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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 ....

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.

  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 p.......

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 gen.......

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,

Acknowledgements

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

NameCompanyCatalog NumberComments
1 µm polystyrene microbeadsBang LaboratoriesPS04001Cell size beads
10 µm Streptavidin-coated microbeadsBang LaboratoriesCP01007Aptamer affinity beads
4-inch Silicon Wafer/SU-8 mold4science29-03573-01Components of chip
AptamerIntegrated DNA TechnologiesGN3-6'RNA for bacteria conjugation
Borosilicate glassSchottBOROFLOAT 33Components of chip
CentrifugeDaihanCF-10Wasing particles
Cyanoacrylate glue3MAD100Attach PZT to microchip
Escherichia coli DH5αKCTCKCTC2571Target bacteria
Functional generatorGW InstekAFG-2225Generate frequency
High-speed cameraPhotronFASTCAM MiniObservation of separation
Hot plateAs oneHI-1000Heating plate for curing of liquid PDMS
KOVAX-SYRINGE 10 mL SyringeKoreavaccine22G-10MLFill the microfluidic acoustophoresis channel with bubble-free demineralized water.
Liquid polydimethylsiloxane, PDMSDow Corning Inc.Sylgard 184Components of chip
LB Broth MillerBD Difco244620Cell culture (Luria-Bertani medium)
MicroscopeOlympus Corp.IX-81Observation of separation
PBS bufferCapricorn scientificPBS-1AWasing bacteria
PEEK TubesSaint-Gobain Ppl Corp.AAD04103Inject or collect particles
Piezoelectric transducerFuji CeramicsC-213Generate specific wave in channel
Power amplifierAmplifier Research75A250AAmplify frequency
Pressure controller/μfluconAMEDAMED-μfluconControl of air pressure/flow controller
Tris-HCl bufferinvitrogen15567027Wasing particles
Tube rotatorSeouLin BioscienceSLRM-3Modifiying aptamer and bead

References

  1. Wu, M., et al. Acoustofluidic separation of cells and particles. Microsystem & Nanoengineering. 5 (1), 1-18 (2019).
  2. Lee, S. W., et al. Aptamer affinity-bead mediated capture and displacement of Gram-n....

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