JP2022105961A - Method for detection or quantification of target biomolecule using temperature-responsive fluorescent particle - Google Patents

Abstract

To provide a method and apparatus that can detect or quantify target biomolecules at a low concentration in a simple manner and short time.SOLUTION: Temperature-responsive probe particles have been prepared by modifying surfaces of temperature-responsive fluorescent particles and a detection method using the same, which is a modified ELISA method, has been invented. The temperature-responsive probe particles exhibit fluorescent properties when the temperature of the temperature-responsive probe particles becomes equal to or more than a specific temperature and have strong fluorescent emission. By taking advantage of this property and using a transparent inspection container 1 that transmits excitation light and fluorescence and a transparent hot plate 2, the temperature-responsive probe particles that are fixed at the bottom in a transparent inspection container are heated. A timing when the temperature of the temperature-responsive probe particles exceeds a phase transition temperature and emits fluorescence is measured by a detection unit 4 disposed below the transparent hot plate 2.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for detecting or quantifying a target biomolecule using temperature-responsive fluorescent particles.

In recent years, the ELISA method has been widely used for the detection and quantification of biomolecules (biomarkers). Since the ELISA method uses an antigen-antibody reaction, high specificity can be obtained, but operations such as reagent washing and enzyme reaction are complicated and time-consuming, and the detection concentration of biomolecules is limited to about 1 pM. there were.

That is, in the ELISA method, there is an essential problem that the detection sensitivity is deteriorated because the photolabeling substance floating without being solid-phased becomes background noise. As the light-labeling substance, a fluorescent substance that constantly exhibits fluorescence characteristics regardless of the surrounding environment was used, so that it was not possible to selectively fluoresce only the immobilized light-labeling substance.

Under these circumstances, the cleaning process removes floating photolabeling substances that cause background noise, but this time there is a problem that the inspection process becomes complicated.

Further, the photolabeling substance used in the ELISA method has a problem that fluorescence emission is weak and it is difficult to measure with high sensitivity.

Therefore, a method for detecting biomolecules, which is a further development of the ELISA method, has also been developed. For example, in a method called a digital ELISA method, high sensitivity is realized by devising a reaction device and performing an enzymatic reaction in fine droplets (femtolitre) (Non-Patent Documents 1 and 2). However, the measurement principle of these methods is the same as that of the ELISA method, and there is a problem that the operation is more complicated and takes time than the conventional ELISA method.

Single Molecule Counting (SMCTM) Immunoassay Technology has been developed as a molecular detection technique (Non-Patent Document 3). This technique is also based on the ELISA method, but by devising the optical system of the detector, the detection region is miniaturized and the detection sensitivity is improved. However, there is a problem that a dedicated expensive detector is required and it takes a long time to measure because a minute area is repeatedly scanned and measured.

In addition, although the immunochromatography method is used (Non-Patent Document 4), it cannot be used for early detection of diseases and infections due to its low sensitivity. It has the drawback of being as high as ~ 30%).

Since detection sensitivity of 1 pM or less is required for early diagnosis of epidemics and infections using biomarkers, the development of a detection method that is more sensitive, simple, and can be carried out in a short time is awaited.

Further, although a temperature-responsive fluorescent particle having high detection sensitivity has recently been reported (Patent Document 1), a method for optimally detecting or quantifying using the fluorescent particle has not been specifically disclosed.

Rissin D.M., et al, Nat Biotechnol. 2010: 28 (6): 595-9. Kim S.H., et al, Lab Chip, 2012, 12, 4986-4991. Hwang J., et al, Methods, 2019, 158, 69-79. Hurt C. A., et al, J. Clin. Virol., 2007, 39, 132-135. WO2020 / 241830

Therefore, as a result of diligent studies, the inventors have produced temperature-responsive probe particles in which the surface of the temperature-responsive fluorescent particles is modified, and invented a detection method, which is an improved ELISA method using the same. The temperature-responsive probe particles exhibit fluorescence characteristics when the temperature rises above a specific temperature, and have the property of having strong fluorescence emission. Utilizing this characteristic, the temperature-responsive probe particles immobilized on the bottom surface of the transparent inspection container are first warmed using a transparent inspection container and a transparent hot plate that transmit excitation light and fluorescence, and the phase transition temperature is reached. The timing of fluorescence emission exceeding the above is measured by a detection unit located under the transparent hot plate.

One aspect of the present invention is described above by measuring the fluorescence emission intensity after contacting a temperature-responsive probe particle having a modified surface of the temperature-responsive fluorescent particle, a biological sample, and a biomolecule recognition element for capture. A method for detecting or quantifying target biomolecules contained in a biological sample, in which (1) a transparent inspection container in which a biomolecule recognition element for capture is immobilized inside the bottom surface is prepared, and (2) temperature response. A step of contacting a sex probe particle, a biological sample, and a biomolecule recognition element for capture to obtain a measurement target in a transparent inspection container, (3) a temperature-responsive probe particle, a biological sample, and a biomolecule for capture. A step of contacting a recognition element with a transparent inspection container to obtain a measurement target, (4) a step of arranging the bottom surface of the transparent inspection container on a transparent hot plate, and (5) a step of heating the transparent hot plate, ( 6) The method is characterized by including a step of measuring the fluorescence emission intensity in the vicinity of the bottom surface in the transparent inspection container from the lower side of the transparent hot plate.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a method and an apparatus capable of detecting or quantifying a low concentration target biomolecule easily and in a short time.

It is a schematic diagram of the detection device of this invention. It is a perspective view which shows the situation which the transparent inspection container 1 of this invention is arranged in the inspection container holder 10. It is sectional drawing which shows the situation which the transparent inspection container 1 of this invention is arranged in the inspection container holder 10. It is sectional drawing of an example of the transparent hot plate 2 of this invention. It is a top view of the transparent hot sheet 20 used as an example of the transparent hot plate 2 of this invention. It is a figure (1) explaining the outline of the fluorescence emission intensity measurement among the detection methods of this invention. It is a figure (2) explaining the outline of the fluorescence emission intensity measurement among the detection methods of this invention. It is a figure explaining the quenching and fluorescence emission of the temperature-responsive fluorescent particle of this invention. It is a figure (1) which shows an example of the detection method of this invention. It is a figure (2) which shows an example of the detection method of this invention. It is a figure explaining the time change of the fluorescence emission intensity in a transparent container 1 in the presence of a target biomolecule. It is a figure explaining the time change of the fluorescence emission intensity in a transparent container 1 in the absence of a target biomolecule. It is a figure explaining the principle of the detection method of this invention.

Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments. Further, in each drawing, the dimensional ratio in the drawing and the actual dimensional ratio do not always match.

(Overview of the detection device of the present invention)
The outline of the detection apparatus of this invention will be described with reference to FIG. The entire detection device 100 is shielded from light by the housing 9 so that noise light does not enter from the outside during detection. A transparent hot plate 2 is installed in the upper part of the inside of the detection device 100. A light source unit 3 and a detection unit 4 are arranged under the transparent hot plate 2. A filter 5 may be arranged in order to facilitate measurement of the fluorescence emitted from the detection unit 4.

The light source unit 3 is arranged so that the light emitted from the light source unit 3 reaches the transparent hot plate 2 and the transparent inspection container 1 to be arranged above the transparent hot plate 2. Since the detection unit 4 measures the fluorescence emission intensity near the bottom surface in the transparent inspection container 1 through the transparent hot plate 2, it is installed on the lower side in the substantially gravity direction of the transparent hot plate 2. The light source unit 3 needs to be arranged at a position that does not interfere with the measurement of the detection unit 4.

(Overview of the detection method of the present invention)
In the measurement, the transparent inspection container 1 containing the measurement target is placed on the transparent hot plate 2 to heat the transparent inspection container 1 from the bottom surface, and the excitation light is emitted from the light source unit 3 to perform the transparent inspection by the detection unit 4. This is done by measuring the fluorescence emission intensity near the bottom surface in the container.

(Transparent inspection container)
As the material of the transparent inspection container 1 used in the present invention, a material such as transparent plastic or glass that does not block or absorb the excitation light emitted by the light source unit 3 and the fluorescence emitted by the measurement target is preferable. The shape of the transparent inspection container 1 is not limited to, for example, a cylindrical container having an open upper portion.

(Inspection container holder)
The transparent inspection container 1 may be placed directly on the transparent hot plate 2, but may also be fixed to the inspection container holder 10 as shown in FIGS. 1 and 2. This makes it possible to use many transparent inspection containers 1 for measurement at the same time. The inspection container holder 10 has a structure that does not cover the bottom surface of the transparent inspection container 1 as shown in FIG. 3A so as not to interfere with the detection method of the present invention. As described above, it is preferable that the bottom surface is made of a transparent material. Further, the basic member constituting the inspection container holder 10 is preferably black so as not to interfere with the fluorescence emission intensity measurement due to the reflection of light.

As an example of the former, it is conceivable to provide a raised portion on the upper portion of the transparent inspection container 1 and hook it on the hole portion of the inspection container holder 10 to fix it. When the inspection container holder 10 is placed on the transparent hot plate 2, the transparent inspection container 1 may be designed to be in contact with the transparent hot plate 2 or may be designed to float by about several millimeters.
As an example of the latter, there is a case where the bottom surface is made of a transparent bottom 11 which does not block or absorb the excitation light emitted by the light source unit 3 and the fluorescence emitted by the measurement target, such as those made of transparent plastic or glass.

(Transparent hot plate)
As the material of the transparent hot plate 2, a material such as transparent plastic or glass that does not block or absorb the excitation light emitted by the light source unit 3 and the fluorescence emitted by the measurement target is preferable. Glass is particularly preferable from the viewpoint that the temperature is easily transmitted quickly and uniformly.

FIG. 4 shows an example of the structure of the transparent hot plate 2. The transparent hot sheet 20 is transparent as a whole, but is a sheet that generates heat by conducting electricity. As an example of the structure of the transparent hot plate 2, a three-layer structure in which the transparent hot sheet 20 is sandwiched by the glass plate 21 is adopted.

FIG. 5 is a plan view of the transparent hot sheet 20. The transparent hot sheet 20 is configured by arranging a plurality of thin heating wires 22 on a transparent film 24 serving as a substrate and connecting the heating wires 22 to the electrodes 23. The transparent film 24 may be any material as long as it does not block or absorb the excitation light emitted by the light source unit 3 and the fluorescence emitted by the measurement target. The heating wire 22 is a thin wire made of a heat generating element such as a nichrome wire, and is incorporated or attached to the transparent film 24. By energizing the electrode 23, the heating wire 22 generates heat. A plurality of heating wires 22 are arranged on the transparent film 24, but since they are thin wires, they do not interfere with the measurement.

In addition to the above, the transparent hot plate 2 may be a plate in which a glass or plastic plate is used alone and the whole is heated by heat transfer by heating from the end portion. In addition, the configuration of the transparent hot plate 2 is not limited.

(Light source)
As the light source unit 3, any light source capable of outputting the excitation wavelength of the photolabeling substance is sufficient. For example, it is conceivable to install a filter that can output a specific wavelength such as a light emitting diode, or a filter that can pass only a specific wavelength in the middle of the laser optical path of a laser light source that can output a wide range of wavelengths in advance.

(Detection unit)
As the detection unit 4, in addition to a general commercially available fluorometer, a general-purpose digital camera, a built-in camera of a smartphone or a tablet, or the like is used. According to the inspection method of the present invention, since the temperature-responsive fluorescent particles have strong fluorescence, they can be practically detected by general-purpose devices such as smartphones and tablets depending on the conditions.

(filter)
In the present invention, a filter 5 may be provided on the upper part of the detection unit 4 or the like in order to limit the light entering the detection unit 4. The filter 5 is a film that cuts light that causes noise other than fluorescence emission, such as excitation light.

(Outline of Fluorescence Emission Intensity Measurement of the Present Invention)
FIG. 6 is a diagram illustrating an outline of fluorescence emission intensity measurement in the method of the present invention. The light source unit 3 irradiates the excitation light corresponding to the temperature-responsive fluorescent particles from the lower side of the transparent hot plate 2 in the substantially gravity direction, and the excitation light passes through the bottom surfaces of the transparent hot plate 2 and the transparent inspection container 1 to be measured. Be irradiated.

At this time, as shown in FIG. 7, since the transparent hot plate 2 is gradually heated from the bottom surface of the transparent inspection container 1, a temperature distribution is generated in the liquid of the transparent inspection container 1, and the closer to the bottom surface, the warmer it is. The situation is born. When the temperature-responsive fluorescent particles immobilized on the bottom surface exceed the phase transition temperature described later, the temperature-responsive fluorescent particles exhibit fluorescence characteristics and emit fluorescent light (FIG. 8). That is, by adjusting the temperature distribution, it is possible to create a timing in which the temperature-responsive fluorescent particles immobilized on the bottom surface exhibit fluorescence characteristics and the floating temperature-responsive fluorescent particles do not exhibit fluorescence characteristics.
Returning to FIG. 6, the detection unit 4 measures the intensity of the fluorescence emitted at this time. At the above timing, only the fluorescence emitted by the temperature-responsive fluorescent particles immobilized on the bottom surface can be detected.

(Temperature-responsive fluorescent particles)
The temperature-responsive fluorescent particles contain at least one fluorescent molecule in a molecular assembly composed of at least one amphipathic molecule. That is, in the temperature-responsive fluorescent particles of the present invention, the extinction state due to the aggregation of fluorescent molecules is dissociated with the phase transition (that is, the transition from the solid phase to the liquid phase or the transition from the gel phase to the liquid crystal phase). This utilizes the phenomenon of being in a light emitting state (Fig. 8).

Therefore, the temperature-responsive fluorescent particles used in the present invention can be used without particular limitation as long as the phase transition occurs from the solid phase state to the liquid phase state while maintaining the molecular aggregate morphology. The molecular assembly preferably has a monomolecular membrane or a bilayer, and more preferably, the molecular assembly has a lipid bilayer (also referred to as a lipid bilayer).

The amphipathic molecule (eg, lipid molecule) is not limited to a lipid derived from a living body, but is a small molecule such as a phospholipid produced by semi-synthesis or synthesis, a derivative thereof, a polymer, a peptide, an amino acid, and the like. It contains amphipathic molecules that can be produced synthetically using carboxylic acids and the like as raw materials.

The temperature-responsive fluorescent particles dissociate and emit fluorescence when the molecular aggregate is in the liquid phase due to a solid-phase-liquid phase transition in response to temperature, and aggregate and extinguish when the molecular aggregate is in the solid phase. As a result, the fluorescence emission of fluorescent molecules and the extinction can be reversibly converted in response to temperature (FIG. 8).

That is, the molecular assembly exhibits a solid-phase-liquid phase transition in response to temperature, has fluorescent molecules dissociated during the liquid phase and emits fluorescence, and aggregates with fluorescent molecules aggregated during the solid phase. It is extinguished by aggregation-caused quenching. When the molecular assembly is a molecular assembly having a lipid bilayer, the solid phase is a gel phase and the liquid phase is a liquid crystal phase.

The fluorescent molecule is arranged in a hydrophobic region constructed in an aqueous phase in a molecular assembly state, for example, a region surrounded by a monomolecular membrane, or a bimolecular membrane such as a lipid bimolecular membrane. Therefore, amphipathic or lipophilic fluorescent molecules are suitable. Normally, fluorescent molecules have the property of quenching by aggregation, so any fluorescent molecule that exhibits aggregation and dissociation in response to a solid-phase-liquid phase transition (for example, a gel-liquid crystal phase transition of a lipid bilayer membrane). For example, quenching and emission can be converted.

As an example of the fluorescent molecule, a compound represented by the following general formula (A) can be mentioned.

Some biomolecules, such as nicotine amide adenine dinucleotide phosphate, flavins, proteins, and amino acids, absorb light with a wavelength of 600 nm or less and emit fluorescence with a wavelength of 600 nm or less (autofluorescence). Therefore, if a fluorescent probe having a maximum absorption wavelength and a maximum fluorescence emission wavelength of 600 nm or less is used for detecting biomolecules, interference due to autofluorescence of contaminant molecules may become a problem in fluorescence measurement. Therefore, it is desirable that the compound represented by the general formula (A) of the present invention is a fluorescent molecule having a maximum absorption wavelength and a maximum fluorescence emission wavelength of 600 nm or more, and further, light having a wavelength of 980 nm or more is transferred to water. Considering that the heat generated by the absorption of water acts on the temperature-responsive particles when irradiated, it is more desirable that the fluorescent molecule has a maximum absorption wavelength and a maximum fluorescence emission wavelength in the range of 600 to 900 nm.

As a more specific example of the compound of the formula (A) of the fluorescent molecule, a squaric acid derivative can be mentioned.

Examples of the squaric acid derivative include a compound represented by the following general formula (I).

However, R ₁ and R ₂ represent hydrophobic groups that are independent of each other and may be the same or different.

The compound represented by the general formula (A) or the general formula (I) shall include not only the free form but also a salt thereof. Such salts vary depending on the type of compound, and are, for example, alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), aluminum salts, ammonium salts, and other inorganic base salts, as well. Base addition salts such as organic base salts such as trimethylamine, triethylamine, pyridine, picolin, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N, N'-dibenzylethylenediamine, or hydrochlorides, hydrobromates, sulfates. Inorganic acid salts such as salts, hydroiodide, nitrates and phosphates, citrates, oxalates, acetates, formates, propionates, benzoates, trifluoroacetates, maleates, Examples thereof include acid addition salts such as organic acid salts such as tartrate, methanesulfonate, benzenesulfonate and paratoluenesulfonate.

In the squaric acid derivative, R ₁ and R ₂ may be a linear hydrocarbon, a branched chain hydrocarbon, a saturated hydrocarbon, or an unsaturated hydrocarbon. May be. Preferably, a linear saturated hydrocarbon can be mentioned, but the present invention is not limited as long as the fluorescent molecule dissociates in the liquid crystal phase of the molecular assembly and aggregates in the gel phase.

More specific examples of R ₁ and R ₂ include, for example, a hydrocarbon group having 2 to 10 carbon atoms, and more specifically, from an n-butyl group, an n-pentyl group or an n-hexyl group. It can be selected, but is not limited as long as it dissociates in the liquid crystal phase of the molecular assembly and aggregates in the gel phase.

Examples of the amphipathic molecule include phospholipids.

The type of phospholipid is not particularly limited, but glycerophospholipid (also referred to as diacyl-type phospholipid) is preferable, and in order to form a stable lipid bilayer film showing a phase transition, diacyl having a phosphocholine group in the hydrophilic portion is particularly preferable. Type phospholipids are preferred. The acyl chain lengths of the diacyl-type phospholipids may be the same or different. The diacyl-type phospholipids having a phosphocholine group include saturated phospholipids and unsaturated phospholipids, and any of them can be used in the present invention, and these may be used in combination. Saturated phosphocholine is synthetic or semi-synthetic, for example, natural phospholipids such as hydrogenated egg yolk lecithin and hydrogenated soybean lecithin having a hydrogenation rate close to 100% and their derivatives, as well as dimyristoylphosphocholine and dipentadecanoylphosphocholine. , Dipalmitylphosphocholine, diheptadecanoylphosphocholine, distearoylphosphocholine and the like can be used.

As an example of the molecular assembly, an emulsion having a hydrophobic core surrounded by a monolayer (also referred to as a monolayer), lipid nanoparticles, and a vesicle having a spherical shell-like closed membrane structure are vesicles. Can be mentioned. Examples of such vesicles include, but are not limited to, lipid bilayer membrane vesicles (also referred to as liposomes). As long as the particles that can be used as the temperature-responsive fluorescent particles of the present invention, that is, the particles that can be used as the molecular aggregate, are molecular aggregates that undergo a solid-phase-liquid phase transition or a gel-liquid crystal phase transition at a specific temperature, there are many. It is not limited by a multilayer or a single layer, a particle size, or the like.

In order to prevent aggregation and fusion of molecular aggregates such as lipid vesicles, amphipathic molecules having charged residues or polymer chains in the hydrophilic portion can be included in the constituents. The molecular aggregates such as lipid vesicles thus obtained have high dispersion stability due to electrostatic repulsion due to electric charge and steric exclusion effect due to polymer chains.

As an example of an amphipathic molecule having a charged residue, when the molecule is a lipid molecule, an anionic phospholipid having phosphatidylglycerol, phosphatidylserine, phosphatidic acid in the hydrophilic part, L-glutamic acid, N- (3). -Carboxylic acid-type lipids such as carboxy-1-oxo propyl)-, 1,5-dihexadecyl ester (SA), and amino acid-type lipids having an amino acid as a hydrophilic group can be mentioned.

Further, as an example of an amphipathic molecule having a polymer chain, when the molecule is a lipid molecule, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-monomethoxy poly (ethylene glycol) can be mentioned. .. The molecular weight of Poly (ethylene glycol) is not limited, but a molecular weight of 1000 to 5000 is preferable to prevent aggregation of lipid vesicles.

In addition, as an amphipathic molecule, when the molecule is a lipid molecule, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (PC14), 1,2-dipentadecanoyl-sn-glycero-3-phosphocholine (PC15). ) (Manufactured by Avanti Polar Lipids Inc.), 1,2-diheptade canoyl-sn-glycero-3-phosphocholine (PC17) (manufactured by Avanti Polar Lipids Inc.), 1,2-distearoyl-sn-glycero-3-phosphocholine (manufactured by Avanti Polar Lipids Inc.) PC18), 1,2-dipalmitoy l-sn-glycero-3-phosphocholine (PC16 (also called DPPC)), 1,2-Dioleoyl- sn-glycero-3-phosphocholine (DOPC), 1,5-dihexadecyl- N-succiny-L-gluta mate (DHSG), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), etc. can also be used.

Quenching caused by aggregation of fluorescent molecules in a molecular assembly such as a lipid vesicle depends on the concentration in the molecular assembly, and as the concentration of fluorescent molecules decreases, the quenching caused by aggregation also decreases. Therefore, the temperature-responsive fluorescent particles of the present invention contain the compound represented by the general formula (I) at a concentration of 0.3 mol% or more, preferably 0.7 mol% or more, more preferably 1.0 mol% or more.

The production of the temperature-responsive fluorescent particles can be obtained by coexisting the compound of the formula (I) in the production of the molecular assembly. As an example of the production of the molecular assembly, it can be produced according to a known method for producing a lipid vesicle (Sheng Dong et al, ACS Appl. Nano Mater. 2018, 1, 1009-1013).

The temperature-responsive fluorescent particles of the present invention can be used for producing the following temperature-responsive probe particles.

(Temperature responsive probe particles)
The temperature-responsive probe particles can be obtained by modifying the surface of the temperature-responsive fluorescent particles with a biomolecule recognition element. As used herein, the term "element" means a molecule that exerts a specific function, such as a biomolecule recognition function, and the terms "element" and "molecule" can be used interchangeably. Further, as used herein, "recognition" means recognizing a target molecule and binding to the molecule. Furthermore, the "biomolecule recognition element" is not only an element that directly recognizes a biomolecule, but also a complex of another molecule or a molecular complex (eg, a complex of streptavidin and a biomolecule-recognizing biomolecule). Etc.), those that indirectly recognize biomolecules are also included.

Examples of the biomolecule recognition element include an antibody, a variable region of the antibody, or a fab fragment of the antibody (hereinafter, referred to as “antibody or the like”). In the present specification, such an element, for example, an antibody or the like, is referred to as a "detection biomolecule recognition element" as described below, and when the element is an antibody or the like, it is also referred to as a "detection antibody".

On the other hand, for the purpose of fixing the target biomolecule to the bottom surface of the measurement container or the like, an element for another epitope of the target biomolecule, for example, an antibody or the like may be used. In the present specification, such an element is referred to as a "capture biomolecule recognition element", and when the element is an antibody or the like, it is also referred to as a "capture antibody".

The biomolecule recognition element can form a complex of the target biomolecule and the temperature-responsive fluorescent particle by binding to the target biomolecule to be detected or measured.

When an antibody is used as the biomolecule recognition element as used in the sandwich ELISA method, which is a conventional technique, two types of antibodies, a detection antibody and a capture antibody, which bind to different epitopes of the same antigen are used. The detection antibody binds the antigen to the temperature-responsive fluorescent particles via the antibody. The captured antibody has a role of capturing and fixing the target biomolecule to be tested on the bottom surface of the measuring container or the like by fixing it on the bottom surface of the measuring container such as a well.

Similar to the sandwich ELISA method, in the temperature-responsive probe particles of the present invention, the detection antibody is used as an antibody for binding to the temperature-responsive fluorescent particles, and the captured antibody is placed on the bottom surface of a measurement container such as a well as a target living body. It is used as an antibody for fixing molecules.

That is, when the biomolecule recognition element is an antibody, (a) temperature-responsive probe particles composed of the temperature-responsive fluorescent particles and the detection antibody, and (b) the target biomolecule which is an antigen of the detection antibody and the capture antibody. , (C) The captured antibody immobilized on the bottom surface of the measurement container such as a well is a temperature-responsive probe particle (temperature-responsive fluorescent particle in which the detection antibody is modified on the membrane surface) -target biomolecule (antigen) -capture. When forming the antibody-measurement container complex, the temperature-responsive probe particles are in a state of being fixed to the bottom surface of the measurement container. When the bottom surface of the measuring container is heated in this state, the temperature of the composite rapidly rises due to heat conduction from the bottom surface of the measuring container (FIG. 11 (b)). At this time, the temperature-responsive probe particles in the complex undergo a phase transition from the solid phase to the liquid phase, so that the fluorescent molecules in the temperature-responsive fluorescent particles that have aggregated and quenched in the solid phase dissociate and emit fluorescent light. On the other hand, when the target biomolecule, that is, the antigen, is not present in the test sample, the complex is not formed. Therefore, the temperature-responsive probe particles cannot adhere to the bottom surface of the measuring container, and even if the bottom surface of the measuring container is heated, the temperature rises only slowly due to heat transfer via the solvent, so that the solid phase state is longer. The time is maintained and the dimmed state is maintained (FIG. 12 (b)).

The temperature-responsive probe particles of the present invention, that is, the temperature-responsive fluorescent particles whose surface is modified with a biomolecule recognition element, can be obtained by producing a lipid vesicle modified with a detection antibody according to a known method. (International Publication Pamphlet WO2020 / 241830, WO2000 / 064413, JP-A-2003-73258, JP-A-58-134032, Manjappa S.A., et al, J. Controlled Release 2011, 150, 2-22, Li T., et al, Nanomed .: Nanotechnol. Biol. Medicine, 2017, 13, 1219-1227).

In addition, for the method for producing the temperature-responsive fluorescent particles and the temperature-responsive probe particles of the present invention, known methods (Scientific Reports volume 10, Article number: 18086 (2020), etc.) can be referred to.

In the measurement operation of the ELISA method, which is a conventional technique, an operation of separating an antibody-enzyme conjugate bound to a target biomolecule and an antibody-enzyme conjugate not bound to the target biomolecule, and / or a target biomolecule Requires an operation to clean the antibody-enzyme conjugate that did not bind to. On the other hand, the present invention utilizes an antigen-antibody reaction against an epitope of a target biomolecule to rapidly raise the temperature by heat conduction to temperature-responsive probe particles adhered near the heat source, and a temperature not bound to the target biomolecule. Since the difference in slow temperature change due to heat transfer via the solvent to the responsive fluorescent particles is utilized, it is easy to measure the fluorescence emission intensity without performing the separation operation and cleaning operation required by the ELISA method. The target biomolecule can be detected in a short time and with high sensitivity.

On the other hand, in the sandwich ELISA method, measurement kits for various biomolecules have already been commercially used. That is, the kit includes a microwell plate for measurement in which the captured antibody is fixed on the bottom surface of the well as a measurement container, and a detection antibody. So, for example, such a commercially available sandwich ELISA measurement kit can be utilized. The present invention can be carried out by producing the temperature-responsive probe particles of the present invention and obtaining the detection antibody contained in such a kit, a microwell plate in which the captured antibody is fixed on the bottom surface of the well, or the like. ..

In the sandwich ELISA method, a capture antibody immobilized on the bottom of the well of the microwell plate, an antigen (target biomolecule), a biotinylated detection antibody, and a horseradish peroxidase (HRP) -labeled streptavidin are combined. Measurement kits that measure antigens by forming a body are commercially available (see FIG. 9 (a)). Therefore, as the temperature-responsive probe particles of the present invention, temperature-responsive fluorescent particles whose surface is labeled with biotin are produced, and the captured antibody fixed on the bottom surface of the well of the microplate, the antigen (target biomolecule), and biotin. The converted detection antibody, streptavidin, and temperature-responsive fluorescent particles whose surface is labeled with biotin are mixed to form a complex thereof, and the bottom of the well of the microplate is heated with a heat source to be temperature-responsive. The antigen (target biomolecule) in the test sample can be measured by rapidly undergoing a phase transition of the fluorescent particles to cause fluorescence emission (see FIG. 9 (b)). That is, it can be said that the biological recognition element in this case is not a detection antibody alone, but a complex of a biotin-streptavidin-biotinylated detection antibody modified on the surface of temperature-responsive fluorescent particles. Alternatively, the surface of the lipid vesicle is modified with a maleimide group by introducing a maleimide-terminated polyethylene glycol-type lipid, and the detected antibody is treated with pepsin and then reduced to react with the thiol group of Fab', which is a fragmented antibody obtained. It can be said that the complex thus produced is also a complex of a temperature-responsive fluorescent lipid vesicle-fragment detection antibody. As described above, the biological recognition element is not limited to the antibody alone, nor is it limited to biotin-streptavidin, and various molecules forming a complex having a strong binding property by molecular recognition or the like. By utilizing the above, the temperature-responsive probe particles of the present invention can be produced and used.

Examples of molecular species that can be used for complex formation by molecular recognition include a combination of a sugar chain and a lectin, a complementary base sequence of a nucleic acid, a combination of a receptor and a ligand molecule, a nucleic acid aptamer, and a peptide aptamer. Not limited to.

(Detection or quantification method of target biomolecule)
The method for detecting or quantifying a target biomolecule of the present invention transports a fluorescent probe particle or a fluorescent molecule contained in the particle to a cell membrane by a conventional detection method, for example, endocytosis or membrane fusion, and detects a cell. It is completely different from the method and the method of detecting the fluorescence generated by the emission of fluorescent molecules from fluorescent probe particles.

As described above, the temperature-responsive probe particles and the temperature-responsive fluorescent particles are targeted biomolecules in a short time, easily and with high sensitivity by measuring the fluorescence emission intensity without performing a separation operation or a washing operation. Can be detected or measured. Alternatively, biomolecules can be detected or quantified by a washing operation rather than heating. It can also be used as a molecular detection method for a target biological sample by making the test sample into microdroplets.

(Step (1) of the detection method of the present invention)
The step (1) of the detection method of the present invention is a step of preparing a transparent inspection container in which a biomolecule recognition element for capture is immobilized on the inside of the bottom surface. The method for immobilizing the biomolecule recognition element for capture is a known method. As the step (1), a commercially available transparent inspection container in which the biomolecule recognition element for capture is immobilized on the inside of the bottom surface can also be used.

(Step (2) of the detection method of the present invention)
The step (2) of the detection method of the present invention is a step of bringing the temperature-responsive probe particles, the biological sample, and the capture biomolecule recognition element into contact with each other to obtain a measurement target in the transparent inspection container. be.

The concentration of the temperature-responsive probe particles used in the method for detecting or quantifying biomolecules of the present invention is not particularly limited, but the temperature-responsive probe particles are 1.0 μg / g / in the solution at the time of contact with the biomolecule recognition element for capture. It is preferably contained in a concentration of ml or more, more preferably 2.0 μg / ml or more, still more preferably 5.0 μg / ml or more.

Examples of the biological sample used in the above-mentioned method for detecting or quantifying a biomolecule of the present invention include a sample collected from an animal or a cell. Further, the sample may be a sample known to contain a target biomolecule, or may be a sample in which it is unknown whether or not the target biomolecule is contained. Such animals or cells include, for example, mammals such as mice, rats, hamsters, guinea pigs, dogs, monkeys, oran wootans, chimpanzees, humans, or cells derived from such animals. Examples of animal-derived samples include blood, serum, plasma, saliva, urine, tears, sweat, milk, nasal juice, semen, pleural effusion, gastrointestinal secretions, cerebrospinal fluid, intertissue fluid, and lymph. , Preferably blood, serum or plasma. These samples can be obtained by a method known per se, for example, serum or plasma can be prepared by collecting blood from a test animal according to a conventional method and separating humoral components, and cerebrospinal fluid can be prepared. , Can be collected by known means such as spinal puncture.

The method of bringing the temperature-responsive probe particles and the biological sample into contact with the capture biomolecule recognition element fixed to the substrate is not particularly limited. For example, a solution containing a biological sample and temperature-responsive probe particles can be added onto the substrate in this order, but the method is not limited to this method. Alternatively, a solution prepared by previously mixing the biological sample and the temperature-responsive probe particles may be added onto the substrate (that is, the biological sample and the temperature-responsive probe particles may be brought into contact with the substrate at the same time. ). The conditions for the above contact are not particularly limited, but are usually contacted at a temperature of 0 to 45 ° C, preferably at a temperature of 0 to 40 ° C, and more preferably at a temperature of 4 to 37 ° C. , More preferably, the contact is carried out at a temperature of 25 ° C to 37 ° C. The contact time is not particularly limited, but is typically 6 hours or less, preferably 2 hours or less, and more preferably 1 hour or less. Further, the lower limit of the contact time is not particularly limited, but is typically 10 seconds or longer, preferably 1 minute or longer, and more preferably 10 minutes or longer.

Further, an element that recognizes the biomolecule recognition element (eg, streptavidin) by adding the same biomolecule recognition element (eg, biomolecules, etc.) of the temperature-responsive probe particles to the detection biomolecule recognition element. The temperature-responsive probe particles may be bound to the biomolecule recognition element for detection via (avidin or the like). Therefore, in one embodiment, step (2) is a capture biomolecule in which a temperature-responsive probe particle, an element that recognizes a biomolecule recognition element, a biomolecule recognition element for detection, and a biosample are fixed to a substrate. It may be a step of contacting the recognition element. The contact method can be the same as the method described above. For example, a solution containing a biological sample and a solution containing a biomolecule recognition element for detection, an element for recognizing a biomolecule recognition element, and temperature-responsive probe particles can be added to the substrate in this order. , Not limited to this order. Further, the biomolecule recognition element for detection, the element for recognizing the biomolecule recognition element, and the temperature-responsive probe particles may be separately brought into contact with the biomolecule recognition element for capture. Alternatively, a solution prepared by mixing a biological sample, a biomolecule recognition element for detection, an element for recognizing a biomolecule recognition element, and temperature-responsive probe particles in advance may be added onto the substrate (that is, the biological sample, Temperature-responsive fluorescent probe An element that recognizes a biomolecule recognition element and a biomolecule recognition element for detection may be brought into contact with the substrate at the same time). Schematic diagrams of this method are shown as FIGS. 10 (a) to 10 (c). By such a method, as shown in FIG. 10 (c), a plurality of temperature-responsive probe particles are bound to one biomolecule, and it is possible to improve the detection sensitivity. In a preferred embodiment, the biomolecule recognizing element is biotin, and the element recognizing the biomolecule recognizing element is streptavidin, avidin or neutravidin.

(Step (3) of the detection method of the present invention)
The step (3) of the detection method of the present invention is a step of arranging the bottom surface of the transparent inspection container on the transparent hot plate. In this aspect, one or more transparent inspection containers may be placed directly on the transparent hot plate, or may be placed in the inspection container holder and then placed on the transparent hot plate. As long as the heat of the transparent hot plate is transferred to the transparent inspection container, there is no limitation, and there is no problem even if the transparent inspection container floats from the transparent hot plate or a transparent sheet or the like is pinched. However, it is necessary that the excitation light emitted by the light source unit 3 and the fluorescence emitted by the measurement target are not blocked or absorbed.

(Step (4) of the detection method of the present invention)
The step (4) of the detection method of the present invention is a step of heating a transparent hot plate. As for the timing of heating, the bottom surface of the transparent inspection container may be placed on the transparent hot plate and then heated, or the heating may be performed from the stage before the placement.

The transparent hot plate may generate heat by itself, generate heat by the action of electricity or light, or obtain heat by heat conduction from another heat source, regardless of the method. The temperature (phase transition temperature) at which the temperature-responsive fluorescent particles used undergo a phase transition (that is, a transition from a solid phase to a liquid phase or a transition from a gel phase to a liquid crystal phase) is measured in advance, and a transparent hot plate is used. Needs to be heated above the phase transition temperature.

At that time, the larger the difference between the set temperature of the transparent hot plate and the phase transition temperature, the faster the bottom surface of the transparent inspection container warms up, and the faster the phase transition occurs. If the difference is too large, the temperature will rise sharply and the entire transparent inspection container will quickly exceed the phase transition temperature. Since the time difference between the temperature-responsive probe particles floating without being captured in the transparent inspection container and the temperature-responsive probe particles exceeding the phase transition temperature becomes small, there arises a problem that it becomes difficult to measure the fluorescence emission intensity.
On the other hand, if the difference is too small, the temperature-responsive probe particles to be measured, which are captured on the bottom surface of the transparent inspection container, do not easily warm up, and it takes too much time to measure the fluorescence emission intensity.
Therefore, it is preferable that the set temperature of the transparent hot plate is set so that the fluorescence emission intensity measurement is completed within a few seconds to one minute.

(Step (5) of the detection method of the present invention)
The step (5) of the detection method of the present invention is a step of measuring the fluorescence emission intensity near the bottom surface in the transparent inspection container from the lower side of the transparent hot plate.

The method for detecting or measuring fluorescence emission is not particularly limited, but a detector equipped with a general optical fiber type fluorescence detector, a microplate reader, a fluorescence microscope, and a camera (eg, a camera equipped with a CMOS element) (eg, Gerui). It can be detected or measured by using a major) or the like. Further, as in the method of the ELISA method, a calibration curve is prepared using a sample (standard product) having a known concentration of the target biomolecule, and the calibration curve is used to determine the concentration of the target biomolecule in the biological sample. It can also be quantified. Furthermore, it can also be quantified by the quantification method for biomolecules described below. That is, the biomolecule detection method of the present invention can be applied as a biomolecule quantification method.

The basic principle of detection is that by irradiating excitation light from the light source located under the transparent hot plate toward the inside of the transparent inspection container, the extinction state due to aggregation of fluorescent molecules exceeds the phase transition temperature. The temperature-responsive probe particles that have been dissociated and are in a light-emitting state are selectively fluorescently emitted, and the intensity thereof is measured by a detection unit.

At that time, since the temperature-responsive probe particles to be measured are captured on the bottom surface of the transparent inspection container, they are transparent as compared with the temperature-responsive probe particles floating in the transparent inspection container without being captured. The hot plate will exceed the phase transition temperature at an earlier timing.

FIG. 11 shows the relationship between the passage of time and the fluorescence intensity when the target biomolecule is present in the biological sample. (A) At the moment when the transparent inspection container is placed on the transparent hot plate, the temperature of the temperature-responsive probe particles to be measured is lower than the phase transition temperature, so that even if the excitation light is applied from the light source, fluorescence is not emitted. .. After that, (b) when the transparent inspection container is warmed by the transparent hot plate, the temperature-responsive probe particles to be measured, which are previously captured on the bottom surface of the transparent inspection container, are warmed and exceed the phase transition temperature. , Will emit fluorescent light. However, at this point, the temperature-responsive probe particles floating in the transparent inspection container without being captured do not exceed the phase transition temperature, so that they do not emit fluorescence. Further, after that, (c) the temperature-responsive probe particles floating in the transparent inspection container without being captured are also warmed and exceed the phase transition temperature, so that they emit fluorescent light. When this is measured from the lower detection unit, it becomes as shown in the graph at the lower part of FIG.

FIG. 12 shows the relationship between the passage of time and the fluorescence intensity when the target biomolecule is not present in the biological sample. Compared with FIG. 11, since there is no temperature-responsive probe particle to be measured captured on the bottom surface of the transparent inspection container, most of the temperature-responsive probe particles in the transparent inspection container are suspended. Therefore, even in the state of (b) in which only the bottom surface of the transparent inspection container is warmed, the fluorescence emission intensity is weak. After that, when the temperature-responsive probe particles floating in the transparent inspection container without being captured are also warmed, the fluorescence emission intensity becomes the maximum value (c).

FIG. 13 is an overlay of the graphs at the bottom of FIGS. 11 and 12. Depending on whether the target biomolecule is present or not in the biological sample, the fluorescence emission intensity when the temperature-responsive probe particles floating without being captured in the transparent inspection container are also warmed is It is the same. However, if the target biomolecule is present in the biological sample, it is not present because the temperature-responsive probe particles to be measured are captured and localized on the bottom surface of the transparent inspection container. The fluorescence emission intensity becomes stronger at an earlier timing as compared with the above. By measuring this time difference, it is possible to determine whether or not the target biomolecule is present.
It is also possible to quantify the amount of the target biomolecule contained by calibrating by using a sample in which a specific amount of the target biomolecule is known to be present.

1 Transparent inspection container 2 Transparent hot plate 3 Light source unit 4 Detection unit 5 Filter 9 Housing 10 Inspection container holder 40 Antigen 41 Capturing antibody 42 Photolabeling substance

Claims (5)

Targets contained in the biological sample by measuring the fluorescence emission intensity after contacting the temperature-responsive probe particles having the surface of the temperature-responsive fluorescent particles modified, the biological sample, and the biomolecule recognition element for capture. A method for detecting or quantifying biomolecules.
The step of preparing a transparent inspection container in which the biomolecule recognition element for capture is immobilized on the inside of the bottom surface,
A step of bringing the temperature-responsive probe particles, the biological sample, and the capture biomolecule recognition element into contact with each other to obtain a measurement target in the transparent inspection container.
The step of placing the bottom surface of the transparent inspection container on the transparent hot plate,
The step of heating the transparent hot plate,
A step of measuring the fluorescence emission intensity near the bottom surface in the transparent inspection container from the lower side of the transparent hot plate.
A method characterized by including. The method according to claim 1, wherein the step (2) does not include a step of cleaning the inside of the transparent inspection container when obtaining the measurement target. The method according to any one of claims 1 or 2, wherein the transparent hot plate is made of glass. A device for detecting a target biomolecule contained in a biological sample, which comprises a light source unit, a transparent hot plate, and a detection unit arranged under the transparent hot plate. The detection device according to claim 5, wherein the detection device is used in the method according to any one of claims 1 to 3.

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