JP2007155603A - Analyzing method using fluorescent depolarization method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enhance the measuring sensitivity of a low-molecular weight substance to be measured in measurement using a fluorescent depolarization method. <P>SOLUTION: In a preferable embodiment, an anti-IL-4 antibody for forming a solid phase is labelled with an Ru complex fluorescent agent to form a fluorescence probe and an anti-IL-4 antibody for use in detection is added to a sample to be bonded to IL 4 being the substance to be measured. The molecular weight of a sandwich link at the time when the fluorescent probe is bonded to IL-4 is increased because the anti-IL-4 antibody for use in detection is bonded to IL-4 and the difference between the fluorescent anisotropy only of the fluorescent probe and the fluorescent anisotropy of the sandwich link is increased to enable the detection of high sensitivity. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an analytical method used to study the behavior of a biological sample in the development field of new crops, new agricultural chemicals, functional foods, pharmaceuticals, etc. And an analytical method for measuring the bound measurement object in a sample by fluorescence depolarization.

  In order to efficiently develop advanced new products in development fields such as new crops, new agricultural chemicals, functional foods, and pharmaceuticals, the effects of substances on cells (for example, proteins produced by cells into which genes have been introduced) are analyzed over time. There is a request to analyze the mechanism and capture the mechanism of its effects.

  One method for analyzing cellular responses over time is fluorescence depolarization. The fluorescence depolarization method uses a fluorescent probe and knows the mobility of the target labeled with the fluorescent probe by eliminating the polarization property of the fluorescence generated from the fluorescent probe excited by the excitation light.

The fluorescence depolarization method was theorized by Perin in the 1950s, and the mobility of fluorescent agents immobilized in solution or in the membrane can be evaluated. It is applied to the detection of trace amounts of biomolecules by observing changes. The fluorescence depolarization method is characterized in that the interaction of target molecules in a solution can be analyzed without separating the fluorescent probe from the detection system (see Patent Document 1 and Non-Patent Document 1).
JP 2004-279143 A T. Sakamoto et al., Study on structure of ribosomal RNA by time-resolved luminescence anisotropy analysis, Nucleic Acids Research Supplement No.1, pp.143-144 Anal. Chem. Vol.70, No.3, pp.632-637,1998

  When the measurement object is detected by the fluorescence depolarization method, the difference in fluorescence anisotropy between the “molecular weight of the fluorescent probe” and the “molecular weight of the fluorescent probe bound to the measurement object” is used as an index. Here, as the molecular weight of the substance to be measured is smaller than the molecular weight of the fluorescent probe, the difference in the fluorescence anisotropy value becomes smaller, and as a result, the sensitivity is low and the detection becomes difficult.

  Of course, in other methods such as enzyme immunoassay (ELISA), it is possible to detect a low-molecular-weight target substance with high sensitivity by using a so-called competitive method. The method requires sampling the substance to be measured. For this reason, measurement over time, which is one characteristic of the fluorescence depolarization method, cannot be performed.

Furthermore, when using the ELISA method, a considerable amount of sample is required, and it is difficult to apply for the purpose of, for example, analyzing the function of cells in a minute space in the microchip.
An object of the present invention is to increase the measurement sensitivity of a low-molecular-weight measurement target substance in measurement using a fluorescence depolarization method.

  The present invention is directed to a method in which an object to be measured in a sample is bound to a fluorescently labeled probe, and the bound object to be measured is analyzed in the sample by measurement using a fluorescence depolarization method. In the present invention, a molecular weight substance that specifically binds to the measurement target and does not bind to the fluorescent agent in the fluorescently labeled probe is added to the sample to form a conjugate of the measurement target and the fluorescently labeled probe. It is characterized by increasing the molecular weight.

An example of the measurement object is an antigen. In that case, the fluorescently labeled probe and the molecular weight increasing substance are antibodies. And those substances are combined by the sandwich method.
The volume of the sample reaction solution is preferably 1 μL or less.

  The fluorescence depolarization method is a “time-resolved fluorescence depolarization method” in which polarization is excited using a pulsed light source, and the temporal change in the degree of polarization of the fluorescence that is delayed is detected within 10 nanoseconds to several microseconds. There is a “fluorescence depolarization method by stationary light excitation” in which polarized light is excited using stationary light as a light source. In the time-resolved fluorescence depolarization method, the fluorescence lifetime of the fluorescent agent can be measured, and in the fluorescence depolarization method by stationary light excitation, the time average value of the degree of polarization obtained by the time-resolved fluorescence depolarization method is obtained. The fluorescence depolarization method used in the present invention includes both methods.

The fluorescence anisotropy r is obtained by irradiating a sample with linearly polarized excitation light having a specific polarization direction, and receiving each of the linearly polarized light components of the received fluorescence having a polarization direction equal to the polarization direction of the excitation light and a polarization direction orthogonal thereto. Ask based. The fluorescence anisotropy r is a value obtained by dividing the difference in fluorescence intensity between the component parallel to the excitation polarization and the component orthogonal to the total fluorescence intensity. Since the time correlation function r (t) of the fluorescence anisotropy r and the rotational correlation time (閘) representing the rotational motion of the molecule have the following relationship, the measurement object coupled to the fluorescently labeled probe Rotational motion can be read. As shown here, even when there are a plurality of rotational motion components, it is possible to individually evaluate by approximating r (t) with a multicomponent exponential function.
r (t) = (I _VV (t) −I _VH (t)) / (I _VV (t) +2 I _VH (t))
= Σa _i exp (−t / θ _i )
θ = 1 / 6D
= ΗV / kT
here,
I _VV : Intensity of longitudinal polarization component of fluorescence I _VH : Intensity of transverse polarization component of fluorescence D: Rotational diffusion coefficient 轗: Viscosity V: Volume of molecule k: Boltzmann constant T: Absolute temperature (K)
i: represents the individual molecules (or binding site), the rotational movement component in the above description a _i: proportional coefficient (ratio of the existing ratio) becomes 1 when plus all the a _i.

  Although the fluorescence anisotropy r is attenuated by the rotational movement of the molecule, the change in the value after a predetermined time from the irradiation of the excitation light or the generation of the fluorescence represents an increase or decrease in the amount of the measurement object. Therefore, in a preferred embodiment of the present invention, the measurement is performed after a predetermined time from irradiation of excitation light or generation of fluorescence by the time-resolved fluorescence depolarization method.

In addition to fluorescein, various fluorescent agents can be used for the fluorescent probe.
When the objective is to detect the substance with which the measurement object interacts with the fluorescent probe over time, the fluorescence depolarization method can in principle measure the interaction quantitatively. In addition, since autofluorescence (background light) generated from the culture medium or lens in the sample may be included as noise, it is advantageous if it can be removed. By utilizing the fact that many autofluorescences have a short lifetime, noise due to autofluorescence can be removed by detecting the target fluorescence after the autofluorescence has disappeared.

As the method, it is preferable to use a time-resolved fluorescence depolarization method. At that time, it is preferable to measure the fluorescence anisotropy within the fluorescence lifetime by using a fluorescent probe having a fluorescence lifetime in the range of 200 nanoseconds to 2 microseconds. Examples of the fluorescent agent having a fluorescence lifetime in the range of 200 nanoseconds to 2 microseconds include a Ru (ruthenium) complex. An example of a Ru complex is [Ru (phen) ₃ ] Cl ₂ (tris-1,10-Phenanthroline ruthenium (II) dichloride).

In the present invention, the apparent molecular weight of the substance to be measured is increased by binding the molecular weight substance to the substance to be measured, thereby increasing the fluorescence anisotropy obtained by the measurement by the fluorescence depolarization method, and the high sensitivity of the measurement. Can be realized.
As a result, for example, it becomes possible to analyze a low molecular weight protein typified by cytokines over time, thereby enabling functional analysis of cells over time.

  When the volume of the sample reaction solution is 1 μL or less, the measurement object is not inadvertently diluted, and a trace amount measurement is possible. The effect in the analysis using a minute space is great, and it can be expected to be an effective analysis technique in cell function analysis, particularly in the field of signal transduction, and to contribute to investigation of cancer and allergy aspects and drug discovery.

  The time-resolved fluorescence depolarization method is adopted as the fluorescence depolarization method, and a fluorescent probe having a fluorescence lifetime in the range of 200 nanoseconds to 2 microseconds is used. If measurement is performed, noise due to autofluorescence can be removed.

An apparatus for measuring fluorescence anisotropy will be described.
The fluorescence depolarization method includes a fluorescence depolarization method by stationary light excitation and a time-resolved fluorescence depolarization method. The former fluorescence depolarization method by steady light excitation can be carried out by a normal fluorescence microscope. An example of an apparatus that implements the time-resolved fluorescence depolarization method as the preferred fluorescence depolarization method of the present invention is shown in FIG.

  The microchip 2 is used as a reaction container in which a sample is accommodated. The microchip 2 is held on the sample holder of the microscope 4. A pulse laser device 6 is provided as a pulse excitation light source unit that outputs pulsed excitation light. In order to irradiate the sample of the microchip 2 with the pulse excitation light from the pulse laser device 6, the light of the excitation light wavelength is reflected. A dichroic mirror 8 that transmits fluorescence from the sample is provided. A polarizing plate 10 is used as a polarization means for converting the pulse excitation light output from the pulse laser device 6 into linearly polarized light having a specific polarization direction, for example, a longitudinal polarization direction, on the optical path between the pulse laser device 6 and the dichroic mirror 8. Is arranged. Thereby, the pulse excitation light whose polarization direction is defined by the polarizing plate 10 is irradiated through the microscope 4 to the sample in the minute space in the microchip 2. The dichroic mirror 8 and the microscope 4 constitute an irradiation optical system.

  Fluorescence generated from the sample by irradiation with pulsed excitation light passes through the microscope 4, passes through the dichroic mirror 8, passes through the analyzer (polarizing element) 12 set in the same polarization direction as the polarizing plate 10, and then excited. The light is removed and guided to the streak scope 16 of the detection means via the cut-off filter 14 for removing the light component. The microscope 4, the analyzer 12, and the cut-off filter 14 constitute a light receiving optical system.

  The computing means (not shown) evaluates the depolarization of fluorescence using a detection signal within a range of, for example, 200 nanoseconds to 2 microseconds after a predetermined time from the generation of fluorescence among the detection signals detected by the streak scope 16. It is supposed to be. The calculation means can be included in the function of the data processing apparatus.

  The streak scope 16 used for detection has a feature that high-sensitivity detection is possible in the photon counting mode when the amount of light to be detected is small, and high-speed detection can be performed by analog measurement when the amount of light is large. However, instead of the streak scope 16, a TCSPC (time correlation single grid counting method) type detector using a TAC (time-voltage converter) method may be used.

When the analyzer 12 is installed in the same direction as the polarizing plate 10, I _VV, that is, the intensity of the longitudinal polarization component of fluorescence is obtained, and when the analyzer 12 is installed in the direction orthogonal to the polarizing plate 10, I _VH, that is, fluorescence. Is obtained. Therefore, as another method, a rotation mechanism for rotating the analyzer 12 can be provided in the light receiving optical system. I _VV and I _VH can be obtained by rotating the analyzer 12. The analyzer 12 may be rotated manually or automatically.

As another method, a polarization beam splitter that can separate the longitudinal polarization and the lateral polarization of incident light without using an analyzer is provided in the light receiving optical system, and the polarization polarization splitter of the fluorescence is used by using the polarization beam splitter. Alternatively, the horizontal polarization component may be guided to a detector to detect I _VV and I _VH at the same time. In this case, two detectors such as a streak scope are required.
The fluorescence anisotropy can be obtained by calculating the obtained I _VV and I _VH by the calculation means. This computing means can also be included in the function of the data processing apparatus.

  In a preferred mode, the binding reaction between the measurement object and the fluorescently labeled nucleic acid or the fluorescently labeled sugar chain and the measurement by the time-resolved fluorescence depolarization method are performed in a state where the sample volume is 1 μL or less. Therefore, a microchip formed so that the sample volume is 1 μL or less can be used as a reaction container. Such a reaction vessel can be formed using, for example, a transparent glass substrate or transparent quartz glass on the upper and lower bottom surfaces by forming a reaction chamber and a flow path by fine processing on a silicon substrate.

  In recent years, such microchips have been used for chemical analysis and chemical synthesis on glass and silicon substrates using micromachining technology called μTAS (Micro Total Analysis Systems) and LOC (Laboratory on a Chip). Research into integrating functions into chips has been actively conducted, and they can be easily produced or obtained.

  For μTAS and LOC, researches are mainly focused on ultra-miniaturized analyzers and chemical reactions in microspaces. Recently, research on cell manipulation is gaining attention. All of them share the following features: 1) Significant reduction in reagents and samples to be used, 2) Faster analysis and reaction (shorter time), 3) High throughput of analysis and synthesis operations by parallel processing, 4) There are high functions, automation, labor savings and 5) downsizing of the whole system, and it is expected to form new future markets in various fields.

An example of the microchip 2 is shown in FIG. (A) is a top view, (B) is sectional drawing along the flow path.
The microchip 2 is configured by bonding three glass substrates 20, 22, and 24. The glass substrate 20 is a quartz glass having a thickness of 1.0 mm, the glass substrate 22 is a quartz glass having a thickness of 0.5 mm, and the glass substrate 24 is a cover glass having a thickness of 0.17 mm. The material of the cover glass is not limited, but a material with less autofluorescence such as quartz, BK-7, Pyrex (registered trademark) is desirable.

  On one side of the glass substrate 20, there are a minute channel groove 26 having a width and depth of several hundred μm or less, and sample introduction (Inlet) and discharge (Outlet) positioned at both ends of the channel groove 26. Through holes 28 and 30 are formed. A through hole 32 for a reaction chamber having a diameter of 1 mm is formed in the glass substrate 22 at a position corresponding to the central portion of the flow channel groove 26. The glass substrate 24 is a flat plate that has not been processed.

In a state where the surface of the glass substrate 20 on which the flow channel grooves 26 are formed and one surface of the glass substrate 22 face each other and are in close contact with each other, and the glass substrate 24 is in close contact with the back surface of the glass substrate 22, The microchip 2 is configured by bonding the liquids in a liquid-tight manner by means such as bonding with a hydrofluoric acid solution.
The reaction chamber 32 in the microchip 2 has a diameter of 1 mm, a depth of 0.5 mm, and a capacity of about 0.4 μL.

  As one embodiment, for example, a case where it is detected over time that cells produce interleukin-4 (IL-4), which is said to be closely related to IgE production in type I allergy, by external stimulation. I will explain.

First, the relationship between the fluorescence anisotropy r and the molecular weight M is expressed by the following equation (see Non-Patent Document 2).
r = r ₀ / (1 + τ / θ),
θ = ηM (v + h) / RT,
r: fluorescence anisotropy,
θ: rotational correlation time,
τ: fluorescence lifetime,
M: molecular weight,
r ₀ : Anisotropy in the absence of molecular motion (assuming non-patent document 2 to 0.3)
η: Viscosity (assuming 1 cp from Non-Patent Document 2)
v + h: Hydration volume (assuming non-patent document 2 to 1.9)
T: Absolute temperature (assuming non-patent literature 2 to 293)

Here, with reference to Non-Patent Document 2, assuming that r ₀ = 0.3, η = 1 cp, (v + h) = 1.9, T = 293 ° K, the fluorescence lifetime τ is 5 nanoseconds, 500 nanoseconds FIG. 3 shows the relationship between the fluorescence anisotropy r and the molecular weight M for each of the second and 50 μsec fluorescent probes.

IL-4 has a molecular weight of about 15,000. Commercially available antibodies that specifically bind IL-4 are relatively readily available, but have a molecular weight of about 150,000. Depending on the recognition site, it is possible to suppress the molecular weight of the fluorescent probe by cleaving the Fab ′ fragment and fluorescently labeling it, but the molecular weight is still about 50,000.
Assume that a fluorescent probe is prepared by fluorescently labeling an antibody having a molecular weight of about 150,000.

  First, as a reference, let us consider detecting the presence of IL-4 from the change in fluorescence anisotropy value r by binding a fluorescent probe to IL-4. The molecular weight of the fluorescent probe is about 150,000, and the molecular weight of the fluorescent probe bound to IL-4 is about 165,000.

In that case, when the fluorescence anisotropy value r is calculated when a fluorescent probe having a fluorescence lifetime of about 500 nanoseconds is used, from FIG. 3, only the fluorescent probe: r = 0.057 (a in FIG. 3)
A combination of a fluorescent probe and IL-4: r = 0.061 (b in FIG. 3)
It becomes. As a result, since the difference is small, it turns out that highly sensitive detection is difficult.

  Note that even if a fluorescent probe with a fluorescence lifetime of about 5 nanoseconds or a fluorescent probe with about 50 microseconds is used here, there is almost no difference between the fluorescence anisotropy value of the fluorescent probe alone and the fluorescence anisotropy value of the conjugate. Therefore, it is difficult to use in this system, and those having a fluorescence lifetime of about 5 nanoseconds are not suitable for measurement over time because they are affected by contaminating fluorescence from the medium and the like.

  On the other hand, although sandwich ELISA is not possible over time, in the present invention, IL-4 can be detected by using two types of antibodies that use IL-4 as an antigen and antibodies that do not bind to each other. Is possible. In many cases, antibodies used in this sandwich ELISA method are available as commercially available reagents. For example, from IL-4 targets, anti-IL-4 antibodies for solid-phase immobilization and high molecular weight substances for detection Prepare anti-IL-4 antibody.

  Then, the anti-IL-4 antibody for immobilization is labeled with, for example, a Ru complex fluorescent agent to form a fluorescent probe, and the anti-IL-4 antibody for detection, which is a molecular weight substance, is added to the sample in advance and measured. It is combined with the target substance IL-4. In this case, the molecular weight of the fluorescent probe is about 150,000, and the molecular weight of the sandwich conjugate when the fluorescent probe is bound to IL-4 is 315 because the anti-IL-4 antibody for detection is bound to IL-4. 1,000.

When the fluorescence anisotropy value is calculated using a fluorescent probe having a fluorescence lifetime of about 500 nanoseconds as described above, as shown in FIG.
Only fluorescent probe: r = 0.057 (a in FIG. 4)
Fluorescent probe and IL-4 combined: 0.099 (B in FIG. 4)
Thus, the difference increases, and it can be seen that detection with higher sensitivity is possible.

  The measurement object, the fluorescently labeled probe, and the molecular weight increasing substance to which the present invention is applied are not limited to those shown in the examples, and even other biological substances can specifically bind to increase the molecular weight. If it is a thing, it can apply similarly. For example, an antibody, a part of the antibody, a protein, an aptamer and the like can be mentioned. Moreover, the substance which couple | bonded the protein of the suitable molecular weight etc. couple | bonded with a measuring object directly may be sufficient. For the combination of a substance (antibodies, a part of an antibody, etc.) that is fluorescently labeled to detect the substance to be measured and a high molecular weight substance, the combination of “solid phase antibody and detection antibody” used in sandwich ELISA is good. It is an example. At this time, a substance in which the detection antibody, which is a high molecular weight substance, and the measurement target are bound to each other is further bound to a fluorescently labeled solid phase antibody to measure fluorescence anisotropy. Of course, the combination of “solid phase antibody and detection antibody” may be reversed.

  The present invention can be used when studying the behavior of biological samples in the development field of new crops, new agricultural chemicals, functional foods, pharmaceuticals, and the like.

It is a schematic block diagram which shows an example of the analyzer used for implementation of this invention. It is a figure which shows the microchip of one Example which can be used in the analyzer, (A) is a top view, (B) is sectional drawing along the flow path. It is a graph explaining the case where IL-4 is detected in a reference example. It is a graph explaining the case where IL-4 is detected in one Example.

Explanation of symbols

2 Microchip 20, 22, 24 Glass substrate 26 Channel groove 28, 30 Through hole for sample introduction or discharge 32 Through hole for reaction chamber

Claims (5)

A method of binding a measurement object in a sample with a fluorescently labeled probe, and analyzing the bonded measurement object in the sample by measurement using a fluorescence depolarization method,
A molecular weight substance that specifically binds to the measurement object and does not bind to the fluorescently labeled probe or the fluorescent agent is added to the sample to increase the molecular weight of the conjugate of the measurement object and the fluorescently labeled probe. An analysis method characterized by that. The analysis method according to claim 1, wherein the measurement object is an antigen, the fluorescently labeled probe and the molecular weight substance are antibodies, and these substances are bound by a sandwich method. The analysis method according to claim 1 or 2, wherein the volume of the sample solution is 1 μL or less. The analysis method according to claim 1, wherein the fluorescence depolarization method is a time-resolved fluorescence depolarization method. 5. The analysis method according to claim 4, wherein the measurement is performed within the fluorescence lifetime using a fluorescent agent having a fluorescence lifetime in the range of 200 nanoseconds to 2 microseconds as the fluorescent agent of the fluorescently labeled probe.

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