JP2011196879A - Detection method and kit of target biomolecule - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect biomolecules simply and sensitively by using a chemosynthetically producible aptamer.SOLUTION: This method is described as follows: a sample solution containing target biomolecules 41 are supplied to a device wherein a first nucleic acid chain 21 functioning as an aptamer is immobilized on a first electrode 2 provided in a reaction cell 1, and a second nucleic acid chain 22 which is a comparison object is immobilized on a second electrode 3; further, a sixth nucleic acid chain 26 which is a complementary chain for the nucleic acid chain 21 is supplied thereto; and thereafter, a difference of each signal amount from the first and second electrodes 2, 3 is determined, to thereby detect the target biomolecules 41.

Description

  The present invention relates to a method and a kit for detecting a biomolecule.

  In recent years, there has been a demand for a method for easily monitoring a biomolecule related to a disease, such as a tumor marker. In general, biomolecules including proteins are known to perform sensing using an antigen-antibody reaction. In an actual medical field, an immunochromatography kit based on an antigen-antibody reaction is used. When using an antigen-antibody reaction, it is necessary to prepare an antibody as a biological sensor to detect an antigen and an antigen to detect an antibody. However, it is necessary to immunize living organisms such as mice and rabbits and generate them biologically, and there is a risk of mass production and quality variations.

  In recent years, aptamers that are nucleic acid molecules that recognize and specifically bind to biomolecules such as proteins have been known, as in the case of antigen-antibody reactions. Since aptamers are nucleic acid molecules, chemical synthesis is possible, and quality-controlled substances can be industrially mass-produced. Therefore, it is considered useful if necessary biomolecules can be easily detected using an aptamer.

  Patent Document 1 discloses a biomolecule detection method using an aptamer. However, in any case, it is necessary that the aptamer is labeled in advance by a fluorescent label, dielectric, or intercalator, and these substances are affected by the interaction with the target biomolecule, There is a risk that the detection is difficult or the detection sensitivity is lowered.

JP 2008-278837 A

  An object of the present invention is to provide an apparatus and a method for easily detecting a biomolecule with high sensitivity using a chemically synthesized aptamer.

  The first target biomolecule detection method of the present invention includes a reaction cell, first and second electrodes provided in the reaction cell, and one end immobilized on the first electrode. A first nucleic acid chain that functions as an aptamer and binds to the target biomolecule, and a second nucleic acid that has one end immobilized on the second electrode and does not function as an aptamer for the target biomolecule A device comprising: a strand; and a third nucleic acid strand that does not form a double strand with the first and second nucleic acid strands and their complementary strands in the reaction cell, and the second A step of supplying a region forming a double strand with a nucleic acid strand and a fourth nucleic acid strand having a region forming a double strand with the third nucleic acid strand, and may include the target biomolecule A target sample solution is supplied, and the target biomolecule and the first nucleic acid strand are bound to each other. A step of combining them, a fifth nucleic acid strand that does not form a double strand with the first to fourth nucleic acid strands, and a double strand with the first nucleic acid strand can be formed in the reaction cell A first nucleic acid strand that is not bound to the target biomolecule, and a sixth nucleic acid strand having a single-stranded region and a region that forms a double strand with the fifth nucleic acid strand And a step of forming a double strand with the single-stranded region of the sixth nucleic acid strand, from the first electrode, the first nucleic acid strand and the fifth nucleic acid strand; A first electrochemical signal derived from a double strand formed between the sixth nucleic acid strands, the second and third nucleic acid strands from the second electrode, and the fourth Detecting a second electrochemical signal derived from a double strand formed between the nucleic acid strands of the first nucleic acid strand, the first electrochemical signal, and the second electrical signal. From the difference between the chemical signal, and performing the step of calculating the amount of the target biomolecule. Supplying the third nucleic acid strand and the fourth nucleic acid strand to the reaction cell; supplying a sample solution that may contain the target biomolecule; and The above-described step that is performed under the condition that one nucleic acid chain binds may be performed simultaneously.

  The second target nucleic acid detection method of the present invention includes a reaction cell, first and second electrodes provided in the reaction cell, one end immobilized on the first electrode, and a target living body. A first nucleic acid chain that functions as an aptamer for a molecule, can bind to the target biomolecule, and a second nucleic acid that has one end immobilized on the second electrode and does not function as an aptamer for the target biomolecule. A nucleic acid strand, a third nucleic acid strand that does not form a double strand with the first and second nucleic acid strands and their complementary strands, a region that forms a double strand with the second nucleic acid strand, and Providing a device comprising a fourth nucleic acid strand having a region forming a double strand with a third nucleic acid strand, and supplying a sample solution that may contain the target biomolecule, A step of subjecting the target biomolecule and the first nucleic acid strand to a binding condition; In the reaction cell, a fifth nucleic acid strand that does not form a double strand with the first to fourth nucleic acid strands, a single-stranded region that can form a double strand with the first nucleic acid strand, and the fifth nucleic acid strand A first nucleic acid strand that has a region that forms a double strand with the nucleic acid strand, and that is not bound to the target biomolecule and the one of the sixth nucleic acid strand Formed between the first nucleic acid strand, the fifth nucleic acid strand, and the sixth nucleic acid strand from the first electrode, under the condition of forming a double strand with the single strand region. First electrochemical signal derived from the double strand, and two formed from the second electrode between the second nucleic acid strand and the third nucleic acid strand and the fourth nucleic acid strand Detecting a second electrochemical signal derived from a chain, and the difference between the first electrochemical signal and the second electrochemical signal, And performing the step of calculating the amount of body molecules. The second nucleic acid chain and the third nucleic acid chain may be linked.

  In the first and second target nucleic acid detection methods described above, the step of detecting the first and second electrochemical signals supplies an intercalating agent solution that specifically binds to the reaction cell in a double-stranded manner. A step of binding an intercalating agent to the double strand in the reactive cell, and an electrochemical signal generated by oxidation or reduction reaction of the intercalating agent is detected by the first electrode and the second electrode, respectively. It is desirable to perform the process.

  In addition, the first target biomolecule detection kit of the present invention is a target biomolecule immobilized on a reaction cell, first and second electrodes provided in the reaction cell, and the first electrode. A first nucleic acid chain that functions as an aptamer and binds to the target biomolecule, and a second nucleic acid that has one end immobilized on the second electrode and does not function as an aptamer for the target biomolecule A first nucleic acid strand, a third nucleic acid strand that does not form a double strand with the first and second nucleic acid strands and their complementary strands, and a double strand with the second nucleic acid strand. A fourth nucleic acid strand having a single-stranded region and a region that forms a double strand with the third nucleic acid strand, and the first to fourth nucleic acid strands do not form a double strand. 5 nucleic acid strands, a single-stranded region capable of forming a double strand with the first nucleic acid strand, and the fifth nucleic acid strand with two Characterized by comprising a sixth nucleic acid strand having a region forming the chain, the.

  The second target biomolecule detection kit of the present invention comprises a reaction cell, first and second electrodes provided in the reaction cell, one end immobilized on the first electrode, and a target biomolecule A first nucleic acid chain that functions as an aptamer and binds to the target biomolecule, and a second nucleic acid that has one end immobilized on the second electrode and does not function as an aptamer for the target biomolecule A strand, a third nucleic acid strand that does not form a double strand with the first and second nucleic acid strands and their complementary strands, a region that forms a double strand with the second nucleic acid strand, and the first A fourth nucleic acid chain having a region forming a double strand with three nucleic acid strands, and a fifth nucleic acid that does not form a double strand with the first to fourth nucleic acid strands A strand, a single-stranded region capable of forming a double strand with the first nucleic acid strand, and a fifth strand with the fifth nucleic acid strand Characterized by comprising a sixth nucleic acid strand having a region that forms the. The second nucleic acid chain and the third nucleic acid chain may be linked.

  The first and second kits preferably further comprise an intercalating agent solution that specifically binds to double strands.

  According to the present invention, it is possible to detect a biomolecule with high sensitivity and ease using an aptamer.

The schematic diagram which shows the method of this embodiment. The schematic diagram which shows the method of this embodiment. The schematic diagram which shows the method of this embodiment. The schematic diagram which shows the method of this embodiment. The schematic diagram which shows the method of this embodiment. The schematic diagram which shows the method of this embodiment. The schematic diagram which shows the method of this embodiment. The schematic diagram which shows the method of this embodiment. The schematic diagram which shows the method of this embodiment. The schematic diagram which shows the method of this embodiment. The schematic diagram which shows the method of this embodiment. The relationship figure of the signal value from the detection electrode by the method of this embodiment, and the binding rate of a target biomolecule. The characteristic view which shows the detection result by the method of an Example.

  The present invention relates to a method for electrochemically detecting a target biomolecule in a sample using an aptamer that specifically binds to a biomolecule as a probe.

  In the present specification, an aptamer is a nucleic acid molecule having a function of specifically binding to a specific biomolecule. Examples of the nucleic acid molecule include DNA, RNA, PNA and the like.

Examples of target biomolecules include proteins, cells, cell tissues, microorganisms, viruses, bacteria, and toxins.
Hereinafter, a preferred embodiment of the method of the present invention will be described with reference to the drawings.

<First step>
First, an apparatus for electrochemically detecting a target biomolecule is prepared. FIG. 1 is a schematic diagram of an apparatus according to this embodiment. The apparatus includes a reaction cell 1 for reacting a sample solution with an aptamer. In the same reaction cell 1, a first electrode 2 for detecting a target biomolecule and a second electrode 3 for comparison are provided. One end of the first nucleic acid chain 21 that functions as an aptamer for the target biomolecule is immobilized on the first electrode 2. The sequence of the first nucleic acid chain 21 is appropriately selected according to the type of the target biomolecule. One end of a second nucleic acid chain 22 that does not function as an aptamer for the target biomolecule is immobilized on the second electrode 3. For example, the first electrode 2 and the second electrode 3 are made of gold, and one end of the first nucleic acid strand 21 and the second nucleic acid strand 22 is modified with a thiol group, thereby immobilizing the nucleic acid strand. Is possible.

  When a plurality of target biomolecules are to be detected, a plurality of first electrodes 2 may be provided in the reaction cell 1, and aptamers corresponding to the target biomolecules may be immobilized on each electrode. In that case, one second electrode may be provided for each first electrode on which different aptamers are immobilized, or one second electrode on each of a plurality of first electrodes on which different aptamers are immobilized. The second electrode may be shared.

  Note that the substance can move between the first electrode 2 and the second electrode 3.

<Second step>
Next, a third nucleic acid strand 23 and a fourth nucleic acid strand 24 shown below are supplied into the reaction cell 1. FIG. 2 is a schematic diagram showing a third nucleic acid strand and a fourth nucleic acid strand.

  The third nucleic acid strand 23 has a non-complementary sequence with each of the first and second nucleic acid strands 21 and 22 and their complementary strands, and does not form a double strand with them. The fourth nucleic acid strand 24 is complementary to at least a part of the second nucleic acid strand 22, and has a region 31 having a sequence capable of forming a double strand with the second nucleic acid strand 22, and a third nucleic acid strand 23. And a region 32 that is complementary to the third nucleic acid strand 23 to form a double strand. That is, the first nucleic acid strand pair 11 (partial double strand) in which the third nucleic acid strand 23 and the fourth nucleic acid strand 24 are combined to form a double strand is supplied into the reaction cell 1.

  FIG. 3 is a schematic diagram showing a state when the third nucleic acid strand and the fourth nucleic acid strand are supplied to the apparatus. As a result, as shown in FIG. 3, on the second electrode 3, a fourth nucleic acid strand 24 and a double strand of the third nucleic acid strand 23, a fourth nucleic acid strand 24 and a second nucleic acid strand 22, and Will be present.

That is, the fourth nucleic acid strand 24 is bonded to the second nucleic acid strand 22 to form a double strand. In addition, the third nucleic acid chain 23 binds to a region of the fourth nucleic acid chain 24 that does not form a double strand with the second nucleic acid strand 22 to form a double strand. That is, the fourth nucleic acid strand 24 is a region 31 that forms a double strand with the second nucleic acid strand 22 and a region that forms a double strand with the third nucleic acid strand 23. 32.
When the second nucleic acid strand on the second electrode 3 is immobilized at the stage of preparing the first process apparatus, the second nucleic acid strand 22 and the third nucleic acid strand 23 are complementary to them. A device may be prepared in which the fourth nucleic acid strand 24 having the correct sequence is double-stranded and immobilized on the second electrode 3. In this case, the second step of supplying the third nucleic acid strand 23 and the fourth nucleic acid strand 24 is not necessary.

  In order to obtain a device immobilized on the second electrode 3 as a double strand of a second nucleic acid strand 22 and a third nucleic acid strand 23 and a fourth nucleic acid strand 24 complementary thereto, After immobilizing the second nucleic acid strand 22, a fourth nucleic acid strand having a sequence complementary to the nucleic acid strand is supplied for hybridization, and further a third nucleic acid strand 23 is supplied for hybridization. Obtainable. More preferably, as shown in FIG. 2, a first nucleic acid strand pair 11 is prepared in advance, in which a third nucleic acid strand 23 and a fourth nucleic acid strand 24 are formed in a region 31, and a double strand is formed. It can be obtained by hybridization with the second nucleic acid strand 22 immobilized on the second electrode 3.

<Third step>
Next, a sample solution that may contain the target biomolecule 41 is injected into the reaction cell 1, and the reaction cell 1 is placed under conditions where the target biomolecule and the first nucleic acid chain bind to each other.

  FIG. 4 is a schematic diagram showing a state in which a double strand is formed on the second nucleic acid strand 22 on the second electrode, and the target biomolecule 41 is bound to the first nucleic acid 21. When the target biomolecule 41 is contained in the sample solution, the first nucleic acid chain 21 functions as an aptamer, and the target biomolecule 41 binds to the first nucleic acid chain 21 as shown in FIG.

  When the target biomolecule 41 is not contained in the sample solution, it is natural that there is no target biomolecule 41 that binds to the first nucleic acid strand 21.

  The condition for the target biomolecule 41 to bind to the first nucleic acid chain 21 is, for example, about 25 minutes to 30 minutes to 1 hour. By increasing the temperature from 25 ° C. to 37 ° C., the time can be further shortened. However, when the target biomolecule is a protein, if it exceeds 37 ° C., the protein may be denatured, which is not preferable.

Alternatively, the second step may be omitted, and the first nucleic acid strand pair 11 may be included in the sample solution in the third step. In this way, it is possible to monitor whether the sample solution has been supplied into the reaction cell 1. Further, this third step may be performed simultaneously with the fourth step shown below.
<4th process>
Next, the second nucleic acid strand pair 12 partially forming a double strand is supplied to the reaction cell 1. FIG. 5 is a schematic diagram of the second nucleic acid strand pair 12. FIG. 6 is a schematic diagram showing a state in which the second nucleic acid chain pair 12 is added to the reaction cell 1 when the target biomolecule 41 is bound to the first nucleic acid chain 21. FIG. 7 is a schematic diagram showing a state where the second nucleic acid chain pair 12 is added to the reaction cell 1 when the target biomolecule 41 does not bind to the first nucleic acid chain 21.

  The second nucleic acid strand pair 12 to be added includes a fifth nucleic acid strand 25, a single-stranded region 33 capable of forming a double strand with the first nucleic acid strand 21, and a fifth nucleic acid as shown in FIG. And a sixth nucleic acid strand 26 having a region 34 forming a double strand with the strand 25. The fifth nucleic acid strand is a sequence that does not form a double strand with the first to second nucleic acid strands 21 and 22, the fourth nucleic acid strand region 31, and the sixth nucleic acid strand 26 region 33. It is.

  After supplying the second nucleic acid strand pair 12, the single stranded region 33 of the sixth nucleic acid strand 26 and the first nucleic acid strand 21 are hybridized, and the second nucleic acid strand pair 12 in the second nucleic acid strand pair 12 is hybridized. The conditions are such that the single-stranded region 33 of the six nucleic acid strands 26 and the first nucleic acid strand 21 form a double strand.

  When a sufficient amount of the target biomolecule 41 is contained in the sample solution and the binding between the first nucleic acid chain 21 and the target biomolecule 41 occurs in the third step, as shown in FIG. The single-stranded region 33 of the sixth nucleic acid strand 26 in the second nucleic acid strand pair 12 does not form a double strand with the first nucleic acid strand 21. The sufficient amount here means that at least the number of target biomolecules 41 needs to be larger than the number of first nucleic acid strands 21 immobilized on the first electrode 2.

  When the target biomolecule 41 is not included in the sample solution, the first nucleic acid strand 21 on the first electrode 2 is the sixth nucleic acid strand of the second nucleic acid strand pair 12 as shown in FIG. 26 single-stranded regions 33 and double strands are formed.

  In addition, even if the target biomolecule 41 is contained in the sample solution, in the case of a small amount, the target biomolecule 41 binds to a part of the first nucleic acid chains 21, so that the first nucleic acid 41 The first nucleic acid strand 21 in which the strand 21 and the single-stranded region 33 of the sixth nucleic acid strand 26 of the second nucleic acid strand pair 12 do not form a double strand and the target biomolecule 41 is not bound to the first nucleic acid strand 21 The single-stranded region 33 of the sixth nucleic acid strand 26 in the second nucleic acid strand pair 12 forms a double strand.

  The nucleic acid hybridization conditions are, for example, holding at 25 ° C. for 30 minutes.

  The fourth step may be performed simultaneously with the third step, that is, the supply of the second nucleic acid strand pair 12 and the supply of the sample solution to the reaction cell 1 may be performed simultaneously.

<5th process>
Next, an electrochemical signal derived from the double strand present on the first and second electrodes is detected. As a method for detection, a method in which an intercalator molecule 51 that specifically binds to a double strand is bound to a double strand and an oxidation or reduction reaction of the intercalator molecule is detected electrochemically has high detection efficiency. Although this method is desirable, any method that can detect double strands may be used.

  FIG. 8 is a schematic diagram showing a state in which, when the target biomolecule 41 is bound to the first nucleic acid 21, the intercalating agent molecule 51 is added and the intercalating agent molecule 51 is bound to the double-stranded forming region. FIG. 10 is a schematic diagram showing a state in which, when the target biomolecule 41 is not bound to the first nucleic acid 21, the intercalating agent molecule 51 is added and the intercalating agent molecule 51 is bound to the double-stranded forming region. .

  A specific procedure is shown below.

  First, a solution containing an intercalator molecule 51 that specifically binds to a double-stranded nucleic acid is added to the reaction cell 1.

  When a sufficient amount of the target biomolecule 41 is present in the sample solution, since no double strand is formed on the first electrode 2 as shown in FIG. 8, the intercalating agent molecule 51 does not bind. On the other hand, since the second nucleic acid strand 22, the third nucleic acid strand 23, and the fourth nucleic acid strand 24 form a double strand on the second electrode 3, the intercalator molecule 51 is placed in the double strand here. Join.

  When the target biomolecule 41 is not present in the sample solution, the first nucleic acid strand 21, the fifth nucleic acid strand 25, and the sixth nucleic acid strand 26 are also formed on the first electrode 2 as shown in FIG. Forms a double strand. Therefore, the intercalator molecule 51 is bound to this double strand. Also on the second electrode 3, the second nucleic acid strand 22, the third nucleic acid strand 23, and the fourth nucleic acid strand 24 form a double strand. The intercalator molecule 51 also binds to this double strand.

  When a small amount of the target biomolecule 41 is present in the sample solution, a case where no double strand is formed on the first electrode 2 as shown in FIG. 8 and a case where the first electrode 2 is shown as shown in FIG. The case where a double strand is formed is mixed. When a double strand is formed, the intercalating agent molecule 51 binds, and when no double strand is formed, the intercalating agent molecule 51 does not bind.

  As the intercalating agent, Hoechst 33258 is desirable, but it is not limited to Hoechst 33258. Double-strand recognizers such as acridine orange, quinacrine, dounomycin, metallointercalators, bisintercalators such as bisacridine, trisintercalators, and polyintercalators may be used. Furthermore, even if these substances are modified with substances showing an electrochemical response such as ferrocene and viologen, the same detection can be performed.

  Next, each of the first electrode 2 and the second electrode 3 is used as a working electrode, and a counter electrode and a reference electrode separately provided are used, and the potential of the reference electrode provided in the reaction cell is fed back as necessary. However, the electric potential is swept between the working electrode and the counter electrode in the reaction cell, and the current detected from the working electrode is measured. Electrochemical signals derived from these double strands can be easily and automatically inspected by using an automatic base sequence analyzer disclosed in Japanese Patent Application Laid-Open Nos. 2004-61426 and 2004-125777. Is possible.

  FIG. 9 is a schematic diagram showing a state in which current detection is performed when the target biomolecule 41 is bound to the first nucleic acid chain 21. FIG. 11 is a schematic diagram showing a state in which current detection is performed when the target biomolecule 41 does not bind to the first nucleic acid chain 21.

  When a sufficient amount of the target biomolecule 41 is present in the sample solution, as shown in FIG. 9, no double strand exists on the first electrode 2 for detection. The electrochemical signal 61 from the intercalator molecule 51 is not observed. Since a double strand exists on the second electrode 3 for comparison, an electrochemical signal 61 from the intercalating agent molecule 51 is observed from the second electrode 3. Here, the direction of the electrochemical signal 61 indicates the direction of electron flow when Hoechst 33258 is used as the intercalating agent molecule.

  When the target biomolecule 41 does not exist in the sample solution, double strands are present on the first electrode 2 for detection as shown in FIG. An electrochemical signal 62 from the molecule 51 is observed. In addition, since a double strand exists also on the second electrode 3 for comparison, an electrochemical signal 62 from the intercalating agent molecule 51 is observed from the second electrode 3.

  When the target biomolecule 41 is present in a very small amount in the sample solution, a part of the first nucleic acid strand 21 on the first electrode 2 for detection becomes a second nucleic acid strand pair as shown in FIG. 12 and a double strand are formed, and a part of the double strand is not formed as shown in FIG. Therefore, an electrochemical signal is observed from the nucleic acid strand in which the double strand is formed, and no electrochemical signal is observed from the nucleic acid strand in which the double strand is not formed. Accordingly, in this case, as the first electrode 2, the value of the electrochemical signal when all the first nucleic acid strands 21 form a double strand and the first nucleic acid strand 21 are completely two. A value between the values of the electrochemical signal when the chain is not formed will be observed.

  As described above, the magnitude of the electrochemical signal obtained from the first electrode 2 differs depending on the presence / absence of the target biomolecule 41 in the sample solution and the abundance. On the other hand, the magnitude of the electrochemical signal obtained from the second electrode 3 for comparison is constant regardless of the presence or absence of the target biomolecule 41. Therefore, it was bound to the first nucleic acid 21 by calculating the difference of the electrochemical signal obtained from the first electrode for detection from the magnitude of the electrochemical signal obtained from the second electrode 3 for comparison. The presence / absence of the target biomolecule 41 and the abundance can be determined. If the target biomolecule is present in a large amount, the difference value is large. If the target biomolecule is small, the difference value is small. It is practical to determine the magnitude of the difference by comparison with a threshold value obtained in advance through experiments or the like.

    Further, when a sufficient amount of the target biomolecule 41 is present in the added sample solution and the target biomolecule 41 is bound to all the first nucleic acid chains 21 on the first electrode 2, Ideally, the electrochemical signal is not detected, and the electrochemical signal 61 is detected only from the second electrode 3. Actually, the number of first nucleic acid chains 21 to which the target biomolecule 41 is bound varies depending on the number of target biomolecules 41. Since the electrochemical signal 62 from the first electrode 2 is observed by an amount corresponding to the amount of the first nucleic acid strand 21 to which the target biomolecule 41 is not bound, the sample is sampled by the above method. It is also possible to measure the abundance of target biomolecules therein. That is, from the value of the difference between the value of the electrochemical signal obtained from the second electrode 3 and the value of the electrochemical signal obtained from the first electrode 2, the target bound to the first electrode 2 for detection The amount of the biomolecule 41 can be calculated. It is practical to determine the magnitude of the difference by comparison with a calibration curve obtained in advance through experiments or the like.

  FIG. 12 is a relationship diagram between the signal value from the detection electrode and the binding ratio of the target biomolecule according to the method of this embodiment.

In actual measurement, even when the target biomolecule binds to all the first nucleic acids 21 and no double strand exists on the first electrode 2, the electrochemical signal does not become zero, and other than the double strand An electrochemical signal due to the oxidation / reduction reaction of the intercalating agent molecules 51 adsorbed nonspecifically on is detected. In that case, it is necessary to subtract such a background signal to evaluate the magnitude of the signal. (Fig. 12)
In the method of the present invention, in the situation where the target biomolecule 41 is not present at all, the intercalating agent molecule 51 is supplied into the reaction cell 1 and the electrochemical signal obtained from the first electrode 2 for detection is obtained as a result of the voltage sweep. The fourth and fifth nucleic acid strands and the second nucleic acid strand forming the first nucleic acid strand pair 11 so that the value and the value of the electrochemical signal obtained from the second electrode 3 for comparison are substantially equal. It is desirable to select the sequence and length of the fifth and sixth nucleic acid strands that form pair 12. Further, in principle, the first nucleic acid strand pair 11 has only the region 31 of the fourth nucleic acid strand 24, and the second nucleic acid strand pair 12 has only the region 33 of the sixth nucleic acid strand 26. Although it is possible to detect whether the nucleic acid strand 21 or the second nucleic acid strand 22 has formed a double strand, in order to clearly detect an electrochemical signal derived from the double strand, as described above, the first strand A double-stranded region in the region 32 of the third nucleic acid strand 23 and the fourth nucleic acid strand 24 in advance, and a fifth nucleic acid strand 25 and a sixth in advance in the second nucleic acid strand pair 12. It is very useful to prepare a double-stranded region in the region 34 of the nucleic acid strand 26.

  Specifically, the length of the first nucleic acid chain is 10 to 30 bases, more preferably 13 to 20 bases, and the length of the second nucleic acid chain is 10 to 30 bases, more preferably It is desirable that it is 13 bases or more and 20 bases or less.

  The fourth nucleic acid strand or the sixth nucleic acid strand is preferably longer, and the third nucleic acid strand is complementary to the fourth nucleic acid strand other than the complementary region of the second nucleic acid strand. It is preferable that the fifth nucleic acid strand is complementary to the region other than the complementary region of the first nucleic acid strand in the sixth nucleic acid strand. Here, since each double-stranded region is a region to which an intercalator molecule binds, the longer the length, the more binding regions, resulting in a larger electrochemical signal. Can be taken and desirable.

  Further, here, a fourth nucleic acid strand that is a single strand and a third nucleic acid strand that is a single strand form a first nucleic acid strand pair, and a sixth nucleic acid that is a single strand. A configuration has been described in which a second nucleic acid strand pair is formed by a fifth nucleic acid strand that is a single strand and a strand, and each is hybridized with the second nucleic acid strand or the first nucleic acid strand. It is not necessarily limited to. That is, the first nucleic acid strand pair only needs to have a single-stranded region complementary to the second nucleic acid strand and a double-stranded region. Therefore, the first nucleic acid strand pair is a region complementary to the second nucleic acid strand. May be a nucleic acid amplified by LAMP (Loop-Mediated Isolation Amplification) having a single-stranded structure. Similarly, the second nucleic acid strand pair may be a LAMP-amplified nucleic acid having a structure in which a region complementary to the first nucleic acid strand is a single strand.

  In the method of this embodiment, when reacting a target biomolecule, it is not necessary to label the target biomolecule or the second nucleic acid chain pair with a fluorescent dye or the like in advance. Therefore, there is no fear that problems such as undetectability and detection sensitivity decrease due to inhibition of the reaction by these labeling substances. In addition to the first and second nucleic acid strands, the third to sixth nucleic acid strands are used, the absolute amount of the double strand formed on the electrode is increased, and the amount of signal obtained from each electrode is amplified. Therefore, accurate measurement is possible.

  In carrying out the method of the present invention, it is practically desirable to make a kit of the apparatus according to the present invention and the nucleic acid chain and reagents to be supplied. Examples of kit forms include the following kits A and B.

[Kit A] Device A, nucleic acid chain set A, other reagents
Apparatus A: Reaction cell, first and second electrodes provided in the reaction cell, immobilized on the first electrode, functions as an aptamer for a target biomolecule, A first nucleic acid strand capable of binding, and a second nucleic acid strand which is immobilized on the second electrode and does not function as an aptamer with respect to the target biomolecule.

Nucleic acid strand set A: As the first nucleic acid strand pair, the first nucleic acid strand and the third nucleic acid strand that does not form a double strand with the complementary strand thereof, and the second nucleic acid strand and the second nucleic acid strand A fourth nucleic acid strand having a single-stranded region capable of forming a strand and a region forming a double strand with the third nucleic acid strand;
As a second nucleic acid strand pair, a fifth nucleic acid strand that does not form a double strand with the first to fourth nucleic acid strands, a single-stranded region that can form a double strand with the first nucleic acid strand, and A nucleic acid chain set comprising: a sixth nucleic acid chain having a region forming a double strand with the fifth nucleic acid chain.

Reagents: intercalating agent solution (when measuring using intercalating agent), buffers, etc.
[Kit B] Device B, nucleic acid chain set B, other reagents
Apparatus B: a reaction cell, first and second electrodes provided in the reaction cell, one end fixed to the first electrode, functioning as an aptamer for a target biomolecule, and the target living body A first nucleic acid strand that can bind to a molecule, a second nucleic acid strand that has one end immobilized on the second electrode and does not function as an aptamer for the target biomolecule, and a first nucleic acid strand pair, A third nucleic acid strand that does not form a double strand with the first and second nucleic acid strands and their complementary strand, a region that forms a double strand with the second nucleic acid strand, and the third nucleic acid And a fourth nucleic acid chain having a region forming a double strand with the chain (may be a linked nucleic acid chain in which the second nucleic acid chain and the third nucleic acid are linked).
Nucleic acid strand set B: As a second nucleic acid strand pair, a fifth nucleic acid strand that does not form a double strand with the first to fourth nucleic acid strands and a double strand with the first nucleic acid strand can be formed A nucleic acid chain set comprising a single nucleic acid region and a sixth nucleic acid strand having a region forming a double strand with the fifth nucleic acid strand.

  Reagents: intercalating agent solution (when measuring using intercalating agent), buffers, etc.

  In addition, all methods and kits that include elements of the present invention and that can be appropriately modified by those skilled in the art are included in the scope of the present invention.

  Below, the specific example which detected thrombin as a target biomolecule using the above-mentioned detection apparatus is demonstrated.

  A silicone resin structure having a groove formed on a glass substrate was loaded so that the groove was disposed on the substrate, and the reaction cell 1 was obtained. Both the first electrode for detection 2 and the second electrode for comparison 3 are arranged so as to be accommodated in the reaction cell 1. The electrode is composed of gold formed on a glass substrate and has a diameter of 200 μm. Twelve electrodes were formed on the glass substrate.

  Electrode numbers 1 to 4 were assigned to the negative control electrodes, electrode numbers 5 to 8 were assigned to the second electrode 3 for comparison, and electrode numbers 9 to 12 were assigned to the first electrode 2 for detection.

  A thrombin aptamer probe (SEQ ID NO: 1) was immobilized on the first electrode 2 as the first nucleic acid chain 21. This is an aptamer sequence known to bind to the fibrinogen binding site of thrombin.

  A positive control probe (SEQ ID NO: 2) was immobilized on the second electrode 3 as a second nucleic acid chain 22 having a sequence different from that of the thrombin aptamer probe (SEQ ID NO: 1).

  Furthermore, a negative control probe (SEQ ID NO: 3) having a different sequence from the thrombin aptamer probe (SEQ ID NO: 1) and the positive control probe (SEQ ID NO: 2) was immobilized on the negative control electrode.

Here, each nucleic acid chain was immobilized on a gold electrode by modifying a thiol group at the 3 ′ end. Table 1 shows the nucleic acid probe sequences used.

  Such a chip was placed in a reaction cell, and a solution containing thrombin at a concentration of 2.7 μM was supplied onto the chip and left at 25 ° C. for 60 minutes. Thrombin is dissolved in a buffer of 10 mM Tris HCl, 150 mM NaCl, 5 mM KCl, 0.05% Tween.

Thereafter, target nucleic acid 1 for detection electrode (SEQ ID NO: 4) as the sixth nucleic acid strand, target nucleic acid 2 (SEQ ID NO: 5) common as the fifth and third nucleic acid strands, and positive as the fourth nucleic acid strand A solution in which the control target nucleic acid 3 (SEQ ID NO: 6) was mixed was supplied onto the chip. These nucleic acids are dissolved in a buffer of 2 × SSC, 10 mM Tris HCl, 5 mM KCl, 0.05% Tween, and the final concentrations of the target nucleic acids are 0.4 μM for the target nucleic acid 1 and 1 for the target nucleic acid 2, respectively. 6 μM and target nucleic acid 3 are prepared to be 0.4 μM.

  Thereafter, using an automatic base sequence analyzer disclosed in Japanese Patent Application Laid-Open No. 2004-61426 and Japanese Patent Application Laid-Open No. 2004-125777, after being left at 25 ° C. for 30 minutes, an insertion agent Hoechst 33258 is supplied onto the chip, Chemical measurements were taken.

  In FIG. 13, the measurement result by said method is shown. Electrode numbers 1 to 4 detect the current from the electrode immobilized from the negative control probe as the background current. Electrode numbers 5 to 8 indicate currents from electrodes on which positive control probes are immobilized for comparison. Electrode numbers 9 to 12 indicate currents from electrodes on which aptamer probes are immobilized for detection. (A) shows the case where the target biomolecule thrombin is not included, and (b) shows the experimental result when thrombin is included. As can be seen from this, it has been shown that when thrombin is contained, the current value decreases, and the effect of detection by this method was demonstrated.

1: Reaction cell 2: First electrode (for detection)
3: Second electrode (for comparison)
11: first nucleic acid strand pair 12: second nucleic acid strand pair 21: first nucleic acid strand 22: second nucleic acid strand 23: third nucleic acid strand 24: fourth nucleic acid strand 25: fifth nucleic acid Strand pair 26: Sixth nucleic acid strand 41: Target biomolecule 51: Intercalator molecule 61, 62: Electrochemical signal

Claims (9)

A reaction cell, first and second electrodes provided in the reaction cell, one end fixed to the first electrode, function as an aptamer for a target biomolecule, and bind to the target biomolecule Providing a device comprising: a possible first nucleic acid strand; and a second nucleic acid strand, one end of which is immobilized on the second electrode and not functioning as an aptamer for the target biomolecule;
In the reaction cell, a first nucleic acid strand that does not form a double strand with the first and second nucleic acid strands and a complementary strand thereof, a region that forms a double strand with the second nucleic acid strand, and Supplying a fourth nucleic acid strand having a region forming a double strand with the third nucleic acid strand;
Supplying a sample solution that may contain the target biomolecule, and subjecting the target biomolecule and the first nucleic acid strand to a binding condition;
In the reaction cell, a fifth nucleic acid strand that does not form a double strand with the first to fourth nucleic acid strands, a single-stranded region that can form a double strand with the first nucleic acid strand, and the fifth strand A first nucleic acid strand that has a region forming a double strand with the first nucleic acid strand, and the first nucleic acid strand not bound to the target biomolecule and the sixth nucleic acid strand A step of forming a double strand with a single-stranded region; and
From the first electrode, a first electrochemical signal derived from a double strand formed between the first nucleic acid strand and the fifth nucleic acid strand and the sixth nucleic acid strand, and the first Detecting a second electrochemical signal derived from a double strand formed between the second nucleic acid strand and the third nucleic acid strand from the second electrode and the fourth nucleic acid strand;
A method for detecting a target biomolecule, comprising the step of calculating an amount of the target biomolecule from a difference between the first electrochemical signal and the second electrochemical signal. Supplying the third nucleic acid strand and the fourth nucleic acid strand to the reaction cell;
2. The step of supplying a sample solution that may contain the target biomolecule and placing the target biomolecule and the first nucleic acid strand under a condition for binding is performed simultaneously. Method for detecting target biomolecules. A reaction cell, first and second electrodes provided in the reaction cell, one end fixed to the first electrode, function as an aptamer for a target biomolecule, and bind to the target biomolecule A possible first nucleic acid strand, a second nucleic acid strand, one end of which is immobilized on the second electrode, which does not function as an aptamer for the target biomolecule, the first and second nucleic acid strands, and A third nucleic acid strand that does not form a double strand with the complementary strand, a region that forms a double strand with the second nucleic acid strand, and a region that forms a double strand with the third nucleic acid strand Providing a device comprising a fourth nucleic acid strand having:
Supplying a sample solution that may contain the target biomolecule, and subjecting the target biomolecule and the first nucleic acid strand to a binding condition;
In the reaction cell, a fifth nucleic acid strand that does not form a double strand with the first to fourth nucleic acid strands, a single-stranded region that can form a double strand with the first nucleic acid strand, and the fifth strand A first nucleic acid strand that has a region forming a double strand with the first nucleic acid strand, and the first nucleic acid strand not bound to the target biomolecule and the sixth nucleic acid strand A step of forming a double strand with a single-stranded region; and
From the first electrode, a first electrochemical signal derived from a double strand formed between the first nucleic acid strand and the fifth nucleic acid strand and the sixth nucleic acid strand, and the first Detecting a second electrochemical signal derived from a double strand formed between the second nucleic acid strand and the third nucleic acid strand from the second electrode and the fourth nucleic acid strand;
A method for detecting a target biomolecule, comprising the step of calculating an amount of the target biomolecule from a difference between the first electrochemical signal and the second electrochemical signal.   The method for detecting a target biomolecule according to claim 3, wherein the second nucleic acid chain and the third nucleic acid chain are linked. Detecting the first and second electrochemical signals comprises:
Supplying an intercalator solution that specifically binds to a double strand in the reaction cell, and binding the intercalator to the double strand in the reactive cell;
5. The target biomolecule according to claim 1, wherein a step of detecting an electrochemical signal generated by oxidation or reduction reaction of the intercalating agent with the first electrode and the second electrode is performed. Detection method. A reaction cell;
First and second electrodes provided in the reaction cell;
A first nucleic acid chain immobilized on the first electrode, functioning as an aptamer for a target biomolecule, and capable of binding to the target biomolecule; and one end immobilized on the second electrode; A second nucleic acid chain that does not function as an aptamer for a biomolecule, and a device comprising:
A third nucleic acid strand that does not form a double strand with the first and second nucleic acid strands and their complementary strands;
A fourth nucleic acid strand having a single-stranded region capable of forming a double strand with the second nucleic acid strand and a region forming a double strand with the third nucleic acid strand;
A fifth nucleic acid strand that does not form a double strand with the first to fourth nucleic acid strands;
A sixth nucleic acid strand having a single-stranded region capable of forming a double strand with the first nucleic acid strand and a region forming a double strand with the fifth nucleic acid strand;
A kit for detecting a target biomolecule, comprising: A reaction cell, first and second electrodes provided in the reaction cell, one end fixed to the first electrode, function as an aptamer for a target biomolecule, and bind to the target biomolecule A possible first nucleic acid strand, a second nucleic acid strand, one end of which is immobilized on the second electrode, which does not function as an aptamer for the target biomolecule, the first and second nucleic acid strands, and A third nucleic acid strand that does not form a double strand with the complementary strand, a region that forms a double strand with the second nucleic acid strand, and a region that forms a double strand with the third nucleic acid strand A device comprising: a fourth nucleic acid strand having:
A fifth nucleic acid strand that does not form a double strand with the first to fourth nucleic acid strands;
A sixth nucleic acid strand having a single-stranded region capable of forming a double strand with the first nucleic acid strand and a region forming a double strand with the fifth nucleic acid strand;
A kit for detecting a target biomolecule, comprising:   The method for detecting a target biomolecule according to claim 7, wherein the second nucleic acid chain and the third nucleic acid chain are linked.   The kit for detecting a target biomolecule according to any one of claims 6 to 8, further comprising an intercalating agent solution that specifically binds to double strands.

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