Virus Enrichment for Single Virus Infection by Using 3D Insulator Based Dielectrophoresis
Citation: Masuda T, Maruyama H, Honda A, Arai F (
Virus Enrichment for Single Virus Infection by Using 3D Insulator Based Dielectrophoresis
Taisuke Masuda 0
Hisataka Maruyama 0
Ayae Honda 0
Fumihito Arai 0
Suryaprakash Sambhara, Centers for Disease Control and Prevention, United States of America
0 1 Department of Micro-Nano Systems Engineering, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya, Japan, 2 Department of Frontier Bioscience, Hosei University , Koganei, Tokyo , Japan
We developed an active virus filter (AVF) that enables virus enrichment for single virus infection, by using insulator-based dielectrophoresis (iDEP). A 3D-constricted flow channel design enabled the production of an iDEP force in the microfluidic chip. iDEP using a chip with multiple active virus filters (AVFs) was more accurate and faster than using a chip with a single AVF, and improved the efficiency of virus trapping. We utilized maskless photolithography to achieve the precise 3D grayscale exposure required for fabrication of constricted flow channel. Influenza virus (A PR/8) was enriched by a negative DEP force when sinusoidal wave was applied to the electrodes within an amplitude range of 20 Vp-p and a frequency of 10 MHz. AVF-mediated virus enrichment can be repeated simply by turning the current ON or OFF. Furthermore, the negative AVF can inhibit virus adhesion onto the glass substrate. We then trapped and transported one of the enriched viruses by using optical tweezers. This microfluidic chip facilitated the effective transport of a single virus from AVFs towards the cellcontaining chamber without crossing an electrode. We successfully transported the virus to the cell chamber (v = 10 mm/s) and brought it infected with a selected single H292 cell.
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Funding: This work was partially supported by Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corporation
(JST). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional funding was
received for this study.
Competing Interests: The authors have declared that no competing interests exist.
Conventional analysis of viral functions is performed by using
virus-infected cultured cells and this method has been considered
as the most precise technique of analysis. This method provides
information that is an average result of data generated from all of
the cells in the population. However, the physiological state and
cell cycle stage of each infected cell is different [1]. In order to
quantitatively analyze viral effects, analysis of specific cell that is
infected by a single virus is required. To satisfy this requirement,
we previously constructed a system for the manipulation of a single
virus using optical tweezers [2,3]. This single-virus infection system
enabled the effective transport of a single virus from the periphery
towards the cell-containing chamber. Recently this system was
used to characterize the difference in influenza virus susceptibility
between G1- and S/G2/M-phase cells [4]. However,
bionanoparticles such as the influenza virus (shape: sphere, diameter:
approximately 100 nm) tend to be present in samples at a low
concentration, and a low virus number provides a limitation to the
method. We therefore fabricated an active virus filter (AVF) that
could enrich for viruses and modulate virus distribution, by using a
dielectrophoretic force.
A dielectrophoretic force is a force that is exerted on a
polarizable particle under conditions of a non-uniform electric
field [5,6,7]. Dielectrophoretic manipulation and accumulation of
micro- and nanoparticles as well as its theoretical background were
first advocated by Pohl [8]. Biological particles such as cells,
bacteria, macromolecules, DNA and viruses have been extensively
studied using this method [9]. There is now considerable effort
being directed toward applying dielectrophoresis (DEP) for
biomedical and biotechnological applications [10,11,12]. DEP
has been used to trap and analyze individual cells, immobilize cells
in an array format, separate different cell types (e.g. viable from
dead cells), detect bacteria and manipulate viruses. Conventional
electrode-based systems generate electric field gradients by
applying an AC signal across two or more metallic electrodes.
These systems typically use a coplanar electrode [13,14] or an
interdigitated castellated microelectrode [15,16,17], and trap
particles at or near the electrode surfaces. Electrode-based DEP
systems have been used in the analysis of various particles for the
purpose of concentration of the particles in the samples, and they
exhibit high selectivity [18,19].
In insulator-based dielectrophoresis (iDEP), remote electrodes
apply an electric field within a fluidic volume while insulating
structures are used to distort the electric field thereby producing
spatial non-uniformities [20,21,22]. They can be (...truncated)