Summary
This paper describesthe fabrication and operation of microfluidic acoustophoretic chips using the microfluidic acoustophoresis technique and aptamer-modified microbeads that can be used for fast, efficient isolation of Gram-negative bacteria from a medium.
Abstract
This article describes the fabrication and operation of microfluidic acoustophoretic chips using a microfluidic acoustophoresis technique and aptamer-modified microbeads that can be used for the fast, efficient isolation of Gram-negative bacteria from a medium. This method enhances the separation efficiency using a mix of long, square microchannels. In this system, the sample and buffer are injected into the inlet port through a flow controller. For bead centering and sample separation, AC power is applied to the piezoelectric transducer via a function generator with a power amplifier to generate acoustic radiation force in the microchannel. There is a bifurcated channel at both the inlet and outlet, enabling simultaneous separation, purification, and concentration. The device has a recovery rate of >98% and purity of 97.8% up to a 10x dose concentration. This study has demonstrated a recovery rate and purity higher than the existing methods for separating bacteria, suggesting that the device can separate bacteria efficiently.
Introduction
Microfluidic platforms are being developed to isolate bacteria from medical and environmental samples, in addition to methods based on dielectric transfer, magnetophoresis, bead extraction, filtering, centrifugal microfluidics and inertial effects, and surface acoustic waves1,2. The detection of pathogenic bacteria is continued using polymerase chain reaction (PCR), but it is usually laborious, complex, and time-consuming3,4. Microfluidic acoustophoresis systems are an alternative to address this through reasonable throughput and non-contact cell isolation5,6,7. Acoustophoresis is a technology that separates or concentrates beads using the phenomenon of material movement through a sound wave. When sound waves enter the microchannel, they are sorted according to the size, density, etc., of the beads, and cells can be separated according to the biochemical and electrical properties of the suspension medium7,8. Accordingly, many acoustophoretic studies have been actively pursued9,10,11, and recently, 3D numerical simulations of acoustophoretic motion induced by boundary-driven acoustic streaming in standing surface acoustic wave microfluidics have been introduced12.
Studies in various fields are examining how to replace antibodies2,3. Aptamer is a target material having high selectivity and specificity, and many studies are being conducted2,9,10,13. Aptamers have advantages of small size, excellent biological stability, low cost, and high reproducibility compared to antibodies and are being studied in diagnostic and therapeutic applications2,3,14.
Here, this article describes a microfluidic acoustophoresis technology protocol that can be used for the rapid, efficient separation of Gram-negative (GN) bacteria from a medium using aptamer-modified microbeads. This system generates a two-dimensional (2D) acoustic standing wave through single piezoelectric actuation by simultaneously stimulating two orthogonal resonances within a long rectangular microchannel to align and focus aptamer-attached microbeads at the node and anti-node points for separation efficiency2,11,15,16. There is a bifurcated channel at both the inlet and outlet, enabling simultaneous separation, purification, and concentration.
This protocol can be helpful in the field of early diagnosis of bacterial infectious diseases, as well as a rapid, selective, and sensitive response to pathogenic bacterial infections through real-time water monitoring.
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Protocol
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.
- 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.
- 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) - 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.
- 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.
- 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. - 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.
- 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.
- 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.
- For Enterobacter (GN) and Bacillus megaterium (GP; KCTC 1021), incubate in nutrient broth medium at 37 °C and 220 rpm for 16 h.
- For Enterococcus thailandicus (GP), incubate in de Man, Rogosa, and Sharpe (MRS) medium at 37 °C and 220 rpm for 16 h.
- For Listeria grayi (GP), incubate in brain heart infusion medium at 37 °C and 220 rpm for 16 h.
- Centrifuge (9056 x g) the cultured bacteria for 1 min at room temperature (RT), then wash twice with 1x phosphate-buffered saline (PBS) buffer.
- Prepare the selected GN and GP bacteria for analysis by resuspending in PBS buffer.
4. Microbeads and immobilization of aptamer onto microbeads
- Resuspend the streptavidin-coated microbeads (10 µm) mixture (Table of Materials) before use (mix via vortexing for 20 s).
- Prepare aptamer by denaturing at 95 °C for 3 min and then refolding at 0 °C for 2 min.
- 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.
- Incubate the mixture at RT for 30 min while rotating (25 rpm).
- After centrifugation (9056 x g), wash the tube twice with 200 µL of Tris-HCl buffer at RT.
- Add 10 µL of BSA (100 mg/mL) to the washed sample tube and incubate for 30 min at RT with rotation (25 rpm).
- Finally, wash the aptamer-modified microbeads twice by centrifugation (9056 x g) in Tris-HCl buffer at RT.
5. Acoustophoresis setup and operation
- Connect PEEK tubes to the two inlets for injecting two samples and buffer and the two outlets for collecting and discharging waste (Figure 4).
- Manually fill the microfluidic acoustophoresis channel with bubble-free demineralized water using a 10 mL syringe.
- 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. - 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. - 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. - 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. - Observe the separation and enrichment processes on the acoustofluidic chip with a fluorescence microscope and a high-speed camera operating at 1,200 fps.
- 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.
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Representative Results
Figure 5 shows the image of bead flow as a function of PZT voltage (OFF, 0.1 V, 0.5 V, 5 V). In the case of the acoustophoretic chip introduced in this study, it was confirmed that as the voltage of the PZT increased, the central concentration of the 10 µm-sized beads increased. Most of the 10 µm-sized beads were concentrated in the center at 5 V of the PZT voltage. Through this result, a resonant frequency of 3.66 MHz was generated in a single channel function generator, and a general signal was amplified by 16 dB (about 9 times) using a power amplifier.
Table 3 shows the microbeads mixture of 1 µm (cell size) and 10 µm (aptamer attached bead) were injected into the acoustic fluid chip used in this study, and the chip separation performance according to the outlet flow rate (400, 450, and 475 µL/min) is the result of the evaluation. The recovery rate is the ratio of the number of beads collected at the outlet to the total number of injected beads for 10 µm-sized beads, which were 98% ± 2.2%, 98% ± 2.5%, and 90% ± 9.8%, respectively. Purity is the ratio of the number of 10-µm size beads to the total number of beads collected, which were 97.1% ± 3.6%, 97.6% ± 2.4%, and 99.4% ± 0.6%, respectively. This shows that the device has a high separation efficiency for beads with a size of 10-µm.
Figure 6 shows images and a graph of bacteria-bound numbers per bead.Data pertaining to acoustophoresis chip operation regarding the target and waste samples were collected at the outlet and inlet, respectively. All samples were subjected to 10-µL sampling in collection tubes, and the number of bacteria binding to microbeads was observed under a fluorescence microscope. Many GN bacteria (e.g., E. coli DH5α) are bound to all beads, while a few GP bacteria (e.g., Listeria grayi) are bound to some beads. The numbers of GN and GP bacteria bound to each aptamer-modified microbead (of 25 beads) were measured using a microscope with a high-speed camera. All five GN bacteria were bound to beads(4.96 ± 0.77 each), while significantly fewer GP bacteria were bound to the substances (0.08 ± 0.08 each) tested. Signal strength differed significantly between the GN and GP bacteria. These data confirm that this device was successful in isolating GN bacteria.
Figure 1: Schematic of the microchannels, central alignment of microbeads via acoustophoresis, and collection of the targets. Please click here to view a larger version of this figure.
Figure 2: CAD image of the Microfluidic acoustophoresis chip for separation of GN bacteria using aptamer affinity beads. (A) Silicon channel layer. (B) Upper glass layer. (For the dimensions of 1 to 4, see Table 1). Please click here to view a larger version of this figure.
Figure 3: Structure of the acoustophoresis microdevice. (A) Floor plot, (B) cross-section plan, and (C) photograph of the device. Please click here to view a larger version of this figure.
Figure 4: Schematic of the acoustofluidic system used to separate and collect cells. The sample or buffer solution is injected into the inlet port through the flow controller. For bead centering and sample separation, AC power is applied to the PZT via a function generator with a power amplifier to generate an acoustic radiation force in the microchannel. The separated target sample is collected through a collection tube, and the remaining waste fluid is recovered through another outlet. A high-speed camera module is used to visualize the separation. Please click here to view a larger version of this figure.
Figure 5: Image of beads flow as a function of PZT voltage. (A) PZT OFF, (B) PZT 0.1 V, (C) PZT 0.5 V, (D) PZT 5 V. Please click here to view a larger version of this figure.
Figure 6: Numbers of GN (E. coli DH5α) and GP (Listeria grayi) bacteria bound to aptamer-modified microbeads (n = 25, error bar = standard error). Please click here to view a larger version of this figure.
| Number | Device | Width (W) or Diameter (D) (µm) |
| 1 | Inlet and Outlet port (silicon) | 1000 (D) |
| 2 | Microfluidic channel | 200 (W) |
| 3 | Inlet and Outlet channel | 400 (W) |
| 4 | Inlet and Outlet port (glass) | 1500 (D) |
Table 1: Dimensions of the pneumatic microfluidic platform (1 to 4 in Figure 2).
| GN bacteria | GP bacteria |
| Escherichia coli (DH5α) | Bacillus megaterium (KCTC 1021) |
| Enterobacter cloacae | Staphylococcus epidermidis |
| Sphingomonas insulae | Listeria grayi |
| Escherichia coli KCTC 2571 | Enterococcus thailandicus |
| Pseudomonas pictorum | Staphylococcus pasteuri |
Table 2: The GN and GP bacteria studied.
| Flow rate at wate outlet (µL/min) | Recovery rate (%) | Purity (%) |
| 400 (5x volumetric concetnration) | 98 ± 2.2 | 97.1 ± 3.6 |
| 450 (10x volumetric concetnration) | 98 ± 2.5 | 97.6 ± 2.4 |
| 475 (20x volumetric concetnration) | 90 ± 9.8 | 99.4 ± 0.6 |
Table 3: Recovery rate and purity of separation.
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Discussion
We developed a sonic levitation microfluidic device for capturing and transferring GN bacteria from culture samples at high speed based on a continuous running method according to their size and type, and aptamer-modified microbeads. The long, square microchannel enables a simpler design and greater cost-efficiency for 2D acoustophoresis than previously reported20,21,22,23,24,25,26. The device has a recovery rate of >98% up to a 10x dose concentration. This study shows a higher recovery rate and purity than the existing label-free methods20,21,22,23,24 and affinity bead methods25,26 for separating bacteria. This suggests that the device can separate bacteria efficiently. However, as the 10-µm beads can flow into the side channels, the recovery rate is reduced to 90% at a 20x dose concentration due to the strong withdrawal force arising from the closed outlet blocking acoustic radiation in a performance test. While the recovery rate on the chip can reach 98%, several factors reduce the recovery rate, such as the precipitation of beads in the inhaler or clogging of the tube connecting the inhaler to the chip.
There are a few key points needed to make this acoustophoretic chip and make it work. The adhesive used to attach the PZT should be used as little as possible for accuracy of corrugation and the PZT and microchannel should be parallel. Errors in this step result in the transmission of incorrect waveforms through the PZT to the chip, which manifests as beads misalignment. Also, be careful when operating, as several factors can worsen the recovery rate, such as sedimentation of beads from the syringe or blockage of the connecting tube with chips from the syringe.
This device can not only be used to detect live bacteria or bacterial-derived biomarkers in the field of early diagnosis of bacterial infectious diseases but can also be extended to monitoring water contamination, which will help identify bioterrorism and pathogenic bacterial infections.
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Disclosures
The authors have no conflicts of interest to disclose.
Acknowledgments
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT). (No. NRF-2021R1A2C1011380)
Materials
| Name | Company | Catalog Number | Comments |
| 1 µm polystyrene microbeads | Bang Laboratories | PS04001 | Cell size beads |
| 10 µm Streptavidin-coated microbeads | Bang Laboratories | CP01007 | Aptamer affinity beads |
| 4-inch Silicon Wafer/SU-8 mold | 4science | 29-03573-01 | Components of chip |
| Aptamer | Integrated DNA Technologies | GN3-6' | RNA for bacteria conjugation |
| Borosilicate glass | Schott | BOROFLOAT 33 | Components of chip |
| Centrifuge | Daihan | CF-10 | Wasing particles |
| Cyanoacrylate glue | 3M | AD100 | Attach PZT to microchip |
| Escherichia coli DH5α | KCTC | KCTC2571 | Target bacteria |
| Functional generator | GW Instek | AFG-2225 | Generate frequency |
| High-speed camera | Photron | FASTCAM Mini | Observation of separation |
| Hot plate | As one | HI-1000 | Heating plate for curing of liquid PDMS |
| KOVAX-SYRINGE 10 mL Syringe | Koreavaccine | 22G-10ML | Fill the microfluidic acoustophoresis channel with bubble-free demineralized water. |
| Liquid polydimethylsiloxane, PDMS | Dow Corning Inc. | Sylgard 184 | Components of chip |
| LB Broth Miller | BD Difco | 244620 | Cell culture (Luria-Bertani medium) |
| Microscope | Olympus Corp. | IX-81 | Observation of separation |
| PBS buffer | Capricorn scientific | PBS-1A | Wasing bacteria |
| PEEK Tubes | Saint-Gobain Ppl Corp. | AAD04103 | Inject or collect particles |
| Piezoelectric transducer | Fuji Ceramics | C-213 | Generate specific wave in channel |
| Power amplifier | Amplifier Research | 75A250A | Amplify frequency |
| Pressure controller/μflucon | AMED | AMED-μflucon | Control of air pressure/flow controller |
| Tris-HCl buffer | invitrogen | 15567027 | Wasing particles |
| Tube rotator | SeouLin Bioscience | SLRM-3 | Modifiying aptamer and bead |
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