JP2009250960A - Detection method of biomolecule, biomolecule trapping substance, and biomolecule detection device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection method of a biomolecule with high sensitivity and high accuracy utilizing surface plasmon resonance excitation fluorescence, and provide a biomolecule trapping substance, and a biomolecule detection device. <P>SOLUTION: The detection method of the biomolecule is a method for detecting the biomolecule by detecting the surface plasmon resonance excitation fluorescence, and uses a biomolecule trapping substance which bonds a substance that can control arrangement by receiving external force. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a biomolecule detection method, biomolecule capture substance and biomolecule detection apparatus using surface plasmon resonance excitation fluorescence.

  A spectroscopic analysis method using surface plasmon resonance (hereinafter also referred to as SPR) is known as a method for investigating the interaction of biomolecules and a method for detecting specific biomolecules thereby. For example, Patent Document 1 discloses an apparatus and method for determining the binding of an analyte to a solid phase by individually analyzing signals obtained by surface plasmon resonance measurement and fluorescence measurement.

  Patent Documents 2, 3 and 5 below disclose a two-dimensional imaging apparatus using surface plasmon resonance, and Patent Document 4 discloses surface plasmon resonance measurement and second plasmon resonance in which generated fluorescence is generated. The surface plasmon fluorescence microscope used is disclosed, and Patent Document 6 discloses a biomolecule interaction detection apparatus and detection method using plasmon resonance fluorescence.

  In these surface plasmon resonance excitation fluorescence methods, enhanced excitation light generated by surface plasmon resonance is applied to a fluorescent material fixed on a metal substrate via a carrier, a biomolecule capturing material, or the like, and fluorescence emitted is detected. At this time, if the distance between the fluorescent material and the metal substrate is short, energy transfer occurs from the excited fluorescent material to the metal substrate, and the fluorescence quantum yield is greatly reduced. In order to detect a very small amount of biomolecules with high sensitivity and accurately quantify them, it is necessary to control the distance from the base of the labeled fluorescent substance.

When arranging biomolecule-capturing substances such as antibody molecules on a metal substrate, a polymer layer SAAM layer is usually formed, and antibody molecules are bound thereon, but it is difficult to arrange them with good orientation, and therefore Similarly, it is difficult to control the distance of the labeled fluorescent substance captured by the antigen-antibody reaction from the substrate.
JP-A-10-307141 JP 2001-255267 A JP 2002-116149 A JP 2004-156911 A JP 2004-271337 A JP 2006-208069 A

  An object of the present invention is to provide a highly sensitive and highly accurate biomolecule detection method, biomolecule detection method, biomolecule capture substance, and biomolecule detection device using surface plasmon resonance excitation fluorescence.

The present invention is achieved by the following configurations.
1. A method for detecting a biomolecule by detecting surface plasmon resonance excitation fluorescence, characterized by using a biomolecule-capturing substance combined with a substance capable of controlling the sequence by receiving an external force Detection method.
2. 2. The biomolecule detection method according to 1 above, wherein the external force is a magnetic force.
3. 3. The method for detecting a biomolecule according to 1 or 2, wherein the biomolecule-capturing substance is an antibody.
4). 4. The biomolecule detection method according to any one of 1 to 3, wherein the biomolecule-capturing substance is fluorescently labeled.
5. A biomolecule-capturing substance used for detecting a biomolecule by detecting surface plasmon resonance excitation fluorescence is combined with a substance capable of controlling the arrangement by receiving an external force. Molecular trapping material.
6). A biomolecule detection apparatus that detects a biomolecule by detecting surface plasmon resonance excitation fluorescence using a biomolecule capture substance combined with a substance whose sequence can be controlled by receiving an external force. A biomolecule detection apparatus comprising a generation means, a laser light detection means, a fluorescence detection means, and an external force generation means.

  According to the present invention, a highly sensitive and highly accurate biomolecule detection method, biomolecule capture substance, and biomolecule detection apparatus using surface plasmon resonance excitation fluorescence can be provided.

[Method for detecting biomolecules]
The biomolecule detection method of the present invention will be described in detail.

  In the present invention, biomolecules (hereinafter also referred to as analytes) are detected using surface plasmon resonance excitation fluorescence (Surface Plasma Fluorescence Spectroscopy, hereinafter also referred to as SPFS).

  A general protocol for surface plasmon resonance excitation fluorescence is shown below. This method includes a prism (A), an object detection means (B) having a metal thin film on the prism surface and an object detection layer, a laser generation means (C), and first and second light detection means (D, E) is used to detect the reflection intensity of the laser light incident on the thin film from the laser generating means (C) by the first light detecting means (D) and enter the thin film from the laser generating means (C). The second light detection means (E) detects the fluorescence output from the fluorescent molecules excited by the plasmon light generated on the back surface of the thin film by the emitted laser light.

  Next, the characteristics of plasmon light will be described below.

  Plasmon light is generated on the back surface of the thin film in the incident angle region where the intensity of the reflected light detected by the first light detection means (D) attenuates when the laser light is irradiated to the metal thin film under total reflection conditions. The intensity varies depending on the incident angle of the laser beam. Assuming that the incident angle of the laser beam when the attenuation of the reflected light intensity is maximum is the first incident angle, the intensity of the plasmon light is maximized in the vicinity, but strictly speaking, the intensity of the plasmon light is the maximum. The incident angle of the laser light (second incident angle) is slightly lower than the first incident angle. In calculating this theoretical value, the document: T.W. Liebermann, W.M. Knoll, Surface-plasmon field-enhanced fluorescence spectroscopy, Colloids and Surfaces A: Physicochemical and Engineering Aspects 171 (2000) 115-130 and the like can be referred to. Plasmon light is near-field light, and its electric field strength has a characteristic that it attenuates as the distance from the metal thin film increases. When a fluorescent molecule is excited by plasmon light, the fluorescent molecule needs to exist in a range where the near-field light can reach. In addition, when the fluorescent molecule is located very close to the metal thin film, the transition of excitation energy to the metal thin film occurs.

  Therefore, it can be seen that in order to efficiently excite fluorescent molecules by plasmon light, it is necessary to control the distance of the fluorescent molecules from the metal substrate.

[Biomolecule-capturing substance binding substance capable of controlling sequence by receiving external force]
In order to detect a biomolecule (also expressed as an analyte) by surface plasmon resonance excitation fluorescence, generally a substance (F) that specifically recognizes and adsorbs a biomolecule (both a biomolecule capture substance or an analyte capture substance) For example, an antibody against an antigen is used. By binding a fluorescent molecule to the biomolecule capturing substance (F), the analyte can be detected by measuring fluorescence.

  The present invention is characterized in that a substance (G) whose sequence can be controlled by receiving an external force is bound to the biomolecule capturing substance (F).

  Examples of the biomolecule include DNA, RNA, protein, sugar chain, or a degradation product thereof. Examples of the biomolecule-capturing substance include complementary DNA and RNA, an antibody, a lectin, or an imprint polymer that can specifically recognize and capture them.

  As a method for capturing biomolecules, a conventionally known assay may be used. References: Development of biotest drugs (CMC Publishing Co., Ltd.), development / evaluation of biodiagnostic drugs and companies (CMC Publishing Co., Ltd.) The cited literature, immunoassay lecture (WEB information, http://www.shibayagi.co.jp/IA-LEcture/Contents.htm), etc. are helpful. In the present invention, an immunoassay is preferred, and a sandwich type assay is preferred.

  For example, when a gold substrate is selected as the sensor substrate used for the detection layer and sandwich immunoassay is performed, an alkane having a reactive group (or a functional group that can be converted into a reactive group) such as a carboxyl group or an amino group on the gold substrate. A SAM film is formed by allowing thiol to act, and a primary antibody is allowed to act on and immobilize the SAM film appropriately through a polymer or the like. Alternatively, a polymer having a reactive group for the primary antibody (or a functional group that can be converted into a reactive group) may be directly immobilized on a gold substrate, and the primary antibody may be immobilized thereon. When an antibody or a polymer is bound using various reactive groups, an amidation condensation reaction through succinimidylation, an addition reaction through maleimidation, or the like is common. By flowing a solution containing the antigen through the thus configured detection layer, the antigen can be captured by the immobilized primary antibody. On the other hand, the captured antigen can be labeled by applying a solution containing a labeled secondary antibody. The primary antibody may be allowed to act after the antigen and the secondary antibody have been reacted in advance.

  The external force of the present invention is, for example, a force that the device can work on without contacting the substance, such as magnetic force (magnetic attractive force, magnetic repulsive force), coulomb force (electric attractive force, electric repulsive force), buoyancy, etc. In view of ease of handling, magnetic force is preferable.

  A substance whose arrangement can be controlled by receiving an external force is, for example, a magnetic substance (paramagnetic substance, ferromagnetic substance, superparamagnetic substance, etc.) when the external force is a magnetic force. A particle-type magnetic body or molecular magnetic body containing chromium oxide, cobalt, ferrite, neodymium, or the like can be used. When the external force is a Coulomb force, a charged substance such as polyatomic ions or cluster ions can be used. When buoyancy is used as an external force, particles having a specific gravity lighter than that of a solvent or particles enclosing a substance having a light specific gravity can be used.

  On the other hand, what gives an external force is required, but when a magnetic force is used for the external force, a permanent magnet, an electromagnet, or the like can be used. When a Coulomb force is used for the external force, an electrode or the like can be used.

  In the normal fluorescence method, detection is performed from the direction perpendicular to the substrate plane. If a magnet or electrode is placed, detection may be hindered. For example, a transparent magnet or electrode is used, or a detection window is prepared on both sides. This can be solved by arranging a magnet on the screen or by detecting the fluorescence from the lateral direction.

  In the present invention, the substance whose arrangement can be controlled by receiving an external force binds to an analyte capturing substance that binds (or binds) to an analyte capturing substance or a fluorescent molecule that is immobilized on the substrate, and binds to the analyte capturing substance. It is used by binding to the fluorescent molecule. At this time, the fluorescent molecule itself may be a substance capable of controlling the arrangement by an external force.

  In the case of bonding, the bonding may be performed using a covalent bond, an ionic bond, a coordination bond, a hydrogen bond, or the like, and the bonding can be performed through an appropriate high molecular material or low molecular material.

  As a method for controlling the arrangement of fluorescent molecules by an external force, for example, a substance that captures an analyte (also referred to as an analyte capturing substance) is bound from a substrate via a SAM film, a coating layer, etc. After that, an analyte capturing substance bound with a fluorescent molecule and a substance whose sequence can be controlled by an external force is added to capture the analyte immobilized on the substrate. In the case of a normal assay, the chain molecules that have captured the analyte are oriented in various directions or bent, so the distance from the substrate is very short or fluctuates with respect to the chain length of the molecules. Is also big. In the present invention, an external force is applied to a substance that can be controlled by receiving an external force that is bound to the substance that has captured the subject, and its position is controlled. For example, by placing a magnet on the upper part of the detection layer and using magnetic attraction, it is possible to lift the magnetic material upward, and it is possible to stretch the chain molecule group that captured the bound analyte, The distance of the bound fluorescent molecule from the substrate can be controlled. At this time, by using the difference in adsorption force, it is possible to move away only the analyte capturing substance that has not captured the analyte that causes noise from the range where the plasmon light reaches.

[Biomolecule detector]
Hereinafter, although the structural example of the biomolecule detection apparatus of this invention is shown, this invention is not limited to this.

  FIG. 1 is a block diagram showing a schematic configuration of a biomolecule detection apparatus according to an embodiment of the present invention. The biomolecule detection apparatus according to the present embodiment includes an analyte detection unit (hereinafter, referred to as “analyte”) that holds a sample including a biomolecule to be detected (hereinafter referred to as “analyte”) formed on the prism 1. 2), a laser generator 3, and a polarizing plate 41, a diaphragm 42, a shutter 43, a rotating mirror 44, and a lens 45 arranged on the optical path of the laser light L output from the laser generator 3. The first and second CCD cameras 5 and 6 serving as light detection means, and the first and second filters 7 and 8 are provided.

  FIG. 2 is a cross-sectional view showing an example of the configuration of the detection unit 2 formed on the prism 1. As the prism 1, a 45-degree right-angle prism having a high refractive index can be used. The detection unit 2 includes a gold thin film 21 formed on the surface of the prism 1 by a sputtering method or a vapor deposition method to a thickness of 30 to 60 nm (preferably 40 to 60 nm, more preferably 43 to 53 nm), and a surface of the gold thin film 21. And an analyte detection layer (hereinafter, also referred to as a detection layer) 24 formed by being surrounded by a spacer 23 and containing an antibody that reacts with an antigen, a quartz substrate 25, and a fixture 22 for fixing them. ing. Here, a buffer space 27 is formed by the spacer 23, the detection layer 24, and the quartz substrate 25, and flow paths 26a and 26b are formed in the quartz substrate 25 by through holes. The flow paths 26a and 26b are used to inject the sample solution into the buffer space 27. For example, the sample solution is injected from the flow path 26a into the buffer space 27 via a tube (not shown), and the flow path 26b. From the buffer space 27 through another tube (not shown). For the detection layer 24, a polymer material to which an antibody is bound can be used. The antibody is bound to one end of the polymer material, and the other end of the polymer material is directly or indirectly fixed to the surface of the gold thin film 21. A plurality of types of polymer materials may be present. Examples of the polymer material include polyethylene glycol (hereinafter referred to as PEG) and MPC polymer. This may be prepared at the time of use, or a substrate on which these are bonded in advance may be used.

  The first CCD camera 5 is for observing the reflected laser beam from the gold thin film, and is arranged at a position where the reflected laser beam can be received through the first filter 7. On the other hand, the second CCD camera 6 is for detecting the fluorescence from the fluorescent molecules contained in the detection layer 24. The high-sensitivity CCD camera suitable for the observation wavelength and the second filter 8 detect the fluorescence. It is installed above the part 2. Here, the first CCD camera 5 only needs to be able to measure at least the reflected light intensity, and may not be capable of two-dimensional imaging, and may be a photodiode.

  For the laser generator 3, for example, a He—Ne laser can be used. The rotating mirror 44 can change the angle of the mirror, and the incident angle θ of the laser light L to the gold thin film 21 can be changed, for example, in the range of θ = 28 to 62 (degrees). 1, the bending of the optical path on the prism surface through which the laser beam L passes is omitted.

  Laser light L output from the laser generator 3 is P-polarized by the polarizing plate 41, passes through the aperture 42, and opens the shutter 43 while the detection unit 2 passes through the rotating mirror 44, the lens 45, and the prism 1. Is reflected by the first CCD camera 5 through the lens 45 and the filter 7. The fluorescence from the fluorescent molecules in the detection layer 24 is measured by the second CCD camera 6.

[Detailed description of biomolecule detection method]
Next, a biomolecule detection method using the biomolecule detection apparatus will be described with a specific example, but the present invention is not limited to this.

  First, a PBS (or other buffer solution) solution is injected into the buffer space 27 of the detection unit 2.

The laser beam L output from the laser generator 3 is fixed to the angle of the mirror surface of the rotating mirror 44 and is incident on the gold thin film 21 of the detector 2 via the prism 1 as described above. Measure with 1 CCD camera 5 and record reflection intensity. As is well known, when the P-polarized laser beam L is coupled with the electronic vibration in the gold thin film 21 to cause the SPR phenomenon, the intensity of the reflected light decreases. The angle of the rotating mirror is adjusted so that the intensity of the reflected light becomes the minimum value. (From here, the mirror angle may be changed so that the plasmon light has the maximum incident angle.)
An antibody is immobilized on the detection layer 24 in the buffer space 27 in advance, and a solution containing an antigen for the antibody is flowed, followed by a solution containing a secondary antibody in which fluorescent molecules and magnetic particles are bound.

  As a result, the fluorescent molecules in the detection layer 24 are excited by the plasmon light generated by SPR and generate fluorescence. At this time, by bringing the magnet closer from the top, the antigen captured by the secondary antibody and the antibody bound thereto are changed. Extends to the top and can increase fluorescence intensity.

  The generated fluorescence is measured, that is, two-dimensionally imaged, by the second CCD camera 6 located above the detection unit 2. The luminance of each pixel in the obtained two-dimensional image corresponds to the fluorescence intensity at each location on the detection layer.

  As described above, the antigen in the solution injected into the detection layer 24 can be detected with high sensitivity and high accuracy by measuring the fluorescence intensity with the second CCD camera 6.

  In this measurement, an appropriate fluorescent molecule may be used as the fluorescent molecule in consideration of the wavelength of the laser beam or the like. For example, when a He-Ne laser having a wavelength of 632.8 nm is used as the light source, Cy5, Alexa Fluor 647, or the like can be used.

  Moreover, although the case where PEG was used was demonstrated above, it is not limited to this, A fluorescent molecule | numerator and an antibody can be couple | bonded with an edge part, A soft chain-like high molecular material whose length is several to several dozen nm If it is. An appropriate polymer material can be used according to the antibody or fluorescent molecule to be used. For example, polyglutamic acid, 2-hydroxyethyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, or the like can be used.

In addition, the thin film formed on the prism surface is not limited to a gold thin film, but may be a thin film of other metal (such as silver) or metal oxide (such as SiO ₂ , TiO ₂ , Al ₂ O ₃ , Ag ₂ O). There may be.

  Moreover, although the detection part provided with the quartz substrate and spacer which have a flow path was demonstrated above, it is not limited to this. Any structure may be used as long as the analyte detection layer is formed on the gold thin film surface and can hold the biomolecules of the analyte.

  Moreover, although the case where one detection layer was provided was demonstrated above, you may form a detection part by arranging the several detection layer containing a different antibody on an gold | metal thin film in an array form.

  In the above description, the case where one kind of fluorescent molecule is used has been described. However, the present invention is not limited to this. It may be formed. In this case, SPR measurement and SPFS measurement are performed using a plurality of laser beams having different wavelengths according to the type of fluorescent molecule or a lamp having various filters inserted therein.

  Further, the first light detecting means is not limited to the CCD camera, and any means capable of measuring laser light having a predetermined wavelength may be used. For example, a photodiode or the like can be used. Similarly, the second light detection means is not limited to the CCD camera described above, and may be any means that can measure fluorescence of a predetermined wavelength. For example, a photomultiplier tube or the like may be used.

  The light source to be irradiated may be any light source that can extract light in a straight line form, and may be a semiconductor laser, an LED, or a lamp.

  The method of scanning the irradiation light is not limited to the rotating mirror, and the light source itself may be scanned by a stage controller or the like.

  The shape of the prism is not limited to a 90-degree triangular prism, and a 60-degree triangular prism or a semi-cylindrical prism may be used.

  The present invention will be described with reference to specific examples, but the present invention is not limited thereto.

Example 1
(Preparation of detector)
As shown in FIG. 2, LaSFN9 (refractive index n = 1.85) manufactured by SCHOTT GLASS, which is a high-refractive 45-degree right-angle prism, is used as the prism 1, and a film thickness of about 48 nm is formed on the bottom by sputtering. A quartz substrate 25 on which the flow paths 26a and 26b were formed was sandwiched between silicon rubber spacers 23 having a thickness of 2 mm and placed on the gold thin film 21. A PBS buffer (phosphate buffer, pH = 7.2) was injected into this using a pump. The cell volume at this time was approximately 160 μl.

  Next, a 0.5 mM PBS buffer solution of HS-PEG-COOH (MW5000, nanoc) is prepared, and this PBS buffer solution is injected by a pump into the detection part at the stage where the quartz substrate 25 is formed and left overnight. A self-assembled film was formed on the surface of the gold thin film 21.

(Preparation of labeled antibody solution of the present invention)
Nanomag-D-spio (registered trademark, PEG-COOH, 50 nm, 5 mg / ml, manufactured by Corefront Co., Ltd.) 100 ml, which is a magnetic particle, N-hydroxysuccinimide is 50 mM, WSC (1-Ethyl-3 -After adding 5 ml of PBS buffer prepared so that (3-dimethylaminopropyl) carbodiimide, hydrochloride, condensing agent) is 100 mM, and stirring for 20 minutes, anti-AFP monoclonal antibody (1D5, 2.5 mg / ml, Japanese medical clinic) 40 μl of 1M sodium bicarbonate aqueous solution and 0.5 mg of AlexaFluor 647 NHS ester were added and reacted for 2 hours, so that the magnetic particles and the fluorescent dye were combined. To obtain a PBS buffer 識抗 body.

(Preparation of labeled antibody solution for comparison)
Anti-AFP monoclonal antibody (1D5, 2.5 mg / ml, sold by Nippon Medical Laboratory), 40 μl, 1 M sodium bicarbonate aqueous solution 10 μl, AlexaFluor 647 NHS ester 0.5 mg, PBS 5 ml mixed and reacted for 2 hours Thus, a PBS buffer solution of a comparative labeled antibody to which only a fluorescent dye was bound was obtained.

(Detection of labeled antibody of the present invention)
The actual measurement will be described. The laser used was a 633 nm HeNe laser.

  After the detection unit is manufactured as described above, the laser incident light angle is adjusted so that the reflected light intensity applied to the first CCD is minimized, and the laser irradiation range of the detection unit from above using the second CCD. A 0.2 mm square at the center was observed, and the count value of the CCD at this time was adjusted to 0 point.

  Thereafter, a PBS buffer prepared so that N-hydroxysuccinimide is 25 mM and WSC is 50 mM is injected into the detection part and allowed to act for 30 minutes, and then a PBS buffer containing the labeled antibody of the present invention is injected and circulated. Then, the labeled antibody was immobilized. One hour after the injection, the plate was washed with 0.05% by mass Tween-PBS buffer for 10 minutes, and the count value when observed from the second CCD was measured. The count value at this time was 8000 au (arbitrary unit). The second CCD was confirmed in advance that the light intensity and the count value were in direct proportion up to 500,000 au.

  Next, two commercially available neodymium magnets were set 3 cm above the detector (fixed on both sides of the cell so as not to interfere with light detection from above), and the second CCD count was confirmed to be 13000 au. Yes, ie, the observed signal was 1.6 times.

(Detection of comparative labeled antibody)
Measurement was performed in the same manner as in the detection of the labeled antibody of the present invention described above except that a comparative labeled antibody solution was used instead of the labeled antibody of the present invention.

  The count value at this time was 9000 au (arbitrary unit).

  Next, two commercially available neodymium magnets were set 3 cm above the detector (fixed on both sides of the cell so as not to prevent light detection from above), and the count value of the second CCD was confirmed again to be 9000 au. There was no change in the observed signal.

Example 2
(Preparation of detector)
As shown in FIG. 2, LaSFN9 (refractive index n = 1.85) manufactured by SCHOTT GLASS, which is a high-refractive 45-degree right-angle prism, is used as the prism 1, and a film thickness of about 48 nm is formed on the bottom by sputtering. A quartz substrate 25 on which the flow paths 26a and 26b were formed was sandwiched between silicon rubber spacers 23 having a thickness of 2 mm and placed on the gold thin film 21. A PBS buffer (phosphate buffer, pH = 7.2) was injected into this using a pump. The cell volume at this time was about 160 μL (microliter).

  Next, a 0.5 mM PBS buffer solution of HS-PEG-COOH (MW5000, manufactured by nanoc) is prepared, and this PBS buffer solution is injected into the detection unit at the stage where the quartz substrate 25 is formed by a pump overnight. Then, a self-assembled film was formed on the surface of the gold thin film 21.

  PBS buffer solution prepared so that N-hydroxysuccinimide is 25 mM and WSC is 50 mM is injected into this and allowed to act for 30 minutes, and then anti-AFP (α-fetoprotein) monoclonal antibody (1D5, 20 μg / ml, Japanese medical clinical examination) PBS buffer solution was infused and was allowed to act for 1 hour, and 1% BSA-PBS buffer solution was infused to act for 1 hour, and finally PBS buffer solution was infused to produce a detector.

(Preparation of labeled secondary antibody)
100 ml of nanomag-D-spio (registered trademark, PEG-COOH, 50 nm, 5 mg / ml, manufactured by Corefront Co., Ltd.), 5 ml of PBS buffer prepared so that N-hydroxysuccinimide is 50 mM and WSC is 100 mM In addition, after stirring for 20 minutes, 40 μl of anti-AFP monoclonal antibody (6D2, 2.5 mg / ml, sold by Japan Medical Laboratory), 10 μl of 1M aqueous sodium bicarbonate solution, and 0.5 mg of AlexaFluor 647 NHS ester were mixed. A labeled secondary antibody PBS buffer solution was obtained by adding the mixture and reacting for 3 hours.

(Assay)
The actual measurement will be described. The laser used was a 633 nm HeNe laser.

  After the detection unit is manufactured as described above, the laser incident light angle is adjusted so that the reflected light intensity applied to the first CCD is minimized, and the laser irradiation range of the detection unit from above using the second CCD. A 0.2 mm square at the center was observed, and the count value of the CCD at this time was adjusted to 0 point. AFP PBS buffer solution (10 ng / ml, 5 ml) was injected, reacted while circulating for 30 minutes, and then washed with 0.05 mass% Tween-PBS buffer for 10 minutes. Next, 5 ml of the labeled secondary antibody PBS buffer was circulated for 30 minutes and washed again with 0.05 mass% Tween-PBS buffer. The count value when observed from the second CCD 20 minutes after the start of washing was measured. The count value at this time was 38000 au (arbitrary unit). The second CCD was confirmed in advance that the light intensity and the count value were in direct proportion up to 500,000 au.

  Next, two commercially available neodymium magnets were set 3 cm above the detector (fixed on both sides of the cell so as not to prevent light detection from above), and the second CCD count was confirmed to be 53000 au. That is, the observed signal was 1.4 times.

  In addition, the standard deviation of the count value of 5 minutes before and after the count value of 38000 au (20 minutes after the start of washing) was 1000 au, but after setting the magnet, the standard deviation was about 53,000 au. The standard deviation of the 5-minute count value was 800 au, which was 0.8 times. In the detection method of the present invention, variation in the count value is suppressed, and it can be said that the measurement is more accurate.

Example 3
The assay was carried out under the same conditions as in Example 2 except that PBS buffer not mixed with AFP was sent instead of PBS buffer mixed with AFP.

  The count value of the second CCD was 5000 au 20 minutes after feeding the labeled secondary antibody PBS buffer and washing. Thereafter, when the magnet was set in the same manner as in Example 1, the count value of the second CCD showed 3800 au.

  As described above, by using the present invention, the fluorescence intensity per biomolecule is increased, the variation can be suppressed, and the noise due to non-specific adsorption can be reduced. Compared to this, it has become possible to detect biomolecules with higher sensitivity and higher accuracy.

It is a block diagram which shows schematic structure of the biomolecule detection apparatus of this invention. It is sectional drawing which shows an example of a structure of the detection part used with the biomolecule detection apparatus of this Embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Prism 2 Test object detection part 3 Laser generator 4 Light control part 5 1st CCD camera 6 2nd CCD camera 7 1st filter 8 2nd filter 21 Gold thin film 22 Fixture 23 Spacer 24 Test object detection layer 25 Quartz substrate 26a, 26b Flow path 27 Buffer space 41 Polarizing plate 42 Aperture 43 Shutter 44 Rotating mirror 45 Lens 46 External force generator (magnet)

Claims (6)

A method for detecting a biomolecule by detecting surface plasmon resonance excitation fluorescence, characterized by using a biomolecule-capturing substance combined with a substance capable of controlling the sequence by receiving an external force Detection method. The biomolecule detection method according to claim 1, wherein the external force is a magnetic force. The method for detecting a biomolecule according to claim 1 or 2, wherein the biomolecule-capturing substance is an antibody. The method for detecting a biomolecule according to any one of claims 1 to 3, wherein the biomolecule-capturing substance is fluorescently labeled. A biomolecule-capturing substance used for detecting a biomolecule by detecting surface plasmon resonance excitation fluorescence is combined with a substance capable of controlling the arrangement by receiving an external force. Molecular trapping material. A biomolecule detection apparatus that detects a biomolecule by detecting surface plasmon resonance excitation fluorescence using a biomolecule capture substance combined with a substance whose sequence can be controlled by receiving an external force. A biomolecule detection apparatus comprising a generation means, a laser light detection means, a fluorescence detection means, and an external force generation means.

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