Dielectrophoresis apparatus and dielectrophoresis method

Abstract

To locally provide electrodes having different conductivities and apply a specific frequency to a solvent having a high dielectric constant such as bioactive environment, and thereby operate a vector of dielectrophoresis in an arbitrary direction, when dielectrophoresis is performed in the solvent.SOLUTION: A dielectrophoresis device 1 operates an operation object X by dielectrophoresis, and includes an operation electrode 10 and a counter electrode 20 which are arranged at an interval so as to store a solvent including the operation object X and have translucency, a light conductive part arranged so as to face the solvent, whose conductivity is changed by irradiation with light, a light irradiation part for locally irradiating the light conductive part with light, a function generator 30 for applying an AC voltage to the operation electrode 10 and the counter electrode 20, and changing the frequency of the AC voltage, and a control part 80 for controlling the frequency of the AC voltage applied by the function generator 30.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present 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 or microparticles in a solvent. [Background technology]

[0002] 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. 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.

[0003] In the apparatus shown in Figure 1, when an AC voltage is applied to the upper and lower electrodes, the AC voltage is applied to the solvent, such as water, sandwiched between the upper and lower electrodes. Microscopic material particles are mixed into the solvent, and the application of an AC voltage to the solvent induces a charge in these microscopic particles. Simultaneously, when light of a desired wavelength is incident from the lower electrode side, the light irradiates a photoconductive material (such as amorphous silicon). In the irradiated portion of the photoconductive material, the light is absorbed and electron-hole pairs are formed, which increases the conductivity of the amorphous silicon (decreases its resistance). As a result, the conductivity of the electrodes differs between the irradiated and unirradiated regions, causing a change in the voltage applied to the solvent and creating a difference in the electric field. A charge is induced in the microscopic material, and this difference in the electric field causes the microscopic material to move due to dielectrophoretic force. If the dielectric constant of the microscopic material is smaller 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 larger than that of the solvent, the microscopic material moves in the direction of higher electric field strength.

[0004] Figure 2 shows the behavior of dielectrophoresis when the dielectric constant of the micromaterial is lower than the dielectric constant of the solvent. Figure 2 is a diagram showing the dielectrophoresis described in Non-Patent Literature 1, in which 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.

[0005] The dielectric constant of micromaterials is approximately 2.4 for polystyrene beads and approximately 47.5 for cells (see, for example, Non-Patent Literature 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-buffered saline) are used, and their dielectric constant is approximately 80, which is higher than that of the cells or polymer materials to be manipulated.

[0006] When dielectrophoresis is applied to a material with a dielectric constant lower than that of such a solvent, the dielectrophoretic force is expressed by the following equation 1: RE[F cm (ω)] (a coefficient reflecting the polarization of the particle) becomes negative, resulting in repulsion (negative dielectrophoresis).

[0007]

number

[0008] Furthermore, in highly polar materials such as cells and bacteria (materials that easily become polarized), as shown in Figure 3, 1 × 10 6 By applying high frequencies of [Hz] or higher, the vector of the dielectrophoretic force can be changed. Figure 3 shows the relationship between the Clausius-Mossotti coefficient and frequency as described in Non-Patent Document 3. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Wenfeng Lianga et al., “A Review on Optoelectrokinetics-Based Manipulation”, Micromachines January 2020, 11, 78 [Non-Patent Document 2] Keita Yamamoto et al., “Development of intracellular delivery technology for biomolecules using electric field driving force (Part 6) -Influence 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 [Non-Patent Document 3] Yamakawa, Retsu et al., Separation of living cells using dielectrophoresis, Electrochemistry, 82(11), 1000-1006 (2014) [Non-Patent Document 4] 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). [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] When performing dielectrophoresis in a high dielectric constant solvent, such as a physiologically active environment, a repulsive force acts on micromaterials such as cells and polymer particles. This results in manipulation based solely on repulsion, limiting the freedom of manipulation of these micromaterials.

[0011] 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, so only repulsive force can be exerted. Figure 4 is a diagram showing the frequency characteristics of the dielectric constant of polyethylene as described in Non-Patent Literature 4.

[0012] Therefore, the present invention proposes an apparatus and method that, when performing dielectrophoresis in a solvent with a high dielectric constant, such as a physiologically active environment, can manipulate the dielectrophoretic vector in any direction by creating a region with locally different conductivity and applying a specific frequency. [Means for solving the problem]

[0013] The dielectrophoresis device of the present invention is a dielectrophoresis device that operates an object to be operated by dielectrophoresis, and includes a first electrode and a second electrode having translucency that are arranged at intervals so as to accommodate a solvent containing the object to be operated, a photoconductive portion that is arranged facing the solvent and whose conductivity changes by irradiation with light, a light irradiation portion that locally irradiates the photoconductive portion with light, an AC power supply that applies an AC voltage to the first electrode and the second electrode, and a control portion that controls the frequency of the AC voltage applied by the AC power supply.

[0014] Further, the dielectrophoresis method of the present invention is a dielectrophoresis method that operates an object to be operated by dielectrophoresis, and includes applying an AC voltage to a first electrode and a second electrode having translucency that are arranged at intervals so as to accommodate a solvent containing the object to be operated, locally irradiating a photoconductive portion that is arranged facing the solvent and whose conductivity changes by irradiation with light with light, and changing the frequency of the AC voltage.

Advantages of the Invention

[0015] According to the present invention, when performing dielectrophoresis in a solvent having a high dielectric constant such as a bioactive environment, by providing a region with locally different conductivities and applying a specific frequency, the dielectrophoresis vector can be operated in an arbitrary direction.

Brief Description of the Drawings

[0016] [Figure 1] It is a diagram showing the structure of a conventional dielectrophoresis device. [Figure 2] It is a diagram showing the operation of dielectrophoresis when the dielectric constant of the micro material is lower than the dielectric constant of the solvent. [Figure 3] It is a diagram showing the relationship between the Clausius-Mossotti coefficient and the frequency. [Figure 4] It is a diagram showing the frequency characteristics of the dielectric constant of polyethylene. [Figure 5] It is a configuration diagram of the dielectrophoresis device 1 of the embodiment. [Figure 6] It is a flowchart of the dielectrophoresis method of the embodiment. [Figure 7]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. [Figure 8] 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. [Figure 9] This is an example of manipulating an object using the switching between repulsive and attractive forces via frequency control in the embodiment. [Modes for carrying out the invention]

[0017] Hereinafter, a dielectrophoresis apparatus and dielectrophoresis method relating to embodiments of the present invention will be described with reference to the drawings.

[0018] (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 with 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.

[0019] Each of the operating electrode 10 and the counter electrode 20 is a light-transmitting electrode having 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 arranged with a gap between them so that a solvent containing the object to be operated X can be contained within them.

[0020] 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 irradiation with light.

[0021] 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 a cell, polymer particles, or polyethylene beads. For example, the dielectric constant of the object to be manipulated X is lower than that of the solvent.

[0022] The function generator 30 applies an alternating current voltage of an arbitrary frequency to the operating electrode 10 and the counter electrode 20, and applies the same alternating current voltage to the solvent. The applied alternating current voltage induces a charge in the object X being worked on in the solvent.

[0023] The laser projector 40 irradiates the photoconductive material of the photoconductive section 15 with light of a wavelength that can be photoexcited. The change in the photoconductivity of the photoconductive material is 1 × 10⁻⁶. 2~4 It is desirable that the ratio be around S / m. The laser projector 40 can change the position, shape, and size of the light it emits. For example, the laser projector 40 can change the shape of the light emitted onto the photoconductive part 15 to an annular or circular shape, gradually reduce 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.

[0024] 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 1 × 10⁻¹⁶. 2~4 A value of approximately S / m is desirable.

[0025] 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 having a processor 81 and memory 82. The processor 81 is a CPU (Central Processing Unit) or DSP (Digital Signal Processor), which loads a program stored in the auxiliary storage unit 83 into the memory 82 and executes it. The program stored in the auxiliary storage unit 83 is, for example, a frequency control program that controls 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.

[0026] 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.

[0027] (Operation of the 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).

[0028] 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).

[0029] (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.

[0030] The control unit 80 instructs the function generator 30 to apply an AC voltage of default AC voltage value and frequency to electrodes 10 and 20 (step S601). The function generator 30 applies an AC voltage of default AC voltage value and frequency to electrodes 10 and 20 in accordance with the instruction.

[0031] 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 the instruction.

[0032] 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 the instruction.

[0033] 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 the 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 the instruction.

[0034] Then, the control unit 80 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 the instruction.

[0035] In this example, the size of the annular light was reduced, and the shape of the light was changed from an annular to a circular shape. However, the shape, size, and position of the light emitted by the laser projector 40 may be appropriately selected depending on the operation being performed on the object X.

[0036] (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.

[0037]

number

[0038] And each impedance is calculated by the following mathematical formulas 3 to 5.

[0039]

Number

[0040]

Number

[0041]

Number

[0042] If the impedance of ITO is small and ignored, Zt is calculated by the following Mathematical Formula 6.

[0043]

Number

[0044] Assuming that the voltage applied to the whole is V, the voltage Vm applied to the solvent is calculated by the following Mathematical Formula 7.

[0045] [[ID=5৮]]

Number

[0046] The electric field strength Em applied to the solvent is calculated by the following Mathematical Formula 8.

[0047]

number

[0048] 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 the values ​​shown in Table 1 below.

[0049] [Table 1]

[0050] 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.

[0051]

number

[0052]

number

[0053] The resistance Rm and volume Cm of pure water in the unirradiated region (region B) are calculated using equations 11 and 12.

[0054]

number

[0055]

number

[0056] 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.

[0057]

number

[0058]

number

[0059] The resistance Rml and volume Cml of pure water in the light-irradiated area (area A) are calculated using equations 15 and 16.

[0060]

number

[0061]

number

[0062] 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 10kHz. The results of the verification experiments are shown in Figure 8. Similar to the calculation results in Figure 7, below 10kHz, the number of particles inside or around the laser pattern increased, confirming that an attractive force acts on the light pattern (local electric field). On the other hand, below 1kHz, the number of particles inside or around the laser pattern decreased, confirming that a repulsive force acts. From the results in Figures 7 and 8, it is possible to manipulate the dielectrophoretic vector using a device that can form regions with locally different conductivity.

[0063] (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.

[0064] As shown in Figure 9(a), multiple target objects X are scattered throughout the solvent.

[0065] As shown in Figure 9(b), a ring-shaped laser pattern (light irradiation area A) is irradiated so as 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.

[0066] As shown in Figure 9(c), the size of the annular laser pattern (light irradiation area A) is reduced in diameter so that multiple objects X are concentrated in one place.

[0067] 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.

[0068] As shown in Figure 9(e), the circular laser pattern (light irradiation area A) is reduced in diameter so that multiple objects X are concentrated in an even narrower area.

[0069] Then, as shown in Figure 9(f), a circular laser pattern (light irradiation area A) is moved in order to move the multiple objects X that are concentrated in a narrow area to any desired position.

[0070] 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.

[0071] (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.

[0072] Furthermore, in this embodiment, the shape, position, and size of the light irradiation area A can be changed, allowing the object to be manipulated X to be gathered or moved to a desired position.

[0073] 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.

[0074] Furthermore, as an example of its application, the dielectrophoresis apparatus of the present invention can be used in situations where it is necessary to position minute materials in a liquid at a desired location, such as in experiments and production processes in the biological and chemical fields (microfluidic devices, etc.). This makes it possible to concentrate, diffuse, or trap arbitrary cells or microparticles in the laser-irradiated area and move them while keeping them trapped.

[0075] Although the present invention has been described above along with its embodiments, 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.

[0076] 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. [Explanation of symbols]

[0077] 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 to be manipulated

Claims

1. 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.

2. The dielectric constant of the object being operated on is lower than the dielectric constant of the solvent. The dielectrophoresis apparatus according to feature 1.

3. 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, the first frequency is changed to the second frequency. The dielectrophoresis apparatus according to feature 1.

4. 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. Locally irradiating light onto a photoconductive portion that is positioned facing the solvent and whose conductivity changes upon irradiation with light, and This includes 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.

5. The dielectric constant of the object being operated on is lower than the dielectric constant of the solvent. The dielectrophoresis method according to feature 4.

6. In controlling the frequency of the AC voltage, the annular light is made smaller in diameter, and when the elapsed time for making the light smaller 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.

Images