Trapping of viruses in high-frequency electric field cages

Dielectrophoresis , Cambridge: Cambridge University Press. Schnelle, Th. Gimsa, J. Download references. You can also search for this author in PubMed Google Scholar. Reprints and Permissions. High frequency electric fields for trapping of viruses.

Biotechnol Tech 10, — Download citation. The cells of influenza virus attached and unattached cells were assayed by immunostaining using anti-PB1. We used inverted microscope Ti-E, Nikon Co. The laser is focused by objective lens Magnification: with high numerical aperture NA: 1. The virus is trapped at the laser focal point by Lorentz force because the size of the virus nm is smaller than that of laser wavelength.

Trapping force becomes bigger according to the increase of laser power. However, laser power is limited within 1 W to avoid photo-bleaching of Syto 21 during manipulation. In fact, even if laser power output 1 W, input to a sample is decreased to about mW since an objective lens is passed. The white region represents PDMS microchannel. When 20 Vp-p was applied, the values of the maximum electric field strength around the constricted part of these 3D multi-constricted flow channels were The conductivity and permittivity of the influenza virus that was employed in this work was previously reported by Schnelle et al.

By employing Eq. Analysis of the data shown in Fig. The reason why the gradient generation in each height is considered that by increasing the channel height, the gradient of the electric field should decrease as the electric field streamlines are less disturbed at the trapping area.

The highest iDEP power observed for the four analytic surfaces was that of 0. When the size of influenza viruses is nm, the driving force was calculated to be 9. Therefore, we decided to turn off an AVF in a single virus transporting since the negative DEP force exceeds the driving force by optical tweezers. After enrichment, we then determined the total number of fluorescent influenza viruses in each section by confocal microscopy Z-scan imaging using z-sections with z-scan steps of nm.

Figure 5 shows the experimental result of the distribution of the viruses at different heights in the microfluidic channel after enrichment. The data in this figure indicate that the viruses exhibit negative dielectrophoretic behavior. Furthermore, the viruses were observed to be trapped at regions of the channel that were at a distance from the glass bottom, since they were trapped closer to the region of the high electric field gradient. As a result of observing around this region using a confocal microscope, about times as much virus concentration was shown.

During the enrichment, convection flow due to the joule heat did not occur in these conditions. Thus, the use of an AVF appears to inhibit adhesion of the viruses to the glass substrate. These results demonstrated that AVF has dual functions of virus trapping and inhibition of virus adherence to the glass substrate. The AVF inhibits adhesion of the virus to the glass substrate. We succeeded in trapping one of the enriched viruses by using optical tweezers. Finding and trap of single virus is difficult because the virus is quite small.

Moreover, Brownian motion of the virus is fast and the virus moves away from the focus plane easily. Moreover, we transported this virus to the cell chamber where it made attachment with the selected H cell for infection Fig. Infection was confirmed by detection of the PB1 subunit of influenza virus by immunostaining of the cell using anti-PB1 serum at 4 h after virus attachment.

As shown in Figure 7 , we succeeded in infection of a virus to the selected H cell. However, the microscopic observations indicated that the infection of influenza virus particles to H cells was not uniform, suggesting the cell cycle-dependent variation in virus susceptibility.

Our previous study indicated that influenza virus binds preferentially to G1-phase cells with higher level of sialic acid content. It is reasonable to think that difference of cell cycle could have an effect on infection virus to three cells. 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 H cell by the virus.

The process of iDEP, virus transport and viral infection of an H 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.

We should note that electrode allocation of iDEP contributed to the safe and easy transportation of viruses using optical tweezers. Our previous study indicated that the direct irradiation of laser was caused since the pectinate electrodes and transportation path crossed [3].

On the other hand, since there are no electrodes located across this transportation path in this study, we can avoid direct irradiation of laser by the electrode. Therefore, this microfluidic chip facilitated the effective transport of a single virus from AVFs towards the cell-containing chamber without crossing an electrode.

This is the greatest advantage of using iDEP. A further important point is that iDEP traps have a high flexibility for the design of a microchannel. As the FEM results, iDEP can determine the position of maximum electric field gradients by the design of a microchannel. The viral-cell infection process is a very intriguing interaction [36]. It starts with attachment between the virus and the cell membrane and finally results in transport of the virus into the nucleus and gene expression.

It is essential to understand these processes for antiviral drug design, as well as for the development of efficient gene therapy vectors. We designed the microfluidic chip presented here to selectively enrich for viruses using iDEP in order to enable single virus infection of a specific single cell. We demonstrated the feasibility of this active virus filter with iDEP for reliable enrichment and distribution of a virus. An additional advantage of this technology is that we utilized maskless photolithography to achieve precise 3D gray-scale exposure at a low cost.

AVF can be quickly turned on or off without a decrease in performance. Since this filter can perform virus enrichment and distribution at will, it will therefore contribute to the future of quantitative analysis of viruses and viral functions.

We developed an active virus filter AVF that enabled virus enrichment and distribution for single virus manipulation by using 3D insulator-based dielectrophoresis iDEP. The design of the 3D constricted flow channel enabled the microfluidic chip to produce an iDEP force. We utilized maskless photolithography to achieve precise 3D gray-scale exposure for construction of the constricted flow channel. The use of the AVF achieved enrichment of the influenza virus via a negative dielectrophoretic force.

In this result, we succeeded in infection of a virus to the selected H cell. Investigation of the relationship between the DEP force and the distance between electrodes by FEM analysis of electric field distribution.

Simulations were carried out in which the distance between electrodes was either 4 mm L 1 or 7 mm L 2. The effect of single or multiple AVFs between electrodes set at a distance of 7 mm was also analyzed.

The results of FEM analyses are shown. Enrichment of influenza virus in AVF. De, Leoni, R. D in press. Saito, F. New York: Plenum. Hoekstra, D. Lowry, O.

Download references. Schnelle, T. Fiedler, S. Shirley, K. Ludwig, A. You can also search for this author in PubMed Google Scholar. We are grateful to Prof. Schmidt for the kind gift of Sendai viruses. Web of Science. Let us know here. System error. Please try again! How was the reading experience on this article? The text was blurry Page doesn't load Other:.

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