JP4637565B2 - Biological sample labeling and biological substance labeling method and biological substance inspection method - Google Patents

Description

  The present invention relates to a probe chip for testing various test items at once, that is, a multi-item sensor. The objects are DNA, RNA, and protein, and in particular, biological materials such as DNA chips for DNA testing that have recently attracted attention. Related to assay chip.

  Along with the progress of the genome project, the movement to understand living organisms at the DNA level, to diagnose diseases and to understand life phenomena has become active. It is effective to investigate the expression status of genes in order to understand life phenomena and investigate the functions of genes. As an effective method, a DNA probe array or a DNA chip in which a large number of DNA probes are classified and fixed on a solid surface (called an oligo chip because it is actually an oligonucleotide derivative) Is also used. Or recently, protein chips fixed in an array as various proteins (generally antibodies as probes) have been used.

  In order to make a DNA chip, a method of synthesizing oligomers of sequences designed in a large number of compartmented cells using photochemical reaction and lithography often used in the semiconductor industry one by one (Non-patent Document 1: Science 251, 767-773 (1991)), or a method of implanting DNA probes and protein probes one by one in each compartment (Non-Patent Document 2: Proc. Natl. Acad. Sci. USA 93, 4613-4918 (1996)) and so on.

  Each of these chips has a structure in which a large number of probes such as a slide glass are arranged in a plane by dividing a plurality of probes into sections. In general, which probe is located at which position is indexed only at a physical position where the probe is fixed.

  The method of use is to introduce a fluorescent substance that is introduced onto a substrate by hybridizing a sample polynucleotide (hereinafter simply referred to as a sample polynucleotide) such as a fluorescently labeled DNA fragment or mRNA to a probe on a chip substrate, or cDNA reversely transcribed from this. Is detected with a fluorescence scanner. Alternatively, after hybridizing the sample polynucleotide, a fluorescently labeled oligo complementary to the sample polynucleotide adjacent to the probe is linked by ligation, or a fluorescently labeled dNTP substrate is reacted using DNA polymerase. Thus, it is the mainstream to detect the phosphor to be introduced onto the substrate.

  Recently, an electrochemical detection method using a redox reaction has been put into practical use. In the case of protein, using an affinity reaction such as an antigen-antibody reaction to capture a specific protein on the substrate and then analyzing with a mass spectrometer, a sandwich reaction is performed with a fluorescently labeled antibody or enzyme labeled antibody, and the substrate There are a method for detecting the phosphor and enzyme activity remaining above, and a detection method using electrochemiluminescence.

  In the electrochemiluminescence method, an antigen capturing antibody is present on the electrode surface. A ruthenium complex is used as a label for the sandwich antibody (Non-patent Document 3: Clin. Chem., 37, 1534-1539 (19991)). Ruthenium is oxidized on the electrode surface, and emits light when the ruthenium electrons, which are excited when they are reduced by being coupled with the redox reaction of TPA, fall to the ground state.

  As a detection method aiming at high-sensitivity and quantitative detection, an immunoassay is used in which a normal microscopic detection particle of about 700 μm is used as a label, and the number of reacted particles is counted with a counter to quantitatively detect the target substance. (Non-Patent Document 4: Anal. Biochem. 202, 120-125 (1992)).

Science 251, 767-773 (1991) Proc. Natl. Acad. Sci. USA 93, 4613-4918 (1996) Clin. Chem., 37, 1534-1539 (19991) Anal. Biochem. 202, 120-125 (1992))

  In the current chip technology, particularly a DNA probe array or a DNA chip, probes corresponding to one-to-one with respect to thousands to tens of thousands of DNA fragments to be measured are all fixed independently by dividing the section on the substrate surface. Each block is assigned an address, and the probe at which address the target DNA fragment is bound is detected using a fluorescent label. Such a structure is the same for a protein chip that fixes a protein such as an antibody on a substrate, and will be described below in the case of a DNA probe array. In order to create such a DNA probe array, as described in the above prior art, there is only a method of synthesizing a DNA probe for each section using a mask or planting a pre-synthesized DNA probe. It was. The manufacturing process is required depending on the type of probe, and the number of processes is large, and it is difficult to prepare the reaction conditions for each section because it is necessary to fix the probe to another position on the substrate. It takes a lot of effort to create.

  Therefore, it is desired to develop a technique that can measure the same number of measurement objects with fewer sections. As a matter of course, it is possible to multiplex a sample label and detect a plurality of measurement objects in the same section. However, in the conventional method using a phosphor as a label, the excitation light source and the fluorescence wavelength Because of the relationship, there are large restrictions when performing multi-labeling.

  In general, even when coherent light is used as an excitation light source, the obtained fluorescence wavelength has a wavelength dispersion of several tens of nm. For this reason, when a single excitation wavelength light source is used, it is the limit to measure fluorescence by exciting about four types of phosphors. Even if a plurality of excitation wavelength light sources are used, only about 6 types of phosphors can be used in a practical range of 500 to 700 nm. For this reason, when detecting a plurality of target polynucleotides in the same element by the fluorescence method, about 4 types are considered to be the limit.

  For this reason, in a multi-item biological material detection method such as a DNA probe array, a probe that minimizes the probe compartment and can identify several tens to thousands of sample molecules in the same compartment, and multi-inspection of the biological material using the label The law is desired.

  Furthermore, in a conventional DNA probe array, it is necessary to react a probe fixed on a flat substrate with a sample in a solution. Since the reaction takes place at the interface between the solid phase and the liquid phase, the probe cannot be dispersed in the solution. For this reason, any of the uniformity, reproducibility, quantitativeness, and speed of the reaction is disadvantageous compared to the reaction in the solution state.

For example, if multiple substrates can be immersed in a uniform probe solution at the same time and fixed at the same time, at least the reproducibility when fixing the probe can be secured, but different types of probes are fixed on the substrate. Therefore, it is impossible to adopt this method with a conventional DNA microarray or protein chip. The only solution is to separate the probe fixing elements of the probe array individually and fix the probes in a homogeneous solution for each element. Basically, since one lot of elements is reacted to each type of probe, variations in probe fixing can be minimized. Various probe fixing elements prepared in this way can basically measure for each item, but it is necessary to devise in order to bring them into another item simultaneous measurement system like a probe array. That is, in order to obtain information by reacting multiple probe elements simultaneously, it is a big problem whether to distinguish each probe-fixing element and measure the amount of DNA hybridized on it (or the amount of protein bound on a protein chip). Become. In the prior art, after preparing each probe fixing element, a method of arranging in an array and two cases of using the element as a suspension have been developed. In either case, the indexing of each probe element is an important issue. There are two methods: arraying elements in which the types of probes are known in advance in an array, and indexing by labeling each element with various phosphors. The street is in practical use. However, in the former, it takes time to mechanically arrange the elements one by one. For this reason, the number of elements arranged is substantially several hundred elements or less. In the latter, beads are fluorescently labeled by changing the ratio of two types of fluorescent dyes. For example, 64 types of elements can be indexed by preparing one with the amount of each fluorescent substance changed to 8 levels. Increasing the types of fluorescent dyes allows more elements to be indexed, but in the case of fluorescent dyes, there are restrictions on the wavelength dispersion of excitation light and fluorescence, and there are substantially three types that can be indexed to several hundreds. Both the method of arranging elements after probe fixation and indexing with fluorescent dyes are considered to be extremely effective due to their fast reactivity and reproducibility in measurement systems that require only a limited number of measurement items, such as clinical tests. .

However, as the needs of the world, in the array technology, at least several thousand kinds of measurement objects, preferably the number of measurement objects that can cover all mRANs, can be measured simultaneously and quantitatively with high reproducibility. In the present invention, in a multi-item biological material detection method such as a DNA probe array, reproducibility of the probe fixing amount is ensured by preparing probe fixing elements at once in a uniform solution, and each probe fixing element is mixed and suspended. By reacting with a sample in a turbid state at a time, the reaction is performed at a high speed and reproducibility is ensured. The indexing of each probe element is devised for easy identification, and thousands to tens of thousands of sample molecules can be identified. Therefore, a biological sample label used in a multi-testing method for biological materials that can be quantitatively detected by the method, a biological material labeling method, and a biological material testing method are desired.

  In the first embodiment of the present invention, nanoparticles prepared by changing the ratio of different elements to the label are used for labeling. For example, a description will be given with a mixture of a small amount of palladium and chromium based on gold. When the composition ratio of palladium and chromium is changed by 8 gradations, 64 types of gold nanoparticles can be obtained. When the number of elements added to gold is three, 512 kinds of gold nanoparticles are obtained. If the particle size is changed between about 10 to 50 nm and about 5 types at every 10 nm, about 2500 types of gold nanoparticles having different compositions and sizes can be obtained.

  Since this particle is a conductor, it is easy to illuminate the particle with a scanning electron microscope, measure the energy distribution of the secondary electron beam, and obtain an SEM image that identifies the position and size of the particle. Can be detected. Further, the characteristic X-rays generated when the particles are irradiated with electrons by a scanning electron microscope are used to obtain the position and size by obtaining an elemental analysis image with an energy dispersive characteristic X-ray detector. In this way, it is detected at what position on the partition of the substrate what kind of element and what size of the nanoparticles are contained. Particles having different compositions and particle sizes have a structure having probe DNAs that bind to different DNA sequences, whereby thousands of target DNA fragments can be detected on the same plane.

  Since the nanoparticle is based on gold, the probe can be immobilized on the particle surface by using a DNA probe having an alkyl sulfide group.

  The present invention is basically based on an alloy manufacturing technique, and there are a wide variety of elements that can be mixed, and it is also possible to combine four or more elements. For example, by combining five kinds of elements, it is possible to obtain 30,000 or more different kinds of nanoparticles having different compositions comparable to the number of sections of an existing DNA chip. Alternatively, it is possible to distinguish and detect thousands of types of DNA by preparing 250 combinations of three elements as a group and preparing a plurality of groups of combinations of other elements.

  As a combination of elements that change the composition, three to five kinds of alloys may be selected from gallium, aluminum, yttrium, erbium, holonium, cesium, cobalt, titanium, nickel, iron, and the like. For the probe immobilization, in addition to the reaction between gold and SH groups described above, in the case of an alloy having an oxidized surface, a functional group may be introduced using a silane coupling reaction to immobilize the probe DNA.

  Thus, the present invention overturns the concept of a DNA chip that conventionally requires different probes to be fixed to a large number of compartment elements. Simply trap the mRNA on a chip with poly-T immobilized, hybridize a synthetic DNA probe with a sequence complementary to each mRNA, labeled with nanoparticles of different composition, and analyze with a scanning electron microscope. It is possible to analyze mRNA expression over time.

  If a DNA probe is changed to an antibody and used on a biological material (for example, a protein) immobilized on a substrate, an analysis method capable of analyzing thousands of epitopes at once can be established.

  The second embodiment of the present invention develops the first embodiment and uses particles produced by changing the ratio of different elements to fix a specific probe. That is, a particle having a specific probe fixed is prepared for each particle having a predetermined element ratio. On the other hand, the target DNA fragment to be hybridized to the probe fixed to each particle is labeled with gold fine particles for counting. Particles fixing the probe and the target DNA fragment are mixed in a solution, and the probe and the target DNA fragment are hybridized. This process is performed in a predetermined section on the container or the substrate, and then the particles are washed and collected. For washing and collection, the particles may be collected by centrifugation and the supernatant may be exchanged. When the particles contain a magnetic substance, the particles may be collected by a magnet and the supernatant may be exchanged. The particles are fixed to the compartments on the substrate by drying after treatment in predetermined compartments on the substrate. As a result, the target DNA fragment can be evaluated by particle indexing and gold fine particle count as a label.

  According to this second embodiment, the hybridization between the probe immobilized on the particle and the target DNA fragment can cause the particle to react in a solution suspension state, so that the hybridization is a boundary between the solid phase and the liquid phase. The problem of the reaction non-uniformity generated by the surface, the low reaction rate and the low reaction rate due to the diffusion-controlled molecular diffusion can be considerably solved. Handling as a suspension has the advantage of not requiring special containers, pipettes or special techniques. And the technique used as the label | marker of the above-mentioned measuring object is utilized for the indexing of this particle | grain. That is, a scanning electron microscope irradiates particles with electrons to obtain an SEM image in which the positions and sizes of the particles are identified, and the characteristic X-rays generated when the electrons are irradiated are converted into elemental elements by an energy dispersive characteristic X-ray detector. An analysis image is obtained, and the particles fixed on the section of the substrate are indexed from the comparison of both. And the particle | grain is fixed on the division of a board | substrate. Here, an energy dispersive characteristic X-ray detector is used, but in other embodiments of the present invention, other methods with higher sensitivity such as wavelength dispersive X-ray spectroscopy (WDX) are used. Can also be used. Rather, wavelength dispersive X-ray spectroscopy may be more suitable for the present invention because of its excellent X-ray wavelength resolution.

  When the detection target is a protein or sugar chain, an immunoassay technique is applied. That is, an antibody in which an antibody molecule that reacts with a specific epitope is immobilized on an indexing particle and an antibody immobilized on a gold nanoparticle for labeling are used. In this case, the antibody immobilized on the indexing particle and the labeling particle is an indexing particle-antigen-labeled gold particle hybrid by sandwiching antigen molecules using an antibody specific to the molecule to be measured. Alternatively, sandwiching is performed using indexing particles to which a specific antibody is immobilized, an unlabeled antibody, and a second antibody that reacts universally with the unlabeled antibody. In any method, a large number of antigens can be indexed and quantitatively detected.

  For immobilization of the indexing particle-mRNA-labeled gold particle hybrid or the indexing particle-antigen-labeled gold particle hybrid on the substrate, a suspension of these hybrids may be dropped on the substrate and dried, or more rationally. Specifically, the problem can be solved by using a magnetic substance for the indexing particles, drawing the solution on a substrate with a magnet and then drying the solution with a blower or the like.

  In this way, the concept of the DNA chip in which different probes must be fixed to a large number of compartment elements in the past is overturned. By simply reacting the indexing particles, the specimen sample, and the labeled particles, and analyzing them with a scanning electron microscope, an analysis method is established that can analyze the types and amounts of thousands to tens of thousands of mRNAs and proteins at once.

  According to the present invention, elemental analysis and shape analysis of particles are performed during scanning with a scanning electron microscope. Since thousands to tens of thousands of kinds of particles can be identified and detected on the same section, thousands to tens of thousands of kinds of biological substances can be simultaneously examined without being separated by pretreatment.

  The present invention will first be described from the first embodiment.

Example 1
FIG. 1 is a conceptual view showing a part of the DNA chip of Example 1 of the present invention in a perspective view. The chip 1 is formed on a silicon substrate 101 having an oxide film surface. The size is 20 × 20 mm. One probe fixing region 102 for fixing the DNA probe is 10 mmφ. The periphery is coated with a Teflon (registered trademark) water-repellent resin 103. The thickness of the coating is approximately 50 μm. In the probe fixing region 102, a 5 base long random sequence oligo DNA is bound to the 3 ′ end of a 26 base long poly T at the 5 ′ end. This is because poly-T alone cannot sufficiently secure the stability of mRNA hybridization. The probe is made of PNA (Peptide Nucleic Acid) so that it can easily interact with intracellular mRNA. Since PNA does not have a negative charge derived from a phosphodiester bond unlike ordinary DNA, an electrostatic repulsive force does not act on the target DNA. This has the effect of increasing the efficiency of hybridization.

  Furthermore, using PNA as a probe and having no charge property does not generate electrostatic repulsion, so the poly A portion of the target mRNA forms a partial double strand with other sites in the molecule. Even in such a case, the DNA can be competitively submerged in the double strand and can be hybridized competitively, so that the hybridization proceeds without a denaturing operation.

  The probe immobilization method is as follows. The method described by Kumar et al. (Nucleic Acids Research (2000) 28, No. 14 e71) was modified by introducing a silanized DNA probe in which a trimethoxysilane residue was previously introduced into the amino terminus of a synthetic PNA into an element on a chip substrate. The probe is fixed by applying to the part.The silanized DNA probe can be obtained, for example, by binding the glycidoxy group of 3-glycidoxypropyltrimethoxysilane to the amino terminus of PNA, or the surface of silicon oxide Aminopropyltrimethoxysilane is reacted with amino group to introduce amino group by silane coupling reaction, phenylene diisothiocyanate is reacted to the amino group, and isothiocyanate group is introduced to the silicon substrate surface. A DNA probe having a nate group and an amino group introduced at the 5'-end is reacted to fix it. (Nucleic Acids Research (1999) 27, No. 9, 1970-1977).

  As an example, gold in which the ratio of gallium, aluminum, yttrium, and chromium is changed so that a sequence of about 40 bases complementary to various mRNAs can be distinguished from 50 μl of total RNA solution extracted from a resected colon cancer tissue fragment according to a conventional method. A mixture of base nanoparticles (10 μm) in a 0.1% (w / v) solution is added to the probe fixing region 102 of the chip 1 without any special treatment. The chip is kept warm at 45 ° C in advance. A 40 mmφ glass plate is placed on the upper surface of the probe fixing region with a gap of about 1 mm, and the glass plate is moved while drawing a circular arc so that the edge of the probe fixing region 102 substantially coincides with the edge of the glass plate. The speed of movement is once every 5 seconds. Thus, hybridization can be performed at high speed. In the probe fixing region 102 of the chip 1, the probe, mRNA, and particle-labeled probe fixed to the chip 1 are bonded in this order in a sandwich shape. Unreacted particle-labeled probe and RNA are washed away.

  FIG. 2 is a conceptual diagram illustrating an aspect in which the probe tip 1 described in FIG. 1 is observed with a scanning electron microscope.

  A large number of probes are fixed on the surface of the probe fixing region 102 of the chip 1. Here, for simplification, the DNA fragments 201-204 are hybridized to the fixed probes 11-14, respectively. Are labeled with gold nanoparticles 21-24. The gold nanoparticles 21, 22, 23, and 24 have (gallium: aluminum: yttrium: chromium) ratios (1: 1: 1: 0), (1: 1: 0: 1), (1: 0), respectively. : 1: 1) and (1: 1: 1: 1). The maximum mixing ratio of each metal to gold is 20%. This is because the bond between alkanethiol and gold introduced at the 5 'end of the probe is used to immobilize the mRNA-specific probe on the nanoparticle surface. Gold and thiol are vulnerable to oxidative conditions and UV irradiation, but a very stable binding force can be obtained under normal hybridization conditions. For this reason, in Example 1, alkanethiol previously introduced into the 5 'end of the probe and gold nanoparticles are mixed 10 to 1 to obtain a series of gold nanoparticles having the mRNA sequence-specific probe immobilized on the surface.

  An electron gun 300-1, a focusing lens 300-2, and a scanning coil 300-3 of a scanning electron microscope 300 for detecting gold nanoparticles are provided on the surface of the probe fixing region 102 of the chip 1. Electrons of the electron beam 300-4 emitted from the electron gun 300-1 collide with the gold nanoparticles 21, 22, 23, and 24, and the gold nanoparticles emit secondary electrons 300-5. The detector 300-6 captures the secondary electrons. A so-called SEM image is obtained based on the secondary electrons detected by the detector 300-6, and the position and size of the gold nanoparticles are specified.

  On the other hand, in the present invention, when the electrons of the electron beam 300-4 collide with the gold nanoparticles 21, 22, 23, and 24, the gold nanoparticles 21, 22, 23, and 24 emit constituent elements of the gold nanoparticles. An energy dispersive characteristic X-ray detector 300-8 for detecting X-rays 300-7 having a specific wavelength. That is, an elemental analysis image is obtained from wavelength signals corresponding to constituent elements detected by the energy dispersive characteristic X-ray detector 300-8. Alternatively, an elemental analysis image is obtained using wavelength dispersive X-ray spectroscopy that is detected by spectroscopic analysis with an X-ray spectroscopic crystal.

  The elemental analysis image obtained from the energy dispersive X-ray detector 300-8 has information on the position of gold nanoparticles and constituent elements. Therefore, if the SEM image and the elemental analysis image are viewed in correspondence, the mRNA molecule hybridized with the gold nanoparticle labeled probe can be identified.

  FIG. 3 is a conceptual diagram illustrating a method for identifying mRNA molecules hybridized with a gold nanoparticle labeled probe from the correspondence between SEM images and elemental analysis images.

  As an example, on the surface of the probe fixing region 102, oligo PNA (28 bases) having a sequence corresponding to the EpCAM mRNA sequence, and a sequence corresponding to the mRNA sequence of CD44, which is also said to be highly expressed in cancer cells. Oligo PNA (26 bases) and oligo PNA (29 bases) having sequences corresponding to the mRNA sequence of CEA are respectively immobilized as probes. The ratio of gallium: aluminum: yttrium: chromium (1: 1: 1: 0), (1: 1: 0: 1), (1: 0: at the amino terminus of mRNA hybridizing to these probes, respectively. The case where gold nanoparticles (10 nm diameter) containing 1: 1) are added as a label will be described.

  In FIG. 3, 30 is an SEM image. The SEM image shows all particles. Reference numerals 31, 32, 33, and 34 denote a gallium image, an aluminum image, an yttrium image, and a chromium image, respectively. When the SEM image 30 is compared with the gallium image 31, the aluminum image 32, the yttrium image 33, and the chromium image 34, the SEM image 30 and the gallium image 31 are the same, but the aluminum image 32, the yttrium image 33, and the chromium image 34 are the same. In the SEM image 30, particles at positions indicated by broken lines do not appear. That is, since gallium is contained in all of the gold nanoparticles serving as labels, all particles appear in the gallium image 31 like the SEM image 30. In the aluminum image 32, particles at positions indicated by broken lines do not appear. That is, it can be seen that the mRNA hybridized to the probe at this position is a CEA molecule labeled with gold nanoparticles not containing aluminum. This is a gold nanoparticle indicated by reference numeral 37 in the SEM image 30. Similarly, in the yttrium image 33, two particles at positions indicated by broken lines do not appear. That is, it can be seen that the mRNA hybridized to the probe at this position is a CD44 molecule labeled with gold nanoparticles not containing yttrium. This is a gold nanoparticle indicated by reference numeral 36 in the SEM image 30. Further, in the chrome image 34, three particles at positions indicated by broken lines do not appear. That is, it can be seen that the mRNA hybridized to the probe at this position is an EpCAM molecule labeled with gold nanoparticles not containing chromium. This is a gold nanoparticle indicated by reference numeral 35 in the SEM image 30.

  In FIG. 3, in order to simplify the explanation, all the particles have the same size. However, if particles having different particle sizes are used in combination, more particles can be identified. Of course, it goes without saying that this identification can be performed by image processing of a computer.

  Thus, according to the present invention, a plurality of mRNAs can be distinguished and measured quantitatively on a DNA chip that simply captures mRNA having a poly A tail. Since the energy dispersive characteristic X-ray detector or wavelength dispersive X-ray spectrometer attached to the SEM can identify an element ratio of several percent, the same element can be identified with about 8 gradations. When four kinds of elements are used, 4096 kinds of particles can be identified. When five elements are used, 32768 particle identifications can be achieved by taking only five images.

  In this way, by using particles with different element ratios of the present invention for labeling, it is possible to use particle counting technology without using a conventional probe tip for compartmentalization that has a complicated structure and is difficult to manufacture. This enables mRNA profiling at the single molecule level.

(Example 2)
Example 2 describes multi-detection of biological material by antigen-antibody reaction. Here, the same substrate as that described with reference to FIG. 1 is used. In Example 2, the probe fixing region 102 is used as a reaction part of an antigen-antibody reaction, and an IgG fraction obtained by affinity-purifying anti-human antiserum is fixed to the reaction part 102. Since the serum to be measured contains a large amount of human albumin and human IgG, the reaction unit 102 needs to remove the anti-human albumin antibody and the anti-human IgG antibody in advance so that they do not react. is there. For this purpose, an IgG fraction in which the affinity purified anti-human antiserum IgG fraction is absorbed with human albumin and human IgG is prepared, and this is fixed to the reaction part 102, and the extra adsorbing site is masked with phosphatidylcholine. . Next, the reaction part 102 is washed with 0.15 M NaCl, 50 mM sodium phosphate buffer (PBS: pH 7.4) containing 10 mg / ml bovine serum albumin to remove unreacted human IgG.

  When 100 μl of a human serum sample is added to the reaction unit 102 and stirred for 10 minutes in the same manner as in Example 1, an antigen-antibody reaction occurs and the antibody on the reaction unit 102 captures the protein present in the serum.

Separately, a nanoparticle-labeled antibody is prepared. For example, an SH group is introduced into an F (ab ′) ₂ fragment obtained by papain degradation of an affinity purified polyclonal anti-AFP antibody and an anti-CEA antibody. The number of SH groups to be inserted is 3 to 4 molecules per F (ab ′) ₂ molecule. Anti-AFP and anti-CEA antibodies containing SH groups prepared as described above on gold nanoparticles with a ratio of palladium to chromium (20:80) and (30:70) based on 20 nmφ gold The derived F (ab ′) ₂ is mixed to obtain gold nanoparticles having F (ab ′) ₂ bonded to the surface.

  As a sample, prepare AFP alone at a concentration of 1 zmol / μl, CEA alone at a concentration of 5 zmol / μl, and a control containing nothing. The solvent is PBS (pH 7.4) containing 0.1% Tween 20 and 0.5% BAS.

Each solution is reacted with a protein chip, and further, gold nanoparticle label F (ab ′) ₂ is reacted. The reaction time is 5 minutes for the first sample reaction and 5 minutes for the reaction of the gold nanoparticle labeled F (ab ′) ₂ . After completion of the reaction, it is washed with a buffer solution containing 0.1% Tween 20 and 0.5% BAS, gold nanoparticles are detected with a scanning electron microscope and an energy dispersive X-ray detector, and the abundance ratio of palladium and chromium. Count AFP and CEA molecules.

When only AFP is reacted with the protein captured by the antibody on the reaction part 102 described above, the gold nanoparticles that can be recognized as AFP bound to the protein are 120 particles / μm ² , and the other gold nanoparticles are 2 particles / μm ² is detected. Similarly, when only CEA is reacted, 1580 particles / μm ^{2 of} gold nanoparticles that can be recognized as CEA bound to protein and 6 particles / μm ^{2 of} other gold nanoparticles are detected. Only 2-4 particles / μm ² of gold nanoparticles are detected in the control solution.

  In the present invention, the metal or semiconductor constituting the alloy of the particles to be labeled is a transition metal excluding No. 43 up to No. 79 in the periodic table and No. 13, No. 31, No. 32, No. 49, No. 50, It can be selected from any of No. 51, No. 81, No. 82, No. 83 metal, No. 14, No. 33, No. 34, No. 52 semiconductor.

  As described above, the present invention can be implemented in the form of several examples. In any case, the hybridized sample DNA and the like are detected substantially every molecule, so that the sensitivity can be increased. Can achieve sensitivity exceeding conventional methods. Since a trace amount of DNA (RNA) can be detected in a very small volume, the target DNA (RNA) can be detected without performing pretreatment amplification by PCR, which was impossible in the past. Since the size and shape of the labeled particles can be changed for detection, it is possible to perform multi-analysis of 6 to 10 different samples using the same element. In addition to the use for conventional differential hybridization, this technique enables the sample polynucleotide to be captured on the element with the same probe and detected with a different labeled probe. The multi-analysis technique of the present invention has an advantage that alternative splicing detection and multiple SNP typing can be performed with one element.

(Example 3)
A method using a low-resolution SEM or an X-ray detector with a resolution of about 0.1 μm will be described as a simple detection version of the first embodiment. Such a low-resolution apparatus is small enough to be installed on a desktop, and has the effect of being cheaper than a normal SEM with an X-ray detector. Alternatively, an analysis example by an electron probe X-ray microanalyser (EPMA) using an electron microprobe that performs elemental analysis in the range of about 1 μm ² will be described. When the resolution is about 0.1 μm, it is not possible to obtain a gold nanoparticle count or elemental analysis image. In this case, the elemental analysis value of each element in the range to which X-rays are applied is obtained. In this embodiment, can not be labeled with too many kinds of elements for each DNA probe, in element 2-3 or for use as a label on one particle, resolution of ratio of addition is 3 It is about a seed. Or, in the case of labeling with one kind of element, it is possible to label as many as the number of elements used, and there are several tens of kinds of DNA and biological substances that can be analyzed simultaneously.

  Next, a second embodiment will be described.

Example 4
FIG. 4 is a diagram showing the concept of biological sample measurement according to Example 4 of the present invention. The difference from Example 1 resides in the use of indexing particles. A probe is not fixed to the silicon substrate 101. The silicon substrate 101 is simply a measurement container with a defined area, and is used to fix indexing particles during measurement. The size is 20 × 20 mm. The indexing particle fixing region 102 for fixing the indexing particles is 3 mmφ in one place. SU8 is applied on the substrate 101, and the bank 103 is formed by ultraviolet curing. Of course, the substrate may be directly dug by etching. The bank 103 only needs to have a structure capable of holding a liquid. However, as described later, the liquid to be held may be an aqueous solution containing 70% alcohol. In this case, the height should be at least 150 μm.

  41 to 44 are indexing particles each having a different elemental composition. The indexing particles correspond to the gold nanoparticles 21 to 24 for labeling having different element compositions in correspondence with the first embodiment, but are different in the following points. In the first embodiment, the elemental composition of the gold nanoparticles 21 to 24 for labeling is different, and this generates X-rays having wavelengths specific to the constituent elements of the gold nanoparticles by irradiation with an electron beam. Attention was focused on the gold nanoparticles for labeling, and specific biological substances (for example, target DNA fragments) were detected. On the other hand, in the second embodiment, the indexing particles 41 to 44 are particles for fixing the probe on the surface thereof, and separately from this, the amount of the specific biological material captured by the probe is counted. Labeled particles were used. That is, in the second embodiment, focusing on the fact that the indexing particles 41 to 44 irradiated with the electron beam generate X-rays having wavelengths specific to the constituent elements of the particles, the position of the indexing particles is specified, The specific biological substance trapped by the indexing particle was detected. For example, the indexing particles include a plurality of elements other than gold, and the coefficient label particles are gold nanoparticles.

The particle serving as the base of the indexing particle is made of polystyrene that does not contain an element used for indexing, so that the elemental analysis does not hinder the detection of the identifying element in the indexing particle. Alternatively, an identification element may be vapor-deposited and fixed on this surface using polystyrene magnetic particles in which a paramagnetic substance such as iron or cobalt is embedded in polystyrene. In addition to vapor deposition, there are many ways to prepare particles containing an identification element. For example, a predetermined number of each identification element may be kneaded into a polystyrene sphere. In this case, the element signal of each particle can be obtained by increasing the energy of the electron beam to be irradiated to the inside of the polystyrene sphere.

  The use of magnetic particles has the advantage of facilitating the operation of the particles in the reaction operation and particle detection operation with a magnet as will be described later. In the case of ordinary polystyrene, operation can be performed without any problem by a method of collecting particles by centrifugation or a filter.

Black circle attached to the indexing particles 41-44 is labeled particles for counting, as will be described later, it is labeled particles of a specific biological substance captured on the probe which is fixed to the indexing particles 41-44 . The labeled particles are used to measure the amount of specific biological material trapped on the surface of the indexing particles 41-44. The indexing particles 41 to 44 are fixed to the indexing particle fixing region 102 (3 mmφ) of the substrate 101. The indexing particles 41 to 44 and a sample containing a target specific biological material labeled with the labeling particles were mixed in a solution, and the specific biological material was captured by the probe fixed to the indexing particles 41 to 44. Thereafter, a predetermined amount of this mixed solution is dropped onto the probe fixing region 102 and dried to fix the indexing particles to the indexing particle region 102.

  When the base of the indexing particles is polystyrene particles, 1 μl of the mixed solution is dropped on the probe fixing region 102 and then dried under reduced pressure to fix the indexing particles. For this reason, it is necessary to add a mechanism for holding droplets to the indexing particle fixing region 102. In the third embodiment, SU8 is applied on the substrate 101, and the bank 103 is formed by ultraviolet curing. Of course, the substrate 101 may be directly dug by etching. The bank 103 only needs to have a structure capable of holding the liquid. However, as described later, the liquid to be held may be an aqueous solution containing 70% alcohol. In this case, the height should be at least 150 μm.

  With the indexing particles fixed to the indexing particle region 102, the substrate 101 is mounted on a scanning electron microscope 300 having an energy dispersive X-ray detector or a wavelength dispersive X-ray spectrometer, as in the first embodiment. . The scanning electron microscope 300 includes an electron gun 300-1, a focusing lens 300-2, and a scanning coil 300-3. The electrons of the electron beam 300-4 emitted from the electron gun 300-1 collide with the indexing particles 41 to 44. The indexing particles 41 to 44 emit secondary electrons 300-5. The detector 300-6 captures the secondary electrons. A so-called SEM image is obtained based on the secondary electrons detected by the detector 300-6, and the positions and sizes of the indexing particles 41 to 44 are specified. Further, at this time, the labeled particles bonded to the surfaces of the indexing particles 41 to 44 are detected. On the other hand, when the electrons of the electron beam 300-4 collide with the indexing particles 41 to 44, the energy dispersion for detecting the X-rays 300-7 having a wavelength specific to each indexing particle constituent element emitted by the indexing particles 41 to 44 is generated. A type characteristic X-ray detector or a wavelength dispersive X-ray spectrometer 300-8 is provided. That is, an elemental analysis image is obtained from wavelength signals corresponding to constituent elements detected by the energy dispersive X-ray detector or the wavelength dispersive X-ray spectrometer 300-8. Thereby, the indexing particle can distinguish the kind of probe fixed on the surface by its size and element.

  FIG. 5 shows the position and size of the indexing particles 41 to 44 from the SEM image obtained by the detector 300-6 and the elemental analysis image obtained from the energy dispersive characteristic X-ray detector 300-8. It is a figure explaining the evaluation of the specific biological material to which the labeling particle | grains hybridized to the indexing particle | grains 41-44 were added. Here, the elemental composition of the indexing particles 41 to 44 is the same as in Example 1, the ratio of gallium: aluminum: yttrium: chromium is the indexing particle 41 (1: 1: 1: 0), and the indexing particle 42 (1: 1: 0: 1), indexing particle 43 (1: 0: 1: 1), and indexing particle 44 (0: 1: 1: 1), and the particle size is A case where the thickness is 0.5 to 5 μm will be described.

  In FIG. 5, 50 is an SEM image. In the SEM image, all the indexing particles 41 to 44 and the labeled particles of the specific biological substance captured by these are shown. 51, 52, 53, and 54 are elemental analysis images of a chromium image, an yttrium image, an aluminum image, and a gallium image, respectively. When the SEM image 50 is compared with the chromium image 51, the yttrium image 52, the aluminum image 53, and the gallium image 54, in the chromium image 51, the particle image at the position corresponding to the indexing particle 41 at the position indicated by the broken line does not appear. . Similarly, in the yttrium image 52, the aluminum image 53, and the gallium image 54, particle images at positions corresponding to the indexing particles 42, 43, and 44 do not appear in the SEM image 30. That is, since each of the indexing particles 41, 42, 43 and 44 does not contain chromium, yttrium, aluminum and gallium, the element of the composition of the particles does not appear in the elemental analysis image. is there. In the elemental analysis image, when the labeled particle is a gold nanoparticle, it does not appear in principle. However, when the labeled particle emits an electron beam and emits an X-ray having a specific wavelength close to that of the element of the indexing particle composition, it appears in the elemental analysis image. Therefore, although it includes noise in contrast with the SEM image 50, it is not fatal.

  Therefore, the indexing particles 41 to 44 can be specified by comparing the SEM image 50 and the elemental analysis images 51 to 54. Moreover, since the labeled particles appear in the SEM image 50, if this is counted and evaluated together with the specific results of the indexing particles 41 to 44, how much labeled particles are present in which indexing particles, In other words, it can be evaluated which specific biological material was present in the sample. Here, for simplicity, a simple schematic diagram of an example of whether or not a specific element is included in four particles of the same size is used. However, the particle size of the indexing particle is at a level that can be identified by image recognition with an SEM. By using various combinations of indexing particle composition at a level that allows elemental analysis images to be identified by image recognition, various types of particle size x number of element combinations x amount ratio of each element can be used. Can be a thing. For example, if the particle size of the indexing particles is 4 to 4 in the range of 0.5 to 5 μmφ, and the amount of 4 elements is changed to 10 in each, 40000 kinds of indexing particles can be obtained.

  Here, the elements that can be used for the indexing particles will be described. Elements that can be analyzed by the energy dispersive characteristic X-ray detector 300-8 are number B from number B to number 92 U in the periodic table. If it is 1% or more by weight if it is an element in this, it can detect. Since it is used by being mixed with the problem of apparatus resolution and spectrum assignment and basically with magnetic particles and polystyrene particles, it can be distinguished by deterministic analysis in about 10 steps in the range of about 1% to 20%. If it is based on magnetic particles, elements involved in magnetism cannot be used for indexing. Therefore, Fe, Co, and Ni are excluded from the indexing elements. C, N, and O are also impossible because they are polystyrene materials. In the first place, these Fe, Co, Ni, C, N, and O are elements that are abundant in nature, and are often mixed due to contamination. Therefore, they should be avoided for indexing. For the same reason, alkali metals (Group I), alkaline earth metals (Group II), Groups 15, 16 and 17 up to As should be excluded, and all 18 genera should be excluded because they are gases. . Al, Si, Mo, and Sn are excluded because they exist around us. Five V genera are excluded because they are relatively large in the living body. Exclude Tc, Pm, Ac, Pa, and U with no or little stable isotopes. Hg is excluded because it is a liquid in the carrier. Other than those excluded are used for indexing. That is, as an indexing element, Sc, Ti, Ga, Ge, Y, Zr, Nb, Ru, Rh, Pd, Ag, Cd, In, Sb, La, Ce, Pr, Nd, Sm, Eu, Gd , Tb, Dy, Ho, Er, Tm, Yb, Lu, Hf, Ta, W, Re, Os, Ir, Pt, Au, Tl, Bi, and Th can be used.

  FIG. 6 (A) is a diagram schematically showing the indexing particles 41 to 44 of Example 3 and probes 41a, 42a, 43a and 44a fixed to the surface, and FIG. 6 (B) is a probe to which labeled particles are added. The figure which shows typically the specific biological material 41b, 42b, 43b and 44b which hybridizes to 41a, 42a, 43a, and 44a, and FIG.6 (C) show a mode that the probe and the specific biological material hybridized. It is a figure shown typically. Here, an example in which a DNA probe is used as the probe will be described.

  As shown in FIG. 6A, specific probes are fixed to the surfaces of the indexing particles 41 to 44, respectively. An existing method is used to fix the probe to the indexing particle. For example, the indexing particle is irradiated with oxygen plasma to generate an active group on the surface, then reacted with 3-aminoethylaminopropyltrimethoxysilane to introduce an amino group on the surface, and the amino group is converted to a carboxyl group with succinic anhydride. The probe DNA having an amino group at the 5 ′ end may be immobilized using this carboxyl group in the form of a succinimide ester. When PNA is used for the probe DNA, it can be obtained by reacting the amino terminus of the probe DNA in the same manner. Naturally, the specific biological substances 41b, 42b, 43b and 44b which hybridize to the probes 41a, 42a, 43a and 44a shown in FIG. 6B have a sequence complementary to the probes 41a, 42a, 43a and 44a. is there. Immobilization of labeled particles such as gold nanoparticles (20 nm) on specific biological substances is prepared by synthesizing those having SH groups at the 5 'end and mixing them with gold nanoparticles. The indexing particles 41 to 44 are mixed with the sample solution containing the specific biological material to which the labeling particles are added, and the specific biological material is hybridized to the probes 41a, 42a, 43a and 44a. A predetermined amount is dropped onto the fixed region, dried, and scanned with an electron beam with the scanning electron microscope 300 as described above to obtain an SEM image and an elemental analysis image, which can be evaluated by the method described in FIG. .

(Example 5)
An example will be described in which the above-described method of Example 3 is applied directly or directly from a mixture of mRNAs to detect these quantitatively.

  FIG. 7A schematically shows the indexing particles 41 to 44 of Example 5 and the probes 41a, 42a, 43a and 44a fixed to the surface, and FIG. 7B shows the probes 41a, 42a, 43a and FIG. 7C schematically shows a specific biological material added with poly A that hybridizes to 44a, and FIG. 7C schematically shows poly T added with a label that hybridizes with poly A. is there.

  As shown in FIG. 7A, in Example 4, the configuration of the indexing particles 41 to 44 is the same as that in Example 3. That is, the unique probes 41a, 42a, 43a and 44a are fixed to the indexing particles 41 to 44, respectively. As shown in FIG. 7B, poly A is added to the specific biological materials 41b, 42b, 43b and 44b which are samples. Here, it is natural that the probes 41a, 42a, 43a and 44a and the specific biological substances 41b, 42b, 43b and 44b are in a complementary relationship. 1) A sequence complementary to a sequence of 30 to 50 bases spanning the exon closest to the poly A terminus of mRNA and the exon closest to the second is used as a probe. Alternatively, when measuring as cDNA, 2) When using mRNA as a sample in accordance with a standard method, a single-stranded cDNA (sequence obtained by synthesizing cDNA and removing mRNA sequence with RNase H and complementary to mRNA) is used as a sample. A 30-50 base sequence spanning the exon closest to the poly A terminus and the exon closest to the second is used as a probe on the same side as the mRNA. In this case, (A) 41a, 42a, 43a is a probe having a sequence complementary to cNDA, (B) 41b, 42b, 43b is a single-stranded cDNA, and (C) is a poly-T bound to a labeled particle. Instead, poly A will be used. Alternatively, 3) When the double-stranded cDNA is a sample, for example, probe a part of the sequence (30 to 50 bases) near the 3 ′ end from the poly A end of the human mRNA sequence to the place where the first MboI sequence appears Used as In this case, either (A) 41a, 42a, 43a is a combination of a probe having the same sequence as the mRNA and poly (T) bound to the labeled particle (C), or (A) 41a, 42a, 43a Any combination of a probe with a complementary sequence and poly A bound to the labeled particles of (C) can be used. Alternatively, only when a double-stranded dDNA is used, an arbitrary synthetic DNA is introduced into the MboI cleavage end by a ligation reaction using DNA ligase, and a sequence complementary thereto is bound to a labeled particle instead of poly-T (C ) Can also be used.

  Here, the case of 1) will be described as a representative example. An existing method is used to fix the probe to the indexing particle in FIG. For example, the indexing particle is irradiated with oxygen plasma to generate an active group on the surface, then reacted with 3-aminoethylaminopropyltrimethoxysilane to introduce an amino group on the surface, and the amino group is converted to a carboxyl group with succinic anhydride. The probe DNA having an amino group at the 5 ′ end may be immobilized using this carboxyl group in the form of a succinimide ester. When PNA is used for the probe DNA, it can be obtained by reacting the amino terminus of the probe DNA in the same manner.

  As shown in FIG. 7C, separately from this, poly T-gold nanoparticles (20 nm) to which poly T (T30) is fixed are prepared. The immobilization of poly-T on gold nanoparticles is prepared by synthesizing those having SH groups at the 5 'end and mixing with gold nanoparticles.

  (B) The sample mRNA mixed solution (including the RNase inhibitor) described in (A), the indexing particle suspension described in (A), and the poly T-gold nanoparticle suspension described in (C) are mixed and heated to 70 ° C. . As the reaction solution, a 50 mM citrate buffer solution (pH 7) containing 0.1 to 1 M NaCl and a surfactant as a dispersant is used. Gently stir at 45 ° C. for 1 hour to keep the particles always in suspension. Then, each of the mRNAs 41b, 42b, 43b and 44b is captured by the indexing particles 41 to 44 having complementary DNA probes 41a, 42a, 43a and 44a, and the poly A portion of the captured mRNA has a poly T-gold nanoparticle. Particles combine.

  FIG. 8 shows that a hybrid of the DNA probes 41a, 42a, 43a and 44a, the mRNAs 41b, 42b, 43b and 44b and the poly T-gold nanoparticles of the indexing particles 41 to 44 is obtained by mixing the particles, the sample and the label. It is a figure which shows the result. In each hybrid, the indexing particle and the surface probe correspond, and the probe and individual mRNA correspond. In this case, poly T of some poly T-gold nanoparticles may bind reversely to poly A of mRNA, or may be bound with a shift of one base. However, from the effect of the present invention, There is no hindrance. In FIG. 8, probes 41a, 42a, 43a and 44a to be fixed to 41 to 44 are fixed at the 3 'end, and gold nanoparticles in which poly T is kneaded at the 5' end are used. For this reason, the complex structure as shown in FIG. 8 is obtained, but it is also possible to use a probe in which probes 41a, 42a, 43a and 44a are fixed at the 5 'end.

  The substrate having a hybrid obtained in Example 5 is scanned with an electron beam by the scanning electron microscope 300 to obtain an SEM image and an element analysis image, and can be evaluated by the method described with reference to FIG.

  FIG. 9 is a diagram showing a state in the middle of the process that expects higher accuracy than the homogeneous reaction described in FIG. 8 in which the indexing particles, the sample mRNA, and the poly T-gold nanoparticles are reacted at the same time. First, the DNA probes 41a, 42a, 43a and 44a of the indexing particles 41 to 44 are reacted with the mRNAs 41b, 42b and 43b to prepare the indexing particles and the sample mRNA hybrid, and then unnecessary components are washed away. Next, poly T-gold nanoparticles shown in FIG. 7C may be reacted.

  In Examples 4 and 5, a plurality of biological substances contained in a sample can be simultaneously detected by being distributed to indexing particles on which a specific probe is fixed. At this time, since the reaction between the sample and the indexing particles can be performed in a suspended state at a time, the advantage that the reaction can be performed uniformly compared to the DNA microarray (DNA chip) or protein array that performs the reaction on the substrate surface, the particles are contained in the solvent. Because it is dispersed, simultaneous measurement of multiple items is possible by taking advantage of quick response.

  For example, a colon cancer excision tissue fragment frozen in liquid nitrogen and directly added to phenol chloroform and homogenized to extract total RNA, but prepared in a 50 μl total RNA solution to be adjusted, a sequence of about 40 bases complementary to various mRNAs Here, 0.1% (w / v) of magnetic particles (2.8 μm) in which the contents of gallium, yttrium, cesium, osmium, and platinium with various probes fixed thereto are changed to 8 levels each are fixed. ) Add the mixed solution as indexing particles. The conditions are such that the salt concentration is 1 M and the citric acid concentration is 50 mM, and the mixture is allowed to stand at 70 ° C. for 1 minute and then gently stirred at 45 ° C. for 1 hour to perform hybridization. The magnet is brought close to the outside of the container, the magnetic particles are attracted and the supernatant is removed, followed by washing with 1M NaCl, 50 mM citrate buffer (pH 7). Suspend gold nanoparticle labeled poly T in the same buffer and stir for 1 hour at room temperature.

The indexing particle-mRNA -gold nanoparticle-labeled poly-T complex that has undergone the hybridization reaction is collected on the wall of the container with a magnet, washed with 1M NaCl, 50 mM citrate buffer (pH 7), washed with 70% aqueous ethanol, 70 Suspend in 100 μl% ethanol. 1 μl is dropped into the container 102 (FIG. 4) of Example 4 and dried under reduced pressure for 3 hours. The depressurization time is increased because it does not affect the high vacuum of the scanning electron microscope.

  The particles thus prepared are observed with a scanning electron microscope. At this point, the number of colloidal gold particles captured on the surface of the magnetic particles can be counted. Next, the detection is switched to the energy dispersive characteristic X-ray detection mode. When the electron beam 300-4 hits the indexing particle, X-rays having a wavelength specific to the element on the surface are emitted. This specific wavelength X-ray is detected by an energy dispersive X-ray detector or a wavelength dispersive X-ray detector 300-8, and elemental analysis is performed. By this operation, the magnetic particles to which the gold nanoparticles are bound can be indexed, and the amount of the mRNA molecule hybridized with the corresponding gold nanoparticle labeled probe is identified. Here, one gold nanoparticle corresponds to one mRNA molecule.

  As in Example 5, when 2 elements having different compositions of gallium, yttrium, cesium, osmium and platinium are used as the indexing elements, 25,000 kinds of human mRNAs can be comprehensively measured.

  In this way, by using the particles with different element contents according to the present invention, it is possible to use a particle counting technique without using a conventional probe tip for compartmentalized division with a complicated structure. Single-molecule mRNA profiling is possible.

(Example 6)
In Examples 4 and 5, indexing particles to which individual probe DNAs are immobilized and particles for detection and quantification having a common sequence of poly T are used, but in Example 6, in order to further increase the specificity, individual sequence probes 41 a to 41- It can be developed into a system using indexing particles 41 to 44 having 44a and coefficient labeling particles having probes 41d to 44d corresponding to the indexing particles. This will be described.

  FIG. 10A schematically shows the individual probes 41a, 42a, 43a, 44a and 45a fixed to the surfaces of the indexing particles 41 to 45, as in Examples 4 and 5, and FIG. ) Is a sample having the mRNA sequence to be measured, the specific biological material 41b, 42b, 43b and 44b having poly A hybridizing to the probes 41a, 42a, 43a and 44a, and the same specific biological material. FIG. 10C schematically shows a state in which the probes 41c, 42c, 43c and 44c of the portion of the sequence are added, FIG. 10C shows the probes 41c, 42c, 43c, 44c and 45c (here, 45c) of the above sequence Synthetic oligonucleotides (20-50 bases) 41d, 42d, 43d, 44d complementary to (B) not in the sample) The example labeled with gold nanoparticles (20 nm) is a diagram schematically showing. FIG. 10 (D) is a diagram showing the state of the indexing particle, sample, and gold nanoparticle oligonucleotide after hybridization.

  Indexing particles 41 to 45 to which probes 41a, 42a, 43a, 44a and 45a are immobilized are reacted with the mRNA mixed sample described in (B), and indexing particles with the sequences of probes 41b, 42b, 43b and 44b of each mRNA. Synthetic oligonucleotides (20 to 50 bases) 41d, 42d, 43d complementary to the sequence corresponding to the indexing particle, that is, another portion 41c, 42c, 43c, 44c of the same mRNA sequence. , 44d with gold nanoparticles (20 nm). Magnetic particles are used as the indexing particles. First, indexing particles for mRNA that is abundant in mRNA such as β-globin and β-actin are added, and only the reacted particles are attracted and removed by a magnet. Since only unnecessary mRNA is roughly removed here, the hybridization reaction time may be as short as about 15 minutes. Next, after adding indexing particles corresponding to mRNA of the item to be actually measured and reacting for 30 minutes, unreacted substances are removed by washing. Further, gold nanoparticles having a probe corresponding to the mRNA sequence are added and allowed to react for 30 minutes. What is important here is that the synthetic oligonucleotides 41d, 42d, 43d, 44d, and 45d labeled with gold particles are a mixture. Therefore, in the obtained particle hybrid, the gold nanoparticles 46 are detected only in the hybrids 41e, 42e, 43e, and 44e, and the gold nanoparticles are not substantially detected in 45e having no target substance in the sample.

  Advantages of the sixth embodiment will be described. For example, consider the case where mRNA has a similar sequence. Assume that the mRNA 41b in FIG. 10B hybridizes to the probe 42a in addition to the 4-index particle probe 41a. Such cases can occur frequently in DNA hybridization. That is, on the surface of the indexing particle 42, mRNA having the sequence 41b as well as mRNA having the original sequence 42b is captured. At this time, by dividing the probe group of FIG. 10 (C) into two series of those containing 41d and those containing 42d and reacting them separately, even mRNA that cannot be separated and identified only by the probe sequence of the indexing particles is shown in FIG. 10 (C). It can be separated and counted by the difference in the probe sequence. In the indexing particle group to which the gold nanoparticles having the arrangement corresponding to the indexing beads are not added, the gold nanoparticles do not bind to the surface of the indexing particle 42a and are not detected.

(Example 7)
In Example 7, multi-detection of biological material by antigen-antibody reaction will be described with reference to FIG.

F (ab ′) ₂ fragments obtained by papain degradation of various monoclonal antibodies are prepared. Magnetic particles (3 μm) coated with polystyrene containing 0%, 1%, 2%, 3%, 4%, 6%, 8%, and 10% of Hf, Pt, and Ce in weight percent are prepared as indexing particles. . Different F (ab ′) ₂ 91a, 92a, 93a, and 94a are fixed to the indexing particles 91, 92, 93, and 94, respectively, to obtain F (ab ′) ₂ labeled indexing particles. A known method is used as the fixing method. For example, in the fixing method, a small amount of a monomer having a functional group introduced into polystyrene may be mixed to make use of a functional group exposed on the surface, or the surface may be oxidized with oxygen plasma to produce 3-aminoethylaminopropyltrimethoxy. Silane is reacted to introduce an amino group on the surface, the amino group is converted to a carboxyl group with succinic anhydride, and the probe DNA having an amino group at the 5 ′ end is fixed by using this carboxyl group in the form of a succinimide ester. As F (ab ′) ₂ , for example, antibodies against AFP91b, CEA92b, EpCAM93b and the like can be used. The F (ab ′) ₂ labeled particle mixture is coated with sphingophospholipid.

Serum diluted twice with 2 × PBS (1 × PBS: 0.15 M NaCl, 50 mM sodium phosphate buffer (pH 7.4)) containing 0.2% Tween 20 in the F (ab ′) ₂ -labeled indexing particle mixture (From healthy people and from liver cancer patients). After stirring at 37 ° C. for 10 minutes, the indexing particles are sucked into the container wall with a magnet, and the supernatant is discarded. Wash with PBS containing 0.1% Tween20. 91c, 92c, 93c, and 94c labeled with gold nanoparticles are added to a monoclonal antibody of an antigen to be measured (using an epitope different from F (ab ′) ₂ immobilized on indexing particles). This monoclonal antibody is also made into F (ab ′) ₂ , and about 3 molecules of SH groups are introduced per molecule with iminothiolane, and the SH groups are labeled with gold nanoparticles (20 nmφ). A gold nanoparticle labeled antibody against AFP is represented by 91c, a gold nanoparticle labeled antibody against CEA by 92c, a gold nanoparticle labeled antibody by EpCAM by 93c, and a gold nanoparticle labeled antibody by other antigens by 94c. After reacting at 37 ° C. for 10 minutes to obtain indexing particle-antigen-gold nanoparticle hybrids 91d, 92d, 93d, 94d, the supernatant was discarded and washed with PBS containing 0.1% Tween 20, followed by 50% ethanol. Wash with pure water. This step denaturates the protein, but does not disrupt the indexing particle-antigen-gold nanoparticle hybrid. 1 μl is added to the base container 102 of FIG. 4 and dried under reduced pressure, and then subjected to shape and elemental analysis with a scanning electron microscope.

  As a result, the surface of the indexing beads for AFP is more often used when using serum from cancer patients, such as 64 gold nanoparticles from serum from healthy individuals and 3200 from serum from cancer patients. Gold nanoparticles are detected. In CEA and EpCAM, there is no great variation, and in this specimen, only about 30 to 100 solid gold nanoparticles are observed on the surface of the indexing beads for these samples.

  As described above, the present invention can be implemented in the form of several examples. In any case, the hybridized sample DNA and the like are detected substantially every molecule, so that the sensitivity can be increased. Can achieve sensitivity exceeding conventional methods. Since it is possible to detect a very small amount of DNA (RNA) in a very small volume, it becomes possible to detect a target DNA (RNA) without pretreatment amplification by PCR, which was impossible in the past. Since the size and shape of the labeled particles can be changed for detection, it is possible to perform multi-analysis of 6 to 10 different samples using the same element. In addition to the use for conventional differential hybridization, this technique enables the sample polynucleotide to be captured on the element with the same probe and detected with a different labeled probe. The multi-analysis technique of the present invention has an advantage that alternative splicing detection and multiple SNP typing can be performed with one element.

It is a conceptual diagram which shows a part of DNA chip of Example 1 of this invention with a perspective view. It is a conceptual diagram explaining the aspect which observes the probe tip 1 demonstrated in FIG. 1 with the scanning electron microscope. It is a conceptual diagram explaining the method to identify the mRNA molecule | numerator with which the gold nanoparticle label | marker probe hybridized from the correspondence of a SEM image and an elemental analysis image. It is a figure which shows the concept of the biological sample measurement of Example 3 of this invention. From the SEM image obtained by the detector 300-6 and the elemental analysis image obtained from the energy dispersive characteristic X-ray detector 300-8, the position and size of the indexing particles 41 to 44 are specified and the indexing particles 41 to 41 are identified. It is a figure explaining evaluation of the specific biological material to which the labeled particle | grains hybridized to 44 were added. (A) is a diagram schematically showing the indexing particles 41 to 44 of Example 3 and the probes 41a, 42a, 43a and 44a fixed to the surface, and (B) is a probe 41a, 42a, to which labeled particles are added. The figure which shows typically the specific biological material 41b, 42b, 43b, and 44b which hybridizes to 43a and 44a, and (C) is a figure which shows typically a mode that the probe and the specific biological material were hybridized. is there. (A) is a diagram schematically showing the indexing particles 41 to 44 of Example 4 and the probes 41a, 42a, 43a and 44a fixed to the surface, and (B) is hybridized to the probes 41a, 42a, 43a and 44a. The figure which shows typically the specific biological material to which poly A to be added is shown, and (C) is the figure which shows typically the poly T to which the label | marker which hybridizes with poly A was added. The result of having obtained the hybrid of DNA probe 41a, 42a, 43a and 44a of the indexing particles 41-44, mRNA 41b, 42b, 43b and 44b, and poly T-gold nanoparticle by mixing operation of particle | grains, a sample, and a label | marker is shown. FIG. It is a figure which shows the condition of the middle process of the process which anticipates still higher precision than the homogeneous reaction which demonstrated the indexing particle | grains demonstrated by FIG. 8, sample mRNA, and the poly T-gold nanoparticle simultaneously. (A) is a figure which shows typically individual probe 41a, 42a, 43a and 44a currently fixed to the surface of the indexing particle | grains 41-44 similarly to Example 4, 5, (B) is a probe 41a, 42a. , 43a and 44a, and the specific biological material 41b, 42b, 43b and 44b to which poly A is hybridized, and probes 41c, 42c, 43c and 44c of another part of the same specific biological material. FIG. 6C schematically shows a state in which synthetic oligonucleotides (20 to 50 bases) 41d, 42d, 43d, and 44d complementary to the probes 41c, 42c, 43c, and 44c of the above sequence are made into gold nanoparticles. It is a figure which shows typically the example labeled with (20 nm). It is a figure explaining the example of the multi detection of the biological material by an antigen antibody reaction.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Chip | tip, 101 ... Silicon substrate, 102 ... Probe fixing area | region, 103 ... Teflon-type water-repellent resin, 11-14, 41a, 42a, 43a, 44a, 45a, 41d, 42d, 43d, 44d, 45d ... Probe 21-24, 46: Gold nanoparticles, 201-204: DNA fragments, 41b, 42b, 43b, 44b, 45b ... Specific biological substances 300 hybridizing to probes 41a, 42a, 43a, 44a, 45a ... Scanning electrons Microscope, 300-1 ... electron gun, 300-2 ... focusing lens, 300-3 ... scanning coil, 300-4 ... electron beam, 300-5 ... secondary electron, 300-6 ... detector, 300-7 ... element X-rays with specific wavelengths, 300-8 ... Energy dispersive X-ray detectors, 30, 50 ... SEM images, 31, 54 ... Gallium images, 32, 53 ... Aluminum image, 33,52 ... yttrium image, 34,51 ... chromium image, 37 ... gold nanoparticle labeling CEA molecule, 36 ... gold nanoparticle labeling CD44 molecule, 35 ... gold nanoparticle labeling EpCAM molecule, 41-44 ... indexing particles composed of different elemental compositions, 41b, 42b, 43b, 44b, 45b, 41c, 42c, 43c, 44c, 45c ... specific biological sample arrangement sites, 41e, 42e, 43e, 44e, 45e ... Hybrids of indexing particles, specially specified biological materials, and gold nanoparticle labeled probes.

Claims (18)

A plurality of indexing particles of a predetermined size composed of an alloy of at least two kinds of transition metal elements or semiconductor elements, the indexing particles having a probe bound to the surface thereof, and a biological material capable of binding to the probe. A step of contacting the biological material to which the labeled particles are bound,
Fixing the plurality of indexing particles from the step on a substrate;
Obtaining an electron beam scanned image of the indexing particles from secondary electrons obtained by scanning the substrate with an electron beam, and obtaining an electron beam scanned image of the labeled particles;
Obtaining an elemental analysis image from X-rays having specific wavelengths according to the compositional elements of the indexing particles obtained by scanning the plurality of indexing particles fixed on the substrate with an electron beam, and the electron beam scanning Identifying the composition and position of the elements of the plurality of indexing particles by comparing an image and the elemental analysis image,
An inspection method for a biological material, wherein the amount and type of the biological material are detected by matching the amount of the labeled particle counted from the electron beam scanning image of the labeled particle with the composition and position of the identified indexing particle.   A plurality of types of elements used for indexing particles of a predetermined size composed of at least two types of transition metal elements or semiconductor element alloys are Sc, Ti, Ga, Ge, Y, Zr, Nb, Ru, Rh, Pd. , Ag, Cd, In, Sb, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hf, Ta, W, Re, Os, Ir, Pt The method of claim 1, wherein the method is selected from the group consisting of: Au, Tl, Bi, and Th. The label particles are
Gold nanoparticles or transition metals except atomic number up to 79 in the periodic table, metals number 13, number 31, number 32, number 49, number 50, number 51, number 81, number 82, number 83 And a plurality of metal or semiconductor alloy particles selected from the group consisting of No. 14, No. 33, No. 34, and No. 52 semiconductors.   The method according to claim 1, wherein a plurality of elements other than gold are used for the indexing particles, and gold nanoparticles are used for the labeling particles.   The method according to claim 1, wherein the indexing particle has a size of 0.5 to 5 μmφ.   The method according to any one of claims 1 to 5, wherein the size of the labeling particles is 10 nm to 50 nmφ.   The method according to claim 1, wherein the biological material is DNA, RNA, or protein.   The method according to any one of claims 1 to 7, wherein the probe and the biological substance are bonded using a complementary bond between oligonucleotides.   The method according to any one of claims 1 to 7, wherein the binding between the probe and the biological substance utilizes an antigen-antibody reaction. A mixture of a plurality of indexing particles used in the method for inspecting a biological material according to any one of claims 1 to 9,
Various probes that are composed of at least two kinds of transition metal elements or semiconductor element alloys, and have affinity for different biological materials for each of a plurality of particles of a predetermined size that have different composition ratios of the elements. only contains a plurality of indexing particles fixed by a one-to-one correspondence, the size of the particles is 0.5～5Myuemufai, mixture. A mixture of a plurality of indexing particles used in the method for inspecting a biological material according to any one of claims 1 to 9,
Various probes that are composed of at least two kinds of transition metal elements or semiconductor element alloys, and have affinity for different biological materials for each of a plurality of particles of a predetermined size that have different composition ratios of the elements. And a plurality of kinds of elements used for the indexing particles are Sc, Ti, Ga, Ge, Y, Zr, Nb, Ru, Rh, Pd, Ag, Cd, In, Sb, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hf, Ta, W, Re, Os, Ir, Pt, Au, Tl, it is selected from the group consisting of Bi, and Th, mixed compounds. The mixture according to claim 10 or 11 , wherein the biological material is DNA and the probe is a DNA probe. The mixture according to claim 10 or 11 , wherein the biological substance is an antigen and the probe is an antibody. Comprising a combination of a mixture according to any one of claims 1 0-13, a mixture of a plurality of label particles,
A plurality of labeled particles composed of an alloy of at least two kinds of transition metal elements or semiconductor elements, each of which has a different biological material fixed to each of a plurality of particles of a predetermined size having different composition ratios of the elements. Containing the mixture. In the periodic table, the metal or semiconductor is a transition metal excluding No. 43 up to atomic number 79 and Nos. 13, 31, 32, 49, 50, 51, 81, 82, 83 The mixture according to claim 14 , wherein the mixture is any one of a metal, a No. 14, a No. 33, a No. 34, and a No. 52 semiconductor. The mixture according to claim 14 or 15, wherein the particles are in the size range of 10 nm to 50 nmφ. Various ligands that are composed of an alloy of at least two kinds of transition metal elements or semiconductor elements and have affinity for different biological materials for each of a plurality of particles of a predetermined size having different composition ratios of the elements. Preparing a first particle group fixed in a one-to-one correspondence,
Bringing the biological material labeled with second particles into contact with the first particles,
The first particle group contacted with the biological material labeled with the second particle is fixed to a substrate surface , the substrate surface is scanned with an electron beam, and the first particle group is obtained from secondary electrons obtained. And obtaining an electron beam scanned image of the second particles,
Obtaining an elemental analysis image from X-rays having specific wavelengths according to the compositional elements of the first particle group obtained by scanning the first particle group on the substrate surface with an electron beam;
The elemental composition and position of each particle of the first particle group is identified by comparing the electron beam scanning image and the elemental analysis image,
The amount of the second particle counted from the electron beam scanning image of the second particle is associated with the elemental composition and position of the identified first particle, and each particle of the first particle group A method for examining a biological material, wherein the biological material bound to the ligand is identified and the amount thereof is evaluated. The method according to claim 17 , wherein the ligand is DNA, RNA, or an antibody or a fragment thereof, and the biological material is DNA, RNA, or an antigen.

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