JP2005249429A - Reaction detection chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve the S/N ratio by increasing stably a signal component, and to improve detection capacity of a DNA chip, by stabilizing the magnitude of the signal component by manufacturing a three-dimensionally arranged probe capable of controlling stably the number of probes and uniformizing specimen supply to the probes in order to improve the detection capacity in a reaction detection chip. <P>SOLUTION: In this reaction detection chip where a spot is formed by gathering and immobilizing on a substrate a plurality of such porous particles that probe molecules are bonded to the surface including pores of each porous particle, the porous particles are transparent to incident light, and the porous particles are immobilized in the monolayer state on the substrate. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a reaction detection chip that enables recognition of a large number of functional molecules used for genetic diagnosis and physiological function diagnosis.

  With recent advances in biotechnology, reaction detection chips that enable recognition of functional molecules are playing an increasingly important role. Of the reaction detection chips, a DNA chip that has recently been attracting attention will be described as an example.

  Detection of gene mutations, particularly single nucleotide polymorphisms (single nucleotide mutations in the nucleotide sequence) is not only effective for diseases caused by mutations, such as cancer diagnosis, but also genes related to the etiology of multifactorial diseases It is also necessary for investigating the degree of drug responsiveness and side effects of individuals. In addition, it is very important to investigate the gene expression status, that is, the status where gene information is read by mRNA and the corresponding protein is made, to understand life phenomena and diseases at the gene level and to develop new drugs. . It is known that a DNA chip is effective as a means for quickly detecting this gene mutation or gene expression status.

  A DNA chip is a probe in which molecules having the property of binding specifically to various types of DNA as specimens are used as probes, and each probe is arranged and fixed on the surface of a carrier corresponding to each compartment. is there. This probe is generally a single-stranded DNA or oligonucleotide having a base sequence complementary to a single-stranded sample DNA.

  The sample solution containing the sample DNA is brought into contact with the surface of the DNA chip on which the probe is fixed, and binding is caused between the sample DNA and the corresponding probe. This binding usually utilizes the property of hydrogen bonding between the paired bases of the probe and the sample DNA, that is, hybridization. Then, by detecting the binding due to the hybridization by some means, the sample DNA bound to the probe can be identified and quantified.

  Here, the contact between the probe and the sample liquid is performed by dropping the sample liquid onto the region of the carrier surface on which the probe is fixed and covering the sample with a cover glass or the like for the purpose of effectively acting a small amount of sample liquid on the probe. The method is generally carried out by a method in which the liquid is evenly distributed, the so-called “cover glass method”.

  Further, as the detection means, at present, it is common to add a fluorescent dye molecule as a label to the sample DNA in advance and measure the intensity of fluorescence from the fluorescent dye molecule of the sample DNA bound to the probe.

  Patent Document 1 discloses that porous carrier particles carrying reactive substances capable of binding to different detection targets on porous inner surfaces of the porous particles are integrally used as a porous carrier particle probe as a porous carrier particle. There has been disclosed a reaction detection chip characterized in that it is arrayed and fixed to one or more of a plurality of micro-sections provided on a substrate while maintaining the reactivity of the pore internal surface. This porous carrier particle probe is used in one embodiment of the present invention.

JP 2001-281251 A

  By the way, the fluorescence measured at the time of detection is not limited to the fluorescence from the fluorescent dye molecule of the sample DNA hybridized with the probe, that is, the “signal component”. Fluorescence from fluorescent dye molecules of sample DNA bound to the substrate and probe by a mechanism other than hybridization, fluorescence from the substrate itself, dust and dirt adhering to the substrate, or fluorescence inside the measurement optical system, and more There is also a fluorescent component (herein referred to as “noise component”) that hinders detection, such as a dark signal that the measurement optical system has.

  If the sum of such noise components is constant for each probe of each DNA chip, a signal component can be obtained by subtracting the constant value from the measured fluorescence value. However, in practice, the sum of noise components is not constant but varies. It is practically impossible to accurately separate the signal component and the noise component in each probe. Therefore, the ratio of the signal component to the noise component in the measured fluorescence (S / N) until the influence of the variation of the noise component is sufficiently reduced so that the magnitude relationship of the measured fluorescence values is consistent with the magnitude relationship of the signal components. Ratio) is necessary. In particular, in the detection of single nucleotide polymorphisms, since a small difference in signal components must be identified, this improvement in the S / N ratio is very important.

  One way to improve the S / N ratio is to reduce the noise component. For this purpose, the confocal method is generally used for fluorescence measurements. The confocal method has an effect of reducing fluorescence emitted from a position whose height differs from the probe position by several μm or more because the depth of focus is narrow. Therefore, it is possible to greatly cut a noise component generated from a position having a height different from that of the probe.

  However, in the confocal method, since it is necessary to focus precisely, the fluorescence measuring apparatus has an elaborate structure and is expensive. The substrate is also expensive because high thickness uniformity is required. In addition, there is no effect on the noise component generated from the position where the height coincides with the probe.

Therefore, a radical method for improving the S / N ratio, which is different from the confocal method, is expected.
Another way to improve the S / N ratio is to increase the signal component. One of the most effective ways to increase the signal component is to place the probe that has been placed two-dimensionally in a three-dimensional manner, including the direction perpendicular to the substrate, and make the signal source three-dimensional. It is to collect light. This idea is contrary to the standard confocal method in fluorescence measurement, which collects signal components only from a narrow range in the direction perpendicular to the substrate surface. Under such circumstances, a DNA chip based on this idea has not been sufficiently developed, and the conventional developed product has the following problems.
(I) If the probe is arranged in a three-dimensional arrangement as compared with the two-dimensional arrangement, it is difficult to stably control the number of probes per projection unit area on the substrate.
(II) When the probe is arranged in a three-dimensional arrangement as compared with the two-dimensional arrangement, it is difficult to make the sample supplied uniformly to the probe when the cover glass method is used.

Therefore, although the signal component is increased by arranging the probes in a three-dimensional arrangement, the detection capability is not improved due to the large variation of the signal component.
Therefore, in order to improve the detection capability, it is indispensable to create a three-dimensionally arranged probe in which the number of probes can be stably controlled and the sample supply to the probe is uniform, and the size of the signal component is stabilized. is there.

  Thus, it is an object of the present invention to stably increase the signal component to improve the S / N ratio and improve the detection ability of the DNA chip.

The present invention has solved the above problems by the following means.
(1) A plurality of porous particles in which probe molecules are bonded to a surface including pores of a porous particle are assembled and fixed to a substrate to form a spot, and the spot is irradiated with incident light, In a reaction detection chip for detecting an analyte molecule bound to a probe molecule, the porous particles are transparent to the incident light, and the porous particles are fixed in a single layer state on the substrate. Characteristic reaction detection chip.
(2) An optically detectable labeling substance, for example, a fluorescent dye molecule, a phosphorescent dye molecule, a functional dye molecule, etc., preferably a fluorescent dye molecule, is added to the analyte molecule. The reaction detection chip according to item 1, wherein the generated optical signal is detected.
(3) The reaction detection chip according to item (1), wherein a linear absorption coefficient of the porous particles with respect to the incident light is 10 μm ⁻¹ or less.
(4) The reaction detection chip according to item 1, wherein a linear absorption coefficient of the substrate with respect to the incident light is 100 μm ⁻¹ or more.
(5) The reaction detection chip according to item 1, wherein the porous particles have a particle size of 0.1 μm or more and 200 μm or less.
(6) A thermoplastic organic film is formed on the substrate, the porous particles are spotted on the organic film, and the substrate is heated to soften the organic film. 2. The reaction detection chip according to claim 1, wherein the porous particles are fixed by being embedded in the organic film, and excess porous particles that are not fixed are removed.
(7) The reaction detection chip according to item 6, wherein the thickness of the organic film is not more than half of the particle diameter of the porous particles.
(8) The reaction detection chip according to item 6, wherein the organic film is formed by dissolving the material of the organic film in an organic solvent by a spin coating method.
(9) The reaction detection chip according to item 6, wherein the spotting is performed by dispersing the porous particles in a liquid.
(10) The reaction detection chip according to item 6, wherein the excess porous particles are removed by ultrasonic cleaning.
(11) The reaction detection chip according to item 6, wherein the organic film is a vinyl acetate film.
(12) The reaction detection chip according to item 1, wherein the probe molecule and the analyte molecule are any one of nucleic acid, protein, and glycoprotein.

  In a normal DNA chip, the probe is directly fixed to a flat substrate, so that the arrangement of the probe is two-dimensional (see FIG. 7). If the arrangement of the probes can be made three-dimensional so that the probes are distributed not only in the direction parallel to the substrate surface but also in the direction perpendicular to the substrate surface, the number of probes per projected unit area with respect to the substrate surface is remarkably high. Will increase. Therefore, if signals relating to the direction perpendicular to the substrate surface from the analyte bound to the probe can be collected, the signal component can be increased by making the probe arrangement three-dimensional.

  First, the means disclosed in Japanese Patent Application Laid-Open No. 2001-281251 is used for the purpose of three-dimensional probe arrangement. That is, porous particles are used as the probe carrier, and the probes are bonded to the entire surface including the pores inside the porous particles. And this porous particle with a probe is fixed on a board | substrate, and the spot of a reaction detection chip | tip is formed (refer FIG. 4). Since the specimen can diffuse into the porous particles via the pores, the probes existing inside the porous particles can also contribute to the binding reaction with the specimen.

  Next, a method for collecting signals from the analyte bound to the probe inside the porous particle is presented. This signal is a response light when an incident light is irradiated onto an optically detectable labeling substance, generally a dye, previously added to the specimen, as in the standard detection method in the current DNA chip. Generally, a fluorescent dye is selected as the dye, excitation light is irradiated as incident light, and fluorescence is detected as response light.

  In the case of a specimen bound to a probe inside the porous particle, in order to excite the fluorescent dye added to the specimen, that is, the fluorescent label, the excitation light is irradiated through the medium called the porous particle. Therefore, if porous particles that are transparent to the excitation light are selected, the excitation light passes through the porous interior without being attenuated and irradiates the fluorescent label. The wavelength of the fluorescence excited by the excitation light irradiation is only about 20 nm longer than the wavelength of the excitation light, and the difference from the wavelength of the excitation light is small. Therefore, the permeability of the fluorescence to the porous particles is almost equal to the permeability of the excitation light to the porous particles, and if the porous particles are transparent to the excitation light, it is considered transparent to the fluorescence. Good. Therefore, the excited fluorescence is transmitted through the porous interior without being attenuated. By selecting porous particles that are transparent to excitation light in this way, it becomes possible to collect signals from fluorescent labels throughout the porous particles, and thus increase the signal component.

Finally, a method for stabilizing the size of the signal component by controlling the number of three-dimensionally increasing probes and making the supply of the analyte to the probes uniform is presented.
Control of the number of probes is achieved by suppressing thickness variations between spots. This is because the number of probes per projected unit area with respect to the substrate is proportional to the thickness of the spot, and the magnitude of the signal component is proportional to the thickness of the spot.

  In order to make the supply of the sample to the probe uniform, it is a spot suitable for the cover glass method used at the time of the binding reaction, that is, a method that spreads evenly a small amount of sample liquid dropped on the spot portion of the substrate by covering the cover glass. It needs to be in form. This is because in order to perform the binding reaction uniformly, it is an essential condition that the thickness of the layer of the specimen liquid spreading on the spot is uniform.

  Regarding the fixing of the probe-attached porous particles to the substrate, for example, JP-A-2001-281251 discloses a method of spotting and fixing a paste in which the particles are dispersed in a sol or polymer to the substrate. Some sols and polymers have a network structure in which analyte molecules can diffuse when solidified. In this case, the analyte molecules can reach the probe. If it is a sol or polymer that becomes transparent when solidified and has a small autofluorescence, there is no problem in measuring the fluorescence intensity.

  However, the problem with this method is that the thickness of the spot, that is, the number of porous particles with a probe in the direction of the thickness of the spot cannot be controlled. If this method is used, the spot will inevitably consist of a stack of porous particles with a probe, but the thickness and cross-sectional shape of the spot will depend on the concentration of the same particle in the paste and the amount of paste deposited. This is because it is difficult to control stably even if is constant. That is, with this method, the thickness of the spot varies greatly between spots and within spots.

  Since the thickness of the spot varies in this way, even when the cover glass method is used, the distance between the lower surface of the cover glass and the upper surface of the spot is not constant, and the thickness of the sample liquid layer spreading on the spot is not uniform. . As a result, the way in which the sample liquid permeates varies, and the binding reaction between the probe and the sample becomes non-uniform. In addition, even if the sample liquid sufficiently penetrates and reaches the probe, the thickness of the porous particle with probe through which fluorescence passes and the thickness of the paste layer for fixation vary depending on the position, causing fluctuation in light transmission and being observed from the outside The fluorescence intensity varies greatly.

Because of such inconvenience, the method of stacking and fixing the probe-attached porous particles is not suitable for the cover glass method used during the binding reaction.
Therefore, it is necessary to adopt a method in which the porous particles with probes are arranged in a single layer and fixed to the substrate so that the spot thickness is constant. For this purpose, an optimum method is to form an adhesive layer on the substrate and bury the probe-attached porous particles in the adhesive layer for fixing. The thickness of the adhesive layer should be less than half of the particle size of the probe-attached porous particle, and more than half of the volume of the probe-attached porous particle should be exposed to the outside of the adhesive layer. Make contact.

  As a specific example of the production method, a thermoplastic organic film is formed as an adhesive layer on a substrate by a spin coat method or the like, and probe-attached porous particles dispersed on the organic film are spotted. Then, the organic film is softened by heating the substrate, and the probe-attached porous particles are buried. After the substrate is cooled and the organic film is cured, excess porous particles with the probe overlaid on the fixed porous particles with the probe are removed by ultrasonic cleaning or the like.

  In this way, a spot in which the probe-attached porous particles are arranged in a single layer and fixed to the substrate is formed. Since the thickness variation of the spot is suppressed, the number of probes per projected unit area on the substrate can be controlled, and the cover glass method used at the time of the binding reaction can be used without any problem.

The higher the transparency of the porous particles, the better. It is desirable that the linear absorption coefficient for the excitation light is 10 μm ⁻¹ or less. Roughly speaking, this corresponds to the fact that excitation light can enter the interior of the porous particles by about 0.1 μm or more. Since the average interval between probe molecules is generally several tens of nanometers, if the transparency is lower than this, the number of probes existing within the excitation light penetration distance will be several or less, and in the excitation light penetration direction, that is, the direction perpendicular to the substrate surface This is because the signal integration effect is reduced.

  In the present invention, the signal from the fluorescent label throughout the entire transparent porous particle is integrated. Therefore, when performing fluorescence detection, the confocal method should be avoided especially when the particle size of the porous particle exceeds 10 μm. It is. This is because the confocal method has a narrow depth of focus, so that signals can be integrated only from a region of several μm in the direction perpendicular to the substrate.

From the viewpoint of improving the S / N ratio, it is also important to reduce the fluorescence of the substrate itself, which is one of the noise components. In a conventional DNA chip, a slide glass with small autofluorescence has been used as a substrate. In the present invention, it should be noted that the fluorescence is integrated with respect to the transparent substrate for the same reason as that with which the fluorescence is integrated with respect to the transparent porous particles with a probe. Even if it is made of a material having low autofluorescence, there is a concern that the noise component becomes large due to the effect of this integration in the transparent substrate. Therefore, the substrate is required to be opaque in addition to the conventional request that autofluorescence is small with respect to excitation light, and specifically, the linear absorption coefficient is preferably 100 μm ⁻¹ or more.

  Since the conventional DNA chip directly fixes the probe molecule to the substrate, glass is preferred as the substrate material for ease of bonding. On the other hand, in the present invention, the probe molecule is fixed to the porous particle, and the role of the substrate is to fix the porous particle via the adhesive layer, so that the material of the substrate does not need to be related to glass. Further, since the confocal method is not used, high thickness uniformity of the substrate is not required. Even with the condition of being opaque, the selection range of the substrate is wider than that of the conventional DNA chip.

  As disclosed in JP-A-2001-281251, the particle size of the porous particles is preferably 0.1 μm or more and 200 μm or less, preferably 0.1 μm or more and 100 μm or less, more preferably 1 μm or more and 10 μm or less. Since the average interval between probe molecules is generally several tens of nanometers, if the particle size is less than 0.1 μm, the number of probes existing within the excitation light penetration distance will be several or less, and the signal integration effect will be reduced, which is meaningless.

On the other hand, when the particle diameter exceeds 200 μm, inconvenience occurs when the binding reaction between the probe and the specimen is caused. The reason for this will be described below.
When performing hybridization, which is a binding reaction between a DNA probe and a DNA sample, a cover glass method is generally used for the purpose of effectively acting a small amount of sample solution on the probe. Since the sample solution is difficult to obtain and valuable, the amount of sample solution used per DNA chip is 40 μl or less, usually about 10 μl. If 10 μl of the sample liquid is dropped on the substrate spot and a 20 mm square cover glass is placed on it to spread the sample liquid evenly, the sample liquid will form a 25 μm thick liquid layer between the substrate and the cover glass. Intervene. The thickness of the liquid layer varies depending on the amount of the sample liquid and the cover glass size, but is usually about several tens of μm at most. On the other hand, if the particle diameter is larger than 200 μm and the spot protrudes beyond 200 μm from the substrate, the specimen liquid does not spread evenly even if it is covered with a cover glass, and the manner in which the specimen liquid comes into contact varies between spots. Therefore, from the viewpoint of adaptability to the cover glass method, the particle size is desirably 200 μm or less.

First, the fluorescent dye that labels the analyte molecule is determined. Then, a porous particle having a linear absorption coefficient with respect to excitation light for exciting the fluorescent dye is selected to be 10 μm ⁻¹ or less is selected as a probe carrier.

This linear absorption coefficient reflects the optical characteristics specific to the constituent material of the porous particles and the properties as a porous body, and depends on the pore diameter, pore volume, etc. of the porous particles.
Examples of fluorescent dyes include FITC (fluorescein isothiocyanate), Cy3, and Cy5. The wavelength of excitation light necessary to excite these fluorescent dyes is in the range of 450 to 650 nm. Therefore, as the porous particles, those that are transparent to visible light are candidates. From the viewpoint of having a suitable surface property and chemical stability as a probe carrier, porous particles made of glass or a transparent plastic such as polypropylene or polystyrene are optimal.

  As described above, the particle size of the porous particles is 0.1 μm to 200 μm, preferably 0.1 μm to 100 μm, more preferably 1 μm to 10 μm. Further, the pore diameter of the porous particles is empirically adjusted to be 10 nm or more and 200 nm or less, more preferably 50 nm or more and 100 nm or less.

  Next, probe molecules are bound to the porous particles. This is performed by immersing a porous particle, to which a linker is bound by surface treatment, in a solution of a probe molecule whose end is modified so as to bind to the linker following a known method.

  Alternatively, a technique of directly chemically synthesizing probe molecules on the surface of the porous particles can be used. For example, in the case of oligonucleotide probe molecules, there are established synthetic methods such as the phosphoramidite method.

  Subsequently, a process of fixing the porous particles to which the probe molecules are bonded to the substrate will be described with reference to FIGS. First, as the substrate 1, a plate that is opaque to excitation light and has a size of about 25 mm × 75 mm and a thickness of about 1 mm is prepared. Silicon plates, metal plates, plastic plates that are opaque in the visible light region, and the like can be used.

  A thermoplastic organic film 5 is formed on the substrate 1 as an adhesive layer for fixing the porous particles 3 to which the probes 2 are bonded, that is, the probes-attached porous particles 4. As a forming method, it is preferable to perform spin coating after dissolving the material of the organic film 5 in an organic solvent to obtain an appropriate viscosity. What is required for the material of the organic film 5 is that the autofluorescence is small, the adhesiveness with the substrate 1 is high, the adhesive force to the porous particle 4 with the probe is strong, and the hybridization reaction is not hindered. Specifically, it is water resistant, and specifically, vinyl acetate, polyvinyl alcohol, vinyl chloride, synthetic rubber and the like can be used.

  As shown in FIG. 1, the probe-attached porous particles 4 dispersed in a liquid such as water are spotted on the organic film 5 by an appropriate spotter 6. At this stage, as shown in FIG. 2, the probe-attached porous particles 4 are in a laminated aggregate.

  After the liquid contained in the aggregate has evaporated, the substrate 1 is heated horizontally, and the porous particles 4 with the probe are buried in the softened organic film 5 as shown in FIG. Heating is performed by raising the ambient temperature around the substrate 1 using a thermostat. When a substrate 1 having a high thermal conductivity such as a silicon plate or a metal plate is used, it can be heated by a hot plate. The heating temperature and heating time are determined according to the type of the organic film 5. When the organic film 5 is a vinyl acetate film, a heating temperature of 100 ° C. and a heating time of about 10 minutes are appropriate. By such heat treatment, the probe-attached porous particles 4 naturally settle in the softened organic film 5 and reach the substrate surface.

  At the stage where the organic film 5 is cured by cooling, the probe-attached porous particles 4 embedded in the organic film 5 are fixed to the substrate 1. Subsequently, the non-fixed porous particle 4 with the probe overlaid on the fixed porous particle 4 with the probe is removed by ultrasonic cleaning or the like in water, and the substrate 1 is dried with an air gun or the like.

  In this way, a spot 7 in which the porous particles 4 with probes are arranged in a single layer without any gap and fixed on the substrate 1 is completed. A sectional view of the spot 7 is shown in FIG. 4, and a top view thereof is shown in FIG. For the purpose of visualizing the state in which the probe 2 is bonded to the pores 8 of the porous particle 3, an enlarged view of a part of the probe-attached porous particle 4 is also shown in FIG. 4. Since the porous particles 4 with probes do not overlap each other, the thickness of the spots 7 is stabilized, and the number of probes 2 in the direction perpendicular to the surface of the substrate 1 can be stably controlled. The diameter of the spot 7 should be 10 times or more the particle diameter of the probe-attached porous particle 4 and determined within 10 to 1000 μm according to the spot density required for each DNA chip. Since the number of the porous particles 4 with the probe constituting the spot 7 is at least about 100 or more, even if there is some variation in the particle diameter of the porous particle 4 with the probe, it is averaged in the spot 7 unit. It can be said that the thickness variation between 7 is extremely small. The number of the probe-attached porous particles 4 constituting the spot 7 is allowed to vary to some extent. This is because the fluorescence intensity of the spot 7 depends not on the total projected area of the spot 7 on the substrate 1 but on the number of probes 2 per projected unit area.

  When the whole of the probe-attached porous particles 4 is buried in the organic film 5 at the time of fixation, it is impossible to bond the specimen and the probe 2. Therefore, in order to expose more than half of the volume of the probe-attached porous particle 4 that is fixed in contact with the substrate surface, the thickness of the organic film 5 is less than half of the particle diameter of the probe-attached porous particle 4. To do. If sufficient fixing strength can be obtained, the thickness of the organic film 5 is preferably thin from the viewpoint of increasing the intrusion area of the specimen into the probe-attached porous particles 4 and from the viewpoint of reducing autofluorescence.

  In this way, the reaction detection chip of the present invention is completed. Since the protruding amount of the spot from the substrate is equivalent to one porous particle having a particle size of 200 μm or less, in this reaction detection chip, using a cover glass method that is usually performed in order to spread a small amount of sample liquid uniformly in the spot part, It is possible to cause a uniform binding reaction. The cleaning and detection after the reaction can be handled in the same manner as a conventional reaction detection chip.

  Hereinafter, the present invention will be described in more detail with reference to examples and comparative examples, using a DNA chip using an oligonucleotide probe as an example. However, the present invention is not limited to this example.

[Example]
FITC (fluorescein isothiocyanate) is used as a fluorescent labeling molecule for the specimen. The corresponding excitation wavelength is 494 nm and the fluorescence wavelength is 518 nm.

As the oligonucleotide probe carrier, porous glass particles having a particle diameter of 7 μm, a pore diameter of 50 nm, and a pore volume of 1.3 ml / g are selected. When the absorbance of the porous glass particles was measured, the linear absorption coefficient at a wavelength of 494 nm was 0.1 μm ⁻¹ . The required condition of 10 μm ⁻¹ or less is satisfied, and the transparency is sufficiently high. In order to directly synthesize and carry oligonucleotide probes on the porous glass particles, the following method disclosed in Japanese Patent Application No. 2002-254558 was used.

  Porous glass particles are immersed in a solution of N- (2-aminoethyl) -3-aminopropyltrimethoxysilane dissolved in toluene. By maintaining the solution temperature at 110 ° C. for several hours, amino groups are introduced onto the surface of the porous glass particles. The aminated porous glass particles are immersed in an acetonitrile solution of β-cyanoethyl phosphoramidite corresponding to the first nucleotide of the oligonucleotide to bind the first nucleotide to the particle. Subsequently, an oligonucleotide chain corresponding to each probe is synthesized by an ordinary phosphoramidite method. Then, the porous glass particles with synthetic oligonucleotide chains are immersed in ammonia water for several tens of hours to remove the protecting groups remaining in the chains, thereby completing the porous glass particles with oligonucleotide probes.

  On the other hand, a DNA fragment containing the codon 248 of the tumor suppressor gene p53 is selected as a sample to confirm detection, and two types of samples, a normal type and two types of variants 1 and 2 having a point mutation in codon 248, are prepared. did. Each has a FITC label at the 3 'end and has the following structure.

Normal specimen: 5 'AT GGG CCT CCG GTT CAT GCC 3'- FITC
Mutant 1 specimen: 5 'AT GGG CCT CCA GTT CAT GCC 3'- FITC
Two variants: 5 'AT GGG CCT CTG GTT CAT GCC 3'- FITC
Corresponding to these specimens, the following complementary strand probes and non-complementary negative control probes were directly synthesized and supported on the porous glass particles by the method described above.

Normal probe: PG-3 'TA CCC GGA GGC CAA GTA CGG 5'
Mutant 1 probe: PG-3 'TA CCC GGA GGT CAA GTA CGG 5'
Mutant type 2 probe: PG-3 'TA CCC GGA GAC CAA GTA CGG 5'
Negative control probe: PG-3'TT TTT TTT TTT TTT TTT TTT 5 '
A, C, G, and T represent four types of bases that bind to sugar residues: adenine, cytosine, guanine, and thymine, and PG represents porous glass particles.

    Then, the process of fixing the porous glass particle which combined the oligonucleotide probe molecule | numerator to a board | substrate is demonstrated using FIGS. First, spin-coating an acetone solution of vinyl acetate onto a silicon substrate 1 having a size of 25 mm × 75 mm corresponding to the size of the slide glass and a thickness of 0.73 mm, which is completely opaque to an excitation wavelength of 494 nm. A vinyl acetate layer 5 of about 3 μm is formed. The vinyl acetate layer 5 serves as an adhesive layer for fixing the porous glass particles 3 to which the oligonucleotide probes 2 are bonded, that is, the porous glass particles 4 with oligonucleotide probes, to the silicon substrate 1.

  Next, as shown in FIG. 1, a dispersion in which various porous glass particles 4 with oligonucleotide probes are dispersed in water is spotted on a vinyl acetate layer 5 using an appropriate spotter 6. At this time, the vinyl acetate layer 5 is apparent from the subsequent steps, but may be completely dried, and this is also applicable to the adhesive layers of other materials. Therefore, the substrate with the adhesive layer can be made and stored, which is advantageous for mass production.

At the stage immediately after spotting, as shown in FIG. 2, the porous glass particles 4 with oligonucleotide probes are in a laminated assembly.
After the moisture contained in the aggregate has evaporated, the silicon substrate 1 is heated in an air atmosphere at 100 ° C. for 10 minutes in a horizontal state by a thermostat. The vinyl acetate layer 5 is softened, and the porous glass particles 4 with oligonucleotide probes are embedded in the vinyl acetate layer 5 as shown in FIG. After cooling and the vinyl acetate layer 5 is cured, the silicon substrate 1 is ultrasonically washed in water at a frequency of 28 kHz for 1 minute, and the porous glass particles 4 with an extra oligonucleotide probe not fixed to the vinyl acetate layer 5 are made into silicon. Remove from the substrate 1. Thereafter, the silicon substrate 1 is dried with an air gun to complete a spot 7 in which only one porous glass particle 4 with an oligonucleotide probe is fixed to the silicon substrate 1. A sectional view of the spot 7 is shown in FIG. 4, and a top view thereof is shown in FIG. For the purpose of visualizing the state in which the oligonucleotide probe 2 is bound to the pores 8 of the porous glass particle 3, an enlarged view of a part of the porous glass particle 4 with the oligonucleotide probe is also shown in FIG. Since the thickness of the vinyl acetate layer 5 is less than half of the particle diameter of the porous glass particles 4 with oligonucleotide probes, more than half of the volume of the particles is exposed outside the vinyl acetate layer 5. The part can be in direct contact with the sample liquid.

  The size of the spot 7 is about 1000 μm. The spot 71 corresponding to the normal probe, the spot 72 corresponding to the mutant type 1 probe, the spot 73 corresponding to the mutant type 2 probe, and the spot 74 corresponding to the negative control probe, each of five types, five each, FIG. As shown in FIG. 4, the layers are arranged on the vinyl acetate layer 5 in a square lattice pattern with an interval of 2000 μm.

The DNA chip thus produced can be handled in the same manner as a normal DNA chip.
First, 10 μl of each of the three types of the sample diluted with the hybridization buffer solution is dropped on the spot part of the three DNA chips, and each sample solution is covered with a 20 mm square cover glass. Spread evenly.

Subsequently, the substrate is placed in a hybridization chamber and incubated in a thermostat at 45 ° C. for 12 hours.
Thereafter, the substrate is taken out of the chamber, immersed in a primary cleaning solution in which 0.01% of SDS is dissolved in 2 × SSC solution, and the cover glass is removed. Subsequently, cleaning is performed for 5 minutes while swinging the substrate in the primary cleaning solution.

Next, the substrate is immersed in a secondary cleaning solution in which 0.01% of SDS is dissolved in a 0.2 × SSC solution, and cleaning is performed for 5 minutes while rocking.
Then, in order to remove SDS, the substrate is immersed in a 0.2 × SSC solution and rinsed for 1 minute while rocking.

After rinsing, the substrate is dried with an air gun.
Thus, the spot of the board | substrate which completed hybridization was observed with the normal fluorescence microscope, and the fluorescence intensity of each spot was measured.

  The fluorescence intensity of each spot was calculated by averaging the brightness of the pixels of the digitized spot image. Table 1 shows the results obtained by averaging the values of five spots composed of the same type of probe and summarizing the fluorescence intensity of each probe for each specimen.

  For each specimen, the ratio between the fluorescence intensity of perfect matching and the fluorescence intensity of mismatching, that is, the discrimination ratio is described in parentheses. For normal specimens, the normal probe is perfect matching, and the mutant 1 probe and the mutant 2 probe are mismatched. For the mutant type 1 specimen, the mutant type 1 probe is perfect matching, and the normal type probe and the mutant type 2 probe are mismatched. For the two variants, the variant 2 probe is perfect matching, and the normal probe and the variant 1 probe are mismatched. The frame corresponding to perfect matching is shaded. For each specimen, the intensity of hybridization binding is greater in perfect matching than in mismatching, so the fluorescence intensity should increase. Therefore, the discrimination ratio is greater than 1, and the larger this value, the higher the detection capability.

Table 1 shows that all six discrimination ratios exceed 1, indicating that point mutations can be identified.
Moreover, in perfect matching of the shaded frame, all the combinations showed a fluorescence intensity of 150 or more, whereas in mismatches other than the shaded frame, the maximum is 127, which is significantly lower than the perfect matching. It has become. As a result, it is possible to identify point mutations sufficiently.

  Since the spot fluorescence intensity of the negative control probe does not include a signal component due to hybridization, it can be regarded as consisting of only a noise component. The main cause of noise may be a so-called non-specific adsorption mechanism of analyte molecules other than normal hybridization. This is considered to include the case where the analyte molecule is mechanically attached to the probe molecule itself or the case where it is attached directly to the porous particles mainly due to lack of washing operation.

  As shown in Table 1, the noise component is estimated to be 37.6 by averaging spot fluorescence intensities of three types of negative control probes. Since the average spot fluorescence intensity in perfect matching of three types of specimens is 171.3, the signal component is obtained as 133.7 (= 171.3-37.6). Therefore, the S / N ratio is determined to be 3.56 (= 133.7 / 37.6). In addition, the variation of the signal component of the same kind of spot was 5.5% as a relative standard deviation when the noise component was determined to be constant.

[Comparative Example 1]
For the purpose of comparing the present invention with a DNA chip having a two-dimensional probe arrangement, a two-dimensional DNA chip in a system in which the probe 2 is directly fixed on the surface of the slide glass 9 as shown in FIG. Using the usual spotting method, the above synthetic probe molecule whose terminal was aminated was immobilized on a glass slide treated with an active ester by a covalent bond. The spot diameter and spot arrangement are the same as in this embodiment.

  Hybridization similar to the present invention was performed, and the fluorescence intensity of each spot was digitized. The results are shown in Table 2.

The fluorescence intensity in the shaded frame corresponding to perfect matching in Table 2 shows no significant difference from the fluorescence intensity corresponding to other mismatches.
Although the six discrimination ratios all exceed 1, the difference from 1 is small compared to the present invention, and discrimination is impossible. Further, the signal component obtained in the same manner as in the present invention is 5.3, whereas the noise component is 30.7, which is much larger than the signal component.

[Comparative Example 2]
Subsequently, for the purpose of contrasting the present invention with a DNA chip produced by a conventional method in which the probe arrangement is three-dimensional but the signal component is not controlled, the porous glass particles with oligonucleotide probes are not stacked but thick. A DNA chip consisting of uncontrolled spots was prepared. Specifically, porous glass particles 4 with oligonucleotide probes were dispersed in silica sol 10 and spot-fixed on silicon substrate 1 to form spots 7 having the cross-sectional shape shown in FIG. The cross-sectional shape of the spot 7 becomes an irregular convex shape, and the porous glass particles 4 with oligonucleotide probes vary between about 3 to about 6 layers and the spot 7 in the thick part. The spot size and spot arrangement were the same as in the present invention, hybridization was performed in the same manner as in the present invention, and the spot fluorescence intensity was measured. The results are shown in Table 3.

  Although the six discrimination ratios all exceed 1, the difference from 1 is small compared to the present invention, and discrimination is almost impossible as with the two-dimensionally arranged probe. Further, the signal component obtained in the same manner as in the present invention is 182.3, the noise component is 57.8, and the S / N ratio is 3.15. However, there is an adverse effect that the variation of the signal component of the same kind of spot is as large as 25.6%.

[Summary]
The DNA chip of the present invention in which the probe arrangement is three-dimensional and the spot thickness is controlled, the DNA chip of Comparative Example 1 in which the probe arrangement is two-dimensional, and the probe arrangement is three-dimensional, but has been compared. Table 4 summarizes the detection ability of the DNA chip of Comparative Example 2 in which the thickness is not controlled. The portions related to the above-mentioned inconvenient problems are marked with x.

Thus, it can be seen that the detection ability of the DNA chip of the present invention is improved for the following two reasons.
In the present invention, the S / N ratio is remarkably increased because the noise component is not so much increased while the signal component is remarkably increased as compared to a two-dimensional DNA chip.
In the present invention, the variation in signal components is small compared to a DNA chip in which the probe arrangement is three-dimensional but the spot thickness is not controlled.

In the present invention, the signal component is markedly increased in that the probes are arranged three-dimensionally inside the porous particles, and the number of probes per unit area projected onto the substrate is significantly increased compared to the two-dimensional arrangement. In addition, since porous particles that are transparent to the excitation light are used, the probe light inside the porous particles is irradiated without being attenuated. Here, when the linear absorption coefficient of the porous particles with respect to the excitation light increases beyond 10 μm ⁻¹ , the attenuation of the excitation light in the porous particles increases, so that the fluorescence intensity decreases remarkably and the two-dimensionally arranged probe DNA chip. It has been confirmed that it will be almost the same. Accordingly, it is essential to use porous particles having a linear absorption coefficient for excitation light of 10 μm ⁻¹ or less.

  On the other hand, the increase in the noise component is small because nonspecific adsorption, which is feared to increase when the probe arrangement is made three-dimensional, is sufficiently suppressed. This is considered to be due to the fact that nonspecific adsorption sites on the surface of the probe molecules and the porous particles were sufficiently capped during the probe synthesis and that the washing after the hybridization reaction was appropriately performed.

  In addition, the decrease in the variation of the signal component in the present invention is that when the probe-attached porous particles are fixed to the substrate to form the spot, the thickness of the spot is suppressed by arranging in one layer, so the number of probes is stably controlled, And it originates in that the hybridization reaction using a cover glass can be performed uniformly.

  Thus, in addition to increasing the number of probes by three-dimensional arrangement, the DNA chip of the present invention has a high detection capability by adopting a spot configuration that can stably control the signal component from the increased probe. It became possible to get.

  Based on the results and considerations described above, the use of the DNA chip of the present invention enables single nucleotide polymorphism analysis and the like that need to identify small signal differences to be performed with high detection capability.

  In a reaction detection chip such as a DNA chip, by arranging a probe inside a porous particle that is transparent to excitation light, the excitation light is irradiated to the probe whose number increases three-dimensionally without attenuation. The signal in the detection of the fluorescent dye-labeled specimen is greatly increased. Further, when forming the spots by fixing the probe-attached porous particles to the substrate, the particles are arranged in a single layer, so that signal variations due to spot thickness variations can be suppressed. Therefore, since the S / N ratio increases stably, the detection capability of the reaction detection chip is improved.

It is sectional drawing explaining the process of fixing the porous particle with a probe to a board | substrate. It is sectional drawing explaining the process following FIG. FIG. 3 is a cross-sectional view illustrating a process following the process in FIG. 2. It is sectional drawing explaining the process following FIG. 3, and explanatory drawing which shows the probe fixed to the porous particle. It is a spot composed of porous particles with a probe. It is the arrangement | positioning figure of the spot in the DNA chip of a present Example. It is sectional drawing of the DNA chip which consists of a two-dimensional arrangement | positioning probe. It is sectional drawing of the spot where the porous glass particle with an oligonucleotide probe is piled up and fixed.

Explanation of symbols

1 substrate, silicon substrate 2 probe, oligonucleotide probe 3 porous particle, porous glass particle 4 porous particle with probe, porous glass particle with oligonucleotide probe 5 organic film, vinyl acetate layer 6 spotter 7 spot 8 pore 9 Glass slide 10 Silica sol 71 Spot 72 corresponding to normal probe Spot 73 corresponding to variant 1 probe Spot 74 corresponding to variant 2 probe Spot corresponding to negative control probe

Claims (15)

  In a reaction detection chip in which spots are formed by assembling a plurality of the porous particles obtained by binding probe molecules to the surface including the pores of the porous particles and fixing them to the substrate, the porous particles are used for detection. A reaction detection chip, which is transparent to incident light, and wherein the porous particles are fixed in a single layer state on the substrate.   The reaction detection chip according to claim 1, wherein the spot is irradiated with the incident light to detect an analyte molecule bound to the probe molecule.   The reaction detection chip according to claim 1 or 2, wherein an optically detectable molecule is added to the analyte molecule, and the molecule is detected.   The reaction detection chip according to claim 3, wherein a fluorescent dye molecule is added to the sample molecule, and fluorescence generated from the fluorescent dye molecule is detected. The reaction detection chip according to claim 1, wherein a linear absorption coefficient of the porous particles with respect to the incident light is 10 μm ⁻¹ or less. The reaction detection chip according to claim 1, wherein a linear absorption coefficient of the substrate with respect to the incident light is 100 μm ⁻¹ or more.   The reaction detection chip according to claim 1, wherein the porous particles have a particle size of 0.1 μm or more and 200 μm or less.   A thermoplastic organic film is formed on the substrate, the porous particles are spotted on the organic film, and the organic film is softened by heating the substrate to thereby form the porous particles on the organic film. The reaction detection chip according to claim 1, wherein the porous particles are fixed by being embedded in the substrate, and excess porous particles that are not fixed are removed.   The reaction detection chip according to claim 8, wherein the thickness of the organic film is smaller than half of the particle diameter of the porous particles.   The reaction detection chip according to claim 8, wherein the organic film is formed by dissolving a material of the organic film in an organic solvent by a spin coating method.   The reaction detection chip according to claim 8, wherein the spotting is performed by dispersing the porous particles in a liquid.   The reaction detection chip according to claim 8, wherein the excess porous particles are removed by ultrasonic cleaning.   The reaction detection chip according to claim 8, wherein the organic film is a vinyl acetate film.   The reaction detection chip according to claim 1, wherein the probe molecule is any one of a nucleic acid, a protein, and a glycoprotein.   Probe molecules are bonded to the surface of the porous particles containing pores to form porous particles, a thermoplastic organic film is formed on the substrate, the porous particles are spotted on the organic film, Each step of heating the substrate to soften the organic film, embedding the porous particles in the organic film, fixing the porous particles on the substrate, and removing excess porous particles that are not fixed A method for producing a reaction detection chip comprising: