JP2016121940A - Test and diagnosis method using aptamer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of detecting a detection target, which enables quick, low-cost, easy, and highly accurate detection and quantification of the detection target.SOLUTION: A method of detecting a detection target contained in a sample includes steps of: mixing the sample, an aptamer that can bind to the detection target, and a cohesive substance containing a stimuli-responsive polymer together; subjecting the obtained mixture to an environment that allows the stimuli-responsive polymer molecules to cohere and determining a degree of cohesion of the cohesive substance contained in the stimuli-responsive polymer; and determining the presence of the detection target in the sample if the degree of cohesion has changed compared to when the sample is not mixed with the cohesive substance.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a detection method and a quantification method of a detection target.

  Conventionally, a latex agglutination method has been performed as a method for detecting a detection target in a specimen. The latex agglutination method is a method for detecting the antigen in a fluid such as a biological sample by mixing the fluid with a latex carrying an antibody or fragment thereof that specifically binds to the antigen to determine the degree of latex aggregation. This is a method for detecting or quantifying an antigen by measuring (for example, see Patent Document 1).

  According to this latex agglutination method, an antigen added as a specimen crosslinks a plurality of latex-bound antibodies to promote latex agglutination. Since the procedure is simple as described above, the antigen can be detected easily and rapidly. However, when the antigen is in a trace amount, the crosslinking is difficult to occur, so that the latex does not sufficiently aggregate. For this reason, it was difficult to detect a trace amount of antigen.

  Therefore, methods utilizing enzyme substrate reactions such as ELISA and CLEIA are also widely adopted. In these methods, for example, a primary antibody that specifically binds to an antigen is bound to the antigen, and a secondary antibody having an enzyme is bound to the primary antibody. Here, the antigen is detected or quantified by adding the enzyme substrate and measuring the degree of reaction catalyzed by the enzyme.

  According to these methods, for example, when a luminescent reagent is used as the substrate, the detection sensitivity of luminescence after the addition of the substrate is high, so that a trace amount of antigen can be detected.

Japanese Patent Publication No.58-ll575

  However, in the method using the enzyme substrate reaction, a large number of special reagents such as secondary antibodies and luminescent reagents are essential, and the operation cost is high.

  In addition, this method has multiple steps such as a step of incubating the sample and each reagent, a step of washing, and a step of measuring luminescence, and the operation is complicated. Moreover, the time required for each stage is extremely long, and is not suitable for large-scale processing.

  On the other hand, in recent years, the number of patients with age-related macular degeneration has increased remarkably due to the aging of the population and the Westernization of life. Age-related macular degeneration is considered to involve vascular endothelial growth factor (VEGF), and VEGF inhibitors are used for treatment, but VEGF inhibitors are approved in Japan. Is said to have high patient risk due to the effects of drug efficacy and high price. Since effective treatment can be performed by early detection, the possibility of maintenance and improvement of visual function is increased. Therefore, a rapid diagnosis method for VEGF is desired.

  This invention is made | formed in view of the above situation, and makes it a subject to provide the detection method and quantification method of the detection target which can detect and quantitate a detection target rapidly, cheaply, simply and with high precision.

The present inventors have found that when a charged substance or a hydrophilic substance is bound to a stimulus-responsive polymer, aggregation of the stimulus-responsive polymer is inhibited or promoted, and the present invention has been completed.
Specifically, the present invention has the following configuration.

1. A method for detecting a detection target in a specimen, wherein the specimen, an aptamer that can bind to the detection target, and an aggregating substance containing a stimulus-responsive polymer are mixed, and the resulting mixture is used as the stimulus response. When the degree of aggregation of the aggregating substance containing the stimulus-responsive polymer is determined under the condition that the agglutinable polymer aggregates, and the degree of aggregation changes compared to the absence of the specimen, A method for detecting a detection target, including a procedure for determining that a detection target exists in a specimen.
2. 2. The method according to item 1 above, wherein the aptamer capable of binding to the detection target is DNA, RNA, or peptide.
3. 3. The method according to item 1 or 2, further comprising separating the agglomerated magnetic substance by applying a magnetic force, wherein the aggregating substance further contains a particulate magnetic substance.
4). 4. The method according to any one of items 1 to 3, wherein the change in the degree of aggregation is a change in which aggregation inhibition of the stimulus-responsive polymer is alleviated.
5). 5. The method according to any one of items 1 to 4, wherein the detection target is VEGF.
6). 5. The method according to any one of items 1 to 4, wherein the detection target is thrombin.
7). A method for quantifying a detection target in a sample, wherein the sample, an aptamer capable of binding to the detection target, and an aggregating substance containing a stimulus-responsive polymer are mixed, and the mixture is used as the stimulus-responsive polymer. Measuring the turbidity of the mixture, and calculating the amount of the detection target in the sample based on the correlation equation between the amount of the detection target and the turbidity under the condition Including a method for quantifying a specimen.
8). 8. The method according to item 7 above, wherein the aggregating substance further contains a fine particle magnetic substance, and further includes separating the agglomerated magnetic substance by applying a magnetic force.
9. A kit for detecting and / or quantifying a detection target, comprising an aptamer capable of binding to the detection target and an aggregating substance containing a stimulus-responsive polymer.

  According to the present invention, when the detection target is present in the specimen, the aptamer and the detection target are combined, and when the complex approaches an aggregating substance containing the stimulation responsive polymer, the stimulation responsive polymer aggregates. Under the conditions, the inhibition of aggregation of the stimuli-responsive polymer by the aptamer is alleviated or promoted by the influence of the charge portion or the hydrophobic portion to be detected. Therefore, the presence or absence of the detection target can be detected by observing the presence or absence of this aggregation inhibition. Moreover, the detection target can be quantified by measuring the degree of aggregation inhibition.

  The above procedure is achieved by using only an aggregating substance containing an aptamer and a stimulus-responsive polymer, and is performed without using any special reagent or device, so that it is inexpensive and simple. In addition, it is only a measure of the degree of aggregation inhibition, and since it is not a system that utilizes a reaction catalyzed by an enzyme, it can be carried out rapidly.

FIG. 1 is a schematic configuration diagram of a method according to an embodiment of the present invention. FIG. 2 is a schematic diagram illustrating determination of presence / absence of a detection target in the method according to the embodiment. FIG. 3 is a photograph showing a measurement cell and a magnetic force application mode of the method according to one embodiment of the present invention. FIG. 4 is a graph showing the correlation between reaction time and turbidity in the method according to Example 1 of the present invention. FIG. 5 is a graph showing the correlation between reaction time and turbidity in the method according to Example 2 of the present invention.

  Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

<First embodiment> Detection method (mixing / aggregation)
In the detection method of the present invention, the aptamer and the specimen are mixed, and the obtained mixture is mixed with an aggregating substance containing a stimuli-responsive polymer, and then subjected to conditions under which the stimuli-responsive polymer aggregates. When the detection target is not present, the aggregation of the stimulus-responsive polymer remains inhibited, whereas when the detection target is present, the degree of aggregation of the stimulus-responsive polymer changes more than in the absence of the detection target. Specifically, for example, the inhibition of aggregation of the stimulus-responsive polymer is alleviated, and conversely, the inhibition of aggregation of the stimulus-responsive polymer is further promoted. Preferably, aggregation inhibition is mitigated by the influence of the charge to be detected or the hydrophobic portion.

(Aptamer)
The aptamer may be an aptamer that recognizes a specific site to be detected, or may be a mixture of aptamers that recognize different sites to be detected. The aptamer used here may be any type of aptamer, and may be a DNA aptamer or an RNA aptamer that forms a Hairpin-loop or G-quadruplex structure. In addition, a peptide aptamer may be used. Moreover, the bivalent type which connected two molecules of the aptamer which recognizes the specific site | part of a detection target with a linker, and the bispecific type which connected two types of aptamers which recognize a different site | part with respect to a detection target with a linker may be sufficient. . By using the aptamer, the detection target is specifically recognized. Compared to the case of using an antibody, for example, a DNA aptamer can be easily modified in various ways, such as biotin labeling at the 5 ′ end and 3 ′ end, and has the ability to bind to an antigen even after such processing. Hold high. Moreover, since it is easy to design a DNA aptamer having high binding ability and specificity for antigen (dissociation constant lower than that of an antibody), detection can be performed with high sensitivity and in a short time.

(Aggregating substance)
The aggregating substance used in the present invention contains a stimulus-responsive polymer. This stimulus-responsive polymer is a polymer that undergoes structural changes in response to external stimuli and can adjust aggregation and dispersion. The stimulus may be any physical or chemical signal such as, but not limited to, temperature, light, acid, base, pH or electricity.

  In particular, in the present invention, as the stimulus-responsive polymer, a temperature-responsive polymer that can be aggregated and dispersed by temperature change can be used. Examples of the temperature-responsive polymer include a polymer having a lower critical solution temperature (hereinafter also referred to as LCST) and a polymer having an upper critical solution temperature.

  Examples of the polymer having a lower critical solution temperature used in the present invention include Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, and N-acryloylpiperidine. N-acryloylmorpholine, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine, N-methacryloylmorpholine, etc. Polymer consisting of N-substituted (meth) acrylamide derivative; hydroxypropyl cellulose, polyvinyl alcohol partially acetylated product, polyvinyl methyl ether, (polyoxyethylene-polyoxy Propylene) block copolymers, polyoxyethylene alkylamine derivatives such as polyoxyethylene laurylamine; polyoxyethylene sorbitan ester derivatives such as polyoxyethylene sorbitan laurate; (polyoxyethylene nonylphenyl ether) acrylate, (polyoxyethylene octylphenyl) (Polyoxyethylene alkylphenyl ether) (meth) acrylates such as ether) methacrylate; and (polyoxyethylene alkyl ether) (meth) acrylate such as (polyoxyethylene lauryl ether) acrylate and (polyoxyethylene oleyl ether) methacrylate And other polyoxyethylene (meth) acrylic acid ester derivatives. Furthermore, for example, a copolymer composed of these polymers and at least two kinds of these monomers can be mentioned. In addition, for example, a copolymer of N-isopropylacrylamide and Nt-butylacrylamide can also be used. When a polymer containing a (meth) acrylamide derivative is used, another copolymerizable monomer may be copolymerized with the polymer within a range having a lower critical solution temperature. In the present invention, among others, Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N-acryloylmorpholine, Nn- From at least one monomer selected from the group consisting of propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine, N-methacryloylmorpholine Or a copolymer of N-isopropylacrylamide and Nt-butylacrylamide can be preferably used.

  As the polymer having the upper critical solution temperature used in the present invention, for example, a polymer comprising at least one monomer selected from the group consisting of acryloylglycinamide, acryloylnipecotamide, acryloyl asparagine amide, acryloyl glutamine amide and the like. Is mentioned. Moreover, the copolymer which consists of these at least 2 types of monomers may be sufficient. These polymers include other copolymers such as acrylamide, acetylacrylamide, biotinol acrylate, N-biotinyl-N′-methacryloyl trimethylene amide, acroyl sarcosine amide, methacryl sarcosine amide, acroyl methyl uracil, etc. Possible monomers may be copolymerized in a range having an upper critical solution temperature.

In the present invention, as the stimulus-responsive polymer, a pH-responsive polymer that can be aggregated and dispersed by pH change can be used.
Examples of such a pH-responsive polymer include a polymer containing a group such as carboxyl, phosphoric acid, sulfonyl, and amino as a functional group. More specifically, for example, acrylic acid, methacrylic acid, maleic acid, vinyl sulfonic acid, vinyl benzene sulfonic acid, phosphoryl ethyl (meth) acrylate, aminoethyl methacrylate, aminopropyl (meth) acrylamide, dimethylaminopropyl (meth) Examples include polymers containing acrylamide or a salt thereof as a copolymerization component.

  The aggregating substance preferably further contains a particulate magnetic substance in that detection accuracy can be improved by applying a magnetic force described later. Such a magnetic substance may be composed of a polyhydric alcohol and magnetite.

  The polyhydric alcohol is not particularly limited as long as it is an alcohol structure having at least two hydroxyl groups as a structural unit and capable of binding to iron ions, and examples thereof include dextran, polyvinyl alcohol, mannitol, sorbitol, and cyclodextrin. For example, Japanese Patent Application Laid-Open No. 2005-82538 discloses a method for producing a particulate magnetic material using dextran. Moreover, the compound which has an epoxy like a glycidyl methacrylate polymer and forms a polyhydric alcohol structure after ring-opening can also be used.

  The fine particle magnetic material (magnetic fine particles) prepared using such a polyhydric alcohol preferably has an average particle size of 0.9 nm or more and less than 1000 nm so as to have good dispersibility. The average particle size is preferably 2.9 nm or more and less than 200 nm, in particular, in order to increase the detection sensitivity of the target detection target.

(Bonding of aptamer and aggregating substance)
The binding method of the aptamer and the aggregating substance is not particularly limited. For example, both the aptamer side and the aggregating substance side (for example, the stimulus-responsive polymer moiety) have mutually affinity substances (for example, biotin and streptavidin or Avidin) is bound, and aptamer and aggregating substance are bound via these substances.

  Specifically, as described in International Publication No. 01/09141, the binding of biotin to the stimulus-responsive polymer is performed by adding biotin or the like to a polymerizable functional group such as methacryl or acrylic. A monomer is used and copolymerized with other monomers to immobilize streptavidin. The aptamer is labeled with biotin or the like according to a conventional method, and labeled on the 5 'side or 3' side during oligo DNA synthesis. When the biotin-labeled aptamer and the streptavidin-immobilized stimulus-responsive polymer are mixed, the aptamer and the stimulus-responsive polymer are bonded through the binding between biotin and streptavidin.

  In this specification, a material in which a stimulus-responsive polymer and a particulate magnetic substance are combined is referred to as a stimulus-responsive magnetic fine particle, and when the stimulus is temperature, it is referred to as a temperature-responsive magnetic fine particle.

  The fine magnetic substance can be bonded to the stimulus-responsive polymer via a reactive functional group, or a polymerizable unsaturated bond is introduced into the active hydrogen or polyhydric alcohol on the polyhydric alcohol in the magnetic substance. The graft polymerization may be carried out by a method known in the art such as graft polymerization (for example, ADV.Polym.Sci., Vol.4, p111, 1965, J. Polymer Sci., Part-A, 3, p1031). 1965).

  When a mixture of the aggregating substance, aptamer, and specimen as described above is placed under conditions where the stimuli-responsive polymer related to the aggregating substance aggregates, the aggregation of the stimuli-responsive polymer occurs when there is no target to be detected. Does not occur. On the other hand, when a detection target is present, aggregation inhibition of the stimulus-responsive polymer is mitigated by the charge portion or hydrophobic portion of the detection target, and aggregation occurs.

  Among these, the phenomenon in which aggregation inhibition is alleviated will be described with reference to FIGS. 1 and 2.

  As shown in FIG. 1, the stimulus-responsive magnetic fine particle 1 includes a fine particle-like magnetic substance 2, and a stimulus-responsive polymer 3 is bonded to the surface of the magnetic substance 2. Streptavidin 4 is bound to the stimulus-responsive polymer 3, and an aptamer 6 for the detection target 7 is bound via biotin 5. As a result, the detection target 7 approaches the stimulus-responsive polymer 3 via the aptamer 6, and the inhibition of aggregation of the stimulus-responsive polymer 3 is alleviated due to the influence of the positively charged portion or the hydrophobic portion of the detection target 7.

  In order to agglomerate the stimulus-responsive polymer, for example, when a temperature-responsive polymer is used, the container containing the mixture may be transferred to a thermostatic bath at a temperature at which the temperature-responsive polymer aggregates. There are two types of temperature-responsive polymers: a polymer having an upper critical solution temperature (hereinafter sometimes abbreviated as “UCST”) and a polymer having a lower critical solution temperature (hereinafter sometimes abbreviated as “LCST”). There is. For example, when a polymer having a lower critical solution temperature of LCST of 37 ° C. is used, the temperature-responsive polymer can be agglomerated by moving the container containing the mixture to a constant temperature bath of 37 ° C. or higher. When a polymer having an upper critical solution temperature with a UCST of 5 ° C. is used, the temperature-responsive polymer can be agglomerated by moving the container containing the mixture to a thermostatic bath of less than 5 ° C.

  Moreover, what is necessary is just to add an acid solution or an alkaline solution to the container containing a mixture, when using a pH-responsive polymer. Specifically, an acid solution or an alkaline solution is added to a container containing a dispersed mixture outside the pH range where the pH-responsive polymer undergoes a structural change, and the pH-responsive polymer undergoes a structural change in the container. What is necessary is just to change to the pH range to raise. For example, when a pH-responsive polymer that aggregates at a pH of 5 or less and disperses at a pH of more than 5 is used, an acid solution may be added to a container containing a mixture dispersed at a pH of more than 5 so that the pH is 5 or less. When a pH-responsive polymer that aggregates at pH 10 or higher and disperses at a pH lower than 10 is used, an alkaline solution may be added to a container containing a mixture dispersed at a pH lower than 10 so that the pH becomes 10 or higher. The pH at which the pH-responsive polymer undergoes a structural change is not particularly limited, but is preferably pH 4-10, more preferably pH 5-9.

Further, when a photoresponsive polymer is used, light having a wavelength capable of aggregating the polymer may be irradiated to a container containing the mixture. The preferred light for aggregation varies depending on the type and structure of the photoresponsive functional group contained in the photoresponsive polymer, but generally ultraviolet light or visible light having a wavelength of 190 to 800 nm can be suitably used. At this time, the strength is preferably 0.1 to 1000 mW / cm ² . In addition, it is preferable that a photoresponsive polymer is a thing which is hard to produce dispersion | distribution in other words, when it is irradiated with the light used for a turbidity measurement in the point which can improve a measurement precision. When using a photoresponsive polymer that generates dispersion when irradiated with light used for turbidity measurement, the measurement accuracy can be improved by shortening the irradiation time.

  When the aptamer binds to the stimulus-responsive polymer and this conjugate is subjected to a condition where the stimulus-responsive polymer aggregates, inhibition of aggregation occurs due to the negative charge of the aptamer. That is, when there is no detection target, dispersion is maintained [(A) of FIG. 2]. On the other hand, when the detection target exists, the inhibition of aggregation of the stimulus-responsive polymer is alleviated and the aggregation is promoted by the influence of the positively charged portion or the hydrophobic portion of the detection target [(B) of FIG. 2]. As can be seen from FIG. 2, the absorbance in (A) is higher than that in (B) at the end of the measurement, and the turbidity is increased.

  Here, the lower critical solution temperature and the upper critical solution temperature are determined as follows, for example. First, a sample is put into a cell of an absorptiometer and the sample is heated at a rate of 1 ° C./min. During this time, the change in transmittance at 550 nm is recorded. Here, when the transmittance when the polymer is transparently dissolved is 100% and when the transmittance when the polymer is completely aggregated is 0%, the temperature at which the transmittance is 50% is obtained as LCST. In the case of UCST, the sample is cooled at a rate of 1 ° C./min.

(Judgment)
The determination of the degree of aggregation, in the above example, can be performed by visual observation or turbidity measurement, for example. The turbidity can be calculated from the light transmittance of the light scattering device. If the turbidity is low, aggregation of the stimulus-responsive polymer is inhibited, which indicates the presence of the detection substance. Here, the wavelength of the light to be used may be appropriately set so as to obtain a desired detection sensitivity according to the particle size of the magnetic substance. The wavelength of light is preferably within the range of visible light in that a conventional general-purpose device can be used.

  Visual observation or turbidity measurement may be performed intermittently at a certain point in time or continuously over time. Further, the determination may be made based on the difference between the turbidity measurement value at a certain time point and the turbidity measurement value at another time point.

(Separation)
When the aggregating substance contains a particulate magnetic substance, it is preferable that the detection method of the present invention further includes separating the aggregated magnetic substance by applying a magnetic force. Thereby, the agglomerated magnetic substance is separated from impurities including the non-aggregated magnetic substance. For this reason, the measurement values such as the amount of the separated magnetic substance and the light transmittance when dispersed in the solvent exclude the influence of foreign substances and more accurately reflect the presence of the detection substance.

  The magnetic force can be applied by bringing a magnet close to the magnetic substance. The magnetic force of this magnet varies depending on the magnitude of the magnetic force of the magnetic substance used. An example of the magnet is a neodymium magnet (manufactured by Neomag).

  Further, the addition of magnetic force may be performed before the determination or in parallel with the determination. However, the simultaneous parallel is preferable in that the time spent for the process can be shortened. It should be noted that when magnetic force is applied, the aggregated magnetic substance is separated by inclusion of impurities, so the turbidity of the mixture after separation is presumed to be rather smaller when the impurities are present. .

(Detection target)
Examples of the target that can be detected by the above detection methods include substances used in clinical diagnosis. Specifically, for example, vascular endothelial growth factor (VEGF) contained in body fluid, urine, sputum, feces, saliva, etc. , Thrombin, human immunoglobulin G, human immunoglobulin M, human immunoglobulin A, human immunoglobulin E, human albumin, human fibrinogen (fibrin and degradation products thereof), α-fetoprotein (AFP), C-reactive protein (CRP) ), Myoglobin, carcinoembryonic antigen, hepatitis virus antigen, human chorionic gonadotropin (hCG), human placental lactogen (HPL), HIV virus antigen, allergen, bacterial toxin, bacterial antigen, enzyme, hormone (eg, human thyroid stimulation Hormones (TSH), insulin, etc.), drugs, etc. It is. In addition, secretory fluids collected from every part of the body are also targeted, and causative substances related to all diseases can be mentioned.

  In many cases, a sample suspected of containing these detection targets contains many types and a large amount of contaminants, but the measured magnetic field is not greatly affected by the contaminants in the detection targets. For this reason, it is not always necessary to perform a preliminary procedure of removing impurities before measurement.

(Function and effect)
According to 1st Embodiment of this invention, the following effects are obtained.
When the detection target exists in the specimen, the aptamer binds to the detection target. When the complex between the detection target and the aptamer binds to an aggregating substance having a stimulus-responsive polymer, the charge or hydrophobic portion of the detection target is placed in the vicinity of the stimulus-responsive polymer, and the aggregation inhibition of the stimulus-responsive polymer is alleviated. Is done. Therefore, the presence or absence of the detection target can be determined by observing this change in aggregation inhibition.

  The above procedure is achieved using only an aggregating substance having an aptamer and a stimulus-responsive polymer, and is performed without using any special reagent or device, so that it is inexpensive and simple. In addition, only the change in the degree of aggregation is measured, and since the reaction catalyzed by the enzyme is not used, it can be performed rapidly.

Second Embodiment
(Quantitative method)
In the quantification method of the present invention, first, an aptamer and a specimen are mixed. Next, the mixture and an aggregating substance having a stimulus-responsive polymer are mixed, and the mixture is subjected to predetermined conditions in which the stimulus-responsive polymer aggregates. Subsequently, the turbidity of the mixture is measured, and the amount of the detection target in the sample is calculated based on a correlation equation under a predetermined condition between the amount of the detection target and the turbidity. Since the procedure of the first half is similar to the detection method described above, the description is omitted.

(Correlation formula)
A correlation equation between the amount of detection target and turbidity under the same condition as the predetermined condition is created. In the measurement of the amount of detection target and the turbidity constituting the correlation formula, the more reliable the correlation formula is obtained as the amount of data increases. Therefore, the data may be related to the amount of two or more detection targets, and is preferably related to the amount of three or more detection targets.
Here, the correlation equation between the amount of detection target and turbidity is not only an equation showing a direct correlation between the amount of detection target and turbidity, but also the correlation between the amount of detection target and a parameter reflecting turbidity. It may be a formula.

(Calculation)
By substituting the turbidity measurement value of the mixture into the created correlation equation, the amount of the detection target in the sample can be calculated.
The “turbidity measurement” in the detection method or the quantitative method includes not only measuring the turbidity directly but also measuring a parameter reflecting the turbidity. Such parameters include differences in turbidity measurement values at multiple time points, the amount of separated aggregates, the turbidity of non-aggregates after separation, and the like. Here, one point of the plurality of time points is preferably a time point at which the turbidity reaches a maximum value when, for example, a magnetic force is applied to a negative control in which the detection target is absent. Thereby, the difference from the turbidity measurement value at another time point becomes large, and the amount of the detection target can be quantified more accurately.

(Function and effect)
According to 2nd Embodiment of this invention, the following effect is obtained. As in the first embodiment, since the degree of turbidity depends on the amount of the detection target, the detection target can be quantified by substituting the turbidity measurement value into the correlation equation between the detection target amount and the turbidity. . In addition, this procedure is achieved using only an aggregating substance having an aptamer and a stimulus-responsive polymer, and is performed without using any special reagent or device, so that it is inexpensive and simple. Moreover, it is possible only to measure the degree of aggregation inhibition and not to use a reaction catalyzed by an enzyme.

<Example 1>
(Preparation of thrombin sample)
Bovine α-thrombin (Thrombin; manufactured by Thermo scientific) 0.1% (w / v) BSA (manufactured by Wako Pure Chemical Industries), 10 mM Tris-HCl (pH 7.5, manufactured by Wako Pure Chemical Industries), 100 mM NaCl, Solutions containing 5 mM KCl diluted to 100 nM, 80 nM, 60 nM, 40 nM, 20 nM, and 10 nM were prepared as thrombin samples, respectively.

(Preparation of thrombin-binding DNA aptamer)
An oligonucleotide having biotin at the 5 ′ end (SEQ ID NO: 1) was diluted to 1 μM with a solution containing 10 mM Tris-HCl (pH 7.5, manufactured by Wako Pure Chemical Industries), 100 mM NaCl, 5 mM KCl, A temperature cycler (DNA Engine, manufactured by Bio-Rad) was used to incubate at 95 ° C. for 10 minutes, and then a temperature of 25 ° C. over 30 minutes was prepared as a thrombin-binding DNA aptamer.

(SEQ ID NO: 1)
5′-agtccgtggttagggcagggtggggtgactttttttggtttgtgtgtggtttgg-3 ′

(Sample mixing)
100 μL of the thrombin sample and 10 μL of the thrombin-binding DNA aptamer were placed in a 1.5 mL microtube and mixed by pipetting, and incubated at 37 ° C. for 5 minutes.

(Mixing of sample and magnetic fine particles)
To the above mixed sample, 100 μL of Thermo-Max (trade name) LSA Streptavidin (0.02 mass%) manufactured by JNC Petrochemical Co., Ltd., which is magnetic fine particles in which streptavidin and a temperature-responsive polymer are bonded, is mixed by pipetting. And kept at 21 ° C. for 5 minutes.

(Sample measurement)
As shown in FIG. 3, a 10 mm × 5 mm × 3 mm neodymium permanent magnet B (manufactured by Neomag) was attached to the cell adapter A attached to a commercially available microcell for spectrophotometer (UVette, manufactured by Eppendorf). . The microcell C was put into this cell adapter, installed in an ultraviolet-visible spectrophotometer V-660DS (manufactured by JASCO Corporation) equipped with a cell temperature controller, and kept at 37 ° C. for 10 minutes or more.

  200 μL of the mixed solution of the above sample and magnetic fine particles was dispensed into a microcell, and the absorbance was immediately measured continuously for 600 seconds at a wavelength of 420 nm and a bandwidth of 2.0 nm. FIG. 4 shows the measurement results of the magnetic fine particles, magnetic fine particle-aptamer complex, and magnetic fine particle-aptamer-thrombin (20 nM, 60 nM, 100 nM) complex.

  As shown in FIG. 4, the absorbance increased until about 200 seconds after the start of measurement, and thereafter the absorbance decreased. This is presumably because the turbidity increases due to aggregation of the stimulus-responsive polymer of the magnetic fine particles, and then the magnetic fine particles are adsorbed and separated by the magnet.

  It was also found that the absorbance after 600 seconds after the start of measurement was higher for the magnetic fine particle-aptamer complex than for the magnetic fine particles alone. This is presumed to be because the stimulus-responsive polymer was dispersed due to aggregation inhibition by the aptamer and became difficult to adsorb to the magnet.

  Furthermore, it was found that the absorbance after 600 seconds after the start of measurement decreases as the thrombin concentration increases. This is presumed to be because thrombin bound to the complex of magnetic fine particles and aptamer alleviated aggregation inhibition by the aptamer.

  Next, for each sample, the difference between the measured values at two points immediately after the start and after 600 seconds was calculated. The results are shown in Table 1.

  As shown in Table 1, the difference between the measured values at the two points immediately after the start and after 600 seconds was dependent on the amount of thrombin. That is, the higher the thrombin concentration, the greater the difference between the measured values at the two points immediately after the start and after 600 seconds. Thus, it was found that thrombin can be detected or quantified with high sensitivity by measuring the difference between the measured values at two points immediately after the start and after 600 seconds.

  Further, the detection time of 600 seconds or less after the start is completed within 15 minutes including the incubation time, and is much shorter than conventional techniques such as ELISA that required several hours. In addition, the procedure is simple because the detection target can be detected or quantified simply by performing the reaction between the aptamer and the sample, mixing the temperature-responsive magnetic fine particles, and measuring the turbidity.

<Example 2>
(Preparation of VEGF sample)
VEGF (manufactured by R & D) was diluted with 50 mM acetic acid, 100 mM NaCl, 5 mM KCl (pH 4.5) to 0, 10, 100, 495 fM. At that time, human serum was added to a final concentration of 10% to prepare a VEGF sample.

(Preparation of VEGF-binding DNA aptamer)
An oligonucleotide having biotin at the 5 ′ end (SEQ ID NO: 2) was diluted with 50 mM acetic acid, 100 mM NaCl, 5 mM KCl (pH 4.5) to 1 μM, heated at 95 ° C. for 10 minutes, and then over 30 minutes. Cooled slowly to 25 ° C. This was diluted with 50 mM acetic acid, 100 mM NaCl, 5 mM KCl (pH 4.5) to 75 nM to obtain a VEGF-binding DNA aptamer.

(SEQ ID NO: 2)
5′-tgtgggggggtggacggggccggggtag-3 ′

(Sample mixing)
50 μL of the sample and 50 μL of the VEGF-binding DNA aptamer were put in a 1.5 mL microtube, and shaken at 20 ° C., 1,200 rpm for 10 minutes using a thermomixer.

(Mixing of sample and magnetic fine particles)
Thermo-Max (trade name) LSA manufactured by JNC Petrochemical Co., Ltd., which is magnetic fine particles in which streptavidin diluted with 50 mM acetic acid, 100 mM NaCl, 5 mM KCl (pH 4.5) and a temperature-responsive polymer are bound to the above mixed sample. 50 μL of Streptavidn (0.02 mass%) was added, and shaken at 20 ° C., 1,200 rpm for 10 seconds using a thermomixer.

(Sample measurement)
As shown in FIG. 3, a 10 mm × 5 mm × 3 mm neodymium permanent magnet B (manufactured by Neomag) was attached to the cell adapter A attached to a commercially available microcell for spectrophotometer (UVette, manufactured by Eppendorf). . The microcell C was put into this cell adapter, installed in an ultraviolet-visible spectrophotometer V-660DS (manufactured by JASCO Corporation) equipped with a cell temperature controller, and kept at 37 ° C. for 10 minutes or more.

  150 μL of the mixed solution of the above sample and magnetic fine particles was dispensed into the microcell, and the absorbance was immediately measured continuously for 600 seconds at a wavelength of 420 nm and a bandwidth of 2.0 nm. The measurement results of the magnetic fine particle-aptamer complex and the magnetic fine particle-aptamer-VEGF complex are shown in FIG. The numerical value beside the graph in FIG. 5 indicates the VEGF concentration.

  As shown in FIG. 5, it was found that the absorbance after 300 seconds from the start of the measurement decreases as the VEGF concentration increases. This is presumed to be because aggregation inhibition by the aptamer was alleviated by binding of VEGF to the complex of the magnetic fine particles and the aptamer.

  Next, for each sample, the difference between the measured values at two points immediately after the start and after 600 seconds was calculated. The results are shown in Table 2.

  As shown in Table 2, the difference between the measured values at the two points immediately after the start and after 600 seconds was dependent on the amount of VEGF. That is, the higher the VEGF concentration, the greater the difference between the measured values at the two points immediately after the start and after 600 seconds. Thus, it was found that VEGF can be detected or quantified with high sensitivity by measuring the difference between the measured values at two points immediately after the start and after 600 seconds.

<Example 3>
SARS Coronavirus Nucleocapsid Protein (manufactured by RayBiotech) was detected in the same procedure as in Example 1.

  As a SARS Coronavirus Nucleocapsid Protein binding aptamer, an oligonucleotide having biotin at the 5 'end (SEQ ID NO: 3) was used.

(SEQ ID NO: 3)
5'- gcaatggtacggtacttccggatgcgaaactggctaattggtgaggctggggcgtcgtgcagaaaagtgcacgctactttgcta-3 '

  Table 3 shows the difference in measured values at two points immediately after the start and after 600 seconds at the SARS Coronavirus Nucleocapsid Protein sample concentrations of 0 nM, 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, 100 nM.

  As shown in Table 3, the difference in the measured values at the two points immediately after the start and after 600 seconds was dependent on the amount of SARS Coronavirus Nucleocapsid Protein. That is, the higher the SARS Coronavirus Nucleocapsid Protein concentration, the smaller the difference between the measured values at two points immediately after the start and after 600 seconds. Thus, it was found that the SARS Coronavirus Nucleocapsid Protein can be detected or quantified with high sensitivity by measuring the difference between the measured values at two points immediately after the start and after 600 seconds.

<Example 4>
Influenza virus hemagglutinin (H5N1-HA, A / Anhui / 1/2005, manufactured by Abnova) was detected in the same procedure as in Example 1.

  As an Influenza virus hemagglutinin-binding aptamer, an oligonucleotide (SEQ ID NO: 4) having biotin at the 5 'end was used.

(SEQ ID NO: 4)
5'-gaattcagtcggacagcgggggtcccatgcgatgtttaataagcagtcgcttataagggaggagatatatcgtccc-4 '

  Table 4 shows the difference between the measured values at two points immediately after the start and 600 seconds after the Influenza virus hemagglutinin sample concentrations of 0 nM and 100 nM.

  As shown in Table 4, the measured values 600 seconds after the start of measurement showed a clear difference between the sample concentrations of 0 nM and 100 nM, and the aggregation of magnetic fine particles was observed in the presence of Influenza virus hemagglutinin. Thus, it was found that Influenza virus hemagglutinin can be detected.

<Comparative Example 1>
In the same procedure as in Example 1, VEGF (Human Recombinant VEGF A165: manufactured by Human Zyme) was detected using an antibody instead of an aptamer.

  Anti-VEGF antibody (Biotin) (manufactured by abcam) was used as a VEGF binding antibody. Anti-VEGF antibody (Biotin) was used after diluting to 1 μM with a solution containing 10 mM Tris-HCl (pH 7.5, manufactured by Wako Pure Chemical Industries), 100 mM NaCl, 5 mM KCl.

  Table 5 shows the difference in measured values at two points immediately after the start and 600 seconds after the VEGF sample concentrations of 0 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM.

  As shown in Table 5, the difference between the measured values at the two points immediately after the start and 600 seconds later was dependent on the amount of VEGF. That is, the higher the VEGF concentration, the greater the difference between the measured values at the two points immediately after the start and after 600 seconds. Thus, it was found that VEGF can be detected or quantified by measuring the difference between the measured values at two points immediately after the start and after 600 seconds.

  On the other hand, the difference in the measured value of VEGF 0.1 nM was smaller than the difference in the measured value of VEGF 0 nM. From this, the measurement sensitivity in this detection system is considered to be 0.1 nM or more.

  Thereby, it was confirmed that Example 2 which was able to detect even 10 fM using the VEGF binding DNA aptamer has remarkably higher detection sensitivity than Comparative Example 1 using the antibody.

  From the above results, it was shown that the turbidity changes according to the concentration of the detection target, and that the concentration of the detection target can be quantified by measuring the turbidity. That is, the method according to the present invention is a novel method that can detect and quantify a detection target quickly, inexpensively, and easily without requiring special reagents and equipment such as secondary antibodies, luminescent reagents, and luminescence detection devices. Was confirmed.

  The present invention is not limited to the above-described embodiment, and modifications, improvements, and the like within the scope that can achieve the object of the present invention are included in the present invention.

DESCRIPTION OF SYMBOLS 1 Stimulus responsive magnetic microparticle 2 Magnetic substance 3 Stimulus responsive polymer 4 Streptavidin 5 Biotin 6 Aptamer 7 Detection object A Cell adapter B Neodymium permanent magnet C Microcell

Claims (9)

  A method for detecting a detection target in a specimen, wherein the specimen, an aptamer that can bind to the detection target, and an aggregating substance containing a stimulus-responsive polymer are mixed, and the resulting mixture is used as the stimulus response. When the degree of aggregation of the aggregating substance containing the stimulus-responsive polymer is determined under the condition that the agglutinable polymer aggregates, and the degree of aggregation changes compared to the absence of the specimen, A method for detecting a detection target, including a procedure for determining that a detection target exists in a specimen.   The method according to claim 1, wherein the aptamer capable of binding to the detection target is DNA, RNA, or a peptide.   The method according to claim 1, wherein the aggregating substance further contains a fine particle magnetic substance, and further includes separating the agglomerated magnetic substance by applying a magnetic force.   The method according to any one of claims 1 to 3, wherein the change in the degree of aggregation is a change in which aggregation inhibition of the stimulus-responsive polymer is alleviated.   The method according to claim 1, wherein the detection target is VEGF.   The method according to claim 1, wherein the detection target is thrombin.   A method for quantifying a detection target in a sample, wherein the sample, an aptamer capable of binding to the detection target, and an aggregating substance containing a stimulus-responsive polymer are mixed, and the mixture is used as the stimulus-responsive polymer. Measuring the turbidity of the mixture, and calculating the amount of the detection target in the sample based on the correlation equation between the amount of the detection target and the turbidity under the condition Including a method for quantifying a specimen.   The method according to claim 7, further comprising separating the aggregated magnetic substance by applying a magnetic force, the aggregating substance further containing a particulate magnetic substance.   A kit for detecting and / or quantifying a detection target, comprising an aptamer capable of binding to the detection target and an aggregating substance containing a stimulus-responsive polymer.