JP5541098B2 - Surface plasmon resonance fluorescence analyzer and surface plasmon resonance fluorescence analysis method - Google Patents

Description

  The present invention relates to a surface plasmon resonance fluorescence analysis apparatus and a surface plasmon resonance fluorescence analysis method for detecting an analyte contained in a sample liquid using surface plasmon resonance (SPR).

  Conventionally, surface plasmon resonance fluorescence analysis (surface plasmon excitation enhanced fluorescence spectroscopy: SPFS) method is known as a method for detecting a sample (a substance to be detected) with high sensitivity in bioassay for detecting protein, DNA, and the like.

  In the SPFS method, in a prism in which a metal film made of gold, silver, or the like is formed on a predetermined surface, excitation light is incident on the metal film from the prism side under total reflection conditions, and the prism and the metal film having different refractive indexes are made to enter. Light (evanescent wave) that exudes from the interface when the excitation light is totally reflected at the interface is used. Specifically, in the SPFS method, the specimen contained in the sample liquid on the surface of the metal film or the fluorescent substance (labeling substance) labeled on the specimen is excited by the evanescent wave that is oozed out when the excitation light is totally reflected. The presence or amount of the specimen can be detected by analyzing the fluorescence emitted (excitation fluorescence).

  In the optical measurement using the SPFS method, the specimen can be detected with higher sensitivity and higher accuracy as the intensity of the enhanced electric field (evanescent wave) enhanced by the surface plasmon resonance is larger.

  Therefore, in order to optimize the generation condition of the surface plasmon resonance and improve the intensity of the enhanced electric field, the analyzer described in Patent Document 1 includes the excitation light on the optical path of the excitation light between the excitation light source and the prism. A polarizing element capable of adjusting the polarization state is disposed. Specifically, the excitation light includes a P wave component and an S wave component, and when the excitation light is totally reflected at the interface between the metal film and the prism, the P wave component effectively contributes to the excitation of the surface plasmon. However, the S wave component does not contribute to the excitation of the surface plasmon. Therefore, in the above analyzer, the polarization state of the excitation light before entering the prism is set so that the light amount (intensity) of the P-wave component in the total amount of excitation light reflected by the metal film is maximized. It is adjusted. Thus, the excitation of the surface plasmon in the metal film is suitably performed by reflecting the excitation light in the state in which the light quantity of the P wave component occupying the total light quantity of the excitation light is maximized by the metal film. The strength of the enhanced electric field based on plasmon resonance can be improved.

JP 2009-236709 A

  However, if the prism in which the metal film is formed on a predetermined surface is replaced in the above-described analyzer, the measurement result may vary from prism to prism even if the specimen contained in the same sample solution is detected.

  In view of the above problems, an object of the present invention is to provide a surface plasmon resonance fluorescence analyzer and a surface plasmon resonance fluorescence analysis method capable of suppressing variations in measurement accuracy for each prism.

  As a result of diligent research to solve the above problems, the present inventors have found that when the prism in which the metal film is formed on a predetermined surface is replaced in the above-described analyzer, the excitation light incident on the metal film for each prism. It was found that the variation in the light amount of the P wave component in the total light amount of the excitation light occurs in FIG. That is, in the above analysis apparatus, when the prism is replaced, the amount of the P wave component in the total amount of the excitation light in the excitation light incident on the metal film in each prism is maximized. In some cases, the amount of the P wave component of the excitation light incident on the light beam is not constant.

The present invention has been made based on such knowledge, and is a surface plasmon resonance fluorescence analyzer that measures fluorescence emitted by excitation of an analyte or a fluorescent substance attached to the sample by an electric field based on surface plasmon resonance. A chip holding unit capable of holding an analysis chip including a prism having a metal film formed on a predetermined surface so that the analysis chip can be attached and detached; and an excitation light is emitted to the analysis chip held by the chip holding unit A light source unit that causes the excitation light to enter the prism so as to be reflected by the metal film of the included prism, and a reflected light measurement unit that measures the amount of reflected excitation light that is the excitation light reflected by the metal film; The light amount of the P wave component of the excitation light incident on the metal film is determined based on the light amount of the excitation light emitted from the light source unit and the light amount of the reflected excitation light measured by the reflected light measurement unit. And a P-wave light quantity adjusting unit for adjusting the light source unit changes the amount of excitation light emitted in response to the amount of current supplied, the P-wave light quantity adjusting portion includes a said light source and said portion prism And a half-wave plate disposed on the optical path of the excitation light emitted from the light source section, and a rotational drive that rotates the half-wave plate so that the polarization direction of the excitation light with respect to the metal film changes And a light source current supply unit that supplies current to the light source unit, and the rotational drive unit based on the amount of excitation light emitted from the light source unit and the amount of reflected excitation light measured by the reflected light measurement unit And a control unit that controls the amount of current supplied by the light source current supply unit. The control unit reflects the amount of excitation light emitted from the light source unit and the reflection light measured by the reflected light measurement unit. Metal based on the amount of excitation light A rotation amount adjustment unit for adjusting the rotation amount of the half-wave plate by controlling the rotation drive unit so that the light amount of the P wave component in the total light amount of the excitation light incident on the light beam is maximized; and the excitation light The light source current is based on the light amount of the excitation light emitted by the light source unit and the light amount of the reflected excitation light measured by the reflected light measurement unit in a state where the light amount of the P-wave component in the total light amount is maximum. A current amount adjusting unit that controls the supply unit to adjust the amount of current supplied to the light source unit .

Also, a surface plasmon resonance fluorescence analyzer for measuring fluorescence emitted by excitation of an analyte or a fluorescent substance attached to the sample by an electric field based on surface plasmon resonance, wherein a prism is formed with a metal film on a predetermined surface And a light source unit that emits excitation light and makes the excitation light enter the prism so as to be reflected by the metal film of the prism, and a light amount of reflected excitation light that is excitation light reflected by the metal film. A reflected wave measuring unit to be measured, and a P wave component of excitation light incident on the metal film based on the amount of excitation light emitted from the light source unit and the amount of reflected excitation light measured by the reflected light measurement unit A P-wave light amount adjusting unit that adjusts the light amount of the light source to a specific light amount, the light source unit changes the light amount of the excitation light emitted according to the amount of current supplied, The light source unit and the prism; A half-wave plate disposed on the optical path of the excitation light emitted from the light source unit, and a rotation drive unit that rotates the half-wave plate so that the polarization direction of the excitation light with respect to the metal film changes And a light source current supply unit that supplies current to the light source unit, and the rotational drive unit based on the amount of excitation light emitted by the light source unit and the amount of reflected excitation light measured by the reflected light measurement unit. A control unit that drives and controls the amount of current supplied by the light source current supply unit. The control unit reflects the amount of excitation light emitted from the light source unit and the reflected excitation measured by the reflected light measurement unit. Based on the amount of light, the rotation drive unit is controlled to adjust the amount of rotation of the half-wave plate so that the amount of P-wave component in the total amount of excitation light incident on the metal film is maximized. A rotation amount adjustment unit, and all of the excitation light The light source current supply unit based on the light amount of the excitation light emitted by the light source unit and the light amount of the reflected excitation light measured by the reflected light measurement unit in a state where the light amount of the P wave component occupying the quantity is maximized And a current amount adjusting unit that adjusts the amount of current supplied to the light source unit .

  According to the present invention, the amount of the P wave component of the excitation light incident on the metal film can be made constant even when the prism having the metal film formed on the predetermined surface is replaced. Variation in measurement results is suppressed.

Specifically, even if the amount of the P wave component is constant in the excitation light irradiated into the prism, if the prism is replaced, for example, the P of the excitation light incident on the metal film due to the difference in birefringence of each prism, etc. The amount of wave component may not be constant. Therefore, the amount of P wave component incident on the metal film is obtained based on the amount of excitation light before entering the prism and the amount of reflected excitation light after being reflected by the metal film, and this amount of light replaces the prism. Even if it adjusts so that it may become constant, the dispersion | variation in the measurement accuracy for every prism is suppressed.
By making it possible to adjust the polarization direction and the light amount of the excitation light incident on the metal film, the light amount of the P wave component of the excitation light incident on the metal film can be reliably adjusted to the target light amount, It is possible to improve the S / N.
Specifically, even when the half-wave plate is rotated to maximize the amount of P-wave component in the total amount of excitation light incident on the metal film, the amount of P-wave component of excitation light incident on the metal film is the target. Even if the amount of light does not reach the light amount, the light amount of the excitation light emitted from the light source unit can be increased and the light amount of the P wave component of the excitation light incident on the metal film can be set as the target light amount.
In addition, unnecessary light (noise) such as autofluorescence in the prism can be reduced as the amount of excitation light incident on the prism is reduced. Therefore, the P wave component of the excitation light incident on the metal film is maximized while the light amount of the P wave component in the total light amount of the excitation light incident on the metal film is maximized and the light amount of the excitation light emitted from the light source unit is minimized. By setting the target light amount as the target light amount, it is possible to suppress variations in measurement results for each prism and to reduce noise generated in the prism and improve the S / N. As a result, it is possible to detect the sample with high accuracy and high sensitivity.

  Specifically, the P wave component included in the excitation light emitted from the light source unit is used for excitation of surface plasmon when reflected by the metal film, and the reflected excitation light includes only the S wave component. Therefore, the P-wave light amount adjustment unit is configured to detect the light amount of the P-wave component incident on the metal film from the difference between the light amount of the excitation light emitted from the light source unit and the light amount of the reflected excitation light measured by the reflected light measurement unit. And the state of the excitation light is adjusted based on the amount of light. The state of the excitation light means the output of the excitation light, the deflection direction, and the like.

  For example, the P-wave light amount adjusting unit includes a half-wave plate disposed on an optical path of excitation light emitted from the light source unit between the light source unit and the prism, and excitation light for the metal film. Based on the rotational drive unit that rotates the half-wave plate so that the polarization direction changes, the amount of excitation light emitted by the light source unit, and the amount of reflected excitation light measured by the reflected light measurement unit You may have a control part which drives a rotation drive part.

  As described above, when the half-wave plate is provided between the light source unit and the prism and the half-wave plate is rotated, the P-wave component of the excitation light incident on the metal film is rotated by rotating the deflection direction of the excitation light. The amount of light changes. Therefore, the light amount of the P wave component of the excitation light incident on the metal film is adjusted to the target light amount by rotating the half-wave plate while detecting the light amount of the P wave component of the excitation light incident on the metal film. can do.

  In addition, by rotating the half-wave plate to set the light amount of the P wave component of the excitation light incident on the metal film as a target light amount, the measurement result due to the wavelength variation accompanying the change in the light amount of the excitation light is obtained. The influence of can be suppressed. That is, if the amount of excitation light emitted from the light source unit is changed, the wavelength of the excitation light may change and this wavelength change may affect the measurement result of the specimen. By setting the light amount of the P wave component of the excitation light incident on the metal film as a target light amount, the light amount of the excitation light emitted from the light source unit can be made constant, and as a result, the wavelength change of the excitation light can be reduced. It is possible to suppress the influence on the measurement result.

  The light source unit changes the amount of excitation light emitted according to the amount of current supplied, the P-wave light amount adjustment unit includes a current supply unit that supplies current to the light source unit, and the light source unit You may have a control part which controls the electric current amount which the said electric current supply part supplies based on the light quantity of the excitation light to inject | emits, and the light quantity of the reflected excitation light measured by the said reflected light measurement part.

  Thus, even if the amount of excitation light incident on the metal film is changed, the amount of P wave component of the excitation light incident on the metal film changes. Therefore, by adjusting the light amount of the excitation light emitted from the light source unit while detecting the light amount of the P wave component of the excitation light incident on the metal film, the light amount of the P wave component of the excitation light incident on the metal film is targeted. The amount of light to be adjusted can be adjusted.

  Specifically, even when the half-wave plate is rotated to maximize the amount of P-wave component in the total amount of excitation light incident on the metal film, the amount of P-wave component of excitation light incident on the metal film is the target. Even if the amount of light does not reach the light amount, the light amount of the excitation light emitted from the light source unit can be increased and the light amount of the P wave component of the excitation light incident on the metal film can be set as the target light amount.

  In addition, unnecessary light (noise) such as autofluorescence in the prism can be reduced as the amount of excitation light incident on the prism is reduced. Therefore, the P wave component of the excitation light incident on the metal film is maximized while the light amount of the P wave component in the total light amount of the excitation light incident on the metal film is maximized and the light amount of the excitation light emitted from the light source unit is minimized. By setting the target light amount as the target light amount, it is possible to suppress variations in measurement results for each prism and to reduce noise generated in the prism and improve the S / N. As a result, it is possible to detect the sample with high accuracy and high sensitivity.

  The surface plasmon resonance fluorescence analyzer according to the present invention includes a fluorescence measurement unit capable of measuring the intensity of the fluorescence generated on the surface of the metal film opposite to the prism by reflecting the excitation light on the metal film. An intensity changing unit that changes the intensity of the excitation light that enters the prism based on the amount of fluorescent light measured by the fluorescence measuring unit, and the fluorescence measuring unit that is based on the intensity change of the excitation light by the intensity changing unit It is preferable to include a correction unit that corrects the fluorescence measurement result obtained by the above.

  According to such a configuration, it is possible to obtain a measurement range that is wider than the range of light intensity that can be measured by the fluorescence measurement unit. Specifically, when a specimen having a maximum detection amount or more is present on the metal film, the amount of fluorescence excited by the enhanced electric field based on the surface plasmon resonance generated in the metal film is the amount of light that can be measured by the fluorescence measurement unit. Cannot measure beyond the upper limit. However, since the intensity of the enhanced electric field is reduced by lowering the light amount of the excitation light incident on the metal film (specifically, the light amount of the P wave component of the excitation light incident on the metal film), the fluorescence excited by this enhanced electric field. Becomes smaller than the upper limit of the measurable amount of light of the fluorescence measuring unit, and can be measured. In this case, since the amount of the excitation light incident on the metal film has changed (that is, the intensity of the enhanced electric field that excites the fluorescence has changed), the measurement result in the fluorescence measurement unit is corrected in accordance with this change (for example, When the light quantity of the excitation light is reduced to 1/10, the measurement result (light quantity) obtained by the fluorescence measurement part is multiplied by 10 times, and when the light quantity of the excitation light is reduced to 1/100, the measurement obtained by the fluorescence measurement part. It is necessary to multiply the result (light quantity) by 100).

  A fluorescence measurement unit capable of measuring the intensity of the fluorescence generated on the surface of the metal film opposite to the prism as a result of the excitation light being reflected by the metal film, wherein the specific amount of light is obtained when the excitation light is a metal It is preferable that the amount of fluorescent light generated from the specimen or the fluorescent substance attached to the specimen by reflection on the film is not less than the lower limit of the amount of light that can be measured by the light measuring unit, and the specific light amount is It is preferable that the amount of fluorescent light generated from the sample or the fluorescent material attached to the sample by reflecting the excitation light on the metal film is equal to or less than the upper limit of the amount of light that can be measured by the light measurement unit. Thereby, the fluorescence emitted from the specimen or the fluorescent substance attached to the specimen can be reliably measured by the fluorescence measuring section.

The present invention also relates to a surface plasmon resonance fluorescence analysis method for measuring light emitted from a specimen or a fluorescent substance attached to the specimen by being excited by an electric field based on surface plasmon resonance, wherein a metal film is formed on a predetermined surface. Preparing a formed prism and flowing a sample solution containing the specimen onto the metal film; and reflecting the light from the metal film to cause surface plasmon resonance in the metal film, A step of irradiating excitation light to the light source, and a state of surface plasmon resonance occurring in the metal film, and the amount of excitation light incident on the prism and reflection excitation that is reflected by the metal film reflected light amount and the metal film of the excitation light source unit to be incident and measurement step of measuring P-wave light amount of excitation light from the light amount of the light incident on the metal film, the excitation light into said prism is emitted The light source unit and the prism are configured so that the light amount of the P-wave component in the total amount of excitation light incident on the metal film is maximized based on the amount of reflected excitation light measured by the reflected light measurement unit. The amount of rotation of the half-wave plate arranged on the optical path of the excitation light emitted from the light source unit is adjusted by the control unit, and the light quantity of the P wave component in the total light quantity of the excitation light is maximized In this state, the control unit adjusts the amount of current supplied to the light source unit based on the amount of excitation light emitted from the light source unit and the amount of reflected excitation light measured by the reflected light measurement unit. A method for adjusting a light quantity of light and an adjustment process for adjusting at least one of the light quantity and the deflection direction of the excitation light irradiated in the excitation light irradiation light range based on the measurement of the P-wave light quantity in the measurement process. .

  According to the present invention, the amount of the P wave component of the excitation light incident on the metal film can be made constant even when the prism having the metal film formed on the predetermined surface is replaced. Variation in measurement results is suppressed.

  As described above, according to the present invention, it is possible to provide a surface plasmon resonance fluorescence analysis apparatus and a surface plasmon resonance fluorescence analysis method capable of suppressing variation in measurement accuracy for each prism.

It is a functional block diagram which shows the structure of the state by which the analysis chip was installed in the surface plasmon resonance fluorescence analyzer which concerns on this embodiment. It is an enlarged view showing a configuration of a chip holding unit and an analysis chip held by the chip holding unit in the surface plasmon resonance fluorescence analyzer. It is a functional block diagram of the control processing part of the surface plasmon resonance fluorescence analyzer. It is a flowchart when detecting a specimen in the surface plasmon resonance fluorescence analyzer. It is a figure which shows the relationship between a resonance angle and an excitation incident angle (angle which an enhancement electric field becomes the maximum). In the surface plasmon resonance fluorescence analyzer according to another embodiment, (A) is a functional block diagram of an excitation light emitting unit, and (B) is a functional block diagram of a control processing unit. It is a figure which shows the relationship between the output and measurement range of the optical measurement part in the measurement of the sample in the surface plasmon resonance fluorescence analyzer which concerns on other embodiment, and the detected sample amount (light quantity of excitation fluorescence).

  Hereinafter, an embodiment of the present invention will be described with reference to the accompanying drawings.

  The surface plasmon resonance fluorescence analyzer (hereinafter also simply referred to as “analyzer”) according to the present embodiment uses an evanescent wave (enhanced electric field) that exudes from the reflection interface of excitation light incident on the prism under total reflection conditions. The substance to be detected (hereinafter also simply referred to as “specimen”) or a fluorescent substance labeled (attached) to the specimen is excited, and the amount of fluorescence (excitation fluorescence) generated thereby is detected, thereby detecting the amount of the specimen. It is a device that performs detection.

As shown in FIG. 1, the analyzer includes a chip holding unit 12 that holds an analysis chip 50, an excitation light emitting unit 20 that emits excitation light α to the analysis chip 50 that is held by the chip holding unit 12, and The reflected light measurement unit 30 that measures the amount of light emitted from the analysis chip 50 (reflected excitation light α _s ) and the light measurement unit that measures the intensity of light (excitation fluorescence) generated by the analysis chip 50 (fluorescence measurement) Unit) 32 and a control processing unit (not shown) that controls each component of the analyzer 10 such as the chip holding unit 12, the excitation light emitting unit 20, the reflected light measuring unit 30, and the light measuring unit 32 and performs various arithmetic processes. (Control part) 40 and the display part 16 which displays various information, such as a calculation result. The analyzer 10 also includes a preprocessing unit (not shown) that performs preprocessing of blood from the patient. The pretreatment unit accepts a reagent chip (not shown), performs pretreatment (blood cell separation, dilution, mixing, etc.) of blood injected into the reagent chip to generate a sample liquid, This is a portion to be injected into the analysis chip 50. The reagent chip is provided with a plurality of storage units, and each storage unit is individually sealed with a reagent, a diluent, a cleaning solution, and the like in addition to blood.

  As shown in FIG. 2, the analysis chip 50 includes a prism 51, a metal film 55 formed on the surface of the prism 51, and a sample liquid and a cleaning liquid containing a specimen while contacting the metal film 55 on the metal film 55. And a flow path member 57 that forms a flow path 58 through which and the like flow. The analysis chip 50 of this embodiment is replaced every time a sample is detected (analyzed).

The prism 51 includes an incident surface 52 on which the excitation light α from the excitation light emitting unit 20 is incident, and a film formation surface (predetermined surface) on which the metal film 55 on which the excitation light α incident on the inside is reflected is formed. ) 53 and the exit surface 54 on which the excitation light (reflected excitation light) α _s reflected by the metal film 55 is emitted to the outside of the prism 51, and is made of transparent glass or resin. The emission surface 54 is a surface that first strikes after the excitation light α is reflected by the metal film 55 (specifically, the interface between the metal film 55 and the film formation surface 53), and the reflected excitation light α reflected by the metal film 55. as the light of S-wave component of the _s it does not remain inside the prism 51, similarly to the incident surface 52, is formed on the optical surface. The prism 51 includes an incident surface 52, a film formation surface 53, and an emission surface 54, and the excitation light α incident on the inside from the incident surface 52 is totally reflected by the metal film 55 on the film formation surface 53. The reflection excitation light α _s (specifically, the S wave component of the excitation light α) may be shaped so as to be emitted outside from the emission surface 54 without being irregularly reflected inside.

  The metal film 55 is a metal thin film formed (formed) on the film formation surface 53 of the prism 51, and is formed of gold in this embodiment. The metal film 55 is a member for amplifying an evanescent wave (enhanced electric field) generated when the excitation light α incident on the prism 51 under total reflection conditions is totally reflected at the interface between the metal film 55 and the film formation surface 53. It is. That is, by providing the metal film 55 on the film formation surface 53 and causing surface plasmon resonance in the metal film 55, the excitation light α is totally reflected on the surface without the metal film 55 (film formation surface 53), and evanescent. Compared with the case of generating a wave, the formed evanescent wave can be amplified (that is, an enhanced electric field can be formed in the vicinity of the surface 55a of the metal film 55).

  The material of the metal film 55 is not limited to gold, and may be any metal that causes surface plasmon resonance, and may be, for example, silver, copper, aluminum, or the like (including an alloy).

  A capturing body 56 for capturing a specific antigen is fixed on the surface (surface opposite to the prism 51) 55a of the metal film 55. The capturing body 56 is fixed to the surface 55a of the metal film 55 by surface treatment.

  The flow path member 57 is provided on the film formation surface 53 of the prism 51, and forms a flow path 58 through which the sample liquid flows together with the film formation surface 53. The flow path member 57 is formed of a transparent resin, and is joined to the prism 51 by an adhesive, laser welding, ultrasonic welding, pressure bonding using a clamp member, or the like.

  When the analysis chip 50 configured as described above is installed in the pretreatment unit of the analyzer 10, the sample liquid that has been pretreated in the pretreatment unit is injected (supplied) into the flow path 58. The analysis chip 50 into which the sample liquid has been injected is transported to the chip holding unit 12 when the reaction between the capturing body 56 fixed on the metal film 55 and the specimen (specific antigen) is completed. Are held in a predetermined posture.

  The chip holding unit 12 holds the analysis chip 50 when detecting the specimen. Specifically, in the chip holding unit 12, the excitation light α emitted from the excitation light emitting unit 20 enters the prism 51 from the incident surface 52 under total reflection conditions, and the incident excitation light α is converted into the metal film 55. The analysis chip 50 is detachably held in such a posture as to be reflected by the lens.

  The excitation light emitting unit 20 makes the excitation light α enter the prism 51 so as to be reflected by the metal film 55 of the prism 51 included in the analysis chip 50 held by the chip holding unit 12. Specifically, the excitation light emitting unit 20 includes a light source unit 21, a linear polarization unit 22 that converts the excitation light α emitted from the light source unit 21 into linearly polarized light, and polarization that adjusts the polarization direction of the excitation light α with respect to the metal film 55. A direction adjusting unit 26. The excitation light emitting unit 20 sets the incident angle θ of the excitation light α with respect to the metal film 55 in a state where the emitted excitation light α enters the prism 51 from the incident surface 52 and is totally reflected by the metal film 55. As can be changed, the attitude of the analysis chip 50 (specifically, the metal film 55 of the prism 51) held by the chip holding unit 12 can be changed (see arrow A in FIG. 1). Specifically, the excitation light emitting unit 20 is held by the chip holding unit 12 so as to change the incident angle θ of the excitation light α to the metal film 55 without changing the position where the excitation light α is reflected on the metal film 55. The posture with respect to the analysis chip 50 in such a state can be changed. The excitation light emitting unit 20 of the present embodiment is connected to the control processing unit 40, and changes the posture with respect to the analysis chip 50 in accordance with an instruction signal from the control processing unit 40.

  The light source unit 21 includes an excitation light source (not shown) that emits excitation light α and a temperature control circuit (not shown), and changes the amount of the excitation light α that is emitted according to the amount of current supplied. The light source unit 21 is connected to the control processing unit 40 and supplied with a current from a current supply unit 43 of the control processing unit 40 (see FIG. 3). In this embodiment, a laser diode is used as an excitation light source. This excitation light source is constantly temperature-controlled by a temperature control circuit and maintained at a constant temperature in order to output output light having a stable wavelength with little wavelength fluctuation.

  The linear polarization unit 22 is disposed on the optical path of the excitation light α emitted from the light source unit 21, and filters the excitation light α so that the polarization direction becomes a linear polarization with a unique polarization direction. Specifically, the light source unit 21 is arranged so that the linear polarization unit 22 has a polarization direction in which P-polarized light is incident on the metal film 55 of the prism 51 of the analysis chip 50 held by the chip holding unit 12. Filter the exiting excitation light α.

  The polarization direction adjusting unit 26 includes a half-wave plate 27 and a rotation driving unit 28 that rotates the half-wave plate 27.

  The half-wave plate 27 is disposed on the optical path of the excitation light α emitted from the light source unit 21 and is used as a polarization rotator that continuously rotates the polarization direction of the excitation light α.

  The rotation driving unit 28 rotates the polarization direction of the excitation light α with respect to the metal film 55 by rotating the half-wave plate 27. The rotation drive unit 28 of the present embodiment has a step motor. Then, the rotation drive unit 28 rotates the half-wave plate 27 by the step motor based on the instruction signal from the control processing unit 40. When the half-wave plate 27 is rotated in this way, the polarization direction of the excitation light α linearly polarized in the linear polarization unit 22 is rotated, and thereby the P wave component of the excitation light α incident on the metal film 55 is changed. The amount of light and the amount of S wave component change. That is, the rotation drive unit 28 rotates the half-wave plate 27 to maximize the evanescent wave in the metal film 55 (that is, the electric field enhancement of the enhanced electric field formed in the vicinity of the surface 55a of the metal film 55). It is possible to freely change the polarization direction from the condition where the degree is the maximum) to the condition where no exudation occurs (that is, the condition where no enhanced electric field is formed near the surface 55a of the metal film 55).

The reflected light measurement unit 30 measures the amount of reflected excitation light α _s reflected from the metal film 55 and emitted from the emission surface 54. Specifically, the reflected light measurement unit 30 receives the reflected excitation light α _s emitted from the prism 51 of the analysis chip 50 and outputs an intensity signal corresponding to the intensity (light quantity). The reflected light measurement unit 30 is connected to the control processing unit 40 and outputs the intensity signal to the control processing unit 40. The reflected light measurement unit 30 is disposed at a position facing the emission surface 54 of the analysis chip 50 prism 51 held by the chip holding unit 12 so that the reflected excitation light α _s emitted from the prism 51 can be received. Has been. The reflected light measurement unit 30 of the present embodiment is configured by, for example, a photodiode (PD), but is not limited thereto. That is, the reflected light measurement unit 30 may be configured to include a photoelectric conversion element that converts light energy into electrical energy.

  The light measurement unit 32 measures the intensity of fluorescence generated in the analysis chip 50. Specifically, the light measuring unit 32 receives fluorescence (excitation fluorescence) generated in the vicinity of the metal film 55 of the analysis chip 50 and outputs an intensity signal corresponding to the intensity (light quantity). The light measurement unit 32 is connected to the control processing unit 40 and outputs the intensity signal to the control processing unit 40. In this embodiment, in order to detect weak light such as fluorescence (excitation fluorescence) emitted when a fluorescent substance labeled on the specimen is excited, the photometric multiplier 32 (Photomultiplier Tube) having high sensitivity and high S / N : PMT) is used. The light measurement unit 32 is not limited to the PMT, and may be any device including a photoelectric conversion element that converts light energy into electrical energy, such as a cooled CCD image sensor.

The control processing unit 40 outputs an instruction signal to each component constituting the analysis apparatus 10 to perform control, and performs a calculation based on an output signal from each component. As shown in FIG. 3, the control processing unit 40 includes, for example, a P-wave light quantity deriving unit 41, a rotation amount adjusting unit 42 that controls the rotation driving unit 28 of the polarization direction adjusting unit 26, and the light source unit 21. A current supply unit (light source current supply unit) 43 that supplies current, a current amount adjustment unit 44 that controls the current supply unit 43, and a calculation unit 45 are included. In the present embodiment, the P-wave light amount deriving unit 41, the rotation amount adjusting unit 42, the current supply unit 43, and the current amount adjusting unit 44 together with the polarization direction adjusting unit 26 constitute a P-wave light amount adjusting unit. The P-wave light amount adjusting unit is based on the light amount of the excitation light α emitted from the excitation light emitting unit 20 (specifically, the light source unit 21) and the light amount of the reflected excitation light α _s measured by the reflected light measuring unit 30. The light quantity of the P wave component of the excitation light α incident on the metal film 55 is adjusted to a target light quantity (target light quantity).

The P-wave light quantity deriving unit 41 uses the light quantity of the excitation light α emitted from the light source unit 21 and the light quantity of the reflected excitation light α _s measured by the reflected light measurement unit 30 to generate a P wave of the excitation light α incident on the metal film 55. Obtain the light intensity of the component. Specifically, P-wave light amount derivation unit 41, the difference between the light intensity P _a of the excitation light alpha to the light source unit 21 is emitted, the light amount P _s of the reflected excitation light alpha _s as measured by the reflected light measuring unit 30 (i.e. , P−P _s ), the light amount P _p of the P wave component incident on the metal film 55 is obtained. This is because the P wave component contained in the excitation light α emitted from the light source unit 21 is used to excite surface plasmons when reflected by the metal film 55, and the reflected excitation light α _s has almost no P wave component. This is because only the S wave component is included. That is, in the analyzer 10, the amount of P wave component contained in the reflected excitation light α _s is negligible.

  The P-wave light amount deriving unit 41 obtains the amount of current supplied from the current supply unit 43 to the light source unit 21 from the current amount adjustment unit 44, and calculates the amount of excitation light α emitted from the light source unit 21 from this current amount. To derive.

The rotation amount adjustment unit 42 adjusts the rotation amount of the half-wave plate 27 so that the light amount of the P wave component in the total light amount of the excitation light incident on the metal film is maximized. Specifically, the rotation amount adjustment unit 42 causes the rotation driving unit 28 to rotate the half-wave plate 27 to a rotation position where the light amount P _{s of} the reflected excitation light α _s measured by the reflected light measurement unit 30 is minimized. . That is, the P wave component included in the excitation light α contributes to the excitation of the surface plasmon when the excitation light α is reflected by the metal film 55 and is hardly included in the reflected excitation light α _s. Therefore, when the polarization direction is rotated to change the ratio of the P wave component and the S wave component in the excitation light α, the reflected excitation light α is obtained when the amount of the P wave component in the total amount of the excitation light α is maximum. The light quantity of _s is minimized.

The rotation amount adjustment unit 42 may adjust the rotation amount of the half-wave plate 27 by driving the rotation drive unit 28 based on the P wave component light amount P _p obtained by the P wave light amount deriving unit 41. Good. Specifically, the rotation amount adjusting unit 42 is based on the P wave component light amount P _p obtained by the P wave light amount deriving unit 41, and the light amount of the P wave component in the total light amount of the excitation light α incident on the metal film 55. P _p may adjust the amount of rotation of the half wave plate 27 so as to maximize. In other words, the rotation amount adjustment unit 42 rotates the half-wave plate 27 to a position where the light amount P _p of the P wave component obtained by the P wave light amount deriving unit 41 is maximized.

The current amount adjustment unit 44 adjusts the amount of current supplied to the light source unit 21 by controlling the current supply unit 43 based on the light amount P _s of the P wave component obtained by the P wave light amount deriving unit 41. Specifically, the current amount adjusting unit 44 is supplied to the light source unit 21 such that the light intensity P _t of P-wave component light amount P _s of P-wave component determined by the P-wave light quantity deriving part 41 is a target Adjust the amount of current.

  When analyzing the sample, the calculation unit 45 performs calculation based on the output signal sent from the light measurement unit 32 and performs analysis on the excitation fluorescence measured by the light measurement unit 32. For example, the calculation unit 45 performs counting of the number of excitation fluorescences per unit area detected by the light measurement unit 32, calculation of an increase amount of excitation fluorescence over time, and the like. Then, the calculation unit 45 outputs the calculation result to the display unit 16 connected to the control processing unit 40.

  When the control processing unit 40 configured in this way, for example, when the analysis apparatus 10 analyzes a sample, the preprocessing unit, the light source unit 21, the polarization direction adjustment unit 26, the reflected light measurement unit 30, and the light measurement unit. By controlling 32 and the like, the analyzer 10 performs a pretreatment process, a resonance angle scanning process, a P-wave light quantity adjustment process, an excitation fluorescence measurement process, and the like.

  Details of specific control by the control processing unit 40 will be described later.

  The display unit 16 displays the calculation result based on the output signal from the control processing unit 40. The display unit 16 may display a calculation result or the like on a screen like a liquid crystal display, or may print out the calculation result or the like like a printer. A combination of these may also be used.

  The analysis of the sample in the analyzer 10 configured as described above will be described below with reference to FIG. Details of control of each component of the analyzer 10 by the control processing unit 40 will also be described.

<Pretreatment process>
Blood or the like is collected from the patient, and the collected blood or the like is injected into the reagent chip. The reagent chip into which the blood or the like has been injected is set in the pretreatment unit of the analyzer 10. The control processing unit 40 performs preprocessing (blood cell separation, dilution, mixing, etc.) of the set reagent chip blood and the like by the preprocessing unit to generate a sample solution. In this state, when the analysis chip 50 is installed in the preprocessing unit, the control processing unit 40 injects the pretreated sample solution into the flow path 58 of the analysis chip 50 by the preprocessing unit, and the metal film. The specimen (specific antigen) is captured by the capturing body 56 fixed to the surface of 55 (that is, the capturing body 56 and the specimen are reacted). In this embodiment, the sample labeled with a fluorescent substance (in this embodiment, a fluorescent dye) is captured by the capture body 56, but the present invention is not limited to this, and the analysis chip is captured after the capture body 56 captures the sample. The fluorescent substance may be injected into the fluorescent substance 50 and labeled with respect to the specimen captured by the capturing body 56. In addition, when the specimen itself is excited by the enhanced electric field based on the surface plasmon resonance generated in the metal film 55 and emits fluorescence, the specimen need not be labeled with a fluorescent substance.

  The analysis chip 50 subjected to the reaction in this way is transported to the chip holding unit 12 and held by the chip holding unit 12 (step S1).

<Resonance angle scanning process>
When the analysis chip 50 is held by the chip holding unit 12, the control processing unit 40 performs scanning (resonance angle scanning) under the optimum surface plasmon resonance condition in the analysis chip 50. Based on the result of this scanning, the control processing unit 40 causes the excitation light α to be incident on the metal film 55 at the excitation incident angle θ ₁ that is the incident angle at which the electric field strength of the enhanced electric field generated in the metal film 55 is the largest. Further, the posture of the excitation light emitting unit 20 with respect to the metal film 55 is changed.

Specifically, the control processing unit 40 reflects and excites the reflected light measurement unit 30 while changing the posture of the excitation light emitting unit 20 in a state where the excitation light α is emitted from the excitation light emitting unit 20 (light source unit 21). by measuring the amount of light P _s of light alpha _s, to scan the incident conditions of the excitation light alpha to the metal film 55 of the prism 51 included in the analysis chip 50 (excitation incident angle theta _1).

The light amount P _s of the reflected excitation light α _s measured in this manner rapidly falls when the incident angle θ of the excitation light α to the metal film 55 becomes a predetermined angle, as shown in FIG. . The incident angle of the excitation light alpha to the metal film 55 the light amount P _s of the reflected excitation light alpha _s is the smallest (i.e., the incident angle of reflectance in the metal film 55 is smallest) is the resonance angle theta ₂ . Here, the resonance angle θ _{2 at} which surface plasmon resonance occurs in the metal film 55 and the incidence of the excitation light α on the metal film 55 where the intensity of the enhanced electric field formed near the metal film 55 based on the surface plasmon resonance is maximized. There is a deviation between the angle (excitation incident angle) θ ₁ , that is, the resonance angle θ ₂ and the excitation incident angle θ ₁ do not match. Therefore, the control processing unit 40 adds or subtracts a predetermined angle from the obtained resonance angle θ ₂ (for example, ± 0.5 °) to determine the excitation incident angle θ ₁ (step S2).

When the control processing unit 40 determines the excitation incident angle θ ₁ , the control processing unit 40 changes the posture of the excitation light emitting unit 20 with respect to the metal film 55 so that the incident angle θ of the excitation light α on the metal film 55 becomes the excitation incident angle θ _1. Change (step S3).

<P-wave light intensity adjustment process>
Next, the control processing unit 40 adjusts the light amount P _p of the P wave component of the excitation light α incident on the metal film 55 to be a target light amount (target light amount) P _t .

Specifically, the control processing unit 40 sets all of the excitation light α incident on the metal film 55 so that the light amount P _p of the P wave component of the excitation light α incident on the metal film 55 becomes the target light amount P _t. adjusting the light intensity _{P p} P-wave component occupying in light amount _{P a.} Then, if not thereby the light amount P _t of the light amount P _p P-wave component is the target, the control unit 40 adjusts the light amount P _a of the excitation light alpha.

Specifically, first, the control processing unit 40 rotates the half-wave plate 27 by the rotation amount adjustment unit 42 to rotate the polarization direction of the excitation light α, so that the total amount of excitation light α incident on the metal film 55 is increased. to change the light intensity _{P p} P-wave component occupying in P _a. Then, if the light quantity P _p of the P wave component of the excitation light α incident on the metal film 55 by the rotation of the half-wave plate 27 becomes the target light quantity P _t , the control processing unit 40 performs the half-wave plate. This process is terminated by stopping the rotation of step 27. If the target light quantity _Pt is not reached, the process proceeds to the next step (step S4). Amount this time, the control unit 40, based on the amount P _p P-wave component of the excitation light α incident on the metal film 55 obtained in P-wave light amount derivation unit 41, the light amount P _p P-wave component is the target It is determined whether or not _Pt has been reached.

The control processing unit 40 rotates the half-wave plate 27 to a position where the amount of the reflected excitation light α _s measured by the reflected light measuring unit 30 is minimized (step S5). When the light amount P _p P-wave component to the total light quantity P _a of the excitation light α this manner is incident to the metal film 55 is maximized, the light amount P _t of the light amount P _p P-wave component is the target if not turned, the next control processing unit 40 changes the light quantity P _a of the excitation light alpha.

Specifically, the control processing unit 40 increases the amount of excitation light α emitted from the light source unit 21 by increasing the amount of current supplied to the light source unit 21, thereby increasing the amount of excitation light incident on the metal film 55. increasing the light amount _{P p} P-wave components up to the light quantity _{P t} to the target (step S6). That is, the control processing unit 40 causes the current amount adjustment unit 44 to change the light source unit from the current supply unit 43 so that the light amount P _p of the P wave component of the excitation light α incident on the metal film 55 becomes the target light amount P _t. The amount of current supplied to 21 is adjusted.

<Excitation fluorescence measurement process>
The control processing unit 40 causes the excitation light α set as described above to enter the metal film 55 to detect the specimen. Specifically, the control processing unit 40 causes the excitation light emission unit 20 to emit the excitation light α in which the light amount P _p of the P wave component in the metal film 55 is adjusted. As a result, the excitation light α causes surface plasmon resonance in the metal film 55, and the fluorescent substance labeled on the specimen captured by the capturing body 56 of the metal film 55 is excited by the enhanced electric field based on this to emit excitation fluorescence. . And the control processing part 40 measures excitation fluorescence by the light measurement part 32 (step S7).

  Specifically, the control processing unit 40 counts the number of excitation fluorescence per unit area based on the output signal sent from the light measurement unit 32 by the calculation unit 45, and obtains the amount of excitation fluorescence.

<Memory / display process>
As described above, the control processing unit 40 obtains the amount of excitation fluorescence, stores it in association with the specimen number, and erases the other storage (step S8). In addition, the control processing unit 40 outputs information based on the amount of excitation fluorescence stored in association with the specimen number to the display unit 16, and the display unit 16 displays the information.

  Finally, the control processing unit 40 returns the excitation light emitting unit 20 to the initial posture (step S9) and ends a series of measurements.

According to the analyzer 10 of the present embodiment, the P wave component of the excitation light α incident on the metal film 55 even if the analysis chip 50 (the prism 51 having the metal film 55 formed on the predetermined surface 53) is replaced. The amount of light P _p can be made constant, thereby suppressing variations in measurement results for each prism 51.

More specifically, when the amount of light P _p of the P wave component is constant in the excitation light α irradiated into the prism 51, if the prism 51 is replaced, for example, the metal film 55 due to a difference in birefringence for each prism 51 or the like. In some cases, the amount of light P _p of the P wave component of the excitation light α incident on the light does not become constant. Therefore, the light quantity of the light quantity P _a and P-wave components incident on the metal film 55 on the basis of the light amount P _s of the reflected excitation light alpha _s after being reflected by the metal film 55 of the excitation light alpha before entering the prism 51 seeking P _p, the light amount P _p is constant by replacing the prism 51 (i.e., the light amount P _t to the _target) by adjusting so that the variation in the measurement accuracy of each prism 51 is suppressed.

Further, according to the present embodiment, since the polarization direction and the light amount of the excitation light α incident on the metal film 55 can be adjusted, the light amount P _p of the P wave component of the excitation light α incident on the metal film 55 is both can be reliably adjusted to be the light amount P _t to the target, it is possible to improve the S / N.

For more information, even if the maximum light amount P _p P-wave component to the total light quantity P _a of the excitation light α to rotate the 1/2-wavelength plate 27 and enters the metal film 55, the excitation light incident on the metal film 55 Even when the light amount P _p of the P wave component of α does not reach the target light amount Pt, the P wave of the excitation light α incident on the metal film 55 by increasing the light amount of the excitation light α emitted from the light source unit 21. it can be a quantity P _t to the target light amount P _p components.

Further, unnecessary light (noise) such as autofluorescence in the prism 51 can be reduced as the amount of the excitation light α incident on the prism 51 is smaller. Therefore, in a state where a light amount P _a and the minimum of the excitation light α of light intensity P _p up and to and the light source unit 21 of the P-wave component to the total light quantity P _a is emitted excitation light α incident on the metal film 55, a metal By setting the light quantity P _p of the P wave component of the excitation light α incident on the film 55 as the target light quantity P _t , variation in measurement results for each prism 51 is suppressed, and noise generated in the prism 51 and the like is reduced. S / N can be improved. As a result, it is possible to detect the sample with high accuracy and high sensitivity.

  The surface plasmon resonance fluorescence analysis apparatus and the surface plasmon resonance fluorescence analysis method of the present invention are not limited to the above-described embodiments, and various changes can be made without departing from the scope of the present invention. is there.

In the analysis apparatus 10 according to the above-described embodiment, the control processing unit 40 controls the entire excitation light α in order to set the light amount P _p of the P wave component of the excitation light α incident on the metal film 55 to the target light amount P _t. after the light amount P _a occupying the P-wave component of the light intensity P _{p (the} ratio of P-wave component to the total light quantity P _a) to a maximum, but is adjusted by changing the amount itself of the pumping light alpha, thereto It is not limited.

For example, the control processing unit rotates the polarization direction of the excitation light α incident on the metal film 55 in a state where the light amount of the excitation light α emitted from the light source unit 21 is constant. The light amount P _p of the P wave component of the excitation light α incident on the metal film 55 may be adjusted by adjusting the rotation amount.

As described above, the excitation light α emitted from the light source unit 21 is kept constant, and the half-wave plate 27 is rotated to set the P-wave component light quantity P _p to the target light quantity P _t. The influence on the measurement result due to the wavelength change caused by changing the light quantity of the light α can be suppressed. That is, if the amount of the excitation light α emitted from the light source unit 21 is changed, the wavelength of the excitation light α may change and this wavelength change may affect the measurement result of the specimen. , And the light amount P _p of the P wave component of the excitation light α is set to the target light amount P _t , the light amount of the excitation light α emitted from the light source unit 21 can be made constant. It is possible to suppress an adverse effect on the measurement result due to the wavelength change of the excitation light α.

Further, in the analyzer that adjusts the light amount P _p of the P wave component in a state where the light amount of the excitation light α emitted from the light source unit 21 is constant, as shown in FIGS. 6 (A) and 6 (B). Preferably, the excitation light emitting unit 20A is provided with the light reducing unit 23 and the control processing unit 40A is provided with the light amount adjusting unit 46 so that the light amount of the excitation light α emitted from the excitation light emitting unit 20A can be reduced. . By adopting such a configuration, the excitation emitted by the light source unit 21 when the light intensity P _p of the P wave component of the excitation light α incident on the metal film 55 is too large to measure the excitation fluorescence by the light measurement unit 32. The amount of excitation light α incident on the metal film 55 can be reduced without changing the amount of light α. That is, while suppressing the wavelength change of the excitation light α and the amount of excitation light α of the light source unit 21 emits a constant, it is possible to reduce the amount of light P _a of the excitation light α incident on the metal film 55.

  Specifically, the light reduction unit 23 includes an ND filter 24 and a position switching unit 25 and adjusts the amount of excitation light α emitted from the light source unit 21. The ND filter 24 is a so-called attenuating filter, which attenuates incident light by a predetermined amount and emits it. The position switching unit 25 switches the position of the ND filter 24 between a filtering position and a retracted position. The position switching unit 25 switches the position of the ND filter 24 in accordance with an instruction signal from the control processing unit 40. The filtering position is a position on the optical path of the excitation light α, and the retracted position is a position deviating from the optical path of the excitation light α. The initial position of the ND filter 24 is a retracted position, and is switched to the filtering position by the position switching unit 25 when the excitation light α incident on the metal film 55 needs to be reduced.

The control processing unit 40 </ b> A includes a P-wave light amount deriving unit 41, a rotation amount adjusting unit 42, a light amount adjusting unit 46, and a calculating unit 45. Light amount adjusting unit 46, based on the amount P _p P-wave component determined by the P-wave light amount derivation unit 41, when it is determined that it is necessary to lower the amount of light P _a of the excitation light α is the position switching unit 25 Driven to switch the ND filter 24 to the filtering position, the light amount P _p of the P wave component of the excitation light α incident on the metal film 55 is reduced by a predetermined amount. On the other hand, the light amount adjustment unit 46, if it is determined that based on the amount P _p P-wave component determined by the P-wave light amount derivation unit 41, there is no need to reduce the amount of light P _a of the excitation light α is located switching unit The ND filter 24 is left in the initial position without driving 25.

In addition, for example, the control processing unit 40 increases or decreases the light amount P _p of the P wave component of the excitation light α incident on the metal film 55 by changing the light amount Pa of the excitation light α emitted by the excitation light emitting unit 20. it may be to the amount of light P _t of the target Te. In this case, the linear polarization unit 22 and the polarization direction adjustment unit 26 (the half-wave plate 27 and the rotation drive unit 28) may be omitted, and the apparatus can be simplified and the cost can be reduced.

In the above embodiment, the linearly polarized light portion 22 and the half-wave plate 27 are disposed between the light source portion 21 and the prism 51, and the half-wave plate 27 is rotated, thereby exciting the light incident on the metal film 55. Although the light quantity P _p of the P wave component of the light α is adjusted, the present invention is not limited to this. For example, by arranging a linearly polarizing portion 22 and a quarter wavelength plate composed of a linearly polarizing plate or the like between the light source 21 and the prism 51 and rotating the linearly polarizing portion (linearly polarizing plate), the metal film The light quantity P _p of the P wave component of the excitation light α incident on 55 may be adjusted.

  In the embodiment described above, the measurement range (the range of the measurement range) of the amount of excitation fluorescence (fluorescence from the fluorescent substance labeled on the specimen) in the analyzer 10 is the same as the measurement range of the light measurement unit 32. The light amount of the excitation light α emitted by the excitation light emitting unit 20 may be switched stepwise so that the measurement range of the light amount of excitation fluorescence in the analyzer 10 may be wider than the measurement range of the light measurement unit 32. In such an analyzer 10, for example, the control processing unit 40 changes the intensity of the excitation light α emitted by the excitation light emitting unit 20 based on the amount of excitation fluorescence measured by the light measurement unit 32. 47 and a correction unit 48 that corrects the measurement result of the excitation fluorescence by the light measurement unit 32 based on the intensity change of the excitation light α by the intensity change unit 47. In the above embodiment, the current supply unit 43 and the current amount adjustment unit 44 also function as the intensity changing unit 47, and the calculation unit 45 also functions as the correction unit 48 (see FIG. 3).

  In this analyzer, for example, a sample is measured as follows.

  The control processing unit 40 sets the output (light amount) of the excitation light α emitted by the excitation light emitting unit 20 to 100 μW that achieves the minimum detection sensitivity of the light measurement unit 32. This minimum detection sensitivity is the minimum amount of light that can be measured by the light measurement unit 32 when measuring the excitation fluorescence generated by exciting the fluorescent substance labeled on the specimen with an enhanced electric field based on surface plasmon resonance. It is. On the other hand, the maximum detection sensitivity is the maximum amount of light that can be measured by the light measurement unit 32 when the excitation fluorescence is measured.

  The control processing unit 40 measures the detection of the specimen using the above-excited excitation light α, that is, the measurement of the excitation fluorescence emitted from the fluorescent substance labeled on the specimen by the enhanced electric field, by the light measurement section. At this time, if the control processing unit 40 determines that the amount of excitation fluorescence is larger than the measurement range (measurement range) of the light measurement unit 32, the excitation light emitted by the excitation light emitting unit 20 as shown in FIG. The amount of α is adjusted to 1 μW. When the amount of the excitation light α decreases, the intensity of the enhanced electric field formed in the vicinity of the metal film 55 decreases, so that the amount of fluorescence emitted from the fluorescent material excited by the enhanced electric field also decreases. Thus, when the amount of excitation fluorescence falls within the measurement range of the light measurement unit 32, the light measurement unit 32 measures the amount of excitation fluorescence, and the control processing unit 40 (calculation unit 45, etc.) detects the specimen. .

  On the other hand, even if the light amount of the excitation light α is lowered in this way, when the light amount of the excitation fluorescence is larger than the measurement range of the light measurement unit 32, the control processing unit 40 further performs excitation that the excitation light emitting unit 20 emits. The amount of light α is adjusted to 0.01 μW. Thus, when the amount of excitation fluorescence falls within the measurement range of the light measurement unit 32, the specimen is detected as described above.

  As described above, the control processing unit 40 switches the light amount of the excitation light α emitted by the excitation light emitting unit 20 in a stepwise manner so that the measurement range of the analyzer 10 itself is wider than the measurement range of the light measurement unit 32. be able to.

  In this case, since the amount of the excitation light α incident on the metal film 55 is changed (that is, the intensity of the enhanced electric field that excites the fluorescence is changed), the control processing unit 40 (in detail, The correction unit 48) corrects the measurement result of the light measurement unit 32. For example, when the light amount of the excitation light α is reduced to 1/10, the correction unit 48 increases the measurement result (excitation fluorescence amount) obtained by the light measurement unit 32 by 10 times, and the light amount of the excitation light α is reduced to 1/100. In this case, the measurement result (excitation fluorescence amount) obtained by the light measurement unit 32 is multiplied by 100.

  Further, as shown in FIG. 7, the light quantity to be switched step by step is lower in the minimum detection sensitivity in the light quantity after the light quantity is lowered by one step than in the maximum detection sensitivity in the light quantity before the light quantity is lowered. It is preferable to set each light quantity (output) so as to be slightly smaller in the axial direction. In the horizontal axis direction in the figure, when the minimum detection sensitivity at the light amount after lowering the light amount by one step is larger than the maximum detection sensitivity at the light amount before lowering the light amount, a continuous measurement range cannot be obtained.

  In the analysis apparatus 10 of the above embodiment, the analysis chip 50 is replaced every time the sample is detected, but the present invention is not limited to this. That is, the analyzer 10 may be an analyzer that incorporates an analysis chip in a part of the analyzer 10 and detects a sample by repeatedly using one analysis chip.

DESCRIPTION OF SYMBOLS 10 Surface plasmon resonance fluorescence analyzer 12 Chip holding part 21 Light source part 27 1/2 wavelength plate 28 Rotation drive part 30 Reflected light measurement part 32 Light measurement part (fluorescence measurement part)
40, 40A Control processing unit (control unit)
50 Analysis chip 51 Prism 53 Deposition surface (predetermined surface)
55 incident angle of the excitation light to the metal film P _a pumping light quantity P _p the pumping light quantity alpha excitation light alpha _s reflected excitation light θ metal film to light intensity P _t target light amount P _s reflected excitation light of a P-wave component of the

Claims (9)

A surface plasmon resonance fluorescence analyzer that measures fluorescence emitted by excitation of an electric field based on surface plasmon resonance by a fluorescent substance attached to the sample or the sample,
A chip holder that can hold an analysis chip including a prism having a metal film formed on a predetermined surface so as to be detachable;
A light source unit that emits excitation light and causes the excitation light to enter the prism so as to be reflected by the metal film of the prism included in the analysis chip held by the chip holding unit;
A reflected light measurement unit that measures the amount of reflected excitation light that is excitation light reflected by the metal film;
Based on the amount of excitation light emitted from the light source unit and the amount of reflected excitation light measured by the reflected light measurement unit, the amount of P wave component of the excitation light incident on the metal film is adjusted to a specific amount of light. A P-wave light amount adjustment unit that
The light source unit changes the amount of excitation light emitted according to the amount of current supplied,
The P-wave light amount adjusting unit includes a half-wave plate disposed on an optical path of excitation light emitted from the light source unit between the light source unit and the prism, and a polarization direction of excitation light with respect to the metal film Measured by a rotation drive unit that rotates the half-wave plate so that the wavelength changes, a light source current supply unit that supplies current to the light source unit, and the amount of excitation light emitted from the light source unit and the reflected light measurement unit And a controller that controls the amount of current supplied by the light source current supply unit and drives the rotation drive unit based on the amount of reflected excitation light that is
The control unit includes a P-wave component of the total amount of excitation light incident on the metal film based on the amount of excitation light emitted from the light source unit and the amount of reflected excitation light measured by the reflected light measurement unit. A rotation amount adjustment unit for adjusting the rotation amount of the half-wave plate by controlling the rotation drive unit so that the amount of light is maximized;
Based on the amount of excitation light emitted by the light source unit and the amount of reflected excitation light measured by the reflected light measurement unit in a state where the amount of P wave component in the total amount of excitation light is maximized. A surface plasmon resonance fluorescence analyzer , comprising: a current amount adjustment unit that controls the light source current supply unit to adjust an amount of current supplied to the light source unit . A surface plasmon resonance fluorescence analyzer that measures fluorescence emitted by excitation of an electric field based on surface plasmon resonance by a fluorescent substance attached to the sample or the sample,
A prism having a metal film formed on a predetermined surface;
A light source unit that emits excitation light and causes the excitation light to enter the prism so as to be reflected by the metal film of the prism;
A reflected light measurement unit that measures the amount of reflected excitation light that is excitation light reflected by the metal film;
Based on the amount of excitation light emitted from the light source unit and the amount of reflected excitation light measured by the reflected light measurement unit, the amount of P wave component of the excitation light incident on the metal film is adjusted to a specific amount of light. A P-wave light quantity adjustment unit that
The light source unit changes the amount of excitation light emitted according to the amount of current supplied,
The P-wave light amount adjusting unit includes a half-wave plate disposed on an optical path of excitation light emitted from the light source unit between the light source unit and the prism, and a polarization direction of excitation light with respect to the metal film Measured by a rotation drive unit that rotates the half-wave plate so that the wavelength changes, a light source current supply unit that supplies current to the light source unit, and the amount of excitation light emitted from the light source unit and the reflected light measurement unit And a controller that controls the amount of current supplied by the light source current supply unit and drives the rotation drive unit based on the amount of reflected excitation light that is
The control unit includes a P-wave component of the total amount of excitation light incident on the metal film based on the amount of excitation light emitted from the light source unit and the amount of reflected excitation light measured by the reflected light measurement unit. A rotation amount adjustment unit for adjusting the rotation amount of the half-wave plate by controlling the rotation drive unit so that the amount of light is maximized;
Based on the amount of excitation light emitted by the light source unit and the amount of reflected excitation light measured by the reflected light measurement unit in a state where the amount of P wave component in the total amount of excitation light is maximized. A surface plasmon resonance fluorescence analyzer , comprising: a current amount adjustment unit that controls the light source current supply unit to adjust an amount of current supplied to the light source unit .   The P wave light amount adjustment unit obtains the light amount of the P wave component from the difference between the light amount of the excitation light emitted from the light source unit and the light amount of the reflected excitation light measured by the reflected light measurement unit, and based on the light amount The surface plasmon resonance fluorescence analyzer according to claim 1 or 2, wherein the state of the excitation light is adjusted.   The P-wave light amount adjusting unit includes a half-wave plate disposed on an optical path of excitation light emitted from the light source unit between the light source unit and the prism, and a polarization direction of excitation light with respect to the metal film The rotational drive unit that rotates the half-wave plate so that the light source changes, and the rotational drive based on the amount of excitation light emitted from the light source unit and the amount of reflected excitation light measured by the reflected light measurement unit The surface plasmon resonance fluorescence analyzer according to any one of claims 1 to 3, further comprising a control unit that drives the unit. The light source unit changes the amount of excitation light emitted according to the amount of current supplied,
The P-wave light amount adjustment unit is based on a light source current supply unit that supplies current to the light source unit, a light amount of excitation light emitted from the light source unit, and a light amount of reflected excitation light measured by the reflected light measurement unit. 4. The surface plasmon resonance fluorescence analyzer according to claim 1, further comprising a control unit that controls an amount of current supplied by the light source current supply unit. 5. A fluorescence measuring unit capable of measuring the intensity of the fluorescence generated on the surface of the metal film opposite to the prism by reflecting the excitation light on the metal film;
An intensity changing unit that changes the intensity of the excitation light that enters the prism based on the amount of fluorescent light measured by the fluorescence measuring unit;
Surface according to any one of claims 1 to 5, characterized in that and a correcting unit for correcting the fluorescence measurement result by the fluorescence measurement unit based on the intensity change of the excitation light by the intensity changing portion Plasmon resonance fluorescence analyzer. A fluorescence measurement unit capable of measuring the intensity of the fluorescence generated on the surface of the metal film opposite to the prism by reflecting the excitation light on the metal film;
The specific light amount is such that the amount of fluorescent light generated from the fluorescent material attached to the specimen or the specimen by reflecting the excitation light on the metal film is equal to or greater than the lower limit of the quantity of light that can be measured by the light measurement unit. The surface plasmon resonance fluorescence analyzer according to any one of claims 1 to 6, wherein The specific amount of light is such that the amount of fluorescent light generated from the specimen or a fluorescent substance attached to the specimen by reflecting the excitation light on the metal film is less than or equal to the upper limit of the quantity of light that can be measured by the light measuring unit. 8. The surface plasmon resonance fluorescence analyzer according to claim 7 , A surface plasmon resonance fluorescence analysis method for measuring light emitted when a specimen or a fluorescent substance attached to the specimen is excited by an electric field based on surface plasmon resonance,
  Preparing a prism in which a metal film is formed on a predetermined surface, and a step of flowing a sample solution containing the specimen on the metal film;
  An excitation light irradiation step for causing excitation light to enter the prism after the preparation step so that surface plasmon resonance occurs in the metal film by being reflected by the metal film;
  Excitation incident on the metal film from the amount of excitation light incident on the prism and the amount of reflected excitation light reflected by the metal film in a state where surface plasmon resonance occurs in the metal film A measurement process for measuring the amount of P wave light;
  Excitation incident on the metal film based on the amount of excitation light emitted from the light source unit that causes the excitation light to enter the prism and the amount of reflected excitation light reflected by the metal film and measured by the reflected light measurement unit A half-wave plate arranged on the optical path of the excitation light emitted from the light source unit between the light source unit and the prism so that the light amount of the P wave component in the total light amount is maximized. The amount of rotation is adjusted by the control unit, and the light amount of the excitation light emitted by the light source unit and the reflected light measurement unit are measured with the light amount of the P wave component occupying the total light amount of the excitation light being maximized. A P-wave light amount adjustment step of adjusting the amount of current supplied to the light source unit by the control unit based on the amount of reflected excitation light;
  A surface plasmon resonance fluorescence characterized by comprising: an adjustment step of adjusting at least one of the light amount and the deflection direction of the excitation light irradiated in the excitation light irradiation range based on the measurement of the P-wave light amount in the measurement step. Analysis method.

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