Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads

1. Microfluidic acoustophoresis chip design

NOTE: Figure 1 shows a schematic of the separation and collection of target microbeads from microchannels by acoustophoresis. The microfluidic acoustophoresis chip is designed with a CAD program.

1. Design a microfluidic acoustophoresis chip that uses a mixture of aptamer-modified beads and streptavidin-coated polystyrene (PS) beads corresponding to the size of bacteria to study the separation performance of the device.
2. Design a microfluidic acoustophoresis chip that collects PS beads at the target outlet and discards the rest through the outlet after injecting a PS sample mixture into the sample inlet (see Figure 2 and Table 1).
   NOTE: For this purpose, the acoustofluidic chip is designed consisting of two inlets for injecting samples and buffer, a main channel with an attached piezoelectric transducer (PZT) to allow the microbeads to be aligned centrally, and two outlets through which specimens are collected and waste are discharged (Figure 3A)
3. Design a microfluidic acoustophoresis chip in which microbeads, buffer, bacteria, and aptamers pass through the main channel, with the microbeads aligned centrally via in-chip acoustophoresis induced by the PZT (Figure 3B,C)

2. Microfluidic acoustophoresis chip fabrication

NOTE: Assemble four layers in the following order: a borosilicate glass-silicon layer, a silicon layer, a borosilicate glass layer, and a PZT layer, as shown in Figure 3A,B.

1. Prepare borosilicate glass (the uppermost layer) with 2-mm-diameter holes by sandblast¹⁷ at the inlet and outlet for connecting polyetheretherketone (PEEK) tubes. The borosilicate glass measures 20 × 80 × 0.5 mm³.
2. Prepare a 200-µm-thick silicon channel layer with a microchannel with a cross-sectional area of 0.2 × 0.2 mm² formed using a photoresist and a silicon pattern obtained via deep reactive ion etching (RIE)¹⁸. Drill 1 mm diameter holes for the sample and buffer inlet channels and the collection and waste outlet channels during reactive ion etching.
   NOTE: Here, ion-etching uses the RIE process to form microchannels. A photoresist was applied in the shape of a channel on a silicon wafer for the silicon channel layer. The PR-coated silicon wafer was etched with plasma generated by applying 13.56 Mhz to fluorine¹⁸.
3. Prepare a chip with the layers above and below the silicon layer (20 × 80 × 0.5 mm³) prepared in step 2.2 (20 × 80 × 0.5 mm³) bonded to the borosilicate glass in step 2.1 and a third borosilicate glass layer using anodization at 1,000 V and 400 °C¹⁹.
4. Attach a PZT (20 × 40 mm²) to the borosilicate glass layer along the microfluidic channel using a cyanoacrylate adhesive (10 µL or less).
   ​NOTE: Apply the adhesive as a very thin layer in the channel using a cotton swab to minimize any change in height. Figure 3C is a picture of the device.

3. Bacterial strains and culture

NOTE: Refer to Table 2 to select and incubate GN and Gram-positive (GP) bacteria for experiments. For the culture method, refer to steps 3.1-3.4. All bacteria should be incubated under aerobic conditions until an absorbance of 0.4 at 600 nm (OD₆₀₀) is obtained.

1. For GN bacteria such as Escherichia coli DH5α, Escherichia coli KCTC2571, Sphingomonas insulae, and Pseudomonas pictorum and GP bacteria such as Staphylococcus epidermidis and Staphylococcus pasteuri, incubate in Luria-Bertani medium at 37 °C and 220 rpm for 16 h.
2. For Enterobacter (GN) and Bacillus megaterium (GP; KCTC 1021), incubate in nutrient broth medium at 37 °C and 220 rpm for 16 h.
3. For Enterococcus thailandicus (GP), incubate in de Man, Rogosa, and Sharpe (MRS) medium at 37 °C and 220 rpm for 16 h.
4. For Listeria grayi (GP), incubate in brain heart infusion medium at 37 °C and 220 rpm for 16 h.
5. Centrifuge (9056 x g) the cultured bacteria for 1 min at room temperature (RT), then wash twice with 1x phosphate-buffered saline (PBS) buffer.
6. Prepare the selected GN and GP bacteria for analysis by resuspending in PBS buffer.

4. Microbeads and immobilization of aptamer onto microbeads

1. Resuspend the streptavidin-coated microbeads (10 µm) mixture (Table of Materials) before use (mix via vortexing for 20 s).
2. Prepare aptamer by denaturing at 95 °C for 3 min and then refolding at 0 °C for 2 min.
3. Transfer 250 µL of the resuspended streptavidin-coated microbeads mixture to a 1.5 mL tube and wash with Tris-HCl buffer (50 mM Tris, pH 7.4, 1 mM MgCl₂, 5 mM KCL, 100 mM NaCl) at RT. Then add 100 µL of biotinylated DNA aptamer to the tube.
4. Incubate the mixture at RT for 30 min while rotating (25 rpm).
5. After centrifugation (9056 x g), wash the tube twice with 200 µL of Tris-HCl buffer at RT.
6. Add 10 µL of BSA (100 mg/mL) to the washed sample tube and incubate for 30 min at RT with rotation (25 rpm).
7. Finally, wash the aptamer-modified microbeads twice by centrifugation (9056 x g) in Tris-HCl buffer at RT.

5. Acoustophoresis setup and operation

1. Connect PEEK tubes to the two inlets for injecting two samples and buffer and the two outlets for collecting and discharging waste (Figure 4).
2. Manually fill the microfluidic acoustophoresis channel with bubble-free demineralized water using a 10 mL syringe.
3. Prepare a precision pressure controller with two or more output channels to control the fluid flow. Then, half-fill the vials with sample and buffer with two holes in their caps, respectively, and connect to the chip inlet.
   NOTE: A precision pressure controller with two or more output channels can be replaced with multiple precision pressure controllers. At the buffer inlet, a buffer capable of generating a laminar flow that prevents the sample from moving to the center during sample injection is injected through a flow controller.
4. After preparing the device, inject the sample and buffer by applying a pressure of 2 kPa to the sample inlet and 4 kPa to the buffer inlet using the precision pressure control device.
   NOTE: At this time, for smooth laminar flow, the injection pressure of the buffer should be higher than the injection pressure of the sample. The flow is controlled by a flow controller fixed to the aspirator connected to the inlet channel.
5. Focus on a bead to move it into the center of the microfluidic channel using the PZT while checking through the microscope.
   NOTE: The larger the bead, the greater the effect on the waveform, so it is easier to align with the node point. Function generator with amplifier applies power to PZT to generate sine wave in the microchannel. Since the upper and lower portions of the microchannel are made of glass, the generated sine wave is reflected and creates a node point⁷.
6. Generate a resonance frequency of 3.66 MHz using a single-channel function generator and amplify a typical signal by 16 dB (about nine-fold) using a power amplifier (Figure 4).
   NOTE: The resonance frequency of the actuator must match the size of the channel; because the channel is square, the PZT operates at an accurate frequency to create a single node.
7. Observe the separation and enrichment processes on the acoustofluidic chip with a fluorescence microscope and a high-speed camera operating at 1,200 fps.
8. Quantify and analyze the presence or absence of GN bacteria and GP bacteria by checking the images taken with the fluorescence microscope camera of the bacteria-bound beads and bacteria discharged through the collection and waste outlet samples.
   NOTE: Microbeads with buffer, bacteria, and aptamers pass through the main channel, and the microbeads are aligned centrally via in-chip acoustophoresis induced by the PZT. Finally, microbeads that have bound GN bacteria are collected at the collection outlet, and the uncollected bacteria are discharged through the waste outlet.