JP2006029953A - Solidification carrier for living body-related substance detection, probe solidification method, and living body-related substance analysis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a solidification carrier for living body-related substance detection, a probe solidification method, and a living body-related substance analysis method, for efficiently analyzing living body-related substances in a specimen with high reliability. <P>SOLUTION: As to this solidification carrier for living body-related substance detection, a plurality of probes for detecting living body-related substances are solidified at the same address and a ratio at which the substances are solidified is known. The probe solidification method comprises a first step for preparing a plurality of probes for detecting one or more living body-related substances, a second step for mixing the plurality of probes to obtain a mixed probe, and a third step for bringing the mixed probe into contact with the same address of the carrier. The ratio of the plurality of probes is measured between the first and second steps, or between the second and third steps, or after the third step. The living body-related substance analysis method uses the solidification carrier. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a solid-phase support for detecting a biological substance, a method for immobilizing a probe, and a biological substance analysis method.

In recent years, a technique for detecting and analyzing a biological substance in a sample using a microarray or the like has been used. In a microarray, thousands to hundreds of thousands of probes such as known DNA fragments for detecting biologically related substances are usually arranged on the surface of a carrier made of glass or silicon having a size of about 1 to several tens of centimeters. , Solid-phased.
A biological substance (target) such as unknown DNA is bound to these probes by utilizing specificity due to, for example, complementation of nucleic acid chains. By labeling the target in advance and examining which probe the target is bound to, information on the biological substance such as what the target is can be analyzed (for example, refer to Patent Document 1).

  In Patent Document 1, as a method for immobilizing a probe, a functional group is added to the 3′-end or 5′-end of a probe consisting of a DNA fragment, and this functional group is reacted with a functional group on a carrier, so that the probe is supported on the carrier. Describes a method for solid-phase.

Using such a microarray, for example, the expression of several thousand to several tens of thousands of genes can be examined simultaneously. At this time, in order to improve the reliability of the data, in addition to the target spot on which the probe specific to the target is immobilized, a control spot on which the probe for detecting the control gene is immobilized is prepared. Correction is performed based on the expression state of (see, for example, Non-Patent Document 1).
JP 2000-295990 A Stein Knudsen / Author, Atsushi Shiojima, Osamu Matsumoto, Gozo Enomoto / Director, “Introduction to DNA Microarray Data Analysis”, Yodosha, 2002, p. 18-20

In addition, since various probes for the purpose of analyzing one type of biological substance are solid-phased at different addresses, a factor that makes it impossible to simply compare the signal intensities corresponding to the various probes occurs. .
For example, in the case of a microarray, since the probe spot is different for each type of probe, an error derived from the operation of creating the spot cannot be avoided. For example, differences in the amount of probe immobilized per spot, the amount of spot, the shape of the spot, and the like are error factors that affect the signal intensity. In addition, there is an error factor derived from the detection system due to the different probe spots.

Even when correction is performed based on the expression state of the control gene, the above-mentioned factors become a problem. In particular, these factors have a particularly serious effect when detecting low-expressed genes.
At present, in order to increase the reliability of the analysis of biologically relevant substances, more accurate quantitativeness is required. However, it is difficult to accurately identify low-expressing genes because the fluorescent intensity of the spot is small. For example, even when this low-expressed gene is corrected using a control gene, a slight error in the measured value may cause a large error in the measurement result. Therefore, in particular, when analyzing low-expressing genes, contrivances have been sought for the measurement of the expression level and the correction of the test gene analysis value by the control gene analysis value.

  In the case of an ideal detection optical system, for example, a control probe can be arranged somewhere in the microarray, and the signal corresponding to the target probe can be corrected based on this intensity. However, in an actual optical system, the illumination is not always uniform, and the lens system also has peripheral dimming in the periphery. Therefore, accurate correction over the entire spot of the microarray is difficult. In particular, when a signal of the entire microarray spot is acquired using a CCD as a detector, accurate correction is difficult.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a solid phase for detecting a bio-related substance capable of analyzing a bio-related substance in a sample with high reliability and efficiency. An object is to provide a method for immobilizing a carrier and a probe, and a method for analyzing a biological substance.

In the solid-phase support for detecting a bio-related substance of the present invention, a plurality of types of probes for detecting a bio-related substance are immobilized at the same address, and the plurality of types of probes are immobilized on the solid-phase support. Characterized by the known ratio.
The ratio is determined by measuring the absorbance of the probe, measuring the label of the labeled probe, measuring the concentration of the first reactant in the probe bound to the first reactant specifically corresponding to the probe, or It is preferably obtained by measuring the concentration of the second reactant that can specifically react with the first reactant or probe and is fixed at a predetermined address of the carrier.

In the solid-phase support for detecting a biological substance according to the present invention, at least one of the plural types of probes is preferably for detecting a control biological substance.
In addition, it is preferable that a plurality of types of probes are solid-phased at the same address for one type of biological substance to be detected.

Moreover, it is preferable that the said biological substance is a nucleic acid.
In addition, a plurality of types of probes immobilized on the same address include a first probe in which the 3 ′ end is immobilized on a carrier, the same base sequence as that of the first probe, and 5 ′ on the carrier. It is preferable to include a second probe whose end is immobilized.
Here, it is more preferable that the first probe and the second probe are solid-phased at a ratio of about 1: 1.

The probe immobilization method of the present invention is a probe immobilization method for detecting one or more biological substances,
Preparing a plurality of types of probes for detecting one or more biological substances;
A second step of mixing the plurality of probes to obtain a mixed probe;
And a third step of bringing the mixed probe into contact with the same address of the carrier,
Have
In addition, the ratio of the plurality of types of probes is measured between the first step and the second step, or between the second step and the third step, or after the third step.

Specifically, a method for immobilizing a probe for detecting one or more biological substances,
Preparing a plurality of types of probes for detecting one or more biological substances;
A second step of mixing the plurality of probes to obtain a mixed probe;
A third step of contacting a carrier with a plurality of reactants that specifically react with each of the plurality of probes;
And a fourth step of contacting the mixed probe with the carrier,
Have
In addition, the ratio of the plural kinds of reactants is measured before the third step or between the third step and the fourth step.

Alternatively, a method for immobilizing a probe for detecting one or more biological substances,
A first step of preparing a plurality of types of probes to which a plurality of types of reactants specifically corresponding to each of the one or more biological substances are detected,
A second step of mixing the plurality of probes to obtain a mixed probe;
And a third step of bringing the mixed probe into contact with the carrier,
Have
In addition, the ratio of the plural kinds of reactants is measured between the first step and the second step, or between the second step and the third step, or after the third step.

Alternatively, a method for immobilizing a probe for detecting one or more biological substances,
A first step of preparing a plurality of types of probes to which a plurality of types of reactants specifically corresponding to each of the one or more biological substances are detected,
A second step of mixing the plurality of probes to obtain a mixed probe;
A third step in which a plurality of reactants specifically reacting with a plurality of reactants bound to a probe are brought into contact with a carrier;
And a fourth step of contacting the mixed probe with the carrier,
Have
And the ratio of the several types of reactive material contacted with a support | carrier is measured before a 3rd step or between a 3rd step and a 4th step, It is characterized by the above-mentioned.

It is preferable that the plurality of types of probes include a first probe having the same base sequence and having a 3 ′ end on the side immobilized on a carrier and a second probe having a 5 ′ end.
The biological material is preferably a nucleic acid.

The method for analyzing a biological substance of the present invention is a method for analyzing a biological substance using the solid-phase support of the present invention,
A first step of supplying a sample containing one or more biological substances to the solid-phase support;
A second step of measuring a signal intensity of a reactant obtained by reacting the biological substance with the probe;
And a third step of correcting the intensity based on the ratio,
It is characterized by having.
Here, the signal intensity of the reactant is preferably measured by causing fluorescence resonance energy transfer between the fluorescent label labeled on the probe and the fluorescent label labeled on the biological substance.

  ADVANTAGE OF THE INVENTION According to this invention, the solidification support | carrier for solid-phase detection of a biological substance-related substance and a probe which makes it possible to analyze the biological substance-related substance in a sample with high reliability and efficiency, and the biological substance-related substance An analysis method can be provided. Therefore, the present invention is also suitable for automation of analysis.

<Solid phase support for detection of biological substances>
The solid-phase support for detecting a biological substance (hereinafter referred to as “solid-phase carrier”) of the present invention has a plurality of types of probes for detecting a biological substance, immobilized at the same address, A plurality of types of probes have known solid-phase ratios.
(Biological substances)
The biological substance in the present invention means a substance extracted or isolated from a living body, but includes not only those extracted directly from a living body but also those obtained by chemical treatment, chemical modification and the like. For example, substances such as hormones, tumor markers, enzymes, antibodies, antigens, sugar chains, abzymes, other proteins, nucleic acids, cDNA, DNA, mRNA, RNA, artificial nucleic acids, and other nucleic acids.

(probe)
Probes used in the present invention are hormones, tumor markers, enzymes, antibodies, antigens, sugar chains, abzymes, other proteins, nucleic acids, cDNA, DNA, RNA, artificial nucleic acids, other nucleic acids, etc. It means a substance that can specifically bind to any biological substance.
A biological substance and a probe specifically bind to each other when, for example, a stable duplex is formed between complementary nucleotide sequences found in DNA or RNA (hybridization), or an antigen and an antibody. It means a highly specific bond that selectively reacts only with a specific substance such as biotin and avidin.

The type of probe is distinguished by the type of a biological substance to which the probe specifically binds and the structure of the probe itself (for example, the base sequence in nucleic acid or the like). Furthermore, in the present invention, the type of probe is distinguished in a solid phase. For example, when the probe is a nucleic acid, even if the nucleic acid probes have the same base sequence, if the site immobilized on the carrier is different at the 3 ′ end or the 5 ′ end, they are different from each other. Considered as a probe.
That is, in the present invention, when referring to a plurality of base sequences for detecting at least one type of target nucleic acid, the site to be captured is different for one type of gene, and there are a plurality of types of probe base sequences. Even if the site to be captured is the same for one type of gene, the orientation of immobilization on the substrate etc. is different (for example, immobilization at the 3 ′ end or immobilization at the 5 ′ end) It is.
A reactive substance involved in immobilization may be bound to the probe.

(Address, solid phase)
In the present invention, the target for immobilizing the probe is not particularly limited as long as the probe can be identified, and can be immobilized on various carriers. Examples of the carrier include a plate-like substrate (substrate), beads and the like. More specifically, glass substrates, silicon wafers, various porous substrates, gels, microtiter plates, capillaries, magnetic beads, hollow fibers, and the like can be used. Here, it is preferable to use a porous substrate having a fine pore structure penetrating as the carrier. In this case, when the biologically related substance is detected using the solid-phased carrier of the present invention, the supplied sample solution or the like is repeatedly moved in the porous substrate using the driving means, thereby It has the effect of greatly promoting the reaction with biological substances.
The address referred to in the present invention may be any means for identifying and detecting the unit on which the probe is immobilized, for example, the coordinate range on the substrate surface when the probe is immobilized on the substrate surface, Examples include the type of label when the probe is immobilized on a carrier comprising labeled beads.

To immobilize multiple types of probes at the same address, for example, prepare probe solutions for various probes, mix a plurality of probe solutions to prepare a mixed probe solution, and then apply the mixed probe solution to the surface of the carrier. The first method of supplying and solidifying is possible. Alternatively, a second method may be used in which a solution of each probe is prepared, and the plurality of probe solutions are sequentially or simultaneously supplied to the same address to be solid-phased.
In order to solidify, for example, a desired reactant may be bound to the probe in advance, and the reactant that reacts with the reactant may be immobilized on the surface of the carrier, and the above supply may be performed. Alternatively, the above-described supply may be performed by fixing a reactive substance that reacts with the probe itself on the surface of the carrier.
When a reactive substance is bound to the probe, for example, various functional groups can be used as the reactive substance. When the probe is composed of a nucleic acid, an amino group or a thiol group is preferably used as the functional group.

  When immobilized on the surface of the carrier, the reactant is appropriately selected according to the functional group inherent to the probe, the reactant previously bound to the probe, the material of the carrier, and the treatment on the surface of the carrier. The A preferable reactive substance to be immobilized on the surface of the carrier is a crosslinking reagent. Examples of the crosslinking reagent are those having reactive substances such as NHS (succinimide) ester, maleimide group, carbodiimide, pyridyldithio group, hydrazide group, sulfo NHS ester, phenyldiazo, and biotin at the end of the molecule. Specific examples of cross-linking reagents include cross-linking agents such as N-hydroxysuccinimide, bis [2- (succinimideoxycarbonyloxyethyl)] sulfone, N- (3-maleimidopropionyloxy) succinimide, carbodiimide, peptides, etc. Is mentioned.

  For solid-phase immobilization, a reactive substance is bound to the probe in advance, and a reactive substance capable of reacting specifically with the reactive substance is immobilized on the surface of the carrier, so that the reaction between the reactive substances is stable. Solid phase immobilization is achieved. In addition, estimation of the in-address solid phase immobilization ratio described later becomes easy.

(Solid phase ratio)
In the solid-phased carrier of the present invention, the ratio of the plural types of probes solid-phased at the same address (solid-phase ratio within the address) is known.
In order to obtain such a solid-phase support, for example, the first estimation method for estimating the solid-phase ratio in the address after solid-phase using the ratio of the probe before solid-phase and solid-phase A second estimation method that measures the ratio of probes in the address later is given.
Also, the immobilization ratio in the address is determined by, for example, measuring the absorbance of the probe, measuring the label of the labeled probe, or the first reaction of the probe bound to the first reactant corresponding specifically to the probe. It can be obtained by measuring the concentration of a substance or by measuring the concentration of a second reactant that can specifically react with the first reactant or probe and is fixed at a predetermined address of the carrier.
The combination of the first reactant and the second reactant that can be specifically reacted will be described in detail in the probe immobilization method described later.

In the solid-phased carrier of the present invention, if at least one of a plurality of types of probes immobilized at the same address is for detecting a control biological material, the control biological material is used as a reference. In addition, the correction (control correction) of the analysis result of the target living body related substance is accurately performed, and it is possible to make an analysis in which the reliability of the analysis result is improved compared to the conventional one.
That is, in a conventional system in which the address where the probe specific to the control biological substance is immobilized (for example, the probe spot region) and the address where the probe specific to the target biological substance is immobilized are different. In the control correction, the result after correction was not always constant. On the other hand, in the present invention, if at least one of a plurality of types of probes solid-phased at the same address is used for detecting a control biological substance, a control biological substance probe and a target biological substance Since related substance probes can exist in the same address and their ratios are known, the difference in the amount of solid phase of each probe, the amount of spot liquid to be contacted, the shape of the spot, detection An analysis with accurate control correction can be realized without being affected by variations in irradiation amount in the system.

The control biological substance (hereinafter sometimes referred to as “control”) can be appropriately selected according to the type of the target biological substance (hereinafter also referred to as “target”). Examples of the control include an internal control biological substance (IC) and an external control biological substance (EC).
As the IC, a bio-related substance whose abundance in the sample is substantially constant can be employed. More specifically, for a given sample, a substance that does not vary in abundance between cells and tissues, which are specimens from which it is collected, and between collection times, can be set as an IC.
As the EC, a biological substance that cannot be contained in the sample can be used. For example, a biological substance derived from another biological species that is not related to the biological species of the specimen from which the sample is collected can be used. When comparing multiple specimens, adding EC in an accurate ratio (for example, 1: 1) with respect to the amount of biological substances, such as nucleic acids in the sample, between different samples, between different chips, etc. Comparison with is easy and accurate.

For example, when the target biological substance is a nucleic acid, a nucleic acid or the like can be set as a control, and this control does not cause cross-hybridization with the target nucleic acid, that is, a sequence specific to the target. It is necessary to be unable to hybridize with the probe region.
Examples of nucleic acids that can be set as IC include glycodehyde-3-phosphate dehydrogenase (GAPD), actin, beta (ACTB), similar to polyubiquitin (LOC34641), and ribosomal protein (S7'RP7). It is also possible to use genes other than these contained in the sample as IC. As the EC nucleic acid, for example, when the specimen is human, a plant gene may be used. For example, a Renilla luciferase gene can be used as a gene that can be set as an EC nucleic acid.

In addition, for example, when the target biological substance is a protein, a protein or the like can be set as a control, and this control needs not to cause non-specific binding with the probe region.
Examples of proteins that can be set as IC include various proteins produced from the housekeeping genes exemplified above. Other proteins contained in the sample may be IC. Examples of proteins that can be set as EC include proteins produced from EC nucleic acids exemplified above, for example, Renilla luciferase.

  In the solid-phased carrier of the present invention, the plurality of types of probes solid-phased at the same address include a probe specific to the target biological substance, a probe for detecting IC, and a probe for detecting EC. Can be made. Since the in-address solid phase ratio of these three types of probes is known, more accurate correction can be performed using both controls. In addition, it is possible to correct the detection result of the target biological substance using only one control that is considered to be appropriate for the correction.

In the solid-phased carrier of the present invention, in a mode in which a plurality of types of probes are solid-phased at the same address with respect to one type of biological substance to be detected, the efficiency and reliability of the analysis of the biological substance are improved. In particular, a particularly remarkable effect can be realized.
That is, in the conventional solid-phase support system, as the number of probes used for detection of one type of biological substance increases, the number of addresses corresponding to various probes is secured, and the number of operations and time associated therewith. Cost and other problems increase. Moreover, even if the probes are for the purpose of analyzing one kind of biological substance, the factor that makes it impossible to simply compare the signal intensities corresponding to them cannot be excluded. On the other hand, in the present invention, since a plurality of types of probes used for detecting one type of biologically related substance coexist at the same address, the number of addresses to be secured is at least one type of biologically related substance. One is enough. Therefore, the number of operations, time, cost, etc. can be reduced and analysis efficiency can be increased. Further, for example, error factors derived from the amount of immobilized probe, the amount of spot, the shape of the spot, and the detection system per spot are eliminated. Therefore, if the biological material is analyzed using the solid-phase support of the present invention, the accuracy of correction, that is, the reliability of the analysis result after correction is improved.
This embodiment is a case where the biological substance is a nucleic acid, and is suitable for capturing the target detection gene or when an optimal probe sequence is not known, or a plurality or one polymorphism in the target detection gene This is particularly preferable in the case of detection when (variation) is present. The reason is that, as described above, complementarity studies with a plurality of types of probes can be performed simultaneously on one target nucleic acid with one address.

In the solid-phase support of the present invention, a preferred embodiment is illustrated for the case where the biological substance is a nucleic acid. In this case, a plurality of types of probes immobilized on the same address include a first probe in which the 3 ′ end is immobilized on a carrier, the same base sequence as that of the first probe, and 5 in the carrier. It is preferable to include a second probe whose end is immobilized.
The substance used as the first and second probes may be any substance that can form a complementary strand specifically with the nucleic acid to be detected (target nucleic acid), such as cDNA, DNA, RNA, artificial nucleic acid, other Nucleic acids are exemplified.

Conventionally, when nucleic acid is detected, for example, as shown in Patent Document 1, a functional group is added to the 3 ′ end or 5 ′ end of a probe comprising a DNA fragment, and this functional group is added to a functional group on a carrier. The probe was immobilized on a carrier by reacting with.
However, the efficiency of the hybridization reaction between the target nucleic acid and the probe depends on which end of the probe is immobilized (probe immobilization direction) even if the base sequence of the immobilized probe is substantially the same. May vary. In addition, since the efficiency of the hybridization reaction is affected by the length and site when the target nucleic acid is fragmented, it is not possible to predict which end of the probe should be immobilized before the hybridization reaction. Have difficulty. When detection is performed using a solid-phase support in which the probe is solid-phased in such a direction that the hybridization reaction efficiency is low, only a very low intensity signal is obtained, and the analysis result is lower than the actual value. There was a case that became.
On the other hand, in the solid phase support of the present invention, it is possible to adopt a mode in which two types of probes differing only in the solid phase direction are allowed to coexist at the same address. it can. According to this aspect, even when the hybridization reaction efficiency between the probe and the sample differs depending on the solid phase direction of the probe, the hybridization reaction efficiency at the address can be averaged and a signal can be obtained stably. . In this way, it is possible to reliably detect the target nucleic acid without the signal intensity being extremely reduced.

Furthermore, it is preferable that the first probe and the second probe are solid-phased at a ratio of about 1: 1 in the same address. This is because, in detecting the target nucleic acid, it is advantageous for minimizing the possibility that the variation in the hybridization reaction efficiency depending on the solid phase direction of the probe affects the analysis result. The ratio between the first probe and the second probe is defined by the substance amount ratio, that is, the ratio of the number of probe molecules.
In addition, it is possible to confirm the high-order structure of the detection target substance and the probe in advance by experiments, simulations, etc., and change the ratio of the plurality of probes to be immobilized at one address in consideration of this. For example, it is possible to make the capture efficiency 1: 1, and the control correction becomes easy. For example, the ratio of homo and hetero can be evaluated at the time of analysis of SNP or the like.

<Method of immobilizing the probe>
According to the method for immobilizing a probe of the present invention (hereinafter referred to as “solid phase immobilization method”), a plurality of types of probes for detecting a biological substance are immobilized at the same address, and the plurality of types of probes are detected. Measure the ratio of the solid phase.
The solid-phase immobilization method of the present invention is a method for immobilizing a probe for detecting one or more biological substances, and first preparing a plurality of types of probes for detecting one or more biological substances. A second step of mixing the plurality of types of probes to obtain a mixed probe, and a third step of bringing the mixed probe into contact with the same address of the carrier, and determining the ratio of the plurality of types of probes. , Measured between the first step and the second step, or between the second step and the third step, or after the third step.

In order to measure the ratio of a plurality of types of probes between the first step and the second step, for example, the following means can be used. That is, first, probe solutions for various probes are prepared, and the probe concentration in each probe solution is measured. Next, the mixed probe may be obtained by mixing these probe solutions at a predetermined volume ratio. The probe concentration in each probe solution can be measured by measuring the absorbance of the probe, measuring the label of the labeled probe, or the like.
In order to measure the ratio of the probe between the second step and the third step, it is sufficient to use a means capable of discriminating the plurality of types of probes from each other. Measurement of the label of the probe, measurement of the concentration of the reactant (hereinafter referred to as “first reactant”) of the probe to which the reactant specifically corresponding to the probe is bound Thus, the ratio can be measured.
The probe ratio may be measured after the third step. In the case where the probe ratio is measured after the third step, the ratio of the plurality of types of probes in the contacted address is measured. In this way, after immobilizing the probe, even if the ratio of multiple probes is estimated, when measuring between the first step and the second step, and when measuring between the second step and the third step Similarly to the above, it is possible to provide a solid-phase support capable of improving accuracy such as control correction.
Examples of the label include a fluorescent substance.

Examples of the solid-phase immobilization method of the present invention include the following first to third aspects.
Hereinafter, in the first to third aspects, examples of the biological substance and probe are the same as those exemplified in the above “immobilized carrier”. Further, by bringing the mixed probe into contact with the carrier, a plurality of types of probes are immobilized at the same address.
In the solid phase immobilization method of the present invention, the method for bringing the mixed probe or the reactant into contact with the carrier is not particularly limited. Examples of such a method include an adhesion method for adhering a liquid and a mixing method for mixing a carrier such as a bead and a mixed probe solution.

(First aspect)
A first aspect is a method for immobilizing a probe for detecting one or more biological substances, and a first step of preparing a plurality of types of probes for detecting one or more biological substances, A second step of mixing a plurality of types of probes to obtain a mixed probe, a third step of bringing a plurality of types of reactants that specifically react with each of the plurality of types of probes into contact with a carrier, and the mixed probe comprising: And a method of measuring a ratio of the plurality of types of reactants before the third step or between the third step and the fourth step.
In the first aspect, the reactant to be brought into contact with the carrier in the third step (hereinafter, the reactant to be brought into contact with the carrier is referred to as “second reactant”) is a substance that specifically reacts with the probe itself, It binds to the probe specifically corresponding to the probe.
In this aspect, the order in which the first and second steps and the third step are performed is not limited.

In the first aspect, the ratio of the second reactant is measured before the third step or between the third step and the fourth step.
The concentration of the reactant can be measured, for example, by measuring absorbance.
When the ratio of the multiple types of the second reactant is measured before the third step, the ratio of the multiple types of probes immobilized on the same address through the fourth step is calculated from the measured ratio of the second reactant. The ratio (solid phase ratio in the address) can be estimated.

  That is, the measured ratio is stored in the third step, ie, the second reactant contact operation, and the ratio of the second reactant immobilized within one address of the carrier is known, and this immobilized second reactant is Specifically, various probes are immobilized. Here, when preparing probes for detecting a plurality of biological substances, mixing them, and supplying them to the carrier, it is not necessary to estimate the ratio of the plurality of probes, and the solid phase within the address of the immobilized probe is used. The conversion ratio can be known.

  The ratio of the plural kinds of reactants may be measured between the third step and the fourth step. That is, after the second reactant is brought into contact with the carrier or the like, the concentration of the plurality of second reactants can be measured to estimate the ratio of the plurality of second reactants. In this case, the ratio in the address where the plural kinds of second reactants are contacted is measured.

(Second embodiment)
The second aspect is a method for immobilizing a probe for detecting one or more biological substances, and a plurality of kinds of reactants corresponding specifically to each other for detecting the one or more biological substances. A first step of preparing a plurality of types of probes bound to each other, a second step of mixing the plurality of types of probes to obtain a mixed probe, and a third step of bringing the mixed probe into contact with a carrier, And it is the method of measuring the ratio of this multiple types of reactants between the first step and the second step, or between the second step and the third step, or after the third step.
That is, it is a method of measuring the ratio of a plurality of types of probes by measuring the concentration of the first reactant bound specifically to each probe.

(Third embodiment)
A third aspect is a method of immobilizing a probe for detecting one or more biological substances, and a plurality of kinds of reactants corresponding specifically to each other for detecting one or more biological substances A first step of preparing a plurality of types of probes bound to each other, a second step of mixing the plurality of types of probes to obtain a mixed probe, and a plurality of types that specifically react with a plurality of types of reactants bound to the probes. A third step of bringing the reactants into contact with the carrier, and a fourth step of bringing the mixed probe into contact with the carrier; It is a method of measuring before a step or between a third step and a fourth step.
In this embodiment, the second reactant that is brought into contact with the carrier in the third step is a reactant that can specifically react with the first reactant bound to each probe.

In the third aspect, examples of the combination of the first reactant and the second reactant include a combination of an amino group and an active ester (such as succinimide) and a combination of a thiol group and a maleimide group in this order. The The combination of the first reactant and the second reactant only needs to specifically correspond to the type of the probe. For example, in selecting the second reactant, if the substance is different for each probe, the active ester-containing compound Any compound selected from:
When the reaction efficiency of the first reactant and the second reactant corresponding to each probe is different or unknown, it is preferable to measure the ratio fixed on the substrate by absorption or the like. Measure the concentration of the reactant solution to calculate the ratio, then measure the ratio fixed on the substrate by absorption, etc., calculate the reaction efficiency, and make the two reactant solutions to the desired ratio The concentration can be adjusted and processed. After that, when the probes to which the first reactants different from each other are mixed and supplied to a specific position on the surface of the carrier, each probe is immobilized on the surface of the carrier at a ratio reflecting the ratio of the reactants immobilized on the surface of the carrier. Be phased.

  Probes for detecting biological substances are often expensive, and only a small amount may be prepared. On the other hand, since the reactive substance is cheaper than the probe, it can be prepared in a large quantity and the concentration and the like can be measured accurately. Therefore, the third aspect has a particularly remarkable effect in terms of providing a solid-phased carrier that contributes to improving the reliability of analysis of biological materials.

  In the solid phase immobilization method of the present invention, the biological material is preferably a nucleic acid.

In the solid phase immobilization method of the present invention, the plurality of types of probes have the same base sequence, and the first probe having the 3 ′ end on the side to be immobilized on the carrier and the second probe having the 5 ′ end. And a probe.
In this case, in order to control the side of the probe to be immobilized on the carrier, for example, a reactive substance involved in the immobilization on the carrier can be selectively bonded to either end of the probe. Good.
That is, a probe in which a reactive substance such as a functional group is bonded to the 3 ′ end is prepared as the first probe, and a probe in which a reactive substance such as a functional group is bonded to the 5 ′ end is prepared as the second probe.
In this case, in the solid phase method, prepare probe solutions of the first probe and second probe, measure the probe concentration in these solutions by absorbance, etc., and mix at a predetermined volume ratio to prepare a mixed probe solution can do. The mixed probe solution may be supplied to the carrier. At this time, it is preferable to fix a reactive substance such as a functional group that reacts with the reactive substance bound to the probe at a predetermined address.

As in the third aspect described above, the immobilization is performed by the reaction between the first reactant bound to the probe and the second reactant immobilized at a predetermined address, and the probe In the case where the ratio of a plurality of types of probes or a plurality of types of reactants is measured before the solid phase is immobilized, it is preferable that the first reactant is the same substance for the plurality of types of probes.
Thus, if the type of the first reactant is common among the probes, the immobilization efficiency of each probe becomes almost the same, so that the ratio of mixing the probes is maintained and each probe is immobilized. .
For example, if the types of reactants to be bound to the first probe and the second probe are the same, the immobilization efficiency will be the same, so the ratio of the probes mixed in the preparation of the mixed probe solution is maintained. The first and second probes are preferably immobilized.
In addition, for example, when a plurality of types of probes include a probe specific to the target biological substance and a probe specific to the control biological substance, it contributes to further improvement in the accuracy of control correction, which is preferable. . This is particularly effective when providing a solid-phase support for nucleic acid detection.

  In addition, a method for immobilizing a probe using a method in which a solution of each probe exemplified in the above-mentioned “immobilized carrier” is prepared, and these plural probe solutions are sequentially or simultaneously supplied to the same address for immobilization. Has an advantageous effect over the conventional method. In this case, instead of the preparation of the mixed probe and the contact of the mixed probe exemplified in the first to third aspects, the operation of preparing various probe solutions and sequentially or simultaneously contacting the probe solutions may be performed. Good. As a result, it is possible to provide a solid-phase support having a known solid-phase ratio within the address.

<Analyzing method of biological substances>
The method for analyzing a biological substance of the present invention (hereinafter referred to as “analysis method”) is a method using the solid-phased carrier of the present invention described above, and a sample containing one or more biological substances is fixed. A first step of supplying to the phase support, a second step of measuring the signal intensity of a reactant reacted with the biological substance and the probe, and the intensity to the ratio of the probe or the ratio of the reactant And a third step of correcting based on the third step.
In the analysis method of the present invention, it is preferable to further perform a step of removing a biological substance not forming a reactant between the first step and the second step.
Hereinafter, embodiments of the analysis method of the present invention will be exemplified.

(Embodiment 1)
In this embodiment, the probe is a nucleic acid, the biological substance to be detected is a predetermined nucleic acid region (target nucleic acid), the carrier is a substrate, and the address corresponds to a spot region on the substrate, that is, a probe region. . Furthermore, in the solid-phased carrier, multiple types of probes are solid-phased for one type of biological substance to be detected.
As shown in FIG. 1 (a), in the solid-phase support used in this example, a plurality of types of probes A having different base sequences with respect to one type of target nucleic acid on one spot on the substrate surface. , B are immobilized. Each of these probes is immobilized on a substrate via a linker. Here, the ratio of probes A and B per spot area (in-address solid phase ratio) is known.

In this embodiment, the sample is previously subjected to a treatment for binding a labeling substance to the nucleic acid.
First, a sample is supplied to the solid-phase support shown in FIG. When the target nucleic acid is contained in the sample, as shown in FIG. 1B, the target nucleic acid and the probe hybridize to form a specific binding pair (hybrid). Next, a washing operation is performed. Then, as shown in FIG.1 (c), the substance which did not form the hybrid body among samples is removed, and it will be in the state by which only the hybrid body was hold | maintained in the probe area | region.
Thereafter, the signal strength of the hybrid body is measured. In this embodiment, the signal intensity of the labeling substance that is previously bound to the nucleic acid may be detected corresponding to the spot region.
Further, the intensity is corrected based on the known solid phase ratio in the solid phase carrier.
As described above, according to Embodiment 1, the detection sensitivity of the target nucleic acid can be improved while minimizing the number of spot regions.

(Embodiment 2)
Embodiment 2 is that, in the immobilized carrier, one of the two probes immobilized on the same address is a probe specific to the target nucleic acid, and the other is a probe specific to the control nucleic acid, This is an example similar to the first embodiment. That is, in this example of the immobilized carrier, the in-address solid phase ratio between the probe specific to the target nucleic acid and the probe specific to the control nucleic acid is known.
According to the second embodiment, the control correction can be performed more accurately than ever before.

In this aspect, in the same manner as in the first embodiment, after supplying the sample and measuring the signal intensity of the hybrid, the signal intensity obtained is controlled with a probe specific for the target nucleic acid (probe for the target nucleic acid) and the control. Correction is performed based on the solid phase ratio in the address with a probe specific to nucleic acid (probe for control nucleic acid). In this case, the signal strength measurement of the hybrid is performed by distinguishing the signal strength of the hybrid of the target nucleic acid probe and the nucleic acid in the sample from the signal strength of the hybrid of the target nucleic acid probe and the nucleic acid in the sample. It is necessary to.
For correction, for example, for each spot region, the ratio of the signal intensity of the hybrid corresponding to the target nucleic acid probe and the signal intensity of the hybrid corresponding to the control nucleic acid probe is obtained, and the amount of the target nucleic acid in the sample and the control nucleic acid are determined. This ratio can be calculated by calculating the ratio with the amount of the above, and the value of this ratio can be adopted as the analysis result after correction.

In the second embodiment, when an internal control is set as a control, one of the control probe and the target nucleic acid probe is fluorescently labeled, and this fluorescently labeled substance is used as an internal control. It is necessary to use a substance that causes fluorescence resonance energy transfer (FRET) described later with another fluorescent label labeled on the sample to be contained. Thereby, the signal intensity of the hybrid of the target nucleic acid probe and the nucleic acid in the sample can be detected by distinguishing the signal intensity of the hybrid of the control probe and the nucleic acid in the sample.
In Embodiment 2, when an external control nucleic acid is set as a control, it is not always necessary to use FRET in the detection of signal intensity.

In the analysis method of the present invention, the signal intensity of the reactant can be measured by causing fluorescence resonance energy transfer between the fluorescent label labeled on the probe and the fluorescent label labeled on the biological substance. It is.
Fluorescence resonance energy transfer (FRET) is a phenomenon in which an acceptor absorbs light energy emitted by a donor when two types of fluorescent substances called a donor and an acceptor approach a predetermined distance or less. FRET detection is usually performed by irradiating the excitation wavelength of the donor and measuring the fluorescence intensity of the donor or acceptor. When measuring the fluorescence intensity of the donor, the light intensity is absorbed by the acceptor if the two types of fluorescent substances are close to each other, and the fluorescence intensity of the donor decreases, and the fluorescence intensity increases as the distance increases. Conversely, when measuring the fluorescence intensity of the acceptor, the fluorescence intensity increases when the distance is short, and the intensity decreases when the distance is far away.

In the analysis method of the present invention, at least one of the plurality of types of probes on the immobilized carrier is for detecting a control biological substance, the carrier is a substrate, and the address corresponds to a spot region on the substrate. In addition, the case where the signal intensity of the reactant is measured using FRET will be described in more detail.
In this case, for example, a fluorescent label is applied to any one of the probe for the control biological substance and the probe for the target biological substance, and the fluorescent labeling substance is fluorescently labeled on the sample containing the control biological substance. A substance that causes FRET between the label and the label may be used.
For example, a biological substance, which is a sample labeled with a donor fluorescent substance, is reacted with a probe labeled with an acceptor fluorescent label, and then washed to obtain a signal from the label.

When the excitation wavelength of the donor fluorescent substance is used for detection, FRET occurs between the donor and the acceptor, and light emission from the acceptor is detected. For example, if the fluorescent substance of the acceptor is labeled on the probe for the control biological substance, the intensity of the luminescence corresponds to the abundance of the control biological substance. On the other hand, since the probe spot region (spot region) for detecting the target biological substance is not fluorescently labeled to cause FRET, the fluorescently labeled specimen biological substance that specifically binds to the spot region is The signal can be obtained by directly irradiating the excitation wavelength of the fluorescent material used as the FRET donor during detection. For example, this corresponds to the abundance of the target biological substance.
Here, even if the fluorescent label serving as a donor and the fluorescent label serving as an acceptor are exchanged, it is possible to separate and detect signals of a control biological substance (for example, an internal control nucleic acid) and a target biological substance. It is also possible to detect by labeling a fluorescent substance as an acceptor on the probe.

  In the present invention, particularly when the biological substance is a nucleic acid, a hybrid reaction of an internal control nucleic acid is preferably performed to correct an error not only between addresses, for example, between spot regions, but also between samples and a solid-phase support. It is desirable to align the conditions. Therefore, more accurate results can be obtained by labeling the fluorescent substance serving as an acceptor with a probe specific to the internal control nucleic acid.

As mentioned above in the section of “Solid phase support”, a probe specific to a target biological substance, an internal control substance, a plurality of types of probes immobilized at the same address in the solid phase support If you include probes that detect substances and probes that detect external control substances, you can make more accurate corrections using both controls, or use only one of the controls to correct detection results for target biological substances. It becomes possible.
In this case, the external control nucleic acid can be detected by washing with another label (a label that can be detected other than the combination of labels used for FRET) after hybridization.

For example, the probe A for detecting the nucleic acid to be detected and the probe B for detecting the external control are immobilized on the same address as shown in FIG.
Here, for example, first, an external control nucleic acid labeled with a fluorescent substance is hybridized (first hybridization). In the next step, washing is performed with a buffer solution or the like in order to remove excess external control nucleic acid in which hybridization has not occurred. After this, a signal from the label of the external control nucleic acid is obtained. Since the external control nucleic acid hybridizes only to the probe B, the signal acquired here reflects the amount of immobilized probe, that is, data for performing correction when comparing data between different samples (detection target nucleic acids). It can be.
In the next reaction, the sample nucleic acid labeled different from the label of the external control nucleic acid is hybridized (second hybridization), and then washed with a buffer solution or the like in the same manner as the first hybridization. To obtain a signal from the label of the nucleic acid to be detected.
In this way, it is possible to detect the control and detection target nucleic acids separately by acquiring the fluorescence signal of the sample by washing after obtaining the fluorescence signal of the control and then hybridizing the target sample. Become. Therefore, the reaction can be performed under optimum conditions such as hybridization time, temperature, buffer composition, and the like. Furthermore, interaction between the control nucleic acid and the nucleic acid to be detected and non-specific reaction can be avoided, and more accurate analysis can be performed.
At this time, using FRET, one of the sequence region A for detecting the nucleic acid to be detected and the probe B for detecting the external control is labeled with a donor or acceptor fluorescent substance, and the acceptor or donor fluorescent substance is labeled. Can be labeled with a nucleic acid sequence to be detected or an external control nucleic acid to obtain a signal.

  Corrections using internal control and external control as described above are expected to improve accuracy even in protein detection arrays, antibody arrays, etc., that is, in the case where biological substances to be detected are proteins, antibodies, etc. In the case where a biological substance to be detected is a protein, an antibody, or the like, it is particularly effective to apply the present invention to perform correction when comparing between chips or samples.

The signals from these controls can be used not only to correct the data, but also to confirm whether the preparation of the solution containing the test sample was appropriate. For example, when a predetermined signal strength is obtained, it can be estimated that the preparation of the solution is appropriate, and when the predetermined signal strength is not obtained, it is estimated that the preparation of the solution is inappropriate.
Furthermore, since the ratio between the control and the probe for detecting the detection target is constant as described above, the illumination system of the detection apparatus is not uniform even if the state between the addresses of the immobilized carrier, for example, between the spot areas is not uniform. Even if there is a change in the illumination system over time, it is possible to perform highly reliable correction even if the optical system for acquiring the signal has a peripheral dimming. In particular, when a CCD is used as a detector or the like, it is preferable to apply the present invention because a signal is often obtained by illuminating all spot areas of a microarray at one time without performing scanning.

In the analysis method of the present invention, the probe may be immobilized on a carrier and then contacted with a sample to cause specific binding, or specific binding may be formed between the probe and the sample. After that, it is also possible to immobilize on a carrier. For example, in the case of a nucleic acid probe, the nucleic acid probe and the sample can be hybridized and then immobilized on a substrate or the like, or the nucleic acid probe can be immobilized on a substrate or the like and then contacted with the sample. Thus, a hybridization reaction may be performed.
To form a specific bond between a probe and a sample and then immobilize it on a carrier, for example, after hybridization of a nucleic acid probe having a tag sequence and a sample containing nucleic acid, the tag A solid phase may be formed on a carrier such as a substrate through the arrangement.

In the first and second embodiments, the case where nucleic acid is detected has been described as an example. However, the present invention can be similarly applied to the detection of other biological substances such as proteins, antigens, and antibodies. is there.
As described above, the solid-phase support of the present invention, the solid-phase immobilization method of the present invention, and the analysis method of the present invention are all, for example, analysis of biological materials using microarrays in which probes are solid-phased on various types of substrates. It can use suitably for uses, such as.

・ Experimental example 1
<Preparation of sample solution>
FITC-labeled single-stranded cDNA was prepared by reverse transcription using oligo dT primer using 25 μg of total RNA derived from human prostate cancer cell line LNCap as a template. This FITC-labeled cDNA was dissolved in 37.5 μl of sterilized distilled water to obtain a labeled cDNA solution, denatured by heating at 95 ° C. for 5 minutes, and then rapidly cooled in ice water. 12.5 μl of 20 × SSPE solution (pH 6.6) was added to this labeled cDNA solution (final salt concentration 5 × SSPE solution) to obtain a sample solution.

<Preparation of DNA probe and immobilization>
As a gene (target) to be analyzed, one gene (“KLK-3”) that was predicted to specifically increase in LNCap was selected. Β-Actin, which is a typical housekeeping gene, was selected as a gene used as an internal control (IC).

Based on the base sequences specific to the cDNA sequences of the target and IC, 60-mer DNA probe fragments were respectively designed and synthesized as target probes and IC probes. And what added the amino group to the probe for a target was melt | dissolved in the buffer solution. Similarly, an amino group was added to the IC probe and dissolved in a buffer solution. The absorption at 260 nm of these solutions was measured, and a buffer solution was added so as to have the same concentration, thereby obtaining a final target probe solution and an IC probe solution, respectively.
The anodic oxide film of aluminum on the substrate was coated with aminosilane and further treated with an N-hydroxysuccinimide solution.
A three-dimensional microarray was prepared, in which one spot for the IC probe, five spots for the target (detection target gene “KLK-3”) probe and each substrate were separately immobilized (chip “1” shown in FIG. 2) .)

Separately, a probe solution for a target (that is, KLK-3) was prepared in the same manner as described above. Further, an IC (that is, β-actin) probe synthesized in the same manner as described above, labeled with tetramethylrhodamine, and further added with an amino group was dissolved in a buffer solution. The absorption at 260 nm of this solution was measured, and a buffer solution was added so as to have the same concentration as the above probe solution to obtain a final probe solution. Next, this probe solution and the above target probe solution were mixed at a volume ratio of 1: 1 to prepare a mixed probe solution containing two types of sequences.
This mixed probe solution was solid-phased on the same substrate as that of the chip “1” with 5 spots to produce a three-dimensional microarray (shown in FIG. 2 as chip “2”).

<Sample hybridization>
The following operations 1) and 2) were performed on the chips “1” and “2” produced above. The hybridization analysis was performed using an apparatus capable of automatically driving the solution and controlling the temperature around the reaction filter provided on the substrate.
1) The sample solution was supplied to the surface of the chip (three-dimensional microarray), and the liquid was driven 150 times to form a hybrid body. The reaction temperature at this time was set to 50 ° C. After completion of the reaction, the sample solution was removed, 50 μl of 6 × SSPE solution was added, and the liquid driving process was repeated three times to wash and remove the labeled nucleic acid that did not form a hybrid.
2) After completion of washing, a fluorescent image was obtained using a U-MWIBA2 mirror unit (Olympus) and U-MWIG2 mirror unit (Olympus) using a CCD camera mounted on a fluorescence microscope. At this time, fluorescence images were acquired by irradiating the microarray with light having wavelengths of 460 to 490 nm and 520 to 550 nm, respectively, as excitation light.
Moreover, the fluorescence intensity of FITC and tetramethylrhodamine corresponding to each probe spot was measured.

As a result, the fluorescence of only FITC was observed in the chip “1”, and the fluorescence of FITC and tetramethylrhodamine was observed in the chip “2”.
Here, the FITC fluorescence in the chip “2” is fluorescence from the KLK-3 gene contained in the sample hybridized with the probe for detecting the KLK-3 gene. The fluorescence of tetramethylrhodamine is hybridized between the probe detecting IC-β-actin and the β-actin gene contained in the sample, and the probe is labeled from the FITC labeled on the sample. This is a fluorescence in which FRET has occurred in tetramethylrhodamine.

FIG. 3 shows the hybridization image analysis results of the chips “1” and “2”.
In FIG. 3, the expression ratio (Y axis) is a correction value of the fluorescence intensity of each KLK-3 spot when the fluorescence intensity value of the IC probe spot is 1.
For chip “1”, spot no. This is a value obtained by correcting the FITC fluorescence intensity values of all KLK-3 spots (spot Nos. 1 to 5) with respect to the FITC fluorescence intensity value of 6 (IC probe spot).
In the chip “2”, for each spot, the FITC fluorescence intensity value on the spot is corrected based on the fluorescence intensity value of tetramethylrhodamine on the spot.
In this experiment, the measured values were expected to be the same at every spot. However, when standardized using an IC of a spot different from the target as in the chip “1”, variation in data was observed. This is considered to be due to the fact that the amount of solidification at each spot and the density of solidification vary, so that the amount of hybridization differs depending on the spot and the illumination is not necessarily uniform. In the chip “2”, no variation was observed in the data, and the variation in the expression ratio observed between the spots of the chip “1” was improved.

Thus, after measuring the abundance ratios of various probes in the probe solution, supplying the probe solution for the target (detection target) probe array and the IC array to the same address, the abundance ratio in the solution is substantially reduced. Since the solid phase is maintained, the solid phase ratio of the target and IC probes at the address is known. By this, even when there is a difference in the amount of solid phase between the spots, even if the illumination light is unstable or non-uniform, by performing normalization using the control, It becomes possible to detect the exact expression level of the target.
In this experimental example, after the concentration of the probe solution was measured, the concentration was adjusted so that the final probe concentration would be the same. However, it is not always necessary to adjust the concentration, and the analysis result can be corrected using the ratio from the concentration data.

・ Experimental example 2
<Preparation of sample solution>
A sample solution was prepared in the same manner as in Experimental Example 1.
<Preparation of DNA probe / immobilization>
A probe solution labeled with tetramethylrhodamine was added to a β-actin DNA probe whose concentration was measured in Experimental Example 1, and an amino group was added to the 3 ′ end of the KLK-3 probe. A probe solution that has been subjected to concentration measurement and concentration adjustment, and a probe solution that has been subjected to concentration measurement and concentration adjustment in the same manner as in Experimental Example 1 by adding an amino group to the 5 ′ end of the KLK-3 probe, By mixing at a volume ratio of 1: 1, a mixed probe solution containing three types of sequences was prepared, and the probe solution was “2”.
In addition, the β-actin probe and the probe solution (concentration adjusted) with an amino group added to the 3 ′ end of the KLK-3 probe were mixed at a volume ratio of 1: 1 to obtain two types of sequences. A mixed probe solution was prepared and designated as probe solution “1”.
Three spots each of the probe solutions “1” and “2” were immobilized on an anodized aluminum substrate in the same manner as the chip “1” to prepare a three-dimensional microarray. FIG. 4 shows the arrangement of DNA probes in the produced microarray.

<Sample hybridization>
Similar to Experimental Example 1, the sample solution was subjected to a hybridization reaction. FIG. 5 shows the results of hybridization image analysis using the above chip.
The expression ratio (Y-axis) is a value obtained by correcting the FITC fluorescence intensity value of KLK-3 at the same spot, assuming that the fluorescence intensity value of IC tetramethylrhodamine on the probe is 1 for each spot.
As is clear from FIG. 5, the variation was eliminated both when the 3 ′ end of the KLK-3 probe was solid-phased and when both the 3 ′ end and the 5 ′ end were solid-phased. The analysis result was obtained. Furthermore, stronger signal intensity was obtained when both the 3 ′ end and the 5 ′ end were immobilized at the same address than when the 3 ′ end of the KLK-3 probe was immobilized. This was presumed to be due to the low reaction efficiency between the probe solid-phased at the 3 ′ end and KLK-3 in the sample.

  In the above experimental examples, an example was described in which a fluorescent label serving as a donor was applied to a sample and a fluorescent substance serving as an acceptor was labeled on an IC array. Detection can also be performed by labeling a fluorescent substance as an acceptor with a sequence for detecting a gene to be detected. It is also possible to exchange donors and acceptors.

Experimental example 3
<Preparation of DNA probe and immobilization>
A probe solution in which an amino group is added to the 3 ′ end of the β-actin probe and a probe solution in which a thiol group is added to the 5 ′ end of the KLK-3 probe are mixed at a volume ratio of approximately 1: 1, A mixed probe solution containing two types of sequences was prepared and designated as probe solution “3”.

As reactants to be immobilized on the substrate, a solution of “BS3” (Bis [Sulfosuccinimidyl] suberate) manufactured by Pierce and a solution of “EMCS” (N- (6-Maleimidocaproyloxy) succinimide) manufactured by Dojindo Chemicals were prepared. Concentrations were adjusted so that these solutions had the same concentration, and these solutions were mixed in equal amounts (volumes) to obtain a mixed reactant solution.
The concentration of the “BS3” solution was measured by infrared absorption spectrum of C═O vibration around 1700 cm ⁻¹ . As for the concentration of the “EMCS” solution, the vibration of C═C near 1670 cm ⁻¹ was measured by a Raman spectrum.
The mixed reactant solution was brought into contact with an anodized aluminum substrate that had been treated with aminosilane.

The probe solution “3” containing the two types of sequences described above was supplied to the aluminum anodic oxide film substrate subjected to the above-described treatment, and a three-dimensional microarray in which the probes were solid-phased was produced.
In general, an amino group and an active ester (such as succinimide) react specifically, and a thiol group and a maleimide group react specifically.
Therefore, the above-mentioned two kinds of reactants are immobilized on the substrate through the amino group of the aminosilane treated on the substrate, maintaining the ratio in the mixed reactant solution when the reactants have the same reaction efficiency with the amino group. Is done. If the reaction efficiency is different or unknown, the ratio fixed on the substrate may be measured by absorption or the like, and the ratio is calculated by measuring the concentration of the two reactant solutions, then the substrate The ratio fixed above can be measured by absorption or the like, the reaction efficiency can be calculated, and the concentration of the two reactant solutions can be adjusted to achieve the desired ratio.
Next, when the probe to which the amino group is added and the probe to which the thiol group is added are mixed and supplied to the substrate, each probe is immobilized on the substrate at a ratio that reflects the ratio of the reactants immobilized on the substrate. Is done.

<Confirmation of ratio of immobilized probe>
Cy3 was labeled on the counter oligo DNA of the β-actin probe (oligo DNA having a complementary base sequence), and Cy5 was labeled on the counter oligo DNA of the KLK-3 probe.
These oligo DNAs were dissolved in SSPE buffer and the concentration was adjusted to mix the two types of solutions so that the ratio was 1: 1. This solution was supplied to the prepared three-dimensional microarray and allowed to hybridize. Then, the light for exciting Cy3 and Cy5 was irradiated, and the generated fluorescence was detected.
When the detection result was corrected by the luminous efficiency, it was confirmed that the fluorescence intensity of Cy3 and Cy5 was 1: 1, and the β-actin probe and the KLK-3 probe were immobilized on a 1: 1 basis. It was.

  Thus, without measuring the concentration of the probe, by measuring the concentration of the reactant immobilized on the substrate for immobilizing the probe on the substrate surface, a plurality of probes can be obtained at a predetermined ratio. It was shown that it is possible to immobilize at one address.

It is a conceptual diagram which shows one Embodiment in the bio-related substance analysis method of this invention. FIG. 6 is a plan view showing a DNA probe arrangement (corresponding to address-probe type) in Experimental Example 1. 6 is a graph showing a hybridization image analysis result (after correction) in Experimental Example 1. It is a top view which shows DNA probe arrangement | positioning (corresponding to an address-probe type) in Experimental Example 2. 10 is a graph showing a hybridization image analysis result (after correction) in Experimental Example 2.

Claims (15)

  A plurality of types of probes for detecting a biological substance are immobilized on the same address, and the ratio of the immobilized probes is known. Solid phase support for substance detection.   The ratio is determined by measuring the absorbance of the probe, measuring the label of the labeled probe, measuring the concentration of the first reactant in the probe bound to the first reactant specifically corresponding to the probe, or 2. The biorelevant substance detecting solid body according to claim 1, which is obtained by measuring the concentration of a second reactive substance that can specifically react with the first reactive substance or probe and is fixed at a predetermined address of the carrier. Phase support.   The solid-phase support for detecting a biological substance according to claim 1 or 2, wherein at least one of the plural types of probes is for detecting a control biological substance.   The solid-phase support for detecting a bio-related substance according to any one of claims 1 to 3, wherein a plurality of types of probes are immobilized at the same address for one type of bio-related substance to be detected.   The solid-phase support for detecting a biological substance according to any one of claims 1 to 4, wherein the biological substance is a nucleic acid.   Plural types of probes that are immobilized at the same address include a first probe having a 3 ′ end immobilized on a carrier, the same base sequence as the first probe, and a 5 ′ end on the carrier. The solid-phase support for detecting a biological substance according to claim 4 or 5, comprising a second probe that is solid-phased.   7. The solid-phase support for detecting a biological substance according to claim 6, wherein the first probe and the second probe are solid-phased at a ratio of about 1: 1. A method for immobilizing a probe for detecting one or more biological substances,
Preparing a plurality of types of probes for detecting one or more biological substances;
A second step of mixing the plurality of probes to obtain a mixed probe;
And a third step of bringing the mixed probe into contact with the same address of the carrier,
Have
The ratio of the plurality of types of probes is measured between the first step and the second step, or between the second step and the third step, or after the third step. Phase method. A method for immobilizing a probe for detecting one or more biological substances,
Preparing a plurality of types of probes for detecting one or more biological substances;
A second step of mixing the plurality of probes to obtain a mixed probe;
A third step of contacting a carrier with a plurality of reactants that specifically react with each of the plurality of probes;
And a fourth step of contacting the mixed probe with the carrier,
Have
A method for immobilizing a probe, wherein the ratio of the plurality of types of reactants is measured before the third step or between the third step and the fourth step. A method for immobilizing a probe for detecting one or more biological substances,
A first step of preparing a plurality of types of probes to which a plurality of types of reactants specifically corresponding to each of the one or more biological substances are detected,
A second step of mixing the plurality of probes to obtain a mixed probe;
And a third step of bringing the mixed probe into contact with the carrier,
Have
And a ratio of the plurality of reactants is measured between the first step and the second step, or between the second step and the third step, or after the third step. Solid phase method. A method for immobilizing a probe for detecting one or more biological substances,
A first step of preparing a plurality of types of probes to which a plurality of types of reactants specifically corresponding to each of the one or more biological substances are detected,
A second step of mixing the plurality of probes to obtain a mixed probe;
A third step in which a plurality of reactants specifically reacting with a plurality of reactants bound to a probe are brought into contact with a carrier;
And a fourth step of contacting the mixed probe with the carrier,
Have
A method for immobilizing a probe comprising measuring a ratio of a plurality of types of reactants brought into contact with a carrier before the third step or between the third step and the fourth step.   The plurality of types of probes include the same base sequence, and a first probe having a 3 'end and a second probe having a 5' end on the side to be immobilized on a carrier. A method for immobilizing a probe according to any one of the above.   The method for immobilizing a probe according to any one of claims 8 to 12, wherein the biological substance is a nucleic acid. A method for analyzing a biological substance using the solid-phased carrier according to any one of claims 1 to 7,
A first step of supplying a sample containing one or more biological substances to the solid-phase support;
A second step of measuring a signal intensity of a reactant obtained by reacting the biological substance with the probe;
And a third step of correcting the intensity based on the ratio,
A method for analyzing a biological substance, characterized by comprising: The biological signal of the biological material according to claim 14, wherein the signal intensity of the reactant is measured by causing fluorescence resonance energy transfer between the fluorescent label labeled on the probe and the fluorescent label labeled on the biological material. analysis method.

Images