JP4684915B2 - Biomolecule affinity analyzer and method for analyzing affinity between biomolecules using the device - Google Patents

Description

  The present invention relates to a device for analyzing the affinity of biomolecules and a method for analyzing the affinity between biomolecules using the device.

Conventionally, as a method for analyzing the affinity of a biomolecule, for example, in the case of a protein that interacts with a nucleic acid molecule, when the protein binds to the nucleic acid molecule, the molecular weight becomes larger than in the case of the nucleic acid molecule alone, so a DNA-protein complex A method such as a gel shift assay has been developed in which a gel is electrophoresed on a carrier such as a gel to detect a difference in mobility. (Non-Patent Document 1)
Nucleic Acids Research 23, 6505-6525 (1981)

As other methods for detecting nucleic acid-protein interactions, the following methods have been proposed.
Footprinting method: A method for recognizing a specific gene sequence and determining a gene binding site of a protein to be bound. (Non-patent document 2)
Southwestern method: After separating proteins by SDS polyacrylamide gel electrophoresis, transfer to a nitrocellulose filter. A method of measuring the binding to a target substance using DNA labeled with a radioisotope as a probe. (Non-Patent Document 3)
Double-stranded DNA array: A method in which double-stranded DNA is arrayed on a glass substrate, the protein is reacted on the substrate, and then the protein bound to the DNA is detected using an antibody or the like. (Non-patent documents 4 to 6)
Nucleic Acids Research 5, 3157-3170 (1978) Cell 52, 415-423 (1988) Nature Biotechnology 17,536-537 (1999) Nature Biotechnology 17, 573-577 (1999) Proc Natl Acad Sci USA 98, 7158-7168 (2001)

In addition, as a method for detecting a nucleic acid-protein interaction, there are a surface plasmon resonance (SPR) method (Non-patent Document 7) and a detection method using a microplate. There are commercially available kits for detecting DNA-binding proteins using microplates (for example, Clontech, Activemotif (sold by Funakoshi)), which has DNA immobilized on a 96-well microplate. After the measurement sample is dropped into the well and the DNA is allowed to interact, detection is performed with an antibody.
Nucleic Acids Research 19, 2788 (1999)

Also, in the method of analyzing the affinity of protein-protein interaction, S. Fields et al. Expresses two types of proteins to be examined in yeast and cultured cells, and transcribes only when they interact. Methods such as two-hybrid have been developed in which factors function and detect by activation of expression of specific reporter genes. (See Patent Document 1 and Non-Patent Document 8)
US Pat. No. 5,283,173 Fields and Song, 1989, Nature, 340: 245-246

  As a problem in the method of analyzing proteins that interact with nucleic acid molecules by gel shift assay, it is necessary to examine electrophoresis conditions in which the mobility changes sufficiently with respect to the size of the polyacrylamide gel used for the analysis. is there. In addition, the bands detected by gel shift are often not clear even under the studied electrophoresis conditions, and in particular, when the interaction is weak, even if many nucleic acid molecules and proteins are used, the band of only the nucleic acid molecule itself Is not clear, making it difficult to detect gel shift. In addition, even if the interaction is strong, prepare and prepare a substantial amount of protein and nucleic acid molecules to detect the interaction of the purified protein with RI or fluorescently labeled nucleic acid molecules under many concentration conditions There is a need.

On the other hand, the method of analyzing protein interactions by yeast two-hybrid is time-consuming and has a very high detection sensitivity for interactions and is suitable for exhaustive analysis, but because there are many false positive or non-specific interactions, In order to perform an accurate analysis, the design of fragmented proteins to minimize false positive or non-specific interactions, various molecular biological techniques and data analysis, etc. are combined to produce false positive or non-specific interactions. It is necessary to eliminate the detection of the action.
Furthermore, in the conventional interaction analysis, an antibody against the target protein is required, but a detection method that can be applied to a means for analyzing a novel protein is required.

  Therefore, the present invention solves these problems of the prior art, can use a small amount of sample, and can analyze the affinity between biomolecules quickly and with high accuracy by a simple operation. It is an object of the present invention to provide a device for analyzing the affinity of biomolecules, which can also be applied as a means for performing the same, and a method for analyzing the affinity between biomolecules using the device.

In the present invention, in order to solve the above problems, the following configurations 1 to 12 are adopted.
1. A sample container having at least a pair of electrodes, a light source, a local illumination unit, a high-magnification optical observation unit, a sorting optical filter, and an AC voltage application unit, and at least intends to analyze an affinity interaction in the sample container An affinity analysis apparatus for biomolecules , wherein two types of biomolecules are reciprocated by electrophoresis by applying an alternating voltage between the electrodes .
2. 2. The biomolecule affinity analysis apparatus according to 1, wherein the local illumination unit is a fluorescence excitation unit using fluorescence illumination, and includes a fluorescence excitation light source and a fluorescence selection optical filter.
3. 3. The biomolecule affinity analysis apparatus according to 2, wherein the local illumination means is fluorescence excitation means by evanescent illumination.
4). The sample container has a flow channel shape, a pair or a plurality of pairs of electrodes are arranged in the flow channel, and the biomolecule is electrophoresed by a frequency change of a voltage between the electrodes. 4. The biomolecule affinity analyzer according to any one of 3 above.
5. Interaction is discriminated in the sample container of the biomolecule affinity analyzer comprising at least a pair of electrodes, a light source, local illumination means, high-magnification optical observation means, sorting optical filter, and AC voltage application means By holding a solution of a mixture of at least two types of biomolecules and applying an alternating voltage between the electrodes , the biomolecules are reciprocated by electrophoresis in the solution, and before and after the reaction by affinity interaction. A method for analyzing affinity between biomolecules , wherein the association state of the biomolecules is evaluated by comparing changes in the maximum speed or movement distance of reciprocation of individual biomolecules .
6). 6. The method for analyzing affinity between biomolecules according to 5, wherein one of the biomolecules is a particle having a biomolecule fixed on a surface thereof.
7). 7. The method for analyzing affinity between biomolecules according to 5 or 6, wherein one of the biomolecules is fluorescently labeled.
8 . The sample container forms a flow path shape, when holding the sample solution, using a flow-type or merged-flow channel, the frequency change between the electrodes, a reciprocating movement by electrophoresis the biomolecule The method for analyzing affinity between biomolecules according to any one of 5 to 7 , wherein
(In the present invention, the merged flow channel means a channel in which two or more flow channels are combined and the solution is mixed in the sample container.)
9 . The method for analyzing affinity between biomolecules according to any one of 5 to 8, wherein at least one of the biomolecules is a nucleic acid molecule.
10 . At least one of the biomolecules is a protein, and the pH of the sample solution is adjusted to a pH that is not an isoelectric point of at least one protein, or a pH sandwiched between isoelectric points of two biomolecules. method of analyzing the affinity between bio-molecule of any of 5-9.

  According to the present invention, it is possible to analyze the affinity between biomolecules quickly and with high accuracy by a simple operation using a small amount of sample. The present invention is applicable as a means for analyzing a novel protein, and has high practical value.

  The apparatus for analyzing the affinity between biomolecules of the present invention comprises a sample container having at least a pair of electrodes, a light source, local illumination means, high magnification optical observation means, sorting optical filter, and AC voltage application means. is there. Then, the sample container of the apparatus holds a solution of a mixture of at least two types of biomolecules for discriminating the interaction, and an alternating voltage is applied between the electrodes to reciprocate the biomolecules in the solution, before and after the reaction. By comparing changes in the motion index of individual biomolecules and evaluating the association state of the molecules, the affinity between the molecules is examined.

  In particular, as the local illumination means, it is preferable to use fluorescence excitation means by fluorescence illumination. Only the fluorescent particles or the fluorescently labeled biomolecules immobilized on the surface are detected by the fluorescence excitation light source and the fluorescence selection optical filter. By comparing changes in the maximum moving speed or moving distance (amplitude), the affinity between molecules can be efficiently detected by evaluating the state of association of particles or molecules.

In addition, the sample container has a flow channel shape, a pair or a plurality of pairs of electrodes are arranged in the flow channel, the voltage frequency between the electrodes is changed, and the biomolecules immobilized on the surface or the biomolecules are electrophoresed. By doing so, it becomes possible to detect the affinity of biomolecules with a very small amount of sample.
Furthermore, when the sample container is made into a flow channel shape and a small amount of dilute sample solution is held, the frequency of the voltage between the arranged electrodes is changed using a flow type or combined flow type flow channel so that the biomolecule is placed on the surface. Electrophoresis of fixed particles or biomolecules enables continuous detection.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. However, the following specific examples do not limit the present invention.
1A and 1B are diagrams showing an example of the biomolecule affinity analysis apparatus of the present invention. FIG. 1A is a schematic diagram showing an outline of the apparatus, and FIG. 1B is a state in which an AC voltage is applied to a sample container of the apparatus. It is a schematic diagram which shows. The apparatus 1 includes a sample container 102 in which at least a pair of electrodes 103 are arranged, a light source 105, a local illumination unit 107, a high-magnification optical observation unit 107, a sorting optical filter 109, and an AC voltage application unit 104.

  In order to analyze the affinity between biomolecules using this apparatus 1, a sample container 102 holds particles or a mixture solution of biomolecules 101 on which at least two types of biomolecules for discriminating interactions are fixed on the surface. By applying an AC voltage between the electrodes 103, 103 by the AC voltage applying means 104, the biomolecule to be discriminated or the particle 101 having the biomolecule to be discriminated and the biomolecule fixed on the surface is caused to reciprocate 112 in the solution [ In FIG. 1B, the affinity between molecules is examined by evaluating the state of association of the particles or molecules by comparing changes in the motion index of individual particles or molecules before and after the reaction.

FIG. 2 is a diagram showing another example of the biomolecule affinity analysis apparatus of the present invention.
This device 2 uses fluorescent particles or fluorescently labeled molecules 201 as particles or biomolecules on which biomolecules to be discriminated for interaction are fixed on the surface. In this apparatus 2, the local illumination means and the high magnification optical observation means 107 in the apparatus 1 of FIG. 1 are replaced with the fluorescence illumination by the local illumination means and the high magnification optical observation means 207 arranged below the sample container 202 or the fluorescence by the evanescent illumination 212. As an excitation means, the fluorescence excitation light source 205 and the fluorescence sorting optical filter 209 detect only the fluorescent particles or the fluorescently labeled molecules 213, and evaluate the association state of the particles or molecules by comparing changes in the motion index. This is to check the affinity between molecules.

In this apparatus 2, fluorescent particles to be detected in the sample container 202 are selected by selecting the fluorescence excitation light source 205 and the fluorescence sorting optical filter 209 in accordance with one or more types of fluorescent particles or fluorescently labeled molecules 201. Alternatively, only the fluorescently labeled molecule 213 can be selected and detected.
When the local illumination means and the high magnification optical observation 207 are evanescent illumination, the illumination range 212 in the sample container 202 is a range that is very close to the local illumination means and the high magnification optical observation 207 as compared with the fluorescent illumination. Since it can be limited to (depending on the wavelength and incident angle of the excitation light 206 but generally 100 to 200 nm), fluorescence detection can be performed with lower background noise.

FIG. 3 is a schematic view showing another example of a sample container used in the biomolecule affinity analysis apparatus of the present invention.
FIG. 3A shows the sample container 102 in the apparatus 1 of FIG. 1 as a sample container 302 having a flow path shape. The sample container 302 is provided with a pair of electrodes 304, and the frequency of the voltage between the electrodes is changed by an AC power source 303, and the particles or molecules 301 that discriminate the interaction that has flowed to the pair of electrodes 304 are electrophoresed. By doing so, the reciprocating motion is detected.

FIG. 3B illustrates the other particle or molecule 305 to be mixed with one particle or molecule 301 at the merge portion 306 in front of the pair of electrodes 304 using a merged flow channel as the sample container 302. And join.
FIG. 3C shows a case where a plurality of pairs of electrodes 307 are arranged inside a sample container 302 having a channel shape. Further, FIG. 3D is a diagram in which a plurality of pairs of electrodes 307 are arranged in place of the pair of electrodes 304 in FIG. 3A and FIG. 3E in FIG.

By making the sample container into a flow channel shape as shown in FIG. 3, it becomes possible to make the particles or molecules to be detected a very small and dilute sample solution. When holding the solution, the flow type or the combined flow Continuous detection is possible by using an expression channel.
Also, as shown in FIGS. 3C, 3D, and 3E, a plurality of pairs of electrodes 307 are arranged, and different frequency changes are made between the respective electrodes, thereby comparing with the case of a pair of electrodes. It is possible to obtain a greater change in the maximum moving speed or moving distance (amplitude).

  The light source constituting the biomolecule affinity analysis apparatus of the present invention is not particularly limited, but normally, a mercury lamp or xenon lamp using an arc discharge as a light emitter, particularly an ultrahigh pressure mercury lamp is preferably used. Further, the constituent material of the sample container can be arbitrarily selected, but transparent plastics such as polymethyl methacrylate and polyolefin, glass, and the like are preferably used. As the sorting optical filter, for example, a commercially available fluorescent filter set can be used to select an excitation wavelength range and an observation wavelength range. In addition, an objective lens of various microscopes can be used as the high magnification optical observation means, and a CCD camera or the like can be used as the image sensor for detecting an image.

Examples of biomolecules for which affinity is analyzed according to the present invention include nucleic acid molecules such as proteins, DNA, and RNA.
Suitable analysis targets include proteins having a molecular weight of about 30 to 150 kDa, such as actin, streptavidin, IgG, or double-stranded DNA having a size of about 20 base pairs, such as a transcription factor recognition sequence.
Although these biomolecules can be measured, the biomolecules themselves can be measured, and at least one of the biomolecules can be measured with the surface of fine particles having an average particle size of about 0.1 to 10 μm being fixed. Is possible. As the fine particles for fixing the biomolecule to the surface, for example, any of the fine particles usually used in this field such as plastic beads such as polystyrene and silica beads can be used.
The biomolecule to be measured or the particle having the biomolecule fixed on the surface is usually measured as a dilute aqueous solution using, for example, a phosphate buffer solution containing hydroxypropylmethylcellulose or the like as a solvent. As these biomolecules or particles having the biomolecules immobilized on the surface, it is preferable to use at least one of which is fluorescently labeled.

  A nucleic acid molecule has a negative charge, and the amount of charge does not depend on the base sequence of the nucleic acid molecule, and is proportional to the base length and the number of molecules of the nucleic acid molecule. When at least one of the biomolecules is a nucleic acid molecule, by preparing a sample solution in a pH range of pH 2 to pH 10 such that the surface charge of the particle or molecule changes depending on the association state of the molecule, the maximum moving speed or moving distance It is possible to detect changes in (amplitude). When the nucleic acid molecule is double-stranded DNA, it is necessary to perform measurement at the pH of the sample solution in which the DNA stably forms a double strand.

  Since the apparent charge at a specific isoelectric point of a protein is 0, when at least one of the biomolecules to be measured is a protein, the pH of the dilute sample solution is not the isoelectric point of at least one protein, Alternatively, by adjusting the pH to be sandwiched between two isoelectric points, the change in the maximum moving speed or moving distance (amplitude) of individual particles or molecules before and after the reaction can be increased. When investigating the pH dependence of the affinity of a biomolecule, the measurement is performed while taking into account the isoelectric point.

FIG. 4 is a diagram for explaining the results obtained using the biomolecule affinity analyzer of the present invention. FIG. 4A shows the relationship between the maximum moving velocity vmax with respect to the charge of the particle or molecule for determining the interaction under the two conditions of the frequency f when the amplitude voltage is constant. When measurement with a constant amplitude voltage is performed, the absolute value of the maximum movement speed vmax does not depend on the frequency f but depends on the charge. By performing measurement with a constant amplitude voltage, it is shown that the maximum moving speed vmax changes due to a change in charge when there is affinity.
FIG. 4B shows the relationship of the movement distance (amplitude) A with respect to the charge of the particle or molecule for determining the interaction under the two conditions of the frequency f when the amplitude voltage is constant. When performing measurement with a constant amplitude voltage, the absolute value of the moving distance (amplitude) A decreases as the frequency f increases. By performing measurement with constant amplitude voltage and constant frequency f, it is shown that the movement distance (amplitude) A changes due to a change in charge when there is affinity.
FIG. 4C shows the relationship of the maximum moving speed vmax with respect to the frequency f when the amplitude voltage is constant for the four conditions of the electric charge of the particle or molecule for discriminating the interaction. When measurement with a constant amplitude voltage is performed, the absolute value of the maximum movement speed vmax does not depend on the frequency f but depends on the charge. By performing measurement with a constant amplitude voltage, it is shown that the maximum moving speed vmax changes due to a change in charge when there is affinity.
FIG. 4D shows the relationship of the moving distance (amplitude) A with respect to the frequency f when the amplitude voltage is constant for the four conditions of the electric charge of the particle or molecule for discriminating the interaction. When measuring with a constant amplitude voltage, the absolute value of the moving distance (amplitude) A decreases as the frequency f increases, and the absolute value of the moving distance (amplitude) A increases as the absolute value of the charge increases. By performing measurement with constant amplitude voltage and constant frequency f, it is shown that the movement distance (amplitude) A changes due to a change in charge when there is affinity.
When investigating the affinity of biomolecules using this apparatus, it is possible to obtain a more accurate result by measuring under conditions where the change in maximum movement speed or movement distance (amplitude) is the largest.

Next, the biomolecule affinity analysis apparatus of the present invention and the method of analyzing the affinity of biopolymers using the analysis apparatus will be further described by way of examples. The following specific examples are intended to limit the present invention. is not.
Example 1
In the biomolecule affinity analysis apparatus 2 shown in FIG. 2, a commercially available inverted fluorescent microscope (manufactured by Nikon Instruments Company, TE2000-U) as a light source 205 was used. An excimer laser processing machine (manufactured by Beam Co., Ltd.) forms a groove-like channel having a length of 40 mm, a width of 375 μm and a depth of 400 μm along the width direction in the center of a polymethyl methacrylate plate having a width of 60 mm, a depth of 40 mm, and a thickness of 1 mm After cutting and providing through holes with a diameter of 2.5 mm at both ends in the length direction, the flow path-shaped sample container 202 was configured by covering with a cover glass having a width of 50 mm, a depth of 40 mm, and a thickness of 0.17 mm. . A UV curable adhesive containing mercaptoester as a component was poured between the polymethyl methacrylate plate and the cover glass, and ultraviolet light was irradiated for 10 minutes to cure the adhesive. A pair of electrodes 203 made of a platinum wire having a diameter of 0.5 mm was disposed at both ends of the flow path. Below this sample container 202, a standard filter set for green color detection (G-2A, manufactured by Nikon Instruments Co., Ltd.) is arranged as the fluorescence sorting filter 209, and a cooled CCD camera is arranged as the image sensor 211.
In this apparatus, a dilute solution of at least two types of biomolecules 201 to be measured is injected into a flow path-like sample container 202 from one through hole of the sample container 202, and the dilute solution of the biomolecule 201 is filled in the flow path. Fully filled. An AC voltage having a voltage of 0 to 300 V and a frequency of 0.1 to 1.0 Hz is applied from an AC power source serving as the AC applying means 204 for about 15 seconds, for example, and the biomolecule 201 is reciprocated by the CCD camera while the AC voltage is being applied. A continuous image of motion is acquired, and the affinity of biomolecule interaction is analyzed from the maximum movement distance (amplitude) of the biomolecule.

(Example 2)
In order to measure the voltage dependency and frequency dependency of the AC voltage to be applied, polystyrene fluorescent beads having a carboxyl group introduced on the surface of a spherical particle having a diameter of 1.0 μm (manufactured by Invitrogen, Fluospheres polystyrene microspheres, 1 0 μm, orange fluorescent (540/560)]. A 10 mM phosphate buffer solution (pH 7.0) containing 0.25% hydroxypropylmethylcellulose was used as an aqueous solution for introducing the beads into the measuring apparatus.

The measurement of the voltage dependence and frequency dependence of the AC voltage to be applied is performed by filling these measurement samples in the flow path of the sample container 202 of the analysis apparatus 2 of Example 1, and using a cooled CCD camera in the vicinity of the center of the flow path. After observing fluorescence, an alternating voltage was applied after the movement of the beads in the aqueous solution was sufficiently smaller than the reciprocating motion to measure, and continuous images were acquired. Ten arbitrary beads were selected from the acquired images, and the maximum moving distance, that is, the amplitude of the reciprocating motion of the beads was acquired from the coordinates on each continuous image.
As measurement conditions, an AC voltage of 50, 100, 200, 300 V (12.5, 25, 50, 75 V / cm) was applied to the measurement sample at a frequency of 0.1, 0.2, 0.3 Hz. The data was analyzed. As a result, the amplitude per applied voltage at each frequency is 290 μm / 100 V (correlation coefficient 0.86) at 0.1 Hz, 100 μm / 100 V (correlation coefficient 0.98) at 0.2 Hz, and 54 μm at 0.3 Hz. There was a proportional relationship of / 100V (correlation coefficient 0.98). Moreover, the amplitude per frequency of each applied voltage had an inversely proportional relationship.
In the frequency range where the measurement was performed, the amplitude decreased as the frequency increased, and the correlation of the amplitude per applied voltage was high at 0.2 Hz and 0.3 Hz.

(Example 3)
In order to measure the change in amplitude due to the pH of the aqueous solution, polystyrene fluorescent beads in which carboxyl groups were introduced on the surface of spherical particles having the same diameter of 1.0 μm as in Example 2 were used as measurement targets. As an aqueous solution for introducing the beads into the measuring apparatus, a broad buffer solution containing 0.25% hydroxypropylmethylcellulose (a 0.2 M sodium dihydrogen phosphate aqueous solution is added to a 0.1 M citric acid aqueous solution to obtain a target pH). The buffer solution prepared in (1) was used.

The change in amplitude due to the pH of the aqueous solution is measured by filling these measurement samples in the flow path of the sample container 202 of the analysis apparatus 2 of Example 1, and observing the vicinity of the center of the flow path with a cooled CCD camera for fluorescence observation. After the movement of the beads in the inside was sufficiently smaller than the reciprocating motion to be measured, an alternating voltage was applied to obtain continuous images. Ten arbitrary beads were selected from the acquired images, and the maximum moving distance, that is, the amplitude of the reciprocating motion of the beads was acquired from the coordinates on each continuous image.
As measurement conditions, the pH of the aqueous solution of this measurement sample was set to 4.0, 5.0, 6.0, 7.0, 8.0, the frequency was 0.2 Hz, and an alternating voltage of 200 V (50 V / cm) was applied. The data was analyzed. As a result, the amplitude at each pH was 6.4 μm (standard deviation 1.8 μm) at pH 4.0, 25.6 μm (standard deviation 1.8 μm) at pH 5.0, and 38.7 μm (standard deviation) at pH 6.0. 10.6 μm), pH 7.0 was 55.4 μm (standard deviation 7.8 μm), and pH 8.0 was 164 μm (standard deviation 13.2 μm).
It was found that in the pH range in which the measurement was performed, the amplitude increased as the pH increased, and the amplitude varied depending on the charge of the measurement sample.

Example 4
In order to measure changes in amplitude due to immobilization of heterologous proteins, protein immunoglobulin G (IgG: molecular weight 150 kDa, near isoelectric point pI7) and actin (molecular weight 43 kDa, isoelectric point pI5) are measured as biomolecules. Nearby). As an aqueous solution for introducing these biomolecules into the measuring apparatus, a 10 mM phosphate buffer (pH 7.0) containing 0.25% hydroxypropylmethylcellulose was used.
As particles for immobilizing these biomolecules on the surface, polystyrene fluorescent beads (hereinafter referred to as “non-immobilized beads”) in which carboxyl groups are introduced on the surface of spherical particles having the same diameter of 1.0 μm as in Example 2, A protein immunoglobulin G (IgG: molecular weight 150 kDa) was immobilized on the beads with carbodiimide (hereinafter referred to as “IgG beads”). Similarly, protein actin (molecular weight 43 kDa) was immobilized on the beads with carbodiimide (hereinafter referred to as “actin beads”).

Next, non-fixed beads, IgG beads, and actin beads were mixed with the 10 mM phosphate buffer to prepare a measurement sample.
The measurement of the change in amplitude due to the immobilization of the heterogeneous protein is performed by filling these measurement samples in the flow path of the sample container 202 of the analysis apparatus 2 of Example 1, and observing the vicinity of the center of the flow path with a cooled CCD camera. An alternating voltage was applied after the movement of the beads in the aqueous solution was sufficiently smaller than the reciprocating motion to be measured, and continuous images were acquired. Ten arbitrary beads were selected from the acquired images, and the maximum moving distance, that is, the amplitude of the reciprocating motion of the beads was acquired from the coordinates on each continuous image.
As measurement conditions, an AC voltage of 250 V (63 V / cm) was applied to these measurement samples at a frequency of 0.3 Hz, and data was analyzed. As a result, the amplitude of each sample was 45.3 μm (standard deviation 7.9 μm) for non-fixed beads, 32.0 μm (standard deviation 4.2 μm) for IgG beads, and 63.8 μm (standard deviation 12.8 μm) for actin beads. The amplitude was larger in the order of actin beads, non-immobilized beads, and IgG beads.
In the case of IgG beads, since the pH of the aqueous solution (pH 7.0) and the isoelectric point pI of IgG (near the isoelectric point pI7) are close, the charge becomes small. On the other hand, in the actin beads, the pH of the aqueous solution (pH 7.0) and the isoelectric point pI of actin (near the isoelectric point pI5) are far away, so that the charge becomes large. Therefore, it can be seen that the IgG beads have a smaller amplitude and the actin beads have a larger amplitude than the non-fixed beads.

(Example 5)
As biomolecules to be measured, protein immunoglobulin G (IgG: molecular weight 150 kDa), biotinylated IgG (molecular weight 150 kDa), and streptavidin (molecular weight 60 kDa) were used. As an aqueous solution for introducing these biomolecules into the measuring apparatus, a 10 mM phosphate buffer (pH 7.0) containing 0.25% hydroxypropylmethylcellulose was used.
As particles for immobilizing these biomolecules on the surface, polystyrene fluorescent beads having a carboxyl group introduced on the surface of spherical particles having the same diameter of 1.0 μm as in Example 2 were used, and protein immunoglobulin G (IgG) was used as the beads. : Molecular weight 150 kDa) was immobilized with carbodiimide (hereinafter referred to as “non-biotinylated beads”). Next, beads obtained by biotinylating IgG of non-biotinylated beads with a biotinylation reagent by a conventional method (hereinafter referred to as “biotinylated beads”) were prepared.

Next, for the 10 mM phosphate buffer, only non-biotinylated beads, only biotinylated beads, 0.1 mg / mL streptavidin (molecular weight of about 60 kDa) and non-biotinylated beads, 0.1 mg / mL streptavidin and Biotinylated beads were mixed to prepare measurement samples.
The affinity of the biomolecule is measured by filling these measurement samples into the flow path of the sample container 202 of the analyzer 2 of Example 1, and observing the vicinity of the center of the flow path with a cooled CCD camera to observe the fluorescence in the aqueous solution. After the movement of the beads was sufficiently smaller than the reciprocating motion to be measured, an alternating voltage was applied and continuous images were acquired. Ten arbitrary beads were selected from the acquired images, and the maximum moving distance, that is, the amplitude of the reciprocating motion of the beads was acquired from the coordinates on each continuous image.
As measurement conditions, an AC voltage of 300 V (75 V / cm) was applied to these measurement samples at a frequency of 0.2 Hz, and the data was analyzed. As a result, the amplitude of each sample was 151 μm (standard deviation 18 μm) for the sample containing only non-biotinylated beads, and 143 μm (standard deviation 13 μm) for the sample mixed with streptavidin and non-biotinylated beads, with a difference of about 6%. In contrast, 94.1 μm (standard deviation 4.6 μm) for the sample containing only biotinylated beads and 39.3 μm (standard deviation 8.7 μm) for the sample mixed with streptavidin and biotinylated beads, and streptavidin. In the sample mixed with biotinylated beads, it was about 42% of the amplitude of the sample containing only biotinylated beads.
The amplitude of the sample mixed with streptavidin and non-biotinylated beads is small compared to the amplitude of the sample with only non-biotinylated beads, so there is no significant change in the surface charge, and there is no significant difference between the streptavidin and non-biotinylated beads. It can be seen that the affinity is low. On the other hand, the amplitude of the sample mixed with streptavidin and biotinylated beads is largely different from the amplitude of the sample with only biotinylated beads, so there is a large change in the surface charge, and the affinity between streptavidin and biotinylated beads is large. It turns out that the nature is high.

According to these results, the presence or absence of affinity between the biomolecules to be measured can be easily measured from the changes in the speed and amplitude of the reciprocating motion of the beads that fixed the biomolecules in the aqueous solution to the surface when an AC voltage is applied. It turns out that you can.
When a nucleic acid molecule is used as a biomolecule to be measured, the nucleic acid molecule has a negative charge unlike a protein, and the amount of charge does not depend on the base sequence of the nucleic acid molecule. Since it is proportional to the number of molecules, if protein affinity can be measured as in Example 5, it can be measured in the same manner.

It is a schematic diagram which shows an example of the biomolecule affinity analysis apparatus of this invention. It is a schematic diagram which shows the other example of the biomolecule affinity analysis apparatus of this invention. It is a schematic diagram which shows the other example of the biomolecule affinity analysis apparatus of this invention. It is explanatory drawing of the result obtained using the biomolecule affinity analysis apparatus of this invention.

Explanation of symbols

101, 301, 305 Particles or molecules for discriminating the interaction 102, 202, 302 Sample container 103, 203, 304, 307 Pair of electrodes 104, 204, 303 AC voltage applying means 105 Light source 106 Light 107, 207 Local illumination means and High magnification optical observation 108 Image 109 Sorting optical filter 110 Image 111 sorted by optical filter Image sensor 112 Reciprocating motion of particles or molecules 201, 213 Fluorescent particles or fluorescent labeled molecules 205 Fluorescence excitation light source 206 Excitation light 208 Fluorescence excitation image 209 Fluorescence Selection optical filter 210 Fluorescence excitation image 211 selected by the optical filter Fluorescence image sensor 212 Fluorescent illumination or evanescent illumination 302 Sample container 306 having a flow channel shape Merge portion of a merge flow type flow channel

Claims (10)

At least at least a sample container disposed a pair of electrodes, a light source, a local illumination means, a high magnification optical observation means, comprising a sorting optical filter and alternating-current voltage applying means, in the sample container, to be analyzed for affinity interactions to two biological molecules, by applying an AC voltage between the electrodes, the affinity analyzer of biomolecules, characterized in that to the reciprocating motion by electrophoresis.   The biomolecule affinity analysis apparatus according to claim 1, wherein the local illumination means is fluorescence excitation means by fluorescence illumination, and includes a fluorescence excitation light source and a fluorescence selection optical filter.   3. The biomolecule affinity analysis apparatus according to claim 2, wherein the local illumination means is fluorescence excitation means by evanescent illumination. The sample container has a flow channel shape, a pair or a plurality of pairs of electrodes are disposed in the flow channel, and an alternating voltage is applied between the electrodes, thereby causing the biomolecule to reciprocate by electrophoresis. The biomolecule affinity analyzer according to any one of claims 1 to 3. Interaction is discriminated in the sample container of the biomolecule affinity analyzer comprising at least a pair of electrodes, a light source, local illumination means, high-magnification optical observation means, sorting optical filter, and AC voltage application means By holding a solution of a mixture of at least two types of biomolecules and applying an alternating voltage between the electrodes , the biomolecules are reciprocated by electrophoresis in the solution, and before and after the reaction by affinity interaction. A method for analyzing affinity between biomolecules, wherein the association state of the biomolecules is evaluated by comparing changes in the maximum speed or movement distance of reciprocation of individual biomolecules .   6. The method for analyzing affinity between biomolecules according to claim 5, wherein one of the biomolecules is a particle having a biomolecule fixed on a surface thereof.   The method for analyzing affinity between biomolecules according to claim 5 or 6, wherein one of the biomolecules is fluorescently labeled. The sample container forms a flow path shape, when holding the sample solution, using a flow-type or merged-flow channel, the frequency change between the electrodes, a reciprocating movement by electrophoresis the biomolecule The method for analyzing affinity between biomolecules according to any one of claims 5 to 7 , wherein: The method for analyzing affinity between biomolecules according to any one of claims 5 to 8 , wherein at least one of the biomolecules is a nucleic acid molecule. At least one of the biomolecules is a protein, and the pH of the sample solution is adjusted to a pH that is not an isoelectric point of at least one protein, or a pH sandwiched between isoelectric points of two biomolecules. method of analyzing the affinity between bio-molecule according to any of claims 5-9.

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