JP2006208069A - Apparatus and method for detecting biomolecular interaction using plasmon resonance fluorescence - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus and method for detecting biomolecular interaction capable of detecting precisely the biomolecular interaction. <P>SOLUTION: The apparatus is provided with a prism 1, a specimen detecting means 2 having a metal thin film and a specimen detecting layer on a prism surface, a laser generating means 3, and the first and second photodetecting means 5, 6. The specimen detecting layer contains a linear polymer material coupled with a fluorescent molecule and an antibody in one terminal part, the other terminal part of the polymer material is immobilized to the polymer material, and the biomolecule interaction detector detects the reflection intensity of a laser beam incident into the thin film at the first incident angle from the laser generating means 3, by the first photodetecting means 5, and detects fluorescence output from the fluorescent molecule excited with plasmon light generated by the laser beam incident into the thin film at the second incident angle from the laser generator 3, by the second photodetecting means 6. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention is a surface plasmon resonance (hereinafter also referred to as SPR).
The interaction of biomolecules in the cell differentiation process in cell culture using the surface plasmon excitation enhanced fluorescence spectroscopy (Surface Plasmon Fluorescence Spectroscopy, hereinafter also referred to as SPFS) method The present invention relates to a biomolecule interaction detection apparatus and detection method capable of performing the above.

  As a method for examining the interaction between a biomolecule and a cell, a spectroscopic analysis method using surface plasmon resonance is known. For example, Patent Document 1 below discloses an apparatus and method for determining the binding of an analyte to a solid phase by separately analyzing signals obtained by surface plasmon resonance measurement and fluorescence measurement.

  Patent Documents 2, 3, and 5 listed below disclose a two-dimensional imaging device using surface plasmon resonance.

Patent Document 4 below discloses a surface plasmon fluorescence microscope using surface plasmon resonance measurement and second plasmon resonance in which generated fluorescence is generated.
JP-A-10-307141 JP 2001-255267 A JP 2002-116149 A JP 2004-156911 A JP 2004-271337 A

  However, the above Patent Documents 2, 3, and 5 use SPR, so that there is a problem that the signal is relatively small and the measurement sensitivity is low. Moreover, since the intensity change of the reflected light by adsorption | suction to a thin film is measured, the reaction in the place away from the thin film cannot be detected.

  In the above Patent Document 3, it is possible to improve the sensitivity of measurement by SPR using a conical prism, but it is observable only near the apex of the conical prism, and in order to measure a predetermined area range, the apex of the conical prism is used. It takes a long time to measure.

  In any of the above Patent Documents 1 to 5, even if it can be detected that the adsorption to the thin film is caused by the reaction to be observed, it cannot be distinguished whether it is specific adsorption or non-specific adsorption. .

  In order to solve the above-mentioned problems, the present invention can detect biomolecule interactions such as antigen-antibody reaction with high accuracy and can detect biomolecules generated in the process of cell differentiation. It is an object of the present invention to provide a biomolecule interaction detection apparatus and detection method using the SPFS method and the SPFS method.

  The object of the present invention is achieved by the following means.

That is, the biomolecule interaction detection device (1) according to the present invention includes a prism, a thin film containing a metal formed on the surface of the prism, and an object detection layer formed on the surface of the thin film. A detection means; a laser generation means; a first light detection means; and a second light detection means, wherein the analyte detection layer has a chain shape in which a fluorescent molecule and an antibody are directly or indirectly bound to one end. A polymer material, and the other end of the polymer material is directly or indirectly fixed to the surface of the thin film, and is incident on the thin film at a first incident angle from the laser generating means via the prism. The reflected intensity of the laser beam is detected by the first light detecting means, and excited by the plasmon light generated by the laser light incident on the thin film at a second incident angle from the laser generating means through the prism. The fluorescence fluorescent molecules output which is is characterized by detecting by the second light detecting means.

  Moreover, the biomolecule interaction detection apparatus (2) according to the present invention is the biomolecule interaction detection apparatus (1), wherein the second incident angle is the first when the reflection intensity is minimum. It is characterized by being smaller than the incident angle.

  The biomolecule interaction detection device (3) according to the present invention is the biomolecule interaction detection device (1) or (2) described above, wherein the thin film has a thickness of about 30 nm to 60 nm. The thickness of the film formed of the molecular material is about 50 nm or less.

  The biomolecule interaction detection device (4) according to the present invention is the biomolecule interaction detection device (1) to (3) described above, wherein the polymer material is polyethylene glycol, polyglutamic acid, 2 It is a material selected from the group consisting of -hydroxyethyl methacrylate and 2-methacryloyloxyethyl phosphorylcholine.

  The biomolecule interaction detection device (5) according to the present invention is the biomolecule interaction detection device (1) to (4) described above, wherein the second light detection means is a two-dimensional image imaging means. It is characterized by being.

  The biomolecule interaction detection device (6) according to the present invention is the biomolecule interaction detection device (1) to (5) according to any one of the above-described biomolecule interaction detection devices (1) to (5). The polymer material is polyethylene glycol having a molecular weight of about 200,000 or less, the fluorescent molecule is Cy5 or AlexaFluor 647, bound to the antibody, and the laser beam has a wavelength of 632. .8 nm He—Ne laser, wherein the analyte detection layer has a self-assembled film formed on the thin film, a first biotin bound to the self-assembled film, and the first biotin And the second biotin bound to the streptavidin, and the other end of the polymer material binds to the second biotin. By the polymeric material is characterized in that it is fixed to the thin film indirectly.

The biomolecule interaction detection method (1) according to the present invention comprises an analyte detection means having a thin film formed on the surface of a prism and an analyte detection layer formed on the surface of the thin film. The analyte detection layer includes a chain-like polymer material in which an antibody is bound to one end, a fluorescent molecule is bound to the antibody or the one end, and the other end of the polymer material is A method for detecting an interaction between a biomolecule introduced into the analyte detection means and the antibody using a biomolecule interaction detection device fixed directly or indirectly on the surface of the thin film, comprising: a laser beam Is incident on the thin film through the prism at a first incident angle, and the process of detecting the reflection intensity is repeated by changing the first incident angle, and the reflection intensity is minimized. The first angle of incidence as the candidate angle A laser beam is incident on the thin film through the prism and is excited by plasmon light generated by the incident, with an incident angle defined as an angle obtained by subtracting a predetermined angle from the candidate angle. And a third step of detecting the fluorescence output from the fluorescent molecule.

  The biomolecule interaction detection method (2) according to the present invention further includes a fourth step of washing the biomolecule input to the analyte detection means in the biomolecule interaction detection method (1). Including performing the third step before and after the fourth step to measure the fluorescence output by the fluorescent molecule, comparing the fluorescence intensities obtained before and after the fourth step, and It is characterized by detecting occurrence.

  The biomolecule interaction detection method (3) according to the present invention is the biomolecule interaction detection method (1) or (2) described above, wherein the analyte detection layer includes a plurality of types of antibodies. Fluorescent molecules having different fluorescence wavelengths according to the type of the antibody bound to the one end of the molecular material are bound to the one end, and a plurality of laser beams having wavelengths corresponding to the respective fluorescent molecules And the first to third steps are executed for each wavelength of the laser beam.

  Moreover, the biomolecule interaction detection method (4) according to the present invention is the biomolecule interaction detection method (1) to (3) described above, wherein in the third step, the two-dimensional distribution of the fluorescence is obtained. It is characterized by measuring.

  According to the present invention, the interaction of biomolecules can be detected efficiently with high detection sensitivity using SPR and SPFS.

  Further, only the antibody of the analyte detection layer needs to be fluorescently modified, and the fluorescent modification of the detection target biomolecule or the cell fused with the antigen is unnecessary, and the measurement of the interaction becomes easy.

In addition, among the interactions of biomolecules, specific binding, such as antigen-antibody reaction, in situ,
It is possible to detect with high accuracy. Therefore, it is useful for observation of antigen-antibody reaction on the back side of cells. In particular, for cells that differentiate during the culture process and express different antigens on the cell membrane over time, the present invention was cultured without removing the cells from the medium and examining them as in the conventional method. On the detection part, trace the time course of the appearance of the antigen as it is,
It is effective for identifying an antigen in situ.

  In addition, the fluorescence distribution can be captured as a two-dimensional image, and the interaction of biomolecules can be detected efficiently without scanning.

  In addition, by using multiple antibodies of different types, modifying with different fluorescent molecules for each antibody type, and using multiple laser beams of the corresponding wavelengths, it can be performed simultaneously or for multiple types of antigens. Interaction can be detected over time. Furthermore, a solution containing the analyte may be injected into one entire analyte detection unit, and for each type of antibody, a corresponding small analyte detection layer is divided and arranged in an array. There is no need to inject a solution containing the analyte in the segmented region, and the measurement can be performed very efficiently.

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, embodiments of the invention will be described with reference to the accompanying drawings.

FIG. 1 is a block diagram showing a schematic configuration of a biomolecule interaction detection apparatus according to an embodiment of the present invention. The biomolecule interaction detecting apparatus according to the present embodiment includes an object detection unit (holding a sample including a detection target biomolecule (hereinafter referred to as an object)) formed on the prism 1 and the prism 1. (Hereinafter referred to as a detection unit) 2, a laser generator 3, a polarizing plate 41, an aperture 42, a shutter 43, a rotating mirror 44, and a lens 45 disposed on the optical path of the laser light L output from the laser generator 3. And a first and second CCD cameras 5 and 6 as light detection means, and first and second filters 7 and 8. 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 to a thickness of 30 to 60 nm (nanometer) (preferably 40 to 60 nm, more preferably 43 to 53 nm) by sputtering or vapor deposition, An analyte detection layer (hereinafter referred to as a detection layer) 24 containing an antibody that reacts with an antigen, which is surrounded on the surface of the thin film 21 by a spacer 23, a quartz substrate 25, and a fixture for fixing them. 22. 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). The detection layer 24 includes polyethylene glycol (hereinafter referred to as PEG) to which a predetermined fluorescent molecule and an antibody are bound. Furthermore, both the fluorescent molecule and the antibody are bound to one end of PEG, and the other end of PEG is fixed to the surface of the gold thin film 21 directly or indirectly. The binding order of the antibody, the fluorescent molecule, and the PEG is arbitrary, and the antibody and the fluorescent molecule may be bonded so that they are located at one end of the PEG. Furthermore, another relatively small polymer material may be interposed between them.

  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, may not be able to perform two-dimensional imaging, and may be a photodiode.

  For the laser generator 3, for example, a He—Ne laser can be used. Further, the rotation 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 within a range of θ = 28 to 62 (degrees), for example. it can. In FIG. 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 via 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.

  Next, a biomolecule interaction detection method using the present biomolecule interaction detection apparatus will be described with reference to the flowchart shown in FIG. It is assumed that a solution containing cells having an antigen against the antibody contained in the detection layer 24 is injected into the buffer space 27 of the detection unit 2 in advance, and the detection unit 2 is installed horizontally.

  In step S1, a minimum value θmin is set as the incident angle θ as an initial setting.

  In step S2, the angle of the mirror surface of the rotating mirror 44 is fixed and the laser beam L output from the laser generator 3 is incident on the gold thin film 21 of the detector 2 via the prism 1 as described above. The reflected light is measured by the first CCD camera 5 and the reflection intensity is recorded. 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.

In step S3, it is determined whether or not the incident angle θ is equal to or less than a preset maximum value θmax. When the incident angle θ is equal to or smaller than the maximum value θmax, the process proceeds to step S4, and after increasing the incident angle θ by Δθ, the process returns to step S2. In step S2, the angle of the rotating mirror 44 is changed according to the newly determined θ, and in step S2, the intensity of the reflected light is measured. If the incident angle θ is not less than or equal to the maximum value θmax, the process proceeds to step S5. Thereby, the intensity of the reflected light can be measured by changing the range of θ = θmin to θmax at intervals of a predetermined step Δθ.

In step S5, the incident angle θdip at which the reflection intensity is minimized is obtained from the measurement values obtained in the above steps. FIG. 4 is a diagram illustrating an example of a graph obtained by obtaining the reflectance based on the incident light intensity from the measured reflection intensity and plotting it with respect to the incident angle θ. As shown in FIG. 4, the reflectance has a minimum value at a predetermined angle corresponding to the thickness of the gold thin film 21 and the refractive index of the prism 1. The angle θ at which the minimum value is obtained is determined as θdip. In FIG. 4, a broken line indicates that the angle θdip at which the reflectance becomes a minimum value changes when the thickness of the gold thin film 21 is different or when an adherent is formed on the surface of the gold thin film 21. Show.

In step S6, a predetermined angle δ is subtracted from the angle θdip obtained in step S5,
Find the angle θ1. Here, δ is a value in the range of 0.1 to 2 (degrees). The reason why the angle θ1 that is slightly smaller than the angle θdip obtained in step S5 is obtained is that the peak position of the fluorescence intensity described later occurs at an incident angle that is slightly smaller than the angle θ = θdip.

In step S7, the rotating mirror 44 is adjusted so that the incident angle θ of the laser beam L to the gold thin film 21 becomes the angle θ1 obtained in step S6, and the laser beam L from the laser generator 3 is incident on the gold thin film 21. To do. As a result, the fluorescent molecules in the detection layer 24 are excited by the plasmon light generated by the SPR to generate fluorescence. 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.

  At this time, depending on the distance of the fluorescent molecule from the surface of the gold thin film 21, the fluorescence intensity that can be observed with the second CCD camera 6 changes as shown in the graph of FIG. In FIG. 5, the horizontal axis represents the distance from the gold thin film surface to the fluorescent molecule, and the vertical axis represents the relative fluorescence intensity observed. Therefore, when the antibody attached to PEG in the detection layer 24 binds to the antigen and the PEG is elongated in the interface direction, the fluorescent molecule attached to the same end as the side to which the PEG antibody is attached Since it moves away from the surface of the gold thin film 21, stronger fluorescence is observed.

FIG. 6 schematically shows how fluorescence from fluorescent molecules is greatly observed as PEG expands. As shown on the left side of FIG. 6, when the PEG is contracted and in the vicinity of the surface of the gold thin film 21, the fluorescence emitted from the fluorescent molecules is transferred to the gold thin film 21 as indicated by the downward arrows. It happens to be almost quenched. However, as shown on the right side, when PEG expands and moves away from the surface of the gold thin film 21, the energy transfer efficiency to the gold thin film 21 decreases, and as indicated by the upward arrow, fluorescence is emitted to the outside, It can be detected. From the graph of FIG. 5, the maximum value of the distance that the fluorescent molecule moves away from the surface of the gold thin film due to the extension of PEG is preferably about 50 nm or less, and therefore the length of PEG is preferably about 50 nm or less. That is, the molecular weight of PEG is desirably about 200,000 or less, and more desirably tens of thousands or less. When PEG is indirectly fixed to the gold thin film surface, the value including the thickness of the material used for fixing is preferably about 50 nm or less.

  In step S8, it is determined whether or not the process is finished. If not finished, the process returns to step S1. For example, after a predetermined time elapses, the processes in steps S1 to S7 are performed again and the second CCD camera 6 performs imaging. As a result, a change in fluorescence intensity with time, that is, a change with time in the antigen-antibody reaction can be observed. Further, after the predetermined time has elapsed, the detection unit may be washed (hereinafter also referred to as “rinse”), and the processing of steps S1 to S7 may be performed again, and imaging by the second CCD camera 6 may be performed. If the fluorescence intensity enhanced before the injection of cells having antigen is maintained after washing, it can be said that the interaction is stable, that is, an antigen-antibody reaction.

  As described above, the interaction between the antibody formed in the detection layer 24 and the antigen in the injected solution can be detected by measuring the fluorescence intensity with the second CCD camera 6. Therefore, for example, by bringing a solution containing cells into contact with a detection layer formed using a known antibody, a protein (antigen) produced by the cells can be specified.

  As the fluorescent molecule, an appropriate fluorescent molecule may be used in consideration of the wavelength of laser light. For example, when a He-Ne laser having a wavelength of 632.8 nm is used as the light source, Cy5, AlexaFluor 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, and 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.

In the above description, the laser incident angle θ1 at the time of SPFS measurement is described as being slightly smaller than the incident angle θdip corresponding to the minimum value of the reflection intensity observed by SPR. However, the present invention is not limited to this. It is sufficient that the angle is close to 0, and θ1 ≧ θdip may be satisfied. Although it is desirable that θ1 is slightly smaller than θdip, if θ1 is a value close to θdip (including the case where they are equal), a sufficiently strong signal can be observed.

  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 that case, interaction with a plurality of antibodies can be observed at a time for the same biomolecule.

In the above description, the case of using one type of fluorescent molecule has been described. However, the present invention is not limited to this, and a detection unit is formed by combining multiple types of antibodies and fluorescent molecules having different fluorescence wavelengths for each type of antibody. It may be formed. In this case, the SPR measurement and the SPFS measurement are performed by using a plurality of laser beams having different wavelengths according to the type of fluorescent molecule or a lamp having various filters inserted therein. By using this detection unit, an antigen-antibody reaction can be detected for a plurality of types of antigens simultaneously or over time. In particular, in the case of cells that differentiate during the culturing process, different antigens may be expressed on the cell membrane over time, but this is not the case when cells are removed from the medium and examined as in the conventional method. In the invention, the time course of the appearance of the antigen can be traced as it is on the detection unit for culturing, which is effective for specifying the antigen in situ. In this case, it suffices to inject a solution containing the analyte into one entire detection unit, and for each type of antibody, a corresponding small detection layer is divided and arranged in an array, and each divided region is arranged in an array. An operation of injecting a solution containing the subject is unnecessary, and measurement can be performed very efficiently.

  The means for measuring the two-dimensional distribution of fluorescence (second CCD camera 6) is not limited to the above-described CCD camera, and may be any two-dimensional imaging device capable of measuring fluorescence of a predetermined wavelength.

  Examples are shown below to further clarify the features of the present invention.

(Preparation of the detector according to the present invention)
First, the manufactured detection unit according to the present invention will be described.

  As shown in FIG. 2, LaSFN9 (refractive index n = 1.85) manufactured by SCHOTT GLASS, which is a high-refractive-index 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).

  11-Amino-1-undecanethiol and (+)-Biotin N-hydroxysuccinimide ester (NHS-Biotin) (both manufactured by Dojindo) were reacted at a molar ratio of 2: 1 to obtain 0.25 mM PBS of biotinylated alkanethiol. A buffer was prepared. This PBS buffer solution was injected by a pump into the detection part at the stage where the quartz substrate 25 was formed and left for about 1 hour to form a self-assembled film on the surface of the metal thin film 21.

  Thereafter, it was rinsed with 3 to 4 mL of PBS buffer, followed by injection of 3 μM Streptavidin PBS buffer. After about 1 hour, a layer of Streptavidin bound to Biotin was formed on the self-assembled membrane.

This was rinsed again, and then a PBS buffer solution of Cy5-modified Anti-GFP-PEG-Biotin prepared in advance by the following method was injected. The PBS buffer of Cy5-modified Anti-GFP-PEG-Biotin was first modified with Cy5 as a fluorescent molecule using Cy5 Antibody Labeling Kit (Amersham) and isolated by column Cy5-modified Anti-GFP at a concentration of 0.1 mg / mL
It was obtained by mixing 0.3 mL and 0.34 mg of NHS-PEG-Biotin (molecular weight 3400, Shearwater) and leaving it to stand for about half a day with occasional stirring. Immediately before use, it was diluted 3 times with PBS buffer.

About 1 hour after the injection of Cy5-modified Anti-GFP-PEG-Biotin, rinsing was performed again, leaving only those bound to Streptavidin. As a result, as shown in FIG. 7, a detection layer 24 in which a self-assembled film, Streptavidin, and Cy5-modified Anti-GFP-PEG-Biotin are stacked in this order is formed on a gold thin film. Completed.

(Adjustment of detection target)
Next, preparation of a membrane fraction containing a GFP fusion membrane protein as a detection target will be described.

  The EGFP gene was excised from the EGFP-N1 vector (Clontech), and the Myc tag and EGFP gene were introduced from the amino terminus of GluR2, followed by the signal sequence, and then from the third transmembrane region to the carboxyl terminus of GluR2. A gene encoding the protein to be contained (hereinafter referred to as GFP-tGluR2) was prepared. An expression vector containing this GFP-tGluR2 was transfected into A6 cells to prepare stably expressing cells.

  The stably expressing cells were cultured, and a membrane fraction was prepared as follows. That is, stably expressing cells were suspended in PBS, sonicated and centrifuged (1000 g for 10 minutes). The supernatant was collected, centrifuged again (8000 g for 30 minutes), and the precipitate was suspended again in PBS and used for the experiment.

(SPR measurement and SPFS measurement)
The GFP fusion membrane protein-containing membrane fraction was applied to the detection unit formed as described above at a concentration of 0.69 mg / mL, and as described with reference to FIG. 3, SPR measurement and SPFS measurement (two-dimensional imaging) Was done. A He—Ne laser is used as the laser light source, and the wavelength is 632.8. nm laser light was used. In SPFS measurement, δ = 0.5 (degrees) and θ1 = θdip−0.5 (degrees) using an angle θdip determined by SPR measurement so that the fluorescence intensity can be detected with the highest sensitivity. The incident angle was fixed to, and the fluorescence intensity was measured. In the production of the detection part, the formation kinetics and final film thickness of each layer were confirmed by tracking them by SPR measurement.

  The measurement results are shown in FIG. In FIG. 8, the horizontal and horizontal axes represent time (minutes) and fluorescence intensity, respectively. As indicated by a circle (●) in FIG. 8, the fluorescence intensity increased with time. This is presumably because the PEG chain of a random coil-like soft chain is pulled toward the interface by binding with the membrane fraction, and as a result, the fluorescent molecule is separated from the gold thin film. FIG. 9 is a schematic diagram showing this state. In addition, rinsing was carried out about 1 hour after the start of application of the GFP fusion membrane protein-containing membrane fraction to the detection unit. From FIG. 8, the fluorescence intensity was added to the GFP fusion membrane protein-containing membrane fraction. It can be seen that the value has increased to 200% or more of the value before performing. This means that the cause of the increase in fluorescence intensity, that is, the reaction state between the detection layer and the administered membrane fraction remains without being removed from the detection unit. That is, it can be seen that the reaction was specific binding (antigen-antibody reaction).

  In FIG. 8, as a comparative experiment, a membrane fraction not fused with GFP was prepared and applied to the detection unit according to the present invention prepared above, and SPR measurement and SPFS measurement were performed in the same manner as described above. The results are indicated by squares (□). In FIG. 8, as for the membrane fraction not fused with GFP, the fluorescence intensity is increased from the time of introduction of the membrane fraction, similarly to the membrane fraction fused with GFP. This is presumably because the non-specific adsorption between the membrane fraction not fused with GFP and the detection layer affected the morphology and motility of the PEG chain. The occurrence of non-specific adsorption was confirmed by the fact that a wide-angle shift of the SPR dip, that is, a phenomenon that the angle θdip becomes large was also observed for the membrane fraction not fused with GFP.

  Further, regarding the membrane fraction not fused with GFP, the fluorescence intensity decreases after rinsing. Therefore, in this case, specific binding due to the antigen-antibody reaction did not occur, and it was found that the membrane fraction that had affected the morphology and motility of the PEG chain was removed by rinsing, and PEG returned to its original state. .

As described above, it is possible to detect whether specific binding (antigen-antibody reaction) has occurred by performing SPR measurement and SPFS measurement before and after rinsing using the detection unit according to the present invention. In normal microscopic observation, it is difficult to observe the back side of the cell, which is a binding part, when looking at what is adsorbed or bound to the substrate. Thus, according to the present invention using SPFS measurement, This method is useful for in situ observation of antigen-antibody reaction on the backside of cells, which was not known by the method. In addition, it is only necessary that the detection layer be fluorescently modified, and it is also a great advantage that the fluorescent modification to the cells fused with the antigen is unnecessary.

  A detection unit different from the detection unit according to the present invention described above was produced and a comparative experiment was performed.

(Preparation of detection part for comparative experiments)
Biotinylated PEG alkanethiol, Streptavidin, and Cy5-modified Anti-GFP-Biotin were conjugated as follows.

First, a 1 mM PBS buffer solution of 11-Amino-1-undecanethiol was injected into the detection part at the stage where the quartz substrate 25 was formed in the same manner as described above, and a self-assembled film was formed on the surface of the metal thin film 21. After rinsing, 1 mM NHS-PEG-Biotin (molecular weight 3400, manufactured by Shearwater) was injected and reacted to prepare Biotinylated PEG alkanethiol. After rinsing with PBS buffer, 3 μM Streptavidin was injected and allowed to bind to Biotin. Finally, Cy5-modified Anti-GFP-Biotin modified with Cy5 using a Cy5 Antibody Labeling Kit was injected into Biotin-Anti-GFP (manufactured by Cosmo Bio), rinsed, and completed as a detection unit for comparative experiments. Here, the major difference from Example 1 is that the GFP antibody and PEG are not directly bound in the detection part for comparison experiment.

  In the same manner as in Example 1, the GFP fusion membrane fraction was administered to the detection part for comparative experiments, and SPR measurement and SPFS measurement were performed. As a result, the fluorescence intensity did not increase even after the GFP fusion membrane fraction was added. This is probably because the PEG and the antibody were not directly connected, and the phenomenon that the antibody was pulled toward the cell membrane due to the binding with GFP did not occur.

  From this, in the detection part effective for cell observation in which an antigen-antibody reaction occurs, the PEG chain and the antibody are bound, and the antibody and the fluorescent molecule are bound to the end of the PEG on the same side ( It may be said that it is a detection part (which may be directly fluorescently modified to an antibody).

It is a block diagram which shows schematic structure of the biomolecule interaction detection apparatus which concerns on embodiment of this invention. It is sectional drawing which shows an example of a structure of the detection part used with the biomolecule interaction detection apparatus which concerns on this Embodiment. It is a flowchart which shows the detection method of the biomolecule interaction using the biomolecule interaction detection apparatus which concerns on this Embodiment. It is a graph which shows the relationship between the incident angle in SPR, and a reflectance. It is a graph which shows the relationship between the fluorescence intensity in SPFS, and the distance from a gold thin film. It is a schematic diagram which shows a mode that the fluorescence from a fluorescent molecule is largely observed when PEG expand | extends. It is a schematic diagram which shows the detection part which concerns on this invention formed. It is a figure which shows a measurement result. It is a schematic diagram which shows reaction of the formed detection part concerning this invention, and a to-be-detected film | membrane fraction.

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 Fixing tool 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

Claims (10)

Prism,
An analyte detection means having a thin film containing metal formed on the surface of the prism and an analyte detection layer formed on the surface of the thin film;
Laser generating means;
A first light detection means and a second light detection means;
The analyte detection layer includes a chain-like polymer material in which a fluorescent molecule and an antibody are directly or indirectly bound to one end,
The other end of the polymeric material is directly or indirectly fixed to the surface of the thin film;
The first light detection means detects the reflection intensity of the laser light incident on the thin film at a first incident angle through the prism from the laser generation means,
Fluorescence output from the fluorescent molecules excited by plasmon light generated by laser light incident on the thin film at a second incident angle from the laser generating means through the prism is detected by the second light detecting means. A biomolecular interaction detection device characterized by:   The biomolecule interaction detection apparatus according to claim 1, wherein the second incident angle is smaller than the first incident angle when the reflection intensity is minimized. The thin film has a thickness of about 30 nm to 60 nm;
The biomolecule interaction detection device according to claim 1 or 2, wherein a film formed of the polymer material has a thickness of about 50 nm or less. 4. The polymer material according to claim 1, wherein the polymer material is a material selected from the group consisting of polyethylene glycol, polyglutamic acid, 2-hydroxyethyl methacrylate, and 2-methacryloyloxyethyl phosphorylcholine. The biomolecule interaction detection apparatus according to the section.   The biomolecule interaction detection apparatus according to any one of claims 1 to 4, wherein the second light detection means is a two-dimensional image imaging means. The thin film is formed to a thickness of 40 nm or more and 60 nm or less using gold,
The polymer material is polyethylene glycol having a molecular weight of about 200,000 or less;
The fluorescent molecule is Cy5 or AlexaFluor 647, bound to the antibody,
The laser beam is a He-Ne laser having a wavelength of 632.8 nm,
The analyte detection layer includes a self-assembled film formed on the thin film, a first biotin bonded to the self-assembled film, streptavidin bonded to the first biotin, and the streptavidin A second biotin bound to
6. The polymer material is indirectly fixed to the thin film by binding the other end of the polymer material to the second biotin. The biomolecule interaction detection device according to Item. An analyte detection means having an analyte detection layer formed on the surface of the prism and an analyte detection layer formed on the surface of the thin film, the analyte detection layer having a chain in which an antibody is bound to one end; A biomolecule interaction, wherein a fluorescent molecule is bound to the antibody or the one end, and the other end of the polymer material is directly or indirectly fixed to the surface of the thin film. A method for detecting an interaction between the antibody and a biomolecule charged into the analyte detection means using a detection device,
A first step of repeating laser light incident on the thin film at a first incident angle through the prism and detecting a reflection intensity by changing the first incident angle;
A second step of determining, as a candidate angle, the first incident angle at which the reflection intensity is minimal;
Fluorescence output from the fluorescent molecules excited by the plasmon light that is incident on the thin film through the prism, with the angle obtained by subtracting a predetermined angle from the candidate angle as the incident angle. And a third step of detecting the biomolecule interaction detection method. A fourth step of washing the biomolecule charged into the analyte detection means;
Execute the third step before and after the fourth step, measure the fluorescence output by the fluorescent molecule, compare the fluorescence intensities obtained before and after the fourth step, and generate specific binding. The biomolecule interaction detection method according to claim 7, wherein the detection is performed. The analyte detection layer includes a plurality of types of antibodies,
Fluorescent molecules having different fluorescence wavelengths depending on the type of the antibody bound to the one end of the polymer material are bound to the one end,
Using a plurality of laser beams with wavelengths according to each of the fluorescent molecules,
The biomolecule interaction detection method according to claim 7 or 8, wherein the first to third steps are executed for each wavelength of the laser beam.   The biomolecule interaction detection method according to any one of claims 7 to 9, wherein, in the third step, a two-dimensional distribution of the fluorescence is measured.

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