JP7848655B2 - Dielectrophoresis apparatus and dielectrophoresis method - Google Patents

Description

This invention relates to a dielectrophoresis apparatus and a dielectrophoresis method, and more particularly to a dielectrophoresis apparatus and a dielectrophoresis method for manipulating objects such as cells and microparticles in a solvent.

Figure 1 shows the structure of a conventional dielectrophoresis apparatus. The structure in Figure 1 is the structure of a dielectrophoresis apparatus described in Non-Patent Literature 1, and mainly comprises an upper electrode and a lower electrode, which are glass substrates with ITO, and a photoconductive material such as amorphous silicon or P3HT (poly(3-hexylthiophene)):PCBM (phenyl C61-butyric acid methyl ester) placed between the upper and lower electrodes. Furthermore, micromaterials such as bead particles or cells are suspended in a solvent such as water between the upper and lower electrodes. AC power supplies are connected to the upper and lower electrodes so that an AC voltage can be applied to the solvent. A projector is also provided on the lower electrode side to irradiate light of a wavelength that can be absorbed by the photoconductive material.

In the apparatus shown in Figure 1, applying an AC voltage to the upper and lower electrodes applies the AC voltage to the solvent, such as water, sandwiched between the electrodes. The solvent contains microscopic material particles, and the application of the AC voltage to the solvent induces charge in these particles. Simultaneously, when light of a desired wavelength is incident from the lower electrode, this light irradiates a photoconductive material (such as amorphous silicon). In the irradiated portion of the photoconductive material, the light is absorbed, forming electron-hole pairs, which increases the conductivity of the amorphous silicon (decreases its resistance). Because the conductivity of the electrodes differs between the irradiated and unirradiated regions, the voltage applied to the solvent changes, creating an electric field difference. The induced charge in the microscopic material causes it to move due to dielectrophoretic force caused by this electric field difference. If the dielectric constant of the microscopic material is lower than that of the solvent, the microscopic material moves in the direction of lower electric field strength. Conversely, if the dielectric constant of the microscopic material is higher than that of the solvent, the microscopic material moves in the direction of higher electric field strength.

Figure 2 shows the behavior of dielectrophoresis when the dielectric constant of the micromaterial is lower than that of the solvent. Figure 2 is a diagram illustrating the dielectrophoresis described in Non-Patent Literature 1, depicting how 1 μm beads are attracted to the light-irradiated region and 10 μm beads are attracted to the unirradiated region inside the light-irradiated region.

The dielectric constant of micromaterials is approximately 2.4 for polystyrene beads and approximately 47.5 for cells (see, for example, Non-Patent Document 2). On the other hand, in environments where the physiological activity of cells and proteins is preserved, solvents such as DMEM (Dulbecco's Modified Eagle Medium) and PBS (Phosphate-buffed saline) are used, and their dielectric constant is approximately 80, which is higher than that of the cells or polymer materials to be manipulated.

When dielectrophoresis is applied to a material with a dielectric constant lower than that of the solvent, RE[F _cm (ω)] (a coefficient reflecting the polarization of the particles) in the following equation 1, which represents the dielectrophoretic force, becomes negative, and a repulsive force (negative dielectrophoresis) acts.

The values of each element in formula 1 above are as follows:
F _DEP : Dielectric force, r: particle size of the material, ε _m : dielectric constant of the solvent, ∇E _rms : electric field, RE[F _cm (ω)]: Clausius-Mossotti coefficient (also written as Re[K])

Furthermore, in highly polar materials such as cells and bacteria, the vector of the dielectrophoretic force can be changed by applying a high frequency of 1 × ^10⁶ Hz or higher, as shown in Figure 3. Figure 3 is a diagram showing the relationship between the Clausius-Mossotti coefficient and frequency as described in Non-Patent Literature 3.

Wenfeng Lianga et al. , “A Review on Optoelectrokinetics-Based Manipulation”, Micromachines January 2020, 11, 78 Keita Yamamoto et al., "Development of intracellular delivery technology for biomolecules using electric field driving force (Part 6) - Effect of voltage application conditions on DNA ejection efficiency -", Toyohashi University of Technology, Proceedings of the 2016 Spring Meeting of the Japan Society for Precision Engineering. Yamakawa, Retsu et al., Separation of living cells using dielectrophoresis, Electrochemistry, 82(11), 1000-1006 (2014) Yoneichi Inagaki et al., "Effects of radiation irradiation on the dielectric properties and other aspects of polyethylene," Journal of the Institute of Electrical Engineers of Japan, Vol. 80, No. 862 (July 1960).

When performing dielectrophoresis in a high-dielectric-constant solvent, such as a physiologically active environment, repulsive forces act on micromaterials like cells and polymer particles. This results in manipulation solely based on repulsion, limiting the freedom of control over these micromaterials.

In environments where the physiological activity of cells and proteins is maintained, the dielectric constant of the solvent is higher than that of the cells or polymer materials to be manipulated, resulting in negative dielectric electrophoresis (repulsion). As shown in Figure 4, for low-polarity materials such as polyethylene and polystyrene, the change in dielectric constant with frequency is small, around 3.0 (right axis in Figure 4), and it is not possible to change the sign of the Clausius-Mossotti coefficient Re[K] in Equation 1, thus only repulsive force can be exerted. Note that Figure 4 shows the frequency characteristics of the dielectric constant of polyethylene as described in Non-Patent Literature 4.

Therefore, the present invention proposes an apparatus and method that allows for the manipulation of the dielectric vector in any direction when performing dielectrophoresis in a solvent with a high dielectric constant, such as a physiologically active environment, by creating regions with locally different conductivity and applying a specific frequency.

The dielectrophoresis apparatus of the present invention is a dielectrophoresis apparatus for manipulating an object to be manipulated by dielectrophoresis, comprising: a first electrode and a second electrode, both transparent and spaced apart to contain a solvent containing the object to be manipulated; a photoconductive section, positioned facing the solvent and whose conductivity changes upon light irradiation; a light irradiation section for locally irradiating the photoconductive section with light; an AC power supply for applying an AC voltage to the first electrode and the second electrode; and a control unit for controlling the frequency of the AC voltage applied by the AC power supply.

Furthermore, the dielectrophoresis method of the present invention is a dielectrophoresis method for manipulating an object to be manipulated by dielectrophoresis, comprising: applying an AC voltage to a first electrode and a second electrode, which are transparent and spaced apart to contain a solvent containing the object to be manipulated; locally irradiating a photoconductive portion, which is positioned facing the solvent and whose conductivity changes upon light irradiation, with light; and changing the frequency of the AC voltage.

According to the present invention, when performing dielectrophoresis in a solvent with a high dielectric constant, such as in a physiologically active environment, it becomes possible to manipulate the dielectrophoretic vector in any direction by creating regions with locally different conductivity and applying a specific frequency.

This diagram shows the structure of a conventional dielectrophoresis apparatus. This figure shows the behavior of dielectrophoresis when the dielectric constant of a minute material is lower than the dielectric constant of the solvent. This figure shows the relationship between the Clausius-Mossotti coefficient and frequency. This figure shows the frequency characteristics of the dielectric constant of polyethylene. This is a diagram showing the configuration of the dielectrophoresis apparatus 1 according to the embodiment. This is a flowchart of the dielectrophoresis method according to the embodiment. This figure shows the relationship between the frequency of the AC voltage in the embodiment and the forces (repulsive and attractive forces) acting in the light-irradiated area. This image shows the results of verifying the forces acting on the light-irradiated and un-irradiated regions of the embodiment for each AC voltage frequency. This is an example of manipulating an object using the switching between repulsive and attractive forces via frequency control in the embodiment.

The following describes a dielectrophoresis apparatus and dielectrophoresis method according to embodiments of the present invention, with reference to the drawings.

(Configuration of dielectrophoresis apparatus 1)
Figure 5 is a diagram showing the configuration of the dielectrophoresis apparatus 1 of this embodiment. As shown in Figure 5, the dielectrophoresis apparatus 1 of this embodiment manipulates an object to be manipulated by dielectrophoresis. It comprises a working electrode 10 (first electrode), a counter electrode 20 (second electrode), a photoconductive section 15 provided between the working electrode 10 and the counter electrode 20, a function generator 30 (AC power supply) that applies an AC voltage having an arbitrary frequency to the working electrode 10 and the counter electrode 20, a laser projector 40 (light irradiation section) that irradiates light, a digital mirror device 50 and a lens 60 which are optical systems that irradiate the photoconductive section 15 with light irradiated from the laser projector 40, and a spacer 70 that supports the two electrodes 10 and 20 separated by a predetermined distance.

Each of the operating electrode 10 and the counter electrode 20 is a translucent electrode comprising a glass plate and a transparent electrode, such as ITO (Indium Tin Oxide), formed on the glass plate. The operating electrode 10 and the counter electrode 20 are spaced apart to accommodate the solvent containing the object to be manipulated X.

The photoconductive portion 15 is made of a photoconductive material such as amorphous silicon. The photoconductive portion 15 is positioned facing the solvent, and its conductivity changes upon light irradiation.

A solvent containing the object to be manipulated X is contained between the operating electrode 10 and the counter electrode 20. The solvent is a high-dielectric-constant solvent, such as a physiologically active environment, and the object to be manipulated X is a micromaterial such as cells, polymer particles, or polyethylene beads. For example, the dielectric constant of the object to be manipulated X is lower than that of the solvent.

The function generator 30 applies an AC voltage of a specified frequency to the operating electrode 10 and the counter electrode 20, and applies this AC voltage to the solvent. The applied AC voltage induces a charge in the object X in the solvent.

The laser projector 40 irradiates the photoconductive material of the photoconductive part 15 with light of a wavelength that can be photoexcited. The change in the photoconductivity of the photoconductive material is preferably about 1 × ^{10² to 4} S/m. The laser projector 40 can change the position, shape, and size of the irradiated light. For example, the laser projector 40 can change the shape of the light irradiated onto the photoconductive part 15 to an annular or circular shape, gradually decrease the size of the light, or displace the position of the light. The shape of the light may be other than an annular or circular shape.

A solvent, such as a suspension of a micromaterial (object X), is packed between the two electrodes 10 and 20. In the photoconductive portion 15, a region with locally high conductivity (light-irradiated region) A is formed by irradiation with light from the laser projector 40, and a region with lower conductivity than light-irradiated region A (un-irradiated region) B is formed. The difference in conductivity between light-irradiated region A and un-irradiated region B is preferably about 1 × ^{10² to 4} S/m.

Furthermore, the dielectrophoresis apparatus 1 includes a control unit 80 that controls the frequency of the AC voltage applied by the function generator 30. The control unit 80 is a computer, such as a microcontroller, that includes a processor 81 and memory 82. The processor 81 is a CPU (Central Processing Unit) or DSP (Digital Signal Processor), and it loads programs stored in the auxiliary storage unit 83 into the memory 82 and executes them. The programs stored in the auxiliary storage unit 83 are, for example, frequency control programs that control the frequency of the AC voltage applied by the function generator 30. The processor 81 instructs the voltage value and frequency of the AC voltage applied by the function generator 30 via the I/F (interface) 84.

Furthermore, the control unit 80 controls the operation of the laser projector 40 to change the position, shape, and size of the light emitted by the laser projector 40. The processor 81 instructs the position, shape, and size of the light emitted by the laser projector 40 via the interface 84.

(Operation of dielectrophoresis apparatus 1)
In this embodiment, regions A and B with locally different conductivity are provided, and the impedances of regions A and B with different conductivity are reversed at a specific frequency to manipulate the dielectrophoretic vector. When the function generator 30 applies an AC voltage to electrodes 10 and 20, a potential difference is created between region A (light-irradiated region) with high conductivity and region B (un-irradiated region) with lower conductivity compared to region A. As a result, a local electric field ∇E is formed in region A, and a dielectrophoretic vector acting from region A to region B is generated on the object X being manipulated. In this case, as shown in Figure 5(b), a repulsive force acts on region A (light-irradiated region).

On the other hand, when the function generator 30 applies an AC voltage of a specific frequency to electrodes 10 and 20, the impedance between region A and region B is reversed, the sign of the potential difference between region A and region B is reversed, and a local electric field ∇E is formed in region B. Then, a dielectrophoretic vector acting from region B to region A is generated on the object X being manipulated. In this case, as shown in Figure 5(a), an attractive force acts on region A (the light-irradiated region).

(Dielectrophoresis method)
Figure 6 is a flowchart of the dielectrophoresis method according to the embodiment. The dielectrophoresis method will be described with reference to Figure 6. Each step in the flowchart of Figure 6 is performed by the control unit 80.

The control unit 80 instructs the function generator 30 to apply an AC voltage of default voltage value and frequency to electrodes 10 and 20 (step S601). The function generator 30 applies the AC voltage of default voltage value and frequency to electrodes 10 and 20 in accordance with this instruction.

The control unit 80 instructs the laser projector 40 to emit an annular shape of light (step S602). The laser projector 40 emits an annular shape of light in accordance with this instruction.

The control unit 80 instructs the laser projector 40 to reduce the size of the annular-shaped light (step S603). The laser projector 40 reduces the size of the annular-shaped light according to this instruction.

The control unit 80 instructs the function generator 30 at a predetermined timing to change the frequency of the AC voltage to a specific frequency (step S604). The function generator 30 changes the frequency of the AC voltage to the specific frequency according to this instruction. The predetermined timing may be when the elapsed time for reducing the size of the annular light reaches a predetermined time, or when the size of the annular light reaches a predetermined size. Alternatively, for example, the control unit 80 may change the frequency of the AC voltage from a first frequency (1 kHz) to a second frequency (10 kHz) that is higher than the first frequency, according to this instruction.

The control unit 80 then instructs the laser projector 40 to change the shape of the light to a circular shape (step S605). The laser projector 40 changes the annular shape of the light to a circular shape according to this instruction.

In this example, the size of the annular light beam was reduced, and its shape was changed from an annular to a circular shape. However, the shape, size, and position of the light beam emitted by the laser projector 40 may be appropriately selected depending on the operation being performed on the object X.

(Method for calculating specific frequencies that allow switching between repulsive and attractive forces in dielectrophoresis)
Next, we will explain how to calculate a specific frequency at which the repulsive and attractive forces of dielectrophoresis can be switched. By performing the following calculations, it is possible to calculate a specific frequency at which the attractive and repulsive forces of dielectrophoresis can be switched, as well as the impedance of the electrodes. In this embodiment, we consider photoexcitation dielectrophoresis of polystyrene beads. The impedance of this circuit is a series circuit of three impedances: the impedance of the ITO-coated glass Zi, the impedance of the solvent Zm, and the impedance of α-Si (amorphous silicon) Zα. The impedance Zt of this entire series circuit is calculated by the following equation 2.

Furthermore, each impedance is calculated using the following equations 3 to 5.

However, since ITO is a conductor, its capacitance was ignored.

The values of equations 4 and 5 mentioned above are as follows:
_Wm : electrode area in contact with solvent, _dm : distance from solvent, _σm : conductivity of solvent, _εm : relative permittivity of solvent, _ε0 : permittivity of vacuum, f: frequency of applied voltage

The lowercase letter α in equation 5 is equivalent to the lowercase letter m in equation 4.

Since the impedance of ITO is small and can be ignored, Zt can be calculated using the following equation 6.

If the total voltage applied is V, the voltage Vm applied to the solvent is calculated using the following equation 7.

The electric field strength Em applied to the solvent is calculated using the following formula 8.

Next, the electric field applied to the solvent in the light-irradiated region (region A) and the unirradiated region (region B) is calculated using the formula described above. The constants used are those shown in Table 1 below.

First, calculate the resistance and capacitance of the unirradiated region (region B).
The resistance Rα and capacitance Cα of the α-Si thin film in the unirradiated region (region B) are calculated using equations 9 and 10.

The resistance Rm and volume Cm of pure water in the unirradiated region (region B) are calculated using equations 11 and 12.

Next, calculate the resistance and capacitance of the light-irradiated area (area A).
The resistance Rαl and capacitance Cαl of the α-Si thin film in the light-irradiated region (region A) are calculated using equations 13 and 14.

The resistance Rml and volume Cml of pure water in the light-irradiated area (area A) are calculated using equations 15 and 16.

Using the above results, the frequency dependence of the solvent voltage in the light-irradiated region (region A) and the solvent voltage in the unirradiated region (region B) was calculated. The applied voltage was a sinusoidal wave with an effective value of 5V. The results are shown in Figure 7. A potential reversal was observed around 10 kHz. The results of these verification experiments are shown in Figure 8. Similar to the calculation results in Figure 7, below 10 kHz, the number of particles inside or around the laser pattern increased, confirming an attractive force acting on the light pattern (local electric field). On the other hand, below 1 kHz, the number of particles inside or around the laser pattern decreased, confirming a repulsive force. From the results in Figures 7 and 8, it is possible to manipulate the dielectrophoretic vector using a device capable of forming regions with locally different conductivity.

(An example of manipulating an object using the switching between repulsive and attractive forces)
Referring to Figure 9, an example of operating an object X using the switching between repulsive and attractive forces by frequency control of AC voltage will be explained. Note that the example of operating object X is not limited to the example in Figure 9.

As shown in Figure 9(a), multiple target objects X are scattered throughout the solvent.

As shown in Figure 9(b), a ring-shaped laser pattern (light irradiation area A) is irradiated to surround multiple target objects X. Here, the frequency of the AC voltage is set so that a repulsive force acts on the light irradiation area A.

As shown in Figure 9(c), the size of the annular laser pattern (light irradiation area A) is reduced in diameter so that multiple target objects X converge in one location.

Here, as shown in Figure 9(d), the shape of the laser pattern (light irradiation area A) is changed from an annular shape to a circular shape. At the same time, the frequency of the AC voltage applied to the solvent is changed to the specific frequency described above. This causes an attractive force to act on the light irradiation area A.

As shown in Figure 9(e), the circular laser pattern (light irradiation area A) is reduced in diameter so that the multiple objects X being manipulated converge into an even narrower area.

Then, as shown in Figure 9(f), a circular laser pattern (light irradiation area A) is moved to move the multiple target objects X, which are concentrated in a narrow area, to any desired position.

In this way, it becomes possible to gather multiple objects X scattered in the solvent into one location and move them to any desired location.

(Effects of this embodiment)
In this embodiment, by locally irradiating the photoconductive portion 15 with light, a light-irradiated region A and a light-unirradiated region B with different conductivity are created, and by setting the frequency of the AC voltage applied to electrodes 10 and 20 to a specific frequency, it becomes possible to manipulate the dielectric vector in any direction.

Furthermore, in this embodiment, the shape, position, and size of the light irradiation area A can be changed, allowing the target object X to be concentrated or moved to a desired position.

Furthermore, in this embodiment, by changing the shape of the light-irradiated area A and changing the frequency of the AC voltage to a specific frequency at a predetermined timing, the force acting on the light-irradiated area A can be changed from a repulsive force to an attractive force, or vice versa, in accordance with the change in the shape of the light-irradiated area A. This improves the degree of freedom in manipulating the object X in the solvent.

Furthermore, as an application example, the dielectrophoresis apparatus of the present invention can be used in situations where it is necessary to position microscopic materials in a liquid at a desired location, such as in experiments and production processes in the biological and chemical fields (e.g., microfluidic devices). This allows for the concentration, diffusion, or trapping of specific cells or microparticles in a laser-irradiated area, or their subsequent movement.

The present invention has been described above along with its embodiments. However, these embodiments are merely examples of how the present invention can be implemented, and the technical scope of the present invention should not be interpreted as being limited by them. In other words, the present invention can be implemented in various forms without departing from its technical concept or its main features.

For example, in a dielectrophoresis apparatus capable of forming regions with locally different conductivity, there are no constraints on parameters such as the distance between electrodes or the applied voltage.

1: Dielectrophoresis apparatus, 10: Operating electrode, 15: Photoconductive part, 20: Counter electrode, 30: Function generator, 40: Laser projector, 50: Digital mirror device, 60: Lens, 70: Spacer, 80: Control unit, 81: Processor, 82: Memory, 83: Auxiliary storage unit, 84: Interface, A: Light-irradiated area, B: Un-irradiated area, X: Object being manipulated

Claims (6)

A dielectrophoresis apparatus for manipulating objects by dielectrophoresis,
A first electrode and a second electrode, both being light-transmitting, are arranged at intervals from each other so as to contain the solvent containing the object to be operated on.
A photoconductive portion is positioned facing the solvent and whose conductivity changes upon irradiation with light,
A light irradiation unit that locally irradiates light onto the photoconductive portion,
An AC power supply that applies an AC voltage to the first electrode and the second electrode,
The system comprises a control unit that controls the frequency of the AC voltage applied by the AC power supply ,
The control unit makes the shape of the light emitted by the light irradiation unit annular and sets the frequency of the AC voltage to a first frequency.
The control unit makes the shape of the light emitted by the light irradiation unit circular and changes the frequency of the AC voltage to a second frequency that is higher than the first frequency.
A dielectrophoresis apparatus characterized by the following features. The dielectric constant of the object to be operated on is lower than the dielectric constant of the solvent, as described in claim 1. The control unit reduces the diameter of the annular light, and when the elapsed time for reducing the diameter reaches a predetermined time or when the annular light reaches a predetermined size, it changes the first frequency to the second frequency.
The dielectrophoresis apparatus according to feature 1. A dielectrophoresis method for manipulating an object by dielectrophoresis,
Apply an alternating current voltage to a first and second translucent electrode, which are spaced apart so as to contain the solvent containing the object to be operated.
The method involves locally irradiating a photoconductive portion, which is positioned facing the solvent and whose conductivity changes upon irradiation with light, with light, and changing the frequency of the AC voltage.
In controlling the frequency of the AC voltage, the shape of the light irradiated onto the photoconductive part is made annular, and the frequency of the AC voltage is set to a first frequency.
In controlling the frequency of the AC voltage, the shape of the light irradiated onto the photoconductive part is made circular, and the frequency of the AC voltage is changed to a second frequency that is higher than the first frequency.
A dielectric electrophoresis method characterized by the following features. The dielectric constant of the object to be operated on is lower than the dielectric constant of the solvent, as described in claim 4 . In controlling the frequency of the AC voltage, the annular light is made smaller in diameter, and when the elapsed time for the diameter reduction reaches a predetermined time or when the annular light reaches a predetermined size, the first frequency is changed to the second frequency.
The dielectrophoresis method according to feature 4.