JP3468091B2 - Biochemical measurement device - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a biochemical measuring device for measuring a biochemical reaction by applying a surface plasmon resonance phenomenon.

[0002]

2. Description of the Related Art Surface plasmon resonance (Surface Plasmon Resonance) has recently been used as a method for detecting the amount of physicochemical change accompanying the progress of biochemical reaction.
e, hereinafter abbreviated as SPR. ) A method that applies the phenomenon is being used. In this SPR phenomenon, a metal thin film is formed on the surface of a light transmissive medium such as glass, and the surface of the metal thin film opposite to the light transmissive medium is in contact with the sample.
In an optical system that allows a light beam to pass through an interface with a metal thin film through a light-transmissive medium, the peculiarity that occurs when the light entering the metal thin film through the light-transmissive medium has a specific incident angle It is a phenomenon.

In the above optical system, when a light beam is incident on the metal thin film at an angle of incidence equal to or larger than the total reflection angle, an evanescent wave traveling along the interface between the metal thin film and the light transmissive medium is generated, and this evanescent wave is generated. A surface plasmon wave is excited at the interface between the metal thin film and the sample. When the evanescent wave and the surface plasmon wave resonate, the energy of the incident light beam is absorbed by the metal thin film as the excitation energy of the surface plasmon wave, and the light amount of the reflected light is attenuated. The incident angle at which the above-mentioned SPR phenomenon occurs depends on the density of the medium such as the sample solution in contact with the metal thin film.

On the other hand, the presence or absence of the SPR phenomenon and the SPR phenomenon are generated by detecting the reflection angle and the light amount of the reflected light reflected from this optical system and determining the reflection angle when the light amount specifically decreases. It is possible to find the incident angle when there is. Therefore, the density of the medium can be obtained as a result by detecting the reflection angle and the light amount of the reflected light.

Since the measuring method using the SPR phenomenon can measure a very small amount, it is also applied to the biochemical field dealing with a minute amount that cannot be measured by a general measuring method. According to this method, it is possible to measure a very small amount and to know the progress of the biochemical reaction in real time.

[0006]

By the way, also in the field of drug discovery screening for producing a new drug, it is required to accurately and promptly evaluate the above-mentioned biochemical reaction in a large number of samples. However, conventional SP
The SPR sensor that applies the R phenomenon measures each sample individually. Therefore, when performing tests on many samples at the same time using a microtiter plate, etc., the microtiter plate is used. It was necessary to dispense one sample from each of the many sample holes in the sample container of the SPR sensor. In general, drug discovery screening requires enormous amounts of tests and analyzes targeting a large number of samples. Therefore, if an SPR sensor is to be used in this field, it will be extremely troublesome as described above. However, there was a problem that the efficiency of the measurement work was poor.

Therefore, an object of the present invention is to provide a biochemical measuring device capable of efficiently measuring a biochemical reaction for a large number of samples.

[0008]

According to a first aspect of the present invention, there is provided a biochemical measuring device comprising a sample container provided with a plurality of sample holes for accommodating a liquid sample to be biochemically measured, and an array of the sample holes. A sensor plate of a light-transmissive material provided with a convex portion that fits into the sample hole at a corresponding position, and a thin film made of a metal and / or a semiconductor formed on the surface of the convex portion,
A prism integrally provided with the sensor plate on the surface opposite to the convex portion, a light source unit for irradiating the prism with a light beam, and an incident light on the interface between the sensor plate and the thin film through the prism. Then, a light detecting means for detecting the light quantity of the reflected light of the light beam reflected by the interface is provided.

The biochemical measuring apparatus according to claim 2 is the biochemical measuring apparatus according to claim 1. The sample container was equipped with a temperature adjusting means for maintaining the temperature of the sample container within a predetermined range and a stirring means for uniformly stirring the sample in the sample hole.

According to the present invention, a plurality of sensor plates made of a light-transmitting material having a convex portion having a metal thin film formed on the surface at a position corresponding to a sample hole and having a prism on the reflecting surface of the convex portion are provided. It is attached to a sample container with a sample hole, and the biochemical measurement applying the surface plasmon resonance phenomenon is performed by detecting the reflection angle and the light amount of the reflected light of the light beam incident on this convex part through the prism. Can be efficiently performed on a plurality of samples.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Next, embodiments of the present invention will be described with reference to the drawings. 1 is a side view of a biochemical measuring device according to an embodiment of the present invention, FIGS. 2 and 3 are partial perspective views of the biochemical measuring device, and FIGS. 4 and 5 are partial cross-sectional views of the biochemical measuring device. Is.

First, the structure of the biochemical measuring device will be described with reference to FIG. In FIG. 1, a microtiter plate 2 as a sample container provided with wells 1 that are a plurality of sample holes is placed on a holder 4. A liquid sample 3 is stored in the well 1. The holder 4 is equipped with a temperature control heater 5 to hold the sample 3 in the well 1 at a predetermined temperature. Further, the holder 4 is the swing unit 6
The microtiter plate 2 is rocked by driving the rocking unit 6 to stir the sample in the well 1 to homogenize the composition.

On the microtiter plate 2, a sensor plate 7 made of glass, which is a light transmissive material, is mounted. A convex portion 8 is provided on the lower surface of the sensor plate 7 at a position corresponding to the well 1, and a prism 9 is integrally provided with the sensor plate 7 on the opposite surface. With the sensor plate 7 mounted on the microtiter plate 2, the convex portion 8 is fitted into the well 1 and the lower end portion thereof is immersed in the sample 3 in the well 1.

Above the sensor plate 7, a photodetector 10 is provided.
Is provided. The light detection unit 10 includes a line laser 11 as a light source unit and a PSD (position detection element) unit 1.
Equipped with 2. The laser light projected from the line laser 11 enters the prism 9, is totally reflected by the interface at the lower end, and enters the PSD unit 12. PSD unit 12
Receives the reflected light, and the measuring unit 13 measures the reflection angle and the light amount of the reflected light based on the received light data. That is, the PSD unit 12 and the measuring unit 13 serve as light detecting means. The light detection unit 10 can be moved in the horizontal direction by a feed screw 15 that is rotationally driven by a motor 14.

Next, the sensor plate 7 used for measuring the biochemical reaction will be described with reference to FIGS. 2, 3 and 5. As shown in FIG. 2, the convex portions 8 are provided on the lower surface of the sensor plate 7 corresponding to the positions of the wells 1 provided in the microtiter plate 2 in a grid pattern.
The prisms 9 provided on the opposite surface are formed so as to be located on the row connecting the convex portions 8. A metal thin film 8 a is formed on the lower surface of the convex portion 8. Sensor plate 7 on thin film 8a
When the laser light is transmitted through the laser light and is incident, the laser light is totally reflected according to a geometrical total reflection condition. The thin film 8
In addition to metal, a may be a semiconductor or a combination of metal and semiconductor.

Further, as shown in FIG. 3, the line laser 11
Has a length corresponding to the length of the prism 9 on the sensor plate 7, and the laser light emitted from the line laser 11 as a band-shaped light beam is obliquely directed to the prism 9 over the entire length of the prism 9. Enter into. The direction of this incident light is variable within a predetermined angle range by an optical system (not shown) (see range a shown in FIG. 4). As shown in FIG. 4, of the incident light that is incident on the prism 9 and refracted on the surface, the incident light to the position where the convex portion 8 exists is the thin film 8
Incident angle θ that satisfies the geometrical total reflection condition at the interface with a
Light comes in at 1. The reflected light reflected by this interface is refracted again on the surface of the prism 9 and then received by each PSD 12a of the PSD unit 12.

Here, the SPR phenomenon that occurs specifically when the above laser light is incident at a specific incident angle will be described. As described above, the beam light incident on the interface between the thin film 8a and the sensor plate 7 is totally reflected if the incident angle is equal to or greater than the critical angle of the total reflection condition, but at that time, a light wave is slightly emitted from the interface into the thin film 8a. An evanescent wave that penetrates into the thin film 8a and travels along the interface with the thin film 8a is generated. Then, the evanescent wave excites a surface plasmon wave, which is a dense and dense vibration of free electrons on the surface of the thin film 8a, at the interface between the thin film 8a and the sample 3.

When the evanescent wave and the surface plasmon wave are in a resonance state, the energy of the light beam is transferred to the excitation energy of the surface plasmon wave, so that the light energy of the reflected light is specifically attenuated. Then, there is a specific relationship between the wave number of the surface plasmon wave obtained by the incident angle when the SPR phenomenon occurs and the dielectric constant of the sample 3. Further, if the dielectric constant of the sample 3 is obtained, the concentration of the substance to be measured in the sample 3 can be obtained by a predetermined calibration curve or the like. That is, the concentration of a specific substance to be measured in the sample can be obtained by obtaining the incident angle when the SPR phenomenon occurs based on these relationships.

As shown in FIG. 3, the PSD unit 12 corresponds to the length of the prism 9 similarly to the line laser 11, and at the position corresponding to the convex portion 8 of the sensor plate 7,
Each PSD 12a is arranged. PSD12a
Changes the incident angle by changing the PSD unit 12
Direction orthogonal to the length direction (direction of arrow b in FIG. 4)
The position of the reflected light moving to and the amount of the reflected light are measured. Therefore, by measuring the light receiving amount and the light receiving position of the PSD 12a by the measuring unit 13, the concentration of the specific substance to be measured in the sample 3 can be obtained by applying the SPR phenomenon as described above.

The photodetector 10 is moved to move the line laser 1
When a light beam is irradiated from 1 to a specific prism 9, PS
The D unit 12 simultaneously receives reflected light from a plurality of convex portions 8 fitted in the wells 1 arranged in rows, and the PSDs 12a corresponding to the respective convex portions 8a are reflected at different reflection angles. Received reflected light. Therefore, the measurement unit 13 simultaneously measures different reflection angles and light amounts of the reflected light beams that are totally reflected from the respective convex portions 8a of the row irradiated with the light beam. That is, the PDS unit 12 can simultaneously perform the biochemical measurement of the samples 3 in the plurality of wells 1 in one row. In addition to the method of using the line laser 11 and the PSD unit 12 in combination, an independent laser light source and PSD are used for the well 1.
It may be a method of sequentially moving along the column.

This biochemical measuring device is configured as described above, and the measuring method will be described below. Here, sample 3
A case of measuring the progress of the antibody-antigen reaction in the inside will be described as an example. First, in FIG. 1, each well 1 of the microtiter plate 2 contains a sample 3 to be measured. Prior to measuring the biochemical reaction, the microtiter plate 2 is attached to the holder 4. Then, the temperature control heater 5 and the swing unit 6 are driven to keep the sample 3 at a predetermined temperature and the sample 3 in the well 1 is swung and stirred to mount the sensor plate 7 on the microtiter plate 2. . Thereby, the convex portion 8 of the sensor plate 7
The lower surface of is immersed in the sample 3 and becomes ready for measurement.

As shown in FIG. 5, the lower surface of the thin film 8a formed on the lower surface of the convex portion 8 is pre-coated with dextran 20 which is a gel-like polymer substance to fix the antibody 21 in the sample 3. Thus, the antibody 21 is attached to the surface of the dextran 20 by immersing the convex portion 8 a in the sample 3 in the well 1. Then, as the biochemical reaction proceeds, the amount of the antigen 22 that reacts with the antibody 21 increases, and as a result, the amount of the substance to be measured attached to the thin film 8a increases.

The amount of the object to be measured, which increases with the progress of this biochemical reaction, is measured by the following method. As shown in FIG. 4, laser light is projected onto the prism 9 by the line laser 11 and the convex portions 8 corresponding to the plurality of wells 1 are formed.
A light beam is incident on each. At this time, the incident angle θ
PSD 12 corresponding to each convex portion 8 while changing 1
The amount of reflected light and the reflection angle θ2 are continuously detected by a. Then, the reflection angle θ2 at which the amount of reflected light is specifically minimized, that is, the reflection angle θ2 at which the SPR phenomenon occurs is obtained.

As a result, since the normal incident angle and the reflection angle are the same, the incident angle θ1 when the SPR phenomenon occurs in each convex portion 8 can be specified. Then, by comparing these incident angles θ1 with a predetermined calibration curve, the amounts of the antibody 21 and the antigen 22 attached to the dextran 20 of the thin film 8a immersed in the sample 3 in each well 1, that is, the amount of the biochemical reaction It is possible to measure the amount of the specific measurement target substance generated as a result of the progress. Although the present embodiment shows an example of measuring the reflection angle when the SPR phenomenon is occurring and obtaining the incident angle from this reflection angle, the incident angle is changed without measuring the reflection angle. However, the incident angle when the SPR phenomenon occurs may be directly obtained by measuring the amount of reflected light.

Then, the photodetection section 10 is moved by driving the motor 14, and the above-mentioned measurement operation is performed for all rows of the wells 1 of the microtiter plate 2 to measure one microtiter plate 2. finish. At this time, since it is not necessary to interrupt the biochemical reaction in the well 1 by the dispensing operation in order to perform the measurement operation, the reaction state can be measured while the biochemical reaction is continuously progressing. Real-time measurement is possible. At this time, various operations for the secondary reaction required in the conventional method for measuring the biochemical reaction, that is, a complicated procedure for visualizing the production result of the biochemical reaction by using the coloring, fluorescence, and luminescence by adding the substrate No action needed.

In the measurement of biochemical reaction applying the SPR phenomenon, a convex portion 8 and a prism 9 for measurement corresponding to a large number of wells 1 are provided on one sensor plate 7, and this sensor plate 7 is attached to the microtiter plate 2. By mounting, it is possible to efficiently perform the measurement on the samples in a large number of wells 1 at the same time, while the conventional measurement can be performed on only one sample.

[0027]

According to the present invention, there is provided a sensor plate made of a light transmitting material having a convex portion having a metal thin film formed on its surface at a position corresponding to a sample hole, and a prism provided on the reflecting surface of the convex portion. , The sample was equipped with multiple sample holes, and the prism was transmitted through the prism to detect the reflection angle and quantity of the reflected light of the light beam incident on this convex part, so the surface plasmon resonance phenomenon was applied. Biochemical measurement for small amount of
It can be efficiently performed on many samples of the microtiter plate at the same time. In addition, since it is not necessary to interrupt the biochemical reaction by a dispensing operation, real-time measurement is possible without the need for performing a visualization reaction that requires a complicated operation while the biochemical reaction is continuously progressing. .

[Brief description of drawings]

FIG. 1 is a side view of a biochemical measuring device according to an embodiment of the present invention.

FIG. 2 is a partial perspective view of a biochemical measuring device according to an embodiment of the present invention.

FIG. 3 is a partial perspective view of a biochemical measuring device according to an embodiment of the present invention.

FIG. 4 is a partial cross-sectional view of the biochemical measuring device according to the embodiment of the present invention.

FIG. 5 is a partial cross-sectional view of the biochemical measuring device according to the embodiment of the present invention.

[Explanation of symbols]

1 well 2 microtiter plate 3 samples 7 sensor plate 8 convex 9 prism 10 Photodetector 11 line laser 12 PSD unit 13 Measuring section 21 antibody 22 Source

Front page continued (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 21/00-21/61 Practical file (PATOLIS) Patent file (PATOLIS) ECLA

Claims (2)

(57) [Claims] 1. A sample container provided with a plurality of sample holes for accommodating a liquid sample to be subjected to biochemical measurement, and a convex portion fitted into the sample holes at a position corresponding to the arrangement of the sample holes. And a thin film made of metal and / or semiconductor formed on the surface of the convex portion, a prism integrally provided with the sensor plate on the opposite surface of the convex portion, and the prism A light detecting unit for irradiating a light beam with respect to a light source unit, which is incident on the interface between the sensor plate and the thin film through the prism, and detects the amount of reflected light of the light beam reflected at the interface. A biochemical measuring device characterized by comprising: 2. The biochemical measuring device according to claim 1, further comprising a temperature adjusting means for holding the temperature of the sample container within a predetermined range and an agitating means for uniformly agitating the sample in the sample hole. .