JP4315083B2 - Optical measuring apparatus and optical measuring method - Google Patents

Description

The present invention relates to an optical measurement apparatus that measures information related to the diffusion of particles (for example, biomolecules such as proteins, various fine particles, etc.) existing in a liquid using an optical technique, and more specifically, in a liquid. The present invention relates to an optical measurement apparatus that measures information related to diffusion of particles by using diffracted light by a transient diffraction grating generated by particles existing in the substrate.
The optical measurement apparatus of the present invention can be applied to molecular investigation and research in fields such as drug discovery, biotechnology, and food. Moreover, it can apply to the particle | grain measurement field | area which measures the particle size of particle | grains by measurement of a diffusion coefficient.

One technique for measuring particle diffusion is the transient diffraction grating method. For example, it is disclosed that a diffusion constant is measured using a transient analysis lattice method and protein association is detected by a change in the diffusion constant (see Patent Document 1).
In the conventional transient diffraction method, as shown in FIG. 6, the sample is irradiated with two pulse excitation lights having the same wavelength so as to cross each other, thereby forming interference fringes. Molecules (particles) in the sample present in the bright part of the interference fringe due to pulsed excitation light are locally photoexcited, whereas molecules (particles) in the sample present in the dark part of the interference fringe are not photoexcited. Therefore, in the interference fringe region, excited molecules and non-excited molecules exist so as to be arranged alternately and regularly, and a diffraction grating (referred to as a transient diffraction grating) is temporarily formed.

If the region where the transient diffraction grating is formed is separately irradiated with probe light, the probe light is diffracted by the transient diffraction grating. Then, after the transient diffraction grating with excited and non-excited molecules is formed by irradiating pulsed excitation light, the excited and non-excited molecules are mixed by diffusion over time, and the transient diffraction grating collapses. The intensity of diffracted light of the probe light by the diffraction grating is attenuated. Since the attenuation curve of the diffracted light intensity at this time represents the diffusion constant (diffusion coefficient) of the molecules in the sample, the diffusion coefficient of the molecules in the sample can be calculated by measuring the attenuation curve. In addition, information on the size (particle size) and shape of the particles in the sample and the interaction with the solvent can be acquired from the diffusion coefficient.
JP 2004-85528 A

  In the conventional transient diffraction grating method described above, in order to form an interference fringe by crossing two excitation lights having the same wavelength, the two excitation lights having substantially the same optical path length are guided to the measurement region and generated. Probe light having a specific incident angle is made incident on the diffraction grating formed based on the interference fringes. For this reason, it is necessary to cross two excitation lights and one probe light at one point to be measured, and it is necessary to adjust the three optical axes of the excitation light and the probe light, making adjustment work difficult. It is.

Moreover, since the excitation light used when using molecules (particles) such as proteins as a sample requires a large laser such as an excimer laser with a short wavelength, the apparatus becomes large.
In addition, when a protein or other molecule (particle) is used as a sample, usually the protein molecule (particle) itself is photoexcited because the refractive index, absorption coefficient, and diffusion coefficient are not changed by the excitation light. It is necessary to label the sample substance with a reagent (such as a fluorescent reagent).
However, if the sample is subjected to a labeling process, the properties and characteristics of the protein molecules (particles) to be measured may change.
In general, since the labeling process is an irreversible reaction, the molecules (particles) themselves in the sample are destroyed by the labeling process, and re-measurement using the same sample cannot be performed. It cannot be reused for other purposes. Furthermore, the photoexcitation reaction for forming the transient diffraction grating is also generally an irreversible reaction, and only a weak signal is generated from the sample once irradiated with the excitation light and measured again. I can't.

  In addition, when a sample other than a molecule that can be easily labeled such as a protein is used as a sample, the labeling process cannot be performed depending on the substance, and the photoexcitation of particles by excitation light is difficult. Measurement may be difficult.

Therefore, the present invention provides an optical measurement apparatus that does not require excitation light and that can perform measurement using the transient diffraction grating method without adjusting the optical axis between the two excitation light and probe light. For the purpose.
It is another object of the present invention to provide an optical measuring apparatus that can measure characteristics related to diffusion of a sample using a transient diffraction grating without performing labeling processing of the sample.

  The optical measuring device of the present invention made to solve the above problems includes an AC power source, a container holding a liquid sample, and a region having a high electric field line density in a part of the container by applying a voltage. Generation of a transient diffraction grating using an electrode pair that generates an electric force line distribution regularly aligned with a region having a low electric field line density, and dielectrophoresis of particles in a liquid sample by applying an alternating voltage to the electrode pair; A dielectrophoresis controller that controls the disappearance of the transient diffraction grating due to the diffusion of particles in the liquid sample when the voltage application is stopped, a light source that emits light toward the transient diffraction grating, and a diffracted light detected by the transient diffraction grating And a photodetector, and the particle is evaluated from the change in the intensity of the diffracted light generated by the transient diffraction grating.

According to the optical measuring device of the present invention, by applying an AC voltage from an AC power source to the electrode pair, a region having a high electric force line density and a region having a low electric force line density are partially formed in the container. Generates a distribution of electric field lines regularly arranged. The particles contained in the liquid sample in the container have a dielectrophoretic action due to this electric force line distribution, and the particles move. That is, since the electric force line distribution regularly arranged in the container due to the arrangement of the electrode pairs is generated, the particles in the liquid sample are concentrated in a region where the electric force line density is high due to the dielectrophoretic action. The dense and sparse regions of the particles are regularly arranged, and a transient diffraction grating is formed.
When the transient diffraction grating is irradiated with probe light from a light source, diffracted light is generated in a specific direction by the transient diffraction grating. Strong diffracted light is generated when the transient diffraction grating is stably generated due to the dielectrophoretic action. If the application of the AC voltage is stopped in a state where the transient diffraction grating is stably generated by the application of the AC voltage, the lines of electric force disappear and the dielectrophoresis stops. Therefore, the particles in the container move due to diffusion, and the transient diffraction grating collapses and becomes blurred. As a result, the intensity of the diffracted light generated by the transient diffraction grating attenuates with time, but the attenuation curve at this time shows the diffusion coefficient, so the intensity of the diffracted light is measured with a photodetector. By obtaining an attenuation curve of the diffracted light intensity, information on the diffusion coefficient of the particles, and further, information on the particle shape, particle diameter, and interaction with the solvent is obtained.

  In addition, the optical measurement method of the present invention, which is made from another viewpoint, has an electric force line distribution in which a region having a high electric force line density and a region having a low electric force line density are regularly arranged in a liquid sample by applying a voltage. Using the electrode pair to be generated, an alternating voltage is applied to the electrode pair to induce dielectrophoresis on particles in the liquid sample to form a transient diffraction grating by the particles, and then the voltage application is stopped to form a transient diffraction grating. The particles are evaluated by diffusing particles in the liquid sample and detecting the intensity change of the diffracted light by the transient diffraction grating at this time.

  According to the optical measurement method of the present invention, an alternating voltage is applied to the electrode pair to cause dielectrophoresis on the particles in the liquid sample, and the particles in the sample liquid are concentrated in a region where the electric line density is high. To form a transient diffraction grating. Subsequently, the voltage application is stopped and the particles forming the transient diffraction grating in the liquid sample are diffused, and the transient diffraction grating is destroyed over time. The particle-related evaluation is performed by detecting the intensity change of the diffracted light accompanying the change of the transient diffraction grating at this time.

According to the optical measuring device and the optical measuring method of the present invention, since the transient diffraction grating is formed by using dielectrophoresis, no excitation light is used when generating the transient diffraction grating. . Therefore, it is only necessary to adjust the optical axis of the probe light at the measurement position, and it is not necessary to adjust the optical axis of the excitation light, and the measurement using the transient diffraction grating method can be easily performed.
Further, according to the optical measuring apparatus and the optical measuring method of the present invention, characteristics such as sample diffusibility can be obtained by using a transient diffraction grating without performing labeling processing of the sample and without optical excitation. Since it can be measured, the sample can be measured again, and the sample itself can be reused.

  In the optical measuring apparatus, each electrode constituting the electrode pair includes a plurality of electrode pieces arranged at a predetermined interval and a connection portion that electrically connects the electrode pieces, and each electrode in one electrode If one side end of the piece is arranged so as to face the one side end of each electrode piece in the other electrode with a gap, the region where the electric field line density is high is Concentrating at the position of the opposing gap, and by concentrating the area with low electric field line density in the adjacent area, a transient diffraction grating is generated along the position of the gap where one end of each electrode piece faces. A transient diffraction grating will be generated in the region where there is no electrode pair (the gap between the opposing electrodes), and the change in the diffracted light intensity only by the transient diffraction grating will be measured without being affected by the electrode pair. Can .

  In the optical measuring device, at least a part of the container is formed of a material that transmits light source light, and an electrode pair is formed on the container part that transmits the light source light, and the light source is directed toward the electrode pair. If light is incident and the photodetector detects diffracted light transmitted through the liquid sample or diffracted light reflected by the liquid sample, transmitted diffracted light or reflected diffracted light can be easily obtained. Here, the container portion that transmits the light source light may be a bottom surface or a side wall surface.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the embodiments described below, and it goes without saying that various aspects are included without departing from the spirit of the present invention.

  FIG. 1 is a perspective view showing a configuration of an optical measuring apparatus according to an embodiment of the present invention, and FIG. 2 is a plan view showing a configuration of an electrode portion thereof. This optical measuring apparatus performs optical measurement using a dielectrophoretic action, and includes a container 11 that holds a liquid sample containing particles, and a pair of electrodes 13 that are formed on a bottom plate 12a serving as a bottom surface of the container 11. , 14, an AC power source 15 that applies an AC voltage to the electrode 13 and the electrode 14, a light source 16, a lens optical system 17 that converges the light source light, a photodetector 18 that detects diffracted light, and an AC power source 15. It comprises a dielectrophoresis control unit 19 that controls voltage application to the electrodes 13 and 14.

  The container 11 is formed by sticking a frame body 12b serving as a side wall on the bottom plate 12a. The container 11 is made of a light-transmitting material such as glass, so that incident light can be applied to the gap between the electrodes 13 and 14 through the bottom plate 12a. The container portion other than the portion irradiated with the incident light may be made of a material other than the light transmissive material or provided with a light shielding member to block the incidence of unnecessary light and increase the detection sensitivity. .

The electrodes 13 and 14 are formed on the bottom plate 12a using a mask patterning technique. In the present embodiment, the electrodes 13 and 14 are formed on the bottom plate 12a. However, when the container 11 is sufficiently deep, the electrodes 13 and 14 are formed on the frame 12b serving as the side wall instead of the bottom plate 12a. May be.
The electrode 13 includes parallel linear electrode pieces 13a, 13b, 13c, and 13d arranged in parallel at a predetermined interval, and a connecting portion 13e that electrically connects one side ends of these linear electrode pieces. A so-called comb-shaped electrode is formed.
The same applies to the electrode 14, and parallel linear electrode pieces 14a, 14b, 14c, and 14d are arranged in parallel at a predetermined interval, and one side ends of these linear electrode pieces are electrically connected to each other. A connecting portion 14e is provided to form a so-called comb electrode.
The electrodes 13 and 14 are composed of one end of the linear electrode pieces 13a and 14a, one end of the linear electrode pieces 13b and 14b, one end of the linear electrode pieces 13c and 14c, and a linear electrode piece. One side ends of 13d and 14d are arranged to face each other with a gap S therebetween.

  The dimensions of the electrodes 13 and 14 are preferably about 0.5 μm to 20 μm for each of the electrode width d1 of the linear electrode pieces and the distance d2 between the linear electrode pieces. If each gap S is arranged in a gap, a region having a high electric force line density is generated in a portion of each gap S when a voltage is applied, and a region having a low electric force line density is generated next to the gap S. The dimensions are not particularly limited. For example, the electrode width d1 and the electrode interval d2 may be different dimensions, and the shape of the electrode pieces may not be linear.

  As the AC power source 15, an AC power source having a voltage and a frequency capable of causing dielectrophoresis on particles in the liquid is used. Specifically, an AC power supply capable of applying an AC voltage of about 1 to 100 V, 10 KHz to 10 MHz is used. In general, it is preferable to use a high-frequency power source.

The type of the light source 16 for irradiating the probe light may be selected according to the liquid sample to be measured. For example, a He—Ne laser light source (wavelength 633 nm) or other laser light source is preferably used. .
The lens optical system 17 converges the light source light, and the optical axis is adjusted so that the light source light can be irradiated onto the region A where the transient diffraction grating including the gap S between the electrodes 13 and 14 is formed. In addition, it is preferable that the incident angle of the light source light can be adjusted so that either transmitted diffracted light or reflected diffracted light can be acquired depending on the measurement object and the measurement purpose. For example, when measurement is performed with transmitted diffracted light, the incident angle is set to a condition that does not cause total reflection at the interface between the bottom surface of the container and the liquid sample.

  The photodetector 18 is arranged on the upper side of the liquid sample when detecting transmitted diffraction light. The light detector 18 is provided with an angle adjusting mechanism for measuring the diffraction angle so that the diffraction angle can be detected together with the intensity of the diffracted light. A photodiode or CCD is used for the photodetector 18. Instead of providing the angle adjustment mechanism, the diffraction angle may be measured using an array sensor in which a plurality of elements are arranged.

  The dielectrophoresis control unit 19 is configured by a computer including a so-called CPU, ROM, RAM, and the like, and is necessary for forming a transient diffraction grating from the AC power source 15 to the electrodes 13 and 14 by a program stored in advance. An AC voltage is applied for a necessary time, and then the voltage application is stopped to cause particle diffusion.

Next, the measurement operation of the above apparatus will be described. The optical system is adjusted in advance so that incident light can be irradiated onto the region A from the light source 16.
First, an AC voltage V ₀ is applied between the electrode 13 and the electrode 14 from the AC power supply 15 under the control of the dielectrophoresis control unit 19. When particles (such as proteins) are present in the liquid sample, a dielectrophoretic action due to an alternating voltage works, and the particles move to a region where electric lines of force concentrate. FIG. 3 is a diagram for explaining the state of particles when an AC voltage is applied. As shown in the figure, when the particles move to the gap S where the electric lines of force concentrate, the dense regions B and the sparse regions C are alternately arranged, and a transient diffraction grating is formed by the particles.
At this time, the incident light from the light source 16 that has entered the region A is diffracted by the transient diffraction grating, and diffracted light is generated in a specific direction. When the AC voltage is continuously applied, the transient diffraction grating exists stably, and thus, the diffracted light having a strong intensity by the transient diffraction grating is detected by the photodetector 18. This is measured as a reference value.

  Subsequently, the application of the AC voltage to the electrodes 13 and 14 is stopped under the control of the dielectrophoresis controller 19. As a result, the dielectrophoretic action stops, and the particles concentrated in the gap S gradually spread due to diffusion. As a result, the transient diffraction grating collapses and gradually fades, and eventually the transient diffraction grating disappears as shown in FIG. Since the intensity of the diffracted light becomes weaker as the transient diffraction grating becomes thinner, the intensity change of the diffracted light intensity is measured by the photodetector 18.

  A time chart of the intensity change of the diffracted light obtained by the above measurement operation is shown in FIG. 5 together with the waveform of the applied voltage. Since the attenuation curve after the dielectrophoresis is stopped depends on the diffusion coefficient, information on particle diffusion can be obtained by obtaining the diffusion coefficient from the attenuation curve.

(Other embodiments)
In the above embodiment, transmitted diffracted light is detected, but reflected diffracted light may be detected. If reflected diffracted light is used, it is possible to easily detect diffracted light even for a liquid sample having light absorption.
In the case of an optical measuring apparatus that measures reflected diffracted light, the position of the photodetector 18 is arranged on the lower side of the bottom plate 12a in the apparatus configuration shown in FIG.
In the case of reflected diffracted light measurement, preferably, the incident angle of incident light emitted from the light source 16 is set so that total reflection occurs, and the amount of reflected diffracted light is increased as much as possible. For example, when the container 11 is made of glass and a water-based sample is held as a liquid sample, the incident angle is preferably set to about 46 degrees.

  In the above embodiment, the electrodes 13 and 14 are arranged such that one end of each linear electrode piece is opposed to each other and a transient diffraction grating is formed in a gap portion between the electrodes. This is not limited to this. In short, when a voltage is applied, a region having a high electric force line density and a region having a low electric force line density are regularly arranged alternately. Since it can be formed, the present invention can be implemented.

  The present invention can be used in an optical measurement apparatus that optically measures particles in a liquid sample.

The perspective view which shows the structure of the optical measuring device which is one Embodiment of this invention. The top view which shows the shape of the electrode part of the optical measuring device of FIG. The figure explaining the transient diffraction grating formed when an alternating voltage is applied to an electrode. The figure explaining a state when a voltage application is stopped and a particle | grain is diffused after forming a transient diffraction grating. The figure which shows the time chart of the applied voltage waveform (a) and diffracted light intensity (b) by the optical measuring device of this invention. The figure explaining the conventional transient diffraction grating method.

Explanation of symbols

11: Container 12a: Bottom plate 12b: Frame bodies 13, 14 Electrodes 13a-13d, 14a-14d: Linear electrode pieces 13e, 14e: Connection 15: AC power supply 16: Light source 17: Lens optical system 18: Photo detector 19 : Dielectrophoresis controller

Claims (4)

An AC power source, a container that holds a liquid sample, and an electric force line distribution in which a region having a high electric field line density and a region having a low electric field line density are regularly arranged in a part of the container by applying a voltage. The generation of a transient diffraction grating using the electrode pair to be generated and the dielectrophoresis of particles in the liquid sample by applying an AC voltage to the electrode pair, and the diffusion of the transient diffraction grating by the diffusion of particles in the liquid sample accompanying the stop of voltage application A dielectrophoresis control unit that controls extinction, a light source that emits light toward the transient diffraction grating, and a photodetector that detects diffracted light by the transient diffraction grating,
An optical measuring apparatus for performing particle evaluation from a change in intensity of diffracted light generated by a transient diffraction grating. Each electrode constituting the electrode pair is composed of a plurality of electrode pieces arranged at regular intervals and a connection portion for electrically connecting the electrode pieces,
2. The optical measuring device according to claim 1, wherein one end of each electrode piece in one electrode is arranged to face one end of each electrode piece in the other electrode with a gap. At least a part of the container is formed of a material that transmits light source light, and an electrode pair is formed on the container part that transmits the light source light, and the light source from the container part that transmits the light source light toward the transient diffraction grating The optical measurement apparatus according to claim 1, wherein light is incident, and the photodetector detects diffracted light transmitted through the liquid sample or diffracted light reflected by the liquid sample. Using an electrode pair that generates an electric force line distribution in which a region having a high electric force line density and a region having a low electric force line density are regularly arranged in a liquid sample by applying a voltage, an alternating voltage is applied to the electrode pair to generate a liquid Inducing a dielectrophoresis on the particles in the sample to form a transient diffraction grating with the particles,
Subsequently, the voltage application is stopped to diffuse the particles in the liquid sample forming the transient diffraction grating,
An optical measurement method characterized in that evaluation of particles is performed by detecting a change in intensity of diffracted light by a transient diffraction grating at this time.