JP4367263B2 - Diffusion measuring device - Google Patents

Description

  The present invention relates to a diffusion measurement device that measures the ease of diffusion of particles present in a liquid.

In recent years, a technique for measuring the ease of diffusion of particles in a liquid has attracted attention as one of means for measuring information about biopolymers such as proteins.
When biopolymers are viewed as particles, the mobility of biopolymers, that is, the ease of diffusion varies depending on the size, shape, binding state, etc. of biopolymers. By evaluating the ease, various information on the biopolymer can be obtained.

As a method for measuring the ease of diffusion of particles (biopolymer) in a liquid, for example, there is a micro-fluorescence correlation spectroscopy (see Patent Document 1).
According to the microscopic fluorescence correlation spectroscopy, the measurement target particle (biopolymer) is labeled with a fluorescent molecule, this is excited and illuminated under a microscope field, and the fluorescence intensity associated with the Brownian motion of the measurement target particle emitting fluorescence The change is measured (the number of fluorescent particles is counted) to obtain the diffusion coefficient of the measurement target particles.

  On the other hand, as a measurement method for performing optical measurement without directly irradiating light to the substance to be measured in the liquid, the optical waveguide is disposed so as to contact the object to be measured, and the equivalent refractive index and optical path length of the optical waveguide are set. A sensor device technology that indirectly measures optical characteristics of an object to be measured by measuring a change is disclosed (see Patent Document 2).

This sensor device uses two optical waveguides, a standard waveguide (reference side) and a sensing waveguide (sample side). Among these, the sensing waveguide side is measured in two different states, a state in contact with the object to be measured and a state not in contact with the object to be measured, and the influence of the object to be measured existing in the vicinity of the sensing waveguide. Thus, the equivalent refractive index of the transmitted light on the sensing waveguide side is changed. On the other hand, the reference waveguide side is disposed at a position separated from the object to be measured and is not affected by the object to be measured. The refractive index of light transmitted through the standard waveguide is measured for reference (for standard). Then, the light beams that have passed through these two optical waveguides are caused to interfere. At this time, the position of the interference fringe moves between the interference fringe with the sensing waveguide side in contact with the object to be measured and the interference fringe with no contact with the object to be measured.
The amount of movement of the interference fringes corresponds to the change in the equivalent refractive index of the sensing waveguide, that is, the change in the concentration or thickness of the measurement object existing around the sensing waveguide that causes the equivalent refractive index change. The information on the object to be measured can be measured from the measurement of the movement amount of the interference fringes.
JP-T-11-502608 Special table 2003-515126 gazette

In the microscopic fluorescence correlation spectroscopy of Patent Document 1, it is necessary to label the measurement target particle with a fluorescent molecule as a pre-processing before measurement, and a troublesome work for labeling must be performed.
Further, since the labeling process is performed on the particles during measurement, the particles cannot be measured in a completely natural state.
In addition, when the particle density in the sample liquid increases, fluctuations in fluorescence intensity are inevitably averaged, making analysis difficult.
Furthermore, since it is necessary to measure fluorescence intensity fluctuations of about φ100 μm or more due to Brownian motion under a microscope field of view, the diffusion coefficient is small. 10 seconds to thousands of seconds).

  In the optical measurement method of Patent Document 2, it is possible to measure a change in refractive index due to a change in concentration or thickness of a measurement object without directly irradiating the measurement object with light. For example, the ease of diffusion of the measurement target particles in the sample cannot be evaluated.

Therefore, an object of the present invention is to provide a diffusion measurement apparatus that can evaluate the ease of diffusion of measurement target particles without performing a fluorescence labeling process.
It is another object of the present invention to provide a diffusion measuring device that can evaluate the ease of diffusion even when the particle density is high.
It is another object of the present invention to provide a diffusion measuring device that can measure quickly.

In order to solve the above problems, a diffusion measuring device of the present invention includes a chamber for holding a liquid sample containing particles, a high-frequency power source, and a region having a high electric force line density in the chamber by applying a voltage. A dielectrophoresis control unit that controls the movement of particles using dielectrophoresis by applying an electrode that generates a region with a low electric field line density and applying, stopping, or modulating a high-frequency voltage from a high-frequency power source to the electrode When, a two refractive index detector for measuring the refractive index change following the definitive particles moving in the particle lean region and particle concentration area caused by dielectrophoresis, the refractive index detector, in order to measure the particle movement has a sensor surface consisting disposed in contact with the particle lean region or particle concentration area, the refractive index change is measured from the refractive index change of the sensor surface near both the particle lean region and particle concentration area And to perform an evaluation of the diffusion of the particles based on the difference of the refractive index change in the particle lean region and particle concentration area.

According to the present invention, when a sample containing particles to be measured is held in the chamber and a high frequency voltage is applied from the high frequency power source to the electrode provided in the chamber, the electric field line density is high in the chamber. Create regions and low regions.
In addition, the movement of particles is generated using dielectrophoresis by applying, stopping, or modulating the high-frequency voltage to the electrodes. At this time, a particle concentration region with a high particle density and a particle dilute region with a low particle density are generated according to the electric field line density distribution in the chamber. The refractive index change in the chamber accompanying the particle movement at that time is generated. Measured in both the particle dilute region and the particle concentration region . The two refractive index detectors that measure the refractive index have a sensor surface arranged so as to be in contact with the particle dilute region and the particle concentration region, and measure the refractive index change accompanying the diffusion of particles in the vicinity of the sensor surface. To do. Evaluation of particle diffusion is performed based on the difference in refractive index change between the particle dilute region and the particle concentration region.

According to the present invention, the particles are moved using the dielectrophoresis phenomenon to change from the collected state to the diffused state, or from the diffused state to the collected state, thereby diffusing from the refractive index change caused by the state change. Since the evaluation on ease is performed, it is not necessary to label the particles, and the diffusion measurement can be performed while the particles are in a natural state.
Further, even a liquid sample having a high particle density can measure the refractive index change, so that diffusion measurement can be performed.
Further, since the refractive index is measured from a liquid sample in the vicinity of the sensor surface, the measurement time can be shortened.
Furthermore, since the evaluation of particle diffusion is performed based on the difference in the refractive index change between the particle dilute region and the particle concentration region, it shows a change in the refractive index compared to measurement in either one of the regions. The signal can be magnified and observed, and evaluation can be performed with high measurement sensitivity. Further, by taking the difference between the two refractive index changes, it is possible to eliminate the influence of the temperature change in the liquid sample and the refractive index detection unit (especially the sensor surface).

  In the above invention, the dielectrophoresis control unit applies a high frequency voltage from the high frequency power source to the electrode to move the particles by dielectrophoresis, and generates a particle concentration region and a particle dilute region according to the electric force line density, thereby distributing the particles unevenly If the state is established and the particle voltage is controlled to diffuse from the particle concentration region by stopping or modulating the harmonic voltage applied to the electrode in the particle uneven distribution state, the particle concentration region to the particle dilute region Since the particles can be diffused toward the surface, it is possible to measure the state in which the particles spread by diffusion.

  In addition, if the dielectrophoresis control unit changes the frequency and / or amplitude of the high-frequency voltage applied to the electrode from the high-frequency power source, and measures the refractive index, the particles collected in the particle concentration region will have a frequency. Alternatively, when changing depending on the amplitude, diffusion can be evaluated while taking these into account.

Also, two refractive index detector, one optical waveguide side as the sensor surface and arranged sample side waveguide in contact with the particle lean region, the other one particle concentration area, and the sample-side waveguide A reference-side waveguide formed on the same substrate so as not to be affected by a sample liquid containing particles, a light source that emits light incident on the sample-side waveguide and the reference-side waveguide, and a sample-side waveguide And an interference fringe detector that detects interference fringes generated by interference of light emitted from the reference-side waveguide, and detects the refractive index change in the particle dilute region or particle concentration region from the amount of movement of the interference fringes By doing so, it is possible to obtain a change in refractive index using two-beam interference.
Here, if the electrode is made of an optical waveguide material and this electrode also functions as a sample-side waveguide, the refractive index change due to particles in the region of high electric field line density near the electrode is detected. can do.

Also, two refractive index detector, one waveguide side of the optical waveguide as a sensor surface in the particle lean region, with the other one arranged in contact with the particle concentration area, the optical waveguide comprising a sensor surface An optical resonator constructed by forming a partially transmissive reflection film on each of the optical waveguide entrance end face and the optical waveguide exit end face facing each other with the side face in between, and a transmission spectrum acquisition toward the optical waveguide entrance end face It consists of a light source that emits incident light and a photodetector that detects the transmitted light from the exit end face of the optical waveguide. The refractive index change in the particle-diluted region and the particle-concentrated region is determined by the transmission spectrum of the optical waveguide that constitutes the optical resonator. If it is detected from the data, the optical resonator functions as a so-called etalon type interference filter formed in the optical waveguide. Thus, since a transmission spectrum having a steep transmission peak wavelength is obtained, a change in refractive index can be detected from the shift amount of the peak wavelength of the transmission spectrum.
Here, if the electrode is formed of an optical waveguide material and this electrode also functions as an optical waveguide constituting the optical resonator, particle movement in a region with a high electric force line density near the electrode It is possible to detect a change in refractive index due to.

In addition, if a metal film is used for the electrode and a surface plasmon sensor that uses the metal film used for the electrode as the sensor surface is used for the two refractive index detection units, a change in refractive index is detected by the surface plasmon sensor. be able to.

In addition, if a metal region localized in a granular form is formed adjacent to an electrode, and a local plasmon sensor that uses the metal region localized in a granular form as a sensor surface is used for the two refractive index detection units, The local plasmon sensor can detect a change in refractive index due to particle movement in a region with a high electric field line density near the electrode.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
(Basic configuration of diffusion measurement device)
First, the basic configuration of the present invention will be described with reference to the drawings. 1A and 1B are schematic views for explaining the basic principle of a diffusion measurement apparatus according to an embodiment of the present invention. FIG. 1A is a perspective view of the diffusion measurement apparatus, and FIG. ) Shows a cross-sectional view (YZ cross-sectional view) on the sensor surface 14a of the refractive index detector.

This diffusion measuring device 10 is formed on a chamber 11 for holding a liquid sample S containing particles (for example, protein T), a substrate 12 serving as a bottom surface of the chamber, and a substrate 12 so as to be exposed in the chamber 11. The sensor surfaces 14a and 14b formed so as to be exposed in the chamber 11 in the same manner as the electrodes 13 and 13, and the refractive index detectors 15a and 15b whose refractive indexes are measured using the sensor surfaces 14a and 14b, A high-frequency power source 16 that applies a high-frequency voltage to the electrode 13 and a dielectrophoresis control unit 17 that controls the voltage applied to the electrode 13 from the high-frequency power source 16 to cause particle dielectrophoresis, or to stop or modulate dielectrophoresis. . In FIG. 1, the refractive index detectors 15a and 15b are schematically shown. However, specific examples of the refractive index detectors 15a and 15b (two-beam interference, etalon, surface plasmon, and localized plasmon are shown). The refractive index detection unit used will be described later.

The shape and arrangement position of the electrode 13 are determined so that when the high frequency voltage is applied, a region having a high electric force line density and a region having a low electric force line density are formed in the chamber 11.
As the simplest electrode structure, as shown in FIGS. 1A and 1B, two electrodes 13 are formed in parallel with each other at positions spaced apart from each other on the substrate 12, and the electrodes 13 are formed as follows. It is formed on the substrate by vapor deposition. Thereby, the density of the electric lines of force 18 is increased in the vicinity of the electrode 13, and the density of the electric lines of force 18 is decreased in the vicinity of the center between the two electrodes 13.

When a high frequency voltage is applied to the electrode 13 to cause dielectrophoresis of particles (protein T), an attractive force generally works toward a region where the electric force line density is high, and the particles are attracted. (There are cases where repulsive force acts on a region where the electric force line density is high, but for the sake of convenience, it is assumed that attractive force acts in the following description.)
Therefore, a particle concentration region 19 having a high particle density is generated in the vicinity of the electrode 13, and a particle diluted region 20 having a low particle density is generated in the vicinity of the center between the two electrodes 13 separated from the vicinity. Become.

  The surface of the substrate 12 in contact with the particle concentration region 19 or the particle dilute region 20 is made to be the sensor surfaces 14a and 14b of the refractive index detectors 15a and 15b described above. The sensor surfaces 14a and 14b are optical waveguide side surfaces (refractive index detectors using two-beam interference and etalon) formed on the substrate 12 depending on the types (described later) of the refractive index detectors 15a and 15b. It may be its own surface (a refractive index detector using surface plasmons or localized plasmons).

As the high-frequency power supply 16, a power supply capable of applying a high-frequency voltage having a frequency and amplitude capable of causing the dielectrophoresis of particles is used. In addition, since there are some particles whose dielectrophoretic state changes depending on the frequency and amplitude, the frequency and amplitude are made variable.
The dielectrophoresis control unit 17 is configured by a so-called computer, and automatically performs a series of dielectrophoresis controls by presetting the frequency, voltage, application time, and the like of the high-frequency voltage.

The dielectrophoresis control unit 17 once establishes the particle concentration region 19 by dielectrophoresis, and then stops or modulates the voltage application so that the particle movement starts by diffusion. When the refractive index of the liquid sample S changes as the particles move, the sensor surfaces 14a and 14b are formed so as to be in contact with the particle dilute region 20 and the particle concentration region 19, so that the liquid sample near the sensor surface in these regions The change in the refractive index of S affects the sensor surfaces 14a and 14b.
The original measurement method by the dielectrophoresis control unit 17 is that the dielectrophoretic force disappears or weakens after the particle concentration region is established by the dielectrophoretic force, and the particle movement by diffusion is started. As the process, particles may be collected from a state where there is no dielectrophoretic force or a weak state so that the dielectrophoretic force is increased. Even in the reverse process, information on the ease of diffusion can be obtained. In short, the dielectrophoresis control unit 17 may be controlled so as to cause the movement of particles by using the change in the dielectrophoretic force by applying, stopping, or modulating the high frequency voltage from the high frequency power source to the electrode.
The refractive index detectors 15a and 15b detect the refractive index change of the liquid sample S as an equivalent refractive index change or an optical path length change of the optical system in the refractive index detectors 15a and 15b. Then, from the detected refractive index change, the diffusion coefficient can be obtained by calculation using information on particle diffusion, for example, ease of diffusion, diffusion rate, and diffusion rate.

Next, the refractive index change measurement operation using the diffusion measurement device will be described using a time chart.
FIG. 2 is a diagram showing a time chart when the refractive index detector 15a for measuring the refractive index of the particle diluted region 20 is used alone in the diffusion measurement apparatus of FIG. 1, and FIG. Voltage waveform of the high frequency voltage applied to the electrode, FIG. 2B is a change in transmission refractive index or optical path length obtained by the refractive index detector 15a, and FIG. 2C is a particle obtained from data of the refractive index detector 15a. It is a figure which shows the refractive index change of the diluted area | region 20. FIG. When the refractive index change of the particle diluted region 20 in FIG. 2C is obtained from the equivalent refractive index (optical path length) data in FIG. 2B, a calibration curve method referring to previously prepared calibration curve data is used.

  At time t0, a voltage Vo having a frequency for inducing dielectrophoresis in the particles is applied to the electrode 13 to start dielectrophoresis. As a result, the particles existing near the center of the pair of electrodes 13 move toward the region where the lines of electric force near the electrodes 13 are concentrated, and the particle-diluted region 20 is generated. As the particles in the particle dilute region 20 decrease due to particle movement, the refractive index of the liquid sample S in the particle dilute region 20 decreases and eventually reaches an equilibrium state. The change in refractive index at this time appears as an attenuation curve from t0 to t1.

Subsequently, at time t1 after reaching equilibrium, the voltage application is stopped to eliminate the dielectrophoretic force. This initiates diffusion due to the Brownian motion of the particles. Due to the inflow of particles due to diffusion, the particles in the particle dilute region 20 gradually increase, and eventually the particle dilute region 20 disappears and returns to the steady-state refractive index before dielectrophoresis occurs throughout the liquid sample S.
Information on the diffusion of particles can be obtained from the change in refractive index after the start of diffusion after t1.
Although the measurement in the particle dilute region 20 has been described above, the same time change is observed in the case of the measurement in the particle concentration region 19 except that the direction (sign) of the refractive index change is reversed. And information on the diffusion of the particles can be determined.

FIG. 3 is a time chart when the refractive index change data in the particle concentration region 19 and the particle dilute region 20 are obtained using the two refractive index detectors 15a and 15b of FIG.
As in the case of FIG. 2, at time t <b> 0, a voltage Vo having a frequency that induces dielectrophoresis on the particles is applied to the electrode 13 to start dielectrophoresis. As a result, the particles existing near the center of the pair of electrodes 13 move toward the portion where the electric lines of force near the electrodes 13 are concentrated, and the particle concentration region 19 and the particle diluted region 20 are generated. .
As the number of particles in the particle dilute region 20 decreases due to particle movement, the refractive index of the liquid sample S existing in this region decreases, and eventually reaches an equilibrium state. The change in refractive index at this time appears as an attenuation curve from t0 to t1.
On the other hand, as the number of particles in the particle concentration region 19 increases, the refractive index of the liquid sample S existing in this region increases and eventually reaches an equilibrium state. The change in refractive index at this time appears as an increasing curve from t0 to t1.

Subsequently, at time t1 after reaching equilibrium, the voltage application is stopped to eliminate the dielectrophoretic force. As a result, the diffusion of particles due to the Brownian motion starts, the particles in the particle dilute region 20 gradually increase, the particles in the particle concentrated region 19 decrease, and eventually the particle dilute region 20 and the particle concentrated region 19 disappear. To do. Then, the entire liquid sample S returns to a steady-state refractive index before dielectrophoresis occurs.
By taking the difference data of each refractive index change by the two detectors, the refractive index change can be enlarged and detected, and the fluctuation component depending on the temperature change of the liquid sample or the detector can be obtained by using the difference data. Since it can be removed by taking, accurate measurement excluding errors due to temperature changes becomes possible.

(Configuration of refractive index detector)
Next, a refractive index detector used in the present invention will be described with reference to the drawings. The feature of the refractive index detector used in the present invention is that it has a sensor surface in contact with the liquid sample S in the measurement region (particle dilute region or particle concentration region), and the vicinity of this sensor surface (distance is within about 1 μm from the sensor surface). The refractive index of the liquid sample S is detected.
As such a refractive index detector, (1) a detector using two optical waveguides formed on a substrate and utilizing two-beam interference, and (2) an optical waveguide formed on the substrate, the optical waveguide being an etalon-type optical resonance. Detector used as a detector, (3) Detector using surface plasmon resonance by electrode (metal film), (4) Granular metal film region is provided beside electrode (metal film), and localized plasmon resonance is used A detector will be described as an example. However, the present invention is not limited to these four types, and any refractive index detector that can be used without departing from the spirit of the present invention may be used.

(Two-beam interference detector)
FIG. 4 is a schematic diagram illustrating a configuration when a two-beam interference type refractive index detector 30 is used as the refractive index detector of the diffusion measurement apparatus of FIG. 1, and FIG. 4 (a) is a perspective view. FIG. 4B is a YZ sectional view cut on the optical waveguide 33. In the figure, the same components as those in FIG. In FIG. 4, the refractive index detector 30 is used as a refractive index detector (see the time chart in FIG. 2), but a difference may be taken (see the time chart in FIG. 3).

The refractive index detector 30 includes a sensor unit 35, a light source 36, and an interference fringe detector 37. The sensor unit 35 is made of the glass substrate 12, and the sample-side optical waveguide 32, the reference-side optical waveguide 33, and the separation layer 34 that optically separates the sample-side optical waveguide 32 and the reference-side optical waveguide 33 are formed on the substrate. ing. Among these, the surface 32s of the sample-side optical waveguide 32 is exposed to the chamber 11, and the exposed surface becomes the sensor surface of FIG. The cores of the sample side optical waveguide 32 and the reference side optical waveguide 33 have the same shape.
The material of the substrate is not limited to glass, and a known material that is suitable only for forming an optical waveguide, such as a polymer material (resin or the like) or a semiconductor (silicon or the like), can also be employed.

  Light from the light source 36 is incident from one end face of each of the sample-side optical waveguide 32 and the reference-side optical waveguide 33. And the light which passed through each waveguide is radiate | emitted from the other side end surface. The outgoing lights interfere with each other to generate interference fringes 38. At a position where the interference fringe 38 is generated, an interference fringe detector 37 for detecting the movement of the interference fringe is provided. As the interference fringe detector 37, for example, a diode array sensor is used.

  By adopting such a configuration, when the refractive index of the liquid sample S existing near the surface 32 s (about 1 μm) of the sample-side waveguide 32 exposed to the chamber 11 changes, the equivalent refractive index of the sample-side waveguide 32 is changed. Changes. When the equivalent refractive index of the sample-side optical waveguide 32 changes, the optical path length of the sample-side optical waveguide 32 changes, while the transmission refractive index of the reference-side waveguide 33 is not affected by this, and the interference fringes move. . The interference fringe detector 37 can detect this amount of movement. The amount of movement of the interference fringes depends on the change in the refractive index of the liquid sample S. By obtaining a calibration curve in advance, the change in the refractive index of the liquid sample S from the amount of movement, and further the refraction of the liquid sample S. Information on the diffusion of particles that cause rate changes can be obtained.

(Etalon type detector)
FIG. 5 is a schematic diagram for explaining a configuration when an etalon-type refractive index detector 40 is used as the refractive index detector 15a of the diffusion measurement apparatus of FIG. 1, and FIG. 5 (a) is a perspective view. FIG. 5B is a YZ sectional view on the optical waveguide 43.
In the figure, the same components as those in FIG. In FIG. 5, the refractive index detector 40 is used as the refractive index detector 15a (see the time chart of FIG. 2), but as shown in FIG. 1, the refractive index detector 40 is the same as the refractive index detector 15a. The refractive index detector 15b may be further used to take the difference (see the time chart in FIG. 3).

The refractive index detector 40 includes a sensor unit 41, a light source 46, and a photodetector 47. The sensor unit 41 is made of a substrate 42 such as glass, and an optical waveguide 43 is formed on the substrate. The surface 43s of the optical waveguide 43 is exposed to the chamber 11 and becomes the sensor surface 14a of FIG.
Partially transmissive reflective films 44 and 45 are formed on both end faces of the optical waveguide 43. Of these, one end face faces the light source 46, and the other end face faces the detector 47, and becomes an incident end face and an exit end face, respectively. The partially transmitting / reflecting films 44 and 45 multiplex-reflect incident light that has entered the optical waveguide 43 and radiate the multiple reflected light from the exit end face to the detector. That is, the two partially transmissive reflection films 44 and 45 form an etalon type optical resonator in the optical waveguide.
As a result, when transmission spectrum data from the emitted light is taken, it functions as an etalon type interference filter in which a steep peak wavelength appears. This steep peak wavelength changes depending on the equivalent refractive index of the optical waveguide 43.

The equivalent refractive index of the optical waveguide 43 depends on the refractive index of the liquid sample in the chamber 11 in contact with the optical waveguide 43.
Therefore, when the refractive index of the liquid sample S in the chamber 11 in contact with the surface 43s of the optical waveguide 43 (liquid sample S within about 1 μm from the surface 43s) changes, the peak wavelength of the transmission spectrum of the optical waveguide 43 according to this change. Will shift.
Therefore, a change in the refractive index of the liquid sample can be detected from the peak wavelength shift by obtaining in advance a calibration curve that associates the shift amount of the peak wavelength of the transmission spectrum of the optical waveguide 43 with the refractive index.

(Etalon type detector 2)
FIG. 6 is a schematic diagram for explaining another configuration when an etalon-type refractive index detector is used as the refractive index detectors 15a and 15b of the diffusion measuring apparatus of FIG. 1, and FIG. FIG. 6B is an XZ sectional view taken along a plane crossing the electrodes 52a and 52b, and FIG. 6C is a YZ sectional view on the electrode 52b. In the figure, the same components as those in FIG. In FIG. 6, two refractive index detectors 50a for detecting the particle diluted region 20 and a refractive index detector 50b for detecting the particle concentration region 19 are used (see the time chart of FIG. 3). However, only one of them may be used (see the time chart in FIG. 2). The following describes the refractive index detector 50b, but the same applies to 50a.

    The refractive index detector 50b includes a sensor unit 51b, a light source 55, and a photodetector 56. The sensor unit 51b is formed by forming an electrode 52b having optical waveguide properties on the glass substrate 12 constituting the bottom surface of the chamber 11. In other words, the electrode 52b is configured to have both the function as an electrode and the function as an optical waveguide. As this electrode material, for example, a transparent conductive film such as ITO can be used.

  Then, partially transmissive reflecting films 53 and 54 are formed on both end faces of the electrode 52b. Of these, one end face faces the light source 55 and the other end face opposes the photodetector 56, and becomes an entrance end face and an exit end face, respectively. The partially transmitting / reflecting films 53 and 54 multiplex-reflect incident light incident on the optical waveguide 52b and radiate the multiple reflected light to the photodetector 56 from the emission end face. That is, the two partially transmissive reflection films 53 and 54 form an etalon type optical resonator in the optical waveguide 52b.

Then, in the optical resonator part sandwiched between the partially transmitting and reflecting films 53 and 54, a steep peak wavelength appears by taking transmission spectrum data by the emitted light, similarly to the etalon type detector shown in FIG. It functions as an etalon type interference filter.
Furthermore, the peak wavelength of this steep peak changes depending on the equivalent refractive index of the optical waveguide 52b.

The equivalent refractive index of the optical waveguide 52b depends on the refractive index of the liquid sample S in the chamber 11 in contact therewith. Therefore, when the refractive index of the liquid sample S in the chamber 11 in contact with the surface of the optical waveguide 52b (liquid sample S within about 1 μm from the surface) changes, the peak wavelength of the transmission spectrum of the optical waveguide 52b shifts according to this change. Will be.
Therefore, a change in the refractive index of the liquid sample can be detected from the peak wavelength shift by obtaining in advance a calibration curve that relates the shift amount of the peak wavelength of the transmission spectrum of the optical waveguide 52b and the refractive index.

  The structure in which the electrode has optical waveguide properties and is also used as the optical waveguide can be applied to the electrode in the two-beam interference type refractive index detector described with reference to FIG.

(Surface plasmon detector)
FIG. 7 is a schematic diagram illustrating a configuration when a surface plasmon detector is used as the refractive index detector 15a of the diffusion measurement apparatus of FIG.
In the figure, the same components as those in FIG. In FIG. 7, two refractive index detectors 60a for detecting the particle diluted region 20 and a refractive index detector 60b for detecting the particle concentration region 19 are used (see the time chart in FIG. 3). However, only one of them may be used (see the time chart in FIG. 2). The following describes the refractive index detector 60b, but the refractive index detector 60a has the same configuration except that the high-frequency electrode is not used.

The refractive index detector 60b includes a metal electrode film 61b, a light source 62b, and a photodetector 63b. As the material of the metal electrode film 61b, a material that easily causes surface plasmon resonance and has excellent light reflection characteristics, for example, Au or Ag is used.
In this detector, the metal electrode film 61b is irradiated with incident light while changing the incident angle from the light source 62b, and the reflected light is detected by the photodetector 63b. When the incident angle is an angle condition in which surface plasmon resonance occurs, absorption is observed in the reflected light. On the other hand, this resonance angle changes depending on the refractive index of the liquid sample S in contact with the metal electrode film 61b.
Therefore, the refractive index change can be obtained from the resonance angle change. Also in this case, a calibration curve relating the amount of change in resonance angle and the amount of change in refractive index is obtained in advance.

(Localized plasmon detector)
FIG. 8 is a schematic diagram for explaining a configuration when a localized plasmon detector is used as the refractive index detector 15a of the diffusion measurement apparatus of FIG.
In the figure, the same components as those in FIG. In FIG. 8, two refractive index detectors 70a for detecting the particle diluted region 20 and a refractive index detector 70b for detecting the particle concentration region 19 are used (see the time chart in FIG. 3). However, only one of them may be used (see the time chart in FIG. 2). The following describes the refractive index detector 70b, but the refractive index detector 70a has the same configuration except that the high-frequency electrode is not used.

The refractive index detector 70b includes a metal electrode film 71b, a metal region 74b formed so as to be localized in a granular form adjacent to the metal electrode film 71b, a light source 72b, and a photodetector 73b. The metal electrode film 71b and the metal region 74b are made of a material having excellent light reflection characteristics, such as Au or Ag, in which localized plasmon resonance easily occurs.
The metal electrode film 71b and the metal region 74b are electrically independent of each other so that a high frequency voltage applied to the metal electrode film 71b is not applied to the metal region 74b. Therefore, when a high-frequency voltage is applied to the metal electrode film 71b, the lines of electric force do not go to the metal region 74b but concentrate near the metal electrode film 71b. At this time, the most concentrated portion of the electric lines of force comes immediately above the metal region 74b adjacent to the metal electrode film 71b. Therefore, when dielectrophoresis is caused, the center of the particle concentration region comes directly above the metal region 74b.

  On the other hand, irradiating light from the light source 72b to the metal region 74b localized in a granular manner causes plasmon resonance (in this case, referred to as localized plasmon resonance), similarly to the case of irradiating the metal electrode film 71b. Can be made. Therefore, by causing plasmon resonance in the granular metal region 74b, the center of the particle concentration region 19 where the particles are most concentrated can be matched with the plasmon resonance region, which is higher than in the case of normal surface plasmon resonance in FIG. In addition, measurement sensitivity can be improved.

  As described above, as the embodiment of the present invention, the movement of the particles is controlled by dielectrophoresis and the particles are measured by the refractive index detector. However, in addition to the refractive index, it is known as a means for measuring the particles. It is also possible to use an ellipsometer to which the ellipsometry method is applied.

  INDUSTRIAL APPLICABILITY The present invention can be used in a diffusion measurement device that collects particles by dielectrophoresis and diffuses them after collection to measure the ease of diffusion.

The schematic diagram explaining the basic composition of the diffusion measuring device which is one embodiment of the present invention. The figure explaining an example of the time chart at the time of the measurement by the spreading | diffusion measuring apparatus of FIG. The figure explaining another example of the time chart at the time of the measurement by the spreading | diffusion measuring apparatus of FIG. The schematic diagram explaining the structure of the diffusion measurement apparatus using the refractive index detector of the 2 light beam interference type which is one Embodiment of this invention. The schematic diagram explaining the structure of the diffusion measuring apparatus using the etalon type | mold refractive index detector which is one Embodiment of this invention. The schematic diagram explaining the structure of the diffusion measuring apparatus using the etalon type | mold refractive index detector which is other one Embodiment of this invention. The schematic diagram explaining the structure of the diffusion measuring apparatus using the surface plasmon resonance type | mold refractive index detector which is one Embodiment of this invention. The schematic diagram explaining the structure of the diffusion measuring apparatus using the local plasmon resonance type | mold refractive index detector which is one Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Chamber 12 Glass substrate 13 Electrode 14a, 14b Sensor surface 15a, 15b Refractive index detector 16 High frequency power supply 17 Dielectric migration control part 19 Particle concentration area | region 20 Particle dilute area | region 32 Sample side optical waveguide 33 Reference side optical waveguide 36 Light source 37 Interference fringe Detector 43 Optical waveguide 44, 45 Partially transmitting / reflecting film 46 Light source 47 Photo detector 52a, 52b Optical waveguide electrode 55 Light source 56 Photo detector 61a, 61b Metal film 62a, 61b Light source 63a, 63b Photo detector 71a, 71b Metal electrode films 72a, 72b Light sources 73a, 73b Photo detectors 74a, 74b Metal regions

Claims (9)

A chamber for holding a liquid sample containing particles;
A high frequency power supply,
An electrode that generates a region having a high electric field line density and a region having a low electric field line density in the chamber by applying a voltage;
A dielectrophoresis control unit that controls the movement of particles using dielectrophoresis by applying and stopping or modulating a high frequency voltage from a high frequency power source to the electrode; and
And two refractive index detector for measuring the refractive index change following the definitive particles moving in the particle lean region and particle concentration area caused by dielectrophoresis,
Each refractive index detection unit has a sensor surface arranged so as to be in contact with a particle dilute region or a particle concentration region in order to measure particle movement,
From the refractive index change near the sensor surface , measure the refractive index change in both the particle dilute region and the particle concentrated region, and evaluate the diffusion of particles based on the difference in the refractive index change between the particle dilute region and the particle concentrated region. A diffusion measuring device characterized by that. The dielectrophoresis control unit applies a high frequency voltage from the high frequency power source to the electrode to move the particles by dielectrophoresis, and generates a particle concentration region and a particle dilute region according to the electric force line density to establish a particle uneven distribution state. 2. The diffusion measurement apparatus according to claim 1, wherein particles are diffused from the particle concentration region by stopping or modulating a harmonic voltage applied to the electrode when the particles are unevenly distributed. The diffusion measurement apparatus according to claim 1, wherein the dielectrophoresis control unit measures the refractive index by changing at least one of a frequency and an amplitude of a high-frequency voltage applied to the electrode from a high-frequency power source. Two refractive index detector, one particle lean region optical waveguide side as the sensor surface, and arranged sample side waveguide and the other one in contact with the particle concentration area, the same substrate as the sample side waveguide A reference-side waveguide formed on the sample-side waveguide that is formed so as not to be affected by the sample liquid including particles, a light source that emits light incident on the sample-side waveguide and the reference-side waveguide, and a sample-side waveguide It consists of an interference fringe detector that detects interference fringes generated by interfering with the light emitted from the side waveguide, and detects the refractive index change in the particle dilute region or particle concentration region from the amount of movement of the interference fringes The diffusion measuring apparatus according to claim 1, wherein the apparatus is a diffusion measuring apparatus. 5. The diffusion measurement apparatus according to claim 4 , wherein the electrode is formed of an optical waveguide material, and the electrode is configured to also function as a sample-side waveguide. The two refractive index detectors are arranged so that the waveguide side surface of the optical waveguide is the sensor surface, one is in contact with the particle dilute region , and the other is in contact with the particle concentration region, and the side surface of the optical waveguide that is to be the sensor surface is An optical resonator formed by forming a partially transmissive reflection film on each of the optical waveguide entrance end face and the optical waveguide exit end face that are opposed to each other, and incident light for acquiring a transmission spectrum toward the optical waveguide entrance end face A light source that emits light and a photodetector that detects the transmitted light from the exit end face of the optical waveguide, and the refractive index change in the particle dilute region and the particle concentration region is determined from the transmission spectrum data of the optical waveguide constituting the optical resonator. The diffusion measurement device according to claim 1, wherein the diffusion measurement device is detected. 7. The diffusion measuring device according to claim 6, wherein the electrode is formed of an optical waveguide material, and the electrode also functions as an optical waveguide constituting the optical resonator. The diffusion measurement apparatus according to claim 1, wherein a metal film is used for the electrode, and a surface plasmon sensor that uses the metal film used for the electrode as a sensor surface is used for the two refractive index detection units. . Adjacent to the electrode is metal region localized formation granular, the two refractive index detector, localized plasmon sensor utilizing metal region localized to granular as a sensor surface, characterized by being used The diffusion measurement apparatus according to claim 1.

Images