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

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 waves^1,2. The detection of pathogenic bacteria is continued using polymerase chain reaction (PCR), but it is usually laborious, complex, and time-consuming^3,4. 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 performance of the device.
2. Design a microfluidic acoustophoresis chip that collects PS beads at the target outlet and discards the rest throu....

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 reported^20,21,22,23,

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