JP2001021565A - Fluoroimmunoassay and device therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure the antigen concentration with high sensitivity by selectively exciting only a fluorescent labeled antibody by use of a surface plasmon resonance. SOLUTION: A triangular glass prism 10 having a metallic thin film 11 with an antibody fixed thereon is placed on a rotating table 1 and a sample solution containing an antigen is injected into a flow cell 13 and supplied to the metallic thin film 11 to cause the antigen to bond to the antibody. Further, a fluorescent labeling reagent containing the fluorescent labeled antibody is supplied to cause the fluorescent labeled antibody to bond to the antibody bonded to the antigen. Next, light that is normally absorbed by the fluorescent labeled antibody and light whose wavelength is an integer multiple of the former light are made to impinge on the prism side of the triangular glass prism 10 at the same time to cause two-photon excitation or multi-photon excitation of the fluorescent labeled antibody; fluorescence generated when the light impinges on the prism side of the triangular glass prism with an impinging angle at which reflectivity is lowest is subjected to spectrum analysis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence immunoassay method and apparatus for quantifying an antigen concentration using two-photon or higher multiphoton excitation by plasmon resonance.

[0002]

2. Description of the Related Art In an immunoassay, a very small amount of an antigen present in a sample is analyzed with high sensitivity using an antibody that specifically binds to a certain antigen. Conventionally, various immunoassay methods have been known. Among them, a fluorescent immunoassay method that has a short analysis time, high sensitivity, and does not use a radioactive substance that is difficult to treat a waste liquid has attracted attention. In the fluorescence immunoassay, an antibody labeled with a fluorescent reagent is used to measure the intensity of fluorescence emitted by an antibody bound to an antigen in a solution sample by ordinary light irradiation, and the antigen concentration is quantified from the measured value.

[0003] An immunoassay utilizing surface plasmon resonance, which is advantageous for simplicity and stability, has also been adopted. Surface plasmon resonance, when light is incident on the triangular glass prism with a deposit of metal thin film, a phenomenon that the reflected light intensity is remarkably reduced in the resonance angle theta _R. The resonance angle θ _R changes when an antibody previously bound and immobilized on the surface of the metal thin film binds to an antigen in a solution sample. Therefore, by measuring the resonance angle θ _R when the intensity of the reflected light is minimized, the binding ratio between the antibody and the antigen, and thus the antigen, is analyzed. Fluorescent immunoassays and immunoassays utilizing surface plasmon resonance have been applied to fields such as biochemical analysis and clinical analysis, taking advantage of these advantages.

[0004]

In the conventional fluorescent immunoassay, interfering substances such as serum proteins and enzymes existing in the system are simultaneously excited by excitation light that excites a fluorescently labeled antibody bound to an antigen. The excited interfering substance becomes a fluorescent light source that emits strong background light, and reduces measurement sensitivity. The sources of error caused by the excitation of interfering substances are the method that utilizes the difference in the polarization characteristics between the fluorescent labeling reagent and the interfering substance, and the time-resolved fluorescence analysis method that uses a rare-earth-based labeled antibody that has a longer fluorescence lifetime than the interfering substance. Eliminate to some extent. However, the improvement of sensitivity has already reached its limit, and it is difficult to improve the sensitivity by developing a new fluorescent labeling reagent.

Therefore, two-photon excitation has been considered as a spectroscopic technique that enables even higher sensitivity analysis using an existing fluorescent labeling reagent ("p-Bis (o-methylstyryl) benzen").
e asa Power-Squared Sensor for Two-Photon Absorpti
on Measurements between 537 and 694nm ", SMKenned
y and FE Lytle Anal.Chem., 1986, 58 2643-2647
, "Two-Photon induced fluorescence of biological
markers based on optic al fibers "A. Lago, ATObei
dat, AEKaplan, JBKhurgin, and PLShkolnikov,
Opt. Lett., 1995, 20, 2054-2056, etc.). In ordinary one-photon excitation, an excited state is generated by absorption of a photon having a wavelength corresponding to the electron transition energy of the fluorescent labeling reagent by the fluorescent labeling reagent. On the other hand, in two-photon excitation, the same excited state as one-photon excitation is generated by simultaneously absorbing two low-energy photons having twice the wavelength of the light normally absorbed by the fluorescent labeling reagent. Therefore, the electric field of the excitation light is strong, and localized portions are selectively excited, so that the influence of the background light caused by the interfering substance can be significantly reduced. However, the absorption cross section of two-photon excitation is several tens of orders of magnitude smaller than that of one-photon excitation. As a result, an extremely expensive ultrashort pulse light source must be used to directly irradiate the fluorescent labeling reagent with light. Therefore, there is a need for a method that significantly improves the efficiency of two-photon excitation and enables application to fluorescence immunoassay.

[0006]

SUMMARY OF THE INVENTION The present invention has been developed to meet such a demand, and establishes two-photon excitation by utilizing the electric field enhancement effect and the electric field localization effect of surface plasmon resonance. An object of the present invention is to dramatically increase the excitation efficiency, suppress the influence of background light caused by interfering substances, and quantify the antigen concentration with high sensitivity by increasing it.

In order to achieve the object of the fluorescent immunoassay method of the present invention, a triangular glass prism having a metal thin film on which an antibody is immobilized is placed on a rotary table, and a sample solution containing an antigen is placed on a metal plate of the triangular glass prism. After supplying to the thin film side to bind the antigen to the antibody, and further supplying a fluorescent labeling reagent containing a fluorescently labeled antibody to bind the fluorescently labeled antibody to the antibody bound to the antigen, the fluorescently labeled antibody is normally absorbed Light having a wavelength that is an integral multiple of the light is simultaneously incident from the prism side of the triangular glass prism to excite the fluorescent-labeled antibody by two-photon excitation or multi-photon excitation. It is characterized in that the fluorescence generated upon incidence is spectrally analyzed.

The apparatus used in this method includes a triangular glass prism having a metal thin film on which an antibody is immobilized, a rotating table on which the triangular glass prism is mounted, a sample solution containing an antigen and fluorescence for the antigen. A flow cell that supplies a fluorescent labeling reagent containing a labeled antibody to the metal thin film side, an excitation light source that simultaneously emits light having a wavelength that is an integral multiple of the light normally absorbed by the fluorescently labeled antibody from the prism side of the triangular glass prism, A photodetector for spectrally analyzing the fluorescence emitted when the fluorescent-labeled antibody further bound to the antigen bound to the antibody is excited by two-photon or multiphoton excitation.

[0009]

[Function] Surface plasmons are Au, Ag, Cu, Pt,
Ni, a triangular glass prism with a deposit of thin metal film of Al or the like, a strong local field, which is formed on the metal thin film surface when light is incident from the prism side at a predetermined resonance angle theta _R. Near the resonance angle θ _R , most of the incident light is converted to surface plasmons. Since the electric field of the surface plasmon has almost the same properties as the incident light, the fluorescent labeling reagent placed in the electric field is excited in the same manner as the conventional method of exciting by direct light absorption,
It fluoresces. The most distinctive feature of excitation by surface plasmons is that they exhibit an electric field enhancement effect. That is, the electric field intensity of the surface plasmon is enhanced more than the electric field intensity of the incident light, and reaches several hundred times especially in the near infrared region. Due to this electric field enhancement effect, once light is converted to surface plasmons, an extremely high electric field intensity is obtained compared to direct light excitation using the same light source, and two-photon excitation that was difficult with conventional direct light excitation is possible. .

Moreover, the electric field of the surface plasmon is strongly localized on the metal surface, and decays exponentially according to the distance from the metal surface. Owing to this intensity distribution, interfering substances existing other than the metal surface are not excited, and background light which is an error factor is excluded. Therefore, when surface plasmon resonance is applied to fluorescence immunoassay, only the fluorescently labeled antibody adsorbed and fixed on the metal surface is selectively and efficiently excited, and the influence of interfering substances is minimized. Further, since the electric field of the surface plasmon has a characteristic of p-polarized light, it is possible to reduce the background light by utilizing the difference in the polarization characteristic of the fluorescence. Thus, surface plasmons have ideal advantages as excitation sources for fluorescent immunoassays. Moreover, when surface plasmon resonance exhibiting an electric field enhancement effect and an electric field localization effect is combined with two-photon excitation or multiphoton excitation, a greater effect can be obtained. That is, since the absorption cross section of two-photon excitation is extremely small, 10 ⁻⁴⁸ to 10 ⁻⁵⁰ , excitation cannot be normally performed without irradiating extremely high-intensity excitation light with an ultrashort pulse laser or the like. On the other hand, for example, the intensity of an electric field induced by surface plasmon resonance in the near-infrared light region reaches several hundred times that of incident light. When such electric field intensity is superimposed on the absorption cross section, excitation with a transition probability of two orders of magnitude or more becomes possible, and the advantage of two-photon or multi-photon excitation in which the influence of background light caused by interfering substances is eliminated. Is exhibited. Two-photon excitation is most preferred for exciting the antibody,
Similarly, the background light can be reduced and the fluorescence can be detected with high sensitivity by multiphoton excitation equal to or more than three-photon excitation. In the excitation of three or more photons, excitation is performed using light having a longer wavelength than that of two-photon excitation. In ordinary direct light irradiation, excitation is extremely difficult because the absorption cross-section becomes smaller as compared with two-photon excitation. Here, when surface plasmon resonance in which the electric field intensity increases as the wavelength of the light source becomes longer is added, an effect of increasing the transition probability larger than that of two-photon excitation can be obtained.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In a fluorescence immunoassay apparatus according to the present invention, for example, a triangular glass prism 10 is mounted on a rotary table 1 as shown in FIG. Triangular glass prism 1
A metal thin film 11 on which an antibody is immobilized is provided on one surface of the reference numeral 0, and a flow path is formed by the flow pipe 12 and the flow cell 13 so that the sample solution flows while contacting the metal thin film 11. Of course, a fixed cell can be used instead of the flow pipe 12. The metal thin film 11 is formed directly on one surface of the triangular glass prism 10 or
Is formed by bonding a glass substrate 17 (FIG. 3) on which is formed a triangular glass prism 10. The material of the triangular glass prism 10 or the glass substrate 17 on which the metal thin film 11 is provided is not limited as long as it has a high refractive index having a high transmittance of near ultraviolet rays or near infrared rays, and optical glass BK-7, LaSF -N30, SF-
10 (manufactured by Schott, Germany) or the like is used. Au, Ag, Cu, Pt, Ni, A
1 and the like can be used.

For the flow pipe 12 and the flow cell 13, an acrylic resin which does not react with the sample solution and has high transparency is preferably used. The flow pipe 12 is inserted into a flow cell 13 as shown in FIG. 2, is sandwiched between a triangular glass prism 10 of the same quality, and a glass plate 14, and the whole is pressure-bonded P using a resin adhesive. A part of the flow pipe 12 is opened, and becomes a liquid contact part 15 where the sample solution contacts the metal thin film 11. As the excitation light source 2, a semiconductor laser-induced picosecond Nd-YAG laser (wavelength: 532 nm, pulse width: 800 picoseconds, energy: 1 μJ) is shown in FIG. 1 as a two-photon generation source, but is not limited to this. A laser having a wavelength corresponding to the target fluorescent labeling reagent is appropriately selected.

The laser light emitted from the excitation light source 2 is split by a beam splitter 3a into transmitted light and reflected light.
The transmitted light is further divided into transmitted light and reflected light by the beam splitter 3b, and the transmitted light is guided to the prism surface of the triangular glass prism 10. The transmitted light that has entered the triangular glass prism 10 from the prism side is refracted and projected onto the metal thin film 11, reflected by the metal thin film 11, and then emitted from another prism surface of the triangular glass prism 10. The angle of incidence on the triangular glass prism 10 is set and changed by adjusting the angle of rotation of the turntable 1.

The light emitted from the triangular glass prism 10 is detected by the photodetector 4a, and the reflected light intensity I is measured.
Further, the reflected light split by the beam splitter 3b is detected by the photodetector 4b, measures the incident light intensity I _0. The reflected light intensity I and the incident light intensity I ₀ are output to the I / I ₀ circuit 5 from the light detector 4a and the light detector 4b, respectively. I /
The reflectance I / I ₀ is obtained by the I ₀ circuit 5 and output to the computer 6 and recorded. While rotating the rotary table 1 on which the triangular glass prism 10 is mounted, the reflectance I / I ₀
When measuring the incident angle at which the reflectance I / I ₀ is minimized,
That is, the resonance angle θ _R is obtained.

The reflected light from the beam splitter 3a is input to a photodetector 4c and used to open and close the gate of the photodetector 4d. As the photodetectors 4a to 4c, for example, PIN photodiodes are used. As the light detector 4d, for example, a gated CCD spectral detector having a function of enhancing an image is used. The antibody immobilized on the metal thin film 11 binds to the antigen in the sample solution injected from the flow pipe 12. A fluorescently labeled antibody is further bound to the antigen bound to the antibody. The fluorescent-labeled antibody is excited by light incident on the triangular glass prism 10 and emits fluorescent light FL.

As the reagent containing a fluorescently labeled antibody, many organic fluorescent labeling reagents and rare earth fluorescent labeling reagents can be used as long as they have absorption in the near ultraviolet or near infrared. Rare earth fluorescent labeling reagents include Eu, Tb, Yb, Sm, Tm, Nd, Er, H
There are reagents containing rare earth ions such as o, Pr, and Gd. When a normal organic fluorescent labeling reagent having a short emission life is used, a detection signal is sent from the photodetector 4c to the gate pulser 7 so as to open the gate of the photodetector 4d and detect the fluorescence FL immediately after the laser irradiation. Gate pulsar 7 to photodetector 4
A gate opening / closing signal is output to d. Thereby, the fluorescent light FL emitted from the metal thin film 11 passes through the spectroscope 8 and is detected by the photodetector 4d. The generated fluorescence FL is due to the selective excitation of the antibody, and the background light is suppressed by the two-photon excitation. When a rare earth-based labeling reagent having an extremely long luminescence lifetime is used, after several hundred nanoseconds have elapsed, the photodetector 4
The gate of d is opened, and the fluorescence FL from the metal thin film 11 is detected. When the gate opening timing is adjusted in this manner, the background light due to the interfering substance is greatly reduced, and the fluorescence F
L is measured with higher sensitivity.

[0017]

EXAMPLE A metal thin film 11 having an antibody fixed thereon was provided on a glass substrate 17 in the following procedure. As shown in FIG. 3, Au was vapor-deposited on a glass substrate 17 to form a metal thin film 11, which was then immersed in a 2% acetone solution of γ-aminopropyltriethoxysilane, and washed with methanol and distilled water.
Next, the glass substrate 17 is immersed in a 5% aqueous solution of glutaraldehyde for 3 hours, and washed with distilled water.
An organic layer 21 for immobilizing antibodies was formed on the metal thin film 11. Then, α-, a representative tumor marker in serum,
The glass substrate 17 was immersed in a phosphate buffer solution containing an antibody against fetoprotein (AFP) for 16 hours to bind the anti-AFP antibody 22 to the organic layer 21. Further, in order to prevent non-specific adsorption without involvement of the anti-AFP antibody 22, the active site of the organic layer 21 was blocked with a 0.1 M aqueous solution of ethanolamine and a serum albumin solution.

After the formation of the blocking layer 23, an acrylic resin flow cell 13 is adhered to the glass substrate 17 by an adhesive packing, and the glass side of the glass substrate 17 is adhered to the triangular glass prism 10 on the turntable 1 with a matching oil. . A phosphate buffer solution containing 300 μg / ml of AFP was injected into the flow cell 13 and left for 2 hours in contact with the anti-AFP antibody 22, washed with a phosphate buffer solution, and the anti-AFP antibody 22 on the glass substrate 17. AFP24 to
Was combined. Next, N ¹ -P-isothiosyanatobenzyl) -d which is a typical rare earth fluorescent labeling reagent as a fluorescent labeled antibody
iethylenetriamine-N ¹ , N ² , N ³ , N ³ -tetraaceteic acid
A phosphate buffer solution containing 100 μg / ml of europium (DTTA-Eu) was injected into the flow cell 13, left in contact with AFP 24 for 2 hours, and then washed with a phosphate buffer solution. Thereby, DDTA-Eu25 was bound to AFP24 immobilized with the anti-AFP antibody 22 on the glass substrate 17.

By the above operation, the amount of DTT-E in proportion to the concentration of AFP24 (antigen) contained in the sample solution is
u25 (fluorescently labeled antibody) was immobilized on the glass substrate 17. While rotating the triangular glass prism 10 provided with the glass substrate 17 on which the AFP 24 (antigen) and the DTT-Eu 25 (fluorescence-labeled antibody) are fixed on the turntable 1, the semiconductor laser-excited Nd-YAG laser (excitation light source 2) is turned on. The sample was irradiated with p-polarized light having a wavelength of 532 nm from the prism side, and the reflectance was measured at each incident angle. As a result, the reflectance was minimized when the incident angle was approximately 75 degrees.

When the emission spectrum was measured under the condition that the reflectance was minimized, an emission spectrum characteristic of trivalent europium ion was observed as shown in FIG. Further, when a calibration curve was prepared and quantified using sample solutions having various AFP concentrations, it was found that a close linear relationship was established between the AFP concentration and the emission intensity. From this, it was confirmed that according to the present invention, the concentration of the antigen could be quantified sufficiently.

[0021]

As described above, in the present invention, two-photon excitation or multi-photon excitation of a fluorescent-labeled antibody using surface plasmon resonance, and the fluorescence generated at this time is spectrally analyzed. The antigen bound to the antibody immobilized on the metal thin film is quantified. When using this method, the interfering substances contained in the sample solution are not excited,
Since only the fluorescently labeled antibody immobilized by adsorption can be selectively and efficiently excited, the antigen concentration can be quantified with high sensitivity.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a fluorescence immunoassay apparatus using surface plasmon resonance.

FIG. 2 is a view showing a triangular glass prism and a flow cell provided with a metal thin film on which an antibody is immobilized.

FIG. 3 is an explanatory view of a process for immobilizing an antibody on a metal thin film and a process for measuring the concentration of an antigen bound to the antibody.

FIG. 4 is a fluorescence spectrum of a rare-earth-labeled antibody obtained by surface plasmon excitation obtained in an example.

[Explanation of symbols]

1: Rotary table 2: Excitation light source 3a, 3b: Beam splitter 4a-4d: Photodetector 5: I /
I ₀ circuit 6: Computer 7: Gate pulser
8: Spectroscope 10: Triangular glass prism 11: Metal thin film 1
2: Flow pipe 13: Flow cell 14: Glass plate 15: Liquid contact part 17: Glass substrate 21: Organic layer 22: Anti-AFP antibody 23: Blocking layer 24: AFP (α-fetoprotein: antigen) 25: DTT-Eu (fluorescence Labeled antibody) I: reflected light intensity I ₀ : incident light intensity FL: fluorescence

Claims (2)

[Claims] 1. A triangular glass prism having a metal thin film on which an antibody is fixed is placed on a rotating table, and a sample solution containing an antigen is supplied to the metal thin film side of the triangular glass prism to bind the antigen to the antibody. After supplying a fluorescent labeling reagent containing the fluorescent labeled antibody and binding the fluorescent labeled antibody to the antibody bound to the antigen, light having a wavelength that is an integral multiple of the light normally absorbed by the fluorescent labeled antibody is passed through the triangular glass prism. Two-photon excitation or multi-photon excitation of the fluorescent-labeled antibody by incidence from the prism side, and spectral analysis of the fluorescence generated when the light is incident on the prism side of the triangular glass prism at an incidence angle at which the reflectance is minimized. Fluorescent immunoassay method. 2. A triangular glass prism having a metal thin film on which an antibody is immobilized on one side, a rotary table on which the triangular glass prism is mounted, a sample solution containing an antigen, and a fluorescent label containing a fluorescently labeled antibody against the antigen A flow cell that supplies a reagent to the metal thin film side, an excitation light source that causes light having a wavelength that is an integral multiple of the light normally absorbed by the fluorescently labeled antibody to be incident from the prism side of the triangular glass prism, and an antigen bound to the antibody. A photodetector for spectrally analyzing the fluorescence emitted when the bound fluorescently labeled antibody is excited by two-photon excitation or multiphoton excitation, when the light is incident on the prism side of the triangular glass prism at an incident angle at which the reflectance is minimized. A fluorescence immunoanalyzer characterized by performing spectrum analysis of generated fluorescence.