JP2007225389A - Surface plasmon resonance measuring device and measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To measure a resonance angle change highly accurately even when a light intensity distribution of a light flux irradiated to a surface plasmon resonance sensor is not constant, or when the sensitivity of each light receiving element arranged one-dimensionally or two-dimensionally has dispersion. <P>SOLUTION: This device is equipped with an operation processing device 3 for measuring reflected light intensity of measuring light irradiated to a sensor part 11 by an optical detector 30 when a calibration solution wherein a reflected light intensity change is flat in a measuring domain near a resonance angle of a sample liquid is supplied to the surface plasmon resonance sensor 10, and executing calibration so that the sensitivity of each light receiving element becomes constant based on detection values from each light receiving element arranged one-dimensionally or two-dimensionally. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a surface plasmon resonance measurement method for measuring a change in resonance angle by measuring a change in reflected light intensity from a surface plasmon resonance sensor using an optical detector in which light receiving elements are arranged one-dimensionally or two-dimensionally. .

In recent years, it has been promoted to improve the development efficiency of drug discovery by detecting the state of binding of biomolecules with a sensing device. In addition, it is required to rapidly examine a malignant antigen-antibody reaction.
As a method for detecting the binding and the binding process of these biomolecules, a surface plasmon resonance measurement apparatus (hereinafter simply referred to as “surface plasmon resonance”, hereinafter simply abbreviated as “SPR”) by photoexcitation. (Abbreviated as “SPR measuring device”).
According to this SPR phenomenon, for example, in a state where an antibody or the like is immobilized on a metal thin film, a sample solution containing an antigen is supplied onto the metal thin film, and the resonance angle caused by the change in the refractive index of the antibody due to the antigen-antibody reaction. A change is detected, whereby a specific substance can be quantified.

The SPR measurement device 41 includes a sample solution supply device 43 that supplies a sample solution to the SPR sensor 42, and an optical system 45 that measures the reflected light intensity at the sensor unit 44 that is in contact with the sample solution.
The SPR sensor 42 is a state in which a sensor portion 44 in which a metal thin film is deposited is formed on the flat portion of the semicircular prism 46, and a cell 48 in which a groove-like channel 47 is formed on the bottom surface is in close contact with the sensor portion 44. Then, the sample liquid is supplied to the flow path 47 to perform measurement.

The optical system 45 includes a light source device 49 that emits P-polarized measurement light along an irradiation optical axis Z _L that extends from the arc surface side of the semicircular prism 46 toward the center, and the measurement light in a predetermined center angle range (incident angle). a lens system 50 to be incident at 75 ° ± 5 °), an optical detector 51 such as CCD elements arranged light receiving elements in a two-dimensional matrix on the reflected optical axis Z _R is disposed.
Thereby, the position of the light receiving element corresponds to the incident angle, and the reflected light intensity change corresponding to each incident angle can be measured without moving the light source device 49 and the optical detector 51.
JP-A-7-159319

However, when the optical detector 51 in which the light receiving elements are arranged one-dimensionally or two-dimensionally is used, the light beam irradiated from the light source device 49 to the SPR sensor 42 is different from the measurement using the light receiving elements in general points. If the light intensity distribution is not constant or the sensitivity of the light receiving element varies, there is a problem of adversely affecting the measurement accuracy.
Further, when a cell in which a flow path is formed is used, each time the cell is set, the position of the light receiving element corresponding to the position of the flow path has to be determined, which is troublesome.

  Therefore, the present invention can measure with high accuracy even if the light intensity distribution of the light beam irradiated to the sensor is not constant or the sensitivity of the light receiving element varies. A technical problem is to enable automatic determination of the position of the light receiving element corresponding to the flow path of the cell in which the flow path is formed.

In order to solve this problem, the invention according to claim 1 irradiates the sample liquid supply device for supplying the sample liquid to the surface plasmon resonance sensor and the sensor light in contact with the sample liquid, and reflects the reflected light. A surface plasmon resonance measuring apparatus having an optical system that measures an intensity using an optical detector in which a light receiving element is arranged in one or two dimensions, and the intensity of reflected light in a measurement region near the resonance angle of the sample liquid When a calibration solution having a flat change is supplied to the surface plasmon resonance sensor, the reflected light intensity of the measurement light applied to the sensor unit is measured by the optical detector, and the light receiving element is based on the detection value of each light receiving element. And an arithmetic processing unit that performs calibration so that the sensitivity of the signal becomes constant.
According to a second aspect of the present invention, the arithmetic processing device has a reference value setting means for determining a reference value common to each light receiving element, and the sensitivity of each light receiving element based on the difference between the reference value and the detected value of each light receiving element. A calibration means for adjusting is provided.
In the invention of claim 3, a plurality of flow paths are formed in parallel in the sensor portion of the surface plasmon resonance sensor, and when sample liquid is supplied to an arbitrary flow path of the surface plasmon resonance sensor, the direction is perpendicular to the flow path. And an arithmetic processing unit that measures the reflected light intensity near the resonance angle with the optical detector and determines one or a plurality of measurement light receiving elements corresponding to the center of the flow path based on the reflected light intensity distribution. Yes.

According to the present invention, when the calibration solution is supplied to the surface plasmon resonance sensor, the sensor portion is irradiated with measurement light, and the reflected light intensity is measured by the optical detector. Since the reflected light intensity change is flat in the measurement region near the corner, the reflected light intensity from the sensor unit having a constant reflectance is measured by each light receiving element of the optical detector.
At this time, if the light intensity distribution of the light beam of the measurement light is not constant or the sensitivity of the light receiving element varies, the light intensity is measured by each light receiving element while including such an error.
Therefore, based on the detection value of each light receiving element, for example, the average value of the reflected light intensity detected by each light receiving element is used as a reference value, and based on the difference in the detection value of each light receiving element with respect to this reference value, Calibrate so that sensitivity is constant.
As a result, if the measurement is performed under the same conditions, the light intensity distribution of the measurement light beam and variations in the sensitivity of the light receiving element are canceled out. Therefore, these errors are eliminated, and surface plasmon resonance measurement is performed with high accuracy. be able to.

Further, when a plurality of flow paths are formed in parallel in the sensor unit, after performing the calibration, the sample liquid is supplied to an arbitrary flow path of the surface plasmon resonance sensor and is orthogonal to the flow paths. Then, the reflected light intensity in the vicinity of the resonance angle is measured by the optical detector.
In the vicinity of this resonance angle, the intensity of the reflected light is reduced, so that the portion of the channel through which the sample liquid flows is dark and the portion where the channel is not formed is bright.
That is, if the reflected light intensity is measured in a direction orthogonal to the flow path, the portion showing the minimum value corresponds to the position of the flow path, and therefore the direction passing through the light receiving element where the minimum value is measured is parallel to the flow path. The light receiving elements arranged in the line correspond to the center of the flow path.
Therefore, in this state, if one or a required number of light receiving elements at a position corresponding to an arbitrary incident angle is selected, a change in the resonance angle in the flow path formed in the sensor unit can be accurately measured.

  In this example, in order to achieve the purpose of enabling measurement with high accuracy even when the light intensity distribution of the light beam irradiated to the sensor is not constant or the sensitivity of the light receiving element varies, the sample solution A calibration solution having a flat reflected light intensity change in a measurement region near the resonance angle is supplied to the surface plasmon resonance sensor, and the reflected light intensity is measured by irradiating the sensor unit in contact with the calibration solution with the measurement light. After measuring with an optical detector and performing calibration so that the sensitivity of the light receiving element becomes constant based on the detection value of each light receiving element, supply sample liquid to the surface plasmon resonance sensor to measure the change in resonance angle I tried to do it.

  1 is an explanatory view showing an example of a surface plasmon resonance measuring apparatus according to the present invention, FIG. 2 is an explanatory view showing a surface plasmon resonance sensor, FIG. 3 is an explanatory view showing the relationship between the optical system and the sensor, and FIG. FIGS. 5A and 5B are explanatory diagrams showing the reflected light intensity distribution when the calibration solution and the sample solution are supplied, FIG. 5 is a graph showing the detection result of the light receiving element array orthogonal to the flow path, and FIG. 6 shows the calibration program. FIG. 7 is a flowchart showing a flow path position determination program, FIGS. 8A and 8B are graphs showing the measurement result of reflected light intensity based on data before and after calibration, and FIG. 9 is a graph showing the measurement result of change in resonance angle. It is.

The surface plasmon resonance measuring apparatus 1 of this example measures a change in resonance angle caused by a surface plasmon resonance (hereinafter simply referred to as “SPR”) phenomenon caused by light excitation.
The SPR measurement device 1 includes a sample solution supply device 2 that supplies a sample solution to the SPR sensor 10, and an optical system 20 that measures the reflected light intensity in the sensor unit 11 that is in contact with the sample solution.

The SPR sensor 10 includes a semicircular prism 12 and a cell 13 through which a sample solution flows.
The prism 12 has a sensor portion 11 formed by vapor-depositing a metal thin film on a planar portion thereof.
The cell 13 is attached to the piston 15 of the air cylinder 14 so as to be movable up and down with respect to the sensor unit 11 of the prism 12. Groove-shaped flow paths 16 are formed in parallel on the bottom surface side, and the sample supply device 2 for supplying an arbitrary sample solution to the inlet 16in of each flow path 16 is connected, and the outlet 16out is drained. (Not shown).
As a result, when the cell 13 is brought into close contact with the sensor unit 11 and the sample solution is supplied from the sample solution supply device 2, the cell 13 flows in contact with the sensor unit 11 and is discharged to the drain.

The optical system 20 has a light emitting diode 21 that irradiates light with a central wavelength of 720 nm along an irradiation optical axis Z _L that goes from the arc surface side to the center of the semicircular prism 12, a pinhole 22 with a diameter of 200 μm, and a light beam diameter. Beam expander lenses 23 and 24 to be collimated, an interference filter (center wavelength 720 nm, half width 10 nm) 25, a polarizing prism (extinction ratio 10 ⁻⁶ ) 26 that irradiates the sensor unit 11 with P-polarized light, A cylindrical lens 27 that collects light so as to be irradiated at an incident angle (center incident angle 75 ° ± 5 °) near the resonance angle with respect to the center of the semicircular prism 12 is disposed.

Also, along the reflected optical axis Z _R, it is reflected by the sensor portion 11 and the cylindrical surface lens 28 for collimating the light beam coming emitted extends from the prism 12, an imaging lens for imaging the light 29 And an optical detector 30 such as a CCD element in which light receiving elements are arranged in an i × j two-dimensional matrix on the image plane, and the optical detector 30 is connected to the arithmetic processing unit 3. .

The sample supply device 2 has a supply system with the number of channels corresponding to the flow paths 16 formed in the SPR sensor 10 and is near the resonance angle of the sample liquid (for example, water) with respect to all the flow paths 16. A supply liquid switching device is arranged so that a calibration solution (for example, chloroform) having a flat reflected light intensity change in the measurement region (75 ± 5 °) can be supplied.
Thereby, when calibrating the light receiving element of the optical detector 30, the calibration solution can be supplied to all the flow paths 16 of the SPR sensor 10, and when performing the SPR measurement, all of the SPR sensor 10 can be supplied. A sample solution in which arbitrary samples of the same or different types are mixed is supplied to the channels 16... Or the sample solution is supplied to the arbitrarily selected channels 16. At the same time, the calibration solution is supplied to the channels 16. Can also be supplied.

The arithmetic processing unit 3 determines the light receiving elements corresponding to the calibration execution program PRG ₁ and the flow paths 16 so that the sensitivity of the light receiving elements becomes constant based on the detection value of each light receiving element of the optical detector 30. A road position determination program PRG ₂ is provided.

FIG. 6 is a flowchart showing the calibration execution program PRG _1. First, when the prism 12 and the SPR sensor 10 are set at fixed positions, a calibration solution is supplied from the sample solution supply device 2 to all the flow paths 16 of the SPR sensor 10. Chloroform is supplied.
When chloroform is supplied, the resonance angle does not exist within the range of 75 ± 5 ° and the reflectance is constant. Therefore, the image picked up by the optical detector 30 has the same color as shown in FIG. / Because the screen has the same brightness, the reflected light intensity detected by all the light receiving elements should be constant, but actually, the light intensity distribution of the light emitted from the light emitting diode 21 and the sensitivity of each light receiving element As a result, the detection value is not constant and includes an error.

Therefore, in step STP1, a detection value Eij corresponding to the intensity of reflected light of P-polarized light irradiated from the light emitting diode 21 to the SPR sensor 10 is output from each light receiving element of the optical detector 30, and in step STP2, all light receiving elements are detected. An average value of the values Eij is obtained and set as a reference value Av.
Next, the process proceeds to step STP3, and the difference ΔEij of the detection value Eij of each light receiving element with respect to the reference value Av is set as follows.
ΔEij = Eij / Av
In step STP4, a calibration equation for obtaining a calibration value Kij for an arbitrary detection value Eij of each light receiving element is
Kij = Eij / ΔEij
And the calibration program PRG ₁ is terminated.
According to this, when the detection value Eij of each light receiving element when the calibration solution is supplied is calibrated, the calibration output Kij is
Kij = Eij / ΔEij = Av
Therefore, it can be seen that the same detection value can be obtained from any light receiving element regardless of the light intensity distribution of the light emitted from the light emitting diode 21 and variations in sensitivity of each light receiving element.

FIG. 7 is a flowchart showing the channel position determination program PRG ₂ that is executed after the calibration program PRG ₁ ends.
When only the sample liquid (for example, water) in which the sample is not mixed is supplied from the sample liquid supply device 2 to all the flow paths 16 of the SPR sensor 10, a resonance angle exists within a range of 75 ± 5 °. Since the reflected light intensity of the portion decreases, the image picked up by the optical detector 30 corresponds to the flow paths 16 as shown in FIG. 4B, and the vicinity of the resonance angle is dark.

Therefore, in step STP11, the reflected light intensity of the P-polarized light irradiated from the light emitting diode 21 to the SPR sensor 10 is detected by each light receiving element of the optical detector 30, and in step STP12, it is orthogonal to the flow path 16 in the vicinity of the resonance angle. The detection values of the light receiving elements arranged in the direction X are extracted, and the calibration value of each detection value is calculated in step STP13.
FIG. 5 is a graph of calibration values, where there are minimum values at regular intervals, and the observed portion of the minimum value corresponds to the center of the flow path 16.
Therefore, in step STP14, the light receiving element in which the minimum value is observed is extracted, and the reflected light intensity is measured by the respective light receiving element arrays arranged in the direction Y orthogonal to the direction X through the light receiving element. Thus, the reflected light intensity corresponding to the incident angle can be measured for the sample liquid flowing through each flow path 16.

FIG. 8 shows the measurement of the reflected light intensity corresponding to the incident angle for the sample liquid flowing through one arbitrary flow path 16, and FIG. 8 (a) is a plot of the data before calibration, FIG. 8 (b). Is plotted with the data after calibration.
As described above, even when the detection value of the light receiving element includes an error due to the light intensity distribution of the light emitted from the light emitting diode 21 and the sensitivity variation of each light receiving element, the error is eliminated by executing the calibration. It becomes possible to measure with high accuracy.

The above is one configuration example of the present invention, and the operation thereof will be described next.
First, the SPR sensor 10 is set at a predetermined position by bringing the cell 13 into close contact with a prism 12 to which a reactive substance suitable for a specific substance (sample) to be quantified is fixed to the sensor unit 11.
Then, after supplying chloroform as a calibration solution to each of the flow paths 16 and executing the calibration program PRG ₁ , a sample liquid (water) that does not contain a specific substance is supplied and the flow path position determination program PRG ₂ is executed. Then, the reflected light intensity corresponding to the incident angle is measured by the light receiving element array corresponding to each flow path 16.
When smoothing is subjected to fitting process based on the obtained data, since the resonance curve C ₁ shown in FIG. 9 is obtained, and records the minimum value as a reference resonance angle theta _0.

Subsequently, the sample liquid (water) mixed with the specific substance is supplied to each flow path 16..., And similarly, the reflected light intensity corresponding to the incident angle is measured by the light receiving element array corresponding to each flow path 16.
When smoothing is subjected to fitting process based on the obtained data, since the resonance curve C _2, as shown in FIG. 9 is obtained, and records the minimum value as the resonance angle theta _R.
At this time, since the detection values of the respective light receiving elements are calibrated, the reference resonance angle θ ₀ and the resonance angle θ _R can be obtained with high accuracy, and the changes can be calculated. Based on this, the specific substance is quantified. It becomes possible.

In addition, if SPR measurement is performed while two adjacent flow paths 16 are paired, the sample liquid is flowed through one flow path 16 and the calibration solution is flowed through the other flow path 16, it corresponds to the flow path through which the calibration solution flows. The intensity change of the irradiation light can be monitored by the light receiving element, and the measurement error due to the influence can be reduced even if the state of the measuring apparatus changes due to a change in the outside air temperature or the like.
In addition, the SPR sensor 10 having a plurality of flow paths 16 is described, but one flow path may be provided.
Further, only the case where the optical detector 30 in which the light receiving element is arranged in two dimensions has been described, but when the resonance curve is known, the optical detector in which the light receiving element is arranged in one dimension may be arranged in the vicinity of the resonance angle. When only one flow path 16 is formed in the SPR sensor 10, the present invention can also be applied to an SPR measurement apparatus in which an optical detector having a light receiving element arranged one-dimensionally is arranged along the flow path direction.

  As described above, the present invention measures the reflected light intensity change from the surface plasmon resonance sensor using an optical detector in which a light receiving element is arranged one-dimensionally or two-dimensionally, detects a change in resonance angle, The present invention can be applied to uses for detecting binding and binding processes of biomolecules.

Explanatory drawing which shows an example of the surface plasmon resonance measuring apparatus which concerns on this invention. Explanatory drawing which shows a surface plasmon resonance sensor. Explanatory drawing which shows the relationship between an optical system and a sensor. (A) And (b) is explanatory drawing which shows reflected light intensity distribution when a calibration solution and a sample solution are supplied. The graph which shows the detection result of the light receiving element row | line | column orthogonal to a flow path. The flowchart which shows a calibration program. The flowchart which shows a flow-path position determination program. (A) And (b) is a measurement result of reflected light intensity based on data before and after calibration. The graph which shows the measurement result of the change of a resonance angle. Explanatory drawing which shows a conventional apparatus.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Surface plasmon resonance measuring apparatus 2 Sample liquid supply apparatus 3 Arithmetic processing apparatus 10 Surface plasmon resonance sensor 11 Sensor part 12 Semicircular prism 20 Optical system 30 Optical detector

Claims (5)

Optical detection with sample liquid supply device that supplies the sample liquid to the surface plasmon resonance sensor and the sensor unit that is in contact with the sample liquid is irradiated with measurement light, and the reflected light intensity is arranged in one or two dimensions. A surface plasmon resonance measuring apparatus comprising an optical system for measuring using a measuring instrument,
When a calibration solution having a flat reflected light intensity change in the measurement region near the resonance angle of the sample liquid is supplied to the surface plasmon resonance sensor, the reflected light intensity of the measurement light irradiated on the sensor unit is measured by the optical detector. A surface plasmon resonance measuring apparatus comprising an arithmetic processing unit that performs measurement and performs calibration so that sensitivity of a light receiving element becomes constant based on a detection value of each light receiving element.   The arithmetic processing unit includes a reference value setting unit that determines a reference value common to each light receiving element, and a calibration unit that adjusts the sensitivity of each light receiving element based on a difference between the reference value and a detection value of each light receiving element. The surface plasmon resonance measuring apparatus according to claim 1, comprising: A plurality of flow paths are formed in parallel in the sensor portion of the surface plasmon resonance sensor,
When the sample liquid is supplied to an arbitrary flow path of the surface plasmon resonance sensor, the reflected light intensity near the resonance angle is measured along the direction orthogonal to the flow path with the optical detector, and based on the reflected light intensity distribution The surface plasmon resonance measuring apparatus according to claim 1, further comprising an arithmetic processing unit that determines one or a plurality of measurement light receiving elements corresponding to the center of the flow path. Using an optical detector that supplies sample liquid to the surface plasmon resonance sensor, irradiates the sensor part in contact with the sample liquid with measurement light, and changes the reflected light intensity in one or two dimensions. A surface plasmon resonance measurement method for measuring a change in resonance angle caused by the sample liquid by measuring,
Supply the calibration solution whose reflected light intensity change is flat in the measurement region near the resonance angle of the sample liquid to the surface plasmon resonance sensor,
Irradiate measurement light to the sensor unit in contact with the calibration solution and measure the reflected light intensity with the optical detector,
After performing calibration so that the sensitivity of the light receiving element is constant based on the detection value of each light receiving element,
A surface plasmon resonance measuring method, comprising: supplying a sample liquid to the surface plasmon resonance sensor to measure a change in resonance angle. Using a surface plasmon resonance sensor in which a plurality of flow paths are formed in parallel in the sensor unit, after supplying the calibration solution to each flow path and executing the calibration,
Supply sample liquid to an arbitrary flow path of the surface plasmon resonance sensor, measure the reflected light intensity near the resonance angle along the direction orthogonal to the flow path with the optical detector,
Determining one or more light-receiving elements for measurement corresponding to the center of the flow path based on the reflected light intensity distribution;
5. The surface plasmon resonance measurement method according to claim 4, wherein a change in resonance angle caused by the sample liquid flowing through each flow path is measured by the light receiving element.

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