JP2015055568A - Biomolecule analysis method and biomolecule analyzer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To achieve a biomolecule analysis by a digital-count system with easy operation without using a magnetic field.SOLUTION: A biomolecule analysis method includes: fixing a composite which is made of a support member to be detected, a fixing reaction molecule, and analysis object biomolecule on a supporting substrate through a tie molecule; optically detecting the composite fixed on the supporting substrate based on the support member to be detected; and counting the composite fixed on the supporting substrate based on this.

Description

  The present invention relates to a biomolecule analysis method and a biomolecule analyzer that measure a biomolecule at a single molecule level as an analysis target.

  There is an increasing need to analyze a very small amount of biomolecules contained in a sample with high accuracy. For example, in recent years, in the field of cancer diagnosis, various cancer markers have been studied and put into practical use in order to know signs of cancer onset early. A cancer marker is a secreted biological factor derived from cancer cells, and increases in the progression of cancer and appears in blood and urine. For example, proteins such as hormones and cytokines, and nucleic acids such as microRNA are known as cancer markers. Early cancers are difficult to detect because the amount of these cancer markers is small. Further, in order to realize postoperative progress judgment and prognosis prediction, it is required to detect a minute amount change of a very small amount of cancer marker, and realization of a highly sensitive detection technique is required.

When detecting a cancer marker in blood, the amount of blood that can be collected from a patient is limited, and it is required to capture and detect as much of a very small amount of cancer marker as possible. For example, when detecting from 50 μl of plasma, cancer markers are in the concentration range of 10 ^-16 to 10 ^-12 M in early cancer, so detection sensitivity to quantify about 3000 target molecules present in 50 μl Is required. Thus, there is a need for an ultrasensitive detection technique that can detect a very small amount of cancer marker having only a few thousand molecules in 50 μl with high quantitativeness. However, currently, the most sensitive cancer marker detection methods that are the mainstream are known immunoassay methods using antibodies, such as ELISA and nanoparticle assay, but the detection sensitivity is at the pM level, as described above. It cannot be applied to detection of a very small amount of cancer marker.

  A conventional analysis method employs a method for obtaining a physical property value proportional to the total number of molecules. For example, a method is a method in which a biomolecule to be analyzed is fluorescently labeled, a fluorescence intensity generated from a whole predetermined amount of sample is measured, and a biomolecule contained in the sample is quantified based on the fluorescence intensity. In such an analysis method, there is no analysis object, so-called background intensity and fluctuation cannot be ignored in a low concentration region of 1 pM or less, the problem that the measurement itself is difficult cannot be avoided, and a large bottleneck for high sensitivity It was. On the other hand, a so-called digital counting method has been developed that counts biomolecules to be analyzed at a single molecule level. With the digital counting method, it is not necessary to accurately measure individual signal strengths, it is only necessary to count the number of signal bright spots exceeding a predetermined threshold, and background intensity and fluctuations can be ignored even in low concentration regions. It is. Therefore, the digital count method is promising as a high sensitivity detection technique. Recently, a digital ELISA has been proposed as an immunoassay method using a digital count method (Non-patent Documents 1-2 and 1).

  The procedures for capturing and labeling the antigen used in Non-Patent Document 1 and Patent Document 1 are as follows. First, a biomolecule to be analyzed is captured by reacting with a magnetic bead with a monoclonal antibody using the biomolecule as an antigen. Next, a sandwich reaction is performed with a biotin-modified polyclonal antibody, and the antigen is labeled with biotin. Furthermore, after binding Streptavidin β-Galactosidase Conjugate to this biotin, a chromogenic substrate is introduced to detect a biomolecule. In this method, in order to prevent mixing of reagents, magnetic beads capturing biomolecules are inserted one by one into isolated wells, and each well is sealed after introducing a coloring solution.

  The procedure of antigen capture and labeling used in Patent Document 2 is as follows. After binding the magnetic beads and the biomolecule to be analyzed in the solution, the magnetic beads are collected on the substrate using an external magnetic field, and then the magnetic beads are fixed on the substrate through the biomolecule to be analyzed. To do. Magnetic beads that have not captured the biomolecule to be analyzed are removed from the substrate by a cleaning process. After washing, the amount of magnetic beads fixed on the substrate is obtained by magnetic resonance or engineering techniques, and information on the amount of molecules to be analyzed can be obtained from this amount.

US Patent No. 8,236,574 US Publication No. US 2012/0062219

Rissin DM et al, Nature Biotecnology, Jun 28 (6); p595-599 (2010) Rissin DM et al, Anal. Chem, 83; p2279-2285 (2011)

  In the methods disclosed in Non-Patent Document 1 and Patent Document 1, it is necessary to seal magnetic wells after inserting magnetic beads capturing biomolecules into isolated wells and introducing a coloring solution. As described above, the methods disclosed in Non-Patent Document 1 and Patent Document 1 are complicated in operation, and there are problems that the time required for analysis (turn around time: TAT) becomes long and the cost of consumables is high. . On the other hand, it can be said that the method disclosed in Patent Document 2 is simple because it has fewer steps than the methods disclosed in Non-Patent Document 1 and Patent Document 1. However, in the method disclosed in Patent Document 2, magnetic beads are collected on the substrate surface by an external magnetic field, and the magnetic beads are aggregated (stacked) along the magnetic field lines and fixed on the substrate. In this case, the magnetic beads fixed on the substrate cannot be accurately measured from a scattered image using a microscope. That is, the method disclosed in Patent Document 2 has a problem that it is very difficult to apply to the digital count method described above. Furthermore, the method disclosed in Patent Document 2 has a problem that it is difficult to reduce the size of the analyzer because magnets must be arranged near the substrate.

  Therefore, in view of the circumstances described above, an object of the present invention is to provide a biomolecule analysis method and a biomolecule analyzer that can perform analysis by a digital counting method with a simple operation and without using a magnetic field. .

The biomolecule analysis method according to the present invention that achieves the above-described object includes a detection support having a first capture molecule that selectively captures an analysis target biomolecule, a support substrate having a first binding molecule, Using the immobilized reaction molecule having a second capture molecule that selectively captures the biomolecule to be analyzed and a second binding molecule that binds to the first binding molecule on the surface of the support substrate, Contacting a support, the immobilized reaction molecule and the biomolecule to be analyzed;
Immobilizing a complex composed of the detected support, the immobilized reaction molecule, and the analysis target biomolecule to the support substrate via the first binding molecule and the second binding molecule;
A step of optically detecting a complex fixed on the support substrate based on the support to be detected, and counting the number of complexes fixed on the support substrate based on the detection;

  In the biomolecule analysis method according to the present invention, the first capture molecule and the second capture molecule can be antibodies having different regions of the analysis target biomolecule as epitopes.

  In the biomolecule analysis method according to the present invention, the first binding molecule and the second binding molecule can be avidin and biotin or biotin and avidin, respectively. In the biomolecule analysis method according to the present invention, the second binding molecule may be a nucleic acid molecule having a base sequence that specifically binds to the first binding molecule. In this case, the second binding molecule may be a nucleic acid molecule having a plurality of polynucleotide units each having a base sequence that specifically binds to the first binding molecule.

  In the biomolecule analysis method according to the present invention, the support to be detected can be a fine particle. In particular, the support to be detected is preferably a non-magnetic fine particle. Further, the support to be detected is preferably a fine particle having a specific gravity of 10 or more and an average particle diameter of 40 to 300 nm. More specifically, the detected support can be fine particles containing an element selected from the group consisting of gold, platinum and tungsten.

Moreover, the biomolecule analyzer according to the present invention includes:
A support substrate having a first binding molecule;
Analysis target biomolecule, detection target support having first capture molecule for selectively capturing the analysis target biomolecule, second capture molecule for selectively capturing the analysis target biomolecule, and the support substrate A reaction vessel in contact with an immobilized reaction molecule having a second binding molecule that binds to the first binding molecule on the surface;
A reaction solution supply device for supplying a reaction solution containing a complex composed of the support to be detected, the immobilized reaction molecule, and the biomolecule to be analyzed from the reaction container onto the support substrate;
And a detection device that optically detects the complex fixed on the support substrate based on the detection target support and counts the complex fixed on the support substrate based on the detection target.

  In the biomolecule analyzer according to the present invention, the detection device includes an imaging device that acquires at least one optical image selected from fluorescence, photoluminescence, and scattered light, and detects the detected support by the imaging device. be able to.

  In the biomolecule analysis method and the biomolecule analyzer according to the present invention, a complex containing a biomolecule to be analyzed is immobilized on a support substrate without using an external magnetic field, and the detected support in the immobilized complex is optically detected. Detect. Thereby, according to the biomolecule analysis method and the biomolecule analyzer according to the present invention, the biomolecules to be analyzed can be counted at a single molecule level.

  According to the present invention, even if a sample contains a very small amount of the biomolecule to be analyzed, the biomolecule to be analyzed can be analyzed with high accuracy. In particular, by applying the present invention, an extremely small amount of a biomolecule to be analyzed can be quantitatively analyzed without requiring a special substrate or a complicated process.

The figure for demonstrating an example of the biomolecule analysis method of a present Example. The figure for demonstrating an example of the biomolecule analysis method of a present Example. The figure for demonstrating an example of a structure of the biomolecule analyzer of a present Example. The figure for showing an example of the result obtained with the biomolecule analyzer of a present Example. FIG. 4 is a characteristic diagram showing the relationship between the PSA concentration (horizontal axis) in a sample and the average number of fine gold particles per field (vertical axis).

  Hereinafter, a biomolecule analysis method and a biomolecule analyzer according to the present invention will be described in detail with reference to the drawings.

  In the biomolecule analysis method and the biomolecule analysis apparatus, biomolecules are analyzed. The biomolecules to be analyzed are not particularly limited, and are meant to include all substances produced by organisms such as proteins, peptides, amino acids, nucleic acids, low molecular compounds, sugar chains, lipids, vitamins, hormones and the like.

In the biomolecule analysis method and biomolecule analyzer, it is particularly preferable to analyze proteins, peptides and nucleic acids. Among them, the biomolecule to be analyzed is preferably a biomolecule that increases with the progress of a disease such as cancer, that is, a disease marker to be detected in a clinical test. More specifically, the tumor marker can be a biomolecule to be analyzed. The tumor marker is not particularly limited. For example, AFP (α-fetoprotein), AFP-L3% (AFP lectin fraction), BFP (basic fetoprotein), CEA, BCA225, CA 15-3, CA 19-9 , CA 50, CA 54/61 (CA546 ), CA 72-4, CA 125, CA 130, CA 602, CSLEX ( sialyl Le ^x antigen), DUPAN-2 (pancreatic carcinoma associated glycoprotein antigen), KMO-1, NCC -ST-439, SLX (sialyl Le ^x -i antigen), SPan-1, STN (sialyl Tn antigen), CYFRA (cytokeratin 19 fragment), SCC antigen (squamous cell carcinoma associated antigen), TPA (tissue polypeptide antigen) ), IAP (immunosuppressive acidic protein), ICTP (type I collagen C-telopeptide), CTx (type I collagen cross-linked C-telopeptide), urinary BTA (bladder tumor antigen) urinary NMP22 (nuclear matrix protein 22) , PIVKA-II, PSA (prostate specific antigen), SP1 (pregnancy specific protein), γ-Sm (γ-seminoprotein), ferritin, hCG (human chorionic) Nadtropin), ProGRP (gastrin-releasing peptide precursor), catecholamine, HVA (homovanillic acid), VMA (vanillylmandelic acid), calcitonin (CT), ALP (alkaline phosphatase), PL-ALP (placental ALP), GAT (cancer-related) Galactose transferase), LDH (lactate dehydrogenase), NSE (nerve specific enolase), PAP (prostatic acid phosphatase), pepsinogen (PG) I / II ratio and erbB-2.

These biomolecules to be analyzed are contained in blood, serum, urine, milk, saliva, tissue pieces, cultured cells, and the like. Therefore, in the biomolecule analysis method and the biomolecule analyzer, blood, serum, urine, milk, saliva, tissue pieces, cultured cells, and the like are used as samples depending on the biomolecule to be analyzed. The concentration of the biomolecule to be analyzed in the sample is not particularly limited, but may be 10 ⁻¹⁶ to 10 ⁻¹² M, which is the concentration range of the tumor marker in early cancer. The present biomolecule analysis method and biomolecule analyzer can analyze a biomolecule in such a very small concentration range with high accuracy.

  In this biomolecule analysis method and biomolecule analysis apparatus, a complex containing the above-described biomolecule to be analyzed is formed, this complex is fixed to the surface of the support substrate, and the fixed complex is optically detected. . Here, the complex is composed of a detected support that specifically binds to the biomolecule to be analyzed, a fixed reaction molecule that specifically binds to the biomolecule to be analyzed, and a biomolecule to be analyzed. The The to-be-detected support is a substance to be detected when optically detecting the complex, that is, a substance that is optically detected. An immobilization reaction molecule is a substance having a function of immobilizing a complex on a support substrate.

  An embodiment includes a step of forming a complex composed of a support to be detected, a biomolecule and an immobilized reaction molecule, a step of fixing the formed complex to a support substrate, and an image obtained by imaging the support substrate on which the complex is fixed. As shown in FIG.

  In the example shown in FIG. 1, the support to be detected is composed of fine particles 101 and first capture molecules 102 fixed to the surfaces of the fine particles 101. The first capture molecule 102 is a molecule that specifically captures the analysis target biomolecule 103. For example, an antibody having a predetermined region of the analysis target biomolecule 103 as an epitope can be used. Here, the specific capture of the analysis target biomolecule 103 means that when the first capture molecule 102 is an antibody, the binding constant (Ka) for the analysis target biomolecule 103 is significantly higher than the binding constant for other substances. Means that. As the first capture molecule 102, an antibody or a nucleic acid molecule can be used in accordance with the biomolecule 103 to be analyzed.

  As the fine particles 101, any of fine particles such as metal, silica and polymer, and magnetic fine particles containing ferrite can be used. In particular, it is preferable to use nonmagnetic fine particles as the fine particles 101. The specific gravity and average particle diameter of the fine particles 101 are not particularly limited, but it is preferable that the specific gravity is 10 or more and the average particle diameter is 40 to 300 nm.

  As the material of the fine particles 101, a material that can be easily modified is preferable. For example, if the fine particle 101 made of gold or platinum is used, the thiol group is selectively fixed on the surface, so that the surface can be easily modified, and the first capture molecule 102 is bound to the surface of the fine particle 101. Can be made. For example, when fine particles 101 having aminothiol immobilized on the surface of fine particles made of gold or platinum are used and the first capture molecule 102 is an antibody, the carboxyl group of the antibody and the amino group of the aminothiol are substituted with 1-ethyl-3- It can be easily coupled by a coupling reaction using (3-dimethylaminopropyl) carbodiimide hydrochloride. When tungsten is used as the material of the fine particles 101, phosphoric acid selectively binds to the oxide film on the surface, so that the surface can be modified with a phosphoric acid compound. For example, by using an aminophosphate compound, an antibody can be immobilized as the first capture molecule 102 as in the case of gold or platinum. In this case, the microparticle 101 can be a secondary antibody, protein A or protein G modified microparticle that can be bound while maintaining the activity of the antibody. These fine particles 101 are commercially available and can be easily obtained. For example, when gold fine particles 101 are used, Anti-Mouse IgG (H + L) Antibody, Gold labeled from KPL can be used. By mixing and incubating a primary antibody of about 10 times or more to Anti-Mouse IgG-modified microparticles, microparticles 101 with antibodies immobilized as first capture molecules 102 can be obtained.

  The immobilized reaction molecule 104 is composed of a second capture molecule 105 and a second binding molecule 106. The second capture molecule 105 and the second binding molecule 106 may be directly bonded, but may be bonded through, for example, a hydrocarbon chain having 5 to 20 carbon atoms. An example of the hydrocarbon chain is an alkyl chain, but it may have a branched side chain or a predetermined functional group in the side chain. Here, the second capture molecule 105 is a molecule that specifically captures the analysis target biomolecule 103. Therefore, for the second capture molecule 105, an antibody or a nucleic acid molecule can be used in accordance with the analysis target biomolecule 103, similarly to the first capture molecule 102 described above. However, the first capture molecule 102 and the second capture molecule 105 specifically capture different regions of the biomolecule 103 to be analyzed. That is, for example, when the first capture molecule 102 and the second capture molecule 105 are antibodies, the first capture molecule 102 and the second capture molecule 105 have different regions in the analysis target biomolecule 103 as epitopes, respectively. .

  When the analysis target biomolecule 103 is a nucleic acid molecule, it is preferable to use a nucleic acid as the first capture molecule 102 and the second capture molecule 105. However, when designing the base sequence, the melting temperature, the reaction Needless to say, it is necessary to consider the buffer and reaction temperature.

  In the example shown in FIG. 1, the complex 107 can be formed by bringing a detection support composed of the fine particles 101 and the first capture molecules 102 into contact with the analysis target biomolecule 103 and the immobilized reaction molecule 104. More specifically, the complex 107 can be formed by mixing a sample containing the biomolecule 103 to be analyzed and a solution containing the detection target support and / or the immobilized reaction molecule 104. Here, as described above, the sample containing the biomolecule 103 to be analyzed is a biological sample itself such as blood, serum, urine, milk, saliva, tissue fragment, and cultured cells, dilution, concentration, centrifugation, crushing, and the like. The treatment sample obtained by applying various treatments to a biological sample is meant.

  During the reaction, it is preferable to stir the reaction solution, for example, by rotating a container containing the reaction solution. The reaction time for forming the complex 107 is not particularly limited, but is preferably 30 minutes or less, more preferably 15 minutes or less. By setting the reaction time within this range, it can be used for a normal clinical laboratory test. The reaction time can be appropriately adjusted depending on the concentration of the support to be detected and / or the concentration of the immobilized reaction molecule 104 in the reaction solution.

At this time, it is preferable that only one biomolecule 103 to be analyzed is captured by the microparticles 101. Therefore, it is preferable that the support to be detected composed of the fine particles 101 and the first capture molecules 102 is more than the analysis target biomolecules 103 in the reaction solution, and is 10 times or more than the number of the analysis target biomolecules 103 in the reaction solution. It is more preferable to put the number of the above into the reaction solution. For example, if the volume of the sample solution is 50 μl and the concentration of the target biomolecule 103 to be detected is 1 pM, the number of detected supports to be used is at least 3 × 10 ⁷ or more, more preferably 3 × 10 ^8. It is preferable to use the above. By setting the number of the fine particles 101 within this range, it is possible to prevent a plurality of analysis target biomolecules 103 from binding to one fine particle 101.

  As shown in FIG. 1, the complex 107 formed as described above can be fixed to the support substrate 109 via the fixed reaction molecule 104. As the support substrate 109, a substrate material excellent in smoothness such as a thin quartz glass substrate or a silicon substrate is preferable in optical measurement. The support substrate 109 has a first binding molecule 108 that fixes the fixed reaction molecule 104 by binding to the second binding molecule 106 in the fixed reaction molecule 104. Here, for the first binding molecule 108 and the second binding molecule 106, for example, avidin and biotin can be used. For example, when a thin quartz glass substrate or silicon substrate is used as the support substrate 109, the first binding molecule can be easily formed on the surface of the support substrate 109 by using a silane compound having biotin as a functional group at the terminal. Biotin can be introduced as 108. For example, Silane-PEG-Biotin, MW 5000 manufactured by Nanocs Inc. can be used. When avidin is used as the first binding molecule 108, it can be easily realized by adding excess avidin after introducing biotin into the surface of the support substrate 109. Since the second binding molecule 106 binds to the second capture molecule 105 to form the immobilized reaction molecule 104, the second binding molecule 106 is not preferably a very large molecule. Therefore, a combination using biotin as the second binding molecule 106 and avidin as the first binding molecule 108 is more preferable. When an antibody is used as the second capture molecule 105, a commercially available biotinylated antibody can be used as the immobilization reaction molecule 104.

  After forming the complex 107 as described above, a reaction solution containing the complex 107 is placed on the support substrate 109 and allowed to react for a predetermined time. In order to promote the reaction, it is preferable that air is intermittently blown to the reaction solution on the support substrate 109 and stirred. For the purpose of preventing drying, a method in which the support substrate 109 and the reaction solution are sealed in the flow chamber and reacted is also preferable. In that case, it is also preferable from the viewpoint of promoting the reaction to gently move the reaction solution in the flow chamber for the purpose of stirring. For example, a method of applying an oscillating wave from the outside or a method of heating the mixture with a heater to cause convection in the reaction solution and stirring the solution can be used.

  After the reaction for a predetermined time, other components other than the unfixed fine particles 101, the fixed reaction molecules 104, and the complex 107 are removed from the support substrate 109 by a cleaning process. The cleaning step can be easily performed, for example, by gently flowing a cleaning solution into the flow chamber.

  Note that a general buffer solution such as a phosphate buffer can be used as the reaction solution for forming the complex 107 and the cleaning solution used after the complex 107 is fixed. Furthermore, a so-called blocking agent such as BSA can be added to the reaction solution for forming the complex 107 in order to reduce non-specific adsorption.

  Through the above steps, the complex 107 including the analysis target biomolecule 103 can be fixed to the surface of the support substrate 109. At this time, the composite 107 is fixed to the surface of the support substrate 109 by its own weight without being affected by an external magnetic field. Therefore, the composite 107 can be dispersed and fixed on the surface of the support substrate 109 without overlapping.

  Next, the detected support is optically detected for the complex 107 fixed to the support substrate 109. That is, by acquiring an image of the surface of the support substrate 109 to which the complex 107 is fixed, the fine particles 101 contained in the complex 107 can be optically detected. Note that at this time, it is preferable to perform the image acquisition step in a state where the support substrate 109 on which the complex 107 is fixed is sealed together with the cleaning liquid in a reaction container such as a flow chamber, in order to obtain a clear image.

  At this time, as a method for acquiring an image, acquiring a scattered image is preferable because it is simple. A method in which a fluorescent dye is previously contained in the fine particles 101 to acquire a fluorescent image is also preferable. When gold or platinum fine particles are used as the fine particles 101, photoluminescence from the fine particles is observed from 600 nm to 800 nm, and it is also preferable to obtain a fine particle image by detecting this. In order to acquire an image of the entire surface of the support substrate 109, it is preferable to acquire the image by installing the support substrate 109 on a movable stage so that the entire surface of the support substrate 109 can be scanned.

  Next, the complex 107 fixed to the support substrate 109 is counted from the obtained image. As described above, the fine particles 101 can be optically detected from the obtained image. Therefore, the signal intensity and background based on the fine particles 101 are measured in advance, and a threshold value at which the presence of the fine particles 101 is recognized is set in advance. In the obtained image, the complex 107 fixed to the support substrate 109 can be counted by counting spots showing signal intensity exceeding a preset threshold value. For such numerical image processing, simple image processing software such as Image-J can be used.

  At this time, in the method described above, a plurality of biomolecules 103 to be analyzed can be analyzed simultaneously by using a plurality of different types of microparticles 101. For example, if a plurality of types of microparticles 101 that generate different fluorescence wavelengths are used for a plurality of biomolecules 103 to be analyzed, a plurality of spots that can be distinguished based on the fluorescence wavelength can be detected from the obtained image (fluorescence image). Thereby, a plurality of biomolecules 103 to be analyzed can be analyzed simultaneously by the series of steps as described above.

  As described above, the biomolecule 103 to be analyzed can be analyzed. As described above, according to the present biomolecule analysis method, even a very small amount of the biomolecule 103 to be analyzed contained in a predetermined sample can be quantitatively analyzed. That is, unlike the method in which the fluorescence intensity generated from the entire predetermined sample is measured and the biomolecules contained in the sample are quantified based on the measured fluorescence intensity, this biomolecule analysis method uses a single molecule of the target biomolecule 103. Can be detected by level.

  In this biomolecule analysis method, a calibration curve is prepared in advance by measuring a plurality of samples with known concentrations of the biomolecule 103 to be analyzed as described above. By comparing the measurement result of the unknown sample with the calibration curve, the concentration of the biomolecule 103 to be analyzed in the concentration unknown sample can be determined.

  In particular, in the above-described biomolecule analysis method, when the fine particles 101 having a specific gravity of 10 or more and an average particle diameter of 40 to 300 nm are used, the time required for the complex 107 to settle on the support substrate 109 with its own weight is reduced. It can be 10 minutes or less. Therefore, when the specific gravity and the average particle size of the fine particles 101 are 10 or more and the average particle size is 40 to 300 nm, the entire analysis time can be 30 minutes or less and can be applied to a clinical test apparatus. .

  By the way, the biomolecule analysis method and the biomolecule analysis apparatus are not limited to the embodiment shown in FIG. 1, and may be other embodiments shown in FIG. In the embodiment shown in FIG. 2, a molecule having a plurality of second binding molecules is used as the fixed reaction molecule constituting the complex.

  In the embodiment shown in FIG. 2, the microparticle 201 in which the first capture molecule 202 that specifically binds to the biomolecule 203 to be analyzed is immobilized on the surface is prepared in advance by the same method as in the embodiment shown in FIG. To do. In the embodiment shown in FIG. 2, the support substrate 209 includes a linker molecule 210 introduced on the surface and a first binding molecule 208 immobilized via the linker molecule 210.

  In the embodiment shown in FIG. 2, the immobilized reaction molecule 204 specifically binds to a recognition site different from the first capture molecule 202 with respect to the biomolecule 203 to be analyzed, as in the embodiment shown in FIG. A second capture molecule 205 is provided. The immobilization reaction molecule 204 of the embodiment shown in FIG. 2 includes a nucleic acid molecule 206 having a plurality of base sequence portions 211 as the second binding molecule 204 that specifically binds to the first binding molecule 208. That is, the immobilization reaction molecule 204 of the embodiment shown in FIG. 2 is composed of the second capture molecule 205 and the nucleic acid molecule 206.

  In this embodiment, after the complex 207 is formed, a reaction solution containing the complex 207 is supplied to the support substrate 209 on which the first binding molecule 208 is fixed. Accordingly, the nucleic acid molecule 206 in the complex 207 binds to the first binding molecule 208, whereby the complex 207 can be fixed to the support substrate 209. In particular, in this embodiment, the nucleic acid molecule 206 has a plurality of base sequence portions 211, and each base sequence portion 211 can bind to the first binding molecule 208. Therefore, the binding between the nucleic acid molecule 206 and the first binding molecule 208 is often caused by a plurality of base sequence portions 211 contributing. As a result, the probability of binding of the composite 207 to the support substrate 209 can be improved. For example, when the average particle diameter of the fine particles 201 is increased, it is preferable in that the speed of sedimentation by the weight of the support substrate 209 is high and the measurement time is short, but the second binding molecule introduced into the surface of the fine particles 201 is one molecule. Therefore, it is considered that the probability of coupling to the support substrate 209 decreases as a big problem. However, if a plurality of base sequence portions 211 can be combined as in the present embodiment, the above-described binding probability can be greatly improved.

  More specifically, as the plurality of base sequence parts 211, a specific base sequence that specifically binds to a specific protein, that is, an aptamer sequence can be used. For example, when using a quartz glass substrate as the support substrate 209, biotinylated silane (for example, Silane-PEG-Biotin, MW 5000 manufactured by Nanocs Inc.) as the linker molecule 210 and streptavidin as the first binding molecule 208, An aptamer sequence that binds to streptavidin can be used as the base sequence portion 211. Aptamer sequences that bind to streptavidin are disclosed in the reference Jiangrong Q. et al, Anal. Chem, 81; p5490-5495 (2009). More specifically, as shown in Table 1, examples of aptamer sequences that bind to streptavidin include DNA consisting of the nucleotide sequences of SEQ ID NOs: 1 to 3.

  A nucleic acid molecule 206 composed of a plurality of base sequence parts 211 can be easily obtained using a rolling cycle amplification reaction. The coupling between the nucleic acid molecule 206 and the second capture molecule 205 is a coupling reaction in which the amino group is modified at the end of the nucleic acid molecule 206 and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is used. Can be realized easily.

  In particular, it has been confirmed that by using nucleic acid molecules 206 having about 20 aptamer sequences shown in Table 1, fine particles 201 having a particle size of 100 nm can be bound with a binding probability of about 40%. When the second binding molecule 204 introduced into the microparticles 201 is one biotin molecule, the binding probability is about 5%. By comparing the two, the effect of adding a plurality of second binding molecules, and for that purpose, a nucleic acid molecule 206 having a plurality of base sequence portions 211 that bind to the first binding molecules 208 fixed to the support substrate 209 The effect of using the constituent molecules of the immobilized reaction molecule 204 was confirmed. Furthermore, it has been confirmed that by using the nucleic acid molecule 206 having about 60 aptamer sequences shown in Table 1, fine particles 201 having a particle size of 100 nm can be bound with a binding probability of about 70%. From this, it was confirmed that the use of the nucleic acid molecule 206 having more aptamer sequences shown in Table 1 improves the binding probability of the microparticles 201.

  As described above, the embodiment shown in FIGS. 1 and 2 can be realized, for example, by a biomolecule analyzer having a device configuration as shown in FIG. The biomolecule analyzer shown in FIG. 3 includes the above-described support substrate 307 (the support substrate 109 in the embodiment shown in FIG. 1, the support substrate 209 in the embodiment shown in FIG. 2), the analysis target biomolecule, the detected support, A reaction vessel 304 for forming a complex composed of fixed reaction molecules, a reaction solution supply device 305 for supplying a reaction solution containing the complex from the reaction vessel 304 onto the support substrate 307, and a complex fixed on the support substrate And a detection device for detecting and counting.

  More specifically, the biomolecule analyzer includes a movable stage 308 on which a support substrate 307 is placed, and a measurement flow cell 309 in which a channel member provided with a channel is bonded to the support substrate 307. For example, PDMS (polydimethylsiloxane) can be used as the flow path member.

  The biomolecule analyzer includes a sample solution tank 302 for a biomolecule to be analyzed, a solution tank 301 for supplying a detection target support composed of fine particles and a first capture molecule, and a solution tank 303 for supplying a fixed reaction molecule. The sample solution tank 302, the solution tank 301, and the solution tank 303 are connected to the reaction vessel 304 described above. The biomolecule analyzer also includes valves 320, 321 and 322. By opening these valves 320, 321, and 322, the biomolecule to be analyzed, the support to be detected, and the immobilized reaction molecule can be supplied to the reaction vessel 304.

  In the biomolecule analyzer, a reaction vessel 304 and a reaction solution supply device (hereinafter referred to as a liquid feeding unit) 305 are connected via a valve 315. After the complex formation reaction in the reaction vessel 304 is completed, a predetermined amount can be supplied to the liquid feeding unit 305 by opening the valve 315. After the valve 315 is opened and the reaction solution is sucked by the liquid feeding unit 305 in this manner, the valve 315 is closed and the valves 310 and 316 are opened, and the reaction solution is supplied from the liquid feeding unit 305 to the measurement flow cell 309. Close 316. Accordingly, the composite can be brought into contact with the support substrate 307. By allowing the reaction solution to stand on the support substrate 307 in the measurement flow cell 309 for a predetermined time, the composite is reacted with the support substrate 307. After completion of the reaction, the valve 318 is opened and the cleaning liquid is sent from the cleaning liquid tank 317 to the liquid feeding unit 305, occupies the valve 318, the valves 310 and 316 are opened, and the washing liquid is sent from the liquid feeding unit 305 to the measurement flow cell 309 to be discharged. Unreacted fine particles are removed from the support substrate 307 by sending the cleaning liquid to the tank 315. After performing this cleaning process a predetermined number of times, image observation is performed on the surface of the support substrate 307 in a state where the cleaning liquid is put in the measurement flow cell 309 again.

  The biomolecule analyzer shown in FIG. 3 includes a detection device including an optical system including a light source 312, a dichroic mirror 313, a filter 319, and an objective lens 311, and a two-dimensional CCD camera 314. Note that this detection device can output an image captured by the two-dimensional CCD camera 314 to the controller 306. The controller 306 can count the complex fixed to the support substrate 307 based on the image captured by the two-dimensional CCD camera 314. The controller 306 not only counts the complex, but also opens and closes the valves 310, 316, 318, 320, 321, 322, and 323, sucks and discharges the reaction liquid by the liquid feeding unit 305, and moves the operation stage 308. The operation and the operation of the optical system can be controlled. These controls can be automatically performed based on conditions preset by the operator.

  In the optical system of the biomolecule analyzer, the light from the light source 312 is guided to the objective lens 311 by the dichroic mirror 313 and irradiated onto the support substrate 307. Fluorescence or scattered light emitted from the fine particles is collected by the objective lens 311, passes through the filter 319, passes through the dichroic mirror 313, and forms an image on the photosensitive surface of the two-dimensional CCD camera 314. Whether to observe fluorescence or scattered light can be realized by controlling the transmittance of the filter 319. At this time, a part of the support substrate 307 may be observed, or the entire surface of the support substrate 307 may be observed. In order to observe the entire surface of the support substrate 307, for example, a method of scanning the entire surface of the support substrate 307 by operating the movable stage 308 can be cited. By observing the entire surface of the support substrate 307, the number of bright spots included in the acquired image can be increased, and the quantitativeness in the analysis can be improved. FIG. 4 shows a scattering image of gold fine particles having a diameter of 40 nm actually obtained. The field of view is 430 μm wide and 330 μm long. As shown in FIG. 4, it was confirmed that a clear fine particle image can be acquired.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is not limited to a following example.

[Example 1]
In Example 1, the validity of the analysis method of the present invention was verified as a target protein for detecting PSA (prostate cancer specific antigen). Anti-Mouse IgG (H + L) Antibody, Gold labeled, a KPL gold colloid labeled product, was used for the gold fine particles. About 10 times the amount of primary antibody (PSA mouse mAb PS2 manufactured by Abcam) was mixed and incubated with Anti-Mouse IgG-modified microparticles to obtain PSA primary microparticles with primary antibodies. As the second capture molecule, Human Kallikrein 3 / PSA Affinity Purified Polyclonal Ab, Goat IgG (product number AF1344) manufactured by R & D system was used. As a second binding molecule, a nucleic acid molecule containing 60 aptamer sequences (SEQ ID NO: 1) that bind to streptavidin was synthesized by RCA reaction, and then 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (Dojin) The second capture molecule and the second binding molecule were bound by a coupling reaction using Chemical Co., Ltd. For PSA, Recombinant Human Kallikrein 3 / PSA manufactured by R & D system was used, and the PSA concentration was adjusted to 1 fM, 10 fM, 100 fM, or 1000 fM. In order to measure the baseline, a reaction solution not containing PSA was also prepared. The reaction mixture was reacted for 30 minutes with 2 μM of the gold microparticles with the primary antibody, PSA (1 fM to 1000 fM), and 1 nM of the second capture molecule to which the second binding molecule was bound, with a reaction volume of 50 μl. After the reaction, the fine gold particles were settled with a desktop centrifuge, the unreacted supernatant was removed, and 50 ul of phosphate buffer was added and redispersed. By repeating this washing 5 times, unreacted components were sufficiently removed.

  On the other hand, the quartz substrate washed with a sulfuric acid-hydrogen peroxide washing solution was immersed in an ethanol solution of 1 μM Silane-PEG-Biotin (MW 5000) manufactured by Nanocs Inc. for about 1 hour, dried at 70 ° C. for 1 hour, The substrate was reacted with a streptavidin aqueous solution (1 μM) for 1 hour to prepare a substrate having streptavidin introduced on a quartz substrate.

  The gold fine particle dispersions reacted with the solutions having the respective PSA concentrations were appropriately reacted on the different streptavidin-introduced substrates. After the reaction, the substrate was washed 3 times with a phosphate buffer.

  The substrate was placed on an XY stage, and the scattered image was observed with a fluorescence microscope (light source: 100 W mercury, objective lens: × 20, NA: 0.5). The field size was 330 μm × 430 μm, about 250 fields were observed, image analysis was performed with Image-J, and the average number of fine gold particles per field was calculated. The number of gold fine particles in the reaction solution not containing PSA was judged as the baseline, and was subtracted from the number of gold fine particles at each PSA concentration. FIG. 5 shows a plot of the PSA concentration on the horizontal axis and the average number of fine gold particles per field of view on the vertical axis. As is apparent from FIG. 5, it can be seen that the linearity is maintained up to 1 fM. From this result, it became clear that the PSA concentration of the sample can be quantified to 1 fM level based on the number of observed gold fine particles.

101 ... microparticle, 102 ... first capture molecule, 103 ... analyte molecule, 104 ... fixed reaction molecule, 105 ... second capture molecule, 106 ... second binding molecule, 107 ... complex, 108 ... first Binding molecule, 109 ... support substrate, 201 ... microparticle, 202 ... first capture molecule, 203 ... analyte molecule, 204 ... fixed reaction molecule, 205 ... second capture molecule, 206 ... nucleic acid molecule, 207 ... complex, 208 ... first binding molecule, 209 ... support substrate, 210 ... linker molecule, 301 ... solution tank, 302 ... sample solution tank, 303 ... solution tank, 304 ... reaction vessel, 305 ... liquid feeding unit, 306 ... controller, 307 ... support substrate, 308 ... movable stage, 309 ... measurement flow cell, 310 ... bulb, 311 ... objective lens, 312 ... light source, 313 ... dichroic mirror, 314 ... two-dimensional CCD camera, 315 ... waste liquid tank, 316 ... bulb, 317 Cleaning fluid tank, 318 ... Valve, 319 ... Filter, 320 ... Valve, 321 ... Valve, 322 ... Valve, 323 ... Valve

Claims (11)

A support to be detected having a first capture molecule that selectively captures an analysis target biomolecule, a support substrate having a first binding molecule, and a second capture molecule that selectively captures the analysis target biomolecule And an immobilized reaction molecule having a second binding molecule that binds to the first binding molecule on the surface of the support substrate, and contacts the detected support, the immobilized reaction molecule, and the biomolecule to be analyzed. A process of
Immobilizing a complex composed of the detected support, the immobilized reaction molecule, and the analysis target biomolecule to the support substrate via the first binding molecule and the second binding molecule;
A method for analyzing biomolecules, comprising optically detecting a complex immobilized on the support substrate based on the support to be detected, and counting the complex immobilized on the support substrate based on the detected complex. .   The biomolecule analysis method according to claim 1, wherein each of the first capture molecule and the second capture molecule is an antibody having different regions of the analysis target biomolecule as epitopes.   The biomolecule analysis method according to claim 1, wherein the first binding molecule and the second binding molecule are avidin and biotin, or biotin and avidin, respectively.   The biomolecule analysis method according to claim 1, wherein the second binding molecule is a nucleic acid molecule having a base sequence that specifically binds to the first binding molecule.   The biomolecule analysis method according to claim 1, wherein the second binding molecule is a nucleic acid molecule having a plurality of polynucleotide units each having a base sequence that specifically binds to the first binding molecule.   The biomolecule analysis method according to claim 1, wherein the detection support is a fine particle.   The biomolecule analysis method according to claim 1, wherein the detected support is a nonmagnetic fine particle.   The biomolecule analysis method according to claim 1, wherein the detected support is a fine particle having a specific gravity of 10 or more and an average particle diameter of 40 to 300 nm.   The biomolecule analysis method according to claim 1, wherein the support to be detected is a fine particle containing an element selected from the group consisting of gold, platinum, and tungsten. A support substrate having a first binding molecule;
Analysis target biomolecule, detection target support having first capture molecule for selectively capturing the analysis target biomolecule, second capture molecule for selectively capturing the analysis target biomolecule, and the support substrate A reaction vessel in contact with an immobilized reaction molecule having a second binding molecule that binds to the first binding molecule on the surface;
A reaction solution supply device for supplying a reaction solution containing a complex composed of the support to be detected, the immobilized reaction molecule, and the biomolecule to be analyzed from the reaction container onto the support substrate;
A living body including a detection device that optically detects the complex fixed on the support substrate based on the support to be detected and counts the complex fixed on the support substrate based on the detection target Molecular analyzer.   The biomolecule analyzer according to claim 10, wherein the detection device includes an imaging device that acquires at least one optical image selected from fluorescence, photoluminescence, and scattered light, and the detection support is detected by the imaging device. .

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