Figure 1.
Scheme by which single virus infection of a specific single cell is achieved using an AVF and optical tweezers in a microfluidic chip.
A virus (red dot) is selected from an enriched virus population and is then transported to the cell chamber for infection of a specific single cell. There was an advantage of iDEP that optical tweezers can transport without crossing an electrode as compared to standard (microelectrode based) DEP.
Figure 2.
Scheme of the constricted flow channels and FEM analysis of electrical field intensity.
The constricted flow channel in the analytical model was provided by either two dimensional (2D) microfabrication (a) or 3D microfabrication (b). A non-uniform electric field with a maximum of 0.11 µN was applied and the electric field intensity (left) and the dielectrophoretic force along the center of the microchannel (i.e., at the indicated distance along the line AA’, right) were measured (Input voltage: 20Vp-p). These data indicated that a channel obtained by 3D microfabrication was better than a 2D-constricted channel.
Figure 3.
Fabrication process of the 3D microstructure of the constricted flow channel using maskless gray-scale lithography.
The devices shown are polymer microfluidic chips made using PDMS that were injection molded using photolithography and replica molding techniques. The chip mold was made using a maskless exposure system that achieved synchronous fabrication of the micropattern in the displayed images that were generated by a PC. The light images shown at right are the micropattern image of the maskless photolithography device (top) and a gray-scale exposure (bottom).
Figure 4.
Simulation results of the electric field distribution of an active virus filter (AVF) with height channels.
Analysis of the active virus filter by FEM (a), the electric field distribution intensity in microchannels at heights of 15, 45, 90 and 135 µm from the glass bottom. (b) The electric field intensity and the dielectrophoretic (DEP) force along the centre of the microchannel (i.e., at the indicated distance along the line AA’) at each height are shown.
Figure 5.
Distribution of the virus at various heights from the bottom of the microfluidic channel.
A large number of viruses was trapped at heights of about 10–30 µm from the glass substrate. The AVF inhibits adhesion of the virus to the glass substrate.
Figure 6.
Enrichment, transport and attachment of influenza virus.
Enrichment of the influenza virus in the AVF by a negative DEP force, transport of a single virus to the cell chamber by using optical tweezers and attachment with a selected H292 cell by the virus. The process of iDEP, virus transport and viral infection of an H292 cell is outlined in the top panels. The virus was tracked through these processes by visualization of the green fluorescence of a virus that was co-stained with DiI and SYTO 21 (bottom panels) using confocal microscopy.
Figure 7.
Infection of a virus to the selected H292 cell.
Fluorescent images of cells at 4-PB1 antiserum and anti-rabbit IgG conjugated Cy3 (Red). Nucleus, staining with DAPI (Blue).