JP2001242071A - Surface plasmon resonance angle microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a surface plasmon resonance microscope always irradiating parallel light to a glass substrate for accurately detecting a resonance angle to a plane and widening a light turning angle range for precisely detecting a resonance angle of a various kinds of body to be detected. SOLUTION: A semicylindrical prism with a dimension sufficient for covering the whole of a microscopic area of a metal thin film at least is tightly abutted to the glass substrate on the opposite side to the metal thin film. A light emitting device, which emits light beams so that light from a light source becomes parallel light flux over the whole of the microscopic area toward the light incident side of the semicylindrical prism inside the semicylindrical prism, is supported rotationally within a predetermined angle along the outer circumferential face on the light incident side of the semicylindrical prism. A light receiving device receiving the parallel light flux reflected in the microscopic area on the metal thin film and passed through the semicylindrical prism so as to output an electric signal complying with the reflected light intensity from the whole detection area is supported synchronously rotationally in the opposite direction to the turning direction of the light emitting device along the outer circumferential face of the light outgoing side of the semicylindrical prism. A resonance angle of the whole sample in the microscopic area can be detected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a surface plasmon resonance angle microscope (hereinafter, referred to as an SPR microscope).

[0002]

For example, a surface plasmon resonance angle detection method (hereinafter referred to as SPR method) is known as a method for detecting characteristics of proteins, such as cells, viruses, bacteria, etc., sugars, enzymes and DNA. .

In the SPR method, a glass substrate on which a metal thin film to which an object to be detected is fixed is formed is irradiated with a laser beam through a prism at a predetermined angular width, and the intensity of light reflected from the glass substrate is reduced. The characteristics of the object to be detected are detected based on the incident angle (reflection angle) of the laser beam which is minimum, and thus the absorbance to the object is maximum.

In recent years, as an application of this SPR method, while irradiating parallel light having a width over the entire detection area of a predetermined area on a glass substrate, the irradiation angle of light on the prism is changed to change the resonance angle of each detector in the entire detection area. SP for detecting the characteristic of the object fixed to the detection area by detecting
An R microscope has been proposed.

However, since a triangular prism is used as a prism in the SPR method, when a similar triangular prism is used in an SPR microscope, a light source is rotated on an arc along the outer periphery of the triangular prism. In this case, the angle of incidence of light on the prism surface is constantly changed, and the center of the light irradiated on the detection area is largely shifted, and the light reflected from the detection area is received by the light-receiving member which rotates synchronously in the opposite direction to the light source. Could not.

This disadvantage can be solved to some extent by varying the angle of incidence of the laser beam on the prism with a small angle width around the average resonance angle. Only mirror data is available,
The specimen fixed in the detection area could not be correctly examined.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned conventional drawbacks. The object of the present invention is to change the parallel light irradiated on the detection area at a large angle and fix it on the detection area. S that can accurately observe the sample
An object of the present invention is to provide a PR microscope.

[0008]

SUMMARY OF THE INVENTION The present invention illuminates a predetermined speculum area of a metal thin film formed on a glass substrate with light, and minimizes the intensity of light reflected from the detection area in the metal thin film. In a surface plasmon resonance angle microscope for microscopically examining a sample characteristic based on a resonance angle, a semi-cylindrical prism that is in close contact with a glass substrate opposite to the metal thin film and has a size that covers at least the entire microscopic region of the metal thin film; It is supported rotatably at a predetermined angle along the outer peripheral surface on the light incident side of the cylindrical prism, and the light from the light source is applied to the light incident side of the semi-cylindrical prism in the semi-cylindrical prism over the entire inspection area. A light irradiating device for irradiating light so as to form a parallel light beam, and a metal foil film supported so as to be synchronously rotatable in a direction opposite to the rotation direction of the light irradiating device along the outer peripheral surface on the light emitting side of the semi-cylindrical prism; Speculum area in A light receiving device that receives the parallel light beam reflected from the semi-cylindrical prism and outputs an electric signal according to the intensity of the reflected light from the entire detection area, and can inspect the resonance angle of the sample in the microscopic area It is characterized by the following.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiments of the present invention will be described below with reference to the drawings. 1 is an explanatory front view schematically showing an SPR microscope, FIG. 2 is an explanatory plan view showing an irradiation state of light on a detection area, and FIG. 3 is an explanatory sectional view.

A metal thin film (not shown) such as a gold thin film or a silver thin film is formed on the upper surface of a glass substrate 5 constituting the sensor chip 3 of the SPR microscope 1, and the metal thin film has a flat rectangular shape of a predetermined size. (Indicated by a broken line in FIG. 2) is set. An antibody or a reagent (not shown) corresponding to a sample such as a cell, a bacterium, or a virus to be fixed is adsorbed in advance on the metal thin film in the detection region 5a. The glass substrate 5 is prepared in advance for each type of antibody or reagent to be adsorbed, and is appropriately replaced according to the type of sample to be detected.

A cell block 7 constituting a part of the sensor chip 3 is adhered to the glass substrate 5 on the metal thin film side via a matching oil, a silicon rubber sheet, an O-ring or the like. On the lower surface of the cell block 7 facing the metal thin film, a flow cell 9 is formed with a size corresponding to the detection area 5a and a depth of about 100 μm.

The cell block 7 located on the supply side of the flow cell 9 has first and second supply channels 11 and 13, and the cell block 7 located on the discharge side of the flow cell 9 has a discharge channel 15. It is formed so as to communicate with the inside.

The first supply channel 11 supplies a sample solution fixed on a metal thin film, for example, containing cells, bacteria and viruses into the flow cell 9. In addition, the second supply channel 13 is used to supply a reactant solution containing reactants such as proteins, sugars, enzymes, and DNA bound to a sample of cells, bacteria, viruses, and the like immobilized on the metal thin film in the flow cell 9. To supply. Further, the discharge channel 15 collects the excess sample solution and the reactant solution in the flow cell 9.

On the lower surface of the glass substrate 5 opposite to the metal thin film, a semi-cylindrical prism 17 having a diameter equal to or greater than the width of the glass substrate 5 in the longitudinal direction and having an axial length equal to or greater than the width of the detection area 5a is provided. , A silicone rubber sheet, an O-ring and the like.

A light irradiating device 19 is provided on the left outer peripheral side of the semi-cylindrical prism 17 and a light receiving device 21 is provided on the right outer peripheral side of the semi-cylindrical prism 17 along the outer peripheral surface of the semi-cylindrical prism 17. It is provided so as to be synchronously rotated in the same direction at the same angular width.

The light irradiation device 19 includes, for example, a red laser beam,
A light source 23 for outputting light such as green laser light, a first lens 25 for condensing light from the light source 23, and a light condensing position of the first lens 25 for passing laser light with a predetermined beam diameter A pinhole member 27 having a pinhole, a second lens 29 for converting light passing through the pinhole member 27 into parallel light, and converting the light transmitted through the second lens 29 into at least the region length and axis width of the detection region 5a. A semi-cylindrical lens 31 which is formed into a light beam which coincides with and is incident on the semi-cylindrical prism 17
It is composed of The light beam incident on the semi-cylindrical prism 17 is refracted by the refractive index of the semi-cylindrical prism 17 so as to always become a parallel light beam that can be irradiated over the entire detection area 5a.

A slit plate 3 having a slit 33a having a length equal to the axial width of the detection area 5a is provided in the optical path from the semi-cylindrical lens 31 to the semi-cylindrical prism 17.
3 are arranged to block the influence of disturbance light.

The light receiving device 21 has a semi-cylindrical lens 35 for condensing a light beam that matches the area length and the axial width of the detection area 5a reflected from the glass substrate 5, and converts the light from the semi-cylindrical lens 35 into parallel light. The third lens 37 and the third lens 3
CCD which receives parallel light from 7 and outputs an electric signal;
And a light receiving member 39 such as a photodiode array.

Next, the microscopic action of the SPR microscope 1 will be described. FIG. 4 is an explanatory diagram showing a variable irradiation state of light to the detection area.

The case where the state of binding and dissociation of the reactant with respect to the sample fixed to the detection area 5a is examined by a microscope will be described as an example.
The light receiving device 21 is waiting at a position of, for example, 215 ° and a position of 325 ° with respect to the outer peripheral surface of the light emitting device.

When laser light is output from the light source 23 in this state, the laser light passes through the first lens 25, the pinhole member 27, the second lens 29, and the semi-cylindrical lens 31 and then passes through the semi-cylindrical prism 17 Is formed into a parallel light flux having a length and a width over the entire area of the detection area 5a, and is applied to the boundary between the glass substrate 5 and the metal foil film corresponding to the entire area of the detection area 5a. The parallel light flux of the laser light reflected from the boundary between the glass substrate 5 and the metal thin film corresponding to the detection area 5a is formed by the semi-cylindrical lens 35 and the third lens 37 into parallel light over the entire area of the detection area 5a and received. The light is received by the member 39.

In this state, a sample solution containing, for example, cells as a sample is supplied from the first supply channel 11 into the flow cell 9, and the sample solution overflowing from the flow cell 9 is discharged through the discharge channel 15. While collecting, the light irradiation device 19 is moved from the 215 ° position to the 260 ° position along the outer peripheral surface of the semi-cylindrical prism 17 and the light receiving device 2
1 is synchronously rotated from the 325 ° position to the 280 ° position in a direction opposite to each other with a rotation width of 45 °.

As a result, the parallel luminous flux applied to the entire area of the detection area 5a is received by the light receiving device 21 while changing the reflection angle with respect to the detection area 5a, so that the cells are fixed to the entire area of the detection area 5a. Is detected.

If the resonance angle detected by the reflected light from the detection area 5a fluctuates during this detection operation, the sample is not completely fixed to the antibody or reagent adsorbed on the detection area 5a. Therefore, the supply of the sample solution from the first supply channel 11 is continued. On the contrary, when the resonance angle detected by the reflected light from the detection area 5a is substantially constant, it is determined that the sample is almost fixed to the antibody or the reagent in the detection area 5a, and the supply of the sample solution is interrupted.

In order to observe the binding state of the reactant to the fixed sample, the reactant solution is supplied from the second supply channel 11 into the flow cell 9 with the sample fixed in the entire detection area 5a. The light irradiating device 19 and the light receiving device 21 are respectively supplied along the outer peripheral surface of the semi-cylindrical prism 17 while supplying and collecting the reactant solution overflowing from the flow cell 9 through the discharge channel 15 in the same manner as described above. Synchronously rotate in opposite directions from the initial position.

In this operation, if the binding of the reactant to the sample fixed in the entire area of the detection area 5a is incomplete, the resonance detected with the synchronous rotation of the light irradiation device 19 and the light receiving device 21 When the bond between the sample and the reactant changes, the absorbance (resonance angle) does not become constant. Conversely, when the bond between the two reaches a saturated state and is stabilized, the detected resonance angle becomes constant. Thereby, the binding state between the sample fixed to the detection region 5a and the reactant can be inspected.

In order to observe the dissociation state of the reactant bound to the sample fixed on the entire area of the detection area 5a, a buffer solution is supplied into the flow cell 9 through the second supply channel 13. As described above, the light irradiation device 19 and the light receiving device 21 are synchronously rotated in opposite directions along the outer peripheral surface of the semi-cylindrical prism 17 while dissociating the reactant from the sample, thereby causing resonance in the entire detection region 5a. Detect changes in angle.

Now, when the reactants are not completely dissociated from the sample, the light irradiation device 19 and the light receiving device 2
The resonance angle in the entire detection region 5a detected with the rotation of 1 is not constant. Conversely, when the reactant is completely dissociated from the sample, the resonance angle detected from the entire detection region 5a becomes constant. Thus, the dissociation state of the reactant with respect to the sample can be inspected based on the change in the resonance angle of the entire detection region 5a.

In the present embodiment, the use of the semi-cylindrical prism 17 as the prism causes the light irradiation device 19 to rotate over a large angular width along the outer peripheral surface of the semi-cylindrical prism 17 to detect the resonance angle. The state of the sample fixed over the entire detection region 5a can be accurately inspected.

[0030]

According to the present invention, the specimen fixed to the detection area can be accurately inspected by changing the parallel light applied to the detection area at a large angle.

[Brief description of the drawings]

FIG. 1 is an explanatory front view schematically showing an SPR microscope.

FIG. 2 is an explanatory plan view showing a light irradiation state on a detection area.

FIG. 3 is an explanatory sectional view of an SPR microscope.

FIG. 4 is an explanatory diagram showing a variable irradiation state of light to a detection area.

[Explanation of symbols]

1-SPR microscope, 3-sensor chip, 5-glass substrate, 5a-detection area, 17-semi-cylindrical prism, 19-light irradiation device, 21-light receiving device, 23-light source

Claims (1)

[Claims] 1. A method for irradiating a predetermined speculum area of a metal thin film formed on a glass substrate with light, and obtaining a sample characteristic based on a resonance angle at which the intensity of reflected light from the detection area of the metal thin film becomes minimum. In a surface plasmon resonance angle microscope for microscopy, a semi-cylindrical prism that is in close contact with the glass substrate on the opposite side of the metal thin film and covers at least the entire inspection area of the metal thin film, and the outer periphery of the semi-cylindrical prism on the light incident side It is supported so as to be rotatable at a predetermined angle along the surface, and the light from the light source is directed to the light incident side of the semi-cylindrical prism so that the light from the light source becomes a parallel luminous flux over the entire inspection area in the semi-cylindrical prism. The light irradiating device to irradiate and, supported along the outer peripheral surface on the light emission side of the semi-cylindrical prism so as to be synchronously rotatable in a direction opposite to the rotation direction of the light irradiating device, are reflected from the speculum region in the metal foil film Semi-cylindrical prism Transmitted consists of a light receiving device for outputting an electrical signal corresponding parallel light fluxes in the reflected light intensity from the entire detection area by receiving the surface plasmon resonance angle microscope the resonance angle of the sample in speculum region allowed microscopic examination.