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.
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.
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.
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.
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.
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.
4. Microbeads and immobilization of aptamer onto microbeads
5. Acoustophoresis setup and operation
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.
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.
The authors have nothing to disclose.
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)
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 |