WO2011162150A1 - Fluorescence measurement method - Google Patents

Abstract

The disclosed fluorescence measurement method increases the efficiency of detection of fluorescence emitted from fluorescent molecules generated as a result of an enzyme reaction, thereby providing increased sensitivity and allowing fluorescent substrates from conventional enzyme reactions to be used as is. Said fluorescence measurement method includes: a step (b) wherein water-soluble macromolecules and fluorescent-molecule precursor molecules are introduced onto an enzyme-immobilizing substrate; a step (c) wherein an enzyme reaction is induced on the enzyme-immobilizing substrate and the fluorescent-molecule precursor molecules are converted to fluorescent molecules; a step (d) wherein the fluorescent molecules are bound to the aforementioned water-soluble macromolecules, thereby forming fluorescent-molecule/water-soluble-macromolecule complexes on the enzyme-immobilizing substrate; and a step (e) wherein fluorescence emitted from the fluorescent-molecule/water-soluble-macromolecule complexes is detected.

Description

Fluorescence measurement method

The present invention relates to a fluorescence measuring method for detecting a fluorescent molecule generated by an enzymatic reaction with high sensitivity. More specifically, the present invention relates to a fluorescence measurement method that detects fluorescent molecules with high sensitivity by preventing diffusion of fluorescent molecules generated by an enzyme reaction.

As a method of measuring biomolecules such as nucleic acids and proteins, there is a method of measuring the amount of analyte in a sample by labeling the analyte in the sample with an enzyme and evaluating the amount of the molecule generated by the enzyme reaction. Since this method uses the catalytic activity of an enzyme reaction, signal enhancement through an enzyme substrate is easy and is widely used. Here, various methods are used as methods for evaluating the amount of molecules generated by the enzyme reaction. From the ease of highly sensitive measurement, fluorescence or chemiluminescence from the molecules generated by the enzyme reaction is detected. Many methods are used to evaluate the amount of molecules generated by an enzyme reaction based on the amount of luminescence. Among these, an example of a method for measuring a biomolecule based on fluorescence from a molecule generated by an enzyme reaction is a fluorogenic substrate conversion method. In this fluorogenic substrate conversion method, the amount of analyte in a sample is determined by converting the fluorogenic substrate into a fluorescent molecule with an enzyme labeled with the analyte, and detecting the fluorescence emission from this fluorescent molecule. It is a method of measuring. An example of a method for measuring a biomolecule based on chemiluminescence from a molecule generated by an enzyme reaction is an enzyme chemiluminescence method. This enzyme chemiluminescence method is a method for measuring the amount of analyte in a sample by converting a substrate into a chemiluminescent molecule with an enzyme labeled with an analyte and detecting chemiluminescence from this chemiluminescent molecule. It is.

These fluorogenic substrate conversion methods and enzyme chemiluminescence methods are applied to assay methods such as hybridization assays used for nucleic acid detection and immunoassays used for detection of various biomolecules such as proteins. Here, there are various forms of the presence of the enzyme in the assay method to which the fluorogenic substrate conversion method and the enzyme chemiluminescence method are applied, but the ease of operation and the application to the measurement by an automatic measuring instrument are included. From the viewpoint of easiness, an assay method in which an enzyme reaction is performed on a solid phase is frequently used. A prominent example of such an assay method is a solid-phase enzyme immunoassay (Enzyme-linked Immunosorbent Assay: hereinafter referred to as “ELISA method”). When the fluorogenic substrate conversion method and the enzyme chemiluminescence method are applied to an assay method in which an enzyme reaction is performed on a solid phase, there is an advantage that the enzyme reaction field can be used as it is as a measurement field for fluorescence or chemiluminescence.

However, the analyte measurement method using the catalytic activity of the enzyme has a problem that the detection sensitivity cannot be sufficiently improved because the product of the enzyme reaction diffuses quickly into the solution. Various studies have been made to solve such problems.

As one of such attempts, International Publication No. 2007/052613 pamphlet (Patent Document 1) captures an immune complex containing an enzyme-labeled antibody and an analyte on a support without a solution layer, and this immune complex. A method of measuring the amount of analyte by overlaying a support membrane containing a chemiluminescent substrate on the body and detecting the amount of luminescence generated by the enzyme reaction is disclosed.

As another attempt, Japanese Patent Application Laid-Open No. 2008-139245 (Patent Document 2) describes that an enzyme reaction is performed in a state where an immune complex containing an enzyme and an analyte is present in a solution, and a reaction product is detected as a sensor. A method of measuring the amount of analyte by binding to a surface and detecting light is disclosed.

International publication 2007/052613 pamphlet JP 2008-139245 JP-A-62-234618

However, in the method described in Patent Document 1, it is necessary to prepare a support film containing a chemiluminescent substrate in advance and superimpose it in the enzyme reaction. Conventionally known automatic measuring devices that detect analytes in specimens based on the fluorogenic substrate conversion method or the enzyme chemiluminescence method, in many cases, a substrate having an enzyme immobilized on its surface is arranged in the middle of the flow path, and Since it has a structure that sends various substances in a closed system through this flow path, it will become complicated and cost will increase if a mechanism is introduced to the solid phase from the outside of the flow path. At the same time, there is a problem that restrictions are imposed on miniaturization of equipment.

In the case of the method described in Patent Document 2, in order to bind the reaction product of the enzyme reaction to the sensor surface, it is necessary to bind avidin and biotin or digoxigenin and anti-digoxigenin antibody. Has a problem that the reaction product needs to be modified in advance with avidin, biotin, or digoxigenin.

In order to solve the above-described problems of the prior art, the present invention emits light by preventing a fluorescent molecule or the like generated by an enzymatic reaction from diffusing into a solution in an assay method based on a fluorogenic substrate conversion method. An object to be solved is to provide a fluorescence measurement method that enhances the detection efficiency of fluorescence, thereby improving sensitivity, and that allows a fluorescent substrate obtained by a conventional enzyme reaction to be used as it is.

As a result of diligent research to solve the above-mentioned problems, the present inventors have made a water-soluble polymer coexist in the measurement system when detecting fluorescent molecules or the like generated by an enzyme reaction by an enzyme immobilized on a substrate. If this is done, it is possible to prevent diffusion of the generated fluorescent molecules and the like, and to keep the fluorescent molecules and the like within a limited range. As a result, it is found that the intensity of the detected fluorescence increases. The present invention has been completed.

That is, the fluorescence measurement method of the present invention is:
Step (b): introducing a water-soluble polymer and a fluorescent molecule precursor molecule onto the enzyme-immobilized substrate;
Step (c): causing an enzyme reaction on the enzyme-immobilized substrate to convert the fluorescent molecule precursor molecule into a fluorescent molecule;
Step (d): binding the fluorescent molecule to the water-soluble polymer to form a fluorescent molecule-water-soluble polymer complex on the enzyme-immobilized substrate; and
Step (e): The method includes a step of detecting fluorescence emitted from the fluorescent molecule-water-soluble polymer complex.

In the fluorescence measurement method according to the present invention, the water-soluble polymer used in the present invention preferably has an ionic group in order to suitably form a fluorescent molecule-water-soluble polymer complex. It is more preferable that a cationic group is contained as the ionic group. Examples of such a cationic group include one or more groups selected from a tertiary amino group and a quaternary ammonium group. Such a fluorescent molecule-water-soluble polymer complex is preferably formed by electrostatic interaction between the fluorescent molecule and the water-soluble polymer.

In the fluorescence measurement method according to the present invention, the water-soluble polymer has a property of quickly binding to the fluorescent substance generated by the enzyme reaction, but the fluorescent molecule precursor molecule existing in the system approaches the enzyme. In order to do so, it is preferable not to prevent passage through the inside. One such water-soluble polymer is a linear non-crosslinked water-soluble polymer. The water-soluble polymer may be a polymer matrix gel having thixotropy. These water-soluble polymers preferably have a skeleton composed of one or more selected from linear polyacrylamides and polysaccharides. In one embodiment of the present invention, such a water-soluble polymer is a water-soluble cationized polysaccharide, and in this case, the degree of cation substitution in the water-soluble cationized polysaccharide is preferably 0.01 to 1. . The water-soluble polymer used in the present invention has a weight average molecular weight of usually 10,000 to 2,000,000, preferably 50,000 to 2,000,000.

Steps (c) to (e) are desirably performed in a stationary state in order for the fluorescent molecule-water-soluble polymer complex to remain securely without departing from the region to be subjected to fluorescence detection.

The fluorescence measurement method according to the present invention can be applied for use as a biomolecule assay method. That is, by incorporating the biomolecule in the sample into the enzyme-immobilized substrate, the amount of the biomolecule contained in the sample can be evaluated in the form of the fluorescence amount of the fluorescent molecule-water-soluble polymer complex.

As an embodiment of the enzyme-immobilized substrate in which the biomolecule in the specimen is incorporated in this way, an enzyme-immobilized substrate in which the enzyme is immobilized on the substrate through the bond between the biomolecule and the ligand molecule can be mentioned. As a typical embodiment of such an enzyme-immobilized substrate, a ligand molecule is bound to the substrate, a biomolecule is bound to the ligand molecule, and an enzyme is directly or indirectly bound to the biomolecule. A fixed substrate may be mentioned.

When the fluorescence measurement method according to the present invention is used as an assay method for a biomolecule, typical steps of the fluorescence measurement method according to the present invention include, in addition to steps (b) to (e),
Step (a): a step of producing the enzyme-immobilized substrate using a specimen, an enzyme, a ligand molecule and a substrate; and Step (f): the fluorescence contained in the specimen from the amount of fluorescence detected in Step (e) The method further includes the step of quantifying the biomolecule.

In the fluorescence measurement method according to the present invention, the fluorescence detection in the step (e) is preferably performed by detecting fluorescence excited by near-field light. Here, a typical example of the near-field light used is light enhanced by plasmons.

Further, the fluorescence detection in the step (e) may be performed using a confocal laser microscope or a confocal laser scanner.

In the fluorescence measurement method according to the present invention, an enzyme reaction by an enzyme immobilized on a substrate and detection of a fluorescent molecule or the like generated by the enzyme reaction are performed in the presence of a water-soluble polymer, whereby a fluorescent molecule generated by the enzyme reaction is fluorescent. Since it is possible to suppress the diffusion of the fluorescent molecules to the space outside the measurement range in the measurement system by staying in the vicinity of the detection measurement field, it is possible to provide a highly sensitive fluorescence measurement method.

The schematic diagram of the fluorescence measuring method which concerns on this invention is shown. The relationship between the reaction time after introducing the substrate solution and the fluorescence intensity in Example 1 and Comparative Example 1 is shown.

Hereinafter, the present invention will be specifically described with reference to FIG.

[Fluorescence measurement method]
The fluorescence measurement method according to the present invention includes:
Step (b): introducing the water-soluble polymer 43 and the fluorescent molecule precursor molecule 41 onto the enzyme-immobilized substrate 11;
Step (c): causing an enzyme reaction on the enzyme-immobilized substrate 11 to convert the fluorescent molecule precursor molecule 41 into the fluorescent molecule 42;
Step (d): binding the fluorescent molecule 42 to the water-soluble polymer 43 to form a fluorescent molecule-water-soluble polymer complex 31 on the enzyme-immobilized substrate 11; and
Step (e): The method includes a step of detecting fluorescence emitted from the fluorescent molecule-water-soluble polymer complex 31.

The present invention is most characterized in that the reaction for generating the fluorescent molecules 42 on the enzyme-immobilized substrate 11 is performed in the presence of the water-soluble polymer 43.

<Step (b)>
In the fluorescence measurement method of the present invention, step (b) is a step of introducing the water-soluble polymer 43 and the fluorescent molecule precursor molecule 41 onto the enzyme-immobilized substrate 11.

<Enzyme immobilization substrate>
The enzyme-immobilized substrate 11 used in the fluorescence measurement method according to the present invention is a substrate on which an enzyme 23 is immobilized on the surface. The enzyme-immobilized substrate 11 includes a substrate 21 and an enzyme 23, and has a structure in which the enzyme 23 is bonded to the substrate 21.

Substrate In the enzyme-immobilized substrate 11 used in the fluorescence measurement method according to the present invention, the substrate 21 serves as a base for immobilizing the enzyme 23, and provides a measurement field for performing fluorescence detection in the step (e) described later. Have a role. Therefore, in this specification, the substrate 21 may be referred to as a “base substrate” in order to make the distinction from the “enzyme-immobilized substrate” more clear.

The substrate 21 (that is, “basic substrate”) is not particularly limited in material as long as it does not hinder the fluorescence measurement in the present invention and does not impair the activity of the enzyme 23. Conventionally known materials used for typical assay methods can be used. For example, glass may be used as the substrate 21, or plastic such as polycarbonate (PC) or cycloolefin polymer (COP) may be used.

The substrate 21 may be composed of a plurality of layers as appropriate according to the means for detecting fluorescence.

For example, in the case of detecting fluorescence excited by near-field light, particularly light enhanced by plasmon, a glass or plastic transparent flat substrate formed with a metal thin film of about 10 to 100 nm is used as the substrate 21. It is done. When fluorescence is detected by surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), the metal thin film is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum. More preferably, it consists of. About these metals, the form of the alloy may be sufficient and the thing which laminated | stacked the metal may be sufficient. Examples of a method for forming a metal thin film on a transparent flat substrate include a sputtering method and a vapor deposition method (resistance heating vapor deposition method, electron beam vapor deposition method, etc.).

Further, a surface treatment layer made of a self-assembled monolayer (SAM) used in a general bioassay method or the like may be provided on the surface of the substrate 21. Here, the SAM is used as a base for fixing a coupling portion 22 to be described later to the substrate 21. When a transparent flat substrate having a metal thin film formed on the surface is used as the substrate 21 such as when detecting fluorescence excited by near-field light in the step (e) described later, this surface treatment layer is a fluorescent metal For the purpose of preventing quenching, a spacer layer made of a dielectric may be further included. As a configuration example of the substrate 21 and the surface treatment layer in this case, there is a configuration in which a spacer layer made of a dielectric is present on a metal thin film constituting the substrate 21 and a SAM layer is present on the spacer layer. .

Enzyme In the enzyme-immobilized substrate 11 used in the fluorescence measurement method according to the present invention, the enzyme 23 plays a role of converting a fluorescent molecule precursor molecule 41 described later into a fluorescent molecule 42. The enzyme 23 used in the present invention has the property of changing the fluorescent molecule precursor molecule 41 to the fluorescent molecule 42, but unless it has the property of altering or degrading other components, for example, the water-soluble polymer 43. Any kind of enzyme may be used. Examples of such enzymes 23 include, but are not limited to, oxidoreductases such as horseradish peroxidase (HRP), phosphatases such as alkaline phosphatase, and β-galactosidase.

In the enzyme-immobilized substrate 11 used in the fluorescence measuring method according to the present invention, the enzyme 23 may be directly bonded to the substrate 21. However, in order to ensure that the enzyme 23 is securely fixed on the substrate 21, in the usual case, the enzyme 23 is indirectly connected via a binding portion 22 made of another molecule or molecular complex as shown in FIG. Are connected. Since the coupling portion 22 has a role of linking the substrate 21 and the enzyme 23, the coupling portion inevitably includes a junction portion for coupling with the substrate 21 and a junction portion for coupling with the enzyme 23. It will be.

Depending on the type of substrate 21 used or, if applicable, the surface treatment layer provided on the substrate 21 as the functional group or molecule constituting the bond for bonding to the substrate 21, a conventionally known functional group or molecule Can be used as appropriate.

Moreover, as a functional group or molecule constituting a junction for binding to the enzyme 23, a conventionally known functional group or molecule used in a general bioassay method can be appropriately used.
An example of such a bond at the junction is one that utilizes the bond between biotin and avidin.

Of the binding portion 22, molecules or molecular complexes that constitute portions other than the bonding portion for binding to the substrate 21 and the bonding portion for binding to the enzyme 23 should be an obstacle to fluorescence measurement in the present invention. As long as the activity of the enzyme 23 is not impaired, it does not matter.
If it is sufficient to simply bind the enzyme 23 to the substrate 21, the junction for coupling to the substrate 21 and the junction for coupling to the enzyme 23 may be integrated, or The junction for binding to the substrate 21 and the junction for binding to the enzyme 23 are bonded via a conventionally known molecule generally used as a spacer or linker for a general bioassay method. Also good.

The fluorescence measurement method according to the present invention can be applied for use as a bioassay method. In the fluorescence measurement method according to the present invention as a bioassay method, the content of biomolecules in a specimen is determined from the fluorescent molecules through the amount of enzyme 23 immobilized on the substrate 21 and becoming a part of the enzyme-immobilized substrate 11. This is evaluated as the amount of fluorescence. In order to form such a bioassay system, the biomolecule in the sample constitutes the enzyme-immobilized substrate 11 in a state of being incorporated in the binding portion 22. In other words, in such an enzyme-immobilized substrate 11, the bonding portion 22 includes a bonding portion for bonding to the substrate 21, a bonding portion for bonding to the enzyme 23, a biomolecule, and a ligand molecule.

Biomolecule In the present invention, “biomolecule” means a molecule or molecular fragment to be measured when the fluorescence measurement method according to the present invention is applied to a bioassay method, and is also called an analyte. Examples of such “molecule” or “molecular fragment” include nucleic acids (DNA that may be single-stranded or double-stranded, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), etc., Alternatively, nucleosides, nucleotides and their modified molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modifications thereof Examples thereof include molecules, complexes, and the like. Specifically, it may be a carcinoembryonic antigen such as AFP (α-fetoprotein), a tumor marker, a signaling substance, a hormone, and the like, and is not particularly limited.

Ligand molecule In the present invention, the “ligand molecule” refers to a molecule or molecular fragment capable of specifically recognizing (or recognizing) and binding to the “biomolecule”. Examples of such “molecule” or “molecular fragment” include nucleic acids (DNA that may be single-stranded or double-stranded, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids), etc., Alternatively, nucleosides, nucleotides and their modified molecules), proteins (polypeptides, oligopeptides, etc.), amino acids (including modified amino acids), carbohydrates (oligosaccharides, polysaccharides, sugar chains, etc.), lipids, or modifications thereof Examples include, but are not limited to, molecules and complexes.

Examples of the “protein” include antibodies and the like, specifically, anti-α-fetoprotein (AFP) monoclonal antibody (available from Japan Medical Laboratory), anti-carcinoembryonic antigen (CEA) ) Monoclonal antibody, anti-CA19-9 monoclonal antibody, anti-PSA monoclonal antibody and the like.

In the present invention, the term “antibody” includes polyclonal or monoclonal antibodies, antibodies obtained by genetic recombination, and antibody fragments. In the present invention, a typical example of a ligand molecule is an antibody.

Binding Mode In the enzyme-immobilized substrate 11 used in the fluorescence measurement method according to the present invention, the enzyme 23 is bonded directly to the substrate 21 or indirectly through the bonding portion 22. The binding mode of the enzyme 23 to the substrate 21 is not particularly limited as long as the object of the present invention is achieved. However, in the fluorescence measurement method according to the present invention, it is preferable that the substrate 21 and the enzyme 23 are bonded through a covalent bond or a bond based on an affinity between molecules from the viewpoint of the stability of the bond. Here, a plurality of bonds may be interposed between the substrate 21 and the enzyme 23, and the plurality of bonds may be composed of different types of bonds.

Here, in the case where the binding portion 22 includes a biomolecule and a ligand molecule, the enzyme 23 is fixed on the substrate 21 through a bond between the biomolecule and the ligand molecule in the enzyme-fixed substrate 11.

As one embodiment, in the enzyme-immobilized substrate 11, a ligand molecule is bound to the substrate 21, a biomolecule is bound to the ligand molecule, and an enzyme 23 is directly or indirectly bound to the biomolecule. Here, when the surface treatment layers such as the SAM layer and the spacer layer described above are provided on the substrate 21, the ligand molecules may be bonded to the substrate 21 through such a surface treatment layer. Further, the biomolecule and the enzyme 23 may be directly bonded without interposing the second ligand molecule, or may be indirectly bonded with the second ligand molecule interposed.

As an example of the case where the biomolecule and the enzyme 23 are directly bound, a target DNA in which a part of the base is modified with biotin is used as a “biomolecule” as in the case where the present invention is applied to a DNA hybridization assay. And the target DNA is bound with a biotin-modified enzyme through a biotin-avidin bond. On the other hand, as an example of the case where the biomolecule and the enzyme 23 are indirectly bound via the second ligand molecule, as in the case where the present invention is applied to an immunoassay, the antigen and the antibody are respectively The molecule is used as a “molecule” and a “second ligand molecule”, and there is a case where the enzyme 23 is bound to this antigen via an antibody.

In addition, when it is difficult to directly bind the enzyme 23 to the second ligand molecule, the second ligand molecule is specifically recognized (or recognized) and the third ligand molecule capable of binding. By binding the enzyme 23, the biomolecule and the enzyme 23 may be bound in a form mediated by the second ligand molecule and the third ligand molecule.

In the enzyme-immobilized substrate 11, the biomolecules and ligand molecules interposed between the substrate 21 and the enzyme 23 do not necessarily have to be arranged in series. For example, when the biomolecule and the ligand molecule are bound, the enzyme 23 may be bound to the biomolecule and the ligand molecule via a molecule that is easily incorporated into the binding site. For example, when the biomolecule and the ligand molecule are nucleic acids, the enzyme 23 may be bound to the biomolecule and the ligand molecule by using an appropriate intercalator to which the enzyme 23 is bound.

《Fluorescent molecule precursor molecule》
In the fluorescence measuring method according to the present invention, the fluorescent molecule precursor molecule 41 is used as a substrate for generating the fluorescent molecule 42 by the enzyme reaction by the enzyme 23. This fluorescent molecule precursor molecule 41 is a molecule corresponding to a fluorogenic substrate in a conventionally known fluorogenic substrate conversion method.

In the present invention, the term “fluorescent molecule” means a molecule that emits fluorescence by irradiating with predetermined excitation light or being excited by using an electric field effect. Including.

The fluorescent molecule precursor molecule 41 is a molecule that changes to a fluorescent molecule 42 due to a chemical change caused by an enzyme reaction. Here, the fluorescent molecule precursor molecule 41 used in the present invention has fluorescence in other wavelength regions as long as it does not exhibit fluorescence in the wavelength region near the maximum fluorescence wavelength in the fluorescent molecule 42 generated by the enzyme reaction. You may do it. Moreover, as long as it does not interfere with fluorescence detection, it may be accompanied by generation of chemiluminescent molecules by enzymatic reaction.

In the present invention, as the fluorescent molecule precursor molecule 41, a substrate generally used as a fluorogenic substrate in a conventionally known assay method based on the fluorogenic substrate conversion method can be used. Examples of such fluorescent molecule precursor molecules 41 include oxidoreductase substrates such as horseradish peroxidase (HRP) substrates, phosphatase substrates such as alkaline phosphatase substrates, and glycosidase substrates such as β-galactosidase substrates. However, it is not limited to these.

Table 1 shows a typical enzyme reaction substrate used as the fluorescent molecule precursor molecule 41 and the absorption wavelength (nm) and emission wavelength (nm) of the fluorescent molecule generated from this substrate by the fluorescence reaction.

《Water-soluble polymer》
The greatest feature of the fluorescence measurement method according to the present invention is that the enzyme reaction on the enzyme-immobilized substrate 11 and the detection of luminescence from the fluorescent molecule 42 generated by the enzyme reaction are performed in the presence of the water-soluble polymer 43. Here, the water-soluble polymer 43 is used for the purpose of capturing the fluorescent molecules 42 generated by the enzyme reaction by the enzyme 23 and preventing diffusion into the solution. Since the region to be subjected to fluorescence detection in fluorescence measurement is a very narrow portion in the measurement system, the detection efficiency of fluorescence emission is increased by localizing the fluorescent molecules 42 in the vicinity of the region to be subjected to fluorescence detection. As a result, the detection sensitivity can be improved.

One factor that affects the diffusion of the fluorescent molecules 42 into the solution is the viscosity of the solution. In the enzyme reaction system composed of the enzyme-immobilized substrate 11 and the solution of the fluorescent molecule precursor molecule 41 existing thereon, the water-soluble polymer 43 is present in this solution compared to the case where the water-soluble polymer 43 is not present. The viscosity of the solution is high. In general, the higher the viscosity of the solution, the more difficult it is for the solute in the solution to diffuse. The generated fluorescent molecules 42 tend to be difficult to diffuse.

Another factor that affects the diffusion of the fluorescent molecules 42 into the solution is charge. When a conventionally known enzyme reaction substrate is used as the fluorescent molecule precursor molecule 41, in many cases, the fluorescent molecule 42 generated by the enzyme reaction has a charge. Here, if the water-soluble polymer 43 has a charge opposite to that of the fluorescent molecule 42, the fluorescent molecule 42 is separated from the water-soluble polymer 43 by electrostatic interaction between the fluorescent molecule 42 and the water-soluble polymer 43. As a result, the fluorescent molecules 42 can be prevented from diffusing into the solution. For this reason, it is preferable that the water-soluble polymer 43 has an ionic group. However, in many cases, the fluorescent molecule 42 generated from the fluorescent molecule precursor molecule 41 used in the present invention has an anionic molecular structure under the condition of performing fluorescence measurement. For this reason, in the present invention, it is preferable that a cationic group is included as the ionic group of the water-soluble polymer 43. A particularly preferred ionic group is a cationic group that has a low binding property to the fluorescent molecule precursor molecule 41 and a high binding property to the fluorescent molecule 42 generated by the enzymatic reaction under enzyme reaction conditions. Examples of this cationic group include a tertiary amino group and a quaternary ammonium group. Specific tertiary amino groups and quaternary ammonium groups will be described later because suitable groups differ depending on the type of the water-soluble polymer 43. The water-soluble polymer 43 used in the present invention may contain one kind of such a cationic group or may contain two or more kinds in combination.

The water-soluble polymer 43 used in the present invention may contain an anionic group in addition to a cationic group as an ionic group in order to balance the charge. In this case, preferred anionic groups include sulfonic acid groups, carboxylic acid groups, phosphonic acid groups, and phosphoric acid groups.

In the water-soluble polymer 43 used in the present invention, the larger the number of cationic groups contained in the molecule, the more fluorescent molecules 42 can be captured, which is preferable. The number of cationic groups contained in the molecule varies depending on the type of water-soluble polymer 43 used, and will be described later together with a specific example of the water-soluble polymer 43 used in the present invention.

In the fluorescence measurement method of the present invention, the fluorescent molecule 42 generated in the enzyme reaction on the enzyme-immobilized substrate 11 is combined with the water-soluble polymer 43 to form the fluorescent molecule-water-soluble polymer complex 31. In the fluorescent molecule-water-soluble polymer complex 31, there is at least a bond between the fluorescent molecule 42 and the water-soluble polymer 43 due to the interaction based on the intermolecular force. Here, in the case where the water-soluble polymer 43 includes an ionic group, an electrostatic interaction acts between the fluorescent molecule 42 and the water-soluble polymer 43 in addition to the interaction based on the intermolecular force. . In that case, since a stronger bond based on electrostatic interaction is formed between the fluorescent molecule 42 and the water-soluble polymer 43, the connection between the fluorescent molecule 42 and the water-soluble polymer 43 becomes stronger. The diffusion of the fluorescent molecules 42 into the solution can be more strongly suppressed. Therefore, in the fluorescence measurement method of the present invention, the fluorescent molecule-water-soluble polymer complex 31 is preferably formed by electrostatic interaction between the fluorescent molecule 42 and the water-soluble polymer 43.

By the way, in the step (b), the fluorescent molecule precursor molecule 41 and the water-soluble polymer 43 are introduced onto the enzyme-immobilized substrate 11, and a solution containing the fluorescent molecule precursor molecule 41 and the water-soluble polymer 43 is usually used. It is necessary to show fluidity because it is introduced through the flow path. In order to satisfy such a requirement, in one embodiment of the fluorescence measurement method according to the present invention, a linear non-crosslinked water-soluble polymer is used as the water-soluble polymer 43. When such a water-soluble polymer is used, introduction into the enzyme immobilization substrate 11 in the form of a solution can be performed through a flow path, and in the solution during the enzyme reaction in the step (c). There is an advantage that the fluorescent molecule precursor molecules 41 distributed on the substrate are not prevented from approaching the enzyme 23 in the enzyme-immobilized substrate 11. Here, if a cross-linked polymer is used as the water-soluble polymer 43 instead of the linear non-cross-linked water-soluble polymer, the water-soluble polymer 43 may become a gel and cannot be introduced through the flow path. Examples of this linear non-crosslinked water-soluble polymer include linear polyacrylamide and derivatives thereof, and linear cellulose derivatives.

In another embodiment of the fluorescence measurement method of the present invention, a polymer matrix gel having thixotropy is used as the water-soluble polymer 43. Such a polymer matrix gel exhibits fluidity in the presence of stress and can be introduced through the flow path, while it loses fluidity when no stress is present and it remains stationary. Therefore, there is an advantage that after leaving on the enzyme-immobilized substrate 11, it is not separated from the enzyme-immobilized substrate 11. Examples of the polymer matrix gel having thixotropic properties include water-soluble cellulose derivatives, polysaccharides such as chitosan and derivatives thereof.

The water-soluble polymer 43 used in the present invention is not necessarily limited to the linear non-crosslinked water-soluble polymer and the thixotropic polymer matrix gel as long as the functions and effects of the present invention can be achieved. It is not limited. As the water-soluble polymer 43, the linear non-crosslinked water-soluble polymer and the thixotropic polymer matrix gel can be used singly or in combination of two or more. . In addition, the water-soluble polymer 43 used in the present invention may have both properties of the linear non-crosslinked water-soluble polymer and the thixotropic polymer matrix gel. The skeleton constituting such a water-soluble polymer 43 preferably has one or more skeletons selected from linear polyacrylamide and the above polysaccharides.

As the cationic polymer used as the water-soluble polymer 43, a water-soluble cationized polymer generally used for toiletries such as shampoo and rinse is preferably used. Typical compounds of such water-soluble cationized polymers include, for example, water-soluble cationized polysaccharides such as cationized cellulose derivatives, cationic starches and cationized guar gum derivatives; and diallyl quaternary ammonium salts / acrylamide co-polymers And cationized polyacrylamides such as vinylpyrrolidone / quaternary ammonium modified acrylamide copolymers. As these compounds, for example, compounds described in JP-A-62-234618 (Patent Document 3) can be used.

Among these, as a cationized cellulose derivative that can be used as the water-soluble polymer 43 in the present invention, a cationized cellulose containing an anhydroglucose unit substituted with a polyalkyloxy group having a cationic group as an anhydroglucose unit. Derivatives. The cationized cellulose derivative used in the present invention preferably contains 50 to 20000 anhydroglucose units including an anhydroglucose unit substituted with a polyalkyloxy group having a cationic group.

Examples of the cationic group contained in the cationized cellulose derivative include a quaternary ammonium group. An example of the quaternary ammonium group is a quaternary ammonium group having a structure represented by the following formula (1).

In the above formula (1), R ¹ , R ² , and R ³ are each independently an alkyl group, aryl group, or aralkyl group having 10 or less carbon atoms, R ⁴ is an alkylene or hydroxyalkylene group, and X is negative Ion. Further, two or more of R ¹ , R ² and R ³ may form a heterocyclic ring in a form containing a nitrogen atom in the formula. In one embodiment of the present invention, a quaternary ammonium group having a structure represented by the above formula (1) is bonded to a polyalkyloxy chain bonded to a hydroxyl group not involved in the formation of the cellulose skeleton. Introduced into glucose units.

In the case of polysaccharides such as cationized cellulose derivatives, cationic starch, and cationized guar gum derivatives, the number of cationic groups contained in the molecule is introduced into the constituent monosaccharide units such as the degree of cation substitution, that is, anhydroglucose units. This can be determined by the average number of cationic groups formed. The degree of cation substitution of the cationized cellulose is 0.01 to 1, that is, the average number of cationic groups introduced per anhydroglucose unit is 0.01 to 1, preferably 0.02 to 0.5.

Examples of commercially available cationized cellulose derivatives include Katachiro H-60 (manufactured by Kao), Katchinar (manufactured by Toho Chemical), Leogard (manufactured by Matsumoto Kosho), and the like.

Examples of the cationic starch that can be used as the water-soluble polymer 43 in the present invention include cationized starch in which the hydroxyl group of the starch residue is substituted with a cationic functional group.

As a cationic group contained in this cationic starch, a quaternary ammonium group can be mentioned. An example of the quaternary ammonium group is a quaternary ammonium group having a structure as shown in the above formula (1).

The cationic starch preferably has a cation substitution degree of 0.01 to 1, that is, 0.01 to 1 and especially 0.02 to 0.5 cationic groups introduced per anhydroglucose unit. Among the cationic starches, hydroxypropyltrimonium chloride starch is commercially available. For example, it is sold by Matsumoto Kosho under the trade name “Sensomer CI-50” (molecular weight (GPC-MALLS method): 200,000). Yes.

Examples of the cationized guar gum derivative that can be used as the water-soluble polymer 43 in the present invention include a cationized guar gum derivative in which the hydroxyl group of the guar gum residue is substituted with a cationic group.

Examples of the cationic group contained in the cationized guar gum derivative include a quaternary ammonium group. An example of the quaternary ammonium group is a quaternary ammonium group having a structure as shown in the above formula (1).

The cationized guar gum derivative preferably has a cation substitution degree of 0.01 to 1, that is, 0.01 to 1, particularly 0.02 to 0.5, in which a cationic group is introduced into the sugar unit. Such cationized guar gum derivatives are described in JP-B-58-35640, JP-B-60-46158, and JP-A-58-53996. For example, products from Celanese Stein Hall It is marketed under the name “Jaguar”.

The water-soluble cationized polysaccharide used as the water-soluble polymer 43 in the present invention is not limited to the water-soluble cationized polysaccharide having glucose as a constituent unit as exemplified above, but glucose as a constituent unit. Other types of water-soluble cationized polysaccharides, or water-soluble cationized polysaccharides containing monosaccharides other than glucose as constituent units may be used. In the case of these water-soluble cationized polysaccharides, those having a cation substitution degree of 0.01 to 1 can be preferably used.

The diallyl quaternary ammonium salt / acrylamide copolymer that can be used as the water-soluble polymer 43 in the present invention is a copolymer of diallylammonium-derived cyclic ammonium units and acrylamide units. In this acrylamide unit, some or all of the hydrogen atoms bonded to the nitrogen atom of the amide bond may be substituted with a lower alkyl group (1 to 3 carbon atoms) or a phenyl group.

Examples of the cyclic ammonium unit contained in the diallyl quaternary ammonium salt / acrylamide copolymer include cyclic ammonium units having a structure represented by the following formula (2a) or (2b).

In the formulas (2a) and (2b), R ⁵ and R ⁶ are each independently hydrogen, an alkyl group (having 1 to 18 carbon atoms), a phenyl group, an aryl group, a hydroxyalkyl group, an amidoalkyl group, a cyanoalkyl group. , An alkoxyalkyl group and a carboalkoxyalkyl group, R ⁷ and R ⁸ are each independently a hydrogen atom, a lower alkyl group (having 1 to 3 carbon atoms), a phenyl group, and X is an anion.

The diallyl quaternary ammonium salt / acrylamide copolymer has a molecular weight of 30,000 to 2,000,000, preferably 100,000 to 2,000,000. There is no particular limitation on the ratio of cyclic ammonium units to acrylamide units in the diallyl quaternary ammonium salt / acrylamide copolymer.

Among the diallyl quaternary ammonium salt / acrylamide copolymers as described above, dimethyldiallylammonium chloride / acrylamide copolymer, that is, in the above formula (2a) or (2b), R ⁵ and R ⁶ are methyl groups, R ^A copolymer in which ⁷ and R ⁸ are hydrogen atoms is preferably used as the water-soluble polymer 43. This copolymer is sold by Matsumoto Trading under the trade name “Mercoat 550” (polymerization ratio: dimethyldiallylammonium chloride / acrylamide = 30/70 (molar ratio); molecular weight (GPC-MALLS method): 1.6 million)). ing.

As a vinylpyrrolidone / quaternary ammonium modified acrylamide copolymer that can be used as the water-soluble polymer 43 in the present invention, a quaternary ammonium modified acrylamide in which a vinylpyrrolidone unit and a quaternary ammonium group are bonded to a nitrogen atom of an amide bond. Examples thereof include a copolymer comprising units.

Examples of the quaternary ammonium group contained in the quaternary ammonium-modified acrylamide unit include a quaternary ammonium group having a structure represented by the following formula (3).

In the above formula (3), R ⁹ , R ¹⁰ and R ¹¹ are each independently a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group, an amidoalkyl group, a cyanoalkyl group, an alkoxyalkyl group, a carboalkoxy group. An alkyl group, R ¹² is a linear alkyl group having 10 or less carbon atoms, and X is an anion. Here, in this quaternary ammonium-modified acrylamide unit, the carbon on the main chain adjacent to the carbo group may be further substituted with an alkyl group having 3 or less carbon atoms.

The molecular weight of the vinylpyrrolidone / quaternary ammonium modified acrylamide copolymer is 10,000 to 2,000,000, preferably 50,000 to 1,500,000. The content of the cationic nitrogen derived from the cationic polymer contained in this copolymer is 0.004 to 0.2%, preferably 0.01 to 0.15% in terms of vinyl polymer. The ratio of the vinyl pyrrolidone unit and the quaternary ammonium modified acrylamide unit in the vinyl pyrrolidone / quaternary ammonium modified acrylamide copolymer is not particularly limited.

The cationized polyacrylamide used as the water-soluble polymer 43 in the present invention is not limited to the cationized polyacrylamides exemplified above. In the cationized polyacrylamides used as the water-soluble polymer 43 in the present invention, the cationic group may be introduced into the acrylamide unit in the form of a quaternary ammonium-modified acrylamide unit or the cyclic ammonium unit or the like. In the form, it may be introduced into another monomer unit used for copolymerization with the acrylamide unit, or may be introduced into both the acrylamide unit and the other monomer unit.

The water-soluble polymer 43 used in the present invention may be produced by polymerizing or copolymerizing a corresponding monomer, or chemical modification by a conventionally known method may be performed on an existing polymer compound as necessary. You may manufacture by giving. In the case of the water-soluble polymer 43 having an ionic group, it is produced by copolymerizing a corresponding monomer having no ionic group and a monomer having an ionic group, and further applying chemical modification as necessary. Alternatively, it may be produced by introducing an ionic group into an existing polymer compound having no ionic group. For example, linear polyacrylamide having an ionic group is prepared by a method of copolymerizing acrylamide with a monomer having an appropriate ionic group such as a diallylammonium compound, or a quaternary ammonium-modified acrylamide as necessary. And (co) polymerization with other monomer units. Moreover, the polysaccharide which has an ionic group can be obtained by performing the chemical modification by a conventionally well-known method with respect to the commercially available polysaccharide used as a base | substrate, and introduce | transducing an ionic group.

If applicable, a corresponding commercially available product may be used as the water-soluble polymer 43.

The molecular weight of the water-soluble polymer 43 used in the present invention is usually in the range of 10,000 to 2,000,000, preferably 50,000 to 2,000,000 as a weight average molecular weight so as not to disturb the enzyme reaction.

<Introduction of water-soluble polymer and fluorescent molecule precursor molecule>
In the fluorescence measurement method according to the present invention, the method for introducing the fluorescent molecule precursor molecule 41 and the water-soluble polymer 43 onto the enzyme-immobilized substrate 11 is not particularly limited. However, in a typical embodiment of the fluorescence measurement method according to the present invention, the enzyme-immobilized substrate 11 is disposed in a closed channel system, as in the corresponding conventional fluorescence measurement method that does not use the water-soluble polymer 43, In addition, the measurement is performed in a measurement system in which various drugs, solutions, and the like are supplied through the flow path. From the viewpoint of operability when used in such a measurement system, the fluorescent molecule precursor molecule 41 and the water-soluble polymer 43 are usually introduced onto the enzyme-immobilized substrate 11 in the form of a solution. In a typical introduction mode, it is introduced onto the enzyme-immobilized substrate 11 through a flow path in the form of a liquid feed.

The solvent or buffer used when introducing the fluorescent molecule precursor molecule 41 and the water-soluble polymer 43 as a solution is the same as the solvent or buffer used in the corresponding conventionally known fluorescence measurement method that does not use the water-soluble polymer 43. The same thing can be used according to the kind of the fluorescent molecule precursor molecule 41.

When introducing the fluorescent molecule precursor molecule 41 and the water-soluble polymer 43, an additive generally used in an assay method based on a conventionally known fluorogenic substrate conversion method may be added, if necessary.

The order of introducing the fluorescent molecule precursor molecule 41 and the water-soluble polymer 43 is not particularly limited as long as the action / effect of the present invention is not diminished. Either the fluorescent molecule precursor molecule 41 or the water-soluble polymer 43 may be introduced first, or the fluorescent molecule precursor molecule 41 and the water-soluble polymer 43 may be introduced simultaneously. However, from the viewpoint of suppressing the diffusion of the fluorescent molecule 42 generated by the enzyme reaction, the fluorescent molecule precursor molecule 41 is preferably introduced simultaneously with the water-soluble polymer 43 or after the water-soluble polymer 43 is introduced.

In order to capture more of the fluorescent molecules 42 produced during the enzyme reaction, it is preferable that the amount of the water-soluble polymer 43 present in the solution on the enzyme-immobilized substrate 11 is large. On the other hand, since a certain fluidity is required to introduce the water-soluble polymer 43 as a solution, it is preferable that the viscosity of the solution when the water-soluble polymer 43 is introduced does not become too high. In consideration of these, the water-soluble polymer 43 is usually placed on the enzyme-immobilized substrate 11 so that the concentration in the entire solution immediately before the enzyme reaction is 0.001% to 5%, preferably 0.01% to 1%. be introduced.

The concentration of the fluorescent molecule precursor molecule 41 is not particularly limited as long as the action / effect of the present invention is not diminished, and is approximately the same as the substrate used in the corresponding conventionally known fluorescence measuring method not using the water-soluble polymer 43. The concentration of Specifically, the fluorescent molecule precursor molecule 41 is placed on the enzyme-immobilized substrate 11 so that the concentration in the entire solution immediately before the enzyme reaction is usually 0.01M to 0.000000001M, preferably 0.001M to 0.0000001M. be introduced.

The total amount of the solution containing the fluorescent molecule precursor molecule 41 and the water-soluble polymer 43 is not particularly limited. However, when this solution is introduced onto the enzyme-immobilized substrate 11 in the form of liquid feeding, the total amount of this solution is usually 0.0001 to 20 mL, preferably 0.01 to 1 mL as the volume of the flow path.

The flow rate of liquid feeding when introducing the fluorescent molecule precursor molecule 41 and the water-soluble polymer 43 in the form of liquid feeding is usually 0.1 μL / min to 10 mL / min, preferably 1 μL / min to 1 mL / min.

The flow channel “flow channel” is a rectangular parallelepiped or a tube that can efficiently deliver a small amount of chemical solution and can change the liquid feeding speed or circulate in order to promote the reaction. is there.
Moreover, as this flow path, the vicinity of the location where the substrate 21 or the enzyme-immobilized substrate 11 is installed preferably has a rectangular parallelepiped structure, and the vicinity of the location where the chemical solution is delivered preferably has a tubular shape. About the material which comprises a flow path, and the dimension of a flow path, it can be set to the same thing as the flow path used by the corresponding conventionally well-known method which does not use the water-soluble polymer 43.

In the flow path, the position of the liquid feeding inlet for introducing the liquid feeding from the drug delivery part to the enzyme fixing substrate part and the position of the liquid feeding outlet for discharging the liquid feeding from the enzyme fixing substrate part are both obstructing the fluorescence measurement Unless it becomes, it will not specifically limit. For example, when the flow path of the enzyme-immobilized substrate section has a rectangular parallelepiped structure, it is convenient to create the flow path for both the liquid feeding inlet and the liquid feeding outlet on the ceiling surface. Either one or both of the liquid discharge ports may be provided on the side surface.

The method for fixing the substrate 21 or the enzyme-immobilized substrate 11 to the flow path is not particularly limited as long as the flow path is maintained in a certain shape and the fluorescence measurement is not hindered.

In the present invention, the introduction of the water-soluble polymer 43 and the fluorescent molecule precursor molecule 41 to the enzyme-immobilized substrate 11 is usually performed through the above-mentioned flow path.

<Manufacture of enzyme-immobilized substrate>
When the fluorescence measurement method according to the present invention is applied to a use as a bioassay method, an enzyme is used using a specimen, an enzyme 23, a ligand molecule, and a substrate 21 (that is, a “basic substrate”) prior to the step (b). The fixed substrate 11 is manufactured. The step of manufacturing the enzyme-immobilized substrate 11 can be performed as a step independent of the series of steps (b) to (e) in the fluorescence measurement method of the present invention. However, when the fluorescence measurement method according to the present invention is used in a measurement system in which the enzyme-immobilized substrate 11 is disposed in a closed channel system and various drugs, solutions, and the like are supplied through the channel, the enzyme-immobilized substrate 11 is used. It is advantageous in terms of operation to consistently carry out the manufacturing step and the subsequent steps (b) to (e) under the same flow path system. Therefore, it is preferable that the step of producing the enzyme-immobilized substrate 11 is incorporated in a series of steps in the fluorescence measurement method of the present invention as step (a).

The enzyme-immobilized substrate 11 can be produced by the same method as a general enzyme-immobilized substrate used in enzyme immunoassay methods such as ELISA.

Typically, the enzyme-immobilized substrate 11 is
Step (a-1): A step of forming a surface treatment layer such as a SAM layer on the surface of the substrate 21 as necessary,
Step (a-2): A step of binding ligand molecules directly to the surface of the substrate 21 or indirectly through the surface treatment layer formed in the step (a-1).
Step (a-3): a step of bringing a specimen into contact with the surface of the substrate obtained in the step (a-2) and binding a biomolecule in the specimen to a ligand molecule;
Step (a-4): manufactured through the step of binding the enzyme 23 to the biomolecule bound to the substrate in the step (a-3).

In other words, the enzyme immobilization substrate 11 is
By the step (a-2), a ligand molecule is bound to the surface of the base substrate 21 to obtain a substrate on which the ligand molecule is immobilized (hereinafter sometimes referred to as “ligand-immobilized substrate”).
By the step (a-3), the specimen is brought into contact with the surface of the “ligand immobilization substrate”, and the biomolecule in the specimen is bound to the ligand molecule to thereby immobilize the biomolecule (hereinafter referred to as “biomolecule immobilization”). Sometimes called a "substrate")
In step (a-4), it can be obtained by binding an enzyme to the “biomolecule-immobilized substrate”. In this case, prior to steps (a-2) to (a-4),
By the step (a-1), a surface treatment layer such as a SAM layer can be formed on the surface of the substrate 21 in advance.

Here, in the step (a-4), for example, an enzyme-second ligand molecule complex is prepared in advance by binding the enzyme 23 to a second ligand molecule, and then the step (a-3). The enzyme-second ligand molecule complex can be brought into contact with the surface of the substrate obtained in the above (ie, “biomolecule-immobilized substrate”).

However, when the biomolecule is a nucleic acid, instead of performing the steps (a-3) and (a-4), an enzyme modification treatment is performed on the sample in advance, and then the step (a-2) The enzyme-immobilized substrate 11 can also be manufactured by bringing an enzyme-treated specimen into contact with the surface of the substrate obtained in (i.e., “ligand-immobilized substrate”). The enzyme modification treatment on the specimen can be performed by various conventionally known methods.

As another production method in the case where the biomolecule is a nucleic acid, instead of performing the above steps (a-3) and (a-4), the enzyme 23 is combined with an intercalator in advance and the enzyme-intercalator is used. After preparing the complex, a solution containing the specimen and the enzyme-intercalator complex is brought into contact with the surface of the substrate obtained in the step (a-2) (ie, the “ligand-immobilized substrate”). The enzyme-immobilized substrate 11 can also be manufactured.

Here, “contact” means that the surface on which the ligand molecules and the like are fixed is immersed in the liquid feeding in the substrate obtained through the step (a-2) (that is, “ligand fixing substrate”). In this state, it refers to bringing an object contained in the liquid feeding into contact with the ligand-immobilized substrate. In the present invention, the “contact” between the specimen and the ligand-immobilized substrate means that the specimen is contained in the liquid-feeding liquid circulating in the flow path, and only one side of the ligand-immobilized substrate on which the ligand molecules are immobilized is the liquid-feeding liquid. A mode in which the ligand-immobilized substrate and the specimen are brought into contact with each other in a state of being immersed therein is preferable. In order to prevent non-specific adsorption of the specimen or the like on the ligand-immobilized substrate, the surface of the ligand-immobilized substrate is previously immobilized on bovine serum albumin (BSA) after immobilizing the ligand molecule on the substrate 21 and before contacting the specimen. It is preferable to treat with a blocking agent such as

The solution used for producing the enzyme-immobilized substrate 11 is preferably the same as the solvent or buffer in which the specimen is diluted. For example, phosphate buffered saline (PBS), Tris buffered saline (TBS) However, it is not particularly limited.

The temperature and time for circulating the liquid supply during the production of the enzyme-immobilized substrate 11 vary depending on the type of specimen and are not particularly limited, but are usually 20 to 40 ° C. × 1 to 60 minutes, preferably 37. ° C x 5-15 minutes.

The initial concentration of the biomolecule contained in the specimen being sent may be 100 μg / mL to 0.0001 pg / mL.

The total amount of the liquid delivery, that is, the volume of the flow path is usually 0.0001 to 20 mL, preferably 0.01 to 1 mL.

The flow rate of the liquid supply when producing the enzyme-immobilized substrate 11 is usually 1 to 2,000 μL / min, preferably 5 to 500 μL / min.

In addition, in this invention, it is preferable that the process of wash | cleaning the enzyme fixed substrate 11 after the enzyme fixed substrate 11 is manufactured is included.

As the cleaning solution used in the cleaning step, for example, a surfactant such as Tween 20 or Triton X100 is dissolved in the same solvent or buffer solution as that used in the above-mentioned “liquid feeding used in manufacturing the enzyme-immobilized substrate 11”. Those containing 0.00001 to 1% by weight are desirable.

The temperature and flow rate at which the cleaning liquid is circulated are preferably the same as the above-mentioned “temperature and flow rate at which the liquid feed is circulated when producing the enzyme-immobilized substrate 11”.

The time for circulating the cleaning liquid is usually 0.5 to 180 minutes, preferably 5 to 60 minutes.

Specimen In the present invention, “specimen” refers to various samples to be measured when the fluorescence measuring method according to the present invention is applied to a use as a bioassay method.

Examples of the “specimen” include blood (serum / plasma), urine, nasal fluid, saliva, feces, body cavity fluid (spinal fluid, ascites, pleural effusion, etc.), etc., and appropriately diluted in a desired solvent, buffer solution, etc. May be used. Of these samples, blood, serum, plasma, urine, nasal fluid and saliva are preferred. These may be used alone or in combination of two or more.

<Step (c)>
In the fluorescence measurement method of the present invention, step (c) is a step of causing the enzyme reaction to occur on the enzyme-immobilized substrate 11 to convert the fluorescent molecule precursor molecule 41 into the fluorescent molecule 42.

Fluorescent molecule In the fluorescence measuring method according to the present invention, the fluorescent molecule 42 is a fluorescent molecule that is generated by the fluorescent molecule precursor molecule 41 introduced in the step (b) being changed by an enzyme reaction by the enzyme 23. In the present invention, the amount of the fluorescent molecules 42 generated is evaluated in the form of the amount of fluorescence emitted. The excitation wavelength and emission wavelength of the fluorescent molecule 42 generated from a typical enzyme reaction substrate used as the fluorescent molecule precursor molecule 41 are as described in the section of the fluorescent molecule precursor molecule.

<Step (d)>
In the fluorescence measurement method of the present invention, in the step (d), the fluorescent molecule 42 produced in the step (c) is bonded to the water-soluble polymer 43 to form a fluorescent molecule-water-soluble polymer complex on the enzyme-immobilized substrate 11. This is a step of forming 31.

In the measurement system in which the water-soluble polymer 43 does not exist, the fluorescent molecule 42 generated by the enzyme reaction in the fluorescent molecule-water-soluble polymer complex step (c) is free in the solution existing on the enzyme-immobilized substrate 11. It moves and diffuses to a site away from the enzyme immobilization substrate 11. Here, since the region to be subjected to fluorescence detection in the fluorescence measurement is limited to a very narrow portion around the enzyme-immobilized substrate 11, the light emitted from the fluorescent molecules 42 diffused to a site away from the enzyme-immobilized substrate 11. Fluorescence is not detected, which causes the detection sensitivity not to be sufficiently improved in the fluorescence measuring method based on the conventionally known fluorogenic substrate conversion method.

On the other hand, in the fluorescence measurement method of the present invention, since the water-soluble polymer 43 is present in the solution on the enzyme-immobilized substrate 11, the fluorescent molecule 42 generated by the enzyme reaction is high due to the water-soluble polymer 43. Due to the viscosity, diffusion tends to be suppressed as compared with a solution in which the water-soluble polymer 43 does not exist. Further, when the fluorescent molecule 42 moves through the water-soluble polymer 43, an interaction such as electrostatic interaction acts between the fluorescent molecule 42 and the water-soluble polymer 43. Bonds rapidly to form a fluorescent molecule-water-soluble polymer complex 31. Under such circumstances, since the fluorescent molecules 42 remain more in the region around the enzyme-immobilized substrate 11 that serves as a measurement field for fluorescence detection, the ratio of the fluorescent molecules 42 that contribute to fluorescence detection in the generated fluorescent molecules 42 is As a result, the fluorescence detected is increased. Through such a mechanism, the fluorescence measurement method of the present invention can improve detection sensitivity.

In addition, in the steps (c) to (e) including the step (e) described later, the generated fluorescent molecule-water-soluble polymer complex 31 flows out from the enzyme-immobilized substrate 11 by the flow of the liquid, In order to reduce the possibility of deviating from the region to be subjected to fluorescence detection, it is desirable to carry out in a stationary state.

<Process (e)>
In the fluorescence measurement method of the present invention, step (e) is a step of detecting fluorescence emitted from the fluorescent molecule-water-soluble polymer complex 31 produced in step (d). In the detection of fluorescence in the step (e), the fluorescent molecule-water-soluble polymer complex 31 generated in the step (d) is irradiated with excitation light, and the intensity of fluorescence emission corresponding to the excitation light is measured. Is done by.

The fluorescence detection method used in step (e) is not particularly limited as long as it is a known fluorescence detection method generally used in the field of bioassay.

As a method for generating fluorescence by exciting the fluorescent molecule-water-soluble polymer complex 31, a method for generating fluorescence mainly by directly irradiating the fluorescent molecule-water-soluble polymer complex 31 with excitation light is used. Is mentioned. Various methods are known as means for detecting and measuring fluorescence by this method. In the fluorescence measuring method of the present invention, fluorescence is detected using a confocal laser microscope or a confocal laser scanner. Is preferred. When these fluorescence detection devices are used, images with a high depth of focus can be obtained by acquiring images with high contrast at positions corresponding to the focus at various positions and superimposing them. Therefore, since the region to be subjected to fluorescence detection in the measurement system can be expanded, the ratio of the fluorescent molecules 42 that contribute to fluorescence detection among the generated fluorescent molecules 42 can be increased, thereby improving the detection sensitivity. There are benefits to connect. In addition, as for the members such as the light source, the beam splitter, the objective lens, the pinhole, the photodetector, and the galvanometer mirror constituting the confocal laser microscope and the confocal laser scanner used in the present invention, conventionally known ones should be used. Can do.

The method of exciting the fluorescent molecule-water-soluble polymer complex 31 to generate fluorescence is not limited to the method described above. For example, a method of generating fluorescence by irradiating excitation light indirectly using near-field light may be used. In this case, fluorescence excited by near-field light is detected. An example of such near-field light is light enhanced by plasmons. A specific example is a method of generating fluorescence by exciting the fluorescent molecule-water-soluble polymer complex 31 using surface plasmon or the like by surface plasmon excitation enhanced fluorescence spectroscopy (SPFS). As an advantage of detecting fluorescence excited by near-field light, it is possible to perform highly sensitive measurement while minimizing the influence from incident light during fluorescence detection. Here, when detecting fluorescence excited by near-field light, the region that can be excited by near-field light is extremely limited. For example, the region where the effect of electric field enhancement by SPFS can be obtained is the enzyme-immobilized substrate 11. It is limited to a region from about 100 to 200 nm from the surface of the substrate. In such a measurement system, by allowing the water-soluble polymer 43 to coexist in the solution, the fluorescent molecule 42 is kept in the region near the surface of the enzyme-immobilized substrate 11 in the form of the fluorescent molecule-water-soluble polymer complex 31. This leads to an increase in the ratio of the fluorescent molecules 42 excited by the near-field light in the generated fluorescent molecules 42, which is advantageous in achieving high sensitivity. In the present invention, members such as a light source, a prism, an optical filter, a polarizing filter, a cut filter, a condensing lens, and a light detector used for detecting fluorescence excited by near-field light are conventionally known ones. Can be used respectively.

<Quantification of biomolecules>
When the fluorescence measurement method according to the present invention is applied to a use as a bioassay method, after step (e), the “biomolecule” contained in the specimen is quantified based on the detected fluorescence amount. become. More specifically, it is a step of creating a calibration curve by performing measurement with a biomolecule at a known concentration, and calculating the biomolecular weight in the sample to be measured from the measurement signal based on the created calibration curve.

The step of quantifying the biomolecule contained in the specimen from the detected fluorescence amount can be performed as a step independent of the series of steps (b) to (e) in the fluorescence measurement method of the present invention. However, in the fluorescence measurement method according to the present invention using the water-soluble polymer 43 as well as the corresponding conventionally known fluorescence measurement method that does not use the water-soluble polymer 43, it is usually detected as a step of detecting fluorescence. The step of quantifying the biomolecule contained in the specimen from the amount of fluorescence obtained is performed as a series of steps. Therefore, the step of quantifying the biomolecule contained in the specimen from the amount of fluorescence detected in the step (e) is preferably incorporated as a step (f) in a series of steps in the fluorescence measurement method of the present invention.

In the assay S / N ratio step (f), the “blank fluorescence signal” measured before the step (b), the “measured fluorescence signal” obtained in the step (e), and nothing modified When the signal obtained by measuring the fluorescence emission while flowing the ultrapure water while fixing the substrate 21 to the flow path is “initial noise”, the assay S / N ratio represented by the following formula (4a) is calculated. can do:
Assay S / N ratio = | Ia / Io | / In (4a)
(In the above formula (1a), Ia is the assay fluorescence signal, Io is the blank fluorescence signal, and In is the initial noise).

However, in calculating the assay S / N ratio, instead of the above formula (4a), the following formula is used on the basis of the “assay noise signal” when the concentration of the biomolecule contained in the specimen is 0: You may calculate according to (4b):
Assay S / N ratio = | Ia | / | Ian | (4b)
(In the above formula (4b), Ian is the assay noise signal and Ia is the assay fluorescence signal as in the above formula (4a)).

Fluorescence intensity was measured by SPFS detection method using DDAOP as an enzyme chemical fluorescence reagent and using an alkaline phosphatase labeled secondary antibody.

[Example 1]
After preparing an SPFS sensor substrate with an anti-AFP monoclonal antibody (primary antibody) immobilized on the surface by a conventional method, the surface of the antibody-immobilized side was reacted with AFP (0.1 ng / mL) as an antigen. Alkaline phosphatase-labeled anti-AFP monoclonal antibody (secondary antibody) was bound. As a result, an SPFS sensor having an antigen-antibody sandwich complex formed on the surface, which functions as an enzyme-immobilized substrate, was obtained.

After that, on the surface of the SPFS sensor, 0.05% by weight of a cationized polymer (Catinal HC-100, manufactured by Toho Chemical Co., Ltd.) in 6.0E-6 mol / L DDAOP solution (pH 8.5, Tris-HCl buffer). (Trade name) was added and set at a flow rate of 100 μL / min, and the fluorescence intensity of the generated fluorescent molecules was measured.

[Comparative Example 1]
As a comparative example, the fluorescence intensity of the fluorescent molecule was measured in the same manner as in Example 1 except that a substrate solution to which no cationized polymer was added was used as the substrate solution.

FIG. 2 shows the relationship between the reaction time and fluorescence intensity after introducing the substrate solution on the surface of the SPFS sensor in the SPFS system of Example 1 and Comparative Example 1.

As shown in Fig. 2, in the substrate solution with cationized polymer added, the fluorescence intensity increases linearly after the reaction starts, whereas the substrate without cationized polymer added. In the solution, the fluorescence intensity did not increase until about 5 minutes after the start of the reaction, and thereafter the fluorescence intensity tended to increase gradually. This is because the fluorescent product generated on the surface diffuses quickly into the solution and takes time to be detected as a fluorescent signal, whereas the fluorescent solution generated in the substrate solution to which the cationized polymer is added. This shows that the molecules remain on the outermost surface of the sensor, and as a result, they can be efficiently detected as fluorescent signals immediately after the reaction time.

DESCRIPTION OF SYMBOLS 11 ... Enzyme fixed substrate 21 ... Substrate 22 ... Binding part 23 ... Enzyme 31 ... Fluorescent molecule-water-soluble polymer complex 41 ... Fluorescent molecule precursor molecule 42 ... Fluorescent molecule 43. Water-soluble polymer

Claims (18)

1. A fluorescence measurement method comprising the following steps (b) to (e):
   Step (b): introducing a water-soluble polymer and a fluorescent molecule precursor molecule onto the enzyme-immobilized substrate;
   Step (c): causing an enzyme reaction on the enzyme-immobilized substrate to convert the fluorescent molecule precursor molecule into a fluorescent molecule;
   Step (d): binding the fluorescent molecule to the water-soluble polymer to form a fluorescent molecule-water-soluble polymer complex on the enzyme-immobilized substrate; and
   Step (e): A step of detecting fluorescence emitted from the fluorescent molecule-water-soluble polymer complex.
2. 2. The fluorescence measuring method according to claim 1, wherein the water-soluble polymer has an ionic group.
3. 3. The fluorescence measuring method according to claim 2, wherein the ionic group contains a cationic group.
4. 4. The fluorescence measurement method according to claim 3, wherein the cationic group is one or more groups selected from a tertiary amino group and a quaternary ammonium group.
5. 5. The fluorescence measuring method according to claim 1, wherein the fluorescent molecule-water-soluble polymer complex is formed by electrostatic interaction between the fluorescent molecule and the water-soluble polymer.
6. 6. The fluorescence measuring method according to claim 1, wherein the water-soluble polymer is a linear non-crosslinked water-soluble polymer.
7. 6. The fluorescence measuring method according to claim 1, wherein the water-soluble polymer is a polymer matrix gel having thixotropic properties.
8. 8. The fluorescence measurement method according to claim 1, wherein the water-soluble polymer has a skeleton composed of one or more selected from linear polyacrylamide and polysaccharide.
9. 9. The fluorescence measuring method according to claim 8, wherein the water-soluble polymer is a water-soluble cationized polysaccharide.
10. 10. The fluorescence measuring method according to claim 9, wherein the degree of cation substitution in the water-soluble cationized polysaccharide is 0.01 to 1.
11. 11. The fluorescence measurement method according to claim 1, wherein the water-soluble polymer has a weight average molecular weight of 10,000 to 2,000,000.
12. The fluorescence measurement method according to any one of claims 1 to 11, wherein the steps (c) to (e) are performed in a stationary state.
13. 13. The fluorescence measurement method according to claim 1, wherein the enzyme is immobilized on the substrate through a bond between a biomolecule and a ligand molecule.
14. In the enzyme-immobilized substrate,
    The ligand molecule binds to the substrate;
    The biomolecule binds to the ligand molecule;
    14. The fluorescence measurement method according to claim 13, wherein the enzyme is directly or indirectly bound to the biomolecule.
15. Step (a): A step of producing the enzyme-immobilized substrate using a sample, an enzyme, a ligand molecule and a substrate, and Step (f): The amount of fluorescence detected in the step (e) is included in the sample. 15. The fluorescence measurement method according to claim 13, further comprising a step of quantifying the biomolecule.
16. The fluorescence measurement method according to any one of claims 1 to 15, wherein in the step (e), fluorescence excited by near-field light is detected.
17. The fluorescence measurement method according to claim 16, wherein the near-field light is light enhanced by plasmons.
18. 16. The fluorescence measuring method according to claim 1, wherein in the step (e), fluorescence is detected using a confocal laser microscope or a confocal laser scanner.

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