation bubble will grow larger if the time interval between two adjacent discharges is too short31. This will reduce sorting stability and accuracy. To keep the repeated cavitations stable, we developed an algorithm to dynamically adjust the discharge duration. According to our previous research, there is a linear relationship between cavitation bubble volume and discharge duration.31. When the instantaneous activation frequency increases, the discharge duration will be shortened to reduce the energy deposited on the electrodes. Therefore, it can neutralize the influence of increased conductivity of the aqueous solution, and the bubble size is kept stable during the high-frequency sorting period. The duration of the notth action is adjusted according to its time intervals to the previous three actions, as expressed below.

$$begin {array}{l}t_n = t_{st}left[ {1 – k_1left( {1 – frac{{Delta t_{n – 1}}}{{Delta t_{st}}}} right) cdot Hleft( {1 – frac{{Delta t_{n – 1}}}{{Delta t_{st}}}} right) – k_2left( {2 – frac{{Delta t_{n – 2}}}{{Delta t_{st}}}} right)}right.qquad left.{cdot Hleft( {2 – frac{{Delta t_{n – 2}}}{{Delta t_{st}}}} right) – k_3left( {3 – frac{{Delta t_{n – 3}}}{{Delta t_{st}}}} right) cdot Hleft( {3 – frac{{Delta t_{n – 3}}}{{Delta t_{st}}}} right)} right]end {array}$$

(1)

Hleft( x right) = left{ {begin{array}{*{20}{c}} 1 & {x ,ge, 0} 0 & {x ,

(2)

where younot is the discharge time of the notth sorting operation, youst= 4 μs is the standard discharge time, and (Delta t_{st} = 500upmu {rm{s}}) is the standard time interval between two adjacent actions. (Delta t_{n – 1}), (Delta t_{n – 2}) and (Delta t_{n – 3}) are the time intervals between the current action and the three previous actions. k1, k2 and k3 are the adjustment coefficients, which are determined to be 0.30, 0.10 and 0.05 in our experiments. By applying the dynamic adjustment algorithm, the bubble size is significantly stabilized (Figure S6 shows the stabilizing effect of the algorithm).

### Sample preparation

Green (Ex 470 nm/Em 526 nm) and red (Ex 620 nm/Em 680 nm) fluorescent polystyrene beads of different sizes (BeasLine Tech, China) were used to calibrate the sorter and characterize its sorting ability. HeLa cells were used to assess the impact of sorting on cell viability. HeLa cells expressing green fluorescent protein (GFP) and HeLa cells expressing red fluorescent protein (RFP) were cultured in modified RPMI medium (HyClone, SH30809.01) with 10% fetal bovine serum (Thermo Fisher, 10091148 ) and 1% penicillin-streptomycin (all from HyClone, SV30010) in a cell incubator (HF90, HealForce, China) at 37°C with 5% CO2. Prior to use, HeLa cells were detached from the flask with trypsin (0.25%, HyClone), washed, and resuspended in a mixture of PBS and OptiPrep density gradient medium (D1556, Sigma-Aldrich, St. Louis, MO) in an 88:12 compression ratio. Concentrations are adjusted to ten6 beads per mL for polystyrene beads and 5×106 cells per mL for HeLa cells. To measure viability, HeLa cells were incubated on ice with PI (propidium iodide) solution (500 μg/mL, RuiTaiBio, China) at 4 μL/mL for 5 min. Dead cells stain positively and therefore viability can be measured by counting the percentage of cells that stain negatively.