JP4910132B2 - Surface charge amount measuring apparatus and surface charge amount measuring method - Google Patents

Description

  The present invention relates to a surface charge amount measuring apparatus and a surface charge amount measuring method.

Conventionally, as a method for detecting a chemical reaction or a biological reaction, a method for detecting a change in an electric signal (for example, potential) accompanying the reaction is known. For example, for a highly specific biomolecule, when a target molecule having a high affinity with the biomolecule is detected, the biomolecule is immobilized on a semiconductor substrate, and the binding to the target molecule is detected by an electric signal output from the semiconductor substrate. A method of detecting by a change has been studied (see Patent Document 1).
JP-A-10-332423

  An object of the present invention is to provide a surface charge amount measuring apparatus and a surface charge amount measuring method capable of easily measuring the charge amount on the surface of a sample.

  By the way, it is not enough to verify the binding reaction if the object of detection is only an electric signal. That is, even if a change in potential, for example, can be detected, the reaction cannot be analyzed quantitatively by itself, and further, it is demonstrated that the potential change is actually caused by a binding reaction. Can not.

  Usually, in order to specify the binding reaction, the substance to be measured is directly observed with a fluorescent dye or the like. However, in this case, there is a problem that the labeling operation becomes very complicated.

  For these problems, the inventors of the present application have found that by measuring the amount of charge on the surface of the substrate or the like, it is possible to specify a biological reaction or the like on the surface of the substrate, and also to perform a quantitative analysis. It was. Accordingly, the inventors of the present application have made extensive studies on an apparatus and method that can easily measure the amount of charge on the sample surface in order to contribute to further development of analysis of biological reactions or chemical reactions. As a result, it finally came to the present invention.

  Based on such research results, the surface charge amount measuring apparatus according to the present invention is a surface charge amount measuring apparatus that measures a charge amount on the surface of a charged sample by floating a plurality of charged probe particles upward. A position distribution calculating means for calculating the position distribution of the probe particles, and a charge amount calculating means for calculating the charge amount on the sample surface based on the position distribution.

  In the above surface charge amount measuring apparatus, charged probe particles are distributed above the surface of a charged sample. The charged probe particles are distributed under the influence of electrostatic interaction with the charged sample. Therefore, by obtaining the position distribution of the probe particles, the charge amount on the charged sample surface can be calculated. Further, since only the position distribution of the charged probe particles is obtained, the charge amount on the sample surface can be measured easily and at low cost. In addition, even when liquid or gas is present on the sample surface and the liquid or the like is flowing, the probe particles are floating, so the charge amount on the sample surface is measured without being hindered by the flow. It becomes possible. In addition, it is possible to measure the charge amount with a desired resolution by changing the size of the probe particles.

  A surface charge amount measuring apparatus for measuring the amount of charge on the surface of a sample based on a probe image obtained by picking up an image of probe particles, the probe obtained by picking up an image of probe particles The apparatus further comprises size specification value specifying means for obtaining a measurement value that specifies the size of the probe particle in the image, and the position distribution calculation means includes a value that specifies the size of the probe particle in the probe image and the probe particle from the reference plane. It is preferable to calculate the position distribution of the plurality of probe particles based on the calibration data indicating the relationship with the distance and the measurement value obtained by the size specification value specifying means.

  In this surface charge amount measuring apparatus, the position distribution of the probe particles can be calculated as long as the measurement value that defines the size of each probe particle in the probe image is obtained. Therefore, even if there are a plurality of probe particles imaged in the probe image, it is possible to calculate the position distribution of the plurality of probe particles imaged, and the time required for measuring the charge amount on the surface can be calculated. It can be shortened.

  Alternatively, the surface charge amount described above further includes an intensity measurement unit that measures the intensity of light, and measures the amount of charge on the surface of the sample by causing incident light to enter the first side surface of the sample intersecting the surface obliquely. In the measurement apparatus, the incident light is incident on the first side surface so as to be emitted from the second side surface of the sample intersecting the surface after being propagated through the sample while being totally reflected on the sample surface. The position distribution calculating means preferably calculates the position distribution of the plurality of probe particles based on the intensity difference between the intensity of the incident light and the intensity of the emitted light.

X：界面からの距離
dp：エバネッセント光の強度が界面での強度の1/eになるときの界面からの距離 In this surface charge amount measuring apparatus, since the incident light propagates through the sample while being totally reflected on the surface, the evanescent light penetrates outside the sample near the sample surface. The intensity E of the evanescent light is expressed by Expression (1), where E ₀ is the light intensity at the interface (sample surface).

X: Distance from interface
dp: Distance from the interface when the intensity of the evanescent light is 1 / e of the intensity at the interface

λ：入射光の波長
ｎ_１：試料の屈折率
ｎ_２：試料と界面で接する媒質の屈折率
θ：試料表面の法線に対する入射光の角度 As understood from the equation (1), the intensity E of the evanescent light attenuates exponentially as the distance from the interface increases. The arrival distance dp of the evanescent light is approximately in the order of wavelengths, and is expressed by the following equation (2).

λ: wavelength of incident light n ₁ : refractive index of sample n ₂ : refractive index of medium in contact with sample at interface θ: angle of incident light with respect to normal of sample surface

For example, light with a wavelength of 400 nm is used as incident light, quartz glass (n ₁ = 1.47) is used as a sample, water (n2 = 1.33) is prepared as a medium in contact with the sample at the interface, and the normal of the sample surface is prepared. When the angle of the incident light with respect to is 70 ° (66.8 ° is used in the sample), about 270 nm is obtained as the reach distance dp of the evanescent light from the equation (2). Actually, the maximum reachable distance of the evanescent light contributing to the absorption is 3 dp, so that the light absorption is considered to occur up to about 810 nm. Since the surface charge amount measuring apparatus calculates the position distribution of the probe particles using the evanescent light, the surface of the sample can be measured with a very small scale. In general, when a charged antibody protein is immobilized on the surface of a sample, detection of a target antigen and quantitative analysis thereof are possible.

  The probe particles are preferably spheres. In this case, since the handling of the probe particles is facilitated, the surface charge amount measuring apparatus can more easily measure the charge amount on the sample surface.

  The sample is preferably charged by charging a charged substance on the surface. For example, when charged DNA molecules are immobilized on the surface of a sample, not only detection of target molecules but also quantitative analysis of the binding reaction is possible.

  On the other hand, the surface charge amount measuring method according to the present invention is a surface charge amount measuring method for measuring a charge amount on the surface of a charged sample by floating a plurality of charged probe particles upward, A position distribution calculating step for calculating the position distribution; and a charge amount calculating step for calculating the charge amount on the sample surface based on the position distribution.

  In the above surface charge amount measuring method, charged probe particles are distributed above the surface of a charged sample. The charged probe particles are distributed under the influence of electrostatic interaction with the charged sample. Therefore, by obtaining the position distribution of the probe particles, the charge amount on the charged sample surface can be calculated. Further, since only the position distribution of the charged probe particles is obtained, the charge amount on the sample surface can be measured easily and at low cost. In addition, even when liquid or gas is present on the sample surface and the liquid or the like is flowing, the probe particles are floating, so the charge amount on the sample surface is measured without being hindered by the flow. It becomes possible. In addition, it is possible to measure the charge amount with a desired resolution by changing the size of the probe particles.

  A calibration data preparation step for preparing calibration data indicating a relationship between a value defining the size of the probe particle in the probe image and the distance of the probe particle from the reference plane, and a probe image obtained by capturing an image of the probe particle A size specification value specifying step for obtaining a measurement value that defines the size of the probe particle in the position distribution step, the position distribution calculation step based on the calibration data and the measurement value obtained by the size specification value specifying means It is preferable to calculate the position distribution of a plurality of probe particles.

  In the surface charge amount measuring method, the position distribution of the probe particles can be calculated as long as the measurement value that defines the size of each probe particle in the probe image is obtained. Therefore, even if there are a plurality of probe particles imaged in the probe image, it is possible to calculate the position distribution of the plurality of probe particles imaged, and the time required for measuring the charge amount on the surface can be calculated. It can be shortened.

  Alternatively, a light incident step in which incident light whose intensity is propagated through the sample while being totally reflected by the sample surface is incident obliquely on the first side surface of the sample intersecting the surface, and incident light is incident on the sample. An emission light intensity measuring step for measuring the intensity of the emitted light emitted from the second side surface of the sample that intersects the surface, and the position distribution calculating step includes the intensity of the incident light and the intensity of the emitted light. It is preferable to calculate the position distribution of the plurality of probe particles based on the intensity difference from the intensity.

  In the surface charge amount measuring method, since the incident light propagates through the sample while being totally reflected on the surface, the evanescent light penetrates outside the sample near the sample surface. Since the surface charge amount measuring method calculates the position distribution of the probe particles using the evanescent light, the sample surface can be measured with a very small scale.

  The probe particles are preferably spheres. In this case, since the probe particles can be easily handled, the surface charge amount measuring method can more easily measure the charge amount on the sample surface.

  The sample is preferably charged by charging a charged substance on the surface. For example, when charged DNA molecules are immobilized on the surface of a sample, not only detection of target molecules but also quantitative analysis of the binding reaction is possible.

  ADVANTAGE OF THE INVENTION According to this invention, the surface charge amount measuring apparatus and surface charge amount measuring method which can measure the charge amount in the surface of a sample simply can be provided.

  Hereinafter, preferred embodiments will be described in detail with reference to the accompanying drawings. In the description, the same reference numerals are used for the same elements or elements having the same function, and redundant description is omitted.

(First embodiment)
FIG. 1 is a diagram showing a configuration of a surface charge amount measuring apparatus 1A according to the first embodiment. As shown in FIG. 1, a surface charge measuring device 1A includes a light source 2, a water tank 20, an imaging optical system (imaging means) 3, an imaging device (imaging means) 4, and an image processing device (image processing means). ) 10 and a display device (display means) 5. The surface charge amount measuring apparatus 1 </ b> A measures the amount of charge on the surface 21 a of the sample 21 composed of the transparent substrate 22 and the immobilized DNA molecules 23 immobilized on the surface of the transparent substrate 22.

  The water tank 20 is a container made of a transparent material with one surface opened. The water tank 20 is filled with pure water 25, for example. On the bottom surface of the water tank 20, the sample 21 is placed so that the surface on which the immobilized DNA molecules 23 are not fixed is in contact with the bottom surface of the water tank 20. In the water tank 20, a plurality of spherical probe particles P are suspended above the surface 21 a of the sample 21. If necessary, the detection DNA molecules 24 may also be suspended in the water tank 20. For example, polystyrene latex fine particles having a diameter of about 1 μm are used as the probe particles P. The sample 21, the detection DNA molecule 24, and the probe particle P are charged with the same sign. The sample 21 is charged when the immobilized DNA molecule 23 which is a charged substance is immobilized on the surface thereof.

  The light source 2 is located above the water tank 20, that is, on the side opposite to the bottom surface on which the sample 21 of the water tank 20 is placed. The light source 2 outputs light l that irradiates the sample 21 and the probe particles P. The surface charge amount measuring device 1 </ b> A further includes a condenser lens 2 </ b> A with respect to the light source 2. The condensing lens 2 </ b> A collects the light 1 output from the light source 2 and makes it enter the water tank 20.

  The imaging optical system 3 is located below the water tank 20, that is, on the bottom surface side of the water tank 20. The imaging optical system 3 connects an image (probe image) of the probe particle P irradiated with the light 1 output from the light source 2 on the light receiving surface of the imaging device 4. The sample 21 does not coincide with the focal plane F of the imaging optical system 3 which will be described later. In this embodiment, the sample 21 is arranged so that the focal plane F of the imaging optical system 3 is located in the water tank 20.

  The imaging device 4 is a CCD camera that captures a probe image formed by the imaging optical system 3. The imaging device 4 outputs the probe image obtained by imaging the probe image to the image processing device 10.

  The image processing apparatus 10 inputs the captured probe image from the imaging apparatus 4. The image processing apparatus 10 calculates the position distribution of the probe particles P from the probe image output from the imaging apparatus 4, and calculates the charge amount on the surface 21a of the sample 21 from the position distribution. The image processing apparatus 10 outputs the calculated charge amount on the surface 21 a of the sample 21 to the display device 5. Note that the image processing apparatus 10 may also output a probe image to the display device 5 as necessary.

  The display device 5 displays a charge amount or a probe image on the surface 21 a of the sample 21 input from the image processing device 10. As the display device 5, for example, a CRT monitor, a liquid crystal display, or the like can be used.

  Next, functions of the image processing apparatus 10 will be described with reference to FIG. As illustrated in FIG. 2, the image processing apparatus 10 includes a calibration data storage unit 11, a size specification value specifying unit 12, a position distribution calculation unit 13, and a charge amount calculation unit 14.

  The calibration data storage unit 11 stores calibration data indicating the relationship between the value that defines the size of the probe particle P in the probe image and the distance of the probe particle P from the surface 21a of the sample 21. In this embodiment, since the probe particle P is a sphere, the value that defines the size of the probe particle P is defined as the diameter of the probe particle P on the image.

  FIG. 3 shows a graph obtained from the calibration data. The horizontal axis of the graph in FIG. 3 represents the distance Z (μm) from the focal plane F of the imaging optical system 3 in the direction orthogonal to the surface 21a of the sample 21 (hereinafter referred to as the z direction), and the vertical axis represents the probe image. Represents the diameter d (μm) of the probe particle P in FIG. The focal plane F of the imaging optical system 3 functions as a reference plane for specifying the position of the probe particle. The circle in the graph of FIG. 3 represents actual measurement data, and the curve is the result of fitting to these measurement data. The calibration data storage unit 11 stores, for example, a relational expression obtained by fitting as calibration data. Alternatively, the calibration data storage unit 11 stores, for example, actual measurement data as calibration data. FIG. 4 shows a table of calibration data when the calibration data storage unit 11 stores actual measurement data as calibration data.

The position of the focal plane F of the imaging optical system 3 from the surface 21a of the sample 21 is known. Therefore, as can be understood from the graph of FIG. 3, according to the calibration data, the distance Z of the probe particle P from the surface 21 a of the sample 21 can be obtained by obtaining the diameter d of the probe particle P. . Here, each distance _Z from the focal plane F _0, Z 1, _{Z 2} only separated probe particles _{P 0,} the P 1, _{P 2,} the distance _{Z 0,} Z 1, _{Z 2} and the diameter _d 0 of the probe image, The relationship between d ₁ and d ₂ will be described. In the description, reference is made to FIGS.

FIG. 5 is a diagram for explaining the relationship between the probe particles P ₀ , P ₁ , P ₂ and the focal plane F. FIG. As shown in FIG. 5, the probe particle P ₀ is located at the focal plane F and is separated from the focal plane F by Z ₀ (Z ₀ = 0). The probe particle P ₁ is at a position Z ₁ (<Z ₀ ) with respect to the focal plane F. The probe particle P ₂ is at a position Z ₂ (> Z ₀ ) with respect to the focal plane F.

FIG. 6 is an image of probe particles P ₀ , P ₁ , P _{2 in} the captured probe image. 6A is an image of the probe particle P ₀ , FIG. 6B is an image of the probe particle P ₁ , and FIG. 6C is an image of the probe particle P ₂ . As shown in FIG. 6, the size (diameter size) of the circle in the image of the probe particle P ₀ located in the focal plane F (FIG. 6B) is that of the other probe particles P ₁ and P ₂ . It becomes smaller than the circle (diameter) in the image (FIGS. 6A and 6C). The two-dot chain line T ₂ shown in FIG. 6A, the two-dot chain line T ₀ shown in FIG. 6B, and the two-dot chain line T ₁ shown in FIG. A line that passes through the center point of the particle and traverses the image of each probe particle. Further, the diameter d ₁₂ shown in FIG. 6A, the diameter d ₁₀ shown in FIG. 6B, and the diameter d ₁₁ shown in FIG. 6C are white circles of probe particles in the probe image, respectively. The diameter of the image represented by The diameter d ₂₂ shown in FIG. 6 (a), the diameter d ₂₀ shown in FIG. 6 (b), and the diameter d ₂₁ shown in FIG. 6 (c) are each represented by a black ring of probe particles in the probe image. Is the diameter of the image to be made.

FIG. 7 is a graph showing the intensity of each probe particle P ₀ , P ₁ , P ₂ in the probe image. 7A is a graph of the probe particle P ₀ , FIG. 7B is a graph of the probe particle P ₁ , and FIG. 7C is a graph of the probe particle P ₂ . The horizontal axis of each graph is the position T on the transverse lines T ₂ , T ₀ , and T ₁ shown in FIGS. 6A, 6B, and 6C, and the vertical axis is the intensity I (T) in the probe image. To express. The intensity I ₀ in each graph corresponds to the average intensity of the background image. The diameters d ₀ , d ₁ , d ₂ of each probe particle P ₀ , P ₁ , P _{2 in} the probe image are, for example, of the intensity I (T) located on each transverse line and within the circle of the probe particle on the image The distance between the vertices of the primary peak (convex portion) on both sides of the zero-order peak (convex portion) (the diameter of the white ring in each image of FIGS. 6A to 6C) d ₁₀ , d ₁₁ , and _{d 12.} Alternatively, the diameters d ₀ , d ₁ , d ₂ of the probe particles P ₀ , P ₁ , P ₂ in the probe image are, for example, the intensities I (T) located on the respective transverse lines and within the circle of the probe particles on the image. ) Between the vertices of the concave portions on both sides of the 0th-order peak (convex portion) (diameter of the black ring in each image of FIGS. 6A to 6C) d ₂₀ , d ₂₁ , d _22. May be.

Further, the case where the intensity of the zero-order peak is smaller than the average intensity I ₀ means that the probe particles are located above the focal plane F. On the other hand, the case where the intensity I (T = 0) of the zero-order peak is larger than the average intensity I ₀ means that the probe particles are located below the focal plane F. Thus, from FIG. 7 (a) ~ (c) , the probe particles _{P 2} is positioned above the focal plane F, probe particles _P 0, _{P 1} is shown to be positioned below the focal plane F.

  The description of the function of the image information processing apparatus 10 will be continued with reference to FIG. The size specification value specifying unit 12 inputs the captured probe image from the imaging device 4 and obtains a diameter d that is a measurement value of a value that specifies the size of each of the plurality of probe particles P in the probe image. That is, the diameter d is obtained as a distance between primary peaks based on a graph (see FIG. 7) obtained based on an image as shown in FIG. The size specification value specifying unit 12 outputs the diameter d to the position distribution calculating unit 13.

  The position distribution calculation unit 13 inputs the diameter d of the probe particles P from the size specification value specifying unit 12. The position distribution calculation unit 13 calculates the position information of the probe particles P from the surface 21 a of the sample 21 based on the calibration data stored in the calibration data storage unit 11 and the diameter d of the probe particles P. That is, the position distribution calculation unit 13 is based on the calibration data shown in FIG. 3 obtained from the calibration data storage unit 11 when the diameter of the probe particle P obtained by the size specification value specifying unit 12 is d. A distance (positional information) Z from the focal plane F is obtained. Further, if the distance from the surface 21a of the focal plane F to the surface 21a of the sample 21 is ZF, the ZF is known. Therefore, the position distribution calculation unit 13 adds ZF to the Z thus obtained as necessary, thereby adding the sample 21. The distance ZP of the probe particle P from the surface 21a is calculated.

  The position distribution calculation unit 13 obtains two-dimensional coordinates (x, y) projected on the two-dimensional surface of each probe particle, that is, on the surface 21a of the sample 21, from the two-dimensional probe image. Then, based on the distance from the focal plane F, the position distribution calculation unit 13 takes the desired position as the origin, and the three-dimensional coordinates (x, y, z) (for example, z = Z or z = ZP) of each probe particle P. )

A：定数
φ（ｘ、ｙ、ｚ）：静電的相互作用
ｎ_０：φ（ｘ、ｙ、ｚ）＝０におけるプローブ粒子の個数（ｚが十分に大きいときに成立）
ｋ：Boltzmann定数
Ｔ：絶対温度 The position distribution calculation unit 13 determines the position distribution n (x, y, z) of the plurality of probe particles P floating above the surface 21a of the sample 21 from the three-dimensional coordinates (x, y, z) of the plurality of probe particles P. ) The position distribution n (x, y, z) in the present embodiment is a function that represents the distribution of the number of probe particles P at the position of coordinates (x, y, z) above the surface 21a of the sample 21. It is represented by Formula (3).

A: Constant φ (x, y, z): Electrostatic interaction n ₀ : Number of probe particles at φ (x, y, z) = 0 (Established when z is sufficiently large)
k: Boltzmann constant T: Absolute temperature

  The position distribution calculation unit 13 outputs the position distribution n (x, y, z) that is the position distribution of each probe particle P thus obtained to the charge amount calculation unit 14.

  The charge amount calculation unit 14 inputs the position distribution n (x, y, z) of the probe particles P from the position distribution calculation unit 13. Based on the position distribution of the probe particle P, the charge amount calculation unit 14 obtains an electrostatic interaction φ (x, y, z) between the probe particle P and the surface 21a of the sample 21 from Expression (3).

The charge amount calculation unit 14 obtains the surface potential at the surface 21a of the sample 21 from the equation (4) based on the electrostatic interaction obtained from the equation (3).

The charge amount calculation unit 14 further obtains the charge amount σ per unit area on the surface 21a of the sample 21 from the equation (5) based on the surface potential of the sample 21 obtained from the equation (4).

  Next, a surface charge amount measuring method using the surface charge amount measuring apparatus 1A according to the present embodiment will be described with reference to FIGS. 8 and 9, and the operation of the surface charge amount measuring apparatus 1A will also be described. FIG. 8 is a diagram showing the procedure of the surface charge amount measuring method.

  First, the diameter d that is a value that defines the size of the probe particle P in the image captured by the imaging device 4 and the distance Z of the probe particle P from the focal plane F of the imaging optical system 3 that is the reference surface. Calibration data (see FIGS. 3 and 4) indicating the relationship is prepared (calibration data preparation step S01). FIG. 9 is a diagram for explaining how to obtain calibration data. Calibration data is acquired by the following procedure.

  That is, as shown in FIG. 9, the probe particles P sandwiched between the two cover glasses G1 and G2 are moved in the z direction. That is, the case where it coincides with the focal plane F is set as a reference (Z = 0), and the probe particles P sandwiched between the cover glasses G1 and G2 are moved in the vertical direction (Z <0, Z> 0) with respect to the reference. . At that time, a probe image of the probe particle P at each position is picked up by the image pickup device 4, and the diameter d of the probe particle P in the probe image is obtained. Thus, the diameter d of the probe particle P in the probe image with respect to the distance Z from the focal plane F is obtained, and calibration data is obtained. The obtained calibration data is stored in the calibration data storage unit 11 of the image processing apparatus 10.

  Returning to FIG. 8 again, the description of the surface charge amount measuring method will be continued. Next, the sample 21 is prepared by immobilizing the immobilized DNA molecules 23 on one surface of the substrate 22. Since the immobilized DNA molecule 23 is charged, the sample 21 is charged when the immobilized DNA molecule 23 is immobilized on the surface of the substrate 22.

  Furthermore, a water tank 20 filled with pure water 25 is prepared, and the sample 21 is placed on the bottom surface of the water tank 20 so that the surface opposite to the surface on which the immobilized DNA molecules 23 are fixed is in contact with the bottom surface of the water tank 20. Put. After putting the sample 21 into the water tank 20, a plurality of charged probe particles P are suspended in the pure water 25 of the water tank 20. Thereby, the sample 21 and the probe particle P are arranged so that the probe particle P floats above the surface 21a of the sample 21 (probe particle preparation step S02). Further, the detection DNA molecules 24 may be suspended in the water tank 20 as necessary.

  Subsequently, images of the plurality of probe particles P connected by the imaging optical system 3 are picked up by the image pickup device 4 (image pickup step S03). A plurality of probe particles P having various position maps may be imaged in the probe image. The captured probe image is output from the imaging device 4 to the size specification value specifying unit 12 of the image processing device 10.

  The size specification value specifying unit 12 to which the probe image is input obtains a diameter d that is a measurement value that specifies the size of the probe particle P in the probe image captured by the imaging device 4 (size specification value specifying step). S04). When a plurality of probe particles P are imaged, the diameter d is obtained for them. The diameter size d thus obtained is output from the size specification value specifying unit 12 to the position distribution calculating unit 13.

  In the position distribution calculation unit 13 to which the diameter d is input, based on the calibration data stored in the calibration data storage unit 11 and the diameter d of the probe particle P obtained in the size specification value specifying step S04, a reference is made. The three-dimensional coordinates (x, y, z) of the probe particle P from the focal plane F, which is a plane, are calculated. The position information calculation unit 13 calculates the position distribution n (x, y, z), which is the number distribution of the probe particles P, based on the three-dimensional coordinates (x, y, z) of each probe particle P thus obtained. (Position distribution calculation step S05). The position distribution n (x, y, z) of each probe particle P thus obtained is output from the position distribution calculation unit 13 to the charge amount calculation unit 14.

  In the charge amount calculation unit 14 to which the position distribution n (x, y, z) of the probe particle P is input, the charge amount on the surface 21a of the sample 21 is calculated based on the position distribution n (x, y, z). (Charge amount calculation step S06). In the charge amount calculation unit 14, the electrostatic interaction φ (x, x, x) between the probe particle P and the surface 21 a of the sample 21 is calculated from the position distribution n (x, y, z) of the probe particle P according to Equation (3). y, z) is calculated. Further, the surface potential at the surface 21a of the sample 21 is calculated from the electrostatic interaction φ (x, y, z) by the equation (4). Further, the charge amount σ per unit area of the surface 21a of the sample 21 is calculated from the surface potential on the surface 21a by the equation (5), and the charge amount on the surface 21a is calculated.

  In the charge amount calculation apparatus 1A and the charge amount calculation method according to the present embodiment, a sample constituted by the substrate 22 on which the immobilized DNA molecules 23 are fixed when the plurality of probe particles P are suspended in the water tank 20 in this way. The amount of charge on the surface 21a of 21 is calculated.

  Next, when detecting a DNA molecule having an affinity complementary to the immobilized DNA molecule 23, for example, the calibration data preparation step S01 may be omitted because the calibration data in this measurement system has already been acquired. it can. Then, the solution in the water tank 20 is discarded, and the above-described probe particle preparation step S02 to charge amount calculation step S06 are repeated. That is, first, the sample 21 is again placed on the bottom surface, and the water tank 20 is filled with pure water 25. Thereafter, the same number of probe particles P and detection DNA molecules 24 are suspended in the water tank 20 (probe particle preparation step S02), imaging step S03, size predetermined value specifying step S04, position distribution calculating step S05, and charge amount. Calculation step S06 is executed. By repeating the probe particle preparation step S02 to the charge amount calculation step S06 in this way, various DNA molecules can be detected.

  In the surface charge amount measuring apparatus 1A and the surface charge amount measuring method according to the present embodiment, the probe particles P and the sample 21 are both charged and have an electrostatic interaction with each other. Therefore, the probe particles P are distributed under the influence of the electrostatic interaction, and the charge amount calculation unit 14 obtains the sample 21 by obtaining the position distribution n (x, y, z) of the probe particles P. It is possible to obtain the surface 21a potential of (see equations (3) and (4)). Further, the charge amount calculation unit 14 can calculate the charge amount on the surface 21a of the sample 21 based on the surface potential from the equation (5). Thus, in the surface charge amount measuring apparatus 1A and the surface charge amount measuring method according to the present embodiment, the charge amount on the surface 21 of the sample 21 is calculated from the position distribution n (x, y, z) of the probe particles P. It becomes possible.

  In addition, the surface charge amount measuring apparatus 1B and the surface charge amount measuring method according to the present embodiment that can measure the charge amount on the surface of the sample 21 are considered to be very useful in application to the biosensing field.

  That is, when the immobilized DNA molecule 23 fixed to the substrate 22 of the sample 21 and the detection DNA molecule 24 are bonded, the amount of charge on the surface 21a of the sample 21 changes due to the bonding. Along with this, the surface potential of the sample 21 at the bonding location also changes. Therefore, the probe particle P moves in a direction away from the sample 21 above the portion where the immobilized DNA molecule 23 and the detection DNA molecule 24 are bonded to each other as compared with the case where they are not bonded. Therefore, the position distribution of the probe particles P changes depending on whether or not the binding occurs, and the charge amount also changes. As a result, the amount of charge on the surface 21a of the sample 21 when only the probe particles P and the detection DNA molecules 24 are not suspended in the water tank 20, and the probe particles and the detection DNA molecules 24 are suspended in the water tank 20. The reaction can be specified by comparing the charge amount on the surface 21a of the sample 21 in the case where the sample 21 is present, and the reaction can be quantitatively analyzed.

  In particular, in the surface charge amount measuring apparatus 1B and the surface charge amount measuring method according to the present embodiment, the detection DNA molecules 24 are not suspended in the water tank 20 by repeating the probe particle preparation step S02 to the charge amount calculation step S06. In this case, the amount of charge on the surface 21a of the sample 21 and the amount of charge on the surface 21a of the sample 21 when the detection DNA molecule 24 is suspended in the water tank 20 can be measured very easily. Therefore, in the surface charge amount measuring apparatus 1A and the surface charge amount measuring method according to the present embodiment, the reaction can be specified by a very simple method and can be quantitatively analyzed.

  As described above, what is measured by the surface charge amount measuring apparatus 1A and the surface charge amount measuring method according to the present embodiment is not the electric signal (for example, potential) on the surface 21a of the sample 21, but the charge amount. . Therefore, it is possible to specify that the immobilized DNA molecule 23 and the detection DNA molecule 24 are bound on the surface 21a of the sample 21. Furthermore, it is possible to specify the amount of charge obtained by the binding reaction, thereby enabling quantitative analysis of the reaction. Therefore, the surface charge amount measuring apparatus 1A and the surface charge amount measuring method according to the present embodiment are very useful in application to the biosensing field.

  In the present embodiment, the charge amount is measured only by obtaining the position distribution n (x, y, z) of the charged probe particles P. Therefore, the surface charge amount measuring apparatus 1A and the surface charge amount measuring method according to the present embodiment can measure the charge amount easily and at low cost.

  The sample 21 is disposed in the pure water 25. However, since measurement is performed by the floating probe particles P, the measurement is not hindered by the flow even when the pure water 25 is flowing.

  Further, in the surface charge amount measuring apparatus 1A and the surface charge amount measuring method according to the present embodiment, measurement is performed using the probe particles P floating in the fluid (in this case, pure water 25), so blood It can be applied to biological substances in tissue fluid such as medium, urine, or brain. For this reason, life science research and medical measurement such as detection of trace amounts of specific genes and proteins, response evaluation of cells to chemical substances, and functional evaluation of biological tissues mainly in the brain, or food analysis such as agriculture and fishery products It is thought that it is very useful for such as. As a result, it has led to the development of a target gene / protein sensing system that is quicker and simpler than before, contributing greatly to life science research, medical diagnosis, etc., and also for high-throughput screening of new drugs and environmental pollutants Application is expected.

  Further, by changing the size of the probe particle P, for example, the charge amount in a more local region can be measured.

  Further, in each of the surface charge amount measuring apparatus 1A and the surface charge amount measuring method according to the present embodiment, the position distribution n (x, y, z) of the probe particle P is obtained as long as the diameter d of each probe particle P in the probe image is obtained. ) Can be calculated. Therefore, even if there are a plurality of probe particles P captured in the probe image, it is possible to calculate the position distribution n (x, y, z) of the plurality of probe particles P captured, and the charge It is possible to reduce the time required for measuring the quantity.

  In this embodiment, since the probe particle P is a sphere, the diameter of the probe particle P obtained from the probe image (a value that defines the size of the probe particle P) does not depend on the direction in which the probe particle P is imaged. As a result, the probe particle P can be imaged from any direction, and the charge amount can be easily measured.

  In the present embodiment, since the light source 2 that outputs the light 1 that irradiates the probe particle P is provided, the charge amount can be measured using the probe particle P that does not emit light by itself.

(Second Embodiment)
FIG. 10 is a diagram showing a configuration of a surface charge amount measuring apparatus 1B according to the second embodiment. As shown in FIG. 10, the surface charge amount measuring device 1B includes a light source 2, a water tank 20, an intensity measuring device (intensity measuring means) 6, an arithmetic processing device (arithmetic processing means) 30, and a display device (display means). 5. The surface charge amount measuring apparatus 1B measures the charge amount on the surface 21a of the sample 21 composed of the transparent substrate 22 and the immobilized DNA molecules 23 fixed on the surface of the transparent substrate 22.

  A sample 21 is placed on the bottom surface of the water tank 20. In the water tank 20, a plurality of spherical probe particles P are suspended above the surface 21 a of the sample 21. If necessary, the detection DNA molecules 24 may also be suspended in the water tank 20. For example, gold colloidal particles having a diameter of about 40 nm are used as the probe particles P. The sample 21, the detection DNA molecule 24, and the probe particle P are charged with the same sign. Further, the refractive index of the substrate 22 is higher than any of the refractive indexes of the pure water 25 and the water tank 20 in contact with the substrate 22.

Light source 2 is incident to the incident light _{l 1} obliquely to the first aspect 21b of the sample 21 that intersect the surface 21a of the sample 21. Incident light _{l 1} propagates in the substrate 22 of the sample 21 while being totally reflected at the surface opposite to the surface 21a and the surface 21a of the sample 21. That is, the substrate 22 functions as an optical waveguide with respect to the incident light l _1. Incident light l ₁ propagated through the substrate 22 is emitted as emitted light l ₂ from the second side surface 21 c of the sample 21 intersecting the surface 21 a and is incident on the intensity measuring device 6. The light source 2, the intensity of the incident light l ₁ were measured, and outputs the measured intensity to a processing unit 30.

Strength measuring device 6 has an input of output light l ₂ emitted from the substrate 22 of the sample 21. The intensity measuring device 6 measures the intensity of the outgoing light l ₂ and outputs the measured intensity to the arithmetic processing device 30.

The arithmetic processing device 30 receives the intensity of the incident light l ₁ output from the light source 2 and the intensity of the outgoing light l ₂ output from the intensity measurement device 6 as inputs. The arithmetic processing unit 30 calculates the position distribution of the probe particles P from the intensity difference between the intensity of the incident light l _{1 and} the intensity of the emitted light l ₂ , and calculates the amount of charge on the surface 21 a of the sample 21 from the position distribution. . The arithmetic processing device 30 outputs the calculated charge amount on the surface 21 a of the sample 21 to the display device 5.

  The display device 5 displays the charge amount on the surface 21 a of the sample 21 input from the arithmetic processing device 30. As the display device 5, for example, a CRT monitor, a liquid crystal display, or the like can be used.

  Next, the function of the arithmetic processing unit 30 will be described with reference to FIG. As illustrated in FIG. 11, the arithmetic processing device 30 includes a position distribution calculation unit (position distribution calculation unit) 31 and a charge amount calculation unit (charge amount calculation unit) 32.

Ｉ_１：入射光ｌ_１の強度
Ｉ_２：出射光ｌ_２の強度
ε_ｚ：試料表面（界面）からの距離zにおけるプローブ粒子の吸光係数
Ｃ：プローブ粒子の数密度（濃度）
ｌ_ｚ：全反射光の試料表面（界面）からの距離ｚにおける光路長
ｎ（ｘ、ｙ、ｚ）：試料表面（界面）からの距離ｚに分布するプローブ粒子の数
ｎ_０：全プローブ粒子数
ｎ（ｘ、ｙ、ｚ）／ｎ_０：界面からの距離zにおけるプローブ粒子の分布関数 The position distribution calculating unit 31 inputs the intensity of the incident light l ₁ from the light source 2 and the intensity of the emitted light l ₂ from the intensity measuring device 6. The position distribution calculation unit 31 calculates the position distribution n (x, y, z) of the plurality of probe particles P from Expression (6) based on the intensity difference between the intensity of the incident light l _{1 and} the intensity of the emitted light l _2. Is calculated.

I ₁ : Intensity of incident light l ₁ I ₂ : Intensity of outgoing light l ₂ ε _z : Absorption coefficient of probe particles at a distance z from the sample surface (interface) C: Number density (concentration) of probe particles
l _z : Optical path length at a distance z from the sample surface (interface) of the total reflected light n (x, y, z): Number of probe particles distributed at the distance z from the sample surface (interface) n ₀ : Total probe particles Number n (x, y, z) / n ₀ : Probe particle distribution function at a distance z from the interface

  The charge amount calculation unit 14 inputs the position distribution n (x, y, z) of the probe particles P from the position distribution calculation unit 13. Based on the position distribution of the probe particle P, the charge amount calculation unit 14 obtains an electrostatic interaction φ (x, y, z) between the probe particle P and the surface 21a of the sample 21 from Expression (3).

  The charge amount calculation unit 14 obtains the surface potential at the surface 21a of the sample 21 from the equation (4) based on the electrostatic interaction obtained from the equation (3). The charge amount calculation unit 14 further obtains the amount of charge on the surface 21a of the sample 21 from the equation (5) based on the surface potential of the sample 21 obtained from the equation (4).

  Thus, the charge amount calculation unit 14 calculates the amount of charge on the surface 21a of the sample 21, so that the amount of charge on the surface 21a of the sample 21 is measured in the surface charge amount measuring apparatus 1B.

  Next, with reference to FIG. 12, the surface charge amount measuring method using the surface charge amount measuring apparatus 1B according to the present embodiment will be described, and the operation of the surface charge amount measuring apparatus 1B will also be described. FIG. 12 is a diagram illustrating a procedure of the surface charge amount measuring method.

  First, the sample 21 is prepared by fixing the immobilized DNA molecule 23 on one surface of the substrate 22. Furthermore, a water tank 20 filled with pure water 25 is prepared, and the sample 21 is placed on the bottom surface of the water tank 20 so that the surface opposite to the surface on which the immobilized DNA molecules 23 are fixed is in contact with the bottom surface of the water tank 20. Put. After putting the sample 21 into the water tank 20, a plurality of charged probe particles P are suspended in the pure water 25 of the water tank 20. Thereby, the sample 21 and the probe particles P are arranged so that the probe particles P float above the surface 21a of the sample 21 (probe particle preparation step S12).

Next, the first to be incident obliquely on the side face 21b (light incidence step of the incident light l _1, the sample intersects the surface 21a 21 propagating in the substrate 22 of the sample 21 while being totally reflected by the surface 21a of the sample 21 S12). The refractive index of the substrate 22 is higher than that of any of the pure water 25 and the water tank 20 with which the substrate 22 is in contact. For this reason, by entering the incident light obliquely, the incident light l ₁ propagates toward the second side surface 21 c while causing total reflection on the surface of the sample 21, that is, the surface of the substrate 22.

When the incident light l ₁ causes total reflection at the surface of the substrate 22, the evanescent light is Heading immersion is outwardly from the surface thereof. The thickness of the immobilized DNA molecule 23 is extremely thin as compared with a range where evanescent light oozes out (for example, a range of about 200 nm toward the outside of the substrate 22). Further, the diameter of the probe particle P is very small, for example, about 40 nm. Therefore, the probe particles P are sufficiently distributed within the range where the evanescent light oozes out.

The probe particles P absorb the soaked evanescent light. Therefore, while causing a total reflection incident light l _1, an amount corresponding light intensity of which is absorbed by the probe particles P while propagating toward the substrate 22 to the second side surface 21c is decreased.

Subsequently, the intensity measuring device 6, a second intensity of the outgoing light l ₂ emitted from the side surface 21c of is measured (emission light intensity measuring step S13) of sample 21 that intersect the surface 21a. The intensity of the incident light l ₁ measured by the light source 2 and the intensity of the emitted light l ₂ measured by the intensity measuring device 6 are output to the arithmetic processing unit 30.

In the position distribution calculation unit 31 of the arithmetic processing unit 30 to which the intensity of the incident light l _{1 and} the intensity of the emitted light l ₂ are input, a plurality of values are calculated based on the intensity difference between the intensity of the incident light l _{1 and} the intensity of the emitted light l _2. The position distribution of the probe particles P is calculated (position distribution calculation step S14). The position distribution n (x, y, z) of the probe particle P calculated based on the equation (6) is output from the position distribution calculation unit 31 to the charge amount calculation unit 32.

  In the charge amount calculation unit 32 to which the position distribution n (x, y, z) of the probe particle P is input, the charge amount on the surface 21a of the sample 21 is calculated based on the position distribution n (x, y, z). (Charge amount calculation step S15). In the charge amount calculation unit 32, the charge amount on the surface 21 a of the sample 21 is calculated by the equations (3) to (5) based on the position distribution n (x, y, z) of the probe particles P.

  Next, for example, when a DNA molecule having an affinity complementary to the immobilized DNA molecule 23 is detected, the solution in the water tank 20 is discarded, and the above-described probe particle preparation step S11 to charge amount calculation step S15 are repeated. That is, first, the sample 21 is again placed on the bottom surface, and the water tank 20 is filled with pure water 25. Thereafter, the same number of probe particles P and detection DNA molecules 24 are suspended in the water tank 20 (probe particle preparation step S11), and the light incident step S12, the position distribution calculation step S13, and the charge amount calculation step S14 are executed. Thus, by repeating the probe particle preparation step S12 to the charge amount calculation step S16, various DNA molecules can be detected.

  In the surface charge amount measuring apparatus 1B and the surface charge amount measuring method according to the present embodiment, the probe particles P and the sample 21 are both charged and have an electrostatic interaction with each other. Therefore, the probe particles P are distributed under the influence of the electrostatic interaction, and by obtaining the position distribution n (x, y, z) of the probe particles P, the equations (3) to (5) are obtained. The amount of charge on the surface 21a of the sample 21 can be calculated. Thus, in the surface charge amount measuring apparatus 1B and the surface charge amount measuring method according to the present embodiment, the charge amount on the surface 21 of the sample 21 can be calculated from the position distribution of the probe particles P.

  In addition, the surface charge amount measuring apparatus 1B and the surface charge amount measuring method according to the present embodiment that can measure the charge amount on the surface of the sample 21 are also the surface charge amount measuring apparatus 1A and the surface charge amount measuring method according to the first embodiment. Like the method, it is thought to be very useful in biosensing applications.

  In the present embodiment, the charge amount is measured only by obtaining the position distribution n (x, y, z) of the charged probe particles P. Therefore, the surface charge amount measuring apparatus 1A and the surface charge amount measuring method according to the present embodiment can measure the charge amount easily and at low cost.

  The sample 21 is placed in the pure water 25. However, since measurement is performed by the floating probe particles P, the measurement is not hindered by the flow even when the pure water 25 is flowing.

  Further, by changing the size of the probe particle P, for example, the charge amount in a more local region can be measured. In particular, in this embodiment, a probe particle P having a very small diameter (for example, about 40 nm) is used, and the sample 21 is used as an optical waveguide, and evanescent light is used for measurement. Therefore, it is possible to detect a reaction on a very micro scale.

Further, each surface charge quantity measuring apparatus 1B and surface charge quantity measuring method according to the present embodiment, as long sought even intensity difference of the intensity of the incident light l ₁ and the intensity of the emitted light l _2, the position of the probe particles P Distribution (x, y, z) can be calculated. Therefore, even when a plurality of probe particles P are used, the time required for calculating the position distribution n (x, y, z) is very short. As a result, the time required for measuring the charge amount can be shortened.

  Here, an example for demonstrating that the probe particle P absorbs the evanescent light when the probe particle exists in a range where the evanescent light oozes is shown together with data. A thin glass substrate was used as an optical waveguide, and gold colloid particles having a diameter of 40 nm (charged to a negative charge) were used as probe particles P.

  First, FIG. 17 shows pure water in which colloidal gold particles are suspended on an untreated glass substrate (the surface is negatively charged) and an aminated glass substrate (the surface is charged positively). The graph which arrange | positioned and measured the intensity difference of the incident light and emitted light to a glass substrate is shown. Graph A in FIG. 17 represents the result of the untreated glass substrate, and graph B represents the result of the aminated glass substrate. The horizontal axis of the graph in FIG. 17 represents the absorption spectrum (λ / nm), and the vertical axis represents the intensity difference (A.U.) between the incident light and the emitted light, that is, the amount of absorption of the evanescent light. From the graph of FIG. 17, it can be seen that the untreated glass substrate has a smaller amount of absorbed evanescent light than the aminated glass substrate. This indicates that the negative charge of the untreated glass substrate surface and the negative charge of the gold colloid particles repel each other, and the gold colloid particles are difficult to approach the glass surface. Furthermore, it is understood that the aluminized glass substrate exerts an electrostatic attraction between the gold colloid particles and the gold colloid particles are likely to approach the vicinity of the glass surface.

  In FIG. 18, an untreated glass substrate (the surface is negatively charged) is placed in ion-exchange distilled water in which gold colloid particles are suspended and in a phosphate buffer in which gold colloid particles are suspended, The graph which measured the intensity difference of the incident light and emitted light to a glass substrate is shown. Graph C in FIG. 18 represents the result of placement in ion-exchanged distilled water, and graph D represents the result of placement in phosphate buffer. The horizontal axis of the graph of FIG. 18 represents the absorption spectrum (λ / nm), and the vertical axis represents the intensity difference (A.U.) between the incident light and the emitted light, that is, the amount of absorption of the evanescent light. From the glass of FIG. 18, it is understood that the colloidal gold particles are more likely to approach the glass substrate in the phosphate buffer than in the ion exchange distilled water.

  From the graph of FIG. 17, the ratio of the intensity of the emitted light to the intensity of the incident light changes reflecting the charged state of the glass substrate surface (this ratio decreases when an electrostatic attractive force acts on the probe particles and the substrate surface). ). Further, from the graph of FIG. 18, it was proved that the gold colloid particles, which are the probe particles, can easily approach the glass substrate by the solution in which the glass substrate is arranged. From these results, it is understood that the charge amount can be more easily measured by the measurement using the evanescent light using the glass substrate as the optical waveguide.

(Third embodiment)
With reference to FIG. 13, the structure of the surface charge amount measuring apparatus 1C according to the third embodiment will be described. FIG. 11 is a diagram showing a configuration of a surface charge amount measuring apparatus 1C according to the third embodiment. The surface charge amount measuring apparatus 1C according to the third embodiment relates to the first embodiment in that the substrate 22 of the sample 21 is an opaque substrate 22A and is configured as a reflection microscope for the opaque substrate 22A. Different from the surface charge measuring device 1A.

  The opaque substrate 22A is, for example, a semiconductor device or a colored glass substrate.

  The light source 2 is located obliquely above the water tank 20. The light source 2 outputs the light 1 that irradiates the sample 21 and the probe particles P so that the optical axis thereof is inclined with respect to the substrate 22A.

  On the other hand, the imaging optical system 3 is also positioned obliquely above the water tank 20. The imaging optical system 3 is disposed with an inclination with respect to the substrate 22A so as to tie an image of light reflected on the surface of the substrate 22A on the light receiving surface of the imaging device 4. Thus, the surface charge amount measuring apparatus 1C employs an ultra-illumination system. When using an ultra-illumination system, for example, there are a method using a high-power objective lens having a long working distance, a method combining a low-power objective lens with a zoom optical system, or a method using a dark-field objective lens.

  The surface charge amount calculation method using the surface charge amount calculation apparatus 1C according to the present embodiment is similar to the surface charge amount calculation method using the surface charge amount calculation apparatus 1A, as shown in FIG. It includes a preparation step S02, an imaging step S03, a size prescribed value specifying step S04, a position distribution calculating step S05, and a surface charge amount calculating step S06.

  When a coaxial (bright field) illumination system is used in a reflection type microscope, background reflection is incident on the objective lens and the image of the probe particle becomes unclear. Since the surface charge amount calculation apparatus 1C uses an extraordinary (dark field) illumination system, an image of the probe particle P can be captured even when the sample 21 composed of the opaque substrate 22 is measured. Can do. As a result, the diameter of the probe particle P in the probe image can be specified, so that the charge amount on the surface 21a of the sample 21 can be measured. Thus, the surface charge amount calculation apparatus 1C and the surface charge amount calculation method according to the present embodiment can measure the charge amount on the surface 21a even for the opaque sample 21.

  In the surface charge amount measuring apparatus 1C and the surface charge amount measuring method according to the present embodiment, the probe particles P and the sample 21 are both charged and have an electrostatic interaction with each other. Therefore, the probe particles P are distributed under the influence of the electrostatic interaction, and by obtaining the position distribution n (x, y, z) of the probe particles P, the equations (3) to (5) are obtained. The amount of charge on the surface 21a of the sample 21 can be calculated. Thus, in the surface charge amount measuring apparatus 1C and the surface charge amount measuring method according to the present embodiment, the charge amount on the surface 21 of the sample 21 can be calculated from the position distribution of the probe particles P.

  In addition, the surface charge amount measuring apparatus 1C and the surface charge amount measuring method according to the present embodiment that can measure the charge amount on the surface of the sample 21 are also the surface charge amount measuring apparatus 1A and the surface charge amount measuring method according to the first embodiment. Like the method, it is thought to be very useful in biosensing applications.

  In the present embodiment, the charge amount is measured only by obtaining the position distribution n (x, y, z) of the charged probe particles P. Therefore, the surface charge amount measuring apparatus 1C and the surface charge amount measuring method according to the present embodiment can measure the charge amount easily and at low cost.

  The sample 21 is placed in the pure water 25. However, since measurement is performed by the floating probe particles P, the measurement is not hindered by the flow even when the pure water 25 is flowing.

  Further, by changing the size of the probe particle P, for example, the charge amount in a more local region can be measured.

  In addition, since the positions of a plurality of probe particles can be calculated by one imaging, actual measurement is facilitated, and the time required for measuring the charge amount can be shortened.

  In this embodiment, since the probe particle P is a sphere, the probe particle P can be imaged from any direction, and actual measurement becomes easy. As a result, the charge amount can be easily measured.

  In the present embodiment, since the light source 2 that outputs the light 1 that irradiates the probe particle P is provided, the charge amount can be calculated using the probe particle P that does not emit light by itself.

  The preferred embodiments of the present invention have been described above, but the present invention is not limited to the above-described embodiments, and can be applied to various real-time sensing measurement systems. For example, as in the measurement system shown in FIG. 14, both the surface charge amount measuring device 1 </ b> A according to the first embodiment and the electrochemical measuring device 40 may be provided. That is, in the measurement system shown in FIG. 14, the surface charge amount measuring device 1 </ b> A measures the charge amount on the surface 21 a of the sample 21, and at the same time, the electrochemical measuring device 40 detects the electric signal generated in the sample 21. it can. The electrochemical measurement device 40 includes a reference electrode 41, a counter electrode 42, a potentiostat 43, and an electric signal display device 44. A transparent electrode layer (not shown) is formed on the substrate 22, and the transparent electrode layer functions as a working electrode of the electrochemical measuring device 40.

Alternatively, for example, as in the measurement system shown in FIG. 15, the surface charge amount measuring apparatus 1A according to the first embodiment and the semiconductor measuring apparatus 50 that measures a physical phenomenon or a chemical phenomenon may be provided. That is, in the measurement system shown in FIG. 15, the surface charge amount measuring device 1 </ b> A can measure the charge amount on the surface 21 a of the sample 21, and at the same time, the semiconductor measuring device 50 can detect the electrical signal generated in the sample 21. The semiconductor measurement device 50 includes a reference electrode 61, a semiconductor device SC, and an electric signal display device 62. The semiconductor device SC includes an Si substrate 51 having an n ⁺ layer 51a, an SiO ₂ layer 52, an Si ₃ N ₄ layer 53, a Poly-Si layer 54, an Si ₃ N ₄ / SiO ₂ layer 55, and an Al conductor. A portion 56 and an Au / Ti layer 57 are included. In addition, the semiconductor device SC includes an extended gate type (extended gate type) electrode portion 63. The electrode part 63 of the semiconductor device SC is formed as a transparent electrode layer formed on the substrate 22. The electrode part 63 is connected to the Au / Ti layer 57 by a conducting wire.

  With reference to FIG. 16, the measurement operation of the semiconductor device SC of the semiconductor measurement device 50 will be described. FIG. 16 is a diagram for explaining the measurement operation of the semiconductor device SC of the semiconductor measurement device 50. The semiconductor device SC functionally includes a charge supply unit 71, a charge injection adjustment unit 72, a sensing unit (charge conversion unit) 73, a barrier unit 74, a floating diffusion unit 75, a reset gate 76, and a reset drain 77. In measurement, a pulse voltage is applied to the charge supply unit 71, the barrier unit 74, and the reset gate 76, and a DC voltage is applied to the other electrodes excluding the floating diffusion unit 75.

  First, as shown in FIG. 16A, the potential of the charge supply unit 71 is set high. Next, as shown in FIG. 16B, the potential of the charge supply unit 71 is lowered so that the charge 78 is injected into the sensing unit 73. Subsequently, as shown in FIG. 16C, the potential of the charge supply unit 71 is set high again. As a result, the charge 78 is removed by the charge injection adjusting unit 72 and accumulated in the sensing unit 73.

  Subsequently, as shown in FIG. 16D, the potential of the barrier portion 74 is increased. As a result, the charge 78 stored in the sensing unit 73 is transferred to the floating diffusion unit 75. After all the charges 78 accumulated in the sensing unit 73 are transferred to the floating diffusion unit 75, as shown in FIG. 16E, the potential of the barrier unit 74 is lowered to stop the transfer of the charges 78. The potential of the floating diffusion portion 75 is determined by the amount of charge 78 transferred, and measurement is performed using this potential as an output.

  Thereafter, as shown in FIG. 16 (f), the reset gate 76 is turned on, reset to the potential of the reset drain 77, and the process returns to FIG. 16 (a). By repeating the operations shown in FIGS. 16A to 16F, electric charges are output to the outside and measurement is performed.

  The present invention is not limited to the above-described embodiments and application examples, and various modifications can be made. For example, in the above-described embodiment, the case where the present invention is applied to a DNA reaction that is a biological reaction is illustrated, but the present invention can be applied to various fields. For example, it can be applied to detection of biomolecules such as proteins, saccharides and the like as targets by using antibodies, receptors, and binding proteins, other than DNA reactions. Furthermore, the present invention is not limited to biological reactions, and may be applied to chemical reactions, for example.

  The shape of the probe particle P is not limited to a spherical shape, and may be, for example, a rod shape. The value that defines the size of the probe particle P in the probe image when the rod-shaped probe particle is used is the length in the short direction at both ends in the longitudinal direction of the probe particle. That is, for example, when the lengths in the short direction at both ends of the probe particles in the longitudinal direction are the same in the probe image, the rod-like probe particles are distributed so that the longitudinal direction and the sample surface are parallel. ing. On the other hand, when the lengths in the short direction at both ends in the longitudinal direction of the probe particles are different in the probe image, the rod-like probe particles are distributed with the longitudinal direction having an angle with respect to the sample surface. Therefore, the inclination of the probe particle P with respect to the sample surface can be obtained as positional information of the probe particle P with respect to the sample surface by obtaining the length in the short direction at both ends in the longitudinal direction of the probe particle in the probe image. The inclination of the rod-like probe particle P is determined by being organically related to the sample surface. Therefore, the surface potential can be calculated based on the inclination of the probe particle P with respect to the sample surface.

  The probe particles P are not limited to polystyrene latex or gold, but may be made of other materials. The probe particles are not limited to liquids, and may be suspended in a gas or in a vacuum, for example.

  In addition, for example, the probe particle P itself may emit fluorescence, and an image thereof may be captured to measure the amount of charge on the surface of the sample 21 without having a light source. The light source is not limited and may be a light source that emits light of any wavelength, such as visible light, ultraviolet light, infrared light, X-rays, electron beams, or neutron beams.

It is a figure which shows the structure of the surface charge amount calculation apparatus which concerns on 1st Embodiment. It is a figure for demonstrating the function of an image process part. It is a figure of the graph obtained by calibration data. A table of calibration data when actual measurement data is used as calibration data is shown. It is a figure for demonstrating the relationship between probe particle | grains and the focal plane of an imaging optical system. It is a figure showing the image of the probe particle in the imaged probe image. It is a figure of the graph which shows the intensity | strength of each probe particle in a probe image. It is a figure which shows the procedure of the surface charge amount calculation method which concerns on 1st Embodiment. It is a figure for demonstrating calculating | requiring calibration data. It is a figure which shows the structure of the surface charge amount calculation apparatus which concerns on 2nd Embodiment. It is a figure for demonstrating the function of an arithmetic processing part. It is a figure which shows the procedure of the surface charge amount calculation method which concerns on 2nd Embodiment. It is a figure which shows the structure of the surface charge amount calculation apparatus which concerns on 3rd Embodiment. It is a figure which shows the structure of the measurement system which applied the surface charge amount calculation apparatus which concerns on 1st Embodiment. It is a figure which shows the structure of the measurement system which applied the surface charge amount calculation apparatus which concerns on 1st Embodiment. It is a figure for demonstrating the measurement operation | movement of the semiconductor device of a semiconductor measuring device. The graph which measured the intensity difference of the incident light to a glass substrate and the emitted light from a glass substrate is shown. The graph which measured the intensity difference of the incident light to a glass substrate and the emitted light from a glass substrate is shown.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1A, 1B, 1C ... Surface charge amount measuring device, P ... Probe particle, 2 ... Light source, 3 ... Imaging optical system, 4 ... Imaging device, 5 ... Display device, 6 ... Intensity measuring device, 10 ... Image processing device, DESCRIPTION OF SYMBOLS 11 ... Calibration data storage part, 12 ... Size prescribed value specific | specification part, 13, 31 ... Position distribution calculation part, 14, 32 ... Charge amount calculation part, 30 ... Operation processing apparatus, 40 ... Electrochemical measurement apparatus, 41 ... Reference electrode, 42 ... Counter electrode, 43 ... Potentiostat, 44, 62 ... Electric signal display device, 50 ... Semiconductor measuring device, SC ... Semiconductor device, 61 ... Reference electrode

Claims (10)

A surface charge measurement device that measures the amount of charge on the surface of a charged sample by floating a plurality of charged probe particles upward,
Position distribution calculating means for calculating a position distribution of the plurality of probe particles;
A surface charge amount measuring device comprising: a charge amount calculating means for calculating a charge amount on the surface of the sample based on the position distribution. The surface charge amount measuring device according to claim 1, wherein the charge amount on the surface of the sample is measured based on a probe image obtained by capturing an image of the probe particles.
A size defining value specifying means for obtaining a measurement value that defines the size of the probe particle in the probe image obtained by capturing an image of the probe particle;
The position distribution calculating unit is obtained by calibration data indicating a relationship between a value defining the size of the probe particle in the probe image and a distance of the probe particle from a reference plane, and the size defining value specifying unit. A surface charge amount measuring apparatus that calculates the position distribution of the plurality of probe particles based on the measured value. An intensity measuring means for measuring the intensity of light is further provided, and the amount of charge on the surface of the sample is measured by causing incident light to enter the first side surface of the sample that intersects the surface obliquely. A surface charge measuring device according to claim
The incident light is incident on the first side surface so as to be emitted as emitted light from the second side surface of the sample intersecting the surface after propagating through the sample while being totally reflected on the sample surface. And
The surface charge amount measuring device, wherein the position distribution calculating unit calculates the position distribution of the plurality of probe particles based on an intensity difference between the intensity of the incident light and the intensity of the emitted light.   The surface charge amount measuring apparatus according to claim 1, wherein the probe particle is a sphere.   The surface charge amount measuring device according to claim 1, wherein the sample is charged by fixing a charged substance on the surface. A surface charge measurement method for measuring a charge amount on the surface of a charged sample by floating a plurality of charged probe particles upward,
A position distribution calculating step for calculating a position distribution of the plurality of probe particles;
And a charge amount calculating step of calculating a charge amount on the surface of the sample based on the position distribution. A calibration data preparation step of preparing calibration data indicating a relationship between a value defining a size of the probe particle in the probe image and a distance of the probe particle from a reference plane;
A size defining value specifying step for obtaining a measurement value that defines the size of the probe particle in the probe image obtained by capturing an image of the probe particle, and
The position distribution calculation step calculates the position distribution of the plurality of probe particles based on the calibration data and the measured value obtained by the size specification value specifying means. Method for measuring surface charge. A light incident step in which incident light having a measured intensity propagating in the sample while being totally reflected by the sample surface is incident obliquely on a first side surface of the sample intersecting the surface;
An emitted light intensity measuring step for measuring the intensity of the emitted light emitted from the second side surface of the sample intersecting with the surface after being incident as the incident light and propagating through the sample; and
7. The surface charge amount according to claim 6, wherein the position distribution calculating step calculates a position distribution of the plurality of probe particles based on an intensity difference between the intensity of the incident light and the intensity of the emitted light. Measurement method.   The surface charge amount measuring method according to any one of claims 6 to 8, wherein the probe particle is a sphere.   The surface charge amount measuring method according to claim 6, wherein the sample is charged by fixing a charged substance on the surface.

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