JP5487150B2 - Optical measuring method and optical measuring apparatus - Google Patents

Description

  The present invention relates to an optical measurement method and an optical measurement apparatus for optically measuring a substance to be detected in a sample solution.

  In bio-measurement and the like, the fluorescence method is widely used as a highly sensitive and easy measurement method. The fluorescence method is qualitative or qualitative by irradiating a sample considered to contain a substance to be detected that emits fluorescence when excited by light of a specific wavelength, by irradiating the excitation light of the specific wavelength and detecting the fluorescence emitted at this time. This is a method for quantitatively confirming the presence of a substance to be detected. In addition, when the substance to be detected is not a fluorescent material, the substance to be detected is labeled with a fluorescent label such as an organic fluorescent dye, and then the fluorescence is detected in the same manner. It is a method to confirm.

  In the above-described fluorescence method, there is a technique in which only a specific target substance can be efficiently detected while flowing a sample, and the target substance is fixed to the surface of the sensor unit by the following two methods and then fluorescence detection is performed. It is common. One such technique is, for example, when the substance to be detected is an antigen, the antigen is specifically bound to the primary antibody immobilized on the surface of the sensor unit, and then a fluorescent label is attached. A secondary antibody that specifically binds to the antigen, and further binds to the antigen to form a primary antibody-antigen-secondary antibody binding state, and the fluorescence from the fluorescent label attached to the secondary antibody This is a so-called sandwich method for detection. The other is, for example, when the substance to be detected is an antigen, a secondary antibody in which an antigen and a fluorescent label are attached to the primary antibody immobilized on the surface of the sensor unit (unlike the above-described secondary antibody, Is a so-called competition method in which the primary antibody is bound to the primary antibody competitively and the fluorescence from the fluorescent label attached to the competitively bound secondary antibody is detected. .

  In addition, for the reason that the S / N ratio can be improved in fluorescence detection, an evanescent fluorescence method has been proposed in which a fluorescent label indirectly fixed to a sensor unit by the above-described method is excited by evanescent light. In the evanescent fluorescence method, excitation light is incident from the back surface of the sensor unit, and the fluorescent label is excited by evanescent light that oozes out to the surface of the sensor unit, and fluorescence generated from the fluorescent label is detected.

  On the other hand, in order to improve sensitivity in the evanescent fluorescence method, a method using the effect of electric field enhancement by plasmon resonance has been proposed. In this surface plasmon enhanced fluorescence method, in order to generate plasmon resonance, a metal layer is provided in the sensor portion, surface plasmon is generated in this metal layer, and the signal intensity is increased by the electric field enhancing action to improve the SN ratio. Is.

  As described above, various methods have been proposed as measurement methods in biomeasurement and the like.

JP-A-1-221667

  By the way, in the optical measuring apparatus as described above, generally, the presence of a substance to be detected is confirmed by measuring the amount of fluorescence generated in the sensor unit using a light detection element such as a photodiode or CCD. Is going.

  In this case, if there is an optical distortion in the excitation light incident part of the prism or sensor chip that makes the excitation light incident on the sensor part, not only the fluorescence generated in the sensor part but also the excitation light scattered in this distortion part. There is a risk that accurate measurement cannot be performed because the light enters the light detection element. Further, the same problem occurs when the excitation light is reflected on each surface of the prism and the excitation light introducing portion and unnecessary reflected light is incident on the light detection element.

  In order to eliminate the influence of excitation light on such a detection signal, in Patent Document 1, a phosphorescent label having a long emission duration is used as a photolabel of a photolabel-binding substance to be bound to a substance to be detected. It has been proposed to eliminate the influence of excitation light by stopping the irradiation of excitation light after excitation and detecting the phosphorescence generated from the phosphorescent label in the state where the excitation light is not irradiated.

  However, in order to accurately measure the amount of light having a long emission time, such as phosphorescence, a light detection element having sufficient time resolution with respect to the emission duration is used, and a plurality of times during the emission duration. Since it is necessary to perform measurement and integrate all measurement results, there is a problem that the load during measurement increases.

  The present invention has been made in view of the above problems, and in an optical measurement method and an optical measurement apparatus for optically measuring a substance to be detected in a sample solution, the influence of excitation light is eliminated with a low load. It is intended to enable measurement with a high S / N ratio.

  In the optical measurement method of the present invention, the amount of the substance to be detected contained in the sample liquid is obtained by bringing the sample liquid containing the substance to be detected into contact with the sensor part formed on one surface of the dielectric plate of the sensor chip. The photo-label binding substance corresponding to the amount is bound on the sensor unit, and the sensor unit is irradiated with excitation light at an incident angle at which a total reflection condition is obtained, thereby generating a photoelectric field on the sensor unit. In an optical measurement method that measures the amount of a substance to be detected by exciting the light label of the photolabel binding substance and measuring the light generated by this excitation, the excitation light corresponds to the quantity of the substance to be detected. The signal component indicating the measured value and the noise component due to the stray light of the excitation light are both modulated at a low frequency indicating a synchronized intensity change and irradiated to the sensor unit. Synchronize The component is extracted to obtain a low-frequency measurement signal, and the excitation light is frequency-modulated at a high frequency that shows an intensity change in which only the noise component is synchronized. A high frequency measurement signal is acquired by extracting a component synchronized with the frequency, and a signal having only a signal component is acquired by obtaining a difference between the low frequency measurement signal and the high frequency measurement signal.

In the optical measurement method of the present invention, “noise component caused by stray light of excitation light” means an error component generated by detecting scattered light or reflected light of excitation light.
Further, “frequency modulation” with respect to the excitation light means that the intensity or polarization state of the excitation light is modulated at a constant frequency.

  Further, “low frequency” and “high frequency” mean a relative high-low relationship between frequencies, and do not indicate a specific frequency band. The reason why both the signal component and noise component are detected when the frequency for modulating the excitation light (frequency modulation) is low, and the reason why only the noise component is detected when the frequency is high, is described below. explain.

  Here, processing in the optical measurement method of the present invention will be described in detail with reference to FIG. FIG. 5 is a graph showing the result when the excitation light is modulated at a certain frequency and irradiated to the sensor unit, and the intensity of light that varies in synchronization with the modulation frequency of the excitation light is extracted from the measured light. The horizontal axis of the graph represents the modulation frequency of the excitation light, and the vertical axis represents the effective value of the intensity of light that varies in synchronization with the modulation frequency of the excitation light.

Usually, the response speed of fluorescence as a signal component is slower than the response speed of frequency modulation of excitation light. On the other hand, scattered light or reflected light of excitation light, which is a noise component, is generated by irradiation of excitation light, and therefore responds in synchronization with the modulation frequency of excitation light. When the emission lifetimes of the signal component and the noise component are set as τs and τn, respectively, the real part of the frequency response component of the fluorescence signal obtained from a general fluorescent material with Debye relaxation is obtained as follows. Here, ω represents an angular frequency, and ω = 2πf. H _S , H _n , and H _∞ represent the magnitudes of the signal component, the noise component, and the faster relaxation component among the real parts of the frequency response component. FIG. 5 illustrates a case where H _n = H _S / 10 and H _∞ = 0.

That is, since the noise component has a short emission lifetime and a high relaxation frequency, the noise component is emitted at the same frequency as the excitation light even when the frequency for modulating the excitation light is high. As indicated by the alternate long and short dash line in FIG. 5, the signal intensity of the detection signal synchronized with the modulation frequency of the excitation light is approximately constant at 0.1 to 1000 kHz. The relaxation frequency is defined by the reciprocal of the relaxation time, and is represented by the reciprocal of the light emission lifetime. That is, the relaxation frequency fs of the signal component is expressed as fs = 1 / τs, and the relaxation frequency fn of the noise component is expressed as fn = 1 / τn. Since the fluorescence lifetime of the noise component is short, the relaxation frequency becomes high.
On the other hand, since the signal component has a low relaxation frequency, as shown by the solid line in FIG. 5, the modulation frequency of the pumping light is low in the low frequency region (in the case of FIG. 5, the region is approximately 1 kHz or less). Since the intensity of fluorescence occurs following this, it can be acquired as a detection signal synchronized with the modulation frequency of the excitation light. However, in the high frequency range (in the case of FIG. Since it cannot follow the modulation frequency, the intensity of the fluorescence does not occur and the light always shines. Therefore, it cannot be acquired as a detection signal synchronized with the modulation frequency of the excitation light.

  When measuring the light quantity of the sensor unit, as shown by the dotted line in FIG. 5, it is detected in a state where a noise component is superimposed on the signal component, but the signal component is not included in the high frequency region as described above. By measuring at two points, a low frequency region (for example, 0.1 kHz) that includes both the signal component and the noise component, and a high frequency region (for example, 1000 kHz) that includes only the noise component, the difference between the two is obtained. A signal having only a signal component can be acquired.

  In this case, the slower the response speed of the optical label that is the signal component, the less it becomes possible to follow the modulation frequency of the excitation light at a lower frequency. Therefore, when performing the processing of the present invention, it is easy to separate the signal component and the noise component. become. Therefore, in the optical measurement method of the present invention, it is preferable to use a phosphorescent label as the photolabel.

  Further, frequency modulation may be performed by modulating the intensity of the excitation light.

  In addition, as the sensor chip, the sensor part has a laminated structure including a metal layer adjacent to the dielectric plate, and the plasmon is excited by irradiating excitation light to generate a photoelectric field enhanced by the plasmon. You may make it show.

  In this case, frequency modulation may be performed by modulating the polarization state of the excitation light.

  Furthermore, in this case, phase difference information regarding the phase difference of birefringence that occurs in the excitation light from when the excitation light enters the dielectric plate until it reaches the sensor unit is acquired, and the excitation light is obtained based on the phase difference information. The polarization state of the excitation light may be controlled so that a phase difference opposite to the phase difference is generated. In addition, the reflected light of the excitation light in the sensor unit is detected, and the ratio of the minimum value to the maximum value of the reflected light intensity is minimized based on the intensity of the reflected light modulated in synchronization with the modulation of the polarization state. The polarization state may be controlled.

  An optical measuring device of the present invention is an optical measuring device used in the above optical measuring method, and is provided at a position of a housing portion for housing a sensor chip and a sensor portion of the sensor chip housed in the housing portion. Excitation light irradiation optical system for irradiating excitation light, frequency modulation means for performing frequency modulation on the excitation light, light detection means for detecting light caused by excitation, and detected by the light detection means A frequency analysis means for extracting a component synchronized with a frequency for modulating the excitation light from a signal based on light, and an arithmetic means for obtaining a signal of only the signal component by obtaining a difference between the low frequency measurement signal and the high frequency measurement signal; It is characterized by providing.

  According to the optical measurement method and the optical measurement apparatus of the present invention, when optically measuring a substance to be detected in a sample solution, excitation light is excited with a signal component indicating a value corresponding to the amount of the substance to be detected. Both the noise component due to stray light of the light is modulated at a low frequency that shows a synchronized intensity change and irradiated to the sensor unit, and the component that is synchronized with the low frequency is extracted from the light generated due to excitation. Acquires frequency measurement signal, modulates the excitation light at a high frequency showing the intensity change synchronized only with the noise component, irradiates the sensor part, and extracts the component synchronized with the high frequency from the light caused by the excitation Since the high frequency measurement signal is obtained and the difference between the low frequency measurement signal and the high frequency measurement signal is obtained, the signal of only the signal component is obtained with a small number of measurements and simple calculation. With no load, it is possible to measurement with high SN ratio by eliminating the influence of the excitation light.

  In the optical measurement method of the present invention, when a phosphorescent label is used as the light label, the signal component and the noise component can be easily separated as described above.

1 is a schematic cross-sectional view showing a first embodiment of an optical measuring device of the present invention. The schematic perspective view which shows the sensor chip of the optical measuring device of this invention Schematic sectional view showing a sensor chip of the optical measuring device of the present invention Schematic sectional view showing steps of immunoassay measurement using the optical measurement method of the present invention A graph showing the results when the excitation light is frequency-modulated and applied to the sensor unit, and the fluorescence intensity is measured in synchronization with the modulation frequency. Graph of voltage applied to modulation element when frequency modulation of excitation light Schematic sectional view showing a second embodiment of the optical measuring device of the present invention Schematic sectional view showing a third embodiment of the optical measuring device of the present invention

  Hereinafter, although an embodiment of the present invention is described using a drawing, the present invention is not limited to this. In addition, for easy visual recognition, the scale of each component in the drawings is appropriately changed from the actual one.

“First Embodiment of Optical Measuring Device and Optical Measuring Method”
As shown in FIGS. 1, 2, 3, and 4, the optical measuring device 1 of the present embodiment is disposed in a dry manner on the upstream side of the flow path base material 30 that forms the flow path 33 and the flow path 33. A sensor chip C1 including a phosphorescent label binding substance BF and a detection unit (sensor unit) 38 including a metal film 34a formed in a predetermined region in the channel 33, a light source 10 that emits excitation light Le, a channel group A light guide member 14 for guiding the excitation light Le from one side through the flow path base material 30 to the interface so that the excitation light Le satisfies the total reflection condition at the interface between the material 30 and the metal film 34a; A polarization modulation element 11 that controls the polarization state of the excitation light Le so as to periodically modulate the polarization direction at the interface of Le, and a function generator that generates a periodic clock of a voltage that is a basis of the modulation period of the polarization direction (FG) 21 and this From the polarization modulation element driver 22 that drives the polarization modulation element 11 according to the periodic clock of the voltage generated in the FG 21, and the detection unit 38 or the detection unit 39 that is arranged on the other side with respect to the interface, due to excitation. A photodetector 23 for detecting the generated signal light, a frequency analysis unit 24 for detecting only a signal component synchronized with the period of modulation in the polarization direction, and a calculation unit 25 for calculating a signal obtained by the frequency analysis unit 24 And a control unit 20 for controlling the entire apparatus. Here, a substance B1 (specific binding substance) that specifically binds to the test substance A in the sample is fixed on the surface of the metal film 34a.

  As shown in FIGS. 2 and 3, the sensor chip C1 includes a flow path substrate (dielectric plate) 30 having a flow path 33 having metal films 34a and 34b open in a predetermined region and an open top, and the flow path base. And a lid member 32 mounted on the material 30 so as to form the upper surface of the flow path 33. Here, FIG. 2 is a schematic perspective view showing the entire configuration of the chip C1 according to this embodiment, and FIG. 3 is a schematic cross-sectional view in the zx plane passing through the metal film 34a of the chip C1 in FIG. .

  The flow path base material 30 includes a flow path 33 for flowing a sample containing the test substance A, a transmission surface 30b for transmitting the excitation light Le into the flow path base material 30, and the transmission surface 30b. A protective portion 30a that protects against dirt is formed. In this embodiment, the flow path base material (dielectric plate) 30 also functions as a prism (that is, the entire flow path base material (dielectric plate) 30 also serves as a prism, and a clear boundary between the prism portions). Not) The material of the channel substrate 30 is formed from a transparent material such as transparent resin or glass, for example. The channel substrate 30 is preferably formed from a resin. In this case, polymethyl methacrylate (PMMA), polycarbonate (PC), amorphous polyolefin (APO) containing cycloolefin, polystyrene, and ZEONEX (registered) It is more desirable to use a resin such as a trademark.

  The flow path 33 is formed by attaching the lid member 32 to the flow path base material 30 so as to cover the U-shaped groove formed on the flow path base material 30. In the present specification, the width and height of the flow channel (both lengths in the direction perpendicular to the traveling direction of the sample) are not particularly limited. Furthermore, a liquid reservoir for submerged or waste liquid is formed at both ends of the flow path 33. Further, metal films 34 a and 34 b constituting the detection units (sensor units) 38 and 39 are formed in predetermined regions of the flow path 33. In the present embodiment, a metal film 34a is provided for measurement and a metal film 34b is provided for reference. However, the detection unit need only have one measurement detection unit 38, and the reference detection unit 39 as described above is not necessarily required. The material of the metal films 34a and 34b is not particularly limited, and examples thereof include Au, Ag, Cu, Pt, Ni, Ti and the like from the viewpoint of efficiently inducing plasmons, and Au having a high electric field enhancing effect. , Ag and the like are particularly preferable. The thicknesses of the metal films 34a and 34b are preferably determined as appropriate so that the surface plasmon is strongly excited by the material of the metal films 34a and 34b and the wavelength of the excitation light Le. For example, when a laser beam having a center wavelength of 780 nm is used as the excitation light Le and an Au film is used as the metal films 34a and 34b, the thickness of the metal films 34a and 34b is preferably 50 nm ± 5 nm.

  The lid member 32 is for forming the upper surface of the flow path 33 by being attached to the flow path base material 30. The lid member 32 has an inlet 35a for flowing down a sample or the like connected to a liquid reservoir for submerging, and an air hole 35b for removing air or the like connected to a liquid reservoir for waste liquid. . As the material of the lid member 32, the same material as that of the flow path base material 30 described above can be used. The lid member 32 is attached by ultrasonic fusion or the like after the metal film is formed.

  The light source 10 may be, for example, a laser light source and is not particularly limited, but can be appropriately selected according to detection conditions. Further, as described above, the light source 10 totally reflects the excitation light Le at the interface between the flow path base material 30 of the sensor chip C1 and the metal film 34a, and is incident at a resonance angle that causes surface plasmon resonance at the metal film 34a. Are arranged as follows. The excitation light Le is generally incident on the interface with p-polarized light so as to induce surface plasmons.

  The light guide member 14 is particularly limited as long as it guides the excitation light Le to this interface so that the excitation light Le satisfies the total reflection condition at the interface between the flow path base material 30 and the metal film 34a. Instead, a lens, a mirror, or the like can be used.

  The polarization modulation element 11, the function generator (FG) 21, and the polarization modulation element driver 22 function as frequency modulation means in the present invention as a whole. By this frequency modulation means, the polarization direction of the excitation light Le at the interface between the flow path base material 30 and the metal film 34a is changed with the same period as the signal output from the FG 21. The polarization modulation element 11 of the present embodiment is an element for controlling the polarization state of the excitation light Le by voltage control. Specifically, as shown in FIG. 6, when a first voltage is input, In this element, the polarization direction of the excitation light Le is p-polarized light, and when the second voltage is input, the polarization direction of the excitation light Le is s-polarized light. As such, a Pockels cell using an electro-optic effect can be mentioned. However, the polarization modulation element 11 is not limited to this, and uses a λ / 2 wavelength plate, a λ / 4 wavelength plate, a polarizing plate, a Babinesoleil plate, and a near field as disclosed in JP-A-2006-330105. A polarized light modulation element or the like may be used. The modulation waveform in the polarization direction (more specifically, the modulation waveform of the p-polarized component (or s-polarized component)) is a rectangular waveform in which the first voltage and the second voltage are switched as shown in FIG. Alternatively, it may be sinusoidal. For example, when the Pockels cell is controlled with a rectangular wave voltage, the former is used, and when the wave plate is rotated, the latter is used. The FG 21 receives a trigger from the control unit 20, generates a periodic clock (voltage modulation waveform) of a voltage that is a basis of the modulation waveform in the polarization direction according to the waveform specified by the control unit 20, and supplies the polarization modulation element 11 with the cycle clock. The output is not particularly limited, and other waveform generators may be used. Further, the FG 21 outputs a reference signal having the same cycle as the cycle clock of the voltage output to the polarization modulation element to the frequency analysis unit 24. The modulation waveform of the voltage generated by the FG 21 can be appropriately set such as a rectangular wave shape or a sine wave shape. The polarization modulation element driver 22 is connected to the FG 21 and drives the polarization modulation element 11 according to the waveform of a signal generated by the FG 21. However, the modulation of the polarization direction is not necessarily periodic.

  The photodetector 23 only needs to quantitatively detect the phosphorescence Lf emitted when the phosphorescent label F contained in the sample is excited, and can be appropriately selected according to the detection conditions. A diode), a photomultiplier tube, a c-MOS, or the like can be used. The light detector 23 also detects scattered light and reflected light (not shown) caused by excitation as stray light of excitation light. Since the scattered light and reflected light of the excitation light are detected together with the phosphorescence Lf, they become noise components.

  The frequency analysis unit 24 receives the reference signal output from the FG 21, and varies with a signal component synchronized with the period of the period clock of the voltage output from the FG 21 to the polarization modulation element 11, that is, with the same period as the period clock of the voltage. For example, a lock-in amplifier or the like can be used.

  As will be described later, the calculation unit 25 calculates a difference between the low frequency measurement signal and the high frequency measurement signal obtained by the frequency analysis unit 24.

  The control unit 20 controls the operation content of each component on the apparatus and controls the timing of each operation. Further, the control unit 20 controls the timing of waveform generation of the FG 21 and the signal reception timing of the frequency analysis unit 24, so that the control unit 20 is a synchronization control unit that synchronizes the FG 21 and the frequency analysis unit 24. You can also.

  On the other hand, in the optical measurement method of the present embodiment, the sample containing the test substance A is dropped onto the sensor chip C1 using the measurement apparatus 1, and the sample is brought into contact with the phosphorescent label binding substance BF disposed in a dry state. The detection light is allowed to flow down to the detection unit 38, and the excitation light Le is guided from one side to the interface through the flow path base material 30 so that the excitation light Le satisfies the total reflection condition at the interface between the flow path base material 30 and the metal film 34a. Both the signal component showing a value corresponding to the amount of the test substance A and the noise component caused by the stray light of the excitation light Le are periodically detected, and the polarization direction at the interface of the excitation light Le is periodically changed at a low frequency. In addition to irradiating the excitation unit Le with the excitation light Le and extracting the component synchronized with the low frequency from the measured light to obtain a low frequency measurement signal, only the noise component is detected at a high frequency. Encouragement The polarization direction at the interface of the light Le is periodically modulated to irradiate the detection unit 38 with the excitation light Le, and a component synchronized with the high frequency is extracted from the measured light to obtain a high frequency measurement signal, The presence and / or amount of the test substance A is measured by obtaining a difference between the low-frequency measurement signal and the high-frequency measurement signal.

  Hereinafter, for example, the fluorescence method in the present embodiment for detecting the antigen A from the sample So containing the antigen A as the test substance will be described in detail. In the fluorescence method of the present embodiment, the phosphorescent label F is immobilized on the metal film 34a via the primary antibody B1, the antigen A, and the secondary antibody B2 by performing an assay by the sandwich method described later, and then the light source 10 Excitation light Le emitted from the sensor chip C1 is incident on the interface between the channel substrate 30 and the metal film 34a of the sensor chip C1 at a specific incident angle equal to or greater than the total reflection angle to excite the evanescent wave. By resonating with free electrons in the metal film 34a, surface plasmons are generated in the metal film 34a, and the phosphorescent label F is excited by the enhanced electric field Ew by the surface plasmons to generate phosphorescence Lf. The amount of phosphorus detected by the detector 23 is processed by the frequency analysis unit 24.

  Here, in the above example, it is the phosphorescent label F that is actually confirmed by phosphorescence detection, but it is considered that this phosphorescent label F is not fixed on the metal film 34a without the antigen A. By confirming the presence of phosphorescent label F, the presence of antigen A is indirectly confirmed.

  The primary antibody B1 is a specific binding substance in the present embodiment, and can be appropriately selected according to detection conditions (especially a test substance) without particular limitation. For example, when the antigen is a CRP antigen (molecular weight 110,000 Da), a monoclonal antibody that specifically binds to this antigen (at least an epitope different from the secondary antibody B2) can be used. It can be fixed on the metal film 34a.

  The phosphorescent label-binding substance BF is composed of the phosphorescent label F and the secondary antibody B2 modified thereto. The phosphorescent label F is excited by the surface plasmon-enhanced electric field Ew generated when the excitation light Le is totally reflected by the metal film 34a and emits phosphorescence Lf having a predetermined wavelength. The wavelength can be appropriately selected according to the wavelength of the excitation light. For example, in addition to eosin and auramine, rare earth fluorescent complexes and transition metal fluorescent complexes can be used.

  The enhanced electric field Ew is an electric field formed by surface plasmons generated in the metal film 34a, and is an electric field which is generated in a local region on the metal film 34a and is stronger than a normal evanescent wave. The intensity of the signal such as phosphorescence emitted from the label can be amplified by the enhanced electric field Ew. The surface plasmon is generated in the metal film 34a by resonating the evanescent wave and the free electrons in the metal film 34a.

The assay by the sandwich method for fixing the phosphorescent label F to the metal film 34a is performed, for example, according to the following procedure. Whether or not an antigen as a test substance is contained in blood (whole blood) will be described with reference to FIG. Further, in the following procedure, a sensor chip C1 in which a labeled secondary antibody BF (phosphorescent label binding substance of the secondary antibody B2 and phosphorescent label F) is disposed in a dry state upstream of the detection part of the flow path 33 is used. Yes.
Step 1: Blood (whole blood) So to be examined is injected from the injection port 35a. Here, the case where the antigen A which is a test substance is contained in this blood So is demonstrated. In FIG. 4, blood So is indicated by a shaded area.
step 2: The blood So is filtered by the membrane filter 36, and large molecules such as red blood cells and white blood cells become residues. Subsequently, blood S (plasma) separated by blood membranes by the membrane filter 36 oozes out into the flow path 33 by capillary action. Alternatively, in order to accelerate the reaction and shorten the detection time, a pump may be connected to the air hole, and the plasma S may be caused to flow down by pump suction and push-out operations. In FIG. 4, the plasma S is indicated by a hatched area.
step 3: The plasma S that has oozed into the flow path 33 and the labeled secondary antibody BF disposed in a dry state upstream of the detection section of the flow path 33 are mixed, and the antigen A in the plasma S is labeled with the labeled secondary antibody BF. Combine with.
Step 4: The plasma S gradually flows along the flow path 33 toward the air hole 35b, and the antigen A bound to the labeled secondary antibody BF is combined with the primary antibody B1 fixed on the detection unit 38 for measurement. The so-called sandwich structure in which the antigen A is sandwiched between the primary antibody B1 and the labeled secondary antibody BF is formed.
step 5: A part of the labeled secondary antibody BF that has not bound to the antigen A is an antibody immobilized on the reference detection unit 39, and has a specific binding property with the secondary antibody B2. It binds to antibody B0. Furthermore, even if the labeled secondary antibody BF that has not been bound to the primary antibody B0 may remain on the detection unit, the subsequent plasma plays a role of washing, and the label 2 that floats on the detection unit Wash off secondary antibody BF.

  As described above, after step 1 to step 5 until blood is injected from the injection port and the antigen is combined with the primary antibody and the secondary antibody, as described above, in the apparatus 1 described above, By detecting the signal light, the presence and / or concentration of the antigen can be detected with high sensitivity. Thereafter, the sensor chip C1 is moved so that the signal light from the reference detection unit 39 can be detected, and similarly, the detection signal from the reference detection unit 39 is detected. The signal light from the reference detection unit 39 fixing the primary antibody B0 that binds to the labeled secondary antibody BF is a signal light that reflects reaction conditions such as the amount and activity of the labeled secondary antibody BF. It is believed that there is. Therefore, a more accurate detection result can be obtained by correcting the signal light from the measurement detection unit 38 using this signal light as a reference. In addition, a known amount of a labeling substance (fluorescent label, metal fine particle, etc.) is fixed in advance to the reference detection unit 39, and the signal light from the reference detection unit 39 is used as a reference from the measurement detection unit 38. The signal light may be corrected.

Hereinafter, the operation of the optical measurement apparatus and method of the present embodiment will be described in detail.
In the present embodiment, the polarization modulation element 11 is controlled at a modulation frequency of 0.1 kHz as a low frequency for which both the signal component and the noise component are to be detected, and as a high frequency for which only the noise component is to be detected. explain. Note that these frequencies are not limited to the above, and an appropriate frequency may be selected within a range in which a signal component and a noise component can be separated in consideration of characteristics of a phosphorescent label to be used.

  First, the excitation light Le, the polarization state of which is controlled by the polarization modulation element 11 at a modulation frequency of 0.1 kHz, passes through the flow path base material 30 and is irradiated to the interface between the flow path base material 30 and the metal film 34a. . At this time, when the polarization state of the excitation light Le at the interface becomes p-polarized light within the period of modulation of the polarization direction, the excitation light Le is coupled with the surface plasmon, and the enhanced electric field Ew on the metal film 34a. Will occur. On the other hand, when the polarization state of the excitation light Le at the interface becomes s-polarized light, the excitation light Le does not couple with the surface plasmon, and therefore no enhanced electric field is generated. The phosphor Lf from the phosphorescent label F, which is a signal component to be detected, excited by the enhanced electric field Ew of the surface plasmon is detected by the photodetector 23. The emission time of the phosphorescent label F is shorter than one period in which the excitation light Le is s-polarized when the modulation frequency is 0.1 kHz. Therefore, the phosphorescent label F stops emitting before the excitation light Le is excited again as p-polarized light, and emits again when the excitation light Le becomes p-polarized again. That is, the phosphorescence Lf fluctuates at the same frequency of 0.1 kHz as the excitation light Le. In addition, when there is a scatterer that scatters excitation light in the surface plasmon enhanced electric field Ew, noise components such as stray light of the excitation light Le generated by the scatterer are large when the surface plasmon enhanced electric field Ew is generated. It is small when there is no electric field Ew. That is, the stray light of the excitation light Le caused by the scatterer existing in the surface plasmon enhanced electric field Ew fluctuates at the same frequency of 0.1 kHz as the excitation light Le and is detected by the photodetector 23. In addition, since the autofluorescence generated due to excitation is large only when the surface plasmon enhanced electric field Ew is generated, it fluctuates at the same frequency of 0.1 kHz as the excitation light Le. A signal is transmitted from the photodetector 23 to the frequency analysis unit 24 based on the amount of light detected by the photodetector 23, and a component synchronized with the frequency of 0.1 kHz is detected by the frequency analysis unit 24 from the transmitted signal. The signal (low-frequency measurement signal) detected at this time includes a signal component indicating a value corresponding to the amount of the test substance A, stray light of excitation light Le caused by scatterers existing in the surface plasmon enhanced electric field Ew, and private Both noise components such as fluorescence are included.

  Next, the excitation light Le, the polarization state of which is controlled by the polarization modulator 11 at a modulation frequency of 1000 kHz, passes through the flow path base material 30 and is irradiated to the interface between the flow path base material 30 and the metal film 34a. Similarly to the above, the phosphorescence Lf from the phosphorescent label F, which is the signal component to be detected, excited by the enhanced electric field Ew of the surface plasmon is detected by the photodetector 23. The emission time of the phosphorescent label F is longer than one period in which the excitation light Le is s-polarized when the modulation frequency is 1000 kHz. Therefore, the phosphorescent label F is excited again before the end of the light emission time, and is always in a lighted state. Therefore, the phosphorescence Lf does not vary at a frequency of 1000 kHz. On the other hand, noise components such as stray light of the excitation light Le caused by the scatterers present in the surface plasmon enhanced electric field Ew fluctuate at the same frequency 1000 kHz as the excitation light Le and are detected by the photodetector 23. A signal is transmitted from the photodetector 23 to the frequency analysis unit 24 based on the amount of light detected by the photodetector 23, and a component synchronized with the frequency of 1000 kHz is detected by the frequency analysis unit 24 from the transmitted signal. Since the phosphorescence Lf is not a signal that fluctuates in synchronization with the frequency of 1000 kHz, the detected signal (high frequency measurement signal) includes stray light of the excitation light Le caused by scatterers existing in the surface plasmon enhanced electric field Ew, and private Only noise components such as fluorescence are included.

  And the presence and / or quantity of the to-be-tested substance A are calculated | required by calculating | requiring the difference of a low frequency measurement signal and a high frequency measurement signal by the calculating part 25. FIG.

  As described above, the optical measurement apparatus according to the present embodiment is provided in the surface plasmon enhanced electric field Ew with a small number of measurements and simple calculation, particularly by including the frequency modulation means, the frequency analysis unit, and the calculation unit. Measurement with a high S / N ratio is possible by eliminating the influence of stray light, autofluorescence, etc. of the excitation light Le by the scatterer.

(Design change of the first embodiment)
In the above embodiment, a phosphorescent labeling substance is used as a label for antigen A, but other photoresponsive label (for example, a label such as fluorescence) may be used as the label.

  Elements for adjusting the polarization state, such as a λ / 2 wavelength plate, a λ / 4 wavelength plate, a polarizing plate, and a Babinet Soleil plate, may be added before and after the polarization modulation element.

  By passing the condensing lens through the measurement light so that the measurement light applied to the metal film contains various angle components, surface plasmons can be excited even if the amount of adsorption of the test substance on the metal film varies. Is preferable. However, the presence of a condensing lens is not essential since it is not an essential part of the present invention. Moreover, you may adjust the state of measurement light using a some condensing lens as needed.

  In the above embodiment, the sandwich method using an immune reaction has been described as an example. However, the present invention is not limited to this, and the present invention can be applied to a method using an avidin-biotin reaction or a DNA reaction, or a method using a competition method or the like. It is possible to apply.

In the above-described embodiment, an example in which the present invention is applied to the surface plasmon enhanced fluorescence method has been described. However, the present invention can also be applied to an evanescent fluorescence method. In the evanescent fluorescence method, the metal film 34a may be removed from the channel substrate 30 of the apparatus shown in FIG. 1, and in this case, the excitation light Le can be measured by polarization modulation, but intensity modulation is performed. Is preferred. When intensity-modulating the excitation light Le, it is more preferable to periodically turn on and off the excitation light. Further, the intensity of the excitation light Le may be modulated also in the surface plasmon enhanced fluorescence method. When the intensity of the excitation light Le is modulated, the noise component of the excitation light Le scattered by the scatterers existing outside the surface plasmon enhanced electric field Ew also fluctuates in synchronization with the period of intensity modulation of the excitation light Le. Can be removed.
As for the intensity modulation method of the excitation light Le, the intensity of the excitation light Le may be modulated in synchronization with the function generator, or the excitation light Le is irradiated with a constant intensity and periodically modulated with an optical chopper. May be. For example, the optical chopper transmits the excitation light Le when the first voltage is input, and blocks the excitation light Le when the second voltage is input. That is, the excitation light Le can be periodically turned on / off by the optical chopper. By periodically modulating the intensity of the excitation light, the fluorescent label is excited with a periodically varying intensity. In addition, the stray light of the excitation light is also periodically modulated in intensity.

“Second Embodiment of Optical Measuring Device and Optical Measuring Method”
Next, the optical measuring device 2 and method of the second embodiment will be described. The optical measurement device 2 and method of the present embodiment are configured so that the sensor chip C2 has phase difference information relating to the phase difference of birefringence that occurs in the excitation light Le after the excitation light Le enters the prism portion and reaches the interface. Including an information code 44 for displaying the information code, an information reading unit 43 for reading the information code, and a phase difference control unit 45 for controlling the phase difference of the birefringence generated during propagation of the excitation light Le, It differs from the optical measurement apparatus 1 and method of the first embodiment in that the polarization state of the excitation light Le at the interface is adjusted based on the phase difference information of the information code. Therefore, the description of the same components as those of the optical measurement apparatus 1 and the method of the other first embodiment is omitted unless particularly necessary.

  As shown in FIG. 7, the optical measuring device 2 of the present embodiment includes a flow path base material 30 that forms a flow path 33, a fluorescent label binding substance that is disposed dry on the upstream side of the flow path 33, The sensor unit C2 including the detection unit including the metal film 34a formed in the predetermined region and the information code 44, the light source 10 that emits the excitation light Le, and excitation at the interface between the channel substrate 30 and the metal film 34a. The light guide member 14 that guides the excitation light Le from one side to the interface through the flow path substrate 30 and the polarization direction of the excitation light Le at the interface are periodically set so that the light Le satisfies the total reflection condition. The polarization modulation element 11 that controls the polarization state of the excitation light Le so as to modulate, the FG 21 that generates a periodic clock of a voltage that is a basis of the modulation period of the polarization direction, and the periodic clock of the voltage generated in the FG 21 Depending on polarization modulation A polarization modulation element driver 22 for driving the optical element 11, a photodetector 23 for detecting the signal light generated from the detection unit, disposed on the other side with respect to the interface, and only a signal component synchronized with the period of modulation in the polarization direction. A frequency analysis unit 24 that detects a signal, a calculation unit 25 that performs a calculation on a signal obtained by the frequency analysis unit 24, a control unit 20 that controls the entire apparatus, an information reading unit 43 that reads the information code, and an information processing And a phase difference control unit 45 that controls the phase difference of the birefringence generated during the propagation of the measurement light based on the information read by the unit.

  The information code 44 is obtained by recording phase difference information on the phase difference of birefringence in the flow path base material 30 obtained in advance at the time of shipment of the sensor chip C2, for example, in the form of a barcode or a two-dimensional code. This is attached to the sensor chip C2.

  The information reading unit 43 is, for example, a CCD or a known bar code reader, and transmits the read information to the phase difference control unit 45.

  The phase difference control unit 45 receives the information read by the information reading unit 43, and based on this information, the phase difference opposite to the phase difference of birefringence generated during propagation of the excitation light Le in the flow path base material 30. An instruction is given to the FG 21 so as to impart a phase difference to the excitation light Le. Thereby, the FG 21 generates a voltage modulation waveform reflecting the reverse phase difference.

  The optical measurement method of the present embodiment is attached to the sensor chip C2 when the polarization direction of the excitation light Le is modulated using the measurement device 2 in addition to the processing in the first embodiment. The phase difference information related to the phase difference of birefringence generated during propagation of the excitation light Le in the channel substrate 30 is acquired from the information code 44, and the polarization state of the excitation light Le is controlled based on this phase difference information It is.

Hereinafter, the operation of the optical measurement apparatus and method of the present embodiment will be described in detail.
When birefringence is present in the flow path base material 30 (prism), even if the polarization modulation element 11 performs modulation to periodically switch between p-polarized light and s-polarized light before passing through the flow path base material 30, the flow path While propagating through the base material 30, for example, it becomes elliptically polarized light, and an enhanced electric field is not efficiently generated, resulting in a problem that the SN ratio of the detected signal is deteriorated. Actually, when a prism (Zeonex (registered trademark), polystyrene, PMMA, etc.) using an inexpensive plastic material is used, this birefringence cannot be ignored. Therefore, in actuality, it is necessary to control the polarization state of the excitation light Le in consideration of the birefringence phase difference that occurs during propagation through the flow path base material 30.

  In the present embodiment, phase difference information obtained in advance at the time of shipment is attached to the sensor chip C2, and the information is read at the time of measurement, and the read information is reflected in the modulation waveform generated by the FG 21 to change the polarization state. By adjusting the phase difference of the above birefringence and adjusting the polarization state of the measurement light so as to give a phase difference opposite to this phase difference, a prism portion made of a material having birefringence is provided. Even if the sensor chip is used, measurement with a high S / N ratio can be performed.

(Design change of the second embodiment)
In the second embodiment, the polarization modulation element is directly controlled based on the information code, and the phase difference of birefringence generated during propagation of the measurement light in the flow path base material is adjusted. As in the embodiment, the wave plate may be rotationally controlled by the wave plate control unit.

“Third Embodiment of Optical Measuring Device and Optical Measuring Method”
Next, an optical measurement apparatus and method according to the third embodiment will be described. The optical measuring device 3 and method of the present embodiment include excitation light at the interface between the wave plates 12 and 13 provided between the polarization modulation element 11 and the light guide member 14, and the flow path substrate 30 and the metal film 34a. By including a second photodetector 40 that detects reflected light of Le, a second frequency analysis unit 41, and a wavelength plate control unit 42 that controls rotation of the wavelength plate, by monitoring the reflected light, The optical measurement apparatus 1 and the method of the first embodiment are different in that the polarization state of the excitation light Le at the interface is adjusted. Therefore, the description of the same components as those of the optical measurement apparatus 1 and the method of the other first embodiment is omitted unless particularly necessary.

  As shown in FIG. 8, the optical measuring device 3 of the present embodiment includes a flow path base material 30 that forms a flow path 33, a fluorescent label binding substance that is disposed on the upstream side of the flow path 33, and a flow path 33. All of the excitation light Le is provided at the interface between the sensor chip C3 including the detection unit including the metal film 34a formed in a predetermined region, the light source 10 that emits the excitation light Le, and the flow path base 30 and the metal film 34a. In order to satisfy the reflection condition, the light guide member 14 that guides the excitation light Le from one side to the interface through the flow path base material 30 and the polarization direction of the excitation light Le at the interface are periodically modulated. The polarization modulation element 11 that controls the polarization state of the excitation light Le, the FG 21 that generates a periodic clock of the voltage that is the basis of the modulation period in the polarization direction, and the polarization according to the periodic clock of the voltage generated in the FG 21 Drives the modulation element 11 A polarization modulation element driver 22, a photodetector 23 that is disposed on the other side with respect to the interface and detects signal light generated from the detection unit, and a frequency analysis that detects only signal components synchronized with the modulation period of the polarization direction A wavelength provided between the polarization modulator 11 and the light guide member 14; a calculation unit 25 that performs a calculation on the signal obtained by the unit 24, the frequency analysis unit 24; a control unit 20 that controls the entire apparatus; Plates 12, 13; a second photodetector 40 for detecting the reflected light Lr of the excitation light Le at the interface; a second frequency analyzer 41; and a wavelength plate controller 42 for controlling the rotation of the wave plate. Is provided.

  The sensor chip C3 has the same configuration as that of the first embodiment. However, as shown in FIG. 7, the reflected light Lr of the excitation light Le at the interface can be detected appropriately. It is formed so as to further include a transmission surface 30d for transmitting Lr to the outside of the flow path base material 30 and a protection portion 30c for protecting this from scratches and dirt.

  The second photodetector 40 is arranged to detect the reflected light Lr and transmits a signal of the detected reflected light Lr to the second frequency analysis unit 41. The second photodetector 40 is the same as that of the first embodiment. Similar ones can be used.

  The wave plates 12 and 13, the second frequency analysis unit 41, and the wave plate control unit 42 are further provided as frequency modulation means, and thereby the reflected light detected by the second photodetector 40. The function of adjusting the polarization state of the excitation light Le is achieved based on the intensity. For example, in the wave plates 12 and 13, the wave plate 12 is a λ / 2 wave plate, and the wave plate 13 is a λ / 4 wave plate. The second frequency analysis unit 41 is, for example, a lock-in amplifier, and separates and detects the reflected signal component synchronized with the modulation in the polarization direction of the measurement light from the detected reflected signal light, and the maximum value of the intensity of the signal component The wave plate is controlled so as to control the wave plates 12 and 13 so that the ratio of the minimum value to the wave is minimized (the difference in amplitude change between the maximum value and the minimum value is maximized during periodic modulation). An instruction is given to the control unit 42. The wave plate controller 42 controls the rotation of the wave plates 12 and 13 in accordance with instructions from the second frequency analyzer 41.

  On the other hand, the optical measurement method of the present embodiment uses the measurement device 3 to detect the signal light in addition to the processing in the first embodiment in addition to the processing in the first embodiment. When the reflected signal light including the reflected light Lr of the excitation light Le at the interface is detected, the reflected signal component synchronized with the modulation in the polarization direction is separated and detected from the reflected signal light, and the intensity of the signal component is detected. The polarization state of the excitation light Le is controlled so that the ratio of the minimum value to the maximum value is minimized.

Hereinafter, the operation of the optical measurement apparatus and method of the present embodiment will be described in detail.
As described above, as described above, it is necessary to control the polarization state of the excitation light Le in consideration of the phase difference of birefringence generated while propagating through the channel substrate 30.
When the reflected light Lr of the excitation light Le at the interface between the channel substrate 30 and the metal film 34a is detected by the second photodetector 40, the intensity of the reflected light Lr is the polarization direction of the excitation light Le at the interface. Modulation is performed in response to the modulation. This is because when the polarization state of the excitation light Le at the interface becomes p-polarized light, the excitation light Le is coupled with the surface plasmon, the energy of the excitation light Le is converted into the surface plasmon, and the reflected light Lr is minimized. On the other hand, when the polarization state of the excitation light Le at the interface becomes s-polarized light, the excitation light Le is not coupled with the surface plasmon, and thus the reflected light Lr is maximized.

  Therefore, in this embodiment, the reflected signal component of the reflected signal light is monitored in synchronization with the modulation of the polarization direction of the excitation light Le, and the wavelength is set so that the ratio of the minimum value to the maximum value of the intensity of the signal component is minimized. By adjusting the plates 12 and 13, the phase difference of the birefringence is taken into account, and the polarization state of the excitation light Le is controlled so as to give a phase difference opposite to this phase difference. Even if a sensor chip having a prism portion made of is used, measurement with a high S / N ratio can be performed.

(Design change of the third embodiment)
In the third embodiment, the wavelength plate is rotationally controlled by the wave plate controller, and the phase difference of birefringence generated during propagation of the measurement light in the flow path base material is adjusted. As is the case, the polarization modulation element may be directly controlled instead of the wave plate.

1, 2, 3 Optical measuring device 10 Light source 11 Polarization modulation element (frequency modulation means)
12, 13 Wavelength plate 14 Light guide member 20 Control unit 21 Function generator (FG) (frequency modulation means)
22 Polarization modulation element driver (frequency modulation means)
23 Photodetector 24 Frequency analysis unit 25 Calculation unit 30 Channel substrate (dielectric plate)
32 Lid member 33 Channels 34a and 34b Metal film 40 Second photodetector 41 Second frequency analysis unit 42 Wave plate control unit 43 Information reading unit 44 Information code 45 Phase difference control unit A Test substance BF Phosphorescent label binding Substance C1, C2, C3 Sensor chip Ew Enhanced electric field Le Excitation light Lf Phosphorescence Lr Reflected light

Claims (8)

By contacting the sample liquid containing the substance to be detected on the sensor portion formed on one surface of the dielectric plate of the sensor chip, the amount of the photolabeling corresponding to the quantity of the substance to be detected contained in the sample liquid Binding a substance on the sensor unit;
By irradiating the sensor unit with excitation light at an incident angle at which a total reflection condition is obtained, a photoelectric field is generated on the sensor unit,
In the optical measurement method for measuring the amount of the substance to be detected by exciting the photolabel of the photolabel binding substance with the photoelectric field and measuring the light generated due to the excitation,
The excitation light is frequency-modulated at a low frequency indicating an intensity change in which both a signal component indicating a value corresponding to the amount of the substance to be detected and a noise component caused by stray light of the excitation light are synchronized to the sensor unit. Irradiating and extracting a component synchronized with the low frequency from the light caused by the excitation to obtain a low frequency measurement signal;
The excitation light is frequency-modulated at a high frequency that shows an intensity change synchronized only with the noise component and irradiated to the sensor unit, and a component synchronized with the high frequency is extracted from the light generated due to the excitation. To obtain high frequency measurement signals
An optical measurement method characterized by obtaining a signal of only the signal component by obtaining a difference between the low frequency measurement signal and the high frequency measurement signal.   The optical measurement method according to claim 1, wherein a phosphorescent label is used as the optical label.   The optical measurement method according to claim 1, wherein the frequency modulation is performed by modulating the intensity of the excitation light. As the sensor chip, the sensor unit has a laminated structure including a metal layer adjacent to the dielectric plate,
4. The optical measurement method according to claim 1, wherein plasmons are excited in the metal layer by irradiation with the excitation light to generate a photoelectric field enhanced by the plasmons. 5.   The optical measurement method according to claim 4, wherein the frequency modulation is performed by modulating a polarization state of the excitation light. Obtaining phase difference information regarding a phase difference of birefringence generated in the excitation light from when the excitation light is incident on the dielectric plate until reaching the sensor unit;
6. The optical measurement method according to claim 5, wherein a polarization state of the excitation light is controlled based on the phase difference information so that a phase difference opposite to the phase difference is generated in the excitation light. Detecting reflected light of the excitation light in the sensor unit;
The polarization state is controlled based on the intensity of the reflected light modulated in synchronization with the modulation of the polarization state so that the ratio of the minimum value to the maximum value of the intensity of the reflected light is minimized. The optical measurement method according to claim 5 or 6. An optical measurement device used in the optical measurement method according to claim 1,
An accommodating portion for accommodating the sensor chip;
An excitation light irradiating optical system for irradiating the position of the sensor part of the sensor chip accommodated in the accommodating part with the excitation light;
Frequency modulation means for performing frequency modulation on the excitation light;
A light detection means for detecting light caused by the excitation;
A frequency analysis means for extracting a component synchronized with a frequency for modulating the excitation light from a signal based on the light detected by the light detection means;
An optical measurement apparatus comprising: an arithmetic unit that obtains only a signal component by obtaining a difference between the low frequency measurement signal and the high frequency measurement signal.

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