JP5186675B2 - Measuring method of fine particle state by dielectrophoresis - Google Patents

Description

  The present invention relates to a method for measuring the state of fine particles by dielectrophoresis and an apparatus for use in the method.

In the fields of quality control of biological fine particles (cells, bacteria, cell membranes, etc.), activity measurement, medicinal efficacy determination, tailor-made medicine, etc., measurement of the state of biological fine particles is required. Usually, for measurement of the state of living microparticles such as life and death and activity, a measurement method using intracellular calcium ion concentration, cAMP concentration, extracellular micropH, cell membrane potential, etc. as an index; measuring oxygen, nutrition, or marker uptake A method of contact measurement of cell membrane tension with a needle or the like. However, the former two require a large number of cells and time, and furthermore, since a fluorescent substance or the like is used as a marker, it is difficult to reuse a sample once used for measurement. On the other hand, the latter affects the target sample because of direct contact.
It is known that the dielectrophoretic mobility has different values depending on the sample to be migrated. The sample is polarized by being placed in a non-uniform electric field from the outside, and a dielectrophoretic force is generated to move in a positive or negative direction with respect to the electrode. Dielectrophoresis can also be applied to uncharged samples and large samples. However, for a small sample, the dielectrophoretic force is reduced and the mobility is also reduced. Therefore, the sensitivity required for analysis could not be obtained, and there was no apparatus for observing the dielectrophoretic mobility itself.
In recent years, a method for identifying the presence and / or relative concentration of a specific type of particles in a fluid by determining dielectrophoretic properties and an apparatus therefor have been reported (see, for example, JP 2001-500262 A). ). In this method, a sample is injected into a chamber containing a multi-electrode array, and the internal electrodes are simultaneously excited at different frequencies. The multipole array housed in this chamber consists of a series of comb-like spaced electrodes, and the tip of the electrode is located near the common electrode, so the injected sample is between the electrodes. It can be moved freely. Therefore, when a single type of sample is injected into the chamber and different series of dielectrophoretic fields are simultaneously formed on each electrode, the particles in the sample liquid are preferentially concentrated on one electrode according to its dielectric properties. Will do. This particular frequency can be used as a characteristic frequency for different particle types and can be used, for example, to separate major particles from a solvent by dielectrophoresis. It is also possible to inspect the homogeneity of a sample purified to a single species. Alternatively, if the injected sample is a mixture, the relative concentration of the mixture of known particles can be determined from data and particle counts at the appropriate frequency for certain types of particles present in the mixture.
In addition, a dielectrophoresis method using a combined effect of a dielectrophoretic force by a quadrupole electrode and a flow velocity distribution in a capillary has been reported (see Japanese Patent Application Laid-Open No. 2001-242136). This shows that particles having no charge and particles having the same surface charge can be detected, measured, sorted or separated by an efficient and simple means depending on parameters such as dielectric constant and particle size. This method exceeds the limit of conventional electrophoretic particle separation, that is, separation of particles that cannot be electrophoresed (for example, DNA of 40 kbp or more), separation of non-charged substances, or surface charge. However, the main purpose is to detect, measure, sort, or separate the same substances, not to characterize or identify particles. In order to characterize particles using this capillary, it is necessary to repeatedly perform measurement with different frequencies, which requires a lot of labor and time.
Apart from these, an apparatus for characterizing or identifying a minute amount of particles present in a fluid using dielectrophoresis has been developed (see Japanese Patent Application Laid-Open No. 2004-212272). This device can be used, for example, to identify substances or particles present in a fluid or to analyze the homogeneity of a purified sample. The homogeneity (particle size and chemical composition) of the compounds sampled during the chemical process can also be monitored.
There is also an apparatus for measuring the activity of microorganisms based on an impedance value using dielectric characteristics due to alternating current (Japanese Patent Laid-Open No. 2003-224). When this apparatus is used, only viable bacteria are concentrated by dielectrophoresis, and the number of microorganisms is calculated from the impedance value between the electrodes. With this apparatus, it is possible to determine only the life and death of microorganisms, the activity itself (state) of microorganisms is not measured, and a large amount of sample is required for determination of life and death.
There are also methods that combine the frequency-dependent dielectric and conductive properties of particulate and dissolved materials with the use of the properties of suspension transport media to identify and separate materials (Tokuyo 2000-505545 and F). F. Becker et al., Proc. Natl. Acad. Sci. USA, 1995, 92, pp. 860-864). In this apparatus, substances carried by a fluid flow are separated in the fluid by dielectrophoretic force. For example, it is disclosed that it can be used for separation of a cell mixture such as separation of cancer cells from normal cells, separation of infected erythrocytes from normal erythrocytes, and separation of nucleic acids. As described above, in any of the methods, the purpose is to detect, analyze, or separate / select a substance based on dielectrophoresis, and the measurement of the state of the target sample is not performed.
As the measurement of the state of the target sample based on dielectrophoresis, cell proliferation activity is measured (Yu Hakoda et al., Chemical Engineering Society 69th Annual Conference Proceedings, Q117, 2004, and JP2005). -224171). In these documents, a dielectric property independent of the cell diameter is used as an index of cell activity by quiescing the cell by the balance between the dielectrophoretic force acting on the cell and gravity. That is, the index does not include direct cell surface information.

An object of the present invention is to provide a method for measuring the state of fine particles by dielectrophoresis and an apparatus for use in the method.
The present invention provides a method for measuring the state of microparticles, the method comprising:
Introducing a sample containing fine particles into the dielectrophoresis solution and measuring the position R _{0 of the} fine particles;
Generating a non-uniform electric field for the fine particles in the dielectrophoresis solution and measuring the position R _t of the fine particles after t seconds;
Calculating dielectrophoretic mobility from the positions R ₀ and R _{t of the} obtained fine particles; and correlating the state with the calculated dielectrophoretic mobility;
Including
The state is a group consisting of the structure of the functional group on the surface, the surface specificity, the surface potential, the size, the shape, the activity, the internal ion content distribution, the internal ion component, the internal composition, and the internal structure. At least one selected.
In one embodiment, the non-uniform electric field is generated by a multipole electrode, and the multipole electrodes are arranged symmetrically.
In a further embodiment, the dielectrophoresis solution is in a vertically arranged capillary channel with an inner diameter of 10 μm to 500 μm, and the electrode is 0.1 mm to 2 mm in length, the capillary channel It is arranged along.
In a further embodiment, the surface of the electrode is exposed to the inside of the capillary channel and the wall supporting the electrode is isolated from the capillary channel.
In another embodiment, the microparticle is a bioparticle.
In one embodiment, the measurement is performed on a plurality of microparticles.
In a further embodiment, the plurality of microparticles have the same size and structure. Alternatively, the states of the plurality of fine particles are different.
In one embodiment, the method comprises: after the correlating step,
Comparing the states of the plurality of fine particles;
Further included.
In a preferred embodiment, the condition is active and the activity is assessed by the correlating step.
In another embodiment, the method comprises the step of measuring the position R _t of the microparticle,
Stimulating the microparticles;
Measuring a position R _s of the stimulated microparticle; and calculating a dielectrophoretic mobility from the position R _t and R _{S of the} obtained microparticle;
Further included.
In yet another embodiment, the sample containing the microparticles is prestimulated.
In one embodiment, the stimulus is a chemical addition. More preferably, the amount of the chemical solution added is different.
In a further embodiment, the biological microparticle is a human cell and may be a liver-derived cell.
In one embodiment, the introduction of the sample into the dielectrophoresis solution is performed using an inkjet device, a capillary, a pipette, tweezers, or a dispenser.
In one embodiment, the intensity and frequency of the non-uniform electric field are varied depending on time or sample behavior.
In one embodiment, the method further includes discharging the sample containing the microparticles after measuring the position R _t or R _S.
In one embodiment, the method further comprises analyzing the state based on the dielectrophoretic mobility.
In one embodiment, the state is a surface structure or a functional group of the fine particles.
The present invention also provides a dielectrophoresis device for use in any of the above-described methods for measuring the state of fine particles,
Flow path;
Means for generating a non-uniform electric field provided in the flow path; and means for measuring the position of the fine particles in the flow path;
With
The non-uniform electric field generating means can generate an electric field having a strength sufficient for measuring the state of the fine particles.
In one embodiment, the non-uniform electric field generating means is a multipole electrode, and the multipole electrodes are arranged symmetrically so as to generate a non-uniform electric field having concentric symmetry.
In one embodiment, the apparatus further comprises means for calculating dielectrophoretic mobility from the measured positions of the microparticles and calculating the state of the microparticles from the dielectrophoretic mobility.
According to the present invention, it is possible to integrally measure the state of fine particles such as the surface characteristics and internal structure of the fine particles from the dielectrophoretic mobility. For example, since the dielectric properties depending on the cell diameter obtained by dielectrophoresis include information on the cell surface, not only the inside of the cell but also the state of the cell surface layer can be identified. According to the method of the present invention, it is possible to analyze one individual fine particle without requiring a large amount of sample, and the analysis speed is high. Furthermore, if a database is created, it is possible to identify fine particles and estimate (discriminate) substances. Moreover, since there is no addition of a marker substance or damage due to contact, the sample subjected to the analysis can be reused, and the fine particles subjected to the analysis can be subjected to further precise analysis. Therefore, it can be applied to quality control.

FIG. 1 is a top sectional view schematically showing the configuration of a dielectrophoresis apparatus used in the present invention.
FIG. 2 is a cross-sectional view schematically showing the configuration of the dielectrophoresis apparatus used in the present invention.
FIG. 3 is a graph showing the change over time of the moving distance of each cell cultured under different conditions.
FIG. 4 is a graph showing the relationship between applied frequency and dielectrophoretic mobility for yogurt bacteria and yeast.
FIG. 5 is a graph showing the change over time of the cell movement distance when the HepG2 cells are left in the nutrient medium.
FIG. 6 is a graph showing the change over time of the dielectrophoretic mobility of cells when HepG2 cells are left in a nutrient medium or an oligotrophic medium.
FIG. 7 is a graph showing the relationship between doxorubicin concentration and cell dielectrophoretic mobility α.
FIG. 8 is a graph showing the relationship between doxorubicin concentration and cell dielectrophoretic mobility α when the α value is normalized based on the EC ₅₀ value of doxorubicin.
FIG. 9 is a graph showing EC ₅₀ values of various anticancer agents estimated from the dielectrophoretic mobility α value of cells.
FIG. 10 is a graph showing the relationship between the α value of individual cells derived from each well and the intracellular Ca ²⁺ influx (A) and ATPase activity (B) in each well.
FIG. 11 is a graph showing the change over time of the travel distance obtained for each liposome.

In the present invention, the term “fine particles” refers to various fine particles such as inorganic fine particles, organic fine particles, biological fine particles having a size of about 1 nm to about 1 mm. Examples of such fine particles include inorganic metal oxides such as silica and alumina; metals such as gold, titanium, iron and nickel; inorganic metal oxides into which functional groups are introduced by an operation such as silane coupling treatment; agarose and cellulose , Polysaccharides such as insoluble dextran; polymer particles such as polystyrene latex, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, acrolein-ethylene glycol dimethacrylate copolymer; microorganism (yeast, bacteria, virus), cell (Red blood cells, white blood cells, virus-infected cells, etc.), sugars, nucleic acids (DNA, RNA, etc.), proteins (enzymes, etc.), biological fine particles such as lipids, and the like. For example, non-biological particles such as latex beads may be bound to or coated with biological materials such as microorganisms, cells, viruses, plasmids, or chemically active species. The biological fine particles may be a body fluid such as serum, plasma, cerebrospinal fluid, synovial fluid, lymph fluid, or a processed product of a biological sample such as excrement such as urine and feces. Such a processed product is suitably diluted, dissolved, or suspended as appropriate with water or a buffer solution. Biological fine particles include those chemically synthesized.
In the present invention, the “fine particle state” means not only the surface characteristics of the fine particles but also the total state of the fine particles including the internal composition, internal structure and the like. Surface characteristics refer to surface structure (functional group structure, specificity, etc.), size, dielectric constant, electrical conductivity, surface charge, shape, and the like. The internal composition and internal structure refer to the activity of the fine particles, the ion amount distribution inside the fine particles, the ionic component, the density of the particles, the physical structure, and the like. The overall state refers to the overall state of fine particles including these surface characteristics and internal structure. That is, the total state of the electric potential generated on the surface of the fine particle due to the composition and distribution of the composition of the fine particle and the potential outside the fine particle created by superimposing the potential formed by the substance released from the fine particle and the solvent ions. Show. For example, when the microparticle is a cell, the state may be cell life / death, activity, affinity, and the like.
Since the relative potential varies depending on the activity and structure, the state of the fine particles can be integrally measured by measuring the dielectrophoretic mobility. Further, since the force received from the solvent is changed due to the change in the outer shape, the dielectrophoretic mobility is affected, so that the change in the outer state can also be measured. Specifically, it is as follows. The fine particles are migrated by an electric charge (potential state) formed by the applied electric field and the fine particles by applying the alternating electric field. The electric potential involved in the electrophoretic force is only the surface potential in the initial stage of electric field application. However, the electric field is created by the polarization of the inside of the fine particles as time passes. Charges and charges due to the solvent move and form a unique electric field. Therefore, the potential that affects the electrophoretic force is the total potential created by the superposition of these electric fields. Therefore, by observing the dielectrophoresis, it is possible to integrally measure the surface of the fine particle, the inside of the fine particle, and the outside of the fine particle.
For example, in a cell, the functional group distributed on the cell wall is not constant, and the distribution and type of the functional group changes depending on the degree of cell generation and growth. In addition, the cell membrane itself and the specificity of the cell membrane (selective permeability, selective adsorption, etc.) change with the activity of the cell. Therefore, when a potential is applied to a cell, a change in the state (for example, activity) of the cell can be read based on the dielectrophoretic mobility.
By the way, conventionally, in many dielectrophoresis apparatuses, it has been difficult to perform detailed measurement of individual particles satisfactorily. In a general dielectrophoresis apparatus, since a non-uniform electric field whose intensity varies uniformly is not formed, the dielectrophoretic force varies depending on the particle path, and the final migration distance changes. For this reason, in a general dielectrophoresis apparatus in which it is difficult to grasp the migration path of particles, it is not possible to measure individual states because a linear parameter representing the state of particles cannot be obtained. Also, in such an apparatus, the electric field strength is weak, the measurement accuracy for individual particles is poor, because the distance between the electrodes is large, and a sufficiently uniform electric field cannot be applied to the sample. The state of the particles could not be measured.
Specifically, in the case of biological microparticles of nanometer order such as DNA, there is a large difference in the charge of the microparticles itself with respect to the size, so that even such an apparatus is relatively easy to measure. On the other hand, biological microparticles such as cells, which are in the order of micrometers, are considered to be similar in terms of charge in proportion to size. Therefore, in order to examine individual states, a sufficiently strong electrophoretic electric field is necessary to obtain a large sensitivity. However, when the distance between the electrodes is large, in order to apply a sufficient electric field, a large voltage is required and electrolysis occurs. In addition, a two-dimensional electrode smaller than the sample, which is used in many conventional dielectrophoresis apparatuses, cannot apply a sufficiently uniform electric field to the sample, so that the distance between the electrodes is small. However, as a result, a sufficient migration force cannot be applied. Further, long-time measurement (for example, 10 minutes or more) caused by a weak electric field places a heavy load on the biological sample, which causes deterioration of the sample during measurement and makes it difficult to measure with high accuracy.
In dielectrophoresis, it is necessary to float the target sample. When floating with a dielectrophoretic force, since the electric field is not uniform in the vertical direction (z-axis direction), the electrophoretic force varies depending on the position of the z-axis. In addition, when floating by balancing with an external force such as gravity, measurement can be performed only when the balance is well balanced. If the distribution of the inhomogeneous electric field is unknown, sufficient linear parameters for measurement cannot be obtained. Further, H.C. Watarai et al. (Langmuir, 1997, 13, 8, pp. 2417-2420), H. et al. Watarai et al. (Chem. Lett., 1998, 3, pp. 279-280), S. et al. Tsukahara et al. (Langmuir, 2000, 16, p. 3866); In the case of a planar quadrupole as described in Ikeda et al. (Anal. Sci., 2003, Vol. 19, pp. 27-31), there is the above-mentioned problem of electric field strength, and in the case of a three-dimensional quadrupole. However, in order to eliminate the influence of gravity, the sample and solution are floated with the same density, so it is necessary to adjust the solution for each sample, and it is difficult to compare individual samples under the same conditions. It is. In addition, since a horizontal capillary of several tens mm is used, a plurality of cameras are required for real-time particle movement observation, and only one end surface can be observed with only one camera.
Such a problem is solved by the present invention. In other words, by using a symmetrical three-dimensional multipole electrode and a microcapillary arranged in the vertical direction, it is possible to evaluate the state of the sample linearly and highly sensitively with a dielectrophoretic mobility α that is not position dependent. became.
In the present invention, the dielectrophoretic mobility of the fine particles is represented by α in the following formula:
logR _t = logR ₀ + αt
Here, the center of the circular coordinates is represented by R = 0, the dielectrophoresis start position is represented by R ₀ , and the distance from the center after t seconds is represented by R _t (see FIG. 1). The distance is preferably a horizontal distance on the surface where the non-uniform electric field generating means is provided. Alternatively, the moving speed (moving time) of the sample can be analyzed in consideration of the sedimentation speed due to the gravity of the sample and the flow rate of the electrophoresis solution.
For example, when using a quadrupole electrode, the dielectrophoretic force <F _DEP > is represented by the following formula:
_{<F DEP> = 2πβε m r} dep 3 Re [Ke] ∇ | E rms | 2
Where β is a device coefficient, r _dep is the particle radius, ε _m is the dielectric constant of the medium, and Re [Ke] is the real part of the Clausius-Mossotti factor. Therefore, the dielectrophoretic force is
<F _DEP > ∝ ∇ | E _rms | ²
And is proportional to ∇ | E _rms | ² .
In the case of a quadrupole, the electric field is ∇ | E _rms | ² = 2RV ² _rms / d ⁴ (where V is the applied voltage and d is the interelectrode distance). Therefore, due to the balance with viscosity,
_{F st = 6πηr e dR / dt} = <F DEP> = 2πβε m r dep 3 Re [Ke] 2RV 2 rms / d 4
Here, η is the viscosity of the solvent, and R is the distance from the capillary center R ₀ . If you divide both sides by R,
1 / R dR / dt = 2πβε _m r _dep ³ Re [Ke] 2V ² _rms / d ⁴ = α
It becomes. Since the right side of this equation is uniquely determined by the apparatus and measurement conditions, when α is integrated,
lnR = αt + C (where C is an integral constant)
It becomes. Here, when the position of the sample at the start of electrophoresis (t = 0) is R ₀ ,
lnR ₀ = C
So,
lnR = αt + lnR ₀
It becomes.
As described above, since the dielectrophoretic mobility α is not position-dependent, it can be easily evaluated only by the migration start position and the end position.
The dielectrophoretic mobility is generally measured using a dielectrophoresis apparatus. There is no particular limitation as long as it is a dielectrophoresis apparatus that can generate an electric field with a sufficient strength for measuring the state of fine particles. Usually, the dielectrophoresis apparatus includes a flow path, a non-uniform electric field generating means for generating a non-uniform electric field provided in the flow path, and a means for measuring the position of the fine particles in the flow path. Here, the flow path is a passage of a liquid containing fine particles, and is a chamber portion having a non-uniform electric field generating means and having an inlet and an outlet.
As shown in the upper cross-sectional view of FIG. 1 and the cross-sectional view of FIG. 2, the dielectrophoresis apparatus includes at least a pair of electrodes as non-uniform electric field generating means, for example, inside or outside a channel such as a capillary.
When the channel is a capillary, the inner diameter of the capillary is usually 100 nm to 5 mm, preferably 10 μm to 1 mm, more preferably 10 μm to 500 μm. The material of the capillary is preferably an insulating substance (non-conductive substance). The length of the capillary is usually about 0.1 to 5 mm, more preferably 0.1 mm to 2 mm. A plurality of flow paths may be arranged on one substrate and formed into one chip. In the present invention, the flow path is preferably arranged in the vertical direction.
The width of the electrode itself provided in the channel may be thick or thin like a wire depending on the inner diameter of the channel. It suffices to adopt an electrode structure in which no sample is present in the region where the sample that receives the negative dielectrophoretic force gathers and in the vertical direction thereof. The shape of the electrodes may be any shape that can form a spatially nonuniform electric field between the electrodes. The electrode is preferably a multipole electrode, and the number of electrodes is not limited to a quadrupole, and may be a dipole or an octupole. The cross-sectional shape of each electrode is preferably formed such that the boundary of the electrode cross-section is represented by a function f (x, y) that satisfies the Laplace equation. For example, in the case of a quadrupole, hexapole, and octupole, f (x, y) is a (x ² −y ² ) + bxy, a (x ³ −3xy ² ) + b (y ³ −3x, respectively. ² y) and a (x ⁴ -6x ² y ² + y ⁴ ) + b (x ³ y-xy ³ ). Here, a and b are constants. Specifically, for example, in the case of a quadrupole, a substantially hyperbolic shape is preferable. The formed non-uniform electric field is preferably symmetric with respect to the central axis or the central portion of the flow path. More preferably, the multipole electrodes are arranged symmetrically so as to generate a non-uniform electric field having concentric symmetry. Furthermore, the electrodes are preferably arranged along the capillary channel, ie in the vertical direction.
In the present invention, the electrode is preferably a quadrupole electrode. Among the multipole electrodes, the quadrupole electrode has a sufficiently analyzed electric field, and the state can be measured by measuring only the α value that is the dielectrophoretic mobility as described above. This is because the mobility measured in a non-uniform electric field generated by a normal electrode structure is a tensor amount and is position time dependent. That is, when the electric field is symmetric as in the case of a quadrupole electrode, the α value is a scalar quantity (strictly, in the radial direction), and thus it is easy to handle. In the present invention, since dielectrophoresis is usually performed on a microscale such as a capillary, the mobility in the z-axis direction due to the influence of gravity can be ignored, and the α value can be treated approximately as a scalar quantity. For example, if an electrode having a length of 500 μm to 1 mm is arranged in the vertical direction (parallel to the z axis) along the capillary, the electric field shape does not change and the dielectrophoretic force does not change even if the sample moves in the z axis direction. In addition, it is not necessary to float the sample in order to allow movement in the z-axis direction, and the degree of freedom in the dielectrophoretic solution is high, and it is possible to compare samples under the same conditions. Furthermore, since no external force is acting in the electrode direction (a plane perpendicular to the Z axis), even if the sample is likely to adhere to the electrode or move to the effective measurement range, it is only necessary to stop voltage application. Can stop moving. There is no need to consider the balance between the sample position and external force.
The electrode is made of, for example, a conductive material such as carbon or a noble metal, and its structure may be any as long as the dielectrophoretic force generates a non-uniform electric field in a direction perpendicular to the central axis of the flow path. The diameter of the electrode varies depending on the particulate to be analyzed. Usually, the diameter is 100 nm to 5 mm, preferably 10 μm to 1 mm. The length of the electrode can usually be the same as the length of the channel. The distance between the electrodes depends on the fine processing accuracy, and is usually 500 μm or less and 0.1 μm or more, preferably 75 μm or less and 1 μm or more. The diameter and spacing of the multipole electrodes placed in each channel is the same as that of the microparticles to be measured, such as biological microparticles such as viruses, prions, proteins and DNA, and chemically active particles such as coated latex beads. It can be changed accordingly. If the distance between the electrodes is significantly larger than the size of the substance to be measured, a non-uniform electric field with sufficient electric field strength cannot be formed. In the present invention, the fine particles to be measured have different distances from the central axis of the flow channel when introduced into the flow channel.
Further, it is preferable that the surface of the electrode is exposed inside the capillary channel, and the wall supporting the electrode is isolated from the capillary channel. By separating the walls that support the electrodes from the capillary channel, a strong and less distorted electric field can be formed. This is because, if a non-conductive substance exists between the electrode and the capillary, this substance becomes a dielectric and weakens the electric field. Therefore, the distance between the wall supporting the electrode and the capillary channel is electrically increased. When not isolated, it polarizes and becomes a dielectric to affect the electric field and distort the electric field. When a strong electric field is formed, high-speed migration is performed by the strong electric field, and measurement can be performed in a short time from several tens of seconds to several minutes, so that deterioration of the sample can be prevented and measurement can be performed with high accuracy.
In the present invention, the non-uniform electric field generating means must be able to generate an electric field having a strength sufficient for measuring the state of the fine particles. Here, a sufficiently strong electric field is a disturbance caused by internal factors such as Brownian motion and thermal motion (molecular motion) of the fine particles to be measured, or by external factors such as vibration of the measuring device and flow caused by the turbulent migration liquid. On the other hand, the intensity | strength that measurement object microparticles | fine-particles have a sufficiently large dielectrophoretic mobility, ie, the intensity | strength which S / N becomes high. However, since the dielectrophoretic mobility depends on the dielectric constant, size, electrophoretic liquid, and the like of the fine particles to be measured, the absolute strength of the required electric field cannot be determined. Therefore, the strength of the electric field formed in the flow path can be appropriately determined according to the measurement target fine particles.
The intensity of the electric field formed in the flow path is, for example, usually 3.5 MV / m or less, preferably 1.0 MV / m or less. The applied frequency is usually 10 Hz to 100 MHz, more preferably 100 Hz to 10 MHz. In the present invention, the electric field formed may be either a DC electric field or an AC electric field, but is preferably an AC electric field. When the electric field strength is increased, the analysis may be difficult due to heat generation. If there is such a possibility, the electrode portion may be appropriately cooled. Further, the intensity and frequency of the non-uniform electric field may be changed according to time or the behavior of the sample.
In the case of a typical quadrupole electrode, the alternating voltage to be applied is usually 2 to 90 V, preferably 1 to 20 V, and the frequency is usually 10 Hz to 100 MHz, preferably 100 Hz to 10 MHz.
For example, the sample may be placed in a liquid reservoir provided on the inlet side of the flow path, and introduced into the flow path by sucking with a pump or the like from the outlet side of the flow path. Or you may inject | pour a sample into a flow path using an inkjet device, a capillary, a pipette, tweezers, a dispenser etc., In this case, an inkjet device is preferable. The ejection port of the ink jet device is disposed at the upper part of the flow path inlet, and the position thereof can be adjusted so that the sample can be injected along the central axis of the flow path.
In order to prevent the sample from adhering to the wall surface or electrode of the flow channel, for example, the entire inner surface of the flow channel is preferably covered with a hydrophilic film or a biocompatible film.
The dielectrophoretic liquid is introduced or driven into the dielectrophoresis apparatus by, for example, a syringe pump or an ink jet device. The dielectrophoretic solution introducing means or driving means and the flow path are communicated, for example, with a tube or the like. In order to prevent the generation of bubbles, the dielectrophoresis liquid is preferably defoamed in advance.
The distance (ie, position) and position change from the center of the fine particles in the flow path are measured by any measuring means that can detect the fine particles in the flow path. Examples of such measuring means include a charge coupled device (CCD) camera, a confocal laser microscope, and a fluorescence microscope. As the CCD observation means, observation by visible light, fluorescent light, phase interference, differential interference, or the like is possible. A detection device such as these can be placed under the chip provided with the flow path, and the inside of the flow path can be observed. In this way, since the entire capillary can be observed with only one camera from the bottom, the sample at any position can be measured. In addition, even when an ordinary optical microscope is used, it is possible to focus on the flow path and measure the position of the fine particles. The behavior in the channel cross section may be observed at the entire height. That is, it is possible to observe all positions in the flow path by scanning at a height position or moving the focal point.
Means may be provided for calculating dielectrophoretic mobility from the obtained data and thereby calculating the state. An example of such means is a computer. Specifically, the measured position change is stored in a computer together with the frequency applied to the flow path. The computer calculates the dielectrophoretic mobility from the measured position based on the above formula. Optionally, means may be provided for spectralizing as a function of the high frequency electric field frequency to generate a non-uniform electric field. In order to obtain a spectrum, data at various frequencies is required. Therefore, it is preferable to use an apparatus provided with a plurality of flow paths capable of measuring position changes at different frequencies at the same time. The obtained data can be output as a dielectrophoretic mobility spectrum showing frequency dependence.
A frequency spectrum indicating the dielectrophoretic mobility of the fine particles can be obtained by capturing various frequencies applied to each of the plurality of flow channels and the dielectrophoretic mobility of the fine particles in the flow channels as data. it can. Alternatively, data acquired for various states of various fine particles can be collected by a computer and converted into a database.
When dielectrophoretic mobility or dielectrophoretic mobility spectrum was measured for microparticles in an unknown state, the obtained dielectrophoretic mobility or spectrum was converted into the dielectrophoretic mobility of the known microparticle state in this database using a computer. Alternatively, the state of the fine particles can be identified by collating with the frequency spectrum. The dielectrophoresis apparatus may include a means for such collation.
The method of the present invention is usually a method for measuring the state of fine particles using such a dielectrophoresis apparatus,
Introducing a sample containing fine particles into the dielectrophoresis solution and measuring the position R _{0 of the} fine particles;
Generating a non-uniform electric field for the fine particles in the dielectrophoresis solution and measuring the position R _t of the fine particles after t seconds;
Calculating dielectrophoretic mobility from the positions R ₀ and R _{t of the} obtained fine particles; and correlating the state with the calculated dielectrophoretic mobility;
including. By correlating in this way, the state can be analyzed based on the dielectrophoretic mobility.
Specifically, for example, the measurement can be performed by the following procedure.
First, a medium suitable for the fine particles to be measured is selected as the dielectrophoresis solution. The medium is preferably water, but is not limited to this, and various organic or inorganic solvents can be used. The medium may be used as it is, and it is preferable to adjust the conductivity, density, osmotic pressure, pH and the like in relation to the fine particles. However, it is not preferable to add a substance that hinders the AC electric field.
Next, a sample containing fine particles to be measured is introduced into the flow path or the upper liquid reservoir previously filled with the medium. The fine particles are moved to a predetermined position where the electrodes are provided by causing a flow or settling by a pump.
A sample placed at a predetermined position is observed by means for measuring the above position, for example, a CCD camera, a laser, an electromagnetic measuring means, etc., and stored in a computer.
Next, an alternating electric field is applied to the stationary sample, and the behavior of the fine particles in the sample is observed by the above means and stored in a computer. If necessary, the AC electric field strength or frequency may be changed during electrophoresis. Depending on the observation results, for example, when there is sample position data and flow, the electrode exit passage time is obtained.
Upon completion of the observation, the sample is discharged from the outlet of the flow path by the above suction means. Data correction is performed on the obtained data based on the sample shape, position, flow, electric field distribution, electrode shape, and the like. Based on the corrected data, the dielectrophoretic mobility is calculated. Further, the state of the target fine particles can be analyzed in detail based on the initial acceleration, speed change, and the like. A profile for each frequency is created and a detailed state analysis is performed.
Alternatively, stimulation (electrical, physical, drug-like) can be given to the fine particles before or during migration. As a means for giving such stimulation, for example, means for dropping a drug on a sample containing fine particles by a pipette or an ink jet device; means for arranging a sample containing fine particles in a device for stimulation; a sample containing fine particles Examples include means for inserting a terminal or the like into the containing container; means for inserting a drug dropping pipe, electrode, or antenna into the flow path system or the wall surface. By means for applying such a stimulus, the microparticles are stimulated before and during the analysis. Alternatively, electromagnetic stimulation, impact, or the like can be stimulated by irradiation from the outside of the flow path.
In this case, the method of the present invention is, for example, after the step of measuring the position R _t of the fine particles,
Stimulating the microparticles;
Measuring a position R _S of the stimulated microparticle; and calculating a dielectrophoretic mobility from the position R _t and R _{S of the} obtained microparticle;
Further included. These data are also analyzed in detail and state analysis is performed.
In the case where a plurality of flow paths are installed, it is possible to sequentially or simultaneously perform measurement with changed conditions. In addition, a plurality of samples (fine particles) can be measured by one measurement in the same channel.
The method of the present invention may further include a step of discharging the sample containing the fine particles after measuring the position R _t or R _S. That is, if necessary, the fine particles can be separated based on the calculated data, and the discharged fine particles can be reused. In addition, a database can be created by accumulating data obtained on the state of fine particles. It is also possible to characterize the microparticles by collating the measured data of the individual microparticles with the obtained database.
For example, measurements may be taken on a plurality of microparticles that are the same size and structure to identify or characterize their condition. Alternatively, the states of a plurality of fine particles having the same size and structure but different states can be compared. Examples of such fine particles include biological fine particles such as cells. For example, their individual activities can be evaluated by comparing the obtained measurement results.

In the following examples, a quadrupole microcapillary biological microparticle analysis system having a capillary as shown in FIG. 2 was used. The operation procedure for measuring the dielectrophoretic mobility is as follows.
First, a KCl solution (conductivity 33.5 mS / m) is filled in the chip (chip having a 100 μm diameter capillary), and a sample is injected into a solution reservoir at the top of the chip. The solution is flowed by a syringe pump (10 nl / min to 100 nl / min) to transport the sample to a measurement site in the capillary. Using a Nikon ELWD Plan Fluor 40 × lens installed in the system observation system, the measurement apparatus is operated to start observation of sample behavior as a moving image using the Sony DFW-X700 or PIXERA 600SL-CU digital camera as the system observation system. Next, the set electric field for electrophoresis is generated to start electrophoresis. The applied voltage is stopped after a predetermined time or when the sample reaches the center of the capillary or the electrode. A flow of the solution (about 10,000 nl / min) is generated, and the sample is removed and the inside of the capillary is washed. Next, a new sample is introduced. The sample position in the capillary is calculated by performing image analysis on the data measured in parallel. The dielectrophoretic mobility is calculated from the sample position.
(Example 1: Measurement of the state of cells cultured in different nutritional states)
A part of human brown adipocytes (Chang river strain) is placed in LB nutrient medium (hereinafter sometimes referred to simply as nutrient medium), and the other part in an nutrient medium not containing sodium alginate at 37 ° C. Incubate for hours. On the other hand, the inside of the capillary of the measuring chip having a quadrupole electrode as shown in FIG. 1 was previously filled with each medium. Next, 100 μL of each medium containing cells was injected into the upper reservoir (or inside the capillary) with a microsyringe. The medium in the capillary was moved to the position of the quadrupole electrode by a syringe pump, and a charge of 1 MV / m and 100 Hz was applied for 30 seconds. The flow path was observed from the start to the end of application, and the cell position was measured. Based on the obtained position data, the moving distance was obtained, and the dielectrophoretic mobility α was calculated by the following equation.
logR _t = logR ₀ + αt
(In the equation, when the center of the capillary section is R = 0, the migration start position is R ₀ , and the distance from the center after t seconds is R _t , the dielectrophoretic mobility is α).
The travel distance obtained for each cell is shown in FIG. As shown in FIG. 3, it was found that the direction and mobility of dielectrophoresis were completely different between cells cultured in an oligotrophic state (◯) and cells cultured in a normal nutrient state (□ and Δ). From this, it became clear that the state of the cell surface varies greatly depending on the activity of the cell. The cells remain insulative in the living state, and show a charged state according to the characteristics of various biopolymers having a negative charge on the surface. However, the cell membrane of dead cells has lost insulation, and the results of this example suggest that the state of the cell membrane surface is reflected in the electrophoretic results. Therefore, it is considered possible to identify the cell membrane surface state using the dielectrophoretic mobility by examining the correlation between the dielectrophoretic mobility and the parameter representing the stress state on the cell membrane surface.
(Example 2: Discrimination between yogurt and yeast)
Dielectrophoresis profiles were obtained for commercially available yogurt and yeast. The applied voltage was 5 V, and a KCl solution (conductivity 33.5 mS / m) was used as a solvent. The applied frequency was 1 kHz to 10 MHz. The results are shown in FIG.
At an applied frequency of 1 MHz, yogurt bacteria (◯) were positively migrated and yeast (Δ) were negatively electrophoresed. It is possible to discriminate between yogurt and yeast due to the apparent difference in the profile of dielectrophoretic mobility, and single frequency measurement at places where the migration behavior is clearly different, such as the applied frequency of 1 MHz. It was found that discrimination was possible.
(Example 3: Cell migration behavior in the quadrupole)
HepG2 cells derived from human liver are allowed to stand in a nutrient medium or an oligotrophic medium at 37 ° C. for 60 hours, and the separated cells are introduced into the above-mentioned quadrupole electrode dielectrophoresis measurement chip every 10 hours to move the distance. Was measured. The solvent was each medium, and the applied frequency was 100 Hz. FIG. 5 shows the change over time of the migration distance of HepG2 cells in the nutrient medium. Moreover, the time-dependent change of the dielectrophoretic mobility α accompanying the decrease in the cell activity in the nutrient medium (◯) or the oligotrophic medium (Δ) was calculated and shown in FIG.
The cells in the nutrient medium were negatively migrated when the standing time was short, but changed to normal running as the standing time was long (FIG. 5). As can be seen from FIG. 6, the cells in the nutrient medium had a dielectrophoretic mobility α of 0 in about 40 hours, indicating that the cell activity was decreasing. On the other hand, the cells in the oligotrophic medium in the oligotrophic medium had a slightly larger α than the cells in the normal nutrient state in the nutrient medium, but no significant difference was found from the cells in the nutrient medium.
(Example 4: Examination of drug addition effect)
After adding doxorubicin hydrochloride as an anticancer agent at various concentrations to a culture solution (nutrient medium or oligotrophic medium) containing HepG2 cells and incubating for 24 hours, the HepG2 cells are placed on the above-described quadrupole electrode dielectrophoresis measurement chip. The cells were introduced, the distance of cell movement was measured, and the dielectrophoretic mobility α when each concentration was added was determined. At the same time, the activity of the cells in each medium was measured by ATPase activity, and the concentration corresponding to half the value obtained by standardizing the activity value was defined as the 50% effective concentration (EC ₅₀ ) of doxorubicin hydrochloride. The EC ₅₀ was 0.16 mM for cells in nutrient medium and 0.077 mM for oligotrophic cells. In addition, any cell showed a decrease in activity at 0.5 mM or more.
The relationship between the dielectrophoretic mobility α and the concentration is shown in FIG. The cells cultured in the nutrient medium are indicated by ○, and the cells cultured in the oligotrophic medium are indicated by △. It was found that addition of doxorubicin hydrochloride changed the α value from around 0.1 mM in cells cultured in any medium. Moreover, the change of α value was small at 0.5 mM or more.
Next, in the graph of FIG. 7, the α value corresponding to the obtained EC ₅₀ value concentration is 0.5, the α value when no drug is added is 0, and the drug concentration is when the cell activity is lost. When the α value to be normalized was set to 1 and the α value was normalized, the graph shown in FIG. 8 was obtained. Thus, the possibility that the pharmacological effect of the drug can be screened by the change in the α value was suggested.
(Example 5: Examination of evaluation of addition effect of various drugs)
In the same manner as in Example 4 described above, after culturing by adding various concentrations of doxorubicin hydrochloride, bleomycin sulfate, and mitomycin C, which are anticancer agents, to HepG2 cells in normal or oligotrophic state, The movement distance was measured to determine the dielectrophoretic mobility α. Next, the relationship between the drug addition concentration and the α value was graphed. From the graph for each drug, an α value intermediate between the α value when no drug is added and the α value corresponding to the drug concentration at which the α value no longer changes is obtained, and the drug concentration corresponding to this intermediate α value is determined as EC. It was estimated to be ₅₀ values. The estimated EC ₅₀ values obtained from the α values for each drug are shown in FIG.
(Example 6: Evaluation of biological activity of cells)
HepG2 cells derived from human liver were cultured for 72 hours at 37 ° C. in cell culture wells containing nutrient medium. Three cells were taken out from each well, each cell was introduced into the above-mentioned quadrupole electrode dielectrophoresis measurement chip, the cell migration distance was measured, and the dielectrophoretic mobility α was determined. Then, for each well, intracellular Ca ²⁺ influx and ATPase activity were measured. Intracellular Ca ²⁺ influx was measured by the fluorescent probe Fura-2, and ATPase activity was measured by phosphate concentration. The relationship between the α value of individual cells derived from each well and the intracellular Ca ²⁺ influx and ATPase activity of each well is shown in FIGS. 10A and B, respectively.
As can be seen from these graphs, it was suggested that the α value, intracellular Ca ²⁺ influx and ATPase activity may correspond to each other. Intracellular Ca ²⁺ influx and ATPase activity are both indicators related to cell membrane function. Therefore, it is considered that the state of the cell membrane affects the α value.
(Example 7: Dielectrophoresis of liposome)
An aqueous solution containing 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and POPC / 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) as a fluorescent particle model liposome, calcein (1 mM) as a fluorescent probe In particular, it was prepared by a high temperature hydration method (60 ° C., 3 hours) (particle size: 2 to 20 μm). Next, the inside of the capillary of the above-described quadrupole electrode dielectrophoresis measurement chip was previously filled with the above buffer solution. 100 μL of a buffer solution containing liposomes was injected into each upper reservoir (or in the capillary) with a microsyringe. Each liposome was moved to the position of the quadrupole electrode by a syringe pump through the buffer in the capillary, and a charge of 1 MV / m and 100 Hz was applied for 25 seconds. The flow path was observed from the start to the end of application, and the position of the liposome was measured.
FIG. 11 shows changes with time of the movement distance R obtained for each liposome. The dielectrophoretic mobility α of the liposome made of POPC calculated from the moving distance was 0.00798, and the α value of the liposome made of POPC / POPG was 0.0131. Thus, the α value was different depending on the composition of the liposome.

  According to the method of the present invention, it is possible to integrally measure the state of fine particles such as the surface characteristics and internal structure of the fine particles from the dielectrophoretic mobility. According to the method of the present invention, it is possible to analyze one individual fine particle without requiring a large amount of sample, and the analysis speed is high. Furthermore, if a database is created, it is possible to identify fine particles and estimate (discriminate) substances. Moreover, since there is no addition of a marker substance or damage due to contact, the sample subjected to the analysis can be reused, and the fine particles subjected to the analysis can be subjected to further precise analysis. For example, it is possible to evaluate the difference in the state of the same type of particles (having the same structure) using the dielectrophoretic mobility. Therefore, it is particularly useful for application to fields such as quality control of biological microparticles (cells, bacteria, cell membranes, liposomes, etc.), activity measurement, drug efficacy determination, and tailor-made medicine.

Claims (22)

A method for measuring the state of fine particles,
Introducing a sample containing fine particles into the dielectrophoresis solution and measuring the position R _{0 of the} fine particles;
Generating a non-uniform electric field for the fine particles in the dielectrophoresis solution and measuring the position R _t of the fine particles after t seconds;
Calculating dielectrophoretic mobility from the positions R ₀ and R _{t of the} obtained fine particles; and correlating the state with the calculated dielectrophoretic mobility;
Including
The state is a group consisting of the structure of the functional group on the surface, the surface specificity, the surface potential, the size, the shape, the activity, the internal ion content distribution, the internal ion component, the internal composition, and the internal structure. At least one selected,
The non-uniform electric field is generated by a multipole electrode, the multipole electrode being arranged symmetrically;
The dielectrophoresis solution is in a vertically arranged capillary channel having an inner diameter of 10 μm to 500 μm, and the electrode has a length of 0.1 mm to 2 mm and is disposed along the capillary channel. The way. The method of claim 1, wherein a surface of the electrode is exposed inside the capillary channel and a wall supporting the electrode is isolated from the capillary channel. The method according to claim 1, wherein the fine particles are biological fine particles. The method according to claim 1, wherein the measurement is performed on a plurality of fine particles. The method of claim 4, wherein the plurality of microparticles have the same size and structure. The method according to claim 5, wherein states of the plurality of fine particles are different. After the correlating step,
Comparing the states of the plurality of microparticles;
The method of claim 6, further comprising: 8. A method according to any of claims 1 to 7, wherein the condition is active and the activity is assessed by the correlating step. After the step of measuring the position R _t of the fine particles,
Stimulating the microparticles;
Measuring a position R _S of the stimulated microparticle; and calculating a dielectrophoretic mobility from the position R _t and R _{S of the} obtained microparticle;
The method according to claim 1, further comprising: The method according to any one of claims 1 to 8, wherein the sample containing the fine particles is previously stimulated. The method according to claim 9 or 10, wherein the stimulation is chemical addition. The method according to claim 11, wherein the amount of the chemical solution added is different. The method according to claim 3, wherein the biological microparticle is a human cell. The method of claim 13, wherein the human cell is a liver-derived cell. The method according to any one of claims 1 to 14, wherein the introduction of the sample into the dielectrophoresis solution is performed using an inkjet device, a capillary, a pipette, tweezers, or a dispenser. The method according to claim 1, wherein the intensity and frequency of the non-uniform electric field are changed according to time or sample behavior. The method according to claim 1, further comprising discharging the sample containing the fine particles after measuring the position R _t or R _S. The method according to claim 1, further comprising analyzing a state based on the dielectrophoretic mobility. The method according to claim 1, wherein the state is a surface structure or a functional group of the fine particles. A dielectrophoresis apparatus for use in the method for measuring a state of fine particles according to any one of claims 1 to 19,
Flow path;
A non-uniform electric field generating means for generating a non-uniform electric field provided in a part of the flow path; and a means for measuring the position of the fine particles in the flow path;
With
The non-uniform electric field generating means is a multipole electrode capable of generating an electric field with sufficient strength for measuring the state of the fine particles, the multipole electrodes are arranged symmetrically, and the dielectrophoretic liquid is arranged in a vertical direction. A device in which the inner diameter is 10 μm to 500 μm and the electrode is 0.1 mm to 2 mm long and is arranged along the flow path. 21. The apparatus of claim 20, wherein the non-uniform electric field generating means is a multipole electrode, and the multipole electrode is symmetrically arranged to generate a non-uniform electric field having concentric symmetry. The apparatus according to claim 20 or 21, further comprising means for calculating a dielectrophoretic mobility from the measured position of the microparticle and calculating a state of the microparticle from the dielectrophoretic mobility.

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